Analgesic effects mediated by neuronal nicotinic acetylcholine receptor agonists: correlation with desensitization of α4ß2* receptors.

Nicotinic α4ß2* agonists are known to be effective in a variety of preclinical pain models, but the underlying mechanisms of analgesic action are not well-understood. In the present study, we characterized activation and desensitization properties for a set of seventeen novel α4ß2*-selective agonists that display druggable physical and pharmacokinetic attributes, and correlated the in vitro pharmacology results to efficacies observed in a mouse formalin model of analgesia. ABT-894 and Sazetidine-A, two compounds known to be effective in the formalin assay, were included for comparison. The set of compounds displayed a range of activities at human (α4ß2)(2)ß2 (HS-α4ß2), (α4ß2)(2)α5 (α4ß2α5) and (α4ß2)(2)α4 (LS-α4ß2) receptors. We report the novel finding that desensitization of α4ß2* receptors may drive part of the antinociceptive outcome. Our molecular modeling approaches revealed that when receptor desensitization rather than activation activitiesat α4ß2* receptors are considered, there is a better correlation between analgesia scores and combined in vitro properties. Our results suggest that although all three α4ß2 subtypes assessed are involved, it is desensitization of α4ß2α5 receptors that plays a more prominent role in the antinociceptive action of nicotinic compounds. For modulation of Phase I responses, correlations are significantly improved from an r(2) value of 0.53 to 0.67 and 0.66 when HS- and LS-α4ß2 DC(50) values are considered, respectively. More profoundly, considering the DC(50) at α4ß2α5 takes the r(2) from 0.53 to 0.70. For Phase II analgesia scores, adding HS- or LS-α4ß2 desensitization potencies did not improve the correlations significantly. Considering the α4ß2α5 DC(50) value significantly increased the r(2) from 0.70 to 0.79 for Phase II, and strongly suggested a more prominent role for α4ß2α5 nAChRs in the modulation of pain in the formalin assay. The present studies demonstrate that compounds which are more potent at desensitization of α4ß2* receptors display better analgesia scores in the formalin test. Consideration of desensitization propertiesat α4ß2* receptors, especially at α4ß2α5, in multiple linear regression analyses significantly improves correlations with efficacies of analgesia. Thus, α4ß2* nicotinic acetylcholine receptor desensitization may contribute to efficacy in the mediation of pain, and represent a mechanism for analgesic effects mediated by nicotinic agonists.

Selection of v-abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display.

Methodologies for rapidly identifying cellular protein interactions resulting in posttranslational modification of one of the partners are lacking. Here, we select for substrates of the v-abl tyrosine kinase from two protein display libraries in which the protein is covalently linked to its encoding mRNA. Successive selection cycles from a randomized peptide library identified a consensus sequence closely matching that previously reported for the v-abl tyrosine kinase. Selections from a proteomic library derived from cellular mRNA identified several novel targets of v-abl, including a new member of a class of SH2 domain-containing adaptor proteins. Upon modification, several of the substrates obtained in these selections were found to be effective inhibitors of v-abl kinase activity in vitro. These experiments establish a novel method for identifying the substrates of tyrosine kinases from synthetic and cellular protein libraries.