However, conditions such as those commonly applied to HTS for enzyme inhibitors often violate this approximation and make interpretations based on the MM equation for initial reaction velocity less reliable. Furthermore, such assays are often associated with a high enough level of substrate turnover to render the phenomenon of product inhibition significant, thus complicating interpretation of observed inhibition further. Consequently, interpretation of data from experiments such as HTS, as well as the design of HTS assay conditions, should ideally be founded on progress curve analysis. Since the MM rate law cannot be analytically integrated to explicitly express product concentration as a function of time and in terms of kcat and Km, this has to be achieved by numeric approaches. Due to these issues, a tool in spreadsheet format specifically designed to simplify the analysis and design of HTS assays has been developed. The tool is simple to use and only requires knowledge in standard enzyme kinetics. It provides comparative analysis of the progress of uninhibited versus inhibited reactions for common inhibitory mechanisms and takes reaction reversibility and enzyme half-life into account. Reactions are simulated in response to adjustment of kinetic parameters and key data are automatically deduced. Furthermore, as for any method relying on progress curve analysis, the linearity and range of the response must be accounted for when interpreting and comparing experimental data with simulated data. To further evaluate the validity of the simulations, progress curves were also collected for another enzyme system, peptidolysis by presequence peptidase, with and without bestatin as an inhibitor. Kinetic parameters were derived by leastsquares model fitting. As a comparison, an experiment to determine kinetic parameters with initial reaction rates at increasing substrate concentrations was also performed. Curve fitting gave a good data-to-model agreement, for both progress curves and the initial reaction rate experiment, and kinetic parameters derived with the two methods were very similar. A tool for the simulation and comparative analysis of enzymatic progress curves for common types of inhibition has been developed. The tool can be downloaded as supplemental material or obtained from the author. The tool provides AbMole BioScience kinase inhibitors accurate simulation of experimental progress curves – given that the enzyme system under study can be approximated by the underlying model, as in any simulation approach. Reaction parameters and concentrations can be adjusted to directly R428 1037624-75-1 observe the effects on displayed progress curves and essential data are deduced and clearly presented. The tool is particularly intended to support experimental design and to facilitate interpretation of data obtained in end-point assays in HTS for enzyme inhibitors. In these processes the tool can be used to: study the effect of reaction conditions on the choice of observation window, tune reaction condition in favor of a particular type of inhibition, investigate the amount of substrate turn-over that can be allowed to increase the assay signal without severely affecting observed inhibition, adapt assay conditions to an enzyme with pronounced product inhibition, or to guide the selection of hit cut-off criteria while accounting for product inhibition, reaction reversibility, and substrate turn-over.