An optimized protein kinase C activating diacylglycerol combining high binding affinity (K-i) with reduced lipophilicity (log P)

A small, focused combinatorial library encompassing all possible permutatio ns of acyl branched alkyl chains-small and large, saturated and unsaturated -was generated from the active diacylglycerol enantiomer (S-DAG) to help id entify the analogue with the highest binding affinity (lowest K-i) for prot ein kinase C (PK-C) combined with the minimum lipophilicity (log P). The se lected ligand (3B) activated PK-C more effectively than sn-1,2-dioctanoylgl ycerol (diC8) despite being 1.4 log units more hydrophilic. Compound 3B ind eed represents the most potent, hydrophilic DAG ligand to date. With the he lp of a green fluorescent protein (GFP)tagged PK-C alpha, 3B was able to tr anslocate the full length protein to the membrane with an optimal dose of 1 00 muM in CHO-K1 cells, while diC8 failed to achieve translocation even at doses 3-fold higher. Molecular modeling of 3B into an empty C1b domain of P K-C delta clearly showed the existence of a preferred binding orientation. In addition, molecular dynamic simulations suggest that binding discriminat ion could result from a favorable van der Waals (VDW) interaction between t he large, branched sn-1 acyl group of 3B and the aromatic rings of Trp252 ( PK-C delta) or Tyr252 (PK-C alpha). The DAG analogue of 3B in which the acy l groups are reversed (2C) showed a decrease in binding affinity reflecting the capacity of PK-C to effectively discriminate between alternative orien tations of the acyl chains.