Transition State Inhibitors

Research in the Schramm laboratory has focused on experimental and computational methods to investigate enzymatic transition states for the purpose of developing picomolar inhibitors of enzymes required for the growth and replication of malignant cells. This research is directed to providing fundamental information for enzymatic catalysis and to use information from enzymatic transition state structures to design transition state analogue inhibitors. Transition state theory predicts that analogues of natural substrates with features of the enzymatic transition states should provide unusually powerful inhibitors by capture of the energy of catalysis and its conversion to inhibitor binding energy. This experimental approach has been refined in the Schramm laboratory where transition state structures are being solved for specific enzymes. The procedure involves: (i) Selection of a target enzyme suitable for transition state analysis and important for the survival of malignant cells. (ii) Production and characterization of the enzyme mechanism to isolate the chemical step. (iii) Synthesis of substrates specifically labeled with isotopic probes. (iv) Measurement of intrinsic kinetic isotope effects (KIEs). (v) Quantum chemical calculations to locate a transition state consistent with all KIEs. (vi) Synthesis of chemically stable analogues (geometry and charge) of the transition state. (vii). Evaluation of the transition state analogues against the target enzymes and human tumor models in vitro and in mice. (vii) Based upon these findings, refinements in chemical structures are made based upon target/inhibitor complexes characterized by X-ray crystallography and NMR (in the AECC Structural Biology Shared Resource) by AECC members Steven Almo and Mark Girvin, respectively.