The overall goal of the project is to employ a novel, emerging combinatorial chemistry technology to identify and develop small RNA molecules that bind with high affinity and specificity to selected TRPV channel target sites as potential therapeutic agents for treatment of nociceptive behavior. Two isoforms of the vanilloid receptor subfamily of TRP channels, TRPV1 (the capsaicin receptor) and TRPV4 (an osmoreceptor), display polymodal gating behavior with sensitivity to numerous noxious stimuli. Both channels are widely distributed in small- to medium-sized neurons of the dorsal root and trigeminal ganglia. However, traditional drug discovery efforts to identify specific antagonist are relatively slow and have had limited success, although development efforts are intensifying. The recent discovery of small RNA molecules that fold into unique 3-D structures that bind with high affinity and specificity to protein binding domains, akin to monoclonal antibodies, has opened the door to development of a new, powerful, class of pharmacotherapeutic agent. The purpose of the present study is to generate selective RNA aptamers that bind with high affinity to TRPV1 and/or TRPV4 isoforms and act as antagonist for pharmacotherapeutic applications.
Three specific aims are proposed to accomplish this goal: 1. To generate high affinity RNA aptamers with specificity for TRPV channel isoforms, TRPV1 and TRPV4. RNA aptamers will be identified from a large pool of random RNA molecules by systemic evolution, amplification, and enrichment of high affinity RNA ligands (aptamers). 2. To screen the high-affinity RNA aptamers for function as potential antagonist of the TRPV isoforms. High throughput, high-content, kinetic screening of identified RNA aptamers on calcium influx through TRPV1 or TRPV4 channels will be assessed to evaluate the potential of each identified aptamer as functional antagonist of TRPV1 and TRPV4. 3. To evaluate the potential function of the identified RNA aptamers as pharmacological tools/therapeutic agents. RNA aptamers identified from the kinetic screens will be evaluated as a pharmacotherapeutic tools using patch clamp analysis to directly assess aptamer effects on channel function. Promising aptamers will be further evaluated for therapeutic potential, in vivo, in established models of TRPV1- and TRPV4-dependent pain behavior. The studies will have far reaching impact both in providing a foundation for identification and development RNA aptamers as pharmacotherapeutic agents, and in providing new therapeutic tools for the specific treatment of nociceptive behavior, as well as for numerous other pathophysiological conditions associated with the TRPV ion channels.
The goal of the project is to use a novel, emerging, approach to generate small RNA molecules (RNA aptamers, 3-D folded) that bind with high affinity and specificity (10;26) to target sites on specific channel proteins that are associated with sensing pain (TRPV channels) (40;57;61). RNA molecules that selectively block the function of the TRPV channels and, therefore, the pain sensation, would be specifically targeted. Generating such molecules would open a new platform for development of these, and other, compounds as potential pharmacological tools and therapeutic agents for the treatment of a broad range of painful conditions associated with noxious stimuli or inflammatory states (e.g., pressure, high temperature, nerve damage/tooth decay, hyperalgesia, and exposure to noxious chemicals) (12;13;17;52;78).
| Berrout, Jonathan; Jin, Min; Mamenko, Mykola et al. (2012) Function of transient receptor potential cation channel subfamily V member 4 (TRPV4) as a mechanical transducer in flow-sensitive segments of renal collecting duct system. J Biol Chem 287:8782-91 |
| Jin, Min; Berrout, Jonathan; Chen, Ling et al. (2012) Hypotonicity-induced TRPV4 function in renal collecting duct cells: modulation by progressive cross-talk with Ca2+-activated K+ channels. Cell Calcium 51:131-9 |
| Jin, Min; Wu, Zizhen; Chen, Ling et al. (2011) Determinants of TRPV4 activity following selective activation by small molecule agonist GSK1016790A. PLoS One 6:e16713 |
| Ono, S; Yamauchi, M (1993) Collagen fibril diameter and its relation to cross-linking of collagen in the skin of patients with amyotrophic lateral sclerosis. J Neurol Sci 119:74-8 |
| Ono, S; Yamauchi, M (1992) Glutamate and aspartate are decreased in the skin in amyotrophic lateral sclerosis. Acta Neurol Scand 86:481-4 |
