Development of a cellular mimetic protein kinase assay and a novel methodology for determining the mode of inhibition for multisubstrate enzymes, and rational design and synthesis of protein kinase inhibitors

The protein kinases are enzymes that catalyze the transfer of phosphate from ATP to Ser, Thr, or Tyr within proteins and are intimately involved in signal transduction and the regulation of many cell processes. Since aberrant activity of a variety of protein kinases has been linked to many disease states, specific inhibitors could have a number of therapeutic applications. Based on x-ray crystal structures and molecular modeling technique, we designed inhibitor functionalities at the catalytic site to enhance interactions to the active site residues. The hybrid substrate/product inhibitor design approach was applied, and both solution-phase and solid-phase peptide syntheses were successfully employed.; The structure-based rational design and intracellular mimetic testing of reversible protein kinase inhibitors is a relatively undeveloped area compared with the molecular biology and biochemistry of the kinase field. We found that not only the potency but also the rank order of protein kinase inhibitors is sensitive to the physical chemical conditions of the assay system. Therefore, we have developed our Cellular Mimetic Assay Conditions to simulate the microenvironment inside cells by mimicking the overall intracellular aqueous physical chemistry as closely as possible to reliably predict the potential for inhibitor activity inside cells. The reason for the sensitivity of the results to the assay conditions is that the nature and concentration of the components (e.g. free Mg2+, ATP, ADP) determines the predominant form(s) of the enzyme available for molecular recognition of the inhibitor and also affects the free energy change associated with leaving the aqueous environment to enter the active site (particularly ionic strength/osmolality).; To determine which form of the protein kinase a given inhibitor binds most tightly to, we have developed the novel STAIRe method and identified the tightest binding form for several inhibitors against kinases, which can present themselves in multiple forms to the inhibitors as a class of multisubstrate enzymes. This information can be critical for "structure-based design" in that it can now be determined if the structure/activity data obtained with a series of inhibitors is based upon a single form of the enzyme, and if that form is the structure with which the molecular modeling studies were carried out.