Development of HIV-1 maturation inhibitors and capsid inhibitors

Division Of Basic Sciences - Nci

Investigators

Abstract

Aim 1. Develop highly potent and broadly active HIV-1 maturation inhibitors and define resistance pathways. In our recent studies in collaboration with DFH Pharma, we have identified a number of lead compounds that exhibit markedly superior antiviral activity relative to the parent compound BVM (Urano et al., 2016; 2019 and unpublished). Modifications were introduced at three positions around the BVM scaffold: Carbon-3 (C-3), C-19, and C-28. Replacement of the dimethylsuccinyl (DMS) moiety at C-3 with dimethylcyclobutyl (DMCB) substantially improved antiviral activity, in particular against specific subtype C isolates with highly polymorphic SP1 sequences. In collaboration with Hetero Drugs, we have developed a lead compound known as HRF-10071. This lead MI compound contains additional, distinct modifications at BVM C-3, C-19, and C-28. HRF-10071 exhibits low-nM antiviral activity against a number of strains containing SP1 polymorphisms that reduce or abolish the antiviral activity of the parent compound BVM. Importantly, the DMCB-containing compounds can be formulated and administered as long-acting agents. The development of resistance to therapeutic agents can significantly compromise the effectiveness of HIV-1 treatment. In contrast to BVM, which is rendered inactive by a number of common Gag polymorphisms including SP1-V7A, many second-generation MIs demonstrate potent activity against HIV-1 isolates containing these CA-SP1 boundary region changes (Urano et al., 2016; 2019; Dicker et al., 2022). We originally selected for resistance to BVM and identified a series of mutations in the CA-SP1 boundary region that confer resistance (Adamson et al. 2006). Selection studies using second-generation MIs conducted by us (Urano et al., JVI 2019) and others (Dicker et al., 2022) demonstrated that resistance to these more-potent compounds also maps to substitutions near the CA-SP1 cleavage site. Importantly however, unlike polymorphisms like SP1-V7A that reduce the antiviral activity of BVM, mutations that confer resistance to second-generation MIs, like those currently under development in my lab, are in highly conserved amino acid residues. Thus, pre-existing resistance is not likely to be a problem with the second-generation MI compounds. In summary, the DMCB analogs and HRF-10071 exhibit resistance profiles that make them viable candidates for clinical development. These molecules will also serve as powerful molecular probes for understanding fundamental questions about HIV-1 assembly and maturation. Subaim 1a. Define the antiviral activity and resistance pathways of BVM DMCB analogs. As presented above, BVM analogs containing DMCB modifications at C-3 exhibit markedly superior antiviral potency relative to BVM analogs containing the original DMS moiety at this position. The effect of this C-3 modification is particularly evident with clinically relevant primary subtype C isolates. We will continue elucidating resistance pathways for two DMCB analogs and will build upon these findings by testing an additional set of DMCB analogs provided to us by DFH Pharma. We will also perform long-term selection experiments in human T-cell lines to identify mutations that confer resistance to the DMCB analogs. Subaim 1b. Conduct experiments required to support FDA investigational new drug (IND) filing for HRF-10071. IND filing of the MI HRF-10071 with the FDA will require obtaining several types of virology data that would be supported by this FY25 funding. My lab has been leading the virology effort of this drug development program. First, we need to demonstrate that HRF-10071 exhibits broad potency across HIV-1 subtypes. This analysis will use the CA-SP1 processing and single-round infectivity assays described above. Second, the FDA requires data on viral resistance. This will be conducted using dose-escalation experiments in human T-cell lines. We will carry out a concerted effort to determine the frequency and types of drug-resistance mutations that arise upon long-term propagation of HIV-1 in the presence of HRF-10071. While initial selection experiments have been performed with the lab-adapted subtype B strain NL4-3, it is critical to extend this work to include representatives of HIV-1 subtypes that are prevalent outside North America and Europe. In particular, subtype C currently comprises ~50% of global HIV-1 infections. From a clinical perspective, it is important to demonstrate that HRF-10071 is active against viral isolates that are resistant to approved antiretrovirals. This analysis will be performed with the CA-SP1 processing assay and both single-round and multi-round HIV-1 infectivity assays and using mutants resistant to RT inhibitors, protease inhibitors, integrase strand transfer inhibitors, and LEN. We have all of the required mutants in my lab for these studies. Aim 2. Elucidate HIV-1 capsid inhibitor resistance pathways and develop inhibitors with favorable resistance profiles. As mentioned, LEN exhibits remarkably potent (sub-nM) antiviral activity and has demonstrated efficacy both as a long-acting anti-HIV-1 therapeutic and as an effective agent for PrEP (Segal-Maurer et al., 2022; Gupta et al., 2023; Bekker et al., 2024). Despite these highly favorable properties, resistance to LEN develops with substantial frequency (~20%) in clinical trial participants (Segal-Maurer et al., 2024). These finding indicate that additional capsid inhibitors should be developed that are structurally distinct from LEN and exhibit activity against LEN-resistant variants. We are currently collaborating with several groups - Stefan Sarafianos (Emory University), Peng Zhan (Shandong University, China), and Alexej Dick (Drexel University) - to develop such compounds. With Dr. Sarafianos we have tested a compound referred to as ZW-1261 (Sun et al., 2021), and with Drs. Zhan and Dick we have tested a series of compounds reported by Jiang et al. (Jiang et al. 2022; 2024). We have obtained preliminary data mapping mutations that confer resistance to ZW-1261 and the Zhan lab compounds. Significantly, ZW-1261 retains antiviral activity against several mutants (e.g., CA-M66I) that largely abrogate the activity of LEN. These results demonstrate that capsid inhibitors can be developed that will be active against LEN-resistant mutants. Intriguingly, one of the Zhan lab compounds also exhibits potent antiviral activity against HIV-2, which is only weakly inhibited by LEN. LEN, the first-in-class FDA-approved capsid inhibitor, has demonstrated remarkably potent and long-acting antiviral activity and has shown promise as an agent for PrEP. However, the emergence of LEN resistance will likely require the development of additional capsid inhibitors that are active against LEN-resistant variants. Our work in the area has thus far focused on ZW-1261 and a series of compounds obtained from the Zhan lab (see above). We will continue to evaluate these chemically distinct capsid inhibitors for antiviral activity across diverse subtypes of HIV-1 and will select for high-level resistance using not only the prototypical lab-adapted subtype B strain NL4-3 but also representative viral isolates from other subtypes. Promising preliminary data have been obtained thus far indicating that ZW-1261 maintains antiviral activity against LEN-resistant mutants like M66I that completely abrogate LEN activity. This is clinically significant, as M66I has been observed in participants in LEN clinical trials (Segal-Maurer et al., 2022). More work is urgently needed to explore the phenomenon of LEN resistance and to continue pursuing lead compounds that, like ZW-1261, are able to block the infectivity of LEN-resistant mutants. Additional compounds will be provided by the Sarafianos lab with whom we are collaborating on ZW-1261.