Substrate activity screening with kinases: discovery of small-molecule substrate-competitive c-Src inhibitors

Abstract

Substrate-competitive kinase inhibitors represent a promising class of kinase inhibitors, however, there is no methodology to selectively identify this type of inhibitor. Substrate activity screening was applied to tyrosine kinases. By using this methodology, the first small-molecule substrates for any protein kinase were discovered, as well as the first substrate-competitive inhibitors of c-Src with activity in both biochemical and cellular assays. Characterization of the lead inhibitor demonstrates that substrate-competitive kinase inhibitors possess unique properties, including cellular efficacy that matches biochemical potency and synergy with ATP-competitive inhibitors.

Keywords: drug discovery; inhibitors; kinases; medicinal chemistry; substrate activity screening.

Figure 1

Synergy studies of combinations of substrate-competitive inhibitor 12 with ATP-competitive inhibitors PP2 or PP5. IC₃₅ concentrations are dosed individually and in combination. The dotted line denotes predicted additivity [(eA+eB)-(eA*eB)] of 12 + PP2 (or PP5).^[25] A higher level of inhibition than the predicted additivity indicates synergism.

Scheme 1

Overview of SAS methodology for protein kinases.

Chart 1

Phenol substrates of c-Src and their corresponding K_M values.

Chart 2

Conversion to inhibitors using non-phosphorylatable surrogates of phenol.

Chart 3

Optimized substrate-competitive c-Src inhibitors.