Previously, we demonstrated an apparent cooperativity among interacting groups with a series of thrombin ligands that provides an improvement in the binding affinity from a nanomolar level to a picomolar level. The demonstration of this level of apparent cooperativity can be instructive for the design of super potent ligands more generally. We followed with a kinetic analysis of the kon and koff for the picomolar thrombin inhibitor, which has a benzylsulfonamide moiety that H-bonds with Gly 216 & Gly 219 as well as positioning the phenyl ring in a hydrophobic pocket, along with a series of close analogs. Higher affinity binding is usually achieved mainly by slower dissociation rates. Surprisingly the picomolar binding affinity of this inhibitor, relative to the nanomolar binding affinity of the close analogs, was mainly due to a three order of magnitude increase in kon (to a diffusion controlled binding rate) rather than a slower koff. Crystallographic and modeled studies showed that the thrombin bound conformations for these inhibitors are essentially the same. Detailed NMR studies of the ligands in aqueous buffer indicated that the accelerated kon is due to preformed conformations that are very close to the bioactive conformation within the protein cavity, along with an electrostatic directed binding into the S1 pocket. These structure-kinetic relationship results provide an important new insight into designing super potent ligands for target proteins. Overall, the results obtained from this work provide a better understanding of the underlying interactions involved in biological molecular recognition. The data obtained may be valuable in rational drug design by providing a more accurate prediction of binding affinities.

Picomolar KD ligands can be obtained by increasing the binding rate instead of decreasing the dissociation rate: Surprising structure-kinetic relationship among very similar thrombin inhibitors

249th National Meeting (2015)

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