Design of Inhibitors of Orotidine Monophosphate Decarboxylase Using Bioisosteric Replacement and Determination of Inhibition Kinetics

Abstract

Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and KM = 5 microM) and the molar enthalpy (DeltaH(app) = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 microM and 29 microM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.

Hydrolytic Mechanism of OXA-58 Enzyme, a Carbapenem-hydrolyzing Class D β-Lactamase from Acinetobacter baumannii

“…Determination of the Kinetic Parameters by Isothermal Titration Calorimetry-Isothermal titration calorimetry (ITC) experiments were conducted on a MicroCal VP-ITC instrument (GE Healthcare) using the protocol developed by Kotra and co-workers (22). The kinetic measurements were performed at 25°C.…”

“…Analysis of raw data were done using Origin version 7.0 software and as described previously (22). The data points were corrected for the heat of dilution of the enzyme and mixing.…”

“…Adaptation of ITC for Measurement of Enzyme KineticsThe adaptation of the ITC protocol developed by Kotra and co-workers (22) was an important choice that allowed accurate determination of steady state kinetics parameters of OXA-58 ⌬SP against imipenem and other ␤-lactams with low extinction coefficients. The advantages of this method are high sensitivity, direct rate measurement, and easy data analysis.…”

“…Equations 1-2 were used to convert thermal power into reaction rates (Fig. 2B), which were then plotted against substrate concentration (Equation 3) (22).…”

Determination of the amino acid sequence requirements for catalysis by the highly proficient orotidine monophosphate decarboxylase

“…These values are similar to those obtained previously for the E. coli enzyme as well as the Mt and P. falciparum ODCases while K m is somewhat higher (sixfold) than observed for the yeast enzyme. 9,11,29,32 The D71C enzyme exhibited an 84-fold decrease in k cat and a 1071-fold decrease in catalytic efficiency compared with the wild-type enzyme (Table I). This indicates that mutants exhibiting as low as 0.1% of wild-type activity are recovered in the plasmid Figure 5.…”