Kinetics of small molecule inhibitor binding to p38 kinase

p38 mitogen-activated protein kinase (MAPK) (p38/p38-alpha/ CSBP2/RK) has b een implicated in the regulation of many proinflammatory pathways. Because of this, it has received much attention as a potential drug target for cont rolling diseases such as rheumatoid arthritis, endotoxic shock, inflammator y bowel disease, osteoporosis, and many others. A number of small molecule inhibitors of this kinase have been described, and in this paper we have us ed surface plasmon resonance to directly measure and quantitate their bindi ng to p38. Despite the relatively low molecular mass (approximate to 400 Da ) of these inhibitors, specific binding can be observed. For the two most p otent inhibitors studied, SB 203580 and RWJ 67657, dissociation constants, Kd's, of 22 and 10 nm, respectively, were obtained. These values closely ma tch the IC50 values observed in a cell-based TNF alpha release assay implyi ng that p38 plays a major role in TNF alpha release. The association and di ssociation rates for the binding of these inhibitors to p38 have also been quantitated. SB 203580 and RWJ 67657 have very similar association rates of around 8 x 10(5) M-1.s(-1), and the differences in affinity are determined by different dissociation rates. The weaker binding compounds have dissoci ation rates similar to SB 203580, but the association rates vary by an orde r of magnitude or more. The direct measurement of compounds binding to p38 may help in understanding the difference between potency and efficacy for t hese inhibitors. This in turn may yield clues on how to develop better inhi bitors.