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

Division Of Basic Sciences - Nci

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Abstract

Goal One. 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. We optimized synthetic protocols for the synthesis of our best in-house developed INSTI, designated as 4d or XZ426 and these were employed in the contract synthesis of a multi-gram quantity of XZ426 for use in IDP-sponsored PK studies. The PK studies include 1. Stability in plasma, fasted state simulated gastric fluid and simulated intestinal fluid 2. Thermodynamic solubility in fasted state simulated gastric fluid and simulated intestinal fluid 3. Caco-2 permeability 4. Metabolite profiling in microsomes 5. Plasma protein binding. Under sponsorship of the NCI Invention Development Program (IDP) we are partnering with the Frederick National Laboratory for Cancer Research (FNLCR) NCI Alliance for Nanotechnology in Cancer, a slow-release polymer prodrug formulation of X426 was generated. This prodrug formulation is currently undergoing testing in macaques in collaboration with Dr. Jeff Lifson, director AIDS and Cancer Virus Program (FNLCR). We have partnered with Dr. Lyumkis of the Salk Institute and Dr. Robert Craigie, NIDDK to employ cryo-EM to determine how INSTIs interact with their natural drug target, the HIV intasome (both WT and mutant), and to elucidate the mechanisms by which resistance to these drugs emerges. Our work is extending and building upon efforts to provide a mechanistic understanding of both why and how select viral resistant variants arise in response to the clinically used DTG as well as XZ426. Based on preliminary Cryo-EM data, we have designed new inhibitors that are intended to interrogate the significance of differences observed between the binding of our inhibitors and the best clinical agents. My laboratory is using single round replication assays that employ 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 more than 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. We have determined antiviral EC50 values against viral constructs having the triple mutant E138K/G140A/Q148K and found that our 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). We are currently using this to synthesize next generation inhibitor that are designed to retain greater antiviral potencies against resistant mutant forms of IN. Goal Two.The focus of this goal is to prepare H3.3 histone variants for use in the construction of modified intasomes. We have focused on the 135 amino acid residue H3K36Me3, in which the K36 residue has been replaced by a lysine bearing a NMe3-modified side chain. Initially, we employed a semisynthetic strategy using an expressed 47-135C110A. However, the inherent low solubility of this fragment rendered final TEV tag removal and gel separation to be difficult. Accordingly, we investigated a three segment N to C total synthesis approach using native chemical ligations (NCL) strategies. We optimized the total synthesis and purification of 1-135 H3K36Me3 with an overall yield (22%). We are currently optimizing the desulfurization of Cys residues, which will yield the desired final histone product.