This is important since the rate of drug-target dissociation can occur on the same time scale as clearance of the drug from the body, and thus even small changes in residence time can have a dramatic effect on designing dosing regimens that widen the therapeutic window.5,6 Consequently, the structural and mechanistic factors that control the lifetime of a drug-target complex must be fully understood to deploy the power of drug-target kinetics in selecting and optimizing drug leads. combination of enzyme kinetics and X-ray crystallography to generate a structure-kinetic relationship (SKR) for time-dependent binding. We show that the triazole motif slows the rate of formation for the final drug-target complex by up to three orders of magnitude. In addition, we YH239-EE YH239-EE identify a novel inhibitor with a residence time on InhA of 220 min which is 3.5-fold longer than that of the INH-NAD adduct formed by the tuberculosis drug, isoniazid. This study provides a clear example in which the lifetime of the drug-target complex is controlled by interactions in the transition state for inhibitor binding rather than the ground state of the enzyme-inhibitor complex, and demonstrates the important role that on-rates can play in drug-target residence time. Graphical Abstract Introduction Drug-target interactions often occur under conditions where the concentration of the drug or target is not constant, and thus both the thermodynamics and kinetics of drug binding are required to fully account for time-dependent changes in target occupancy in the human YH239-EE body.1C4 However, often only equilibrium parameters such as IC50 values are used for selecting and optimizing drug candidates, neglecting the potential contribution that kinetic selectivity can make to the therapeutic index. This is important since the rate of drug-target dissociation can occur on the same time scale as clearance of the drug from the body, and thus even small changes in residence time can have a dramatic effect on designing dosing regimens that widen the therapeutic window.5,6 Consequently, the structural and mechanistic factors that control the lifetime of a drug-target complex must be fully understood to deploy the power of drug-target kinetics in selecting and optimizing drug leads. Whilst there is growing IKK-gamma (phospho-Ser376) antibody realization that drug-target binding kinetics can play a major role in improving the therapeutic window, several barriers exist including the lack of extensive structure-kinetic relationships (SKR) to guide the development of compounds with altered drug-target residence times, and insufficient knowledge of the molecular factors that control the lifetime of the drug target complex. InhA, the FabI enoyl-ACP reductase from was cloned into either a pET15b or pET23b plasmid (Novagen) and transformed into BL21(DE3) pLysS cells. Following protein expression, the cells were lysed and the InhA protein was purified via His-bind Ni2+C NTA affinity chromatography (Invitrogen) and size exclusion chromatography. The purified protein was 97% pure by SDS-PAGE and was stored at ?80 C in storage buffer consisting of either 20 mM or 30 mM PIPES pH 6.8, containing 150 mM NaCl and 1 mM EDTA. Progress curve analysis Progress curve kinetics were performed on a Cary 100 UV-Vis spectrophotometer (Varian) at 20 or 25 C as described previously but with minor modifications.28 Briefly, the reaction velocities were measured by monitoring the oxidation of NADH to NAD+ at 340 nm. The enzyme reaction was initiated by adding 100 nM enzyme to C8-CoA (340 M), NADH (250 M), NAD+ (200 M), DMSO (2% v/v), inhibitor (0 C 20 M) and 8% glycerol in 30 mM PIPES pH 6.8 buffer containing 150 mM NaCl and 1 mM EDTA. The reaction was monitored until the progress curve became linear, suggesting the steady state had been reached. A high concentration of substrate and low YH239-EE concentration of enzyme were used to minimize substrate consumption and ensure that progress curves were linear in the absence of inhibitor. The progress curves were analyzed using the Morrison & Walsh integrated rate equation: and kobs from which vales for Kiapp and Ki*app together with their standard errors were calculated using Equations 3 and 4. The values for Kiapp and Ki*app were constrained within the limits set by the standard error and provided input values for global fitting of all data sets to Equations 1C4 which in turn resulted in the optimum values for Kiapp, Ki*app and k6 together with their standard errors. Values for k5 and kon, overall were subsequently determined using Equations 5C8 where it is assumed that k6?k4,k5.41 =?was grown to early mid-log phase in Middlebrook 7H9 media supplemented with 10% OADC and 0.05% Tween 80. Compounds were two fold serially diluted in triplicate in 96-well round bottom microtiter plates. Media.