Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.

Molnupiravir maintains antiviral activity against SARS-CoV-2 variants and exhibits a high barrier to the development of resistance.

Molnupiravir, an oral prodrug of N-hydroxycytidine (NHC), previously demonstrated broad in vitro antiviral activity against multiple RNA viruses and has shown a high barrier to the development of resistance. Here, we present the antiviral activity of NHC against recent SARS-CoV-2 variants and the results of resistance selection studies to better understand the potential for viral resistance to NHC. NHC activity against SARS-CoV-2 variants omicron (BA.1, BA.1.1, BA.2, BA.4, BA.4.6, BA.5, BQ.1.1, XBB.1, and XBB.1.5), alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), and mu (B.1.621) was evaluated in Vero E6 cells using cytopathic effect assays. Resistance selection studies were performed by passaging SARS-CoV-2 (WA1) in the presence of NHC or a 3C-like protease inhibitor (MRK-A) in Vero E6 cells. Supernatants from cultures exhibiting a cytopathic effect score of ≥2 were re-passaged, and IC50 values were estimated. Whole-genome deep sequencing was performed on viral RNA isolated at each passage. NHC demonstrated similar potency against all SARS-CoV-2 variants evaluated. No evidence of SARS-CoV-2 phenotypic or genotypic resistance to NHC was observed following 30 passages. A random pattern of nucleotide changes was observed in NHC cultures, consistent with the drug's mechanism of action. In contrast, resistance was readily selected in all three MRK-A control cultures with the selection of a T21I substitution in the 3C-like protease. In conclusion, molnupiravir maintains antiviral activity across all major SARS-CoV-2 variants. Furthermore, no evidence of viral resistance to NHC was observed, supporting previous reports that NHC has a high barrier to developing resistance.

The HIV-1 capsid core is an opportunistic nuclear import receptor.

The movement of viruses and other large macromolecular cargo through nuclear pore complexes (NPCs) is poorly understood. The human immunodeficiency virus type 1 (HIV-1) provides an attractive model to interrogate this process. HIV-1 capsid (CA), the chief structural component of the viral core, is a critical determinant in nuclear transport of the virus. HIV-1 interactions with NPCs are dependent on CA, which makes direct contact with nucleoporins (Nups). Here we identify Nup35, Nup153, and POM121 to coordinately support HIV-1 nuclear entry. For Nup35 and POM121, this dependence was dependent cyclophilin A (CypA) interaction with CA. Mutation of CA or removal of soluble host factors changed the interaction with the NPC. Nup35 and POM121 make direct interactions with HIV-1 CA via regions containing phenylalanine glycine motifs (FG-motifs). Collectively, these findings provide additional evidence that the HIV-1 CA core functions as a macromolecular nuclear transport receptor (NTR) that exploits soluble host factors to modulate NPC requirements during nuclear invasion.

Potent targeted activator of cell kill molecules eliminate cells expressing HIV-1.

Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.

A Phenotypic Screen Identifies Potent DPP9 Inhibitors Capable of Killing HIV-1 Infected Cells.

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

Islatravir Has a High Barrier to Resistance and Exhibits a Differentiated Resistance Profile from Approved Nucleoside Reverse Transcriptase Inhibitors (NRTIs).

Islatravir (ISL) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) that inhibits human immunodeficiency virus (HIV) reverse transcription by blocking reverse transcriptase (RT) translocation on the primer:template. ISL is being developed for the treatment of HIV-1 infection. To expand our knowledge of viral variants that may confer reduced susceptibility to ISL, resistance selection studies were conducted with wild-type (WT) subtype A, B, and C viruses. RT mutations encoding M184I and M184V were the most frequently observed changes. Selection studies were also initiated with virus containing a single known resistance-associated mutation in RT (K65R, L74I, V90I, M184I, or M184V), and no additional mutations were observed. Antiviral activity assays were performed on variants that emerged in selection studies to determine their impact. M184I and M184V were the only single-codon substitutions that reduced susceptibility >2-fold compared to WT. A114S was an emergent substitution that when combined with other substitutions further reduced susceptibility >2-fold. Viruses containing A114S in combination with M184V did not replicate in primary blood mononuclear cells (PBMCs), consistent with the rare occurrence of the combination in clinical samples. While A114S conferred reduced susceptibility to ISL, it increased susceptibility to approved nucleoside reverse transcriptase inhibitors (NRTIs). This differential impact of A114S on ISL, an NRTTI, compared to NRTIs likely results from the different mechanisms of action. Altogether, the results demonstrate that ISL has a high barrier to resistance and a differentiated mechanism compared to approved NRTIs.

Gag p24 Is a Marker of Human Immunodeficiency Virus Expression in Tissues and Correlates With Immune Response.

We demonstrate that human immunodeficiency virus (HIV) gag p24 protein is more readily detected in gut and lymph node tissues than in blood CD4+ T cells and correlates better with CD4 count during antiretroviral therapy (ART). Gut p24 levels also measurably decline with ART in natural controllers. During ART, gut p24 expression is more strongly associated both with HIV-specific CD8+ T-cell frequency and plasma soluble CD14 levels than gut HIV RNA expression. This study supports using gag p24 as a marker of HIV expression in HIV+ tissues to study effects of viral persistence and to monitor efficacy of treatment in HIV-based clearance studies.

Generation of SARS-CoV-2 reporter replicon for high-throughput antiviral screening and testing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.

Intact Human Immunodeficiency Virus (HIV) Reservoir Estimated by the Intact Proviral DNA Assay Correlates With Levels of Total and Integrated DNA in the Blood During Suppressive Antiretroviral Therapy.

Accurate characterization of the human immunodeficiency virus (HIV) reservoir is imperative to develop an effective cure. HIV was measured in antiretroviral therapy-suppressed individuals using the intact proviral DNA assay (IPDA), along with assays for total or integrated HIV DNA, and inducible HIV RNA or p24. Intact provirus correlated with total and integrated HIV.

Primate immunodeficiency virus proteins Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex.

Host factors that silence provirus transcription in CD4+ memory T cells help HIV-1 escape eradication by the host immune system and by antiviral drugs1. These same factors, however, must be overcome for HIV-1 to propagate. Here we show that Vpx and Vpr encoded by diverse primate immunodeficiency viruses activate provirus transcription. Vpx and Vpr are adaptor proteins for the DCAF1-CUL4A/B E3 ubiquitin ligase that degrade SAMHD1 and increase reverse transcription2-4. Nonetheless, Vpx and Vpr have effects on reporter gene expression that are not explained by SAMHD1 degradation5-8. A screen for factors that mimic these effects identified the human silencing hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN) and MORC29-13. Vpx associated with the HUSH complex and decreased steady-state level of these proteins in a DCAF1/CUL4A/B/proteasome-dependent manner14,15. Replication kinetics of HIV-1 and SIVMAC was accelerated to a similar extent by vpx or FAM208A knockdown. Finally, vpx increased steady-state levels of LINE-1 ORF1p, as previously described for FAM208A disruption11. These results demonstrate that the HUSH complex represses primate immunodeficiency virus transcription, and that, to counteract this restriction, viral Vpx or Vpr proteins degrade the HUSH complex.

HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation.

HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor.

Hybrid and nonhybrid lipids exert common effects on membrane raft size and morphology.

Nanometer-scale domains in cholesterol-rich model membranes emulate lipid rafts in cell plasma membranes (PMs). The physicochemical mechanisms that maintain a finite, small domain size are, however, not well understood. A special role has been postulated for chain-asymmetric or hybrid lipids having a saturated sn-1 chain and an unsaturated sn-2 chain. Hybrid lipids generate nanodomains in some model membranes and are also abundant in the PM. It was proposed that they align in a preferred orientation at the boundary of ordered and disordered phases, lowering the interfacial energy and thus reducing domain size. We used small-angle neutron scattering and fluorescence techniques to detect nanoscopic and modulated liquid phase domains in a mixture composed entirely of nonhybrid lipids and cholesterol. Our results are indistinguishable from those obtained previously for mixtures containing hybrid lipids, conclusively showing that hybrid lipids are not required for the formation of nanoscopic liquid domains and strongly implying a common mechanism for the overall control of raft size and morphology. We discuss implications of these findings for theoretical descriptions of nanodomains.

Competition between line tension and curvature stabilizes modulated phase patterns on the surface of giant unilamellar vesicles: a simulation study.

When prepared in the liquid-liquid coexistence region, the four-component lipid system distearoyl-phospha-tidylcholine-dioleoyl-phosphatidylcholine-palmitoyl,oleoyl-phosphatidylcholine-cholesterol (DSPC-DOPC-POPC-Cholesterol), with certain ratios of DOPC and POPC, shows striking modulated phase patterns on the surface of giant unilamellar vesicles (GUVs). In this simulation study, we show that the morphology of these patterns can be explained by the competition of line tension (which tends to favor large round domains) and curvature, as specified by the Helfrich energy functional. In this study we use a Monte-Carlo simulation on the surface of a GUV to determine the equilibrium shape and phase morphology. We find that the patterns arising from these competing interactions very closely approximate those observed, that the patterned morphologies represent thermodynamically stable configurations, and that the geometric nature of these patterns is closely tied to the relative and absolute values of the model parameters.

Toward a better raft model: modulated phases in the four-component bilayer, DSPC/DOPC/POPC/CHOL.

The liquid-liquid (Ld + Lo) coexistence region within a distearoyl-phosphatidylcholine/dioleoyl-phosphatidylcholine/palmitoyl-oleoyl-phosphatidylcholine/cholesterol (DSPC/DOPC/POPC/CHOL) mixture displays a nanoscopic-to-macroscopic transition of phase domains as POPC is replaced by DOPC. Previously, we showed that the transition goes through a modulated phase regime during this replacement, in which patterned liquid phase morphologies are observed on giant unilamellar vesicles (GUVs). Here, we describe a more detailed investigation of the modulated phase regime along two different thermodynamic tielines within the Ld + Lo region of this four-component mixture. Using fluorescence microscopy of GUVs, we found that the modulated phase regime occurs at relatively narrow DOPC/(DOPC+POPC) ratios. This modulated phase window shifts to higher values of DOPC/(DOPC+POPC) when CHOL concentration is increased, and coexisting phases become closer in properties. Monte Carlo simulations reproduced the patterns observed on GUVs, using a competing interactions model of line tension and curvature energies. Sufficiently low line tension and high bending moduli are required to generate stable modulated phases. Altogether, our studies indicate that by tuning the lipid composition, both the domain size and morphology can be altered drastically within a narrow composition space. This lends insight into a possible mechanism whereby cells can reorganize plasma membrane compartmentalization simply by tuning the local membrane composition or line tension.

Versatile membrane deformation potential of activated pacsin.

Endocytosis is a fundamental process in signaling and membrane trafficking. The formation of vesicles at the plasma membrane is mediated by the G protein dynamin that catalyzes the final fission step, the actin cytoskeleton, and proteins that sense or induce membrane curvature. One such protein, the F-BAR domain-containing protein pacsin, contributes to this process and has been shown to induce a spectrum of membrane morphologies, including tubules and tube constrictions in vitro. Full-length pacsin isoform 1 (pacsin-1) has reduced activity compared to its isolated F-BAR domain, implicating an inhibitory role for its C-terminal Src homology 3 (SH3) domain. Here we show that the autoinhibitory, intramolecular interactions in pacsin-1 can be released upon binding to the entire proline-rich domain (PRD) of dynamin-1, resulting in potent membrane deformation activity that is distinct from the isolated F-BAR domain. Most strikingly, we observe the generation of small, homogenous vesicles with the activated protein complex under certain experimental conditions. In addition, liposomes prepared with different methods yield distinct membrane deformation morphologies of BAR domain proteins and apparent activation barriers to pacsin-1's activity. Theoretical free energy calculations suggest bimodality of the protein-membrane system as a possible source for the different outcomes, which could account for the coexistence of energetically equivalent membrane structures induced by BAR domain-containing proteins in vitro. Taken together, our results suggest a versatile role for pacsin-1 in sculpting cellular membranes that is likely dependent both on protein structure and membrane properties.

HIV-1 Gag protein can sense the cholesterol and acyl chain environment in model membranes.

Membrane binding of the HIV-1 group-specific antigen (Gag) structural protein, a critical step in viral assembly at the plasma membrane, is mediated by the myristoylated, highly basic matrix (MA) domain, which interacts with negatively charged lipids in the inner leaflet. According to a popular model, virus particles bud from membrane rafts, microdomains enriched in cholesterol and high-melting phospholipids with higher order than found outside rafts. How Gag might recognize membrane rafts, if they exist in the inner leaflet, is unknown. Using a liposome flotation assay with proteins translated in vitro, we investigated whether Gag can sense the composition of the hydrophobic part of the bilayer, by fixing lipid head group composition and varying hydrophobic properties. In liposomes composed solely of phosphatidylserine and phosphatidylcholine, and with the same overall membrane negative charge, Gag strongly preferred lipids with both acyl chains unsaturated over those with only one chain unsaturated. Adding cholesterol increased Gag binding and led to closer packing of phospholipids. However, higher membrane order, as measured by electron spin resonance, was not correlated with increased Gag binding. Gag proteins from two other retroviruses gave similar results. These liposome binding preferences were qualitatively recapitulated by purified myristoylated HIV-1 MA. Phosphatidylinositol 4,5-bisphosphate and cholesterol enhanced binding in an additive manner. Taken together, these results show that Gag is sensitive both to the acyl chains of phosphatidylserine and to cholesterol concentration and other details of the membrane environment. These observations may help explain how retroviruses acquire a raft-like lipid composition.

Control of a nanoscopic-to-macroscopic transition: modulated phases in four-component DSPC/DOPC/POPC/Chol giant unilamellar vesicles.

We have found modulated phase morphology in a particular region of composition within the liquid-ordered + liquid-disordered coexistence region in the four-component lipid bilayer mixture DSPC/DOPC/POPC/Chol. By controlling lipid composition, we could see distinct types of modulated liquid-liquid phase morphologies, including linear, irregular, and angular features in giant unilamellar vesicles. We used a combination of confocal, two-photon, wide-field fluorescence, and differential interference contrast microscopies, and used stringent controls to minimize light-induced artifacts. These studies establish that both the size and morphology of membrane rafts can be controlled by the concentration and the type of low-melting lipid in mixtures with cholesterol and a high-melting lipid.

Comparison of three ternary lipid bilayer mixtures: FRET and ESR reveal nanodomains.

Phase diagrams of ternary lipid mixtures containing cholesterol have provided valuable insight into cell membrane behaviors, especially by describing regions of coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. Fluorescence microscopy imaging of giant unilamellar vesicles has greatly assisted the determination of phase behavior in these systems. However, the requirement for optically resolved Ld + Lo domains can lead to the incorrect inference that in lipid-only mixtures, Ld + Lo domain coexistence generally shows macroscopic domains. Here we show this inference is incorrect for the low melting temperature phosphatidylcholines abundant in mammalian plasma membranes. By use of high compositional resolution Förster resonance energy transfer measurements, together with electron spin resonance data and spectral simulation, we find that ternary mixtures of DSPC and cholesterol together with either POPC or SOPC, do indeed have regions of Ld + Lo coexistence. However, phase domains are much smaller than the optical resolution limit, likely on the order of the Förster distance for energy transfer (R(0), ∼2-8 nm).

GUV preparation and imaging: minimizing artifacts.

The components of biological membranes are present in a physical mixture. The nonrandom ways that the molecules of lipids and proteins mix together can strongly influence the association of proteins with each other, and the chemical reactions that occur in the membrane, or that are mediated by the membrane. A particular type of nonrandom mixing is the separation of compositionally distinct phases. Any such phase separation would result in preferential partition of some proteins and lipids between the coexisting phases, and thus would influence which proteins could be in contact, and whether a protein could find its target. Phase separation in a plasma membrane would also influence the binding of molecules from outside the cell to the membrane, including recognition proteins on viruses, bacteria, and other cells. The concept of these and other events associated with membrane phase separation are sometimes grouped together as the "raft model" of biological membranes. Several types of experiments are aimed at detecting and characterizing membrane phase separation. Visualizing phase separation has special value, both because the immiscibility is so decisively determined, and also because the type of phase can often be identified. The fluorescence microscope has proven uniquely useful for yielding images of separated phases, both in certain cell preparations, and especially in models of cell membranes. Here we discuss ways to prepare useful model membranes for image studies, and how to avoid some of the artifacts that can plague these studies.

Molecular mechanism of membrane constriction and tubulation mediated by the F-BAR protein Pacsin/Syndapin.

Peripheral membrane proteins of the Bin/amphiphysin/Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) family participate in cellular membrane trafficking and have been shown to generate membrane tubules. The degree of membrane bending appears to be encoded in the structure and immanent curvature of the particular protein domains, with BAR and F-BAR domains inducing high- and low-curvature tubules, respectively. In addition, oligomerization and the formation of ordered arrays influences tubule stabilization. Here, the F-BAR domain-containing protein Pacsin was found to possess a unique activity, creating small tubules and tubule constrictions, in addition to the wide tubules characteristic for this subfamily. Based on crystal structures of the F-BAR domain of Pacsin and mutagenesis studies, vesiculation could be linked to the presence of unique structural features distinguishing it from other F-BAR proteins. Tubulation was suppressed in the context of the full-length protein, suggesting that Pacsin is autoinhibited in solution. The regulated deformation of membranes and promotion of tubule constrictions by Pacsin suggests a more versatile function of these proteins in vesiculation and endocytosis beyond their role as scaffold proteins.