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Human immunodeficiency virus (HIV) capsid (CA) protein is a promising target for developing novel anti-HIV drugs. Starting from highly anticipated CA inhibitors PF-74, we used scaffold hopping strategy to design a series of novel 1,2,4-triazole phenylalanine derivatives by targeting an unexplored region composed of residues 106-109 in HIV-1 CA hexamer. Compound d19 displayed excellent antiretroviral potency against HIV-1 and HIV-2 strains with EC

Poor metabolic stability of the human immunodeficiency virus type-1 (HIV-1) capsid (CA) inhibitor PF-74 is a major concern in its development toward clinical use. To improve on the metabolic stability, we employed a novel multistep computationally driven workflow, which facilitated the rapid design of improved PF-74 analogs in an efficient manner. Using this workflow, we designed three compounds that interact specifically with the CA interprotomer pocket, inhibit HIV-1 infection, and demonstrate enantiomeric preference. Moreover, using this workflow, we were able to increase the metabolic stability 204-fold in comparison to PF-74 in only three analog steps. These results demonstrate our ability to rapidly design CA compounds using a novel computational workflow that has improved metabolic stability over the parental compound. This workflow can be further applied to the redesign of PF-74 and other promising inhibitors with a stability shortfall.

We previously discovered a class of recombinant lectin conjugates, denoted lectin DLIs (‘dual-acting lytic inhibitors’) that bind to the HIV-1 envelope (Env) protein trimer and cause both lytic inactivation of HIV-1 virions and cytotoxicity of Env-expressing cells. To facilitate mechanistic investigation of DLI function, we derived the simplified prototype microvirin (MVN)-DLI, containing an MVN domain that binds high-mannose glycans in Env, connected to a DKWASLWNW sequence (denoted ‘Trp3’) derived from the membrane-associated region of gp41. The relatively much stronger affinity of the lectin component than Trp3 argues that the lectin functions to capture Env to enable Trp3 engagement and consequent Env membrane disruption and virolysis. The relatively simplified engagement pattern of MVN with Env opened up the opportunity, pursued here, to use recombinant glycan knockout gp120 variants to identify the precise Env binding site for MVN that drives DLI engagement and lysis. Using mutagenesis combined with a series of biophysical and virological experiments, we identified a restricted set of residues, N262, N332 and N448, all localized in a cluster on the outer domain of gp120, as the essential epitope for MVN binding. By generating these mutations in the corresponding HIV-1 virus, we established that the engagement of this glycan cluster with the lectin domain of MVN*-DLI is the trigger for DLI-derived virus and cell inactivation. Beyond defining the initial encounter step for lytic inactivation, this study provides a guide to further elucidate DLI mechanism, including the stoichiometry of Env trimer required for function, and downstream DLI optimization.

The HIV-1 Capsid (CA) is considered as a promising target for the development of potent antiviral drugs, due to its multiple roles during the viral life cycle. Herein, we report the design, synthesis, and antiviral activity evaluation of series of novel phenylalanine derivatives as HIV-1 CA protein inhibitors. Among them, 4-methoxy-N-methylaniline substituted phenylalanine (II-13c) and indolin-5-amine substituted phenylalanine (V-25i) displayed exceptional anti-HIV-1 activity with the EC50 value of 5.14 and 2.57 μM respectively, which is slightly weaker than that of lead compound PF-74 (EC50 = 0.42 μM). Besides, surface plasmon resonance (SPR) binding assay demonstrated II-13c and V-25i prefer to combine with CA hexamer rather than monomer, which is similar to PF-74. Subsequently, molecular dynamics simulation (MD) revealed potential interactions between representative compounds with HIV-1 CA hexamer. Overall, this work laid a solid foundation for further structural optimization to discover novel promising HIV-1 CA inhibitors.

Targeting DNA repair proteins with small-molecule inhibitors became a proven anti-cancer strategy. Previously, we identified an inhibitor of a major protein of homologous recombination (HR) RAD51, named B02. B02 inhibited HR in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. Here, using a medicinal chemistry approach, we aimed to improve the potency of B02. We identified the B02 analog, B02-isomer, which inhibits HR in human cells with significantly higher efficiency. We also show that B02-iso sensitizes triple-negative breast cancer MDA-MB-231 cells to the PARP inhibitor (PARPi) olaparib.

Background We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. Results HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Conclusions Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env’s prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer.

Recently, a novel coronavirus initially designated 2019-nCoV but now termed SARS-CoV-2 has emerged and raised global concerns due to its virulence. SARS-CoV-2 is the etiological agent of "coronavirus disease 2019", abbreviated to COVID-19, which despite only being identified at the very end of 2019, has now been classified as a pandemic by the World Health Organization (WHO). At this time, no specific prophylactic or postexposure therapy for COVID-19 are currently available. Viral entry is the first step in the SARS-CoV-2 lifecycle and is mediated by the trimeric spike protein. Being the first stage in infection, entry of SARS-CoV-2 into host cells is an extremely attractive therapeutic intervention point. Within this review, we highlight therapeutic intervention strategies for anti-SARS-CoV, MERS-CoV, and other coronaviruses and speculate upon future directions for SARS-CoV-2 entry inhibitor designs.

The HIV-1 CA protein has gained remarkable attention as a promising therapeutic target for the development of new antivirals, due to its pivotal roles in HIV-1 replication (structural and regulatory). Herein, we report the design and synthesis of three series of benzenesulfonamide-containing phenylalanine derivatives obtained by further structural modifications of PF-74 to aid in the discovery of more potent and drug-like HIV-1 CA inhibitors. Structure-activity relationship studies of these compounds led to the identification of new phenylalanine derivatives with a piperazinone moiety, represented by compound 11l, which exhibited anti-HIV-1NL4-3 activity 5.78-fold better than PF-74. Interestingly, 11l also showed anti-HIV-2ROD activity (EC50 = 31 nM), with almost 120 times increased potency over PF-74. However, due to the higher significance of HIV-1 as compared to HIV-2 for the human population, this manuscript focuses on the mechanism of action of our compounds in the context of HIV-1. SPR studies on representative compounds confirmed CA as the binding target. The action stage determination assay demonstrated that these inhibitors exhibited antiviral activities with a dual-stage inhibition profile. The early-stage inhibitory activity of compound 11l was 6.25 times more potent as compared to PF-74 but appeared to work via the accelerating capsid core assembly rather than stabilization. However, the mechanism by which they exert their antiviral activity in the late stage appears to be the same as PF-74 with less infectious HIV-1 virions produced in their presence, as judged p24 content studies. MD simulations provided the key rationale for the promising antiviral potency of 11l. Additionally, 11l exhibited a modest increase in HLM and human plasma metabolic stabilities as compared to PF-74, as well as a moderately improved pharmacokinetic profile, favorable oral bioavailability, and no acute toxicity. These studies provide insights and serve as a starting point for subsequent medicinal chemistry efforts in optimizing these promising HIV inhibitors. ispartof: JOURNAL OF MEDICINAL CHEMISTRY vol:63 issue:9 pages:4790-4810 ispartof: location:United States status: published

Acquired Immune Deficiency Syndrome (AIDS) treatment with combination antiretroviral therapy (cART) has improved the life quality of many patients since its implementation. However, resistance mutations and the accumulation of severe side effects associated with cART remain enormous challenges that need to be addressed with the continual design and redesign of anti-HIV drugs. In this review, we focus on the importance of the HIV-1 Gag polyprotein as the master coordinator of HIV-1 assembly and maturation and as an emerging drug target. Due to its multiple roles in the HIV-1 life cycle, the individual Gag domains are attractive but also challenging targets for inhibitor design. However, recent encouraging developments in targeting the Gag domains such as the capsid protein with highly potent and potentially long-acting inhibitors, as well as the exploration and successful targeting of challenging HIV-1 proteins such as the matrix protein, have demonstrated the therapeutic viability of this important protein. Such Gag-directed inhibitors have great potential for combating the AIDS pandemic and to be useful tools to dissect HIV-1 biology.

The HIV-1 capsid (CA) protein plays crucial roles in both early and late stages of the viral life cycle, which has intrigued researchers to target it to develop anti-HIV drugs. Accordingly, in this research, we report the design, synthesis and biological evaluation of a series of novel phenylalanine derivatives as HIV-1 CA protein inhibitors using the Cu(I)-catalyzed azide and alkyne 1,3-dipolar cycloaddition (CuAAC) reaction. Among this series of inhibitors, compound II-10c displayed a remarkable anti-HIV activity (EC(50) = 2.13 μM, CC(50) > 35.49 μM). Furthermore, surface plasmon resonance (SPR) binding assays showed that compounds II-10c and PF-74 (lead compound) have similar affinities to HIV-1 CA monomer. Further investigation showed that the weak permeability and water solubility of representative compounds were probably the important factors that restricted their cell-based activity. Preliminary structure-activity relationships (SARs) were inferred based on the activities of these compounds, and their known structure. The most promising new compound was studied with molecular dynamics simulation (MD) to determine the preferred interactions with the drug target. Finally, the activities of members of this series of inhibitors were deeply inspected to find the potential reasons for their anti-HIV-1 activity from various perspectives. This highlights the important factors required to design compounds with improved potency.

Bioisosteric replacement is a powerful tool for modulating the drug-like properties, toxicity, and chemical space of experimental therapeutics. In this review, we focus on selected cases where bioisosteric replacement and scaffold hopping have been used in the development of new anti-HIV-1 therapeutics. Moreover, we cover field-based, computational methodologies for bioisosteric replacement, using studies from our group as an example. It is our hope that this review will serve to highlight the utility and potential of bioisosteric replacement in the continuing search for new and improved anti-HIV drugs.

HIV-1 CA is involved in different stages of the viral replication cycle, performing essential roles in both early (uncoating, reverse transcription, nuclear import, integration) and late events (assembly). Recent efforts have demonstrated HIV-1 CA protein as a prospective therapeutic target for the development of new antivirals. The most extensively studied CA inhibitor, PF-3450074 (PF-74, discovered by Pfizer), that targets an inter-protomer pocket within the CA hexamer. Herein we reported the design, synthesis, and biological evaluation of a series of 4-phenyl-1H-1,2,3-triazole phenylalanine derivatives as HIV-1 CA inhibitors based on PF-74 scaffold. Most of the analogues demonstrated potent antiviral activities, among them, the anti-HIV-1 activity of 6a-9 (EC50 = 3.13 μM) is particularly prominent. The SPR binding assay of selected compounds (6a-9, 6a-10, 5b) suggested direct and effective interaction with recombinant CA proteins. The mechanism of action studies also demonstrated that 6a-9 displays the effects in both the early and late stages of HIV-1 replication. To explore the potential binding mode of the here presented analogues, 6a-9 was analyzed by MD simulation to predict its binding to the active site of HIV-1 CA monomer. In conclusion, this novel series of antivirals can serve as a starting point for the development of a new generation of HIV-1 treatment regimen and highlights the potentiality of CA as a therapeutic target.

Dual-acting virucidal entry inhibitors (DAVEIs) have previously been shown to cause irreversible inactivation of HIV-1 Env-presenting pseudovirus by lytic membrane transformation. This study examined whether this transformation could be generalized to include membranes of Env-presenting cells. Flow cytometry was used to analyze HEK293T cells transiently transfected with increasing amounts of DNA encoding JRFL Env, loaded with calcein dye, and treated with serial dilutions of microvirin (Q831K/M83R)-DAVEI. Comparing calcein retention against intact Env expression (via Ab 35O22) on individual cells revealed effects proportional to Env expression. "Low-Env" cells experienced transient poration and calcein leakage, while "high-Env" cells were killed. The cell-killing effect was confirmed with an independent mitochondrial activity-based cell viability assay, showing dose-dependent cytotoxicity in response to DAVEI treatment. Transfection with increasing quantities of Env DNA showed further shifts toward "High-Env" expression and cytotoxicity, further reinforcing the Env dependence of the observed effect. Controls with unlinked DAVEI components showed no effect on calcein leakage or cell viability, confirming a requirement for covalently linked DAVEI compounds to achieve Env transformation. These data demonstrate that the metastability of Env is an intrinsic property of the transmembrane protein complex and can be perturbed to cause membrane disruption in both virus and cell contexts.

Further clinical development of PF74, a lead compound targeting HIV-1 capsid, is impeded by low antiviral activity and inferior metabolic stability. By modifying the benzene (region I) and indole of PF74, we identified two potent compounds (7m and 7u) with significantly improved metabolic stability. Compared to PF74, 7u displayed greater metabolic stability in human liver microsomes (HLMs) with half-life (t1/2) 109-fold that of PF74. Moreover, mechanism of action (MOA) studies demonstrated that 7m and 7u effectively mirrored the MOA of compounds that interact within the PF74 interprotomer pocket, showing direct and robust interactions with recombinant CA, and 7u displaying antiviral effects in both the early and late stages of HIV-1 replication. Furthermore, MD simulation corroborated that 7u was bound to the PF74 binding site, and the results of the online molinspiration software predicted that 7m and 7u had desirable physicochemical properties. Unexpectedly, this series of compounds exhibited better antiviral activity than PF74 against HIV-2, represented by compound 7m whose anti-HIV-2 activity was almost 5 times increased potency over PF74. Therefore, we have rationally redesigned the PF74 chemotype to inhibitors with novel structures and enhanced metabolic stability in this study. We hope that these new compounds can serve as a blueprint for developing a new generation of HIV treatment regimens. ispartof: EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY vol:227 ispartof: location:France status: published

HIV-1 capsid (CA) plays indispensable and multiple roles in the life cycle of HIV-1, become an attractive target in antiviral therapy. Herein, we report the design, synthesis, and mechanism study of a novel series of dimerized phenylalanine derivatives as HIV-1 capsid inhibitors using 2-piperazineone or 2,5-piperazinedione as a linker. The structure-activity relationship (SAR) indicated that dimerized phenylalanines were more potent than monomers of the same chemotype. Further, the inclusion of fluorine substituted phenylalanine and methoxyl substituted aniline was found to be beneficial for antiviral activity. From the synthesized series, Q-c4 was found to be the most potent compound with an EC50 value of 0.57 μM, comparable to PF74. Interestingly, Q-c4 demonstrated a slightly higher affinity to the CA monomer than the CA hexamer, commensurate with its more significant effect in the late-stage of the HIV-1 lifecycle. Competitive SPR experiments with peptides from CPSF6 and NUP153 revealed that Q-c4 binds to the interprotomer pocket of hexameric CA as designed. Single-round infection assays showed that Q-c4 interferes with the HIV-1 life cycle in a dual-stage manner, affecting both pre-and post-integration. Stability assays in human plasma and human liver microsomes indicated that although Q-c4 has improved stability over PF74, this kind of inhibitor still requires further optimization. And the results of the online molinspiration software predicted that Q-c4 has desirable physicochemical properties but some properties still have some violation from the Lipinski rule of five. Overall, the dimerized phenylalanines are promising novel platforms for developing future HIV-1 CA inhibitors with considerable potential for optimization.

HIV-1 entry inhibition remains an urgent need for AIDS drug discovery and development. We previously reported the discovery of cyclic peptide triazoles (cPTs) that retain the HIV-1 irreversible inactivation functions of the parent linear peptides (PTs) and have massively increased proteolytic resistance. Here, in an initial structure-activity relationship investigation, we evaluated the effects of variations in key structural and functional components of the cPT scaffold in order to produce a platform for developing next-generation cPTs. Some structural elements, including stereochemistry around the cyclization residues and Ile and Trp side chains in the gp120-binding pharmacophore, exhibited relatively low tolerance for change, reflecting the importance of these components for function. In contrast, in the pharmacophore-central triazole position, the ferrocene moiety could be successfully replaced with smaller aromatic rings, where a p-methyl-phenyl methylene moiety gave cPT 24 with an IC50 value of 180 nM. Based on the observed activity of the biphenyl moiety when installed on the triazole ring (cPT 23, IC50 ∼ 269 nM), we further developed a new on-resin synthetic method to easily access the bi-aryl system during cPT synthesis, in good yields. A thiophene-containing cPT AAR029N2 (36) showed enhanced entropically favored binding to Env gp120 and improved antiviral activity (IC50 ∼ 100 nM) compared to the ferrocene-containing analogue. This study thus provides a crucial expansion of chemical space in the pharmacophore to use as a starting point, along with other allowable structural changes, to guide future optimization and minimization for this important class of HIV-1 killing agents.

Small-molecule HIV-1 entry inhibitors are an extremely attractive therapeutic modality. We have previously demonstrated that the entry inhibitor class can be optimized by using computational means to identify and extend the chemotypes available. Here we demonstrate unique and differential effects of previously published antiviral compounds on the gross structure of the HIV-1 Env complex, with an azabicyclohexane scaffolded inhibitor having a positive effect on glycoprotein thermostability. We demonstrate that modification of the methyltriazole-azaindole headgroup of these entry inhibitors directly effects the potency of the compounds, and substitution of the methyltriazole with an amine-oxadiazole increases the affinity of the compound 1000-fold over parental by improving the on-rate kinetic parameter. These findings support the continuing exploration of compounds that shift the conformational equilibrium of HIV-1 Env as a novel strategy to improve future inhibitor and vaccine design efforts.

Only a minority of patients infected with seasonal influenza A viruses exhibit a severe or fatal outcome of infection, but the reasons for this inter-individual variability in influenza susceptibility are unclear. To gain further insights into the molecular mechanisms underlying this variability, we investigated naturally occurring allelic variations of the myxovirus resistance 1 (MX1) gene coding for the influenza restriction factor MxA. The interferon-induced dynamin-like GTPase consists of an N-terminal GTPase domain, a bundle signaling element, and a C-terminal stalk responsible for oligomerization and viral target recognition. We used online databases to search for variations in the MX1 gene. Deploying in vitro approaches, we found that non-synonymous variations in the GTPase domain cause the loss of antiviral and enzymatic activities. Furthermore, we showed that these amino acid substitutions disrupt the interface for GTPase domain dimerization required for the stimulation of GTP hydrolysis. Variations in the stalk were neutral or slightly enhanced or abolished MxA antiviral function. Remarkably, two other stalk variants altered MxA's antiviral specificity. Variations causing the loss of antiviral activity were found only in heterozygous carriers. Interestingly, the inactive stalk variants blocked the antiviral activity of WT MxA in a dominant-negative way, suggesting that heterozygotes are phenotypically MxA-negative. In contrast, the GTPase-deficient variants showed no dominant-negative effect, indicating that heterozygous carriers should remain unaffected. Our results demonstrate that naturally occurring mutations in the human MX1 gene can influence MxA function, which may explain individual variations in influenza virus susceptibility in the human population.

SummaryHuman myxovirus resistance protein 1 (MxA) is an interferon-induced dynamin-like GTPase that acts as a cell-autonomous host restriction factor against many viral pathogens including influenza viruses. To study the molecular principles of its antiviral activity, we determined the crystal structure of nucleotide-free MxA, which showed an extended three-domain architecture. The central bundle signaling element (BSE) connected the amino-terminal GTPase domain with the stalk via two hinge regions. MxA oligomerized in the crystal via the stalk and the BSE, which in turn interacted with the stalk of the neighboring monomer. We demonstrated that the intra- and intermolecular domain interplay between the BSE and stalk was essential for oligomerization and the antiviral function of MxA. Based on these results, we propose a structural model for the mechano-chemical coupling in ring-like MxA oligomers as the principle mechanism for this unique antiviral effector protein.

Myxovirus resistance (Mx) GTPases are induced by interferon and inhibit multiple viruses, including influenza and human immunodeficiency viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action.

Toscana virus is an emerging bunyavirus in Mediterranean Europe where it accounts for 80% of pediatric meningitis cases during the summer. The negative-strand ribonucleic acid (RNA) genome of the virus is wrapped around the virally encoded nucleoprotein N to form the ribonucleoprotein complex (RNP). We determined crystal structures of hexameric N alone (apo) and in complex with a nonameric single-stranded RNA. RNA is sequestered in a sequence-independent fashion in a deep groove inside the hexamer. At the junction between two adjacent copies of Ns, RNA binding induced an inter-subunit rotation, which opened the RNA-binding tunnel and created a new assembly interface at the outside of the hexamer. Based on these findings, we suggest a structural model for how binding of RNA to N promotes the formation of helical RNPs, which are a characteristic hallmark of many negative-strand RNA viruses.

Human myxovirus resistance protein A (MxA) is an interferon-induced GTPase and part of the host cell defense against influenza viruses. It has a three-domain architecture with an amino-terminal GTPase (G) domain and a carboxy-terminal stalk responsible for oligomerization and viral target recognition. The MX1 gene, encoding MxA, is highly conserved and only a few single nucleotide polymorphisms are described in the human population. In this study we investigate whether and how allelic variations in MxA influence its antiviral function. Two rare nucleotide changes identified in the MX1 gene of healthy individuals result in amino acid exchanges at positions 255 and 268 in the G domain. GTPase and Minireplicon assays revealed that the V268M exchange showed some reduction in GTP hydrolysis, but only a slightly reduced antiviral activity against influenza A virus. However, the G255E exchange caused a complete loss of GTPase and antiviral activity of MxA. Further biochemical analyses of this naturally occurring mutation revealed the central role of GTP binding and hydrolysis for the antiviral mechanism of MxA. Using bioinformatics tools we are currently identifying additional allelic variations in MxA. Their characterization will answer the question how polymorphisms in the MX1 gene influence the antiviral capacity of MxA and whether these are enriched in patients suffering from severe influenza as has been described recently for IFITM3, another interferon-induced antiviral restriction factor.