A drug repurposing screen identifies hepatitis C antivirals as inhibitors of the SARS-CoV-2 main protease

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.

limited by a lack of effective SARS-CoV-2 antiviral drugs, although steroidal anti-inflammatory treatment 23 can be helpful. A variety of potential antiviral targets for SARS-CoV-2 have been considered including 24 the spike protein and replicase. Based upon previous successful antiviral drug development for HIV-1 and 25 hepatitis C, the SARS-CoV-2 main protease (Mpro) appears an attractive target for drug development. 26 Here we show the existing pharmacopeia contains many drugs with potential for therapeutic repurposing 27 as selective and potent inhibitors of SARS-CoV-2 Mpro. We screened a collection of ~6,070 drugs with a 28 previous history of use in humans for compounds that inhibit the activity of Mpro in vitro. In our primary 29 screen we found ~50 compounds with activity against Mpro (overall hit rate <0.75%). Subsequent dose 30 validation studies demonstrated 8 dose responsive hits with an IC50 < 50 M. Hits from our screen are 31 enriched with hepatitis C NS3/4A protease targeting drugs including Boceprevir (IC50=0.95 M), 32 Ciluprevir (20.77M). Narlaprevir (IC50=1.10M), and Telaprevir (15.25M). These results demonstrate 33 that some existing approved drugs can inhibit SARS-CoV-2 Mpro and that screen saturation of all approved 34 drugs is both feasible and warranted. Taken together this work suggests previous large-scale commercial 35 drug development initiatives targeting hepatitis C NS3/4A viral protease should be revisited because some 36 previous lead compounds may be more potent against SARS-CoV-2 Mpro than Boceprevir and suitable for 37 rapid repurposing. 38

Introduction 43
The SARS virus and SARS-CoV-2, the cause of the COVID-19 pandemic, are zoonotic coronaviruses 44 found in bats that can infect humans. Initial symptoms of SARS-CoV-2 infection include fever, myalgia, 45 cough, and headache. Infection usually resolves without active medical intervention, but for a subset of 46 cases infection can progress to viral pneumonia and a variety of complications including acute lung 47 targetable activities for COVID-19, the coronavirus Mpro seems a likely choice for rapid drug 66

development. 67
To accelerate drug development we employed a drug repurposing strategy, an approach of utilizing 68 previously approved drugs for new indications 12,13 . Previous work suggests libraries enriched with known 69 bioactive drug-like compounds provide the best opportunity for finding new lead compounds 14,15 . Thus 70 we attempted the selective optimization of side activities (SOSA) approach 16 as a rapid and cost effective 71 means to identify candidate hits while minimizing the number of compounds screened. The SOSA 72 approach proceeds by two steps. First a limited set of carefully chosen, structurally diverse, well-73 characterized drug molecules are screened; as approved drugs, their bioavailability, toxicity and efficacy 74 in human therapy has already been demonstrated 16,17 . To screen as much of the available approved drug 75 space as possible in an easily accessible format we chose to screen the Broad Institute Drug Repurposing 76 Library (6070 compounds, see Table S1) 18 . This represents about half of the approximately 14,000 77 approved or experimental drugs known to human clinical medicine 19 . There are significant cost and time 78 advantages realized by drug repurposing as it can accelerate the preclinical phase of development and 79 streamline clinical trials to focus on efficacy rather than safety. 80 Repositioning existing approved drugs with the capacity to inhibit COVID-19 virus replication and 81 infection would be of profound utility and immediately impact health care in the current pandemic. There 82 are no drugs in clinical use specifically targeting coronavirus replication. The major advantage of the 83 approach taken here is that by screening drugs with a history of previous clinical use, we will be focusing 84 on compounds with known properties in terms of pharmacokinetics (PK), pharmacodynamics (PD) and 85 toxicity. Thus, the Broad Repurposing Library we screened consists of compounds suitable for rapid 86 translation to human efficacy trials. 87

Development of fluorescent Mpro Assays 89
We began assay development by selecting potentially suitable synthetic Mpro substrates and compared 90 catalyzed hydrolysis curves between 5 fluorescently labeled substrates (Ac-Abu-Tle-Leu-Gln-AFC 20 , 91 DABCYL-VKLQ-EDANS, Ac-VKLQ-AFC, DABCYL-TSAVLQSGFRKM-EDANS 21 , and MCA-92 22 . We chose to use the recently published Ac-Abu-Tle-Leu-Gln-AFC 93 (Abu=2-Aminobutyrate, Tle=tButylglycine) synthetic non-canonical amino-acid containing peptide as 94 Mpro more readily cleaves this preferred sequence as compared to the native VKLQ sequence 20 (Fig 1A). 95 Substrates DABCYL-TSAVLQSGFRKM-EDANS and MCA-AVLQSGFR-K(DnP)-K-NH2 had 96 drastically lower rates of Mpro catalyzed hydrolysis and were not considered further in our assay 97 development ( Fig 1A). To determine concentration ratios between Mpro and substrate, we next preformed 98 a two-dimensional titration and chose 625nM Mpro and 8µM substrate for a balance of relatively modest 99 Mpro protein requirement and a robust fluorescence intensity ( Fig 2B). Before screening the Broad library, 100 we piloted our assay conditions against the NIH Clinical collections library (~650 compounds) and 101 calculated our Z'-factor for each plate at 0.780 and 0.784 (Fig 1C and D). Z'-factor is a score of suitability 102 of assays for high-throughput screening and is derived from the equation Z ' -factor = 1 − 3( + ) | − | , where 103 σ = standard deviation, µ=mean, p=positive controls, and n=negative controls. A score greater than 0.5 104 indicates a screenable assay. Although no promising compounds were identified from this smaller library, 105 it demonstrated that our assay was sufficiently robust for screening the much larger Broad Repurposing 106 library. 107 window was considered at Z-score ≤ -2 and was calculated as the Z-score of ΔRFU at 10 minutes 118 corresponding to the linear portion of the curve. X-axis indicates arbitrary compound number arranged by 119 increasing Z-score. (d) Z'-factor for the two NIH Clinical Collection 384-well plates. Pink circles indicate 120 negative control (DMSO) and black circles represent positive controls (no protein). Z'factor calculated at 121 0.780 and 0.784 for plates 1 and 22 respectively. Y axis represents change in RFU over 10 minutes. 122 123

Drug Repurposing Strategyscreening the Broad Repurposing Library 124
The concept of drug repurposing is to utilize existing therapeutic drugs to treat a new disease indication. 125 This approach is particularly relevant for COVID-19 because of the potential for an accelerated clinical 126 impact as compared to de novo drug development. A systematic approach to facilitate drug repurposing 127 has recently been described ( 18 , http:// www.broadinstitute.org/repurposing) and has made a large collection 128 of drugs with previous history of use in humans available for high throughput screening. We acquired this at 384-well density using the optimized kinetic Mpro assay described in Fig 1. Our overall repurposing 131 strategy is described in Fig 2A. We conducted a single point screen at 50 M compound concentration and 132 observed ~50 compounds with activity against SARS-CoV-2 Mpro for an overall hit rate <0.75%. These 133 compounds were screened in parallel against the natural amino acid substrate (Ac-VKLQ-AFC) as well as 134 a kinetically preferred substrate (Ac-Abu-Tle-Leu-Gln-AFC) ( Fig 2B). Individual compounds are shown 135 in Table 1. virtually. Any hit from the Broad library (Z-score ≤ -2) was validated for dose-responsiveness. All suitable 140 compounds passing this filter with satisfactory curve fitting and potency were ordered as powder and re-141 validated. Future efforts will test for selectivity and in orthogonal assays for suitability. Although outside 142 the scope of this report, determination of viral anti-replicative properties as well as toxic profile at required 143 dosage will be determined. The goal of this paradigm is to find suitable candidates for development both 144 as tools for probing underlying mechanisms of SARS-CoV-2 as well as for translational potential. (b) 145 Screen of the Broad Repurposing Library. Library was screened at a concentration of 50µM against both 146 Ac-VKLQ-AFC (black) and Ac-Abu-Tle-Leu-Gln-AFC (purple). Hit window was considered for 147 compounds falling below Z-score ≤ -2 against both substrates and consisted of 50 compounds. Compounds 148 ordered by average Z-score. 149 150

Analysis of potency 158
We validated the hits from the primary screen by conducting a 10-point dose-response analysis with a drug 159 concentration range from 150 M down to 7.6 nM (3-fold dilution series). From this dose-response analysis, Repurposing library. Using this approach, we derived a docking score for each compound (see Table S1 181 for broad repurposing library with docking scores). We observe a poor correlation (Pearson r=0.02864) 182 between Mpro docking score and Z-score in the protease inhibition assay (Fig 4A). Furthermore, top hits 183 from the screen also exhibit a weak correlation (Pearson r=-0.1503) between compound potency and 184 docking score (Fig 4B). is to complete a survey of approved drugs to identify therapies that can block COVID-19 viral replication 222 by inhibiting the main viral protease. The advantage of this approach is that any approved drug identified 223 can be advanced rapidly to clinical trials without extensive multi-year preclinical development efforts. This 224 is also particularly germane given the limitations of animal models of COVID-19 infection and 225

pathogenesis. 226
A diverse variety of initial hits were identified in our high throughput screen of the broad library. 227 Of these, the most potent hits are all known protease inhibitors and there is strong representation from 228 protease inhibitors developed to inhibit HCV protease NS3/4A (Boceprevir, Ciluprevir, Narlaprevir, and 229 Telaprevir).
Clearly as approved or well-developed clinical candidates, these drugs exhibit 230 pharmacological and pharmacodynamic properties well suited to repurposing as a COVID-19 antiviral 231 therapy. Boceprevir and Narlaprevir appear the most potent against Mpro and may be suitable for 232 repurposing.  (625nM final concentration in reaction buffer detailed above) was added with a MultiFlo FX liquid 301 dispenser using a 5µL cassette. Compounds were incubated with Mpro for 10 minutes at RT after which 10uL of substrate (8uM final concentration of either Ac-VKLQ-AFC or Ac-Abu-Tle-Leu-Gln-AFC) was 303 dispensed into the plate and read using a Cytation 5 multi-mode reader immediately at 380/20 nm excitation 304 and 500/20 nm emission wavelengths every 5 minutes for 30 minutes. Data was analyzed using Biotek 305 Gen5 software, Microsoft Excel, and GraphPad Prism 8. 306 307

Dose validation assays 308
Hit compounds were ordered from the Broad Institute pre-plated in 384-well format (Greiner 781209) as 309 10-point serial dilutions (3-fold) at 300nL per well. Mpro (80nM final concentration) and substrate (Ac-310 Abu-Tle-Leu-Gln-AFC at 32µM final concentration) were dispensed in the same manner described above. Graphs were generated using GraphPad Prism 8. IC50 calculations were performed using GraphPad Prism 323