Small molecule inhibitors of the SARS-CoV Nsp15 endoribonuclease

J Ortiz-Alcantara¹, K Bhardwaj², S Palaninathan¹, M Frieman³, RS Baric³, CC Kao<sup>2

1</sup>Department of Biochemistry and Biophysics, Texas A&M University College Station, TX, USA; ²Department of Molecular Cellular Biochemistry, Indiana University, Bloomington IN, USA; ³Department of Epidemiology, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Abstract: The severe acute respiratory syndrome (SARS) virus encodes several unusual RNA processing enzymes, including Nsp15, an endoribonuclease that preferentially cleaves 3’ of uridylates through a ribonuclease A (RNase A)-like mechanism. Crystal structures of Nsp15 confirmed that the Nsp15 active site is structurally similar to that of RNase A. These similarities and our molecular docking analysis lead us to hypothesize that previously characterized RNase A inhibitors will also inhibit the SARS-CoV Nsp15. Benzopurpurin B, C-467929, C-473872, N-306711, N-65828, N-103019 and congo red were tested for effects on Nsp15 endoribonuclease activity. A fluorescence assay revealed that the IC₅₀ values for inhibiting endoribonuclease activity were between 0.2 µM and 40 µM. These compounds were demonstrated to bind SARS-CoV Nsp15 by a differential scanning fluorimetry assay. Benzopurpurin B also inhibited the endoribonuclease activities of the Nsp15 orthologs from two other coronaviruses: mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV). Benzopurpurin B, C-473872, and congo red reduced infectivity of MHV in L2 cells by 8- to 26- fold. The more effective drugs caused a decrease in MHV RNA accumulation. All three compounds reduced the infectivity of the SARS-CoV in Vero cells.

Keywords: SARS, endoribonuclease, small molecule inhibitors