Now showing items 1-20 of 211

    • Contributions of Hyperactive Mutations in M from SARS-CoV-2 to Drug Resistance

      Flynn, Julia M; Zvornicanin, Sarah N; Tsepal, Tenzin; Shaqra, Ala M; Kurt Yilmaz, Nese; Jia, Weiping; Moquin, Stephanie; Dovala, Dustin; Schiffer, Celia A; Bolon, Daniel N A (2024-03-12)
      The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by the SARS-CoV-2 virus, are major concerns for human health. Many drugs target enzymes, and resistance-conferring mutations impact inhibitor binding or enzyme activity. Nirmatrelvir, the most widely used inhibitor currently used to treat SARS-CoV-2 infections, targets the main protease (Mpro) preventing it from processing the viral polyprotein into active subunits. Our previous work systematically analyzed resistance mutations in Mpro that reduce binding to inhibitors; here, we investigate mutations that affect enzyme function. Hyperactive mutations that increase Mpro activity can contribute to drug resistance but have not been thoroughly studied. To explore how hyperactive mutations contribute to resistance, we comprehensively assessed how all possible individual mutations in Mpro affect enzyme function using a mutational scanning approach with a fluorescence resonance energy transfer (FRET)-based yeast readout. We identified hundreds of mutations that significantly increased the Mpro activity. Hyperactive mutations occurred both proximal and distal to the active site, consistent with protein stability and/or dynamics impacting activity. Hyperactive mutations were observed 3 times more than mutations which reduced apparent binding to nirmatrelvir in recent studies of laboratory-grown viruses selected for drug resistance. Hyperactive mutations were also about three times more prevalent than nirmatrelvir binding mutations in sequenced isolates from circulating SARS-CoV-2. Our findings indicate that hyperactive mutations are likely to contribute to the natural evolution of drug resistance in Mpro and provide a comprehensive list for future surveillance efforts.
    • Direct delivery of stabilized Cas-embedded base editors achieves efficient and accurate editing of clinically relevant targets [preprint]

      Lee, Jeong Min; Zeng, Jing; Liu, Pengpeng; Nguyen, My Anh; Loustaunau, Diego Suchenski; Bauer, Daniel E; Yilmaz, Nese Kurt; Wolfe, Scot A; Schiffer, Celia A (2024-02-08)
      Over the last 5 years, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBE's have the significant liabilities of off-target and bystander editing activities, in part due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study we engineeredhighly stabilized Cas-embedded CBEs (sCE_CBEs) that integrate several recent advances, andthat are highly expressible and soluble for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting sCE_CBE RNP complexes efficiently and specifically target TC dinucleotides with minimal off-target or bystander mutations. Additional uracil glycosylase inhibitor (UGI) protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation to untreated levels. A single electroporation was sufficient to effectively edit the therapeutically relevant locus for sickle cell disease in hematopoietic stem and progenitor cells (HSPC) in a dose dependent manner without cellular toxicity. Significantly, these sCE_CBE RNPs permitted for the transplantation of edited HSPCs confirming highly efficient editing in engrafting hematopoietic stem cells in mice. The success of the designed sCBE editors, with improved solubility and enhanced on-target editing, demonstrates promising agents for cytosine base editing at other disease-related sites in HSPCs and other cell types.
    • Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors

      Boby, Melissa L; Fearon, Daren; Ferla, Matteo; Filep, Mihajlo; Koekemoer, Lizbé; Robinson, Matthew C; Chodera, John D; Lee, Alpha A; London, Nir; von Delft, Annette; et al. (2023-11-10)
      We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.
    • Addressing the dNTP bottleneck restricting prime editing activity [preprint]

      Ponnienselvan, Karthikeyan; Liu, Pengpeng; Nyalile, Thomas; Oikemus, Sarah; Joynt, Anya T; Kelly, Karen; Guo, Dongsheng; Chen, Zexiang; Lee, Jeong Min; Schiffer, Celia A; et al. (2023-10-29)
      Prime editing efficiency is modest in cells that are quiescent or slowly proliferating where intracellular dNTP levels are tightly regulated. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. We report that prime editing efficiency in primary cells and in vivo is increased by mutations that enhance the enzymatic properties of MMLV-reverse transcriptase and can be further complemented by targeting SAMHD1 for degradation.
    • FruitFire: a luciferase based on a fruit fly metabolic enzyme [preprint]

      Adams, Spencer T; Zephyr, Jacqueto; Bohn, Markus F; Schiffer, Celia A; Miller, Stephen C (2023-06-30)
      Firefly luciferase is homologous to fatty acyl-CoA synthetases from insects that are not bioluminescent. Here, we determined the crystal structure of the fruit fly fatty acyl-CoA synthetase CG6178 to 2.5 Å. Based on this structure, we mutated a steric protrusion in the active site to create the artificial luciferase FruitFire, which prefers the synthetic luciferin CycLuc2 to D-luciferin by >1000-fold. FruitFire enabled in vivo bioluminescence imaging in the brains of mice using the pro-luciferin CycLuc2-amide. The conversion of a fruit fly enzyme into a luciferase capable of in vivo imaging underscores the potential for bioluminescence with a range of adenylating enzymes from nonluminescent organisms, and the possibilities for application-focused design of enzyme-substrate pairs.
    • Systematic Analyses of the Resistance Potential of Drugs Targeting SARS-CoV-2 Main Protease

      Flynn, Julia M; Huang, Qiu Yu Judy; Zvornicanin, Sarah N; Schneider-Nachum, Gila; Shaqra, Ala M; Yilmaz, Nese Kurt; Moquin, Stephanie A; Dovala, Dustin; Schiffer, Celia A; Bolon, Daniel N A (2023-06-30)
      Drugs that target the main protease (Mpro) of SARS-CoV-2 are effective therapeutics that have entered clinical use. Wide-scale use of these drugs will apply selection pressure for the evolution of resistance mutations. To understand resistance potential in Mpro, we performed comprehensive surveys of amino acid changes that can cause resistance to nirmatrelvir (Pfizer), and ensitrelvir (Xocova) in a yeast screen. We identified 142 resistance mutations for nirmatrelvir and 177 for ensitrelvir, many of which have not been previously reported. Ninety-nine mutations caused apparent resistance to both inhibitors, suggesting likelihood for the evolution of cross-resistance. The mutation with the strongest drug resistance score against nirmatrelvir in our study (E166V) was the most impactful resistance mutation recently reported in multiple viral passaging studies. Many mutations that exhibited inhibitor-specific resistance were consistent with the distinct interactions of each inhibitor in the substrate binding site. In addition, mutants with strong drug resistance scores tended to have reduced function. Our results indicate that strong pressure from nirmatrelvir or ensitrelvir will select for multiple distinct-resistant lineages that will include both primary resistance mutations that weaken interactions with drug while decreasing enzyme function and compensatory mutations that increase enzyme activity. The comprehensive identification of resistance mutations enables the design of inhibitors with reduced potential of developing resistance and aids in the surveillance of drug resistance in circulating viral populations.
    • HIV-1 protease inhibitors with a P1 phosphonate modification maintain potency against drug-resistant variants by increased interactions with flap residues

      Lockbaum, Gordon J; Rusere, Linah N; Henes, Mina; Kosovrasti, Klajdi; Rao, Desaboini Nageswara; Spielvogel, Ean; Lee, Sook-Kyung; Nalivaika, Ellen A; Swanstrom, Ronald; Yilmaz, Nese Kurt; et al. (2023-05-18)
      Protease inhibitors are the most potent antivirals against HIV-1, but they still lose efficacy against resistant variants. Improving the resistance profile is key to developing more robust inhibitors, which may be promising candidates for simplified next-generation antiretroviral therapies. In this study, we explored analogs of darunavir with a P1 phosphonate modification in combination with increasing size of the P1' hydrophobic group and various P2' moieties to improve potency against resistant variants. The phosphonate moiety substantially improved potency against highly mutated and resistant HIV-1 protease variants, but only when combined with more hydrophobic moieties at the P1' and P2' positions. Phosphonate analogs with a larger hydrophobic P1' moiety maintained excellent antiviral potency against a panel of highly resistant HIV-1 variants, with significantly improved resistance profiles. The cocrystal structures indicate that the phosphonate moiety makes extensive hydrophobic interactions with the protease, especially with the flap residues. Many residues involved in these protease-inhibitor interactions are conserved, enabling the inhibitors to maintain potency against highly resistant variants. These results highlight the need to balance inhibitor physicochemical properties by simultaneous modification of chemical groups to further improve resistance profiles.
    • Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses

      Zvornicanin, Sarah N; Shaqra, Ala M; Huang, Qiu Yu Judy; Ornelas, Elizabeth; Moghe, Mallika; Knapp, Mark; Moquin, Stephanie; Dovala, Dustin; Schiffer, Celia A; Kurt Yilmaz, Nese (2023-03-18)
      With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (Mpro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 Mpro, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 Mpro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors.
    • Selection of HIV-1 for resistance to fifth-generation protease inhibitors reveals two independent pathways to high-level resistance

      Spielvogel, Ean; Lee, Sook-Kyung; Zhou, Shuntai; Lockbaum, Gordon J; Henes, Mina; Sondgeroth, Amy; Kosovrasti, Klajdi; Nalivaika, Ellen A; Ali, Akbar; Yilmaz, Nese Kurt; et al. (2023-03-15)
      Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in high drug concentrations that are achieved in vivo. Little is known about the de novo resistance pathway for DRV. We selected for resistance to high drug concentrations against 10 PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small changes in the inhibitor P1'-equivalent position led to preferential use of one pathway over the other. Changes in the inhibitor P2'-equivalent position determined differences in potency that were retained in the resistant viruses and that impacted the selected mutations. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fifth-generation PIs and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance.
    • Non-canonical pattern recognition of a pathogen-derived metabolite by a nuclear hormone receptor identifies virulent bacteria in C. elegans

      Peterson, Nicholas D; Tse, Samantha Y; Huang, Qiu Yu Judy; Wani, Khursheed A; Schiffer, Celia A; Pukkila-Worley, Read (2023-02-15)
      Distinguishing infectious pathogens from harmless microorganisms is essential for animal health. The mechanisms used to identify infectious microbes are not fully understood, particularly in metazoan hosts that eat bacteria as their food source. Here, we characterized a non-canonical pattern-recognition system in Caenorhabditis elegans (C. elegans) that assesses the relative threat of virulent Pseudomonas aeruginosa (P. Aeruginosa) to activate innate immunity. We discovered that the innate immune response in C. elegans was triggered by phenazine-1-carboxamide (PCN), a toxic metabolite produced by pathogenic strains of P. aeruginosa. We identified the nuclear hormone receptor NHR-86/HNF4 as the PCN sensor in C. elegans and validated that PCN bound to the ligand-binding domain of NHR-86/HNF4. Activation of NHR-86/HNF4 by PCN directly engaged a transcriptional program in intestinal epithelial cells that protected against P. aeruginosa. Thus, a bacterial metabolite is a pattern of pathogenesis surveilled by nematodes to identify a pathogen in its bacterial diet.
    • Structure of the catalytically active APOBEC3G bound to a DNA oligonucleotide inhibitor reveals tetrahedral geometry of the transition state

      Maiti, Atanu; Hedger, Adam K; Myint, Wazo; Balachandran, Vanivilasini; Watts, Jonathan K; Schiffer, Celia A; Matsuo, Hiroshi (2022-11-19)
      APOBEC3 proteins (A3s) are enzymes that catalyze the deamination of cytidine to uridine in single-stranded DNA (ssDNA) substrates, thus playing a key role in innate antiviral immunity. However, the APOBEC3 family has also been linked to many mutational signatures in cancer cells, which has led to an intense interest to develop inhibitors of A3's catalytic activity as therapeutics as well as tools to study A3's biochemistry, structure, and cellular function. Recent studies have shown that ssDNA containing 2'-deoxy-zebularine (dZ-ssDNA) is an inhibitor of A3s such as A3A, A3B, and A3G, although the atomic determinants of this activity have remained unknown. To fill this knowledge gap, we determined a 1.5 Å resolution structure of a dZ-ssDNA inhibitor bound to active A3G. The crystal structure revealed that the activated dZ-H2O mimics the transition state by coordinating the active site Zn2+ and engaging in additional stabilizing interactions, such as the one with the catalytic residue E259. Therefore, this structure allowed us to capture a snapshot of the A3's transition state and suggests that developing transition-state mimicking inhibitors may provide a new opportunity to design more targeted molecules for A3s in the future.
    • Allosteric quinoxaline-based inhibitors of the flavivirus NS2B/NS3 protease

      Zephyr, Jacqueto; Rao, Desaboini Nageswara; Johnson, Colby; Shaqra, Ala M; Nalivaika, Ellen A; Jordan, Aria; Kurt Yilmaz, Nese; Ali, Akbar; Schiffer, Celia A (2022-11-19)
      Viruses from the Flavivirus genus infect millions of people worldwide and cause severe diseases, including recent epidemics of dengue virus (DENV), and Zika virus (ZIKV). There is currently no antiviral treatment against flavivirus infections, despite considerable efforts to develop inhibitors against essential viral enzymes including NS2B/NS3 protease. Targeting the flavivirus NS2B/NS3 protease proved to be challenging because of the conformational dynamics, topology, and electrostatic properties of the active site. Here, we report the identification of quinoxaline-based allosteric inhibitors by fragment-based drug discovery approach as a promising new drug-like scaffold to target the NS2B/NS3 protease. Enzymatic assays and mutational analysis of the allosteric site in ZIKV NS2B/NS3 protease support noncompetitive inhibition mechanism as well as engineered DENV protease construct indicating the compounds likely compete with the NS2B cofactor for binding to the protease domain. Furthermore, antiviral activity confirmed the therapeutic potential of this new inhibitor scaffold.
    • Dual Inhibitors of Main Protease (M) and Cathepsin L as Potent Antivirals against SARS-CoV2

      Mondal, Santanu; Chen, Yongzhi; Lockbaum, Gordon J; Sen, Sudeshna; Chaudhuri, Sauradip; Reyes, Archie C; Lee, Jeong Min; Kaur, Arshia N; Sultana, Nadia; Cameron, Michael D; et al. (2022-11-10)
      Given the current impact of SARS-CoV2 and COVID-19 on human health and the global economy, the development of direct acting antivirals is of paramount importance. Main protease (MPro), a cysteine protease that cleaves the viral polyprotein, is essential for viral replication. Therefore, MPro is a novel therapeutic target. We identified two novel MPro inhibitors, D-FFRCMKyne and D-FFCitCMKyne, that covalently modify the active site cysteine (C145) and determined cocrystal structures. Medicinal chemistry efforts led to SM141 and SM142, which adopt a unique binding mode within the MPro active site. Notably, these inhibitors do not inhibit the other cysteine protease, papain-like protease (PLPro), involved in the life cycle of SARS-CoV2. SM141 and SM142 block SARS-CoV2 replication in hACE2 expressing A549 cells with IC50 values of 8.2 and 14.7 nM. Detailed studies indicate that these compounds also inhibit cathepsin L (CatL), which cleaves the viral S protein to promote viral entry into host cells. Detailed biochemical, proteomic, and knockdown studies indicate that the antiviral activity of SM141 and SM142 results from the dual inhibition of MPro and CatL. Notably, intranasal and intraperitoneal administration of SM141 and SM142 lead to reduced viral replication, viral loads in the lung, and enhanced survival in SARS-CoV2 infected K18-ACE2 transgenic mice. In total, these data indicate that SM141 and SM142 represent promising scaffolds on which to develop antiviral drugs against SARS-CoV2.
    • Mucosal nanobody IgA as inhalable and affordable prophylactic and therapeutic treatment against SARS-CoV-2 and emerging variants

      Li, Qi; Humphries, Fiachra; Girardin, Roxie C; Wallace, Aaron; Ejemel, Monir; Amcheslavsky, Alla; McMahon, Conor T; Schiller, Zachary A; Ma, Zepei; Cruz, John; et al. (2022-09-12)
      Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.
    • 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 Judy; 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 S.; 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 Judy; 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 S.; 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.