title: Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro.
abstract: SARS (severe acute respiratory syndrome) is caused by a newly discovered coronavirus. A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CL(pro) (also termed SARS-CoV 3CL(pro)). We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CL(pro) as well as a truncated form containing only the catalytic domains. The recombinant proteins have been characterized enzymatically using a fluorescently labeled substrate; their structural stability in solution has been determined by differential scanning calorimetry, and novel inhibitors have been discovered. Expression of the catalytic region alone yields a protein with a reduced catalytic efficiency consistent with the proposed regulatory role of the alpha-helical domain. Differential scanning calorimetry indicates that the alpha-helical domain does not contribute to the structural stability of the catalytic domains. Analysis of the active site cavity reveals the presence of subsites that can be targeted with specific chemical functionalities. In particular, a cluster of serine residues (Ser139, Ser144, and Ser147) was identified near the active site cavity and was susceptible to being targeted by compounds containing boronic acid. This cluster is highly conserved in similar proteases from other coronaviruses, defining an attractive target for drug development. It was found that bifunctional aryl boronic acid compounds were particularly effective at inhibiting the protease, with inhibition constants as strong as 40 nM. Isothermal titration microcalorimetric experiments indicate that these inhibitors bind reversibly to 3CL(pro) in an enthalpically favorable fashion, implying that they establish strong interactions with the protease molecule, thus defining attractive molecular scaffolds for further optimization.
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: Usman
authors.initials: U
authors.lastname: Bacha
authors.forename: Jennifer
authors.initials: J
authors.lastname: Barrila
authors.forename: Adrian
authors.initials: A
authors.lastname: Velazquez-Campoy
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Leavitt
authors.forename: Ernesto
authors.initials: E
authors.lastname: Freire
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001897:Boronic Acids / Q000494:pharmacology*; D002152:Calorimetry, Differential Scanning; D020134:Catalytic Domain; D017124:Conserved Sequence; D017934:Coronavirus / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001897:Boronic Acids; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012694:Serine