[Part 1] The DAG-responsive C1 domain as a drug development target [Part 2] Structure–Activity Relationship Studies of a Potent TRPV1 Antagonist

Abstract

[Part 1] Diacylglycerol (DAG) is a lipid second messenger produced through hydrolysis of phosphatidylinositol 4,5-bisphosphate. Increased levels of DAG are translated into cellular signaling via signaling proteins including protein kinase C (PKC), RasGRPs, chimaerin, and protein kinase D which have a C1 domain, which functions as a DAG recognition motif. Based on the significant biological roles of the proteins, the development of selective agents capable of discriminating between them would have great therapeutic significance. Previously, we have demonstrated that DAG-lactones, which are rigidified structures derived from endogenous DAG, function as DAG analogs to potently bind to the C1 domain. Owing to the different lipid requirements of each C1-domain containing protein, the sets of side chains (R1 and R2) function as chemical zip codes in controlling biological activity and protein selectivity by creating a unique microenvironment surrounding the binding site. The overarching theme of this work is design, synthesis, and biological evaluation of DAG-lactone analogs varying in their side chains that have provided inspiration for selective interaction of specific C1-domain proteins based on their known, potent binding affinities. Firstly, a series of DAG-lactones having linoleic acid derivatives were evaluated their selectivity for PKC epsilon under standard lipid conditions (100% phosphatidylserine) as well as in the presence of a nuclear membrane mimetic lipid mixture. We find that selectivity tended to be enhanced in the presence of the nuclear membrane mimetic lipid mixture and, for our lead compound, report a selectivity of 32-fold. Secondly, DAG lactones having α-arylidene (R2) side chain were synthesized and evaluated its selective binding to RasGRP3 as compared to PKC isozymes (PKCα and PKCε). From the preliminary results, the α-methylindolidene DAG-lactone scaffold was selected and modified to enhance the discrimination between PKCs and RasGRPs. This study clearly shows that seemingly small structural modifications in the hydrophobic regions of these DAG analogs strongly contributes to their specific membrane interaction.

[Part 2]

A series of 2-substituted 4-(trifluoromethyl)benzyl C-region analogues of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1 antagonism. The analysis indicated that the phenyl C-region derivatives exhibited better antagonism than those of the corresponding pyridine surrogates for most of the series examined. Among the phenyl C-region derivatives, the two best compounds 43 and 44S antagonized capsaicin selectively relative to their antagonism of other activators and showed excellent potencies with Ki(CAP) = 0.3 nM. These two compounds blocked capsaicin-induced hypothermia, consistent with TRPV1 as their site of action, and they demonstrated promising analgesic activities in a neuropathic pain model. The docking study of 44S in our hTRPV1 homology model indicated that its binding mode was similar with that of its pyridine surrogate in the A- and B-regions but displayed a flipped configuration in the C-region.