Human Immunodeficiency Virus type 1 (HIV-1) is the agent culpable for the malady known as acquired immune deficiency syndrome (AIDS). HIV is a single stranded positive sense RNA virus whose replication is dependent upon the utilization of a DNA intermediate. An indispensable stage in its life cycle is the integration of viral DNA into the host chromatin which is catalyzed by the viral protein integrase (IN). HIV-1 IN interacts with cellular protein lens epithelium derived growth factor (LEDGF)/p75, a cellular cofactor which forms a bimodal tether between chromatin and IN. This interaction has been shown to contribute to HIV-1 integration into host chromatin and has recently become a tantalizing target for antiretroviral therapies. Presently there are more than 35 FDA approved HIV-1 inhibitors, with three of these being IN strand transfer inhibitors (INSTIs). Unfortunately, drug resistance remains problematic in the clinic and the generation of novel inhibitors is essential. Therefore, our work focuses on developing innovative therapies through better understanding of the interactions of current inhibitors, as well as on elucidating the mechanisms of HIV-1 drug resistance. Specifically, my research has centered on better understanding of the mechanism of resistance to allosteric HIV-1 IN inhibitors (ALLINIs) as well as elucidating the role of cellular cofactor LEDGF/p75 on these new inhibitors.
Chapter 1 introduces HIV-1 in its historical context, as the source of the AIDS pandemic and current and developmental biologics. The discussion of the replication cycle of HIV-1 leads to three essential proteins to my work; HIV-1 IN and protease (PR) as well as cellular protein LEDGF/p75. In particular, this chapter focuses on current therapies, drug resistance, and the biological role played by HIV-1 IN. This is done in four distinct sections: (I) analyzing the mechanism of inhibition of the FDA approved INSTIs that target HIV-1 IN’s active site; (II)
evaluation of their drug resistant mutations, (III) development of new therapeutics based on allosteric integrase inhibition and IN oligomerization (ALLINIs), (IV) and how HIV-1 overcomes this new class of inhibitors with novel drug resistance mutations.
Chapter 2 elucidates the mechanistic basis for the evolution of three distinct IN mutations in the presence of novel inhibitor KF116, a representative pyridine-based multimeric IN inhibitor (MINI), a subclass of ALLINIs. Selection of viral strains under KF116 pressure reveals the evolution of HIV-1NL4-3 (IN T124N/V165I/T174I). The T124N and T174I mutations are located at the IN dimer interface in the KF116 binding pocket while the third V165I mutation is located distinct from the pocket. It is revealed that the initial T124N mutation confers a minor level of resistance to KF116 but the mutant virus is unable to endure in the presence of higher concentrations of the drug. This results in the fruition of a second mutation, T174I which increases KF116 resistance, but results in the retardation of viral replication as a consequence of defective viral proteolytic Gag-Pol processing. In order to restore viral replication, HIV-1 develops a third mutation, V165I. We conclude this mutation is compensatory as it serves to restore the diminished viral replication. Secondly, we have demonstrated an unexpected non-catalytic role of HIV-1 IN during maturation due to its role in Gag-Pol proteolytic activity.
Chapter 3 reintroduces class II mutants. The idea of HIV-1 IN mutants was introduced in the second chapter, and further explored with mutants that expressed a phenotype similar to what was observed with the KF116 drug resistant mutant T124N/T174I. With new data that emphasizes the importance of IN-vRNA interactions, the class of mutants was explored further, with initial data that suggest that IN-vRNA binding plays a role in the atypical morphology that is observed.
Chapter 4 presents the role of LEDGF/p75 in relation to ALLINIs during viral replication. ALLINIs exploit a dual mechanism of action, but are most effective in the late stages of viral replication. This is in part because in the early stage of replication they have to compete with endogenous cellular protein LEDGF/p75 for binding IN. This chapter explores the competition between LEDGF/75 and ALLINI, BI-D. Using multiple assays, the alternative IN multimerization patterns between inhibitor and LEDGF/p75 binding are explored as well as the differences in integration site selection when BI-D is present in the early stage of viral replication. We have also investigated the relationship between ALLINIs and LEDGF/p75 during viral particle maturation, and the effects of LEDGF/p75 on MINI KF116’s potency.
Chapter 5 introduces new efforts to evaluate the novel inhibitor KF116 as well as the design and analysis of novel HIV-1 IN small molecule inhibitors. One of the characteristics that makes KF116 very attractive as a therapeutic, is its cooperativity, with hill coefficient >3. This characteristic is important to inhibitors immediate inhibitory potential, and is vital when therapeutics move onto clinical evaluation. KF116 has displayed efficacy to recent HIV-1 IN strand transfer inhibitor Dolutegravir drug resistance mutants N155H/K156N and N155H/K156N/K211R/E212T, which also adds to its potential utility in the clinical setting.
We also wanted to develop possible novel, alternative inhibitors with allosteric mode of action. The first method is the derivation of a natural product that is predicted to bind to the HIV-1 IN allosteric site. The inhibitor, Lavendustin B, is used as the parent molecule to investigate the exploitation of particular interactions using in silico approaches. After modeling and syntheses of candidate molecules, homogenous time-resolved fluorescence (HTRF) based assays are used for screening which identifies compound 2 as the best candidate with an IC50 of 3.78 µM in the LEDGF/p75 dependent IN activity assay. Unfortunately when advanced to cell based assays, it was determined that the compound was cytotoxic at similar low micro molar concentrations. The second approach to design new HIV-1 IN inhibitors was the use of scaffold hopping to determine structurally unique compounds. This led to the innovation of using reverse indole based compounds. Alterations of the substituent groups attached to the reverse indole core have been promising. Although HTRF based assays have reported results in the sub micro molar range, there was a significant loss in potency when tested in cell-based assays. While none of these compounds have displayed clinically relevant levels of inhibition thus far, their structure-activity relationships (SAR) studies will inform future efforts to develop improved ALLINIs.
Finally, chapter 6 is the summation of all that is contained within the dissertation. This includes the findings, their significance, as well as prospective future directions. This dissertation has (I) studied the mechanism of resistance to pyridine-based inhibitor KF116 and uncovered an unanticipated role that IN plays in HIV-1 Gag-pol polyprotein proteolytic processing, (II) explored the relationship between class II mutants and IN-vRNA binding, (III) further dissected the role of LEDGF/75 in ALLINI antiviral activity in the early and late stage of viral replication, (IV) as well as explored some alternative compounds as potential ALLINIs.