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

Division Of Basic Sciences - Nci

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Abstract

Goal One: Second-generation INSTIs are strongly recommended for people living with HIV-1 (PLWH). The emergence of resistance to second-generation INSTIs has been infrequent and has not yet been a major issue in high-income countries. However, the delayed rollouts of these INSTIs in low- to middle-income countries during the covid pandemic combined with increased transmission of drug-resistant mutants worldwide, is leading to an increase in INSTI-resistance. Our development of INSTIs has focused on compounds having of a metal-chelating naphthyridine core scaffold. We have observed that molecular interactions of key 6-substituents are positioned within a defined substrate envelope by an overlapping space region occupied by both the uncleaved viral DNA (vDNA) and the host target DNA (tDNA) substrates. Our recent cryo-EM structures bound to HIV intasomes further reinforce the concept that maximizing shape complementarity within the substrate envelope is a promising strategy for designing new INSTIs with improved resistance profile. We evaluated the antiviral potencies of our lead developmental 6-substituted naphthyridine-based INSTI (4d) and the second-generation INSTIs DTG, BIC and CAB against a panel of viral constructs having IN quadruple mutants. The mutations are centered around G140S/Q148H including positions L74, E92, T97 combined with E138A/K +G140S + Q148H. These IN mutants have been identified in clinical trials where PLWH who have been put on a DTG-containing regimen after failing a first-generation RAL-based therapy. All of the INSTIs we tested lose potency against these IN quadruple mutants as compared with the wild-type IN. In single round infection assays compound 4d retained higher antiviral potencies (EC50 values) than second-generation INSTIs against a subset of quadruple mutants. These findings may advance understanding of mechanisms that contribute to resistance and, in so doing, facilitate development of new INSTIs with improved antiviral profiles. In further work, we have recently discovered a naphthyridine-based INSTI (4a) which lacks the key 6-substituent but contains a 5-hydroxymethyl group. Unexpectedly 4a exhibits potent antivira activity against drug-resistant IN variants. Using cryo-EM structures of 4a bound to HIV-1 intasomes we have shown the 5-hydroxymethyl group of 4a engages the overhanging 3-nucleobase. We substituted the 4-hydroxy group in 4a with a 4-anino group (compound 4b) and found that it exhibits improved retention of antiviral potencies against WT integrase and the well-defined integrase single mutants G118R, E138K, N155H and R263K. As compared to second-generation FDA approved INSTIs, 4b shows enhanced potency against the double mutants G140S/Q148K and G140A/Q148K, and the triple mutant E138K/G140A/Q148K. In cryo-EM structures of HIV intasomes, compound 4b behaves similarly to 4a, showing pi-pi stacking between naphthyridine and the terminal adenine of the vDNA, as well as hydrogen bond interactions between 5-hydroxymethyl group and the terminal 3'-adenine of the vDNA. This work supports the potential importance of enhancing pi-pi-stacking interactions with the 3'-nucleobase as a means of improving retention of antiviral potencies against INSTI-resistant mutant forms of integrase. These structural insights highlight a new strategy for the development of next-generation INSTIs to combat HIV-1 resistance. Goal Two: We are preparing H3.3 histone variants (histones are 135 amino acid proteins) for use in the constructing modified nucleosomes to impart enhanced affinity for binding partners. Our work is primarily concerned with the total synthesis of these modified histones. We have focused on the 135 amino acid residue H3K36Me3 and H3K36Me2 in which the K36 residue has been replaced by a lysine bearing a NMe2- and NMe3- modified side chains. We have optimized the total synthesis and purification of the H3K36Me3 construct using with an overall yield of ~14% yield. Currently, our efforts are focused upon increasing the yield and large-scale synthesis and purification of the H3K36Me3 and H3K36Me2 constructs. We have successfully prepared the modified H3 histones contain acetyl-CoA-modified lysines at the 27 position (H3K27CoA) and the 18 position ((H3K18CoA) with overall yields of 13% and 16%, respectively. These modified histones are being used in the Salk laboratory of Dr. Dmitry Lyumkis to study nucleosome interactions with p300.