Design and Synthesis of HIV Integrase as Potential Anti-AIDS Drugs

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FDA-approved HIV-1 IN inhibitors belong to a class of drugs called "integrase strand transfer inhibitors" (INSTIs), due to their ability to preferentially block the enzymes strand transfer (ST) reaction as related to the enzymes 3-processing (3-P) reaction. The current recommended front-line therapy for HIV-1 infected patients is an INSTI, either Dolutegravir (DTG) or Bictegravir (BIC), in combination with two nucleoside analog reverse transcriptase inhibitors. Both DTG and BIC potently inhibit most of the first generation INSTI-resistant IN mutants. Although little resistance has been selected by either BIC or DTG in treatment-naive patients, patients who have preexisting first-generation INSTI-resistant mutants and have switched to a salvage therapy featuring DTG respond poorly, emphasizing the importance of developing new and improved IN inhibitors. This adds impetus to a continuing need to develop next-generation agents that can retain high antiviral efficacy against emerging strains of INSTI-resistant virus. Utilizing my laboratorys design and synthetic capabilities, we have teamed with pharmacologists (Dr. Yves Pommier, NCI) and virologists (Drs. Hughes and Eric Freed, NCI) to develop a new genre of INSTIs. We have examined our best inhibitors side by side with the clinically relevant INSTIs using a single round infection assay against panel of new IN-resistant mutants that were selected in vitro with DTG, BIC, and CAB. Of these three INSTIs, BIC and our compounds had the broadest efficacy and were superior to DTG. In further collaborations with structural biologists (Dr. Robert Craigie, NIDDK, Dr. Dmitry Lyumkis, the Salk Institute, Dr. Cherepanov, the Francis Crick Institute, UK) we have performed studies to better understand the interactions of INSTIs with intasomes (multimeric integrase with DNA substrate and metal cofactor) and to clarify the roles that mutations play in downregulating these interactions. Cryo-electron microscopy (Cryo-EM) has played a key role in these efforts. Cryo-EM structures of our best INSTIs bound to HIV-1 intasomes revealed a complex and dynamic network of water molecules surrounding bound INSTIs, with many of these waters appearing to be conserved and occupying similar positions in the unliganded and INSTI-bound structures. However, some waters are displaced or shifted as a consequence of binding of our INSTI; others are found only when INSTIs are bound, suggesting that the conformational changes induced by the binding stabilize their position. We concluded that within the "substrate envelope" (the region defined by the binding of host and viral DNA), differences in geometry of the catalytic pockets, their overall volume, the nearby patterns of hydration, among other features, all matter for understanding INSTI interactions. Most recently we have partnered with Dr. Lyumkis to employ cryo-EM to determine how INSTIs interact with INSTI-resistant intasome mutants and elucidate the mechanisms by which resistance to these drugs emerges. The focus of these efforts is to provide a mechanistic understanding of both why and how select viral resistant variants that arise in response to the clinically used DTG as well as our best in-house compound, which is currently under pre-clinical evaluation by the NCI. This collaboration is identifying and analyzing novel mechanisms and pathways of drug resistance that arise in response to treatment with 2nd generation drugs, highlighting both primary and compensatory mutations, and providing strategies to predict future variants. Our work will elucidate the structural basis for mechanisms underlying the superior potency of novel compounds against resistant mutant forms of IN. There are four primary pathways through which IN resistance occurs in response to therapy with the potent INSTI DTG, which involve these changes: Q148H/K/R, N155H, G118R, and R263K. Substitutions at one of these positions usually arise first, both in patients and in cell culture and can cause a major loss of INSTI potency. There are 20 additional positions where a residue can be mutated to give rise to more complex IN mutants. This collectively amounts to hundreds of possible combinations. The Hughes laboratory has determined antiviral EC50 values against viral constructs having the triple mutant E138K/G140A/Q148K and found that our INSTI 4d (XZ426) has an EC50 that is 20-fold lower than that of DTG. To understand the basis of this increased potency, Dr. Craigie has prepared HIV intasomes bearing these three triple mutations. Dr. Lyumkis has determined structures of Dr. Craigies triple mutant intasomes bound to either to DTG or to our current best INSTI. Although the binding modes of both INSTIs and the configuration of individual protein residues are similar, the terminal adenosine of vDNA exhibits a stacked configuration in the context of our INSTI, but an unstacked configuration in the context of DTG. These data suggest that adenosine stacking is a real phenomenon that specifically enhances the binding of our naphthyridine-based INSTIs which may contribute to the improved ability of our INSTI to retain antiviral efficacy against this (and perhaps other) mutant(s).