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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.

Casein kinase 2 (CK2) is an evolutionarily conserved serine/threonine kinase implicated in a wide range of cellular functions and known to be dysregulated in various diseases such as cancer. Compared to most other kinases, CK2 exhibits several unusual properties, including dual co-substrate specificity and a high degree of promiscuity with hundreds of substrates described to date. Most paradoxical, however, is its apparent constitutive activity: no definitive mode of catalytic regulation has thus far been identified. Here we demonstrate that copper enhances the enzymatic activity of CK2 both in vitro and in vivo. We show that copper binds directly to CK2, and we identify specific residues in the catalytic subunit of the enzyme that are critical for copper-binding. We further demonstrate that increased levels of intracellular copper result in enhanced CK2 kinase activity, while decreased copper import results in reduced CK2 activity. Taken together, these findings establish CK2 as a copper-regulated kinase and indicate that copper is a key modulator of CK2-dependent signaling pathways.

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 AIDS pandemic is still of importance. HIV-1 and HIV-2 are the causative agents of this pandemic, and in the absence of a viable vaccine, drugs are continually required to provide quality of life for infected patients. The HIV capsid (CA) protein performs critical functions in the life cycle of HIV-1 and HIV-2, is broadly conserved across major strains and subtypes, and is underexploited. Therefore, it has become a therapeutic target of interest. Here, we report a novel series of 2-pyridone-bearing phenylalanine derivatives as HIV capsid modulators. Compound FTC-2 is the most potent anti-HIV-1 compound in the new series of compounds, with acceptable cytotoxicity in MT-4 cells (selectivity index HIV-1 > 49.57; HIV-2 > 17.08). However, compound TD-1a has the lowest EC50 in the anti-HIV-2 assays (EC50 = 4.86 ± 1.71 μM; CC50= 86.54 ± 29.24 μM). A water solubility test found that TD-1a showed a moderately increased water solubility compared with PF74, while the water solubility of FTC-2 was improved hundreds of times. Furthermore, we use molecular simulation studies to provide insight into the molecular contacts between the new compounds and HIV CA. We also computationally predict drug-like properties and metabolic stability for FTC-2 and TD-1a. Based on this analysis, TD-1a is predicted to have improved drug-like properties and metabolic stability over PF74. This study increases the repertoire of CA modulators and has important implications for developing anti-HIV agents with novel mechanisms, especially those that inhibit the often overlooked HIV-2. ispartof: MOLECULES vol:27 issue:21 ispartof: location:Switzerland status: published

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.

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 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.

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.

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.

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.