Novel PF74-like small molecules targeting the HIV-1 capsid protein: Balance of potency and metabolic stability.
Ontology highlight
ABSTRACT:
SUBMITTER: Wang L
PROVIDER: S-EPMC7982424 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:PF74 (1) is a potent and well-characterized prototypical small molecule targeting human immunodeficiency virus type 1 (HIV-1) capsid protein (CA), but not a viable antiviral lead due to the lack of metabolic stability. We report herein our molecular hybridization-based medicinal chemistry efforts toward potent and metabolically stable PF74-like small molecules. The design of the new sub-chemotype 4 rationally combines binding features of two recently reported PF74-like compounds 2 and 3. The subsequent confirmation and structure-activity relationship (SAR) of hit 4a entailed the chemical synthesis of 37 novel analogs, most of which showed modest but meaningful thermal shift, and low μM antiviral activity. The most potent analogs (4a, 4d, 4o, and 4r) all exhibited noticeably improved metabolic stability over PF74. Molecular modeling suggests that these new analogs bind to the PF74 binding site. Overall, our work demonstrated that the molecular hybridization approach is suitable for designing compounds with balanced potency and metabolic stability.
| S-EPMC8672819 | biostudies-literature
Project description:The PF74 binding site in HIV-1 capsid protein (CA) is a compelling antiviral drug target. Although PF74 confers mechanistically distinct antiviral phenotypes by competing against host factors for CA binding, it suffers from prohibitively low metabolic stability. Therefore, there has been increasing interest in designing novel sub-chemotypes of PF74 with similar binding mode and improved metabolic stability. We report herein our efforts to explore the inter-domain interacting indole moiety for designing novel CA-targeting small molecules. Our design includes simple substitution on the indole ring, and more importantly, novel sub-chemotypes with the indole moiety replaced with a few less electron-rich rings. All 56 novel analogs were synthesized and evaluated for antiviral activity, cytotoxicity, and impact on CA hexamer stability. Selected analogs were tested for metabolic stability in liver microsomes. Molecular modeling was performed to verify compound binding to the PF74 site. In the end, 5-hydroxyindole analogs (8,9 and 12) showed improved potency (up to 20-fold) over PF74. Of the novel sub-chemotypes, α- and β-naphthyl analogs (33 and 27) exhibited sub micromolar antiviral potencies comparable to that of PF74. Interestingly, although only moderately inhibiting HIV-1 (single-digit micromolar EC50s), analogs of the 2-indolone sub-chemotype consistently lowered the melting point (Tm) of CA hexamers, some with improved metabolic stability over PF74.
| S-EPMC7530112 | biostudies-literature
Project description:The capsid protein (CA) of HIV-1 plays essential roles in multiple steps of the viral replication cycle by assembling into functional capsid core, controlling the kinetics of uncoating and nuclear entry, and interacting with various host factors. Targeting CA represents an attractive yet underexplored antiviral approach. Of all known CA-targeting small molecule chemotypes, the peptidomimetic PF74 is particularly interesting because it binds to the same pocket used by a few important host factors, resulting in highly desirable antiviral phenotypes. However, further development of PF74 entails understanding its pharmacophore and mitigating its poor metabolic stability. We report herein the design, synthesis, and evaluation of a large number of PF74 analogs aiming to provide a comprehensive chemical profiling of PF74 and advance the understanding on its detailed binding mechanism and pharmacophore. The analogs, containing structural variations mainly in the aniline domain and/or the indole domain, were assayed for their effect on stability of CA hexamers, antiviral activity, and cytotoxicity. Selected analogs were also tested for metabolic stability in liver microsomes, alone or in the presence of a CYP3A inhibitor. Collectively, our studies identified important pharmacophore elements and revealed additional binding features of PF74, which could aid in future design of improved ligands to better probe the molecular basis of CA-host factor interactions, design strategies to disrupt them, and ultimately identify viable CA-targeting antiviral leads.
| S-EPMC7492092 | biostudies-literature
Project description:Human immunodeficiency virus (HIV) capsid plays important roles at multiple stages of viral replication. At the initial stages, controlled uncoating (disassembly) of the capsid ensures efficient reverse transcription of the single-stranded RNA genome, into the double-stranded DNA. Whereas at later stages, a proper assembly of capsid ensures the formation of a mature infectious virus particle. Hence, the inhibition of capsid assembly and/or disassembly has been recognized as a potential therapeutic strategy, and several capsid inhibitors have been reported. Of these, PF-3450074 (PF74) has been extensively studied. Recently reported GS-CA inhibitors (GS-CA1 and GS-6207), have shown a strong potential and appear to contain a PF74 scaffold. The location of resistance mutations and the results of structural studies further suggest that GS-CA compounds and PF74 share the same binding pocket, which is located between capsid monomers. Additionally, phenylalanine derivatives containing the PF74 scaffold show slightly enhanced capsid inhibiting activity. A comparison of capsid structures in complex with host factors and PF74, reveals the presence of common chemical entities at topologically equivalent positions. Here we present the status of capsid inhibitors that contain PF74 scaffolds and propose that the PF74 scaffold may be used to develop strong and safe capsid inhibitors.
| S-EPMC6956309 | biostudies-literature
Project description:Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification of new variants with balanced antiviral activity and metabolic stability. In the current work, we designed and synthesized three series of PF74-like analogs featuring conformational constraints at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro metabolic stability assays in human and mouse liver microsomes. These studies showed that the two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can provide viable hits. Subsequent SAR identified an improved analog 15 which effectively inhibited HIV-1 (EC50 = 0.31 μM), strongly stabilized CA hexamer (ΔTm = 8.7 °C), and exhibited substantially enhanced metabolic stability (t1/2 = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to increased resistance to oxidative metabolism.
| S-EPMC8000227 | biostudies-literature
Project description:HIV-1 capsid protein (CA) plays an important role in many steps of viral replication and represents an appealing antiviral target. Several CA-targeting small molecules of various chemotypes have been studied, but the peptidomimetic PF74 has drawn particular interest due to its potent antiviral activity, well-characterized binding mode, and unique mechanism of action. Importantly, PF74 competes against important host factors for binding, conferring highly desirable antiviral phenotypes. However, further development of PF74 is hindered by its prohibitively poor metabolic stability, which necessitates the search for structurally novel and metabolically stable chemotypes. We have conducted a pharmacophore-based shape similarity search for compounds mimicking PF74. We report herein the analog synthesis and structure-activity relationship (SAR) of two hits from the search, and a third hit designed via molecular hybridization. All analogs were characterized for their effect on CA hexamer stability, antiviral activity, and cytotoxicity. These assays identified three active compounds that moderately stabilize CA hexamer and inhibit HIV-1. The most potent analog (10) inhibited HIV-1 comparably to PF74 but demonstrated drastically improved metabolic stability in liver microsomes (31 min vs. 0.7 min t1/2). Collectively, the current studies identified a structurally novel and metabolically stable PF74-like chemotype for targeting HIV-1 CA.
| S-EPMC7232165 | biostudies-literature
Project description:Upon infection of susceptible cells by HIV-1, the conical capsid formed by ∼250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host-virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD-CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD-CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD-CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.
| S-EPMC4284599 | biostudies-literature
Project description:UnlabelledDuring HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4(+) T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution.ImportanceThe HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages--cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid.
| S-EPMC4524096 | biostudies-literature
Project description:PF74 and 11L, as potent modulators of the HIV-1 capsid protein, have been demonstrated to act at both early and late stages in the HIV-1 life cycle. However, their clearance is high in human liver microsomes (HLMs). The main goal of this study was to clarify the metabolism of PF74 and 11L in HLMs, and provide guidance for future structural optimization. To accomplish this, the phase-I metabolites of PF74 and 11L, resulting from in vitro incubation with HLMs, were investigated via ultra-performance liquid chromatography-ultraviolet-high-resolution mass spectrometry (UPLC-UV-HRMS). The results show that 17 phase-I metabolites were putatively annotated for PF74, whereas 16 phase-I metabolites were found for 11L. The main metabolic pathways of PF74 in HLMs were oxidation and demethylation, and the secondary metabolic pathway was hydrolysis; thus, the di-oxidation and demethylation products (M7, M9, M11, and M14) were found to be major metabolites of PF74 in HLMs. In comparison, the main metabolic pathways of 11L in HLMs were oxidation, demethylation, dehydrogenation, and oxidative deamination, with M6', M11', M15', and M16' as the main metabolites. We suggest that the indole ring and N-methyl group of PF74, and the aniline group, benzene ring R1', N-methyl, and methoxy group of 11L, were the main metabolic soft spots. Therefore, our research illuminates structural optimization options in seeking improved HIV-1 CA modulators.
| S-EPMC9412436 | biostudies-literature
Project description:Coxsackievirus A16 (CVA16) is an etiologic agent of hand, foot, and mouth disease (HFMD) that affects young children, and although typically self-limited, severe complications, and fatal cases have been reported. Due to the lack of specific medication and vaccines against CVA16, there is currently a need to develop effective antivirals to better control CVA16 infections in epidemic areas. In this study, we identified the tannins chebulagic acid (CHLA) and punicalagin (PUG) as small molecules that can efficiently disrupt the CVA16 infection of human rhabdomyosarcoma cells. Both compounds significantly reduced CVA16 infectivity at micromolar concentrations without apparent cytotoxicity. A mechanistic analysis revealed that the tannins particularly targeted the CVA16 entry phase by inactivating cell-free viral particles and inhibiting viral binding. Further examination by molecular docking analysis pinpointed the targets of the tannins in the fivefold axis canyon region of the CVA16 capsid near the pocket entrance that functions in cell surface receptor binding. We suggest that CHLA and PUG are efficient antagonists of CVA16 entry and could be of value as antiviral candidates or as starting points for developing molecules to treat CVA16 infections.
| S-EPMC6156566 | biostudies-literature