Synthesis, Enantiomeric Resolution and Biological Evaluation of HIV Capsid Inhibition Activity for Racemic, (S)- and (R)-PF74.
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ABSTRACT:
SUBMITTER: Ruddell S
PROVIDER: S-EPMC8272108 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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: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: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: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:1,4-Dihydropyridine (DHP) scaffold holds an outstanding position with its versatile pharmacological properties among all heterocyclic compounds. Although most of the commercially available DHPs are marketed as a racemic mixture, the chiral center at C-4 can lead to even opposite pharmacological activities between the enantiomers. In the present study, enantioseparation of seventeen DHP structural analogues, consisting of either pharmacologically active or newly synthesized derivatives, (M2-4, MD5, HM2, HM10, CE5, N11, N10, N7, M11, MC6-8, MC13, MD23, and 42IIP) by high-performance liquid chromatography was investigated using immobilized polysaccharide-based chiral stationary phase, Chiralpak IC column. Due to the solvent versatility of the covalently immobilized chiral stationary phase in enantiomer separation, multiple elution modes including standard normal phase, nonstandard mobile phase, and reversed phase were used to expand the possibility to find the optimum enantioselective conditions for the tested analytes. Under appropriate separation conditions, complete enantiomeric separation was obtained for nearly all compounds except MC6-8 and MC13 which contained two chiral centers. Additionally, the effects of the polar modifier, the additive, and column temperature on the chiral recognition were evaluated. The thermodynamic parameters calculated according to the linear van't Hoff equation indicated that the chiral separations in this study were enthalpy-driven or entropy-driven. Some parameters of method validation such as linearity, limit of quantitation, and repeatability were also measured for all studied compounds to prove the reliability of the method.
| S-EPMC6566779 | 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:Of all known small molecules targeting human immunodeficiency virus (HIV) capsid protein (CA), PF74 represents by far the best characterized chemotype, due to its ability to confer antiviral phenotypes in both early and late phases of viral replication. However, the prohibitively low metabolic stability renders PF74 a poor antiviral lead. We report herein our medicinal chemistry efforts toward identifying novel and metabolically stable small molecules targeting the PF74 binding site. Specifically, we replaced the inter-domain-interacting, electron-rich indole ring of PF74 with less electron-rich isosteres, including imidazolidine-2,4-dione, pyrimidine-2,4-dione, and benzamide, and identified four potent antiviral compounds (10, 19, 20 and 26) with markedly improved metabolic stability. Compared to PF74, analog 20 exhibited similar submicromolar potency, and much longer (51-fold) half-life in human liver microsomes (HLMs). Molecular docking corroborated that 20 binds to the PF74 binding site, and revealed distinct binding interactions conferred by the benzamide moiety. Collectively, our data support compound 20 as a promising antiviral lead.
| S-EPMC7982424 | biostudies-literature
Project description:Racemic-metal complexes were used to determine identity, enantiomeric excess, and concentration of chiral diamines using metal-to-ligand charge transfer bands in circular dichroism spectroscopy. It takes under just 2 min per sample to determine [G]t and %R with tolerable errors (19% and 4%, respectively). The simplicity of the achiral receptors employed confers to this technique great potential for high-throughput screening.
| S-EPMC2749327 | 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: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