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Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1–5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment—which contributes to virologic failure, resistance generation and viral transmission—as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7–9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV. The small molecule GS-6207, which disrupts the function of the HIV capsid protein, shows potential as a long-acting therapeutic agent for the treatment and prevention of HIV infection.

A tremendous research and development effort was exerted toward combating chronic hepatitis C, ultimately leading to curative oral treatments, all of which are targeting viral proteins. Despite the advantage of numerous targets allowing for broad hepatitis C virus (HCV) genotype coverage, the only host target inhibitors that advanced into clinical development were Cyclosporin A based cyclophilin inhibitors. While cyclosporin-based molecules typically require a fermentation process, Gilead successfully pursued a fully synthetic, oral program based on Sanglifehrin A. The drug discovery process, though greatly helped by facile crystallography, was still hampered by the limitations in the accuracy of predictive computational methods for prioritizing compound ideas. Recent advances in accuracy and speed of free energy perturbation (FEP) methods, however, are attractive for prioritizing and derisking synthetically challenging molecules and potentially could have had a significant impact on the speed of the development of this program. Here in our simulated prospective study, the binding free energies of 26 macrocyclic cyclophilin inhibitors were blindly predicted using FEP+ to test this hypothesis. The predictions had a low mean unsigned error (MUE) (1.1 kcal/mol) and accurately reproduced many design decisions from the program, suggesting that FEP+ has the potential to drive synthetic chemistry efforts by more accurately ranking compounds with nonintuitive structure-activity relationships (SARs).

Treatment of hepatitis C virus (HCV) infection has been historically challenging due the high viral genetic complexity wherein there are eight distinct genotypes and at least 86 viral subtypes. While HCV NS3/4A protease inhibitors are an established treatment option for genotype 1 infection, limited coverage of genotypes 2 and/or 3 combined with serum alanine transaminase (ALT) elevations for some compounds has limited the broad utility of this therapeutic class. Our discovery efforts were focused on identifying an NS3/4A protease inhibitor with pan-genotypic antiviral activity, improved coverage of resistance associated substitutions, and a decreased risk of hepatotoxicity. Towards this goal, distinct interactions with the conserved catalytic triad of the NS3/4A protease were identified that improved genotype 3 antiviral activity. We further discovered that protein adduct formation strongly correlated with clinical ALT elevation for this therapeutic class. Improving metabolic stability and decreasing protein adduct formation through structural modifications ultimately resulted in voxilaprevir. Voxilaprevir, in combination with sofosbuvir and velpatasvir, has demonstrated pan-genotypic antiviral clinical activity. Furthermore, hepatotoxicity was not observed in Phase 3 clinical trials with voxilaprevir, consistent with our design strategy. Vosevi® (sofosbuvir, velpatasvir, and voxilaprevir) is now an approved pan-genotypic treatment option for the most difficult-to-cure individuals who have previously failed direct acting antiviral therapy.

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition (Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

Using available cryo-EM and x-ray crystal structures of the nonstructural proteins that are responsible for SARS-CoV-2 viral RNA replication and transcription, we have constructed an atomistic model of how the proteins assemble into a functioning superstructure. Our principal finding is that the complex is hexameric, centered around nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, where nsp14 is seen to undergo a large conformational change between its two domains. This conformational change facilitates binding of six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Two of the six polymerase subunits are each proposed to carry dimers of nsp13, while two others are proposed to carry monomers. The polymerase subunits that coordinate nsp13 dimers also bind the nucleocapsid, which positions the 5’-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analyzing the path of the viral RNA indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping (the nsp12 NiRAN and the nsp14 and nsp16 methyltransferases). The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping and the role of the endonuclease. It provides a platform to guide biochemical and structural research to address the stoichiometric and spatial configuration of the replication-transcription complex.Author SummaryThe replication of the coronavirus genome and the synthesis of subgenomic mRNA is a complex process involving multiple viral proteins. Despite a fairly complete structural picture of the individual proteins that are believed to coalesce into a larger replication-transcription complex, there is no clear model of how these proteins interact. Here we present the first detailed atomistic model of a complete replication-transcription complex for SARS-CoV-2, made up of the non-structural proteins nsp7-nsp16, as well as the nucleocapsid. Forming a large, hexameric superstructure centered around nsp15, the model provides new perspective on the function of its individual components, including the exonuclease, the endonuclease, the NiRAN site, the helicase, the multiple zinc fingers, and the nucleocapsid. It offers a cohesive view of replication, proofreading, template switching and mRNA capping, which should serve as a guide for future experimental exploration.

Blocking interactions between PD-1 and PD-L1 opens a new era of cancer treatment involving immunity modulation. Although most immunotherapies use monoclonal antibodies, small-molecule inhibitors offer advantages. To facilitate development of small-molecule therapeutics, we implemented a rapid approach to characterize the binding interfaces of small-molecule inhibitors with PD-L1. We determined its interaction with a synthetic macrocyclic peptide by using two mass spectrometry-based approaches, hydrogen-deuterium exchange and fast photochemical oxidation of proteins (FPOP), and corroborated the findings with our X-ray structure of the PD-L1/macrocycle complex. Although all three approaches show that the macrocycle binds directly to PD-L1 over the regions of residues 46-87 and 114-125, the two protein footprinting approaches show additional binding at the N-terminus of PD-L1, and FPOP reveals some critical binding residues. The outcomes not only show the binding regions but also demonstrate the utility of MS-based footprinting in probing protein/ligand inhibitory interactions in cancer immunotherapy.

Cyclophilin inhibition has been a target for the treatment of hepatitis C and other diseases, but the generation of potent, drug-like molecules through chemical synthesis has been challenging. In this study, a set of macrocyclic cyclophilin inhibitors was synthesized based on the core structure of the natural product sanglifehrin A. Initial compound optimization identified the valine-m-tyrosine-piperazic acid tripeptide (Val-m-Tyr-Pip) in the sanglifehrin core, stereocenters at C14 and C15, and the hydroxyl group of the m-tyrosine (m-Tyr) residue as key contributors to compound potency. Replacing the C18-C21 diene unit of sanglifehrin with a styryl group led to potent compounds that displayed a novel binding mode in which the styrene moiety engaged in a π-stacking interaction with Arg55 of cyclophilin A (Cyp A), and the m-Tyr residue was displaced into solvent. This observation allowed further simplifications of the scaffold to generate new lead compounds in the search for orally bioavailable cyclophilin inhibitors.

Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.

Chronic hepatitis C virus (HCV) infection is the cause of an emerging global epidemic of chronic liver disease. Current combination therapies are at best 80% efficacious and are often poorly tolerated. Strategies to improve the therapeutic response include the development of novel interferons, nucleoside analogues with reduced haemolysis compared with ribavirin and inosine 5′-monophosphate dehydrogenase inhibitors. Compounds in preclinical or early clinical trials include small molecules that inhibit virus-specific enzymes (such as the serine proteases, RNA polymerase and helicase) or interfere with translation (including anti-sense molecules, iRNA and ribozymes). Advances in understanding HCV replication, obtaining a sub-genomic replicon and contriving potential small animal models, in addition to solving crystallographic structures for the replication enzymes, have improved prospects for developing novel therapies. This review summarizes current and evolving treatments for chronic hepatitis C infection. In addition, progress in HCV targets and drug discovery tools valuable in the search for novel anti-HCV agents is detailed.

Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site.

The crystal structure of Bacillus subtilis orotidine 5′-monophosphate (OMP) decarboxylase with bound uridine 5′-monophosphate has been determined by multiple wavelength anomalous diffraction phasing techniques and refined to an R -factor of 19.3% at 2.4 Å resolution. OMP decarboxylase is a dimer of two identical subunits. Each monomer consists of a triosephosphate isomerase barrel and contains an active site that is located across one end of the barrel and near the dimer interface. For each active site, most of the residues are contributed by one monomer with a few residues contributed from the adjacent monomer. The most highly conserved residues are located in the active site and suggest a novel catalytic mechanism for decarboxylation that is different from any previously proposed OMP decarboxylase mechanism. The uridine 5′-monophosphate molecule is bound to the active site such that the phosphate group is most exposed and the C5-C6 edge of the pyrimidine base is most buried. In the proposed catalytic mechanism, the ground state of the substrate is destabilized by electrostatic repulsion between the carboxylate of the substrate and the carboxylate of Asp60. This repulsion is reduced in the transition state by shifting negative charge from the carboxylate to C6 of the pyrimidine, which is close to the protonated amine of Lys62. We propose that the decarboxylation of OMP proceeds by an electrophilic substitution mechanism in which decarboxylation and carbon-carbon bond protonation by Lys62 occur in a concerted reaction.

Background: 5′-Deoxy-5′-methylthioadenosine phosphorylase (MTAP) catalyzes the reversible phosphorolysis of 5′-deoxy-5′-methylthioadenosine (MTA) to adenine and 5-methylthio-D-ribose-1-phosphate. MTA is a by-product of polyamine biosynthesis, which is essential for cell growth and proliferation. This salvage reaction is the principle source of free adenine in human cells. Because of its importance in coupling the purine salvage pathway to polyamine biosynthesis MTAP is a potential chemotherapeutic target. Results: We have determined the crystal structure of MTAP at 1.7 A resolution using multiwavelength anomalous diffraction phasing techniques. MTAP is a trimer comprised of three identical subunits. Each subunit consists of a single α / β domain containing a central eight-stranded mixed β sheet, a smaller five-stranded mixed β sheet and six α helices. The native structure revealed the presence of an adenine molecule in the purine-binding site. The structure of MTAP with methylthioadenosine and sulfate ion soaked into the active site was also determined using diffraction data to 1.7 A resolution. Conclusions: The overall quaternary structure and subunit topology of MTAP are similar to mammalian purine nucleoside phosphorylase (PNP). The structures of the MTAP–ligand complexes provide a map of the active site and suggest possible roles for specific residues in substrate binding and catalysis. Residues accounting for the differences in substrate specificity between MTAP and PNP are also identified. Detailed information about the structure and chemical nature of the MTAP active site will aid in the rational design of inhibitors of this potential chemotherapeutic target. The MTAP structure represents the first structure of a mammalian PNP that is specific for 6-aminopurines.

GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC 50 ) of ≤11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.)

The development of JFH1 based intergenotypic recombinants which exploit the unique replication characteristics of JFH1 has made it possible to study infectious HCV encoding the structural genes of additional HCV genotypes including genotype 1b. Although, intergenotypic 1b/2a chimeric genomes replicate efficiently in transfected cells they produce very low viral titers, limiting the utility of this system. Here, intergenotypic 1b/2a variants were generated by serially passaging the virus in a novel highly permissive Huh-7 cell clone. The adapted virus was 1000-fold more infectious than the parental unadapted virus and six adapted mutations were identified throughout the genome. Of the mutations identified, L839S in the NS2 gene was the most critical for the adapted phenotype by enhancing the infectivity of assembled viral particles. Overall, the efficient production of infectious 1b/2a virus particles will facilitate the discovery and characterization of inhibitors targeting steps that involve the structural genes of genotype 1b HCV.

The structural mechanism by which non-structural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 (SF2) helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3’-end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a “spring-loading” mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.

A multilayer monochromator was installed on a bending-magnet beamline at the Cornell High Energy Synchrotron Source (CHESS) and was used to provide an unfocused pseudo-monochromatic X-ray beam for protein crystallography experiments. Datasets were collected from lysozyme at room temperature and human methylthioadenosine phosphorylase at 100 K. The wide energy bandpass of the multilayer allowed short exposure times, typically only a few times longer than on a focused multipole wiggler beamline. The diffraction images were processed using unmodified monochromatic data-processing software to yield datasets of good quality. These first measurements demonstrate that multilayer monochromators can be readily applied to the rapid structure determination of many typical-sized macromolecules.

The three-dimensional structures of orotidine 5′-monophosphate decarboxylases from four different organisms have been determined by X-ray crystallography. The structures reveal an active site in which the pyrimidine base and phosphate groups are rigidly held in place. Surprisingly, both pyrimidine carbonyl groups are hydrogen bonded to amide groups, rather than to strong active site acids, as was previously predicted. The positioning of a conserved aspartate sidechain close to the substrate carboxylate and a conserved lysine ammonium group close to the C6 of the pyrimidine suggests a novel mechanism to explain the extreme catalytic proficiency of this enzyme.