Mode of action of exocarpic acid against mycobacterium tuberculosis

Natural products continue to be a driving force in drug development, particularly for the treatment of infectious diseases. A stunning one third of the world's population (2 billion individuals) is infected by Mycobacterium tuberculosis, the causative agents of pulmonary tuberculosis. While most of these cases are quiescent, about 8 million people suffer from active disease, resulting in a death toll of 2 million people annually worldwide. Exocarpic acid, a polyacetylenic fatty acid isolated from Exocarpos latifolia Santalacea R.Br., native to Papua New Guinea, has shown promising activity against Mycobacterium tuberculosis in vitro. Since several active exocarpic acid analogs were also found in Exocarpos extracts, the decision was made to develop exocarpic acid further as a pharmacophore. The central theme of this dissertation concerns the mechanism of action of exocarpic acid, since it needed to be elucidated before exocarpic acid can be further developed. Mechanistic lead information was developed through microarray data. Inhibition of fatty acid degradation, fatty acid biosynthesis, and amino acid starvation were the primary gene families induced. Physico-chemical consideration also suggested a potential effect on the mycobacterial cell membrane. Experiments designed to address these mechanisms revealed that exocarpic acid exhibits all the signs of a fatty acid biosynthesis system II inhibitor, without any recognizable effects on the mycobacterial membrane. In order to generate lead information for the next step in exocarpic acid drug development, several amide derivatives of exocarpic acid were synthesized. Some of these compounds showed acceptable activity against Mycobacterium tuberculosis without concomitant toxicity against eukaryotic cells. In order to provide initial quantitative structural activity data, a set of exocarpic acid analogs, which lack an unsaturated bond found in exocarpic acid, were also tested. These exhibited a wide array of mechanisms, indicating that the chain-length and unsaturated bond positions are crucial for activity against Mycobacterium tuberculosis. Exocarpic acid's conjugated double-bond acetylenic bond structure may therefore be a valuable pharmacophore for further development.

MODE OF ACTION OF EXOCARPIC ACID AGAINST MYCOBACTERIUM TUBERCULOSIS by Michael Koch A dissertation submitted to the faculty of the University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pharmacology and Toxicology The University of Utah August 2009 Copyright © Michael Koch 2009 All Rights Reserved THE U N I V E R S I T Y OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Michael Koch This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. l> I 3 f Chair: Louis R. Barrows Andrea Bild 9 0<f b f l s l p t j Timothy Bugni Matthew H. Slavvson v Mohammad Sondossi THE U N I V E R S I T Y OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Michael Koch jn jts f m a j form and have found that (1) its format, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. Date Louis R. Barrows Chair: Supervisory Committee Approved for the Major Department Chair/Dean Approved for the Graduate Council C C O f • t r David S. Chapmnaalri Dean of The Graduate School ABSTRACT Natural products continue to be a driving force in drug development, particularly for the treatment of infectious diseases. A stunning one third of the world's population (2 billion individuals) is infected by Mycobacterium tuberculosis, the causative agents of pulmonary tuberculosis. While most of these cases are quiescent, about 8 million people suffer from active disease, resulting in a death toll of 2 million people annually worldwide. Exocarpic acid, a polyacetylenic fatty acid isolated from Exocarpos latifolia Santalacea R.Br., native to Papua New Guinea, has shown promising activity against Mycobacterium tuberculosis in vitro. Since several active exocarpic acid analogs were also found in Exocarpos extracts, the decision was made to develop exocarpic acid further as a pharmacophore. The central theme of this dissertation concerns the mechanism of action of exocarpic acid, since it needed to be elucidated before exocarpic acid can be further developed. Mechanistic lead information was developed through microarray data. Inhibition of fatty acid degradation, fatty acid biosynthesis, and amino acid starvation were the primary gene families induced. Physico-chemical consideration also suggested a potential effect on the mycobacterial cell membrane. Experiments designed to address these mechanisms revealed that exocarpic acid exhibits all the signs of a fatty acid biosynthesis system II inhibitor, without any recognizable effects on the mycobacterial membrane. In order to generate lead information for the next step in exocarpic acid drug development, several amide derivatives of exocarpic acid were synthesized. Some of these compounds showed acceptable activity against Mycobacterium tuberculosis without concomitant toxicity against eukaryotic cells. In order to provide initial quantitative structural activity data, a set of exocarpic acid analogs, which lack an unsaturated bond found in exocarpic acid, were also tested. These exhibited a wide array of mechanisms, indicating that the chain-length and unsaturated bond positions are crucial for activity against Mycobacterium tuberculosis. Exocarpic acid's conjugated double-bond acetylenic bond structure may therefore be a valuable pharmacophore for further development. This dissertation is dedicated to my family and friends, especially my wife Raquel and my daughter Cassandra Rosemarie. TABLE OF CONTENTS ABSTRACT iv LIST OF TABLES x LIST OF FIGURES xii LIST OF ABBREVIATIONS xiii Chapter 1 - BACKGROUND INFORMATION 1 Introduction 1 Pharmacognosy 2 Natural products discovery process 2 Tuberculosis 6 Relevance of tuberculosis treatment to developed countries 8 Current tuberculosis treatments 9 Mycobacterium tuberculosis 10 Mycobacterial cell wall composition and synthesis 11 Drugs in development 22 Compound class: Polyacetylenic fatty acids 22 Exocarpic acid 24 References 25 2 - ISOLATION, ACTIVITY AND STRUCTURE OF EXOCARPIC ACID AND ITS NATURAL ANALOGS 30 Introduction 30 Materials and methods 31 Organisms and chemicals 31 Extraction and isolation 32 Bacterial growth assays 33 Rifampicin and isoniazid potentiation assay 34 Characterization of compounds 1, 2, 3, 4, 5 and 6 34 Results 35 Isolation and characterization of compounds 1, 2, 3, 4, 5 and 6 35 Antimycobacterial activity of compounds 1, 2, 3, 4, 5 and 6 40 Discussion 44 Antimycobacterial activity and structure 44 Potentiation and mode of action 44 Future directions 45 References 45 3 - TRANSCRIPTOME STUDIES 48 Introduction 48 Materials and methods 49 Exocarpic acid transcriptome 49 Hierarchical cluster analysis using Cluster 3.0 50 Gene set enrichment analysis (GSEA) 53 Biological pathway analysis 55 Results 55 Exocarpic acid transcriptome 55 Comparison to published signature gene sets 59 Hierarchical cluster analysis using Cluster 3.0 61 Gene set enrichment algorithm 64 Biological pathway analysis 69 Discussion 69 Hierarchical cluster analysis 70 Gene set enrichment algorithm 71 Biological pathway analysis 72 References 73 4 - MODE OF ACTION STUDIES 75 Introduction 75 Proton gradient dissipation studies 76 Fatty acid biosynthesis inhibition 77 Materials and methods 78 Proton gradient dissipation 78 Fatty acid biosynthesis inhibition 79 Results 81 Proton gradient dissipation 81 Fatty acid biosynthesis inhibition 83 Discussion 88 • Proton gradient inhibition 88 Fatty acid biosynthesis inhibition 88 References 90 5 - EXOCARPIC ACID DERIVATIVES AND ANALOGS 91 Introduction 91 Material and methods 92 Exocarpic acid amides 92 Commercial exocarpic acid analogues 96 Results 99 viii Exocarpic acid amides 99 Commercially available analogs of exocarpic acid 101 Cytotoxicity and activity 101 Proton gradient dissipation 102 Fatty acid biosynthesis inhibition 104 Discussion 107 Exocarpic acid amides 107 Commercially available analogs of exocarpic acid 107 References 108 6 - SUMMARY AND FUTURE DIRECTIONS 110 Summary 110 Future directions 113 ix LIST OF TABLES Table Page 1.1 Primary drugs used to treat tuberculosis in the USA 9 2.1 and 1 3C NMR data for compounds 2, 3, 4 and 6 36 2.2 Antimycobacterial activity of exocarpic acid analogs against Mtb H37Ra..41 3.1 Genes induced in Mycobacterium tuberculosis H37Rv upon treatment with 40 pg/ml exocarpic acid for 4 hours 56 3.2 Comparison of published gene cluster data for cell wall inhibitors to expression data for exocarpic acid 59 3.3 Correlation coefficients derived for exocarpic acid versus other drug treatments 65 3.4 Enrichment scores for exocarpic acid transcriptional data 66 3.5 Top ranked genes identified by GSEA in the INH array that are positively correlated 67 3.6 Biological pathways identified for 40 pg/mL exocarpic acid treatment of Mycobacterium tuberculosis H37Rv 69 4.1 Rhodamine 123 fluorescence in Mycobacterium tuberculosis H37Ra after treatment with INH, EXO, CCCP and TRC 81 4.2 Relative ATP concentration in Mycobacterium tuberculosis H37Rv under microaerophilic conditions 83 5.1 In vitro cytotoxicity of exocarpic acid amides 99 5.2 MIC of exocarpic acid amides against M. tuberculosis H37Ra 100 5.3 In vitro cytotoxicity of GFS series of compounds against TART cells 101 5.4 Activity of GFS series of compounds against M. tuberculosis H37Ra 102 Rhodamine 123 fluorescence in Mycobacterium tuberculosis H37Ra after treatment with GFS series compounds xi LIST OF FIGURES Figure Page 1.1 Participants of the Papua New Guinea ICBG 4 1.2 Overall structural make-up of the mycobacterial cell wall 12 1.3 Various forms of mycolic acids found in mycobacteria 15 1.4 Overall scheme of mycolic acid biosynthesis showing the involvement of the FAS-I and FAS-II systems 17 1.5 Biosynthetic cycle of FAS-II and associated enzymes 20 2.1 Structure of E-octadeca-D-en-^ll-diynoic-acid and derivatives 37 2.2 Exocarpic acid enhances antimycobacterial activity of antibiotics 42 3.1 Overall scheme for preparation of microarray data for HCL and GSEA Analysis 51 3.2 Dendrogram showing hierarchical clustering of arrays 62 4.1 Incorporation of 14C-acetate into the fatty acids and mycolic acids in M. tuberculosis H37Ra 84 4.2 Dose effect on incorporation of 14C-acetate into the fatty acids and mycolic acids in M. tuberculosis H37Ra 86 5.1 Structures of synthesized exocarpic acid derivatives 94 5.2 Structures of exocarpic acid analogs 97 5.3 Incorporation of 14C-acetate after treatment of Mycobacterium tuberculosis H37Ra with exocarpic acid analogs 105 LIST OF ABBREVIATIONS ACP Acyl Carrier Protein ADC Albumin, Dextrose, Catalase enrichment C-# Fatty acid with # of carbons CCCP cynanide-m-chlorophenyl hydrazone CER cerulenin CEM acute lymphoblastic leukemia cell line CEM-TART CEM T-cells expressing both HIV-1 tat and rev COSY Correlation Spectroscopy DCM dichloromethane; methylene chloride DMSO dimethyl sulfoxide DOTS Direct Observed - Short Course EC 100 Effective Concentration 100 percent ETA ehtionamide EXO exocarpic acid FAS-I Fatty Acid Synthesis system I FAS-1I Fatty Acid Synthesis system II HBC High Burden Country HMBC Heteronuclear Multiple Bond Correlation HRESIMS High Resolution ElectroSpray Ionization Mass Spectroscopy INH isoniazid MDR Multiply Drug Resistant MIC Minimal Inhibitory Concentration MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NIAID National Institutes for Allergy and Infectious Diseases NIH National Institutes of Health NMR Nuclear Magnetic Resonance PI propidium iodide R123 rhodamine 123 RIF rifampicin TLM thiolactomycin TRC triclosan XDR Extremely Drug Resistant CHAPTER 1 BACKGROUND INFORMATION Introduction This dissertation summarizes the ongoing work regarding the polyacetylenic fatty acid exocarpic acid (ii-13-ene-octadeca-9,ll-diynoic acid). Exocarpic acid was isolated in the labs of Dr. Louis Barrows and Dr. Chris Ireland at the University of Utah in 2004 from a stem extract of the plant Exocarpos latifolius (Santalaceae), native to Papua New Guinea. Exocarpic acid exhibited bactericidal activity against Mycobacterium tuberculosis H37Ra, a model system for the disease tuberculosis. Few reports of active polyacetylenic fatty against M. tuberculosis exist, hence the decision was made to investigate exocarpic acid further, specifically the mode of action of exocarpic acid against M. tuberculosis. This chapter provides a brief introduction covering the process of drug discovery in the context of the International Cooperative Biodiversity Group Papua New Guinea (ICBG PNG), followed by a discussion of tuberculosis as a public health concern and current treatments against tuberculosis. Many of the currently available drugs against tuberculosis interfere in cell wall biosynthesis of M. tuberculosis, the causative agent of tuberculosis. The cell wall is an important antimycobacterial target; therefore a short overview of the cell envelope structure and biosynthesis of the fatty acid components of the cell wall is provided. A brief summary of compounds which fall into the larger group of polyacetylenic fatty acids and are structurally related to exocarpic acid will be presented as well, followed by the hypothesis and rationale for the experiments presented in Chapters 2 through 5. Pharmacognosy Infectious diseases still rank as one of the major threats to health in the world. The fight against infectious disease is as old as humanity, beginning with ritualistic cures, quarantine, ethnobotanical concoctions and finally progressing to modern targeted medicines. However, many modern medicines derive from the primitive ethnobotanical concoctions, in that they are often highly potent synthetic derivatives or highly purified key ingredients of the natural products found therein (1). This latter relationship in conjunction with rational investigation is now the practice of pharmacognosy. Specifically, this manuscript reports on one outcome of our ongoing search for drugs and drug-like molecules from Papua New Guinean plants with focus on antituberculosis activity. Natural products discovery process Natural products are an important source for drug discovery. Most drugs on the market today are either natural products or are derived from natural products (2). The underlying reason is that natural products display astounding chemical diversity, potentially providing scaffolds/pharmacophores for a vast range of biological targets. Accessing biodiversity in order to discover new pharmacophores has therefore become of importance. To facilitate this process the National Institutes of Health (NIH) has sponsored the International Cooperative Biodiversity Groups (ICBGs) in which US and 3 international academic, corporate and governmental agencies interact in order to facilitate access to plant, marine, and microbial derived chemical entities with bioactive properties. The research described here was conducted under the auspices of an ICBG with the partner country of Papua New Guinea (PNG). The PNG ICBG consists of the University of Utah (Associated Programs (AP) 1, 3, 4), the University of Papua New Guinea (AP 1), the Smithsonian Institute (AP 2) and Wyeth Research (AP 5), an industrial partner. The relationships between the programs is depicted in Figure 1.1. The screening of natural products against disease models of HIV/AIDS, malaria and tuberculosis (M. tuberculosis) was performed in the Barrows laboratory (AP 3). Plant extraction and high-throughput chemical identification was performed by the Ireland Lab (AP4). In general, if a fraction generated by AP4 was shown to be active by AP3 the fraction of interest was then subfractionated, and these subfractions were subsequently re-assayed, resulting in a short discovery loop that leads to the isolation of the active compound(s) in an extract. AP4 also dereplicated initial hits by liquid chromatography coupled mass spectrometry (LCMS) to determine if the constituents of the fractions correspond to known compounds. One of the compounds identified in the tuberculosis model assay was exocarpic acid from an Exocarpos latifolius (Santalaceae) stem extract (voucher specimen U20180-064). Further investigation revealed that several exocarpic acid analogs were also present in the extracts and exhibited antimycobacterial activity. These findings are presented in Chapter 2, along with the determination of activity of exocarpic acid against M. tuberculosis. Figure 1.1. Participants of the Papua New Guinea ICBG. 5 T. M A T A I N A H O , U P N G A P I ASSESSMENT, C O N S E R V A T I O N AND DEVELOPMENT OF P N G BIOLOGICAL RESOURCES P N G I C B G "CONSERVATION AND SUSTAINABLE USE OF BIOLOGICAL RESOURCES IN PAPUA NEW GUINEA" L . BARROWS, UNIV. OF U T A H A P 6 A D M I N I S T R A T I V E C O R E R . B A N K A NATIONAL FOREST RESEARCH INSTITUTE G. C A R T E R , W Y E T H A P S I N D U S T R Y - B A S E D DRUG DISCOVERY AND MICROBE I S O L A T I O N C. IRELAND, UNIV. OF U T A H A P 4 C H E M I S T R Y Tuberculosis Tuberculosis (TB) is most often taken to be a slow progressive pulmonary disease caused by M. tuberculosis. However, this view is not quite correct. Tuberculosis can be a disease of one or more organs, e.g., the lungs, CNS, circulatory system (including the lymphatics), GI tract, bones (Pott's disease), and rarely the skin (lupus vulgaris). This is strongly evidenced by archeological finds showing systemic tuberculosis lesions in Egyptian and Peruvian mummies, often leading to the assertion that tuberculosis is one of the oldest diseases of humans (3). Tuberculosis can also be caused by mycobacteria other than M. tuberculosis, e.g., M. bovis, M. microti, M. africanum and M. canetti. The most common form is pulmonary tuberculosis, which is also the disease state of most concern globally due to its high prevalence, comorbidity with HIV/AIDS, and high mortality (4). Prevalence of tuberculosis is truly staggering with at least one third of the global population, i.e., more than 2 billion people, being positive for M. tuberculosis infection. Most of these individuals have latent, currently nonprogressive disease. Latent infections can become active under certain conditions, e.g., if the immune system becomes weakened by HIV/AIDS. This explains the comorbidity of tuberculosis with HIV/AIDS. Progression from latent to active disease in HIV/AIDS patients has become a devastating public health issue in countries such as Russia and South Africa. In these countries HIV/AIDS and tuberculosis rates have increased dramatically due to a combination of failing health care systems, substance abuse, insufficient public health education, and other contributing influences. While the global tuberculosis burden is estimated at over 2 billion cases, only approximately 8 million individuals currently have active disease, with about half of 7 those being sputum smear-positive, which is a sign of having aggressive progressive disease (5). The global annual death toll is estimated to be approximately 1.6 million individuals. Since even the shortest course of treatment is 6 months in duration the relapse rate is very high, usually due to compliance issues. The World Health Organization (WHO) has established the directly observed treatment short-course (DOTS) as the appropriate strategy to control tuberculosis. DOTS consists of the drug combination of rifampicin, isoniazid and pyrazinamide administered under observation by medical/official personnel over 6 months. In the USA, the drug regimen is shifting towards four drugs: rifampicin, isoniazid, pyrazinamide and either ethambutol or streptomycin (6). While DOTS has been successful it is ultimately, like most large scale antibiotic applications, doomed to fail. As a matter of fact, the 1990 rank order of disease burden in the world for tuberculosis was 7 and in 2020 it is projected to remain at rank 7 (7). The beginning of the end of effective tuberculosis treatment can be seen in the emergence of multiple-drug resistant (MDR) and extremely drug resistant (XDR) strains of M. tuberculosis. The degree of resistance among new cases is about 5%, while that of previously treated cases is 30%. The formal CDC definition for MDR strains is "Multidrug-resistant TB (MDR TB) is TB that is resistant to at least two of the best anti- TB drugs, isoniazid and rifampicin." (8). The formal CDC definition for XDR strains is: disease caused by Mycobacterium tuberculosis that is resistant in vitro to at least isoniazid and rifampin among first-line drugs, and at least three or more of the six main classes of second-line drugs (aminoglycosides, polypeptides, fluoroquinolones, thioamides, cycloserine, and para-aminosalicyclic acid). (9) p.250 Taken together, the long treatment period and emergence of MDR and XDR M. tuberculosis present a formidable challenge to the control of tuberculosis (10). New antitubercular drugs are sought after diligently by researchers worldwide. Considering 8 that the number of druggable targets in M. tuberculosis is estimated to be low, the search for pharmacophores that address new targets, or known targets in a new way, is of paramount importance (11, 12). Relevance of tuberculosis treatment to developed countries The division of the world into developed and developing countries with respect to tuberculosis presents a moving target. The WHO has assigned 22 countries as High Burden Countries (HBCs): Congo DR, Ethiopia, Kenya, Nigeria, Uganda, South Africa, Tanzania (United Rep. of), Zimbabwe, Russian Federation, Brazil, Peru, Afghanistan, Bangladesh, Cambodia, China, India, Indonesia, Myanmar, Pakistan, Philippines, Thailand, Viet-Nam (13). While most fall into the category of developing country, Russia or Brazil, and perhaps even China, are usually not thought of as developing countries in the same manner that one would regard Zimbabwe or Cambodia. High population pressures (e.g., China) and lack of adequate health care facilities (e.g., post-Soviet Russia) are more indicative of tuberculosis burden than developmental status as measured by economic output. Given the ease of communication of tuberculosis, emergence of drug-resistant strains, and global mobility of individuals from HBCs to other countries, it is not surprising that large multicultural cities, such as London, now have rates of tuberculosis that are comparable to HBCs (14). It is becoming clear that tuberculosis is not going to remain a disease of the poor and/or disenfranchised, but rather tuberculosis will make its way into mainstream society, as recently reported in California (15). Tuberculosis remains a major threat to global health, and while countries of high endemicity are still the top priority in the battle against tuberculosis, low burden will see an increased incidence of tuberculosis due to migration, population pressure and drug resistant strains. Current tuberculosis treatments The current highly effective first line drugs used against M. tuberculosis in the US are tabulated in Table 1.1 along with their purported target gene and involvement in metabolic pathways of M. tuberculosis. The number of effective drugs and affected targets is small and clustered around cell wall biosynthesis inhibition. An increase in drug resistant strains of M. tuberculosis will render several of these drugs ineffective as therapeutics in the future. Table 1.1. Primary drugs used to treat tuberculosis in the USA. Drug Target Pathway Ethambutol (E or EMB) Arabinosyl transferase Cell wall biosynthesis: Mycolic acid maturation Isoniazid Cell wall biosynthesis: (I or INH) InhA Long chain fatty acid biosynthesis (FAS-II) Ethionamide Cell wall biosynthesis: (ETH) InhA (?) Long chain fatty acid biosynthesis (FAS-II) Pyrazinamide Cell wall biosynthesis: (Z or PZA) Fas De novo fatty acid biosynthesis (FAS-I) p-aminosalicylic acid (P or PAS) FolK/PobA Folic acid biosynthesis Rifampicin RpoB RNA synthesis (R or RIF) (RNA polymerase inhibition) Ciprofloxacin GyrA DNA replication inhibition (C or CIP) (topoisomerase inhibition) Streptomycin (S or STM) 16S RNA Protein synthesis inhibition 10 Second line drugs include Ciprofloxacin and Moxifloxacin, both of which are fluoroquinolones and target DNA gyrases (topoisomerase II and IV). Para-aminoacetylsalicylic acid (4- aminoacetylsalicylic acid or PAS), which interferes with folic acid metabolism in mycobacteria and was once a first line drug, but since then has been superseded by isoniazid (16). It should be noted that the antibiotics which specifically target M. tuberculosis often interfere with cell wall integrity. This is especially true for isoniazid, an inhibitor of InhA, the enoyl reductase in the fatty acid biosynthesis system II (FAS-II). Ethambutol inhibits the final maturation step of mycolic acids by interfering in attachment of the mycolic acids to arabinosyl residues on the outer envelope as shown below. Thiolactomycin inhibits the KasA/KasB enzymes (17). Mycobacteria have a fatty acid biosynthesis I system (FAS-I) system that is remarkable similar to the eukaryotic FAS-I system. Pyrazinamide inhibits the FAS-I system. Why pyrazinamide is selective for mycobacterial FAS-I and not FAS-I systems in other organisms remains unresolved. Cerulenin, a research drug, inhibits KAS-III, an essential enzyme coupling the FAS-I and FAS-II systems. Mycobacterium tuberculosis M. tuberculosis falls in the actinomycobacteriales and is a nonmotile, slow growing, aerobic, rod-shaped Gram-positive bacterium. However, due to the highly lipid rich cell wall, mycobacteria are usually classified as acid fast bacteria, based on the retention of the dye carbolfuchsin in the cell after rinsing with acid alcohol (18). The lipid rich cell wall sets M. tuberculosis apart from most other bacteria and therefore presents 11 an attractive drug target. As shown in Table 1.1, many of the current tuberculosis drugs target cell wall metabolism (19). The cell wall of M. tuberculosis is critical for survival and virulence, because it bears trehalose dimycolate (chord factor), which allows M. tuberculosis to form caseous granulomas in the lung tissue. M. tuberculosis resides in a dormant state inside these granulomas, which presents the underlying difficulties in eradicating established infection. This dormancy leads to the requirement for long-term multidrug therapy (DOTS, see above). Mycobacterial cell wall composition and synthesis The mycobacterial cell wall contains highly organized functional subunits composed of lipids, peptides, carbohydrate moieties and a special class of long-chain fatty acids termed mycolic acids (Figure 1.2). The peptidoglycan-arabinoglactan portion of the cell wall is not discussed here, the reader is referred to the book by Daffe et al. (20). The peptidoglycan layer is relatively thin in comparison with other Gram-positive organisms. A short linker joins the peptidoglycan layer and a long-chain P-D-galactofuran layer, which in turn serves as the scaffold for several different a-D-arabinofuran core molecules. Every second a-D-arabinofuran core is modified at the head-group to branch three times resulting in eight attachment sites for mycolic acids. The mycolic acids are C-60 to C-90 a-branched p-hydroxylated fatty acids with various chemical modifications on the larger acyl chain. 12 Figure 1.2 Overall structural make-up of the mycobacterial cell wall. Adapted from (21) with permission. 13 Mycotic Acids oc-D-Ara f, a-D-Araf i ct-D-Araf ,1' n-D-Araf s r u-o-Araf Arabinofuran -o-Araf B-D-Araf P-o-Ara/ a-D-Araf a-o-Araf a-o-Araf5 J ^ I IYS a-D-Araf a-D-Araf (a-o-Araf) S u o , c i n y l ( a-o-Araf) a-o-Araf (a-D-Araf) sV ~U o-D-Araf , r ^ a-o-IA 'r af p-D-Gair-^'p-D-Galf -'(i-D-Galf-^-'p-D-Galf -Ip-D-Galf-' P-D-Galf -' P-D-Galf-'p a-L-Rhap 3 Galactofuran ,r a-D-GlcNAcp Linker Peptidoglycan - RK- D-GICNAc- 1P -D-Mu|rjN Ac'- P' -D-GlcNAc B-D-Mur(N'G Iyc'-»p-D-GlcNAc I-- p-D-MutrN5 Ac-• P-D-G Ajpm I /-D-Glu I I.-Ala ,1 L-Ala /A-DzpII-Gm lu A;pm- I /'-D-Glu I L-Ala V L-Ala A2pm P-D-MurNGIyc -^P-D-GICNAC -4P-o-MurNAc ^-D-GICNAC-~P-D-MurNGIyc-JP-D-GICNAC-4P-D-|I Plasma Membrane 14 Figure 1.3 provides an overview of mycolic acids found in mycobacteria. As can be seen from Figure 1.3, the a-branch chain is 26 carbons long and is not modified. It is derived directly from the de novo fatty acid synthesis I (FAS-I) system. The meromycolate branch is synthesized by the fatty acid synthesis II system (FAS-II). The meromycolates are from 48-62 carbons long and decorated with cyclopropanyl rings, keto and/or methoxy groups. As mentioned, the meromycloate branch is the product of the FAS-II system and various modifying enzymes. The structural diversity of meromycolate branch of the mycolic acids differs amongst various members of the mycobacteria, as shown in Figure 1.3, and has been used to type mycobacteria. The a-mycolate and meromycloate branches of the mycolic acid are synthesized separately and eventually joined using a Claisen condensation as depicted in Figure 1.4. Mycobacteria are unusual in that they have a full FAS-I system similar to eukaryotes, which unfortunately makes this system a poor choice for drug targets. Fatty acid synthesis is initiated by one of the many acyl-CoA carboxylases (ACCases) which transfer a carboxyl group to acetyl-CoA to form malonyl-CoA (22). Mycobacterial ACCases exhibit rather broad substrate specificities, which include acetyl, proprionyl and butyryl-CoA. Fas-IA is the essential enzyme that utilizes these precursors to generate C16/C18 or C24/C26 products by successive condensation of acetyl and malonyl subunits. FAS-II initiates synthesis of meromycolates with C16- or CI8-C0A precursors from the FAS-I system. The initial substrates for FAS-II are an acylcarrier protein (AcpM) bound precursor carbon skeleton from FAS-I and an AcpM-bound malonyl group. 15 Figure 1.3 Various forms of mycolic acids found in mycobacteria. Adapted from (20) with permission. Type of mycolate Structure MmaA2 (CmaA1) a cis/cis Methoxy cis Methoxy trans Keto cis Keto trans Hydroxy trans OH COOH OH COOH OH COOH tuberculosis, bovis, leprae, bovis BCG tuberculosis, bovis OH COOH OH COOH OH COOH tuberculosis, bovis, leprae, bovis BCG tuberculosis, bovis, bovis BCG 17 Figure 1.4 Overall scheme of mycolic acid biosynthesis showing the involvement of the FAS-I and FAS-II systems. From (20) with permission. 18 Synthesis of mycolic acid precursors Acetyl-CoA (C2-CoA) Carboxylation Malonyl-CoA Elongation and functional modifications of 'meromycolic' chain C48*C62"X (activated 'meromycolate') carboxylated-C24(26-CoA (a-branch) Mycolic acid condensation 1 Condensation Reduction Mycolic acid ( C 6 0 " C 9 0 ) The synthesis of the malonyl-ACP precursor is catalyzed by mtFabD. The P-ketoacyl ACP synthase III (KasIII) catalyzes the condensation to form a P-ketoacyl-ACP skeleton, which then enters into the FAS-II system (23). The initial FAS-II step is a NADPH dependent reduction of the P-ketoacyl carbonyl to a P-hydroxy group by P-ketoacyl-ACP reductase (MabA). The resulting P-hydroxyacyl- ACP complex is dehydrated by a P-hydroxyacyl dehydratase to form trans- A2-enoylacyl-ACP. The identity of the P-hydroxyacyl dehydratase enzyme was unknown until 2007, but it has now been fully characterized and is encoded by the hadABC genes (24). The second reduction step in the FAS-II cycle is catalyzed by trans-A2-enoylacyl reductase (InhA) using the trans-A2-enoylacyl-ACP as substrate and consuming a NADH molecule in the process. InhA is essential and the target of the antimycobacterial drug isoniazid (INH). The antimicrobial triclosan and the antibiotic ethionamide are also thought to be inhibitors of InhA (25, 26). The next condensation step is catalyzed by the P-ketoacyl ACP synthase A and P-ketoacyl ACP synthase B (KasA/KasB) enzyme pair. KasA is essential for survival and catalyzes the formation of shorter meromycolates, while KasB is not essential, but required for long-chain meromycolate synthesis. KasA has been shown to be the target of the antibacterial drug thiolactomycin (27). For sake of brevity the meromycolate modification steps are not discussed, but are detailed in (20). The FAS-II cycle along with the points of inhibition is provided in Figure 1.5. As most of the enzymes discussed above work in conjunction, it is not surprising that they are found in clusters, i.e., the fabD-acpM-kasA-kasB-accD6 genes form one transcriptional unit (operon) (27). Likewise fadD32-pksl3-accD4 form an operon (28). Figure 1.5 Biosynthetic cycle of FAS-II and associated enzymes. Adapted from (20) with permission. INH E t h i o n a m i d e NADH + H+ InhA (Fabl) R ^ ^ A C P frans-A2-enoyl-ACP TLM 0 O | MtFabD O r A s ' a c p Acyl-ACP KasA/KasB (FabF/FabB) Malonyl-ACP FAS I «- -ACP Pyrazinamide FAS II R ^ S p-ketoacyl-ACP ACP Dehydratase (FabA/Z) OH O P-hydroxyacyl-ACP MabA (FabG) NADP* Acyl-CoA MtFabH (Kas III) C e r u l e n i n XX .ACP NADPH + H* Condensation Export Decoration * 7T Mature Mycolic acid E t t i a m b u t o l Pksl3 catalyzes the mycolic acid condensation step (Figure 1.4), which joins the meromycolate and a-chains, while FadD32 is a fatty acyl AMP ligase involved in fatty acyl activation prior to condensation. Drugs in development New drugs against M. tuberculosis are being actively developed. Currently TMC- 207, OPC 67683, SQ109, nitroimidazopyran PA824, moxifloxacin, and gatifloxacin are promising new drugs that have been developed since 2006 (9). TMC-207 is very promising as it is the only known ATPase inhibitor in M. tuberculosis to date, therefore offering a new target and high potential for treating MDR/XDR infections. Nitroimidazopyran PA824 has been shown to inhibit the synthesis of proteins. The fluoroquinolone class compounds (moxifloxacin and gatifloxacin) are well-known DNA gyrase inhibitors. Less than half a dozen other novel compounds that are in earlier stages of development have also been reported. For example, NAS-91 has been shown to be a weak cell wall synthesis inhibitor (29). A list of natural products with antituberculosis activity has been also published (30). A discussion of the compounds outside of exocarpic acid's compound class would be beyond the scope of a dissertation, hence the discussion below will focus on fatty acids and related compounds with multiple conjugated acetylenic bonds. Compound class: Polyacetylenic fatty acids Prior to the discussion of polyacetylenic compounds one should be aware that many monoacetylenic compounds and conjugated / non-conjugated double and triple bond containing compounds exist in nature and some have been shown to be antibacterial and antifungal. An overview of these and related compounds has been made available on the world wide web (31). As a compound class, polyacetylenic fatty acids, i.e., fatty acids that contain 2 or more acetylenic bonds, are currently underutilized as pharmacophores. These compounds have been isolated from plants, fungi and marine animals and represent a compound class in which interest is currently high due to several promising leads: Oropheic acid (17-octadecene-9,ll,13-triynoic acid) and dihydrooropheic acid (13(£),17-octadecadiene-9,ll-diynoic acid) were isolated from the plant Mitrephora celebica (Scheff) and both had activity against methicillin resistant Staphylococcus aureus and M. smegmatis (32). The MICs were determined to be 25 pg/mL and 12.5 pg/mL for dihydrooropheic acid and oropheic acid against Staphylococcus aureus respectively. The MIC against M. smegmatis was 12.5 pg/mL for both of the compounds. Unfortunately, no M. tuberculosis strain was tested. Plants found to contain marticarial acids were used as a natural curative for pulmonary tuberculosis (33). No further data were presented in the source publication, except that marticarial acids proved to be active at an undisclosed concentration against M. chelonei (34). The diyene falcarinol (1, 9-heptadecadiene-4,6-diyn-3-ol ) and the derivative dihydrofalcarindiol were isolated from Devil's Club (Oplopanax horridus) (35). Disk diffusion assays at 10 pg per disk show activity against both M. tuberculosis and M. avium. Oropheic acid does not seem to have been developed further; however, falcarinol which has shown to have potent tumoricidal activity is being actively pursued for this application (36). Unfortunately, falcarinol is also rather cytotoxic and has been implicated as the causative agent of contact dermatitis from many plants (37). Three allenyldiyonic fatty acids, phomallenic acids A-C, were isolated from a cultured Phoma sp. fungus (38). These compounds exhibited a strong inhibition of Staphylococcus aureus FabF2 and purified FASH enzymes. In summary, several polyacetylenic compounds, which are structurally related to exocarpic acid, have been isolated over the last decade and almost all exhibit activity against some groups of bacteria. Exocarpic acid Exocarpic acid (£-13-ene-octadeca-9,ll-diynoic acid) was first described in the literature in 1963 as an antibacterial agent isolated from the plant family Olacaceae (39). As mentioned above, we have identified exocarpic acid as the active constituent in Exocarpos latifolius (Santalaceae) stem extract when tested against M. tuberculosis H37Ra. The review of the compounds related to exocarpic acid indicated that there is a general antibacterial property associated with polyacetylenic fatty acids. As detailed in Chapter 2, exocarpic acid exhibited high specificity for M. tuberculosis H37Ra, but was essentially inactive against other bacteria we tested. The decision was therefore made to investigate the mode of action of exocarpic acid in order to determine if exocarpic acid could be leveraged as a pharmacophore. The hypothesis was that exocarpic acid interfered with cell wall integrity in M. tuberculosis, either through membrane permeabilization, or cell wall biosynthesis inhibition. The case for membrane permeabilization as a mode of action was derived from the fact that many fatty acids are thought to disrupt the cell membrane, thereby collapsing 25 the proton gradient, which leads to cell death. This is thought of as the canonical mode of action for many "detergent-like" molecules (40). Certainly, exocarpic acid, which has a long hydrophobic aliphatic moiety and a highly hydrophilic head group, fits this molecular pattern and could easily be envisioned to act in such a manner. The specific hypothesis was tested via flow cytometry using a proton gradient sensitive fluorescent dye and is detailed in Chapter 4. Inhibition of cell wall biosynthesis as a mode of action was proposed, since exocarpic acid, as a nonsubstituted 18-carbon fatty acid, could mimic FAS-I precursors fatty acids and enter into the FAS-II cycle, potentially leading to enzyme inhibition (41). Evidence gathered from micro-array analysis of exocarpic acid treated Mycobacterium tuberculosis H37Rv also suggested interference in the fatty acid biosynthesis system. This hypothesized mode of action was tested by following radioactive 14C-acetate incorporation into fatty acids as detailed in Chapter 4. The hypothesis that exocarpic acid derivatives and analogs retain antimycobacterial activity is addressed in Chapter 5. To this end, amide analogs of exocarpic acid were synthesized and tested for antimycobacterial activity. In addition, commercially available polyacetylenic fatty acids, of varied chain length and lacking the conjugated double of exocarpic acid, were assayed to elucidate a structure activity relationship to exocarpic acid. References 1. Ji HF, Li XJ, Zhang HY. Natural products and drug discovery. Can thousands of years of ancient medical knowledge lead us to new and powerful drug combinations in the fight against cancer and dementia? EMBO Reports 2009;10:194-200. 26 2. Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007;70:461-477. 3. Zink AR, Sola C, Reischl U, Grabner W, Rastogi N, Wolf H, Nerlich AG. Characterization of Mycobacterium tuberculosis complex DNAs from Egyptian mummies by spoligotyping. J Clin Microbiol 2003;41:359-367. 4. Gandhi NR, Mol A, Pawinski R, U Lalloo, Sturm AW, Zeller K, et al. High prevalence and mortality from extensively-drug resistant (XDR) TB in TB/HIV coinfected patients in rural South Africa. August 13-18, 2006. XVI International AIDS Conference. Toronto, August 13-18, 2006. 5. WHO tuberculosis fact sheet, http://www.who.int/mediacentre/factsheets/fsl04/en/ Accessed April 2009. 6. CDC core curriculum on Tuberculosis 2000 http://www.cdc.gov/tb/pubs/corecurr/Chapter7/Chapter_7_Regimens- Pulmonary_TB.htm Accessed April 2009 7. The global infectious disease threat and its implications for the United States. http://www.dni.gov/nic/special_globalinfectious.html Accessed April 2009. 8. CDC fact sheet multidrug-resistant tuberculosis (MDR TB) http://www.cdc.gov/tb/publications/factsheets/drtb/mdrtb.htm Accessed June 2009. 9. CDC MMWR morbidity and mortality weekly report March 23 2007;56:245-272. 10. WHO anti-tuberculosis drug resistance in the world Report No. 4 2008. www.who.int/tb/publications/2008/drs_report4_26feb08.pdf Accessed April 2009. 11. Hasan S, Daugelat S, Rao PSS, Schreiber M. Prioritizing genomic drug targets in pathogens: application to Mycobacterium tuberculosis. PLoS Comput Biol 2006;6:e61. 12. Dennis J Murphy, James R Brown. Novel drug target strategies against Mycobacterium tuberculosis. Curr Opin Microbiol 2008;11:422-427. 13. WHO Global Plan to Stop TB 2002 Annex 1 through 4. http://whqlibdoc.who.int/hq/2002/WHO_CDS_STB_2001.16_annexesl-4.pdf. Accessed April 2009. 14. London tuberculosis rates now at third world proportions. http://www.prnewswire.co.uk/cgi/news/release?id=95088. Accessed April 2009. 27 15. San Francisco Department of Public Health: world tb day observed in San Francisco. http://www.sfdph.org/dph/files/newsMediadocs/2009PR/PR03232009.pdf Accessed April 2009. 16. Victor Lorian (ed). Antibiotics in laboratory medicine medicine 5th edition. LW & W Philadelphia, PA. 2005. 17. Kremer L, Douglas JD, Baulard AR, Morehouse C, Guy MR, Alland D, et al. Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting kasa and kasb condensing enzymes in Mycobacterium tuberculosis J. Biol. Chem 2000;275:16857-16864. 18. The Internet Pathology Laboratory for Medical Education. http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/AFB.PDF. Accessed April 2009. 19. http://en.wikipedia.org/wiki/Tuberculosis_treatment Accessed April 2009. 20. Daffe M, Reyrat J-M (eds). The mycobacterial cell envelope. ASM Press Washington, DC. 2008. 21. Crick DC, Schulbach MC, Zink EE, Macchia M, Barontini S, Besra GS, et al. Polyprenyl phosphate biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis. J Bacteriol 2000;182:5771-5778. 22. Zimhony O, Vilcheze C, Jacobs WR. Characterization of Mycobacterium smegmatis Expressing the Mycobacterium tuberculosis fatty acid synthase I (fasl) gene. J Bacteriol 2004;186:4051-4055. 23. Takayama K, WangC, Besra GS. Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin Microbiol Rev 2005;18:81-101. 24. Sacco E, Covarrubias AS, O'Hare HM, Carroll P, Eynard N, Jones TA, et al. The missing piece of the type II fatty acid synthase system from Mycobacterium tuberculosis. PNAS 2007;104:14628-14633. 25. Quemard A, Laneelle G, Lacave C. Mycolic acid synthesis: a target for ethionamide in mycobacteria? Antimicrob Agents Chemother. 1992;36:1316-1321. 26. Slayden RA, Lee RE, Barry III CE. Isoniazid affects multiple components of the type II fatty acid synthase system of Mycobacterium tuberculosis. Mol Microbiol 2000;38:514-525. 27. Bhatt A, Molle V, Besra GS, Jacobs, WR Jr, Kremer L. The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, 28 acid-fastness, pathogenesis and in future drug development. Mol Microbiol 2007;64:1442-1454. 28. Portevin D, de Sousa-D'Auria C, Montrozier H, Houssin C, Stella A, Laneelle MA, et al. The acyl-amp ligase fadd32 and accd4-containing acyl-coa carboxylase are required for the synthesis of mycolic acids and essential for mycobacterial growth: identification of the carboxylation product and determination of the acyl-coa carboxylase components. J Biol Chem 2005;280:8862-74. 29. Gratraud P, Surolia N, Besra GS, Surolia A, Kremer L. Antimycobacterial activity and mechanism of action of NAS-91. Antimicrob Agents Chemother 2008;52:1162- 1166. 30. Copp BR, Norrie PA. Natural product growth inhibitors of Mycobacterium tuberculosis. Nat Prod Rep 2007;24:278-297. 31. http://www.cyberlipid.org/fa/acid0002.htm Accessed April 2009. 32. Zgoda JR, Freyer AJ, Killmer LB, Porter JR. Polyacetylene carboxylic acids from Mitrephora celebica. J Nat Prod 2001;64:1348-1349. 33. Lu T, Cantrell CL, Robbs SL, Franzblau SG, Fischer NH. Antimycobacterial Matricaria esters and lactones from Astereae species. Planta Med 1998;64:665-667. 34. Koyama S, Yamaguchi Y, Tanaka S, Motoyoshiya J. A new substance (yoshixol) with an interesting antibiotic mechanism from wood oil of japanese traditional tree (Kiso-Hinoki), Chamaecyparis obtusa. J. Gen. Pharmacol 1997;28:797-804. 35. Kobaisy M, Abramowski Z, Lermer L, Saxena G, Hancock REW, Towers GHN, et al. Antimycobacterial polyynes of devil's club (Oplopanax horridus), a north american native medicinal plant. J Nat Prod 1997;60:1210-1213. 36. Young J, Duthie S, Milne L, Christensen L, Duthie G, Bestwick C. Biphasic effect of falcarinol on CaCo-2 cell proliferation, DNA damage, and apoptosis. J Agric Food Chem 2007;55:618-623. 37. Hansen L, Hammersh0y O, Boll PM. Allergic contact dermatitis from falcarinol isolated from Schefflera arboricola. Contact Dermatitis 1986;14:9-93. 38. Ondeyka JG, Zink DL, Young K, Painter R, Kodali S, Galgoci A, et al. Discovery of bacterial fatty acid synthase inhibitors from a Phoma species as antimicrobial agents using a new antisense-based strategy. J Nat Prod 2006;69:377-380. 39. Badami RC, Gunstone FD. Vegetable oils. XIII. - the component acids of Isano (Boleko) Oil. J Sci Food Agri 1963; 14:863-866. 29 40. Vaara, M. Agents that increase the permeability of the outer membrane. Microbiol Rev 1992;56:395-411. 41. Wu L, Liu X, Li D. Oct-2-yn-4-enoyl-CoA as a multifunctional enzyme inhibitor in fatty acid oxidation. Organic Let 2008;10:2235-2238. CHAPTER 2 ISOLATION, ACTIVITY, AND STRUCTURE OF EXOCARPIC ACID AND ITS NATURAL ANALOGS Introduction The discovery of exocarpic acid in Exocarpos latifolius (Santalaceae, R. Br.) was a result of the PNG ICBG (1) screening effort mentioned in Chapter 1. Faculty and staff of the University of Papua New Guinea herbarium surveyed Motupore Island, National Capital District, and collected stems and leaves of the plant Exocarpos latifolius. Exocarpos latifolius is a fruit-bearing evergreen shrub capable of living in shaded sandy soils. The plant was identified by Dr. Osia Gideon and Mr. Pius Piskaut of the University of Papua New Guinea herbarium. Voucher specimens for Exocarpos latifolius (U20180) reside at the National Forest Research Institute, Lae, Papua New Guinea and at the University of Papua New Guinea herbarium, Port Moresby, Papua New Guinea. Stem pieces of Exocarpos latifolius were collected, inventoried, dried and submitted to the University of Utah for extraction. Subfractions of a stem extract were found to possess significant antimycobacterial activity against Mycobacterium tuberculosis H37Ra. Bioassay guided fractionation yielded exocarpic acid (1) as the major active agent. Five analogs of exocarpic acid (2 - 6) were present in much smaller quantities in the extract, of which two (3, 4) exhibited activity similar to exocarpic acid. Exocarpic acid belongs to the class of polyacetylenic fatty acids and has been found in several other 31 plants (2, 3). Many polyacetylenic fatty acids have antimicrobial activity, but their activity is not uniform with respect to target organism (4-7), i.e., some exhibit activity against Gram negative strains, others against Gram positive strains. No reports could be found in the literature that describe the antibacterial activity of exocarpic acid against Gram negative bacteria. The following describes the isolation of exocarpic acid and new exocarpic acid analogs from Exocarpos latifolius. The activity of exocarpic acid and analogs against M. tuberculosis, its synergy with first line antimycobacterial drugs and activity against several gram-positive and gram-negative bacteria, is also presented. Materials and methods Organisms and chemicals Albumin/dextrose/catalase (ADC) enriched 7H9 medium (Becton Dickinson, Franklin Lakes, NJ) was used to cultivate Mycobacterium tuberculosis strain H37Ra (ATCC 25177). Escherischia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27953), Streptococcus pneumonia (ATCC 6303), Enterococcus aerogenes (ATCC 13048), and Staphylococcus aureus (ATCC 25923) were obtained from Dr. Mohammad Sondossi, Weber State University and grown in Mueller-Hinton II media (Becton Dickinson, Franklin Lakes, NJ). Rifampicin (>97% pure) and isoniazid (pyridine-4- carbohydrazide, >99% pure) (Sigma Aldrich, St. Louis, MO) served as positive controls in the TB inhibition assay. All other chemicals were purchased from commercial sources (Sigma Aldrich, St. Louis, MO and Thermo Fisher Scientific Inc., Waltham, MA). 32 Extraction and isolation Two hundred grams of ground stems were extracted with 1.0 L methanol using sonication for 30 minutes at room temperature, followed by soaking at 4 °C for 16 hours. The methanol extract was filtered. The ground stems were further extracted with 500 raL methanol using sonication for 1 hour at room temperature. The extract was filtered and combined with the primary methanol extract. The combined extracts were dried using rotary evaporation to yield 10 g of a viscous liquid/oil extract, which may have contained some residual methanol. A portion was redissolved in methanol and coated on a 1 L flask using rotary evaporation followed by high vacuum to give 4.87 g of dry crude extract. The extract was redissolved in methanol (100 mL) followed by addition of 5.00 g of Diaion® HP20SS (Sigma, St. Louis, MO). The extract was dried onto the HP20SS using rotary evaporation. The mixture was pulverized and placed into a 2.5 x 9 cm polypropylene column fitted with a frit. The column was eluted using a step gradient of 30 mL of each solvent (100% H20, 25% isopropanol/H20, 50% isopropanol/H20, 75% isopropanol/H20, and 100% methanol) using a syringe reservoir (60mL) fitted to the top of the column. Five eluates (FW, Fl, F2, F3, F4, respectively) were generated. The solvents were then removed from all samples using centrifugal evaporation. HPLC fractionation of fraction F3 was carried out on a Zorbax XDB C8 column (Agilent, Agilent Technologies Inc, Santa Clara, CA) utilizing an Agilent 1100 HPLC (Agilent Technologies Inc, Santa Clara, CA) with a 50% H20:acetonitrile to 100% acetonitrile linear gradient (20 minutes) and held at 100% acetonitrile for 5 minutes at a flowrate of 3.0 mL/min. Fraction F3 yielded compounds 1 through 6 with elution times of 12.4, 7.3, 8.3, 10.6, 8.9,12.8 minutes, respectively. All compounds were HPLC purified 33 (>99%). A total of approximately 150 mg of exocarpic acid (1) was purified. The yield for compounds 2, 3, 5, and 6 was from 1.2 to 2.0 mg, with compound 4 obtained at 3.0 mg, i.e., approximately l/100th to l/50th of the amount of exocarpic acid. NMR spectra were recorded on a Varian INOVA at 500 MHz for 'H and 125 MHz for 13C. Solvent (CDC13) signals were used as reference (5h 7.24 ppm; 8c 77.0 ppm). Accurate mass measurements were performed on a Micromass Q-tof Micro (Micromass, Manchester, UK) using positive ion mode. Optical rotations were measured on a Perkin Elmer 343 Digital Polarimeter at 580 nm in methanol at 22 °C. Bacterial growth assays Inhibition of M. tuberculosis H37Ra growth was quantified using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay adapted from previously published methods (8, 9). Drugs and plant extracts were dissolved in dimethyl sulfoxide (DMSO) to produce 10 mg/mL stock solutions. DMSO served as a negative control. Mycobacterium tuberculosis H37Ra cultures were dispensed in 200 pL of ADC enriched 7H9 medium into a 96-well culture plates at 100,000 cells per well. One pL of DMSO containing drug or DMSO (control) or extract was added in triplicate wells. After four days incubation at 37°C, 11 pL of sterile MTT (5 mg/mL in PBS) was added and incubation continued overnight. Viable M. tuberculosis metabolized the MTT to the insoluble purple formazan salt that was solubilized by the addition of 50 pL of a solubilization solution (50% DMF v/v, 5% SDS w/v, 45% H20 v/v). Absorbance was measured using a Biorad Model 450 microtiter plate reader at 570 nm (Biorad, Hercules, CA). All data were corrected against media-only blank wells. The percent inhibition was derived as the fraction of the average of the test wells over the average of control wells 34 subtracted from unity and multiplied by 100. Nonmycobacterial bacterial strains were assayed according to CLSI/NCCLS method M07-A5 (10) in Mueller-Hinton II broth. Rifampicin and isoniazid potentiation assay Rifampicin dilutions were added into duplicate columns of a 96 well plate to yield final concentrations at 250 ng/mL (MIC), 83 ng/mL (l/3rd of MIC) and 25 ng/mL (l/10th of MIC). Similarly, isoniazid dilutions were added into duplicate columns of a 96 well plate to yield final concentrations at 1 pg/mL (MIC), 0.3 pg/mL (l/3rd of MIC) and 0.1 pg/mL (l/10th of MIC). The columns then received either DMSO or a dilution series of exocarpic acid of 20 pg/mL final concentration (MIC), 10 pg/mL (1/2 of MIC), 5 pg/mL (l/4th of MIC), or 2.5 pg/mL (l/8th of MIC). The exocarpic acid dilutions were also used as controls without addition of the other drugs. Growth inhibition data analysis was as described above. Characterization of compounds 1, 2, 3, 4, 5 and 6. Exocarpic acid (1): white solid; 'H and l 3C NMR matched those previously published (3). HRESIMS m/z 297.1833 [M+Na]+ (calcd for C18H2602Na 297.1831). £-15-hydroxy- octadeca-13-en-9,ll-diynoic-acid (2): white solid; UV Xmax (log e): 216 (4.5), 254 (3.5), 270 (3.7), 284 (3.5) nm; [a]D 22: 2.31; IR (NaCl) vmax: 2917, 2849, 2359, 1560, 1422,1131 cm"1; HR ESIMS m/z 313.1790 [M+Na]+ (calcd for Ci8H2603Na 313.1780). £-8-hydroxy- octadeca-13-en-9,ll-diynoic-acid (3): white solid; UV Xmax (log s): 216 (4.2), 254 (3.6), 270 (3.7), 284 (3.6) nm.; [a]D 22: 2.60; IR (NaCl) vmax: 2925, 2854, 35 2230, 1706, 1561, 1420, 1093, 954 cm"1. HR ESIMS m/z 313.1779 [M+Na]+ (calcd for QgHaeOsNa 313.1780). E- octadeca-13-en-9,ll-diynoic-acid glycerol ester (4): white solid; UV A,max (log e): 216 (4.3), 254 (3.6), 270 (3.6), 284 (3.6) nm; [a]D 22: 0.57; IR (NaCl) vmax: 3355, 2928, 2852, 2204, 1735, 1671, 1671, 1561, 1421, 1170, 1123, 954, 721 cm"1. HR ESIMS m/z 371.2209 [M+Na]+ (calcd for C2iH3204Na 371.2198). £-octadeca-9,ll,13-trienoic acid (5): white solid; ]H and 13C NMR matched those previously published (11). HRESIMS m/z 301.2144 [M+Na]+ (calcd for Ci8H3o02Na 301.2144). is-15-oxo- octadeca-13-en-9,ll-diynoic-acid (6): white solid; UV A,max (log s): 216 (4.4), 296 (3.61) nm. IR (NaCl) vmax: 3368, 2926, 2853, 2360, 2227, 1561, 1421, 1088 cm"1. HR ESIMS m/z 311.1636 [M+Na]+ (calcd for Qg^OsNa 311.1623). NMR resonances used to elucidate the structure of compounds 2, 3, 4, and 6 are presented in Table 2.1. Results Isolation and characterization of compounds 1, 2, 3, 4, 5 and 6 Screening of HP20SS fractions F3 and F4 of Exocarpos latifolius stem extracts in the MTT system produced a 71%-81% inhibition of M. tuberculosis H37Ra growth at 50 pg/mL. No significant activity against Plasmodium falciparum or HIV could be demonstrated for fractions F3 and F4 and CEM-TART T-cell cytotoxicity was negligible (data not shown). HPLC fractionation of HP20SS F3 resulted in several active compounds. Subsequent NMR, MS and M. tuberculosis bioassay analysis revealed the active constituents as exocarpic acid and related analogs (Figure 2.1.) Table 2.1 *H and 1 3C NMR data for compounds 2, 3, 4 and 6. 36 g-15-hydroxy- octadeca-13-en-9,ll-diynoic-acid (2) 'H NMR (CPC13, 500 MHz) 6 6.25 (1H, dd, / =15.9, 6.3 Hz, H-14), 5.71 (1H, br d ,J = 15.9 Hz, H-13), 4.17 (1H, q , / = 6.3 Hz, H-15), 2.33 (2H, t , / = 7.5 Hz, H-2), 1.62 (2H, quint, J = 7.5 Hz, H-3), 1.52 (2H, quint, J = 7.5 Hz, H-7), 1.50 (2H, m, H-16), 1.38 (2H, m, H-17), 1.36 (2H, m, H-8), 1.35 (6H, m, H-4, H-5, H-6), 0.91 (3H, t, / = 1.2 Hz, H-18) 1 3C NMR (CDCI3, 125 MHz) 8 177.7 (C, C-l), 148.8 (CH, C-14), 109.0 (CH, C-13), 84.6 (C, C-9), 75.3 (C, C-12), 73.0 (C, C-ll), 72.2 (CH, C-15), 65.3 (C, C-10), 39.2 (CH2, C-16), 33.8 (CH2, C-2), 28.9, 28.64, 28.61 (CH2, C-4 or C-5 or C-6), 28.8 (CH2, C-17), 28.3 (CH2, C-l), 24.9 (CH2, C-3),18.6 (CH2, C-8), 14.2 (CH3, C-l8) £-8-hydroxy- octadeca-13-en-9,ll-diynoic-acid (3) 'H NMR (1:1 CD30D:CDC13, 500 MHz) 5 6.27 (1H, dt, J = 16.0, 7.1 Hz, H-14), 5.47 (1H, br d, / = 16.0 Hz, H-13), 4.36 (1H, t, J = 6.7 Hz, H-8), 2.28 (2H, quint, / = 7.5 Hz, H-2), 2.11 (2H, q, J = 7.0 Hz, H-15), 1.66 (2H, m, H- 7), 1.59 (2H, m, H-3), 1.42 (2H, m, H-6), 1.33 (2H, m, H-16), 1.32 (4H, m, H-4 and H-5), 1.28 (2H, m, H-17), 0.86 (3H, t ,J = 1.0 Hz, H-18) 13C NMR (1:1 CD30D:CDC13, 125 MHz) 8 177.6 (C, C-l), 150.0 (CH, C-14), 109.2 (CH, C-13), 83.6 (C, C-9), 77.5 (C, C-12), 72.7 (C. C-ll), 69.8 (C, C-10), 63.1 (CH, C-8), 38.3 (CH2, C-l), 34.9 (CH2, C-2), 33.8 (CH2, C-15), 31.5 (CH2, C-16), 29.87, 29.84 (CH2, C-4 or C-5), 25.9 (CH2, C-6), 25.7 (CH2, C-3), 23.0 (CH2, C-17), 14.4 (CH3, C-18) E- octadeca-13-en-9,ll-diynoic-acid glycerol ester (4) 'H NMR (CPC13, 500 MHz) 8 6.26 (1H, dt, / = 16.0, 7.1 Hz, H-14), 5.46 (1H, br d , / = 16.0 Hz, H-13), 4.20 (1H, dd, J = 11.3, 4.6 Hz, H-19a), 4.13 (1H, dd,/ = 11.3, 6.2 Hz, H-19b), 3.92 (1H, m, H-20), 3.68 (1H, dd, / = 11.4, 3.9 Hz, H-21a), 3.58 (1H, dd, / = 11.4, 5.7 Hz, H-21b), 2.33 (2H, t , / = 7.4 Hz), 2.29 (2H, I, J = 7.1 Hz, H-8), 2.10 (2H, dtd,/ = 7.4, 7.1, 1.4 Hz, H-15), 1.62 (2H, m, H-3), 1.51 (2H, m, H-7), 1.35 (2H, m, H-16), 1.31 (6H, m, H-4, H-5, H-6), 1.29 (2H, m, H-17), 0.87 (3H,t, 7 = 7.1 Hz, H-18) 13C NMR (CPC13, 125 MHz) 8 174.2 (C, C-l), 148.5 (CH, C-14), 108.8 (CH, C-13), 83.7 (C, C-9), 74.1 (C, C-12), 73.5 (C, C-ll), 70.5 (CH, C-20), 65.7 (C, C-10), 65.4 (CH2, C-19), 63.5 (CH2, C-21), 34.2 (CH2, C-2), 33.0 (CH2, C-15), 30.7 (CH2, C-16), 28.9, 28.9, 28.8 (CH2, C-4, C-5, C-6), 28.2 (CH2, C-l), 24.9 (CH2, C-3), 22.3 (CH2, C-17), 19.6 (CH2, C-8), 14.0 (CH3, C-18) ff-15-oxo- octadeca-13-en-9,ll-diynoic-acid (6) 'H NMR (CPC13, 500 MHz) 8 6.62 (1H, d , / = 16.1 Hz, H-13), 6.57 (1H, d , / = 16.1 Hz, H-14), 2.49 (2H, t , / = 7.2 Hz, H- 15), 2.35 (2H, t, / = 7.2 Hz, H-8), 2.35 (2H, t, / = 7.2 Hz, H-2), 1.64 (2H, m, H-3), 1.60 (2H, m, H-17), 1.55 (2H, m, H-7), 1.2-1.4 (6H, m, H-4, H-5, H-6), 0.91 (3H, t, / = 1.2 Hz, H-18) 37 Figure 2.1 Structure of ii-octadeca-lS-en^, 11-diynoic-acid [exocarpic acid] (1) and derivatives: £-15-hydroxy-octadeca-13-en-9,ll-diynoic-acid (2), E-8- hydroxy-octadeca-13-en-9,ll-diynoic-acid (3), f-octadeca-lS-en^ll-diynoic- acid glycerol ester (4), £-octadeca-9,ll,13-trienoic acid (5), and E- 15-oxo-octadeca-13-en-9,ll-diynoic-acid (6). o I o I o II II ~ 0 ~ '" '" ~ '" II ~ ~ II '~" ~ ~ ~ ~ N I 0 ... o II II ~ 0 I 0 II II ~ I ) o 0 I I 0 M o o I o II II ~ 0 ~ ~ ~ I 0 It) 38 Compounds 2, 4, and 6 are new analogs of exocarpic acid, while compound 3 has been described (12). The structures were determined through analysis of NMR data and comparison to the data for exocarpic acid (1). The HRESIMS data for compound 2 indicated an additional oxygen in the molecular formula as compared to exocarpic acid (1). The ]H NMR spectrum showed two features that were distinct from the *H NMR spectrum of exocarpic acid (1). First, the H-14 olefinic proton was a dd for compound 2 instead of a dt as observed for exocarpic acid (1) and indicated that C-15 was oxygenated. Second, the ]H NMR spectrum of 2 showed an oxygenated methine resonance (5H 4.17, 5c 72.2), and the COSY spectrum showed a correlation between H-14 and H-15. Additional support came from the HMBC spectrum that showed HMBC correlations from H-15 to C-13 and C-16 as well as from both olefinic protons (H-13 and H-14) to the oxygenated methine at C-15. HRESIMS data on compound 3 indicated the presence of an additional oxygen compared to exocarpic acid (1). In the *H NMR spectrum, an oxygenated methine was observed at 6 4.36 (H-8) and was clearly distinct from compound 2. The position of the oxygen was identified by analysis of the gHMBC spectrum in conjunction with the gCOSY. In the HMBC spectrum, correlations were observed from the oxygenated methine (H-8) to all four alkyne carbons with only one COSY correlation to a methylene at 5 1.66 (H-7). All other resonances were essentially identical to exocarpic acid (1). HRESIMS data for compound 4 supported a molecular formula of C21H32O4. Analysis of the gHSQC spectrum showed that 4 contained two additional oxygenated methylenes and one additional oxygenated methine as compared to exocarpic acid (1). The gCOSY spectrum showed that the additional signals were part of a contiguous spin system and suggested that compound 4 was the glycerol ester of exocarpic acid (1). Subsequent analysis of the HMBC correlations from H-19a and H-19b to C-l and confirmed the proposed structure as 4. Aside from the signals for the glyceride, all NMR data were identical to that of exocarpic acid (1). The position of the ketone was inferred from the 'H NMR spectrum, which showed the resonance for H-13 was a doublet and had shifted downfield to 5 6.62 compared to 8 5.47 for exocarpic acid (1) indicative of an a,P-unsaturated keto system. The structure of compound 6 was based on the MS data in conjunction with the ]H NMR spectrum. Two-dimensional NMR data were not obtained due to the small amount isolated. The MS data suggested that 6 contained a ketone. Antimycobacterial activity of compounds 1, 2, 3, 4, 5 and 6 The antibacterial activity of exocarpic acid was investigated by determining the MICioo in several strains of bacteria. Exocarpic acid (1) was ineffective against Pseudomonas aeruginosa and Escherichia coli, both Gram negative, at concentrations below 500 pg/mL and 200 pg/mL, respectively. The Gram positive bacteria Staphylococcus aureus and Enterococcus aerogenes were unaffected by exocarpic acid at concentrations below 200 pg/mL, while Streptococcus pneumoniae proved only to be resistant at concentrations below 100 pg/mL. The activity of compounds 1 through 6 against M. tuberculosis H37Ra using the MTT assay is presented in Table 2.2. The MICioo of exocarpic acid in the virulent M. tuberculosis strain H37Rv was performed by Dr. Robert Reynolds at TAACF (13). The aerobic MIC (14) for exocarpic 41 Table 2.2. Antimycobacterial activity of exocarpic acid analogs against Mtb H37Ra. Compound IUPAC Name MICioo (pg/mL) 1 £-octadeca-13-en-9,l 1-diynoic-acid 20 (Exocarpic acid) 2 £-15-hydroxy-octadeca-13-en-9,l 1-diynoic-acid >150 3 £-8-hydroxy-octadeca- 13-en-9,11-diynoic-acid 25 4 E- octadeca-13-en-9,l 1-diynoic-acid glycerol ester 25 5 is-octadeca-9,ll,13-trienoic acid 100 6 £-15-oxo-octadeca-13-en-9,l 1-diynoic-acid >150 acid was 20 pg/mL, while the MIC in the low oxygen recovery assay (LORA) (15), which mimics intracellular M. tuberculosis, was determined to be 87 pg/mL. Exocarpic acid at 20, 10, 5 and 2.5 pg/mL was tested against M. tuberculosis H37Ra in combination with rifampicin and isoniazid at their respective MIC, 1/3 of the MIC and 1/10 of the MIC (Figures 2.2a and 2.2b, respectively). The addition of exocarpic acid increases the efficacy of both rifampicin and isoniazid at the 1/3 and 1/10 of MIC levels. Addition of 50 pg/mL of oleic acid to the 7H9/ADC medium increased the exocarpic acid MICioo to above 150 pg/mL in the MTT assay system (data not shown). 42 Figure 2.2 Exocarpic acid enhances antimycobacterial activity of antibiotics (a) Exocarpic acid enhances antimycobacterial activity of rifampicin (RIF). (b) Exocarpic acid enhances antimycobacterial activity of isoniazid (INH). • No RIF • RifMIC A RIF 1/3 MIC X RIF 1/10 MIC •Linear (No RIF) Linear (RIF 1/10 MIC) Linear (RIF 1/3 MIC) - • Linear (RifMIC) m -• - 2.5 5 10 [Exocarpic acid] ng/mL 20 S 0)0.7 o h § 0.6 u TJ £i0ns ) 0.5 3 0 4 uO- = 0.3 •oa 2 0.2 • No INH • INH MIC A INH 1/3 MIC • INH 1/10 MIC Linear (No INH) Linear (INH 1/10 MIC) - - - Linear (INH 1/3 MIC) - - - Linear (INH MIC) 2.6 5 10 [Exocarpic acid] ng/mL 44 Discussion Antimycobacterial activity and structure Bioactivity-guided extraction of Exocarpos latifolius has yielded exocarpic acid and three new analogs as antimycobacterial compounds. The antibacterial activity of exocarpic acid has been known (2, 3), however here we demonstrate for the first time its antimycobacterial activity. Exocarpic acid displayed high selectivity for M. tuberculosis in comparison to the Gram-negative and Gram-positive bacterial strains tested. The glycerol ester of exocarpic acid (4) and £-8-hydroxy- octadeca-13-en-9,l 1-diynoic-acid (3) exhibited similar antimycobacterial activity when compared to exocarpic acid. Introduction of distal groups into the exocarpic acid pharmacophore, i.e., 1 E-15- hydroxy- octadeca-13-en-9,ll-diynoic-acid (2) and £-15-oxo- octadeca-13-en-9,ll-diynoic- acid (6), markedly reduced activity. Removal of the acetylenic bonds (5) reduced the activity in an intermediate fashion. This suggests that the antimycobacterial activity of exocarpic acid lies in the chemical properties of the acetylenic bonds, similar to the related compound 2-hexadecynoic acid (2-HA) (16). We have expanded on this observation by testing related polyacetylenic compounds (Chapter 5). Potentiation and mode of action Polyacetylenic fatty acids have been reported as antimycobacterial agents from other plant and microbial sources. Falcarindiol was isolated from the Native American medicinal plant Oplopanax horridus (Devil's club) (5) and shown to have activity against M. avium. Oropheic acid was isolated from Mitrephora celebica and shown to be active against methicillin-resistant Staphylococcus aureus (MRSA) and M. smegmatis (4). Both 45 of these compounds also had activity against Gram positive bacteria and fungi. The underlying reason for exocarpic acid's selectivity for M. tuberculosis is not known, but likely involves a target inM tuberculosis that is unique or evolutionarily poorly conserved with respect to the proteobacteria. The potentiating effect of exocarpic acid on both rifampicin and isoniazid is intriguing, since it could mean that exocarpic acid has a target in M. tuberculosis different from either of these drugs. Likewise, that oleic acid, a C-18 monounsaturated fatty acid, was able to suppress activity of exocarpic acid strongly argues that exocarpic acid has a specific target in the mycobacterial cell. It is not clear if the detoxifying effect of oleic acid is based on competition for transport into the cell, or direct competitive inhibition of exocarpic acid's target(s), possibly competing as substrates for the FAS II system. Future directions To date there has been no published investigation into the antimycobacterial mechanism of action of polyacetylenic natural products. It was therefore decided to use exocarpic acid as the basis of such and investigation. The following chapters address the mode of action of exocarpic acid against M. tuberculosis. Since little was know about the mechanism, even with respect to the polyacetylenic fatty acids as a group, microarray analysis of gene induction by exocarpic acid was performed to determine if exocarpic acid's transcription profile resembled those of antimycobacterial drugs with known mechanism (Chapter 3). The lead information from the microarrays provided the rationale to investigate changes in fatty acid metabolism caused by exocarpic acid in M. tuberculosis (Chapter 4). 46 References 1. International Cooperative Biodiversity Groups (ICBG) http://www.fic.nih.gov/programs/research_grants/icbg/. Accessed October 15, 2008. 2. El-Jaber NA, Estevez-Braun AG, Munoz-Munoz RO, Rodriguez-Alfonso A, Murguia JR. Acetylenic acids from the aerial parts of Nanodea muscos. J Nat Prod 2003;66:722-724. 3. Naidoo, LAC, Drewes SE, Van Staden J, Hutchings, A. Exocarpic acid and other compounds from tubers and inflorescences of Sarcophyte sanguined. Phytochem 1992;31:3929-3931. 4. Li X-C, Jacob MR, Khan SI, Ashfaq MK, Babu KS, Agarwal AK, et al. Potent in vitro antifungal activities of naturally occurring acetylenic acids. Antimicrob Agents Chemother 2008;52:2442-2448. 5. Zgoda JR, Freyer AJ, Killmer LB, Porter JR. Polyacetylene carboxylic acids from Mitrephora celebica. J Nat Prod 2001;64:1348-1349. 6. Kobaisy M, Abramowski Z, Lermer L, Saxena G, Hancock REW, Towers GHN, et al. Antimycobacterial polyynes of Devil's Club (Oplopanax horridus), a North American native medicinal plant. J Nat Prod 1997;60:1210-1213. 7. Naidoo LAC, Drewes SE, Drewes FE, Van Staden J, Aken, ME. When is a parasite no longer a parasite? The case of Sarcophyte sanguinea and exocarpic acid. South African J Sci 1994;90:359-61. 8. Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A, et al. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate alamar blue assay. J Clin Microbiol 1998;36:362- 366. 9. Foongladda S, Roengsanthia D, Arjrattanakool W, Chuchottaworn C, Chaiprasert A, Franzblau SG. Rapid and simple MTT method for rifampicin and isoniazid susceptibility testing of Mycobacterium tuberculosis. Int. J. Tuberc. Lung Dis 2002;6:1118-1122. 10. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard - Fifth Edition. NCCLS, 2000. 11. Amakura Y, Kondo K, Akiyama H, Ito H, Hatano T, Yoshida T, et al. Characteristic long-chain fatty acid of Pleurocybella porrigens. J Food Hyg Soc Jap 2006;47:178- 181. 47 12. Badami RC, Gunstone FD. Vegetable oils. XIII. - the component acids of isano (boleko) oil. J Sci Food Agri 1963;14:863-866. 13. The Tuberculosis Antimicrobial Acquisition and Coordinating Facility. http://www.taacf.org. Accessed October 15, 2008. 14. Collins, LA., Franzblau, SG. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 1997;41:1004-1009. 15. Cho HS, Warit S, Wan B, Hwang CH, Pauli GE, Franzblau SG. Low oxygen recovery assay (LORA) for high throughput screening of compounds against non-replicating Mycobacterium tuberculosis. Antimicrob Agents Chemother 2007;51:1380-1385. 16. Morbidoni C, Vilcheze L, Kremer R, Bittman J, Sacchettini W, Jacobs, JR. Dual inhibition of mycobacterial fatty acid biosynthesis and degradation by 2-alkynoic acids. Chem and Biol 2006;13:297-307. CHAPTER 3 TRANSCRIPTOME STUDIES Introduction Polyacetylenic fatty acids present a complete unknown in terms of mode of action against Mycobacterium tuberculosis. It seemed likely that transcriptome data could aid in identification of potential targets. Three preexisting studies (1-3) have described transcriptional signatures for M. tuberculosis upon treatment with various antibiotics/antimicrobials, toxins (e.g., KCN) and physical regimen (e.g., UV light, low pH). Two of the studies (1, 2) showed that treatment of M. tuberculosis H37Rv with compounds thought to have the same mode of action indeed produce repeatable patterns of gene expression. These expression patterns (signatures) can be utilized to group compounds of unknown mode of action into known categories. This approach was taken by Boshoff et al. (2), showing that large scale microarray studies of M. tuberculosis can yield mechanistic information, e.g., ascididemin, a marine pyridoacridine described by our laboratory, was found to interfere in iron metabolism in M. tuberculosis (2). In particular, the study performed by Boshoff et al. (2), showed that triclosan (TRC) affected respiration rather than cell wall biosynthesis (4). It has become clear now that compounds like TRC can exhibit multiple activities quantified in this dissertation: long-chain fatty acid biosynthesis inhibition at low concentration and cellular respiration at high concentration. Concentration dependent changes in mode of action, or perhaps, masking of one mode of action by another, presents a difficulty in generating lead data for compounds of unknown mode of action. The approach taken in this work was two-fold due to the timing of the receiving the array data for exocarpic acid and other antimycobacterial drugs. Initially an informal analysis was performed by simply inspecting gene expression patterns and comparing it to the published clusters. Upon receipt of the full data set, a formal investigation was performed, first, by comparing arrays, i.e., "treatments", against each other using hierarchical clustering (HCL) and second, by gene enrichment study in the data sets using the gene set enrichment algorithm (GSEA) (5). The approach used here differs from those of the previous studies, which used direct comparison of gene expression ratios between treatment groups (1), principal component analysis (4) and k-means clustering (2) for data analysis. We adopted GSEA to take advantage of the previously generated signature sets and to generate a more discriminating analysis by eliminating false positive signals due to large numbers of activated genes. Materials and methods Exocarpic acid transcriptome Prior to performing the array experiments we approached our collaborator Dr. Clifton E. Barry, III at the National Institutes of Health National Institute for Allergy and Infectious Diseases (NIH-NIAID). There was an interest in the Barry lab in fatty acids and their mode of action against M. tuberculosis, hence exocarpic acid was placed in the microarray pipeline at the Berry lab. Dr. Helena Boshoff kindly performed the array experiments with the provided exocarpic acid at 10, 20 and 40 pg/mL. Arrays were of type MTBB GSE1642 (6), with full M. tuberculosis H37Rv genome coverage (3932 ORFs) using oligos from Operon (Eurofins MWG Operon, Huntsville, AL) (7). The platform type was GPL1343 (8). Procedures for growth and processing are provided in Boshoff et al. (2). Briefly, M. tuberculosis was grown in 7H9 medium with albumin/dextrose/catalase (ADC) supplement, and treated with drug, or exocarpic acid at 10, 20 or 40 pg/mL exocarpic acid, or with vehicle (DMSO). Microarray preparation is given in GSE1642 (6). Data were provided to us initially as a reduced set of compounds previously assayed with exocarpic acid included, as a 'fold gene expression over control' in an Excel™ datasheet. The full data set received later contained 188 arrays representing the complete set of previously published microarray data on all antimicrobials tested for comparison (2). Processing of the data for this dissertation was performed with Microsoft Excel™, Cluster 3.0 (9) and GSEA (5). Prior to our analysis the data set was pared down to exclude treatments with ethanol as the solvent, because exocarpic acid was dissolved in DMSO as vehicle, and ethanol was shown to alter gene expression in Dr. Boshoff s data. Also, only the sets of arrays with the treatment time closest to that of exocarpic acid (4h) were retained in our analysis set. For the clustering and gene enrichment data sets the undeveloped spots on these arrays were filled with the average of all expression values for each individual array using Excel™. The arrays were then mean-centered using Cluster 3.0 (Figure 3.1). Annotations and functional descriptions for genes of interest were sourced from BioHealthBase (10) and Tuberculist (11). Hierarchical cluster analysis using Cluster 3.0 Hierarchical clustering (HCL) of arrays was performed using Cluster 3.0 and visualized using jTreeview 1.1.3 (12). Figure 3.1 Overall scheme for preparation of microarray data for HCL and GSEA analysis HCL Gene Enrichment Array Data Array Data Stanford Array Data Stanford Array Data Settings were selected to cluster arrays using Euclidean distance as a similarity metric and complete linkage, i.e., each cluster's similarity is based on their most distant members, as clustering method. Complete linkage clustering results in more compact clusters than other clustering methods such as single-linkage clustering. Euclidean distance was used because it is robust and the arrays were mean centered. The Spearman rank correlation as a similarity metric was also used since it does not assume a linear relationship among data points and could potentially be more predictive of underlying patterns. Direct correlation analysis of arrays was performed using Matlab 2009a (The Math Works, Inc., Natick, MA) using the corrcoef command on the set of arrays used in the HCL analysis. Briefly, array descriptions were removed and loaded into Matlab 2009a as a tab delimited array. Resultant correlation coefficients were saved in text mode, imported into Excel1 M and repatriated with the array descriptions. Gene set enrichment analysis (GSEA) The GSEA tool can determine if a given set of genes is overrepresented in other sets of genes. The method works on the level of a set of significant genes, known a priori. These significant gene sets are then compared against full array data in which gene expression data has been ranked according to their expression level. The occurrence of the signature genes in the full array gene set is evaluated by a running sum, in which the increment of the sum is dependent on the occurrence of the signature genes on either end of the ranked list distribution in the ranked full array data. The output is an "enrichment score" (ES) and a set of genes that are "enriched" in the sample array. The significance statistic is derived by comparison of the ES versus an ES based on a 54 permuted set of genes derived from the input data. The GSEA tool is designed for comparisons of human genome data, since the annotations are based on Human Genome Organization (HUGO) identifiers. However, GSEA also allows for comparisons of raw data without regard to HUGO annotation, as long as the annotations provided are consistent across samples. In order to utilize the tool, two-fold or higher upregulated genes for each antibiotic treatment of the exocarpic acid array data set were selected as 'signatures'. The comparison data was derived from data deposited at the Stanford Microarray Database by Jeanette Barba (submitter JBARBA) (13). The exocarpic acid array set and the comparison set from the Stanford data base were based on several different versions of the array platform. The data therefore was first aligned using the feature array tag information found in the platform ID type. Arrays were then manually edited to remove any inconsistencies, e.g., duplicated or missing genes. Data files were prepared for input into GSEA with Excel™ and modified to conform to the file specifications provided in the GSEA user manual. Simple text files were prepared using Wordpad on a Windows XP Pentium 4 computer. GSEA was performed on the isoniazid (INH), exocarpic acid (EXO), thiolactomycin (TLM), triclosan (TRC), cerulenin (CER), pyrazinamide (PZA), and the protonophore carbonylcyanide-3-chlorophenylhydrazone (CCCP) signature sets with the following settings: 1000 permutations; permutation type: gene set, weighted enrichment statistics. Maximum and minimum gene set sizes were 300 and 5, respectively. All other settings were left at their respective default setting. The phenotype label was set to EXO versus REST. Biological pathway analysis The final analysis was biological pathway analysis using the upregulated genes in the exocarpic acid treatment gene set and was performed online at www.biohealthbase.org using the metabolic pathway tool (10). This tool identifies metabolic pathways up- or down-regulated by the gene products associated with them. Pathways that contain enzymes whose genes are transcriptionally regulated above or below a user selectable threshold-level are flagged. The total data for the exocarpic acid array (40 pg/mL treatment) were loaded into the tool for analysis. The expression ratio cut-off value was chosen to be 2.0-fold, consistent with expression ratio used in the GSEA analysis. Results Exocarpic acid transcriptome The highly upregulated genes, i.e., an expression ratio of two-fold or higher upon exocarpic acid treatment, are tabulated in Table 3.1 with their function annotated, if known. Overall, about 10% of the spots on the array did not yield a signal and these genes are not represented in the exocarpic acid data set. The set of highly induced genes roughly falls into three categories: fatty acid biosynthesis genes, fatty acid degradation genes, and stress response genes. It would be expected that fatty acid degradation genes are induced since exocarpic acid is a fatty acid. The relationship of fatty acid degradation to toxicity to M. tuberculosis is not clear, and no published source exists that connects such metabolism directly to toxicity. 56 Table 3.1 Genes induced in Mycobacterium tuberculosis H37Rv upon treatment with 40 pg/ml exocarpic acid for 4 hours. Rank Fold Activation Gene Description Functional grouping 1 10.93 fadE5 Acyl-CoA_dehydrogenase P-oxidation 2 10.44 Rv0196 TetR/AcrR trx regulator Efflux 3 9.32 Rv0197 Poss0. Oxidoreductase Unknown 4 6.67 efpA Putative efflux channel Efflux 5 5.93 Rv3742c Poss. Monooxygenase Unknown 6 5.50 Rvl856c Poss. Dehydrogenase Unknown 7 4.93 desA3 NADPH dept. desaturase Oleic acid production 8 4.48 Rvl592c Poss. Extracellular lipase Fatty acid degradation 9 4.43 Rv3741c Poss. Oxidoreductase Unknown 10 3.74 aceA Isocitrate lyase Precursor generation 11 3.24 modA molybdate ABC transporter Transport 12 3.08 ORF00540 Unknown unknown 13 3.07 ahpD Alkyl hydroperoxide reduct. Likely stress response 14 2.97 Rv3173c Probb. TetT/AcrR Transport (drug efflux) transcriptional regulator 15 2.90 fadE13 Put. Acyl-CoA deydrogenase p-oxidation 16 2.85 ahpC Alkyl hydroperoxide reduct. Likely stress response 17 2.80 Rv2466c Unkn. - upregulated in Likely stress response oxidative stress response 18 2.64 kasB FAS-II Mycolic acid metab. 19 2.48 glnH Glutamine binding protein Transporter 20 2.34 fadH Putc. 2,4-dienoyl-CoA P-oxidation reductase 21 2.33 fadE12 Put. Acyl-CoA deydrogenase P-oxidation 22 2.32 Rv2971 Put. membrane protein Unknown 23 2.26 Rv2659c Prob. prophage integrase Unknown 24 2.24 trxB2 thioredoxin reductase Detoxification / stress response 25 2.22 Rv0140 Unkown Unknown 26 2.21 rpml 50S ribosomal protein Translation 27 2.20 pksl3 Polyketide synthase Mycolic acid metabolism. 28 2.20 accA3 acetyl-/propionyl-CoA Fatty acid biosynthesis carboxylase precursor generation 29 2.19 fadD32 Fatty-acyl AMP ligase p-oxidation 30 2.18 Rv3683 Unknown Unknown 31 2.17 desAl fatty acid desaturase Fatty acid metabolism 32 2.16 Rv2204c Unknown Unknown 33 2.15 cbhK carbohydrate kinase Purine metabolism 57 Table 3.1 continued. Rank Fold Activation Gene Description Functional grouping 34 2.15 Rv3706c Put. oxalyl-CoA Fatty acid decarboxylase biosynthesis precursor generation 35 2.15 echA8 Prob. enoyl-CoA hydratase (3-oxidation 36 2.15 serC P04-serine Amino acid aminotransferase metabolism 37 2.14 fadA acetyl-CoA Precursor generation acetyltransferase 38 2.09 Rv3835 Unknown Unknown 39 2.09 Rv3252c alkane 1-monooxygenase Fatty acid (AlkB) metabolism (also w-oxidation of carboylic acids) 40 2.08 Rvl460 Prob. Transcriptional Unkown regulator 41 2.06 Rv0726c Put. SAM-dependent Pot. mycolic acid methyltransferase decoration 42 2.05 ORFOH63 Unknown Unknown 43 2.05 Rv3881c Secreted protein Pot. Virulence factor 44 2.04 umaAl Poss. mycolic acid methyl Mycolic acid transferase decoration 45 2.02 Rv0888 Unknown (exported) Unknown 46 2.02 rpsB 30 S ribosomal gene Translation 47 2.02 Rv2494 Unknown Unknown "possible, fcprobably, cputative 58 It is not hard to imagine though that the supply of fatty acids for a growing cell is vital and that exocarpic acid might disrupt this supply. Turn-over of fatty acids from the cell envelope may be critical as well, and exocarpic acid might interfere with that process. However, none of the genes involved in cell wall degradation were upregulated, therefore this pathway seemed to be of low priority. Stress response genes such as Rv0196/Rv3173c, a repressor of an efflux system, Rv0197, a likely oxido-reductase, efpA, an efflux protein (14) and ahpC, an alkyl-hydroperoxidase reductase are all strongly upregulated. Thioredoxin, also a stress response gene, is not in the data presented above; however, it was ranked 50th with a transcriptional activation of 1.99 fold over control. Induction of stress response genes such as efpA, is expected upon treatment with agents toxic to the bacterial cell (15, 16). Most importantly, transcription of fatty acid biosynthesis genes was also strongly increased. The underlying reason may simply be the excess supply of a fatty acid (exocarpic acid) that could be a precursor for cell wall biosynthesis (Chapter 4). No array data exists regarding the effect of fatty acid supplementation, e.g., oleic acid, on the mycobacterial transcriptome, which is a gap in the data sets available which needs to be closed. However, antibiotics known to inhibit fatty acid synthesis induce a fairly distinct set of genes in the fatty acid biosynthesis pathways as shown in Table 3.2. Exocarpic acid induced a large subset of these genes as well. Furthermore, accessory function (mycolic acid decoration) genes such as umaAl, and Rv0726c, both methyl transferases, were also induced. 59 Table 3.2 Comparison of published gene cluster data for cell wall inhibitors to expression data for exocarpic acid. Gene Boshoff Cluster (2) Wilson Cluster Exocarpic acid (1) TLM CER INH INH 20 pg/ml 40 pg/ml moeY 0.3 1.4 0.7 - 1.0 1.03 Fas 0.8 1.7 0.6 - 0.55 0.7 efpA 2.4 1.8 2.3 2.5 3.5 6.7 accA3 1.7 1.5 2.3 - 1.5 2.2 accD4 1.5 1.4 1.6 - 1.3 1.4 pksl3 1.0 1.4 1.3 - 1.5 2.2 fadD32 1.5 1.6 1.5 - 1.5 2.2 Rv0241c 2.1 -0.4 1.8 - 1.5 1.6 fadA2 2.0 -0.3 2.4 - 0.9 0.8 pksl6 2.6 1.7 1.8 - NDb ND fabD 3.3 1.4 2.8 2.8 0.9 1.5 acpM 2.9 1.0 2.7 3.8 1.1 1.4 kasA 2.6 0.6 3.0 2.8 ND ND kasB 2.7 1.0 3.1 2.4 1.6 2.6 accD6 2.9 0.5 3.7 2.5 1.3 1.8 ahpC a - - 2.9 1.1 2.8 " not in cluster, b no data on array Clearly, operon gene expression, in this case the INH inducible KasA/KasB operon (17), could be seen in the microarray data: pksl3 and fadD32 are both upregulated by over two-fold and accD4 is upregulated by 1.4-fold. KasB is upregulated 2.6-fold. The remaining genes in the operon containing kasB were also induced as follows: fabD - 1.6 fold; acpM - 1.4 fold; accD6 - 1.5 fold; kasA - no data on array. Comparison to published signature gene sets A straightforward comparison between the genes induced by mycolic acid synthesis inhibitors and those found in the exocarpic acid data set suggest that there was significant overlap (Table 3.2). 60 It should be noted that the weakness in the exocarpic acid data set is that the spots for kasA and inhA did not develop in the exocarpic acid arrays. However, the fabD-acpM- kasA-kasB-accD6 genes form one transcriptional unit and one can reasonably assume that the "missing" genes were transcriptionally activated as well. INH and EXO both share a significant number of genes in the Boshoff data (efpA (Rv2846c), acpM (Rv2244), kasB (Rv2246), accD6 (Rv2247), fadE5 (Rv0244c)). Furthermore, ahpC (Rv2428), shared with the Wilson cluster was also identified, as well as Rv2229 (ahpD), an enzyme that reduces AhpC (18). Several genes not represented in the published cluster data of mycobacterial cell wall inhibitors for which exocarpic acid increased transcription are: aceA (isocitrate lyase, Rv0467); Rvl057 (a gene of unknown function, but repressed during intracellular growth); Rv0196 (a transcriptional regulator of Rv0197, an oxidoreductase); Rvl592c (an extracellular lipase); Rv2710 (MysB or SigB, a RNA polymerase sigma factor which was upregulated in all cell wall inhibitor treatments and appears to induce transcription of KasA (19)); Rv0251c (hsp, a heat shock protein), and ahpD (an alkyl hydroperoxidase; a stress response gene). A gene not induced in the previously published clusters Rv3645, a predicted adenyl cyclase, was induced by the cell wall inhibitors and exocarpic acid (20). Overall, exocarpic acid induced genes in common with INH and TLM, both known inhibitors of FAS-II enzymes. The INH upregulated gene cluster contains 24 genes, of which exocarpic acid shared 18, thiolactomycin shared 19 and cerulenin (inhibitor of FAS-I) shared 17 genes. Hierarchical cluster analysis using Cluster 3.0 Hierarchical cluster analysis produced the dendrogram in Figure 3.2a. The immediately proximal nodes to exocarpic acid on the dendrogram are cerulenin (CER), ethambutol (EMB), thiolactomycin (TLM), nigericin and pyrazinamide (PZA). All of these compounds interfere in fatty acid metabolism, except nigericin which is a protonophore. Curiously, isoniazid (INH), also a mycolic acid metabolism inhibitor, fell outside of the core cluster, while one of the Rifampicin samples was proximal. Clearly unrelated were tetracycline and proton gradient dissipaters: carbonylcyanide 3- chlorophenylhydrazone (CCCP) and dinitrophenol (DNP). Desfuroylceftiofur cysteine disulfide (DCCD, a cephalosporin type antibiotic; cell wall biosynthesis inhibitor) clusters with other cell wall inhibitors: vancomycin and isoniazid. Amikacin (30S ribosome inhibitor) and streptomycin (16 S ribosome inhibitor) are proximal to each other, but tetracycline (also a 30S ribosome inhibitor) falls outside of the group. Valinomycin, a potassium binding dodecadepsipeptide, which disrupts the potassium gradient, and triclosan, a biocide with partially unknown mode of action likewise cluster together. Overall, there is a reasonable, but not perfect correlation, of the mode of action and treatment target group. For the purpose of lead generation exocarpic acid clearly fell into the group that contained fatty acid biosynthesis inhibitors, with the only outlier in this section of the dendrogram being sodium azide (NaNa), a respiration inhibitor. When the Spearman rank correlation was used as the similarity metric, a correlation resulted (Figure 3.2b) that nearly perfectly placed fatty acid metabolism inhibitors into one group and all other agents into two other clusters. 62 Figure 3.2 Dendrogram showing hierarchical clustering of arrays. Using (a) Euclidian distance or (b) Spearman rank correlation as similarity metric. 63 19-16 lOug/mL Tetracycline 1-43 ImM DNP 2-12 ImM DNP 2-17 50uM CCCP 2-36 50uM CCCP 19-13 lOug/mL Amikacin 19-12 lOug/mL Streptomycin 2-18 20uM DCCD 2-13 20ug/mL KCN 2-38 20uM DCCD 18-31 40ug/mL Vancomycin 19-23 40ug/mL Vancomycin 19-15 SuM INH - 19-11 0.5ug/mL Rifampicin r 2-20 5ug/mL KCN ' 2-39 5ug/mL KCN - 18-20 0.5ug/mL Rifampicin i 8-81 8-81 EMB (10ug/mL)12h | 8-82 8-82 EMB <10ug/mL)12h [i 21-24 20ug/mL exocarpic acid L 2m1t-a2b54 -4104u g0/.m5Lu ge/xmoLc aCrepriucl eanciind 1r 99--8821 113300uugg//mmLL TTLLMM _i1 43~ ~7361 34--7361 11..22mmgg//mmLL PPZZAAppHH55..66 lx r5 5-=336 22mmMM N aNNa3N 3 1r 33--31 0 5500uuMM NNiiggeerriicciinn 5-5 lOug/mL menadione mtab2-19 lOug/mL Triclosan mtabl-26 lOug/mL Triclosan 20-36 5mM Valinomycin 111Iii1i 111111122m3555m2488m212222289909998-tt--- -~t-----------------1aa33a3571821483313211321133bb61b6 8720318926066332112 l4 2l ---24325m28I8O552I510204541ll0l--0Mummu--0u0m09.6.m00O4OuO7u3gM Mgu88guMu5 M5 uuu uguM/6N1 /M21/MM uu gggg/gDlmla Nm 0 Dm gg///V/mN/DONLOCaL1.ND1CLEE//mmmamLPmC3uCu NP 5.CC.M MmmLLLLlL CKgCg 3u2 mCKC2BBLL i DCK/P/ gmPCeDm AnVVTNC mmS /gNg n ((RRmoaaNLe Ltm/ /a1liiinnm t rLmmd0OffkccyreTTL Liuuaaaoocaprr C oggcmmmmictiiPePn//ippyynoyccZrZemmniicc mcAlluA LLiicc ylpoolp))nniiciHssHe11nn in5aa5n22 ne.nn.ihh 6 6n (()) 1i 1223399911-------31881220 12545 5 50112540u330u0uM00MuuM uug g Ngg/I/iN//mNmgmimLHLeLgL re eeTirTxxLciLooMicMcc ni aa nrr ppiicc aacciidd Interestingly, the 10 pg/mL triclosan treatment now clustered with protonophores and respiration inhibitors, as mentioned before triclosan can exhibit multiple mechanisms. Exocarpic acid clearly clustered with known fatty acid metabolism inhibitors, just as in the previous analysis, the only exception in this correlation being the protonophore nigericin. Correlation analysis performed with Matlab 2009a showed that the following arrays had the highest correlation coefficients when compared to exocarpic acid: cerulenin (0.230), thiolactomycin (0.168; 0.160), isoniazid (0.123), one of the repeat DCCD samples (0.123), nigericin (0.104; 0.134), and menadione (0.101). The remaining arrays had correlation coefficients of less than 0.1, or were negatively correlated, e.g., rifampicin and CCCP. The full set of correlation coefficients against the exocarpic acid arrays is presented in Table 3.3. Gene set enrichment algorithm The results of GSEA analysis showed that the EXO gene set is enriched in genes which are found in the INH, CER, TLM array data. The overall enrichment scores obtained for EXO are summarized in Table 3.4. Enrichment score (ES) represents the enrichment score, the larger the value the more enriched the gene set is for signature genes in the rank ordered set of genes for that set. Similarly, the NES (Normalized Enrichment Score) takes into account the differences in gene set sizes. The FDR q-value represents the False Discovery Rate, which indicates the probability of a gene set to be a false positive signal. A lower FDR value represents a more valid gene set enrichment analysis, provided that the nominal p-value (a measure of significance that takes the size of the enrichment set into account) is less than 0.05. 65 Table 3.3 Correlation coefficients derived for exocarpic acid versus other drug treatments. Correlation coefficients >0.1 are highlighted for the 40 pg/mL exocarpic acid treatment. 40 pg/mL Exocarpic acid 20 pg/mL Exocarpic acid 40 pg/mL Exocarpic acid 1.000 0.883 20 pg/mL Exocarpic acid 0.883 1.000 EMB 0.084 0.060 EMB 0.091 0.061 INH 0.123 0.086 TLM 0.168 0.097 TLM 0.160 0.094 TRC 0.026 0.026 TRC 0.023 0.025 CER 0.230 0.196 Nigericin 0.104 0.100 Nigericin 0.134 0.138 PZA 0.073 0.056 PZA 0.066 0.049 DNP 0.030 0.019 DNP 0.027 0.016 Rifampicin -0.091 -0.080 Rifampicin -0.142 -0.136 Streptomycin 0.074 0.065 Amikacin 0.065 0.058 Tetracycline 0.053 0.037 Vancomycin 0.058 0.045 Vancomycin 0.069 0.057 KCN 0.079 0.078 KCN 0.067 0.055 CCCP -0.003 -0.010 CCCP -0.002 -0.010 DCCD 0.094 0.076 DCCD 0 123 0.104 Valinomycin 0.081 0.053 menadione 0.101 0.077 NaN3 0.062 0.059 NaN3 0.090 0.099 66 Table 3.4 Enrichment scores for exocarpic acid transcriptional data. Gene Set Size ES NES Nom. p-value FDR q-val INH 193 0.30 1.31 0.041 0.270 TLM 47 0.23 0.84 0.723 1.000 CER 39 0.20 0.70 0.882 0.940 According to the GSEA manual, FDR q-values of 25% (0.25), or less, are most likely to generate interesting hypotheses, i.e., genes found in these sets are involved in pathways of interest (15). From this point of view only the INH gene set was relevant. The first 50 genes with a positive enrichment score in the INH data set are listed in Table 3.5. Fatty acid metabolism related genes (shaded light grey) constituted 5 of the 50 genes (10%). Most of the remainder of the genes are of unknown function, ribosomal or central metabolism genes. It is not clear why these genes are enriched, but perhaps the safest assumption is that they are activated as a response to stress. The pathways identified as upregulated by exocarpic acid treatment by biological pathways analysis are given in Table 3.6. Fatty acid degradation pathways (P-oxidation) were identified, since the genes involved in these pathways are highly upregulated and therefore easily identifiable (Table. 3.1). Even though most of the genes for fatty acid biosynthesis were upregulated below the cut-off value of 2.0, the metabolic pathway tool still identified these pathways, in agreement with the clustering data above. Both the FAS-I and FAS-II systems are represented. In addition, isoleucine depletion was identified as a possible molecular mechanism for exocarpic acid against M. tuberculosis H37Rv. 67 Table 3.5 Top ranked genes identified by GSEA in the INH array that are positively correlated. Rank Description Rank Metric Running Score ES 1 Rv2055c, 30S_ribosomal_protein_S18 3.37 0.02 2 Rv0073, ABC-transporter_ATP-binding_subunit 2.91 0.04 3 Rv0892, putative_monooxygenase 2.65 0.06 4 Rv2911, penicillin_binding_protein 2.63 0.08 Rv2462c, 5 chaperone_protein,_similar_to_trigger_factor 2.61 0.09 6 Rv2068c, class_A_b-lactamase 2.47 0.11 7 Rv3303c, dihydrolipoamide_dehydrogenase 2.35 0.12 8 Rv3011c, glu-tRNA-gln amidotransferase, subunit B 2.08 0.13 Rv0482, UDP-N- 9 acetylenolpyruvoylglucosaminereductase 2.02 0.14 10 Rv3747, (MTV025.095), len: 120. Unknown, highly 1.87 0.15 11 Rv0888, possible_membrane_protein 1.84 0.16 12 Rv2144c, probable_transmembrane_protein 1.74 0.17 13 Rv3423c, alanine racemase 1.69 0.17 Rv2247, 14 acetyl/propionyl_CoA_carboxylase_b_subunit 1.68 0.19 15 Rv3271c, (MTCY71.11c), len: 222, unknown 1.65 0.19 16 Rv3718c, (MTV025.066c), len: 147. Unknown. 1.56 0.20 17 Rv2075c, (MTCY49.14c) 1.45 0.20 18 Rv0626, (40.0%_id) 1.43 0.21 19 Rv3737, possible membrane_protein 1.43 0.22 20 Rvl560, (MTCY48.05c), len: 72. Unknown 1.26 0.21 21 Rv0879c, (MTCY31.07c), len: 91 unknown, 1.23 0.21 22 Rv2904c, 50S_ribosomal_protein_L19 1.23 0.22 23 Rvl839c, (77aa) (40%_id_in_45aa_overlap) 1.21 0.23 Rv2244, 24 acyl carrier_protein_(meromycolate_extension) 1.19 0.24 Rv2464c, 25 probable_DNA_glycosylase,_endonuclease_VIII 1.17 0.24 26 Rv3481c, possible membrane_protein 1.16 0.25 27 Rv0312, similar_to_MTCY190.09c) 1.08 0.25 28 Rv2721c 1.07 0.25 29 Rv0496 1.07 0.26 30 Rvl240, malate_dehydrogenase 1.01 0.26 31 Rv3368c 0.95 0.26 32 Rv2274c, (MTCY339.36), len: 105. Unknown, 0.91 0.26 Rvl665, polyketide_synthase_(chalcone_synthase- 33 like) 0.90 0.27 34 Rv0647c, Q55884 0.90 0.27 35 Rv3708c, aspartate semialdehyde_dehydrogenase 0.89 0.28 36 Rvl361c, PPE 0.83 0.28 Rvl558, (MTCY48.07c), len: 148. Like 37 MCY03C7.09c, 0.82 0.28 68 Table 3.5 continued Rank Description Rank Metric Running Score ES 38 Rvl592c, similar_Q49629_LEPBl 170_F1_46 0.81 0.29 39 Rv2563, possible membrane_protein 0.79 0.29 40 Rv3893c, (MTCY15F10.19), len: 77. PE 0.78 0.29 41 Rv0530, (31%) 0.78 0.30 42 Rv3524, possible membrane sensor_protein 0.66 0.28 43 Rv0203, (MTV033.11), len: 136. Unknown 0.65 0.28 44 Rvl 144, short-chain_alcohol_dehydrogenase 0.62 0.28 45 Rv0642c, methoxymycolic_acid_synthase_4 0.62 0.29 Rv0566c, 46 similar_to_BSY09476_42_and_YAJQ_ECOLI 0.61 0.29 47 Rv0646c, probable_hydrolase 0.61 0.30 48 Rv 1527c, polyketide_synthase 0.57 0.29 Rv3520c, probable_coenzyme_F420- 49 dependent_enzy me 0.57 0.30 Rv2246, b-ketoacyl-ACP_synthase 50 (meromycolate extension) 0.53 0.29 69 Table 3.6 Biological pathways identified for 40 pg/mL exocarpic acid treatment of Mycobacterium tuberculosis H37Rv. Pathway Class Pathway Upregulated Fatty acid degradation Anapleurotic and degradative pathways Biosynthetic Stress Fatty acid biosynthesis fatty acid (3-oxidation 1 isoleucine degradation I and isoleucine degradation II glyoxylate shunt central metabolism leading to acetate precursors 2-oxobutanoate degradation I octane oxidation serine-isocitrate lyase pathway pyridoxal 5'-phosphate biosynthesis serine biosynthesis thioredoxin pathway fatty acid biosynthesis - initial steps fatty acid biosynthesis - initial steps II (plant) fatty acid elongation - unsaturated I fatty acid elongation - saturated Biological pathway analysis The underlying reason is not quite apparent, but could stem from an increased requirement for branched chain fatty acid biosynthesis secondary to long-chain fatty acid starvation (21). Serine biosynthesis up regulation could conceivably serve to supply precursors to balance isoleucine biosynthesis. Discussion Analysis of the exocarpic acid induced transcriptome data showed that the most reliably identified clusters predict the involvement of fatty acid degradation, stress response pathways and fatty acid biosynthesis (Table 3.1). Our preliminary, non-rigorous analysis performed on a reduced data set found that many of the fatty acid biosynthesis genes were up regulated in the exocarpic acid array very similar to data for isoniazid (INH), thiolactomycin (TLM), and cerulenin (CER). It was on that basis that we hypothesized that exocarpic acid might be a fatty acid biosynthesis inhibitor. Hierarchical cluster analysis Hierarchical cluster analysis of the arrays showed a correlation of exocarpic acid with known fatty acid metabolism inhibitors. Cerulenin (CER), ethambutol (EMB), thiolactomycin (TLM) and pyrazinamide (PZA) all clustered with exocarpic acid. Cerulenin is a KAS-III inhibitor, an enzyme that connects the de novo fatty acid biosynthesis system (FAS-I) and the fatty acid elongation system (FAS-II). Pyrazinamide is a FAS-I inhibitor, while ethambutol and thiolactomycin are known FAS-II inhibitors. This result is in concordance with the initial inspection of the array data and in line with the hypothesis that exocarpic acid is a mycolic acid biosynthesis inhibitor. Reevaluation of the data using Spearman rank correlation as the similarity metric produced a tighter clustering of array with respect to mode of action. In this instance exocarpic acid and all known fatty acid biosynthesis inhibitors, except pyrazindamie (PZA) clustered. Protonophores and oxidative phosphorylation uncouplers (e.g., KCN) clustered, while most of the prototypical antibiotics fell into their own cluster. While more parsimonious with respect to mode of action, the result that exocarpic acid clusters with fatty acid metabolism inhibitors is essentially identical to the Euclidian distance metric derived clusters. It is noteworthy however, that the approach taken to the clustering analysis can produce rather different clustering patterns. It is also clear from the data that the treatment dose has a large influence on the potential mode of action as derived by clustering analysis. This is not unexpected since, for instance, triclosan can exhibit cell membrane effects at high concentration and FAS-II inhibition at low concentration (4). Since HCL analysis are unsupervised and sensitive to outliers the results were confirmed by computing the correlation coefficients between the array data sets used in the HCL analysis. The highest correlation to exocarpic acid were found to be cerulenin and thiolactomycin, both known fatty acid biosynthesis inhibitors. Isoniazid and nigericin were also correlated with coefficients larger than 0.1. Largely these data corroborate the Spearman rank analysis, which clustered exocarpic acid with cerulenin, thiolactomycin, isoniazid and nigericin. Gene set enrichment algorithm The GSEA tool circumvents single gene analysis problems, e.g., a preponderance of significant but unrelated genes or a lack of a pathways concordance of genes, by analyzing a priori known gene signature sets represented in gene expression data. The exocarpic acid array data had to be modified to conform to the program's requirements. Furthermore, a ready-made comparison array data set was not available and therefore had to be constructed from raw expression files. Since the hierarchical clustering analysis predicted that exocarpic acid would be a fatty acid metabolism inhibitor a small group of known inhibitors was chosen, with CCCP, a protonophore, as the "alternate" hypothesis. The GSEA algorithm identified INH as positively correlated with exocarpic acid (Table 3.3) at a statistically significant level. Inspection of the top 50 ranked genes showed that 10% can be characterized as fatty acid/mycolic acid metabolism genes. 72 While not overwhelming, again, the theme that exocarpic acid is a mycolic acid metabolism inhibitor is repeated in the GSEA analysis. Biological pathway analysis As a tool, biological pathway analysis can be tremendously useful in uncovering connections between pathways activated or repressed by a particular drug treatment. The most highly expressed genes in the exocarpic acid data fell into fatty acid degradation pathways. However, fatty acid biosynthesis pathways were also identified. Again, the pattern of mycolic acid biosynthesis inhibition recurred. Unlike any previous analysis however the biological pathway tool also highlighted central metabolism pathways as highly activated. The underlying reason may be the fatty acid biodegradation, which feeds acetyl-CoA precursors through the glyoxylate shunt. This pattern was clearly present in the biological pathways analysis, which identified both the glyoxylate shunt and the related serine-isocitrate lyase pathways; both central enzymes in this anapleurotic (precursor generation) pathway. Reason for the up regulation of serine biosynthesis/isoleucine degradation pathways is not clear, however, an increased requirement for branched fatty acids secondary to long-chain fatty acid biosynthesis inhibition could explain this regulatory pattern. Indeed, the strongest candidate for the mode of action of exocarpic acid is suggested by the fatty acid metabolism data. Here, either degradation, or biosynthesis of fatty acids seems to point to the mode of action. Considering that fatty acid degradation is not known to cause toxicity, the most likely mechanism is mycolic acid biosynthesis inhibition. This mechanism was already a plausible hypothesis before the microarray analysis, and with these data became the favored hypothesis to be tested. Due to the 73 involvement of FAS-I/FAS-II pathways in mycolic acid biosynthesis the mode of action studies focused on these enzymatic systems. References 1. Wilson M, DeRisi J, Kristensen HH, Imboden P, Rane S, Brown PO, et al. Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization. PNAS 1999;96:12833-12838 2. Boshoff HIM, Myers TG, Copp BR, McNeil MR, Wilson MA, Barry III CE. The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism novel insights into drug mechanisms of action. J Biol Chem 2004;279:40174-40184. 3. Betts JC, McLaren A, Lennon MG, Kelly FM, Lukey PT, Blakemore SJ, et al. Signature gene expression profiles discriminate between isoniazid-, thiolactomycin-, and triclosan-treated Mycobacterium tuberculosis. Antimicrob Agents Chemother 2003;47:2903-2913. 4. Slayden RA, Lee RE, Barry III CE. Isoniazid affects multiple components of the type II fatty acid synthase system of Mycobacterium tuberculosis. Mol Microbiol 2000;38:514-525. 5. http://www.broad.mit.edu/gsea/ Accessed April 2009. 6. http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE1642 Accessed April 2009. 7. http://omad.operon.com/download/storage/m_tuberculosis_VLl.2_datasheet.pdf. Accessed April 2009. 8. http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GPL1343. Accessed April 2009. 9. Cluster 3.0 http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/software.htm Accessed April 2009. 10. http://www.biohealthbase.org/GSearch/home.do?decorator=BioHealthBase Accessed April 2009. 11. http://genolist.pasteur.fr/TubercuList/Accessed April 2009. 12. http://jtreeview.sourceforge.net/ Accessed June 2009. 13. http://smd.stanford.edu/cgi-bin/search/QuerySetup.pl Accessed June 2009. 74 14. Doran JL, Pang Y, Mdluli KE, Moran AJ, Victor TC, Stokes RW. Mycobacterium tuberculosis efpA encodes an efflux protein of the qaca transporter family. Clin Diagn Lab Immunol 1997;4:23-32. 15. Fu L, Shinnick T. Understanding the action of inh on a highly INH-resistant Mycobacterium tuberculosis strain using genechips. Tuberculosis 2007;87:63-70. 16. Colangeli R, Helb D, Vilcheze C, Hazbon MH, Lee C-G, Hassan S, et al. Transcriptional regulation of multi-drug tolerance and antibiotic-induced responses by the histone-like protein lsr2 in M. tuberculosis. PLoS Pathog 2007;3:e87. 17. Daffe M, Reyrat J-M (Eds). The Mycobacterial Cell Envelope ASM Press Washington, DC. 2008. 18. Koshkin A, Nunn CM, Djordjevic S, Ortiz de Montellano PR.The Mechanism of Mycobacterium tuberculosis alkylhydroperoxidase ahpd as defined by mutagenesis, crystallography, and kinetics. J Biol Chem 2003;278:29502-29508. 19. Raman S, Hazra R, Dascher CC, Husson RN. Transcription regulation by the Mycobacterium tuberculosis alternative sigma factor SigD and Its Role in Virulence. J Bacteriol 2004;186:6605-6616. 20. Lee J-H, Karakousis PC, Bishai WR. Roles of SigB and SigF in the Mycobacterium tuberculosis sigma factor network. J Bacteriol 2008;190:699-707. 21. http://www.genome.jp/dbget-bin/get_pathway?org_name=mtu&mapno=00280 Accessed June 2009. CHAPTER 4 MODE OF ACTION STUDIES Introduction This chapter details the mode of action studies with exocarpic acid. Chapter 2 showed the specificity of exocarpic acid for Mycobacterium tuberculosis and Chapter 3 suggested the hypothesis that the principal mode of action of exocarpic acid is that of fatty acid biosynthesis inhibition. Although our primary hypothesis was that exocarpic acid inhibits fatty acid biosynthesis, precedent suggested that an alternative hypothesis needed to be explored. It has been established in the literature that fatty acids, fatty acid derivatives and cationic long-chain molecules can act by increasing membrane permeability (1, 2). The physico-chemical character, i.e., the amphiphilicity of these molecules, would be consistent with such a hypothetical mechanism. The end-result of membrane destabilization would be the loss of the proton gradient, which drives cellular adenosine triphosphate (ATP) generation in bacteria (3). The generic mechanism of membrane destabilization would be expected to affect mammalian cells as well. However, many fatty acids exhibit low toxicity against human cells, exocarpic acid included, arguing against such a hypothesis. In addition, other mechanisms of bacterial inhibition have been shown for lauric acid and glycerol monolaurate, both of which interfere with signal transduction in Staphylococcus aureus (4, 5). Similarly, a variety of antimicrobial lipids at the skin surface suggests diverse mechanisms of bacterial 76 inhibition (6). Nevertheless, some anionic and cationic fatty acid derivatives do induce gene expression profiles in M. tuberculosis similar to those of known proton gradient dissipating molecules (1). Therefore it was necessary to first investigate the effect of exocarpic acid on proton gradient in M. tuberculosis, even if just to exclude this alternative mechanism, before proceeding to test fatty acid inhibition by exocarpic acid in M. tuberculosis. Proton gradient dissipation studies The amphiphilic and weak acid characteristics of exocarpic acid, argue that proton gradient dissipation could explain its antimycobacterial action. Dissipation of the proton gradient results in the loss of viability of M. tuberculosis, because the proton gradient is the primary mechanism of ATP and reducing equivalent generation. To measure membrane permeability, we adapted a relatively straightforward slide-deposition assay utilizing the dyes propidium iodide (PI) and rhodamine 123 (R123) which was published by Parish et al. (7). PI functions as a counterstain, to ensure that R123 labeled cells have intact cell membranes and are not simply artifacts. R123 accumulates and fluoresces green (Excitation: 485 nm, Emission: 540 nm) in bacterial cells with an intact proton gradient. Carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), a protonophore which abolishes the proton gradient served as positive control. Vehicle (DMSO) treated cells served as the negative control. If exocarpic acid would indeed dissipate the proton gradient then the expected result would be a decreased fluorescence of R123 stained cells in comparison to vehicle treated cells. The slide-deposition method did not show a difference between control or CCCP labeled cells and, in fact, all cells on a slide showed PI fluorescence, implying that all deposited cells had permeable membranes. Hence, the approach was abandoned after several attempts at varying conditions and dye concentrations. It seemed likely that the failure of the PI/R123 slide-deposition approach was due to the slide deposition process or fixation step. To overcome this, the PI/R123 approach was adapted to flow cytometry. Flow cytometry was deemed as superior to the slide deposition method because no harsh centrifugation treatment or encapsulation of bacteria with silicon oil was required. Furthermore, the analysis was rapid in comparison to counting cells on a slide under a fluorescence microscope. Fatty acid biosynthesis inhibition Fatty acid biosynthesis inhibition was identified early as a candidate for mode of action of exocarpic acid, due to the similarity of the natural saturated 18-carbon acyl fatty acid which is produced by the FAS-I system and utilized as substrate in FAS-II (see Figure 1.4). As the end product of FAS-II is the long-chain component of mycolic acids, an inhibition in FAS-II results in a lack of mycolic acids after treatment. As one might expect from the biosynthetic scheme depicted in Figure 1.4, an inhibition in FAS-II will lead to accumulation of precursors from FAS-I, i.e., C-16/18 and C-24/26 fatty acids. Several methods have been published to determine the amount of newly synthesized mycolic acids in the mycobacterial cell wall. All of these methods rely on radioactive precursor feeding (l4C-acetate) and measuring incorporation of radioactivity into the fatty acid fraction of a cell lysate. Since none of the published methods could be reproduced reliably in our laboratory, the method presented in the materials and methods section is an adaptation of several published methods that we developed in order to generate reliable results. 78 Materials and methods Proton gradient dissipation A log phase culture of M. tuberculosis H37Ra growing in 7H9 medium supplemented with ADC was diluted with fresh prewarmed 7H9 with ADC. Aliquots of 1 mL were withdrawn and placed in 1.5 mL centrifuge tubes in duplicate sets. Each tube received either 1 pL vehicle (DMSO) (4 tubes total), 1 pL of 40 mg/mL exocarpic acid (2x MIC) in DMSO, 2 pL of 2 mg/mL isoniazid (4x MIC) in water, 2 pL of 100 mg/mL triclosan (4x MIC) in DMSO or 20 pL of 7 mM CCCP (Sigma Aldrich, St. Louis, MO). Triclosan was a kind gift of Dr. Mohammad Sondossi. Tubes were incubated for 15 minutes with agitation at 37 °C. All antibiotic treated tubes received 2 pL of 7.5 mM PI and 2 pL of 65 pM R123, both dissolved in DMSO. The R123 dye was diluted with respect to the plate deposition method, which recommends 1 pL of 6.5 mM dye per 49 pL sample, due to the high intensity of the stain. Calibration standards were made as follows: one of the DMSO treated tubes received no dyes, one only PI, one only R123 and the final one both PI and R123. After 15 minutes of incubation at 37 °C with agitation; the cells were pelleted using a centrifuge (15 seconds, at 10,000 rpm) and washed with 1 mL PBS (pH 7.2), pelleted again and resuspended in PBS or fixed with 50 pL of 3% paraformaldehyde in PBS for 1 minute, pelleted again and resuspended in 1 mL PBS. Flow cytometry was performed on a FacScan flow cytometer (Becton Dickinson, San Jose, CA) at the University of Utah Health Core Facilities using CellQuest 3.3 software for analysis. The vehicle treated samples were used as follows: the DMSO sample without dyes added was utilized as the side-scatter control to calibrate the instrument for accurate detection of cells, and the PI and R123 only samples as a fluorescence detection control to set detection limits for each population. Collected data are presented as geometric means of fluorescence of approximately 10,000 counted cells. Fatty acid biosynthesis inhibition A well established method to ascertain if a drug acts as an inhibitor of long-chain fatty acid synthesis is that of 14C-incorporation in de novo synthesized long-chain fatty acids (8-10). The approach presented here is based on modified experimental protocols from Slayden et al. and Gratraud et al. (9, 10). However, the actual isolation and esterification procedure was based on a protocol published by the Centers for Disease Control (CDC) (11) with several modifications. Isoniazid (INH), thiolactomycin (TLM, a kind gift of Dr. Helena Boshoff, NIAID), inhibitors of InhA and KasA/KasB respectively, served as positive controls. DMSO, the solvent for all compounds tested, served as negative control. The expected pattern for InhA inhibition, or rather, inhibition of FAS-II cycle in general, is increased build-up of C-16/C-18 and C-24/26 precursors generated from FAS-I, as these would normally be consumed in the FAS-II cycle. Due to the inhibition of FAS-II less mycolic acids should be synthesized as well. Briefly, mid-log phase cultures of M. tuberculosis H37Ra (4 mL) were treated with the appropriate compound (DMSO, INH, TLM, and EXO) at twice (or occasionally four times) the MIC and incubated for 4 hours at 37 °C with agitation. Each culture then received 20 pL of 1, 2-14C-acetate (approximately 20 pCi) (GE Healthsciences, Piscataway, NJ) and was incubated for an additional 16 hours with agitation. Our initial extraction procedure followed the CDC protocol. Briefly, the bacterial culture was saponified in 40% methanol with 20% potassium hydroxide for 2 hours at 121 °C at 17 psi using an autoclave. The resulting lysate was acidified using 6N hydrochloric acid and extracted with an equal volume of chloroform. The methylation reaction was performed with the following changes: bicarbonate buffer (4% potassium bicarbonate in 50/50 water-methanol) was overlaid on the chloroform fraction (rather than co-evaporated onto the sample). Each reaction received 100 pL of methyl iodide (rather than p-bromophenylacyl bromide), and was incubated overnight under slight refluxing on the edge of a heat plate in a tightly closed 4 mL glass scintillation vial. One mL of 50% methanol in water was added to the lower chloroform layer and vortexed. After phase separation the chloroform layer was withdrawn, dried, dissolved in diethyl ether, transferred to a new vial, dried, and redissolved in dichloromethane. During the latter procedures the vials were usually placed in a -20 °C freezer for 3-5 minutes in order to facilitate pipetting between vials and spotting. A 20 pL aliquot of each extract was scintillation counted in a Tri-Carb 2900TR scintillation counter (PerkinElmer Life And Analytical Sciences, Inc. Waltham, MA). Samples were dried again and dissolved in dichloromethane such as to give equal counts for a 5 pL spotting aliquot. TLC was performed as described (10) using KC18F slide plates (Whatman International, Maidstone, GB) and KieselgeW plates (Merck GmbH, Darmstadt, FRG) with 3:2 chloroform:methanol and 95:5 petroleum ethers:acetone as mobile phase, respectively. The exocarpic acid, INH and TLM treated samples usually showed little incorporation of radioactive precursor, yielding 1500-2000 cpm per pL extract. Spotting volume was 5 pL, approximately 7,000-10,000 cpm total depending on the experiment. TLC plates were placed in a phosphorimager cassette for 5 days and analyzed on a Typhoon 9200 Phosphorimager (GE Healthcare, Chalfont St. Giles, United Kingdom) using ImageQuant TL 7.0. Mycolic acid and palmitic acid (Sigma Aldrich, St. Louis, MO) were used to confirm relative mobility. Methyl esters of mycolic acid had a retention factor (RF) of 0.3 on KC18F plates and 0.1 on Kieselgel60F; the RFs were 0.75 and 0.7 for palmitic acid methyl ester on those same plates. Results Proton gradient dissipation The results of the proton gradient geometric mean quantification are summarized in Table 4.1. Clearly the R123 fluorescence signals of the mycolic acid biosynthesis inhibitor isoniazid and that of exocarpic acid resemble the vehicle only control with fold over DMSO control ratios of 1.1, 1.1 and 1.1, 1.0, respectively, per experiment. Exocarpic acid therefore resembled the vehicle treated control and the INH treated cells. The CCCP treated sample exhibits the expected decrease in R123 fluorescence with fold over control ratios of only 0.1 and 0.3, in fixed and unfixed cells, respectively. Table 4.1 Rhodamine 123 fluorescence in Mycobacterium tuberculosis H37Ra after treatment with INH, EXO, CCCP and TRC. Treatment Fixed Cells Unfixed Cells R123 F/C PI F/C R123 F/C PI F/C DMSO 61.8 1.0 9.6 1.0 35.3 1.0 11.3 1.0 INH 66.6 1.1 9.9 1.0 40.0 1.1 9.2 0.8 EXO 69.1 1.1 10.1 1.1 36.7 1.0 9.9 0.9 CCCP 8.4 0.1 8.9 0.9 11.2 0.3 11.4 1.0 TRC 37.1 0.6 12 1.3 38.1 1.1 10.4 0.9 PI: propidium idiode signal. R123: rhodamine 123 signal. F/C: fold over DMSO control. 82 The triclosan (TRC) treatment in one instance produced a drop in R123 fluorescence and in another resembled the control; fold over control values of 0.6 and 1.1, respectively. For TRC in the fixed cells, the decrease in R123 fluorescence was concomitant with a small increase in PI staining, suggesting an minor artifact. The data indicated that isoniazid did not decrease the proton gradient, which was expected, since it is known to be an InhA inhibitor. The TRC treatment represented more of a challenge for interpretation, since it yielded a partial decreased of 0.6-fold over control in one instance. While TRC is thought to be a specific InhA inhibitor (9) these data suggests that TRC may also alter membrane permeability, as mentioned before. Our result, showing that exocarpic acid does not destabilize the mycobacterial membrane, was in agreement with the data provided by Dr. Boshoff at NIAID using the commercial BacLight assay system (Invitrogen, Carlsbad, CA). The kit measures the internal ATP concentration of M. tuberculosis H37Rv cells by luminescence, expressed as Relative Luminescence Units (RLU) after 1 week of incubation under anaerobic conditions. Metronidazole (MTZ) was used as the positive control, as it is only active under anaerobic conditions. As can be seen from Table 4.2, no dramatic effect on internal ATP concentration was apparent until the anaerobic MIC (see Chapter 2) of exocarpic acid in M. tuberculosis H37Rv was achieved, at which point the luminescence drops to background level. A lack of concentration dependent decrease in the fluorescence signal indicates that exocarpic acid does not act in a physical manner, such as membrane permeabilization. Table 4.2 Relative ATP concentration in Mycobacterium tuberculosis H37Rv under microaerophilic conditions. Metranidazole HM 0 1.563 3.125 6.25 12.5 25 50 100 200 400 RLU 42040 31930 27280 23840 19720 21540 13040 13350 13540 10070 Exocarpic Acid (ig/mL 0 0.3123 0.625 1.25 2.5 5 10 20 40 80 RLU RLU 41680 45440 30350 33240 31760 29790 25200 31280 30080 30440 29860 31220 29050 33070 27810 21150 26220 27760 3400 3730 RLU - Relative Luminescence Units. Fatty acid biosynthesis inhibition Phosphor imaging of TLC resolved radiolabeled fatty acids from the treated M. tuberculosis H37Ra showed that exocarpic acid indeed interferes in fatty acid metabolism Clearly the INH and TLM bands show decreased mycolic acid bands and increased fatty acid bands; both the CI6/18 band and the C-24/26 band (Figure 4.1a). Exocarpic acid displayed a pattern similar to INH and TLM, but with some differences: the fatty acid bands were slightly increased and the decrease in mycolic acids (Figure 4.1b) relative to control was not complete. Repeating the experiment with exocarpic acid at twice, four-fold and six-fold the MIC produced more dramatic results. The treatment with twice the MIC of exocarpic acid consistently showed a decrease in mycolic acid production and increase in fatty acid build-up. Increasing the concentration of exocarpic acid to four fold the MIC clearly shows inhibition of mycolic acid biosynthesis and a large increase in fatty acid accumulation, very similar to INH (Figure 4.2). Treatment with six-fold of the MIC proved too toxic and resulted in no recoverable radioactivity. 84 Figure 4.1 Incorporation of 14C-acetate into the fatty acids and mycolic acids in M. tuberculosis H37Ra. Treatment groups are solvent (DMSO), exocarpic acid (Exo), isoniazid (INH), thiolactomycin (TLM) and rifampicin (RIF). A. Reverse phase KC18 plates were developed using 3:2 chloroform:methanol. B. Normal phase. KieselgeW plate were developed using 95:5 petroleum ethers: acetone m CO A A 00 CD o CO o CO >> XL CO CO^D J CM O •D O CO £ CO o X Lil O w S Q •o o CO o oo>» 86 Figure 4.2 Dose effect on incorporation of 14C-acetate into the fatty acids and mycolic acids in M. tuberculosis H37Ra. Treatment groups are solvent (DMSO), exocarpic acid at twice the MIC (Exo 2x), exocarpic acid at four times the MIC (Exo 4x), and isoniazid (INH). Reverse phase KC18 plates were developed using 3:2 chloroform:methanol. C16/18 Fatty acids C24/26 Fatty acids t • Mycolic Acids ^ DMSO Exo 2x Exo 4x INH2x The pattern observed is exactly what is expected if exocarpic acid were indeed a FAS-II inhibitor. The fact that the fatty acid band was increased, rather than decreased, shows that the FAS-I system is not affected by exocarpic acid. Discussion Proton gradient inhibition There was no discernable difference in R123 fluorescence between the DMSO control and exocarpic acid treatments in the proton gradient dissipation experiments. This result is indistinguishable from INH treatment and argues against membrane permeabilization as the mode of action for exocarpic acid. The lack of large quantities of purified exocarpic acid did not allow us to explore much larger doses, e.g., six times the MIC, or run an extensive time course. Once an abundant amount of exocarpic can be synthesized these experiments will be performed to complete the data set. Fatty acid biosynthesis inhibition The data derived from the fatty acid biosynthesis inhibition experiment argue strongly that exocarpic acid acted as a fatty acid biosynthesis inhibitor, likely in the FAS-II system. The exocarpic acid concentration dependent decrease in mycolic acid production, concomitant with an increase in short chain fatty acids mirrors the pattern observed for isoniazid and thiolactomycin, both known FAS-II inhibitors. One alternative explanation, which also could fit the observations, would be inhibition of KAS-III (Figure 1.4) by exocarpic acid. The build-up of C16/18 and C24/26 fatty acids clearly discounted exocarpic acid as a FAS-I inhibitor. The apparent difference in potency of mycolic acid synthesis inhibition between exocarpic acid at 2x its determined MIC, and INH at 2x its MIC, may be partially explicable by the growth conditions. The growth MICs are determined in a stationary multi-well plate assay, while the radioactive precursor incorporation studies used Erlenmeyer flasks which are incubated under agitation. Furthermore, the volume was only 200 pL for the MIC determination assay, but 4 mL during the cell wall biosynthesi