• Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance

      Shaqra, Ala M.; Zvornicanin, Sarah N.; Huang, Qiu Yu J.; Lockbaum, Gordon J.; Knapp, Mark; Tandeske, Laura; Bakan, David T.; Flynn, Julia M.; Bolon, Daniel N.; Moquin, Stephanie; et al. (2022-06-21)
      Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (M(pro)) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 M(pro) bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of M(pro), map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed M(pro) inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.
    • Comprehensive fitness landscape of SARS-CoV-2 M(pro) reveals insights into viral resistance mechanisms

      Flynn, Julia; Samant, Neha; Nachum, Gily S.; Bakan, David T.; Yilmaz, Nese Kurt; Schiffer, Celia A.; Moquin, Stephanie A.; Dovala, Dustin; Bolon, Daniel N. (2022-06-20)
      With the continual evolution of new strains of SARS-CoV-2 that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs SARS-CoV-2 main protease (M(pro)) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting M(pro) appear promising but will elicit selection pressure for resistance. To understand resistance potential in M(pro), we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high-throughput assays of M(pro) function in yeast, based on either the ability of M(pro) variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to M(pro) function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the M(pro) dimer. The clinical variants of M(pro) were predominantly functional in our screens, indicating that M(pro) is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to M(pro) evolution and that are likely to contribute to drug resistance. This complete mutational guide of M(pro) can be used in the design of inhibitors with reduced potential of evolving viral resistance.
    • HIV-1 VIF and human APOBEC3G interaction directly observed through molecular specific labeling using a new dual promotor vector

      Myint, Wazo; Schiffer, Celia A.; Matsuo, Hiroshi (2022-04-26)
      Over the last few decades, protein NMR isotope labeling methods using E. coli based expression have revolutionized the information accessible from biomolecular NMR experiments. Selective labeling of a protein of interest in a multi-protein complex can significantly reduce the number of cross-peaks and allow for study of large protein complexes. However, limitations still remain since some proteins are not stable independently and cannot be separately labeled in either NMR active isotope enriched or unenriched media and reconstituted into a multimeric complex. To overcome this limitation, the LEGO NMR method was previously developed using protein expression plasmids containing T7 or araBAD promoters to separately express proteins in the same E. coli after changing between labeled and unlabeled media. Building on this, we developed a method to label the Human Immunodeficiency Virus type 1 viral infectivity factor (HIV-1 Vif), a monomerically unstable protein, in complex with CBFbeta, it's host binding partner. We designed a dual promoter plasmid containing both T7 and araBAD promoters to independently control the expression of HIV-1 Vif in NMR active isotope enriched media and CBFbeta in unenriched media. Using this method, we assigned the backbone resonance and directly observed the binding of HIV-1 Vif with APOBEC3G, a host restriction factor to HIV-1.
    • Quantitative structural analysis of influenza virus by cryo-electron tomography and convolutional neural networks

      Huang, Qiu Yu J.; Song, KangKang; Xu, Chen; Bolon, Daniel N.; Wang, Jennifer P.; Finberg, Robert W.; Schiffer, Celia A.; Somasundaran, Mohan (2022-03-14)
      Influenza viruses pose severe public health threats globally. Influenza viruses are extensively pleomorphic, in shape, size, and organization of viral proteins. Analysis of influenza morphology and ultrastructure can help elucidate viral structure-function relationships and aid in therapeutics and vaccine development. While cryo-electron tomography (cryoET) can depict the 3D organization of pleomorphic influenza, the low signal-to-noise ratio inherent to cryoET and viral heterogeneity have precluded detailed characterization of influenza viruses. In this report, we leveraged convolutional neural networks and cryoET to characterize the morphological architecture of the A/Puerto Rico/8/34 (H1N1) influenza strain. Our pipeline improved the throughput of cryoET analysis and accurately identified viral components within tomograms. Using this approach, we successfully characterized influenza morphology, glycoprotein density, and conducted subtomogram averaging of influenza glycoproteins. Application of this processing pipeline can aid in the structural characterization of not only influenza viruses, but other pleomorphic viruses and infected cells.
    • Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance

      Zephyr, Jacqueto; Desaboini, Nageswara Rao; Vo, Sang V.; Henes, Mina; Kosovrasti, Klajdi; Matthew, Ashley N.; Hedger, Adam; Timm, Jennifer; Chan, Elise T.; Ali, Akbar; et al. (2022-02-17)
      Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.
    • Identification of a Permissive Secondary Mutation That Restores the Enzymatic Activity of Oseltamivir Resistance Mutation H275Y

      Jiang, Li; Samant, Neha; Liu, Ping; Somasundaran, Mohan; Jensen, Jeffrey D.; Marasco, Wayne A.; Kowalik, Timothy F.; Schiffer, Celia A.; Finberg, Robert W.; Wang, Jennifer P.; et al. (2022-01-19)
      Many oseltamivir resistance mutations exhibit fitness defects in the absence of drug pressure that hinders their propagation in hosts. Secondary permissive mutations can rescue fitness defects and facilitate the segregation of resistance mutations in viral populations. Previous studies have identified a panel of permissive or compensatory mutations in neuraminidase (NA) that restore the growth defect of the predominant oseltamivir resistance mutation (H275Y) in H1N1 influenza A virus. In prior work, we identified a hyperactive mutation (Y276F) that increased NA activity by approximately 70%. While Y276F had not been previously identified as a permissive mutation, we hypothesized that Y276F may counteract the defects caused by H275Y by buffering its reduced NA expression and enzyme activity. In this study, we measured the relative fitness, NA activity, and surface expression, as well as sensitivity to oseltamivir, for several oseltamivir resistance mutations, including H275Y in the wild-type and Y276F genetic background. Our results demonstrate that Y276F selectively rescues the fitness defect of H275Y by restoring its NA surface expression and enzymatic activity, elucidating the local compensatory structural impacts of Y276F on the adjacent H275Y. IMPORTANCE The potential for influenza A virus (IAV) to cause pandemics makes understanding evolutionary mechanisms that impact drug resistance critical for developing surveillance and treatment strategies. Oseltamivir is the most widely used therapeutic strategy to treat IAV infections, but mutations in IAV can lead to drug resistance. The main oseltamivir resistance mutation, H275Y, occurs in the neuraminidase (NA) protein of IAV and reduces drug binding as well as NA function. Here, we identified a new helper mutation, Y276F, that can rescue the functional defects of H275Y and contribute to the evolution of drug resistance in IAV.
    • Quantitative Structural Analysis of Influenza Virus by Cryo-electron Tomography and Convolutional Neural Networks [preprint]

      Huang, Qiu Yu; Song, KangKang; Xu, Chen; Bolon, Daniel N.; Wang, Jennifer P.; Finberg, Robert W.; Schiffer, Celia A.; Somasundaran, Mohan (2021-12-09)
      Influenza viruses pose severe public health threats; they cause millions of infections and tens of thousands of deaths annually in the US. Influenza viruses are extensively pleomorphic, in both shape and size as well as organization of viral structural proteins. Analysis of influenza morphology and ultrastructure can help elucidate viral structure-function relationships as well as aid in therapeutics and vaccine development. While cryo-electron tomography (cryoET) can depict the 3D organization of pleomorphic influenza, the low signal-to-noise ratio inherent to cryoET and extensive viral heterogeneity have precluded detailed characterization of influenza viruses. In this report, we developed a cryoET processing pipeline leveraging convolutional neural networks (CNNs) to characterize the morphological architecture of the A/Puerto Rico/8/34 (H1N1) influenza strain. Our pipeline improved the throughput of cryoET analysis and accurately identified viral components within tomograms. Using this approach, we successfully characterized influenza viral morphology, glycoprotein density, and conduct subtomogram averaging of HA glycoproteins. Application of this processing pipeline can aid in the structural characterization of not only influenza viruses, but other pleomorphic viruses and infected cells.
    • Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance [preprint]

      Zephyr, Jacqueto; Desaboini, Nageswara Rao; Vo, Sang V.; Henes, Mina; Kosovrasti, Klajdi; Matthew, Ashley N.; Hedger, Adam K.; Timm, Jennifer; Chan, Elise T.; Ali, Akbar; et al. (2021-11-30)
      Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.
    • Viral proteases: Structure, mechanism and inhibition

      Zephyr, Jacqueto; Yilmaz, Nese Kurt; Schiffer, Celia A. (2021-11-17)
      Viral proteases are diverse in structure, oligomeric state, catalytic mechanism, and substrate specificity. This chapter focuses on proteases from viruses that are relevant to human health: human immunodeficiency virus subtype 1 (HIV-1), hepatitis C (HCV), human T-cell leukemia virus type 1 (HTLV-1), flaviviruses, enteroviruses, and coronaviruses. The proteases of HIV-1 and HCV have been successfully targeted for therapeutics, with picomolar FDA-approved drugs currently used in the clinic. The proteases of HTLV-1 and the other virus families remain emerging therapeutic targets at different stages of the drug development process. This chapter provides an overview of the current knowledge on viral protease structure, mechanism, substrate recognition, and inhibition. Particular focus is placed on recent advances in understanding the molecular basis of diverse substrate recognition and resistance, which is essential toward designing novel protease inhibitors as antivirals.
    • Pan-3C Protease Inhibitor Rupintrivir Binds SARS-CoV-2 Main Protease in a Unique Binding Mode

      Lockbaum, Gordon J.; Henes, Mina; Lee, Jeong Min.; Timm, Jennifer; Nalivaika, Ellen A.; Thompson, Paul R.; Yilmaz, Nese Kurt; Schiffer, Celia A. (2021-10-05)
      Rupintrivir targets the 3C cysteine proteases of the picornaviridae family, which includes rhinoviruses and enteroviruses that cause a range of human diseases. Despite being a pan-3C protease inhibitor, rupintrivir activity is extremely weak against the homologous 3C-like protease of SARS-CoV-2. In this study, the crystal structures of rupintrivir were determined bound to enterovirus 68 (EV68) 3C protease and the 3C-like main protease (M(pro)) from SARS-CoV-2. While the EV68 3C protease-rupintrivir structure was similar to previously determined complexes with other picornavirus 3C proteases, rupintrivir bound in a unique conformation to the active site of SARS-CoV-2 M(pro) splitting the catalytic cysteine and histidine residues. This bifurcation of the catalytic dyad may provide a novel approach for inhibiting cysteine proteases.
    • Analyses of HIV proteases variants at the threshold of viability reveals relationships between processing efficiency and fitness

      Nachum, Gily S.; Flynn, Julia; Mavor, David; Schiffer, Celia A.; Bolon, Daniel N. (2021-09-01)
      Investigating the relationships between protein function and fitness provides keys for understanding biochemical mechanisms that underly evolution. Mutations with partial fitness defects can delineate the threshold of biochemical function required for viability. We utilized a previous deep mutational scan of HIV-1 protease (PR) to identify variants with 15-45 per cent defects in replication and analysed the biochemical function of eight variants (L10M, L10S, V32C, V32I, A71V, A71S, Q92I, Q92N). We purified each variant and assessed the efficiency of peptide cleavage for three cut sites (MA-CA, TF-PR, and PR-RT) as well as gel-based analyses of processing of purified Gag. The cutting activity of at least one site was perturbed relative to WT protease for all variants, consistent with cutting activity being a primary determinant of fitness effects. We examined the correlation of fitness defects with cutting activity of different sites. MA-CA showed the weakest correlation (R (2) = 0.02) with fitness, suggesting relatively weak coupling with viral replication. In contrast, cutting of the TF-PR site showed the strongest correlation with fitness (R (2) = 0.53). Cutting at the TF-PR site creates a new PR protein with a free N-terminus that is critical for activity. Our findings indicate that increasing the pool of active PR is rate limiting for viral replication, making this an ideal step to target with inhibitors.
    • Discovery of Quinoxaline-Based P1-P3 Macrocyclic NS3/4A Protease Inhibitors with Potent Activity against Drug-Resistant Hepatitis C Virus Variants

      Desaboini, Nageswara Rao; Zephyr, Jacqueto; Henes, Mina; Chan, Elise T.; Matthew, Ashley N.; Hedger, Adam; Conway, Hasahn L.; Saeed, Mohsan; Newton, Alicia; Petropoulos, Christos J.; et al. (2021-08-26)
      The three pan-genotypic HCV NS3/4A protease inhibitors (PIs) currently in clinical use-grazoprevir, glecaprevir, and voxilaprevir-are quinoxaline-based P2-P4 macrocycles and thus exhibit similar resistance profiles. Using our quinoxaline-based P1-P3 macrocyclic lead compounds as an alternative chemical scaffold, we explored structure-activity relationships (SARs) at the P2 and P4 positions to develop pan-genotypic PIs that avoid drug resistance. A structure-guided strategy was used to design and synthesize two series of compounds with different P2 quinoxalines in combination with diverse P4 groups of varying sizes and shapes, with and without fluorine substitutions. Our SAR data and cocrystal structures revealed the interplay between the P2 and P4 groups, which influenced inhibitor binding and the overall resistance profile. Optimizing inhibitor interactions in the S4 pocket led to PIs with excellent antiviral activity against clinically relevant PI-resistant HCV variants and genotype 3, providing potential pan-genotypic inhibitors with improved resistance profiles.
    • Affinity maturation of SARS-CoV-2 neutralizing antibodies confers potency, breadth, and resilience to viral escape mutations

      Muecksch, Frauke; Hou, Shurong; Schiffer, Celia A.; Nussenzweig, Michel C.; Bjorkman, Pamela J.; Hatziioannou, Theodora; Bieniasz, Paul D. (2021-08-10)
      Antibodies elicited by infection accumulate somatic mutations in germinal centers that can increase affinity for cognate antigens. We analyzed 6 independent groups of clonally related severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) Spike receptor-binding domain (RBD)-specific antibodies from 5 individuals shortly after infection and later in convalescence to determine the impact of maturation over months. In addition to increased affinity and neutralization potency, antibody evolution changed the mutational pathways for the acquisition of viral resistance and restricted neutralization escape options. For some antibodies, maturation imposed a requirement for multiple substitutions to enable escape. For certain antibodies, affinity maturation enabled the neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.
    • Structural basis of substrate specificity in human cytidine deaminase family APOBEC3s

      Hou, Shurong; Lee, Jeong Min.; Myint, Wazo; Matsuo, Hiroshi; Yilmaz, Nese Kurt; Schiffer, Celia A. (2021-08-01)
      The human cytidine deaminase family of APOBEC3s (A3s) plays critical roles in both innate immunity and the development of cancers. A3s comprise seven functionally overlapping but distinct members that can be exploited as nucleotide base editors for treating genetic diseases. Although overall structurally similar, A3s have vastly varying deamination activity and substrate preferences. Recent crystal structures of ssDNA-bound A3s together with experimental studies have provided some insights into distinct substrate specificities among the family members. However, the molecular interactions responsible for their distinct biological functions and how structure regulates substrate specificity are not clear. In this study, we identified the structural basis of substrate specificities in three catalytically active A3 domains whose crystal structures have been previously characterized: A3A, A3B- CTD, and A3G-CTD. Through molecular modeling and dynamic simulations, we found an interdependency between ssDNA substrate binding conformation and nucleotide sequence specificity. In addition to the U-shaped conformation seen in the crystal structure with the CTC0 motif, A3A can accommodate the CCC0 motif when ssDNA is in a more linear (L) conformation. A3B can also bind both U- and L-shaped ssDNA, unlike A3G, which can stably recognize only linear ssDNA. These varied conformations are stabilized by sequence-specific interactions with active site loops 1 and 7, which are highly variable among A3s. Our results explain the molecular basis of previously observed substrate specificities in A3s and have implications for designing A3-specific inhibitors for cancer therapy as well as engineering base-editing systems for gene therapy.
    • Deciphering Antifungal Drug Resistance in Pneumocystis jirovecii DHFR with Molecular Dynamics and Machine Learning

      Leidner, Florian; Yilmaz, Nese Kurt; Schiffer, Celia A. (2021-06-28)
      Drug resistance impacts the effectiveness of many new therapeutics. Mutations in the therapeutic target confer resistance; however, deciphering which mutations, often remote from the enzyme active site, drive resistance is challenging. In a series of Pneumocystis jirovecii dihydrofolate reductase variants, we elucidate which interactions are key bellwethers to confer resistance to trimethoprim using homology modeling, molecular dynamics, and machine learning. Six molecular features involving mainly residues that did not vary were the best indicators of resistance.
    • Report of the National Institutes of Health SARS-CoV-2 Antiviral Therapeutics Summit

      Hall, Matthew D.; Anderson, James M.; Schiffer, Celia A.; Conley, Anthony J.; Davis, Mindy I. (2021-06-10)
      The NIH Virtual SARS-CoV-2 Antiviral Summit, held on November 6, 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for COVID-19, including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss SARS-CoV-2 targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.
    • Deciphering Complex Mechanisms of Resistance and Loss of Potency through Coupled Molecular Dynamics and Machine Learning

      Leidner, Florian; Yilmaz, Nese Kurt; Schiffer, Celia A. (2021-04-13)
      Drug resistance threatens many critical therapeutics through mutations in the drug target. The molecular mechanisms by which combinations of mutations, especially those remote from the active site, alter drug binding to confer resistance are poorly understood and thus difficult to counteract. A machine learning strategy was developed that coupled parallel molecular dynamics simulations with experimental potency to identify specific conserved mechanisms underlying resistance. Physical features were extracted from the simulations, analyzed, and integrated into one consistent and interpretable elastic network model. To rigorously test this strategy, HIV-1 protease variants with diverse mutations were used, with potencies ranging from picomolar to micromolar to the drug darunavir. Feature reduction resulted in a model with four specific features that predicts for both the training and test sets inhibitor binding free energy within 1 kcal/mol of the experimental value over this entire range of potency. These predictive features are physically interpretable, as they vary specifically with affinity and diagonally transverse across the protease homodimer. This physics-based strategy of parallel molecular dynamics and machine learning captures mechanisms by which complex combinations of mutations confer resistance and identify critical features that serve as bellwethers of affinity, which will be critical in future drug design.
    • Introduction: Drug Resistance

      Yilmaz, Nese Kurt; Schiffer, Celia A. (2021-03-24)
      The evolutionary pressure of survival drives the emergence of drug resistance and thereby poses a major challenge to modern medicine. Resistance threatens the longevity of drugs and restricts treatment options for patients, with high prevalence in all areas of oncology and infectious diseases. Any biological entity capable of evolving and creating diversity can develop resistance under selective pressure. This diversity can pre-exist or occur after exposure to the inhibitors. Pathogens evolve to resist antimicrobials, which include antibiotics, antivirals, antifungals, and antiprotozoals. In agriculture, resistance arises with overuse of herbicides and pesticides. In cancer, resistance emerges eventually with most treatment regimens and in infectious diseases with spread of the pathogen to large populations, which is further exacerbated with the overuse of antibiotics. The emergence and spread of drug resistance in this wide range of disease areas severely impact public health, threaten millions of people’s lives, and cause a crippling financial burden, which urges the development of new strategies to unravel and avoid drug resistance.
    • Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies [preprint]

      Muecksch, Frauke; Hou, Shurong; Schiffer, Celia A.; Nussenzweig, Michel; Bjorkman, Pamela J.; Hatziioannou, Theodora; Bieniasz, Paul (2021-03-08)
      Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.
    • Inhibiting HTLV-1 Protease: A Viable Antiviral Target

      Lockbaum, Gordon J.; Henes, Mina; Talledge, Nathaniel; Rusere, Linah; Kosovrasti, Klajdi; Nalivaika, Ellen A.; Somasundaran, Mohan; Ali, Akbar; Mansky, Louis M.; Yilmaz, Nese Kurt; et al. (2021-02-23)
      Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that can cause severe paralytic neurologic disease and immune disorders as well as cancer. An estimated 20 million people worldwide are infected with HTLV-1, with prevalence reaching 30% in some parts of the world. In stark contrast to HIV-1, no direct acting antivirals (DAAs) exist against HTLV-1. The aspartyl protease of HTLV-1 is a dimer similar to that of HIV-1 and processes the viral polyprotein to permit viral maturation. We report that the FDA-approved HIV-1 protease inhibitor darunavir (DRV) inhibits the enzyme with 0.8 muM potency and provides a scaffold for drug design against HTLV-1. Analogs of DRV that we designed and synthesized achieved submicromolar inhibition against HTLV-1 protease and inhibited Gag processing in viral maturation assays and in a chronically HTLV-1 infected cell line. Cocrystal structures of these inhibitors with HTLV-1 protease highlight opportunities for future inhibitor design. Our results show promise toward developing highly potent HTLV-1 protease inhibitors as therapeutic agents against HTLV-1 infections.