10B12

DAG-lactones represent useful templates for the design of potent and selective C1 domain ligands for PKC isozymes. The ester moiety at the sn-1 position, a common feature in this template, is relevant for C1 domain interactions, but it represents a labile group susceptible to endogenous esterases. An interesting challenge involves replacing the ester group of these ligands while still maintaining biological activity. Here, we present the synthesis and functional characterization of novel diacylglycerol-lactones containing heterocyclic ring substituents at the sn-1 position. Our results showed that the new compound 10B12, a DAG-lactone with an isoxazole ring, binds PKCα and PKCε with nanomolar affinity. Remarkably, 10B12 displays preferential selectivity for PKCε translocation in cells and induces a PKCε-dependent reorganization of the actin cytoskeleton into peripheral ruffles in lung cancer cells. We conclude that introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC isozyme selectivity.

Interaction between immunoglobulin-like receptor glycoprotein VI (GPVI) and collagen plays a central role in platelet activation and sequent firm adhesion. Of various antithrombotic agents targeting GPVI, antibody 10B12 is of great potential to block the GPVI-collagen interaction, but less is known about 10B12 paratope and GPVI epitope.

Along the pathway in the computer strategy presented in our previous work, the 10B12/GPVI complex model was constructed through homology modeling and rigid-body docking, and the molecular dynamics (MD) simulation was used to detect the paratope residues on 10B12 and their partners on GPVI. Quantified by free and steered MD simulations, the stabilities of hydrogen bonds and salt bridges were used to rank the contributions of interface residues to binding of 10B12 and GPVI.

We predicted 12 key and seven dispensable residues in interaction of 10B12 to GPVI with present computational procedure. Besides of the 12 key residues, two are epitope residues (LYS⁴¹ and LYS⁵⁹) which had been identified by previous mutation experiments, and others, including four epitope residues (ARG³⁸, SER⁴⁴, ARG⁴⁶ and TYR⁴⁷ on GPVI) and six paratope residues (GLU¹, ASP⁹⁸, GLU¹⁰², ASP¹⁰⁷, ASP¹⁰⁸ and ASP¹¹¹ on 10B12), were newly found and also might be important for the 10B12-GPVI binding. The seven predicted dispensable residues on GPVI were had been illustrated in previous mutation experiments.

The present computer strategy combining homology modeling, rigid body docking and MD simulation was illustrated to be effective in mapping paratope on antithrombotic antibody 10B12 to epitope on GPVI, and have large potential in drug discovery and antibody research.