Refereed Preprints

Review Commons provides authors with a Refereed Preprint, which includes the authors’ manuscript, reports from a single round of peer review and the authors’ response. These Refereed Preprints are transferred on the author’s behalf to bioRxiv. The most recently-completed Review Commons peer-reviews are listed below, with the most recently posted reviews at the top.

Latest Refereed Preprints

Bioinformatics | 07 Apr 2020
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Generating functional protein variants with variational autoencoders

Alex Hawkins-Hooker, Florence Depardieu, Sebastien Baur, Guillaume Couairon, Arthur Chen, David Bikard

The design of novel proteins with specified function and controllable biochemical properties is a longstanding goal in bio-engineering with potential applications across medicine and nanotechnology. The vast expansion of protein sequence databases over the last decades provides an opportunity for new approaches which seek to learn the sequence-function relationship directly from natural sequence variation. Advances in deep generative models have led to the successful modelling of diverse kinds of high-dimensional data, from images to molecules, allowing the generation of novel, realistic samples. While deep models trained on protein sequence data have been shown to learn biologically meaningful representations helpful for a variety of downstream tasks, their potential for direct use in protein engineering remains largely unexplored. Here we show that variational autoencoders trained on a dataset of almost 70000 luciferase-like oxidoreductases can be used to generate novel, functional variants of the luxA bacterial luciferase. We propose separate VAE models to work with aligned sequence input (MSA VAE) and raw sequence input (AR-VAE), and offer evidence that while both are able to reproduce patterns of amino acid usage characteristic of the family, the MSA VAE is better able to capture long-distance dependencies reflecting the influence of 3D structure. To validate the practical utility of the models, we used them to generate variants of luxA whose function was tested experimentally. As further evidence of the practicality of these methods for design, we showed that conditional variants of both models could be used to increase the solubility of luxA without disrupting function. Altogether 18/24 of the variants generated using the AR-VAE and 21/23 variants generated using the MSA VAE retained some luminescence activity, despite containing as many as 35 differences relative to any training set sequence. These results demonstrate the feasibility of using deep generative models to explore the space of possible protein sequences and generate useful variants, providing a method complementary to rational design and directed evolution approaches.
Cell Biology | 10 Feb 2020
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During normal development and response to environmental stress, fungi must coordinate synthesis of the cell wall and plasma membrane. Septins, small cytoskeletal GTPases, colocalize with sterol-rich regions in the membrane and facilitate recruitment of cell wall synthases during dynamic wall remodeling. In this study we show that null mutants of the core septins in Aspergillus nidulans, ΔaspAcdc11, ΔaspBcdc3, ΔaspCcdc12, and ΔaspDcdc10, are sensitive to cell wall-disturbing agents known to activate the cell wall integrity MAPK pathway and that this sensitivity can be remediated by osmotic support. Septin null mutants showed changes in cell wall polysaccharide composition and organization and in chitin synthase localization. Double mutant analysis suggested core septins function downstream of the final kinase of the cell wall integrity pathway. Null mutants of the core septins and of noncore septin AspE were resistant to ergosterol and sphingolipid biosynthesis-disrupting agents. Septins were mislocalized after treatment with sphingolipid biosynthesis-disrupting agents and, to a lesser extent, phosphoinositide biosynthesis- disrupting agents. When septin deletion mutants were challenged with both membrane-disturbing and cell wall-disturbing agents in combination, remediation of the membrane defect restored proper growth, but remediation of the cell wall defect did not. Our data suggest that septins are required for proper coordination of the cell wall integrity pathway and lipid metabolism and that this role requires sphingolipids.
Cell Biology | 18 Nov 2020
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The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility

Miguel Ricardo Leung, Marc C. Roelofs, Ravi Teja Ravi, Paula Maitan, Min Zhang, Heiko Henning, Elizabeth G. Bromfield, Stuart C. Howes, Bart M. Gadella, Hermes Bloomfield-Gadêlha, Tzviya Zeev-Ben-Mordehai

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures rein-forcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.
Cell Biology | 20 Sep 2020
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Loading of the MCM replicative helicase onto origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The number of MCM loaded on origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide characteristics of MCM loading and their direct effect on replication timing remain unclear. In order to probe MCM loading dynamics and its effect on replication timing, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their initiation during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels not limiting for MCM loading. Our results support a model in which the loading activity of origins, controlled by their ability to recruit ORC and compete for MCM, determines the number of helicases loaded, which in turn affects replication timing.
Cell Biology | 05 Oct 2020
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Lumen extension in intracellular tubes can occur by the directed fusion of vesicles with an invading apical membrane domain. Within the C. elegans excretory cell, which contains an intracellular tube, the exocyst vesicle-tethering complex is enriched at the lumenal membrane domain and is required for tube formation, suggesting that it targets vesicles needed for lumen extension. Here, we identify a polarity pathway that promotes intracellular tube formation by enriching the exocyst at the lumenal membrane. We show that the PAR polarity proteins PAR-6 and PKC-3/aPKC localize to the lumenal membrane domain and function within the excretory cell to promote lumen extension, similar to exocyst component SEC-5 and exocyst regulator RAL-1. Using acute protein depletion, we find that PAR-6 is required to recruit the exocyst to the lumenal membrane domain, whereas PAR-3, which functions as an exocyst receptor in mammalian cells, appears to be dispensable for exocyst localization and lumen extension. Finally, we show that the Rho GTPase CDC-42 and the RhoGEF EXC-5/FGD act as upstream regulators of lumen formation by recruiting PAR-6 and PKC-3 to the lumenal membrane. Our findings reveal a molecular pathway that connects Rho GTPase signaling, cell polarity, and vesicle-tethering proteins to promote lumen extension in intracellular tubes.
Cell Biology | 29 Sep 2020
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Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

Meenakshi Basu Shrivastava, Barbara Mojsa, Stéphan Mora, Ian Robbins, Guillaume Bossis, Iréna Lassot, Solange Desagher

NFATc3 is the predominant member of the NFAT family of transcription factor in neurons, where it plays a pro-apoptotic role. Mechanisms controlling NFAT protein stability are poorly understood. Here we identify Trim39 as an E3 ubiquitin-ligase of NFATc3. Indeed, Trim39 ubiquitinates NFATc3 in vitro and in cells, whereas silencing of endogenous Trim39 decreases NFATc3 ubiquitination. We also show that Trim17 inhibits Trim39-mediated ubiquitination of NFATc3 by reducing both the E3 ubiquitin-ligase activity of Trim39 and the NFATc3/Trim39 interaction. Moreover, mutation of SUMOylation sites in NFATc3 or SUMO-interacting motif in Trim39 reduces the NFATc3/Trim39 interaction and Trim39-induced ubiquitination of NFATc3. As a consequence, silencing of Trim39 increases the protein level and transcriptional activity of NFATc3, resulting in enhanced neuronal apoptosis. Likewise, a SUMOylation-deficient mutant of NFATc3 exhibits increased stability and pro-apoptotic activity. Taken together, these data indicate that Trim39 modulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for NFATc3.
Plant Biology | 24 Sep 2020
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Individual plant cells possess a genetic network, the circadian clock, that times internal processes to the day-night cycle. Mathematical models of the clock network have driven a mechanistic understanding of the clock in plants. However, these models are typically either ‘whole plant’ models that ignore tissue or cell type specific clock behavior, or ‘phase only’ models that do not include clock network components explicitly. It is increasingly clear that in order to reveal the design principles of the plant circadian clock, clock network models must address spatial differences. This is because complex spatial behaviours have been observed in tissues and cells in plants, including period and phase differences between cells and spatial waves of gene expression between organs. Here, we implement an up to date clock network model on a spatial template of the plant. In our model, the sensitivity to light inputs varies across the plant, and cells communicate their clock timing locally via the levels of core clock mRNA levels by cell-to-cell coupling. We found that differences in sensitivities to environmental input in the model can explain the experimentally observed differences in clock periods in different organs, and we show using the model that a plausible coupling mechanism can generate the experimentally observed waves in clock gene expression across the plant. We then examined what features of the plant circadian system allow it to keep time under noisy light-dark (LD) cycles. We found that differences in sensitivity to light can allow regional flexibility in phase even under LD cycles, whilst local cell-to-cell coupling minimized variability in clock rhythms in neighboring cells. Thus, local sensitivity to environmental inputs combined with cell-to-cell coupling allows for flexible yet robust circadian timing under noisy environments.
Systems Biology | 27 Aug 2020
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The circadian oscillator analysed at the single-transcript level

Nicholas E. Phillips, Alice Hugues, Jake Yeung, Eric Durandau, Damien Nicolas, Felix Naef

The circadian clock is an endogenous and self-sustained oscillator that anticipates daily environmental cycles and coordinates physiology accordingly. While rhythmic gene expression of circadian genes is well-described in populations of cells, the single-cell mRNA dynamics of multiple core-clock genes remain largely unknown. Here we use single molecule fluorescence in-situ hybridization (smFISH) at multiple time points to measure pairs of core-clock transcripts, Rev-erbα (Nr1d1), Cry1 and Bmal1, in mouse fibroblasts at single-molecule resolution. The mean mRNA level oscillates over 24 hours for all three genes, but mRNA numbers show considerable spread between cells. While transcript number scales with cell size for all genes, gene-to-gene correlations of mRNA number depends on the gene pair. To account for these features of the data, we develop a probabilistic model for multivariate smFISH mRNA counts that quantifies changes in transcriptional bursting across genes and over circadian time. We identify a mixture model of negative binomials as the preferred model of the mRNA count distributions, which accounts for cell-to-cell heterogeneity, notably in cell size. The paired count data and modelling allows the decomposition of mRNA variability into distinct noise sources, showing that circadian clock time contributes only a small fraction of the total variability in mRNA number between cells. Thus, our results highlight the intrinsic biological challenges in estimating circadian phase from single-cell mRNA counts and suggest that circadian phase in single cells is encoded post-transcriptionally.
Developmental Biology | 23 Sep 2020
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Glial Hedgehog and lipid metabolism regulate neural stem cell proliferation in Drosophila

Qian Dong, Michael Zavortink, Francesca Froldi, Sofya Golenkina, Tammy Lam, Louise Y. Cheng

The final size and function of the adult central nervous system (CNS) is determined by neuronal lineages generated by neural stem cells (NSCs) in the developing brain. In Drosophila, NSCs called neuroblasts (NBs) reside within a specialised microenvironment called the glial niche. Here, we explore non-autonomous glial regulation of NB proliferation. We show that lipid droplets (LDs) which reside within the glial niche are closely associated with the signalling molecule Hedgehog (Hh). Under physiological conditions, cortex glial Hh is autonomously required to sustain niche chamber formation, and non-autonomously restrained to prevent ectopic Hh signalling in the NBs. In the context of cortex glial overgrowth, induced by Fibroblast Growth Factor (FGF) activation, Hh and lipid storage regulators Lsd-2 and Fasn1 were upregulated, resulting in activation of Hh signalling in the NBs; which in turn disrupted NB cell cycle progression and reduced neuronal production. We show that the LD regulator Lsd-2 modulates Hh’s ability to signal to NBs, and de novo lipogenesis gene Fasn1 regulates Hh post-translational modification via palmitoylation. Together, our data suggest that the glial niche non-autonomously regulates NB proliferation and neural lineage size via Hh signaling that is modulated by lipid metabolism genes.
Bioinformatics | 10 Jul 2020
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Predicting cell health phenotypes using image-based morphology profiling

Gregory P. Way, Maria Kost-Alimova, Tsukasa Shibue, William F. Harrington, Stanley Gill, Federica Piccioni, Tim Becker, William C. Hahn, Anne E. Carpenter, Francisca Vazquez, Shantanu Singh

Genetic and chemical perturbations impact diverse cellular phenotypes, including multiple indicators of cell health. These readouts reveal toxicity and antitumorigenic effects relevant to drug discovery and personalized medicine. We developed two customized microscopy assays that use seven reagents to measure 70 specific cell health phenotypes including proliferation, apoptosis, reactive oxygen species (ROS), DNA damage, and aberrant cell cycle stage progression. We then tested an approach to predict multiple cell health phenotypes using Cell Painting, an inexpensive and scalable image-based morphology assay. In matched CRISPR perturbations of three cancer cell lines, we collected both Cell Painting and cell health data. We found that simple machine learning algorithms can predict many cell health readouts directly from Cell Painting images, at less than half the cost. We hypothesized that these trained models can be applied to accurately predict cell health assay outcomes for any future or existing Cell Painting dataset. For Cell Painting images from a set of 1,500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts, and by confirming mitotic arrest and ROS phenotypes via PLK and proteasome inhibition, respectively. We provide an intuitive web app to browse all predictions at http://broad.io/cell-health-app. Our approach can be used to add cell health annotations to Cell Painting perturbation datasets.
Infectious Diseases (except HIV/AIDS) | 30 Oct 2020
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A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection

Martin A.M. Reijns, Louise Thompson, Juan Carlos Acosta, Holly A. Black, Francisco J. Sanchez-Luque, Austin Diamond, David A. Parry, Alison Daniels, Marie O’Shea, Carolina Uggenti, Maria C. Sanchez, Alan O’Callaghan, Michelle L.L. McNab, Martyna Adamowicz, Elias T. Friman, Toby Hurd, Edward J. Jarman, Frederic Li Mow Chee, Jacqueline K. Rainger, Marion Walker, Camilla Drake, Dasa Longman, Christine Mordstein, Sophie J. Warlow, Stewart McKay, Louise Slater, Morad Ansari, Ian P.M. Tomlinson, David Moore, Nadine Wilkinson, Jill Shepherd, Kate Templeton, Ingolfur Johannessen, Christine Tait-Burkard, Jürgen G. Haas, Nick Gilbert, Ian R. Adams, Andrew P. Jackson

With the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, there is need for sensitive, specific and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR, with many commercial kits now available for this purpose. However, these are expensive and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (PhHV-1), which monitor sample quality and nucleic acid extraction efficiency respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by nose-and-throat swabbing, and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false negative rates. We demonstrate feasibility of establishing a robust RT-qPCR assay at ∼10% of the cost of equivalent commercial assays, which could benefit low resource environments and make high volume testing more affordable.
Molecular Biology | 08 Oct 2020
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RIOK2 phosphorylation by RSK promotes synthesis of the human small ribosomal subunit

Emilie L. Cerezo, Thibault Houles, Oriane Lié, Marie-Kerguelen Sarthou, Charlotte Audoynaud, Geneviève Lavoie, Maral Halladjian, Sylvain Cantaloube, Carine Froment, Odile Burlet-Schiltz, Yves Henry, Philippe P. Roux, Anthony K. Henras, Yves Romeo

Ribosome biogenesis lies at the nexus of various signaling pathways coordinating protein synthesis with cell growth and proliferation. This process is regulated by well-described transcriptional mechanisms, but a growing body of evidence indicates that other levels of regulation exist. Here we show that the Ras/mitogen-activated protein kinase (MAPK) pathway stimulates post-transcriptional stages of human ribosome synthesis. We identify RIOK2, a pre-40S particle assembly factor, as a new target of the MAPK-activated kinase RSK. RIOK2 phosphorylation by RSK promotes cytoplasmic maturation of late pre-40S particles, which is required for optimal protein synthesis and cell proliferation. RIOK2 phosphorylation facilitates its release from pre-40S particles and its nuclear re-import, prior to completion of small ribosomal subunits. Our results bring a detailed mechanistic link between the Ras/MAPK pathway and the maturation of human pre-40S particles, which open a hitherto poorly explored area of ribosome biogenesis.
Developmental Biology | 09 Jul 2020
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NODAL/TGFβ signalling mediates the self-sustained stemness induced by PIK3CAH1047R homozygosity in pluripotent stem cells

Ralitsa R. Madsen, James Longden, Rachel G. Knox, Xavier Robin, Franziska Völlmy, Kenneth G. Macleod, Larissa S. Moniz, Neil O. Carragher, Rune Linding, Bart Vanhaesebroeck, Robert K. Semple

Activating PIK3CA mutations are known “drivers” of human cancer and developmental overgrowth syndromes. We recently demonstrated that the “hotspot” PIK3CAH1047R variant exerts unexpected allele dose-dependent effects on stemness in human pluripotent stem cells (hPSCs). In the present study, we combine high-depth transcriptomics, total proteomics and reverse-phase protein arrays to reveal potentially disease-related alterations in heterozygous cells, and to assess the contribution of activated TGFβ signalling to the stemness phenotype of PIK3CAH1047R homozygous cells. We demonstrate signalling rewiring as a function of oncogenic PI3K signalling dose, and provide experimental evidence that self-sustained stemness is causally related to enhanced autocrine NODAL/TGFβ signalling. A significant transcriptomic signature of TGFβ pathway activation in PIK3CAH1047R heterozygous was observed but was modest and was not associated with the stemness phenotype seen in homozygous mutants. Notably, the stemness gene expression in PIK3CAH1047R homozygous iPSCs was reversed by pharmacological inhibition of TGFβ signalling, but not by pharmacological PI3Kα pathway inhibition. Altogether, this provides the first in-depth analysis of PI3K signalling in human pluripotent stem cells and directly links dose-dependent PI3K activation to developmental NODAL/TGFβ signalling.
Cell Biology | 01 Oct 2020
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Dynamins are large cytoplasmic GTPases that are targeted to specific cellular membranes which they remodel via membrane fusion or fission. Although the mechanism of target membrane selection by dynamins has been studied, the molecular basis of conferring specificity to bind specific lipids on the target membranes is not known in any of the family members. Here, we report a mechanism of nuclear membrane recruitment of Drp6 that is involved in nuclear remodeling in Tetrahymena thermophila. Recruitment of Drp6 depends on a domain that binds to cardiolipin-rich bilayers. Consistent with this, the nuclear localization of wildtype Drp6 was inhibited by depleting cardiolipin in the cell. Cardiolipin binding was blocked with a single amino acid substitution (I553M) in the membrane-binding domain of Drp6. Importantly, the I553M substitution was sufficient to block nuclear localization without affecting other properties of Drp6. Consistent with this result, co-expression of wildtype Drp6 was sufficient to rescue the localization defect of I553M variant in Tetrahymena. Inhibition of cardiolipin synthesis or perturbation in Drp6 recruitment to nuclear membrane caused defects in the formation of new macronuclei post-conjugation. Taken together, our results elucidate a molecular basis of target membrane selection by a nuclear dynamin, and establish the importance of a defined membrane-binding domain and its target lipid in facilitating nuclear expansion.
Evolutionary Biology | 20 Oct 2020
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On the Emergence of P-Loop NTPase and Rossmann Enzymes from a Beta-Alpha-Beta Ancestral Fragment

Liam M. Longo, Jagoda Jabłońska, Pratik Vyas, Manil Kanade, Rachel Kolodny, Nir Ben-Tal, Dan S. Tawfik

Dating back to the last universal common ancestor (LUCA), the P-loop NTPases and Rossmanns now comprise the most ubiquitous and diverse enzyme lineages. Intriguing similarities in their overall architecture and phosphate binding motifs suggest common ancestry; however, due to a lack of sequence identity and some fundamental structural differences, these families are considered independent emergences. To address this longstanding dichotomy, we systematically searched for ‘bridge proteins’ with structure and sequence elements shared by both lineages. We detected homologous segments that span the first βαβ segment of both lineages and include two key functional motifs: (i) a phosphate binding loop – the ‘Walker A’ motif of P-loop NTPases or the Rossmann equivalent, both residing at the N-terminus of α1; and (ii) an Asp at the tip of β2. The latter comprises the ‘Walker B’ aspartate that chelates the catalytic metal in P-loop NTPases, or the canonical Rossmann β2-Asp that binds the cofactor’s ribose moiety. Tubulin, a Rossmann GTPase, demonstrates the potential of the β2-Asp to take either one of these two roles. We conclude that common P-loops/Rossmann ancestry is plausible, although convergence cannot be completely ruled out. Regardless, both lineages most likely emerged from a polypeptide comprising a βαβ segment carrying the above two functional motifs, a segment that comprises the core of both enzyme families to this very day.
Cell Biology | 03 Jul 2020
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Spatial Variation of Microtubule Depolymerization in Large Asters Suggests Regulation by MAP Depletion

Keisuke Ishihara, Franziska Decker, Paulo Caldas, James F. Pelletier, Martin Loose, Jan Brugués, Timothy J. Mitchison

Microtubule plus end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were almost two-fold higher in the aster interior compared to the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and MAPs in the interior cytosol compared to that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density.
Microbiology | 27 Aug 2020
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A drug repurposing screen identifies hepatitis C antivirals as inhibitors of the SARS-CoV-2 main protease

Jeremy D. Baker, Rikki L. Uhrich, Gerald C. Kraemer, Jason E. Love, Brian C. Kraemer

The SARS coronavirus type 2 (SARS-CoV-2) emerged in late 2019 as a zoonotic virus highly transmissible between humans that has caused the COVID-19 pandemic 1,2. This pandemic has the potential to disrupt healthcare globally and has already caused high levels of mortality, especially amongst the elderly. The overall case fatality rate for COVID-19 is estimated to be ∼2.3% overall 3 and 32.3% in hospitalized patients age 70-79 years 4. Therapeutic options for treating the underlying viremia in COVID-19 are presently limited by a lack of effective SARS-CoV-2 antiviral drugs, although steroidal anti-inflammatory treatment can be helpful. A variety of potential antiviral targets for SARS-CoV-2 have been considered including the spike protein and replicase. Based upon previous successful antiviral drug development for HIV-1 and hepatitis C, the SARS-CoV-2 main protease (Mpro) appears an attractive target for drug development. Here we show the existing pharmacopeia contains many drugs with potential for therapeutic repurposing as selective and potent inhibitors of SARS-CoV-2 Mpro. We screened a collection of ∼6,070 drugs with a previous history of use in humans for compounds that inhibit the activity of Mpro in vitro. In our primary screen we found ∼50 compounds with activity against Mpro (overall hit rate <0.75%). Subsequent dose validation studies demonstrated 8 dose responsive hits with an IC50 ≤ 50 μM. Hits from our screen are enriched with hepatitis C NS3/4A protease targeting drugs including Boceprevir (IC50=0.95 μM), Ciluprevir (20.77μM). Narlaprevir (IC50=1.10μM), and Telaprevir (15.25μM). These results demonstrate that some existing approved drugs can inhibit SARS-CoV-2 Mpro and that screen saturation of all approved drugs is both feasible and warranted. Taken together this work suggests previous large-scale commercial drug development initiatives targeting hepatitis C NS3/4A viral protease should be revisited because some previous lead compounds may be more potent against SARS-CoV-2 Mpro than Boceprevir and suitable for rapid repurposing.