Investigations on alkenyldiarylmethane HIV-1 reverse transcriptase inhibitors: Mechanisms of action, synthesis, and metabolic evaulation of hydrolytically stable analogue

Matthew David Cullen, Purdue University

Abstract

Several drug classes are utilized in the treatment of HIV infections and AIDS, of which the non-nucleoside reverse transcriptase inhibitors (NNRTIs) have seen increased use in highly active antiretroviral therapy over the past decade. However, the rapid emergence of NNRTI-resistant strains of HIV is weakening the utility of the NNRTIs. One of the major goals set before the drug discovery community is to identify novel scaffolds that can be developed into NNRTIs capable of inhibiting both wild type and NNRTI-resistant strains of HIV. As part of this study, the metabolic stability of the alkenyldiarylmethane class of NNRTIs was under investigation for the purpose of increasing the hydrolytic stabilities of potent, lead compounds. To address the metabolic liabilities of the alkenyldiarylmethanes, an array of analogues was synthesized, in which each of the esters present in the pharmacophore was replaced with a variety of ester bioisosteres, and the analogues were evaluated for HIV antiviral activity. Metabolic stabilities of potent analogues were determined by incubating the compounds with rat plasma. The results of this investigation revealed that the metabolic stabilities of the alkenyldiarylmethanes could be improved significantly by replacing the esters with isosteres; however, many of the analogues proved to be particularly cytotoxic and displayed only modest antiviral activity. Fortunately, a sub-set of relatively potent analogues was identified, from which a new pharmacophore model was proposed on the basis of the inhibitors' structures. An additional part of this study included attempts to generate and validate a general interaction model for the alkenyldiarylmethanes and reverse transcriptase. Docking software was utilized to obtain de novo binding orientations for the alkenyldiarylmethanes in the NNRTI binding pocket; unfortunately, a wide range of orientations was observed and the docking results were inconclusive. Eventually a crystal structure was solved for an HIV-1 reverse transcriptase/alkenyldiarylmethane complex. The crystal structure indicated that the alkenyldiarylmethanes possessed a unique binding orientation where a large portion of the inhibitor protrudes from the back of the binding pocket near the polymerase active site. Yet, even with the crystal structure, many of the structure activity relationships for the alkenyldiarylmethanes could not be rationalized, particularly for the esters present in the pharmacophore. A comparative molecular field analysis of a series of fifty-four alkenyldiarylmethanes failed to generate a predictive CoMFA model, further suggesting that the alkenyldiarylmethanes may possess several binding modes in the NNRTI binding pocket. Throughout the history of the project, a general cytotoxicity has been exhibited by the ADAMs. Additionally, many analogues have been identified that are incapable of inhibiting HIV-1 RT, yet are potent inhibitors of the cytopathic effects of HIV in cell culture. Efforts were made to establish the mechanisms through which each of the aforementioned properties occurs. Investigations into tubulin polymerization revealed that two cytotoxic alkenyldiarylmethanes were potent inhibitors of this process, implicating this mechanism as the source of alkenyldiarylmethane cytotoxicity. However, evaluation of supplementary analogues revealed inhibition of tubulin polymerization is unique to specific compounds and not a general property of the alkenyldiarylmethanes. Inhibition of PDE4 by select ADAMs was also investigated as an alternative antiviral mechanism, but many of the compounds showed no inhibition of the enzyme at a 100 μM concentration. Analogues that did exhibit PDE4 inhibitory activity did so at high micromolar concentrations, suggesting that inhibition of the regulatory enzyme did not sufficiently factor into their antiviral activity. Other cellular and viral targets need to be investigated if the ADAMs' alternative antiviral mechanism is to be discovered.

Degree

Ph.D.

Advisors

Cushman, Purdue University.

Subject Area

Pharmacology|Organic chemistry

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