Resiniferatoxin-Amide and Analogues as Ligands for Protein Kinase C and Vanilloid Receptors and Determination of Their Biological Activities as Vanilloids

Abstract: The naturally occurring diterpene resiniferatoxin (RTX) is an ultrapotent analogue of capsaicin. Acting on polymodal afferent neurons, RTX induces a generally similar pattern of responses as does capsaicin. However, the two compounds, as well as other vanilloid derivatives, display different relative potencies for different responses. In the present study, we examined the vanilloid-like activities of two new derivatives, the amide analogue of RTX and phorbol 12,13-dibenzoate 20-homovanillylamide. Structurally, RTX-amide resembles capsaicin more closely than does RTX, and after cleavage of the amide bond the resulting amine would be predicted to not bind to protein kinase C in contrast to resiniferonol 9,13,14-orthophenylacetate, the parent diterpene of RTX. In contrast to our expectations the binding potency of the RTX-amide for the vanilloid receptor present in rat spinal cord was 450-fold lower than that of RTX (K_i values for the RTX-amide and RTX were 10.4 ± 0.7 nM and 23.1 ± 3.2 pM, respectively). In the case of phorbol 12,13-dibenzoate 20-homovanillylamide, there was a further loss of affinity for the vanilloid receptor compared with RTX; nonetheless, the K_i (8.56 ± 0.61 µM) was comparable with that of capsaicin (5.31 ± 0.37 µM). Computer fitting of the binding data yielded Hill coefficient values of 2.25 ± 0.03, 2.33 ± 0.03, and 1.84 ± 0.05 for RTX, RTX-amide, and phorbol 12,13-dibenzoate 20-homovanillylamide, respectively, indicating that both new compounds induced apparent positive cooperativity among vanilloid binding sites. We found that the RTX-amide was also 20- and 300-fold less potent than RTX in inducing chemogenic pain and hypothermia, respectively. The affinities of the compounds for protein kinase C-α were evaluated by competition of [³H]phorbol 12,13-dibutyrate binding. Replacement of the C-20 hydroxyl group in phorbol 12,13-dibenzoate by an amine led to a 750-fold drop in binding affinity, and the conversion of the phorbol 12,13-dibenzoate 20-amine to the amide resulted in a further 60-fold drop in binding activity (K_i values for phorbol 12,13-dibenzoate, phorbol 12,13-dibenzoate 20-amine, and phorbol 12,13-dibenzoate 20-homovanillylamide were 0.96 ± 0.01, 720 ± 80, and 43,500 ± 4,060 nM, respectively). The K_i of the RTX-amide for protein kinase C-α was >100 µM, whereas RTX and the parent alcohol resiniferonol 9,13,14-orthophenylacetate yielded K_i values of 1.49 ± 0.19 µM and 36.5 ± 1.3 nM, respectively.