Identification of cellular targets of marine natural products

Proteases are found in all life forms and regulate many cellular pathways. Proteases have been proven as viable drug targets. Chapter 1 reviews the mechanism of hydrolysis of the amino acid based aspartic, cysteine, serine and threonine proteases. Chapter 1 also highlights the current antiprotease therapeutics in the clinic. Chapter 2 describes studies performed to better understand the mechanism of deubiquitinating enzyme inhibition by the punaglandins, marine derived prostanoids. As hypothesized, altering electronegativity of the a-carbon substituent of prostaglandin A2 derivatives modifies the activity of prostanoids. However, halogenation of the a-carbon does not increase selectivity toward a specific deubiquitinating enzyme. Interestingly, the iodinated prostaglandin A2 derivative was the most potent inhibitor. The iodinated derivative was used to identify the site of adduct formation with deubiquitinating enzymes. As hypothesized, the prostanoids interact with the active site of deubiquitinating enzymes. The triply charged ions representing the tryptic fragments containing the catalytic Cys were observed with addition of the prostanoid. The iodinated derivative forms an adduct that results in the loss of iodine, but this is not a general mechanism for all halogens. The chlorinated prostaglandin A2 derivative forms an adduct but retains the halogen. The prostanoids were also tested for their ability to inhibit ubiquitin ligation. The punaglandins and prostaglandin At derivatives do inhibit the ubiquitination of p53, but the inhibition may be through different mechanisms. A triad protein complex forms during punaglandin 2 treatment of the p53-ubiquitination sample but not during treatment with the iodinated prostaglandin A2 derivative. Chapter 3 presents the identification of the chondropsins as potent inhibitors of the Wnt signaling pathway and cellular proliferation. Their activity in the Wnt signaling pathway was shown to be due to their inhibition of V-ATPases. Inhibition of V-ATPase activity interrupts the recycling of Wntless, which decreases Wnt secretion. V-ATPase inhibition was also shown to influence the mTOR pathway. Since V-ATPase inhibition influenced both signaling pathways, the antiproliferative effects of the chondropsins are most likely due to a combination of many targets rather than one specific target.