Effective SARS-CoV-2 antiviral drugs are desperately needed. The SARS-CoV-2 main protease (Mpro) appears as an attractive target for drug development. We show that 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 and found ~50 compounds with activity against Mpro. 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, ciluprevir. narlaprevir, and telaprevir. 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.

Neurofibrillary tangles composed of aberrantly aggregating tau protein are a hallmark of Alzheimer’s disease and related dementia disorders. Recent work has shown that mammalian suppressor of tauopathy 2 (MSUT2), also named ZC3H14 (Zinc Finger CCCH-Type Containing 14), controls accumulation of pathological tau in cultured human cells and mice. Knocking out MSUT2 protects neurons from neurodegenerative tauopathy and preserves learning and memory. MSUT2 protein functions to bind polyadenosine [poly­(A)] tails of mRNA through its C-terminal CCCH type zinc finger domains, and loss of CCCH domain function suppresses tauopathy in Caenorhabditis elegans and mice. Thus, we hypothesized that inhibiting the poly­(A):MSUT2 RNA–protein interaction would ameliorate pathological tau accumulation. Here we present a high-throughput screening method for the identification of small molecules inhibiting the poly­(A):MSUT2 RNA–protein interaction. We employed a fluorescent polarization assay for initial small molecule discovery with the intention to repurpose hits identified from the NIH Clinical Collection (NIHCC). Our drug repurposing development workflow included validation of hits by dose–response analysis, specificity testing, orthogonal assays of activity, and cytotoxicity. Validated compounds passing through this screening funnel will be evaluated for translational effectiveness in future studies. This preclinical drug development pipeline identified diverse FDA approved drugs duloxetine, saquinavir, and clofazimine as potential repurposing candidates for reducing pathological tau accumulation.

Alzheimer’s disease and related disorders feature neurofibrillary tangles and other neuropathological lesions composed of detergent-insoluble tau protein. In recent structural biology studies of tau proteinopathy, aggregated tau forms a distinct set of conformational variants specific to the different types of tauopathy disorders. However, the constituents driving the formation of distinct pathological tau conformations on pathway to tau-mediated neurodegeneration remain unknown. Previous work demonstrated RNA can serve as a driver of tau aggregation, and RNA associates with tau containing lesions, but tools for evaluating tau/RNA interactions remain limited. Here we employ molecular interaction studies to measure the impact of tau/RNA binding on tau microtubule binding and aggregation. To investigate the importance of tau/RNA complexes (TRCs) in neurodegenerative disease, we raised a monoclonal antibody (mAb TRC35) against aggregated tau/RNA complexes. Here we show native tau binds RNA with high affinity but low specificity, and tau binding to RNA competes with tau mediated microtubule assembly functions. Tau/RNA interaction in vitro promotes the formation of higher molecular weight tau/RNA complexes which represent an oligomeric tau species. Co-expression of tau and poly(A)45 RNA transgenes in Caenorhabditis elegans exacerbates tau related phenotypes including neuronal dysfunction and pathological tau accumulation. TRC35 exhibits specificity for Alzheimer’s disease-derived detergent insoluble tau relative to soluble recombinant tau. Immunostaining with TRC35 labels a wide variety of pathological tau lesions in animal models of tauopathy, which are reduced in mice lacking the RNA binding protein MSUT2. TRC-positive lesions are evident in many human tauopathies including Alzheimer’s disease, progressive supranuclear palsy, corticobasal degeneration, and Pick’s disease. We also identify ocular pharyngeal muscular dystrophy as a novel tauopathy disorder where loss of function in the poly(A) RNA binding protein (PABPN1) causes accumulation of pathological tau in tissue from postmortem human brain. Tau/RNA binding drives tau conformational change and aggregation inhibiting tau mediated microtubule assembly. Our findings implicate cellular tau/RNA interactions as modulators of both normal tau function and pathological tau toxicity in tauopathy disorders, and suggest feasibility for novel therapeutic approaches targeting TRCs.

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. 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 and 32.3% in hospitalized patients age 70-79 years. 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 uM. Hits from our screen are enriched with hepatitis C NS3/4A protease targeting drugs including Boceprevir (IC50=0.95 uM), Ciluprevir (20.77 uM). Narlaprevir (IC50=1.10 uM), and Telaprevir (15.25 uM). 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.

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