Biochemical and Structural Studies of Viral Proteases

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

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Abstract

We made progress in areas 1 to 4 of the goals as summarized below. Expression and purification of recombinant MPro and its analogues and methods to measure catalytic activity, inhibitor binding constant, dimer dissociation constant and global conformational stability were optimized and implemented. Studies of the MPro precursor and its autoprocessing requires a thorough understanding of the mature enzyme. Because of the low micromolar dimer dissociation constant of the wild type mature MPro, which precludes the use of diverse methods, a predominantly monomeric MPro (MProM) was engineered. The binding of a drug, used as a surrogate of a substrate, targeting the active site of MProM favors an equilibrium shift to MProM dimer formation. Upon gradually increasing the concentration of the inhibitor, the activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are described and a modified Michaelis-Menten equation accounts for the results. These results, in addition to providing conclusive evidence that the two active sites of a dimer are catalytically equivalent, also suggest that targeting the monomeric active site and/or the dimer interface to interfere with the conformational rearrangements to active dimer formation as an alternative drug design strategy against MPro. The binding thermodynamics, using isothermal titration calorimetry, of some repurposed and new inhibitors that target the active site of mature dimer and their structures are reported. The enhanced affinity of the inhibitors driven largely by enthalpy point to the importance of the reactive group in the inhibitor, which forms a covalent bond with the active site C145. For the drug nirmatrelvir, which is currently in use for the treatment of COVID-19, lacking this interaction with C145 leads to an 400-fold increase in the dissociation constant. Description of a structure of MPro bearing an active site C145A mutation in complex with an intact substrate will bring timely insights into the nature of catalysis to expedite structure/mechanism-based drug design. Progress in area 5 of the goals is, in principle, accepted for publication and will be included in the next reporting cycle. This pertains to the autoprocessing and active site reorganization upon inhibitor binding of the monomeric catalytic domain of MPro. The local active site reorganization is one of the many conformational equilibria that is tightly coupled to dimerization.