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Yellow fever virus (YFV) transmitted by infected mosquitoes causes an acute viral disease for which there are no approved small-molecule therapeutics. Our recently developed machine learning models for YFV inhibitors led to the selection of a new pyrazolesulfonamide derivative RCB16003 with acceptable in vitro activity. We report that the N -phenyl-1-(phenylsulfonyl)-1 H -1,2,4-triazol-3-amine class, which was recently identified as active non-nucleoside reverse transcriptase inhibitors against HIV-1, can also be repositioned as inhibitors of yellow fever virus replication. As compared to other Flaviviridae or Togaviridae family viruses tested, both compounds RCB16003 and RCB16007 demonstrate selectivity for YFV over related viruses, with only RCB16007 showing some inhibition of the West Nile virus (EC 50 7.9 μM, CC 50 17 μM, SI 2.2). We also describe the absorption, distribution, metabolism, and excretion (ADME) in vitro and pharmacokinetics (PK) for RCB16007 in mice. This compound had previously been shown to not inhibit hERG, and we now describe that it has good metabolic stability in mouse and human liver microsomes, low levels of CYP inhibition, high protein binding, and no indication of efflux in Caco-2 cells. A single-dose oral PK study in mice has a T 1/2 of 3.4 h and C max of 1190 ng/mL, suggesting good availability and stability. We now propose that the N -phenyl-1-(phenylsulfonyl)-1 H -1,2,4-triazol-3-amine class may be prioritized for in vivo efficacy testing against YFV., Competing Interests: The authors declare the following competing financial interest(s): S.E. is owner, A.C.P, T.J. and T.R.L. are employees of Collaborations Pharmaceuticals, Inc. Others have no competing interests. A provisional patent on this work has been submitted., (© 2023 The Authors. Published by American Chemical Society.)

There are very few small-molecule antivirals for SARS-CoV-2 that are either currently approved (or emergency authorized) in the US or globally, including remdesivir, molnupiravir, and paxlovid. The increasing number of SARS-CoV-2 variants that have appeared since the outbreak began over three years ago raises the need for continual development of updated vaccines and orally available antivirals in order to fully protect or treat the population. The viral main protease (M pro ) and the papain-like protease (PL pro ) are key for viral replication; therefore, they represent valuable targets for antiviral therapy. We herein describe an in vitro screen performed using the 2560 compounds from the Microsource Spectrum library against M pro and PL pro in an attempt to identify additional small-molecule hits that could be repurposed for SARS-CoV-2. We subsequently identified 2 hits for M pro and 8 hits for PL pro . One of these hits was the quaternary ammonium compound cetylpyridinium chloride with dual activity (IC 50 = 2.72 ± 0.09 μM for PL pro and IC 50 = 7.25 ± 0.15 μM for M pro ). A second inhibitor of PL pro was the selective estrogen receptor modulator raloxifene (IC 50 = 3.28 ± 0.29 μM for PL pro and IC 50 = 42.8 ± 6.7 μM for M pro ). We additionally tested several kinase inhibitors and identified olmutinib (IC 50 = 0.54 ± 0.04 μM), bosutinib (IC 50 = 4.23 ± 0.28 μM), crizotinib (IC 50 = 3.81 ± 0.04 μM), and dacominitinib (IC 50 = IC 50 3.33 ± 0.06 μM) as PL pro inhibitors for the first time. In some cases, these molecules have also been tested by others for antiviral activity for this virus, or we have used Calu-3 cells infected with SARS-CoV-2. The results suggest that approved drugs can be identified with promising activity against these proteases, and in several cases we or others have validated their antiviral activity. The additional identification of known kinase inhibitors as molecules targeting PL pro may provide new repurposing opportunities or starting points for chemical optimization., Competing Interests: The authors declare the following competing financial interest(s): SE is CEO of Collaborations Pharmaceuticals, Inc. ACP is an employee at Collaborations Pharmaceuticals, Inc. Other authors have no conflicts., (© 2023 The Authors. Published by American Chemical Society.)