ABSTRACT
This study demonstrates the inhibitory effect of 42 pyrimidonic pharmaceuticals (PPs) on the 3-chymotrypsin-like protease of SARS-CoV-2 (3CLpro) through molecular docking, molecular dynamics simulations, and free binding energies by means of molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) and molecular mechanics-generalized Born surface area (MM-GBSA). Of these tested PPs, 11 drugs approved by the US Food and Drug Administration showed an excellent binding affinity to the catalytic residues of 3CLpro of His41 and Cys145: uracil mustard, cytarabine, floxuridine, trifluridine, stavudine, lamivudine, zalcitabine, telbivudine, tipiracil, citicoline, and uridine triacetate. Their percentage of residues involved in binding at the active sites ranged from 56 to 100, and their binding affinities were in the range from -4.6 ± 0.14 to -7.0 ± 0.19 kcal/mol. The molecular dynamics as determined by a 200 ns simulation run of solvated docked complexes confirmed the stability of PP conformations that bound to the catalytic dyad and the active sites of 3CLpro. The free energy of binding also demonstrates the stability of the PP-3CLpro complexes. Citicoline and uridine triacetate showed free binding energies of -25.53 and -7.07 kcal/mol, respectively. Therefore, I recommend that they be repurposed for the fight against COVID-19, following proper experimental and clinical validation.
Subject(s)
COVID-19 Drug Treatment , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Drug Repositioning/methods , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Acetates/chemistry , Acetates/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Cytidine Diphosphate Choline/chemistry , Cytidine Diphosphate Choline/pharmacology , Drug Evaluation, Preclinical , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Uridine/analogs & derivatives , Uridine/chemistry , Uridine/pharmacologyABSTRACT
Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3' of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3' of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA, and will aid in the development of new anti-viral therapeutics.
Subject(s)
Endoribonucleases/metabolism , RNA, Viral/metabolism , SARS-CoV-2/genetics , Uridine/chemistry , Viral Nonstructural Proteins/metabolism , COVID-19/virology , Catalytic Domain/genetics , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Molecular Dynamics Simulation , Protein Multimerization/physiology , RNA, Viral/genetics , Substrate SpecificityABSTRACT
Since herpes simplex virus type 1 (HSV-1) infection is so widespread, several antiviral drugs have been developed to treat it, among which are uracil nucleosides. However, there are major problems with the current medications such as severe side-effects and drug resistance. Here we present some newly synthesized cyclic and acyclic uracil nucleosides that showed very promising activity against HSV-1 compared to acyclovir.
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Herpesvirus 1, Human/drug effects , Uridine/chemical synthesis , Uridine/pharmacology , Acyclovir/pharmacology , Animals , Antiviral Agents/chemistry , Chlorocebus aethiops , Cytopathogenic Effect, Viral/drug effects , Reference Standards , Structure-Activity Relationship , Uridine/chemistry , Vero CellsABSTRACT
Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5'-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type-infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.