Novel Radiolabeled Vanilloid with Enhanced Specificity for Human Transient Receptor Potential Vanilloid 1 (TRPV1).

Transient receptor potential vanilloid 1 (TRPV1) has emerged as a promising therapeutic target. While radiolabeled resiniferatoxin (RTX) has provided a powerful tool for characterization of vanilloid binding to TRPV1, TRPV1 shows 20-fold weaker binding to the human TRPV1 than to the rodent TRPV1. We now describe a tritium radiolabeled synthetic vanilloid antagonist, 1-((2-(4-(methyl-[3H])piperidin-1-yl-4-[3H])-6-(trifluoromethyl)pyridin-3-yl)methyl)-3-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-8-yl)urea ([3H]MPOU), that embodies improved absolute affinity for human TRPV1 and improved synthetic accessibility.

Synthesis and Evaluation of Dimeric Derivatives of Diacylglycerol-Lactones as Protein Kinase C Ligands.

Protein kinase C (PKC) mediates a central cellular signal transduction pathway involved in disorders such as cancer and Alzheimer's disease. PKC is regulated by binding of the second messenger sn-1,2-diacylglycerol (DAG) to its tandem C1 domains, designated C1a and C1b, leading both to PKC activation and to its translocation to the plasma membrane and to internal organelles. Depending on the isoform, there may be differences in the ligand selectivity of the C1a and C1b domains, and there is different spacing between the C1 domains of the conventional and novel PKCs. Bivalent ligands have the potential to exploit these differences between isoforms, yielding isoform selectivity. In the present study, we describe the synthesis of a series of dimeric derivatives of conformationally constrained diacylglycerol (DAG) analogs (DAG-lactones). We characterize the derivatives in vitro for their binding affinities, both to a single C1 domain (the C1b domain of PKCδ) as well as to the conventional PKCα isoform and the novel PKCδ isoform, and we measure their abilities to cause translocation of PKCδ and PKCε in intact cells. The dimeric compound with the 10-carbon linker was modestly more effective for the isolated PKCδ C1b domain than was the monomeric compound. For the intact PKCα and PKCδ, the shortest DAG-lactone dimer had similar affinity to the monomer and affinity decreased progressively up to the 16-carbon linker. The dimeric derivatives did not cause the Golgi accumulation of PKCδ. The present results provide important insights into the development of new chemical tools for biological studies on PKC.

Discovery of N-(3-fluoro-4-methylsulfonamidomethylphenyl)urea as a potent TRPV1 antagonistic template.

A series of homologous analogues of prototype antagonist 1 and its urea surrogate were investigated as hTRPV1 ligands. Through one-carbon elongation in the respective pharmacophoric regions, N-(3-fluoro-4-methylsulfonamidomethylphenyl)urea was identified as a novel and potent TRPV1 antagonistic template. Its representative compound 27 showed a potency comparable to that of lead compound 1. Docking analysis of compound 27 in our hTRPV1 homology model indicated that its binding mode was similar with that of 1S.

Multi-Functional Diarylurea Small Molecule Inhibitors of TRPV1 with Therapeutic Potential for Neuroinflammation.

Transient receptor potential vanilloid type 1 (TRPV1), a heat-sensitive calcium channel protein, contributes to inflammation as well as to acute and persistent pain. Since TRPV1 occupies a central position in pathways of neuronal inflammatory signaling, it represents a highly attractive potential therapeutic target for neuroinflammation. In the present work, we have in silico identified a series of diarylurea analogues for hTRPV1, of which 11 compounds showed activity in the nanomolar to micromolar range as validated by in vitro biological assays. Then, we utilized molecular docking to explore the detailed interactions between TRPV1 and the compounds to understand the contributions of the different substituent groups. Tyr511, Leu518, Leu547, Thr550, Asn551, Arg557, and Leu670 were important for the recognition of the small molecules by TRPV1. A hydrophobic group in R2 or a polar/hydrophilic group in R1 contributed significantly to the activities of the antagonists at TRPV1. In addition, the subtle different binding pose of meta-chloro in place of para-fluoro in the R2 group converted antagonism into partial agonism, as was predicted by our short-term molecular dynamics (MD) simulation and validated by bioassay. Importantly, compound 15, one of our best TRPV1 inhibitors, also showed potential binding affinity (1.39 µM) at cannabinoid receptor 2 (CB2), which is another attractive target for immuno-inflammation diseases. Furthermore, compound 1 and its diarylurea analogues were predicted to target the C-X-C chemokine receptor 2 (CXCR2), although bioassay validation of CXCR2 with these compounds still needs to be performed. This prediction from the modeling is of interest, since CXCR2 is also a potential therapeutic target for chronic inflammatory diseases. Our findings provide novel strategies to develop a small molecule inhibitor to simultaneously target two or more inflammation-related proteins for the treatment of a wide range of inflammatory disorders including neuroinflammation and neurodegenerative diseases with potential synergistic effect.

2-Sulfonamidopyridine C-region analogs of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides as potent TRPV1 antagonists.

A series of 2-sulfonamidopyridine C-region derivatives of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamide were investigated as hTRPV1 ligands. Systematic modification on the 2-sulfonamido group provided highly potent TRPV1 antagonists. The N-benzyl phenylsulfonamide derivatives 12 and 23 in particular showed higher affinities than that of lead compound 1. Compound 12 exhibited strong analgesic activity in the formalin pain model. Docking analysis of its chiral S-form 12S in our hTRPV1 homology model indicated that its high affinity might arise from additional hydrophobic interactions not present in lead compound 1S.