Surface plasmon resonance based assay for the detection and characterization of promiscuous inhibitors.

Promiscuous binders achieve enzyme inhibition using a nonspecific aggregation-type binding mechanism to proteins. These compounds are a source of false-positive hits in biochemical inhibition assays and should be removed from screening hit lists because they are not good candidates to initiate medicinal chemistry programs. We introduce a robust approach to identify these molecules early in the lead generation process using real time surface plasmon resonance based biosensors to observe the behavior of the binding interactions between promiscuous compounds and proteins. Furthermore, the time resolution of the assay reveals a number of distinct mechanisms that promiscuous compounds employ to inhibit enzyme function and indicate that the type of mechanism can vary depending on the protein target. A classification scheme for these compounds is presented that can be used to rapidly characterize the hits from high-throughput screens and eliminate compounds with a nonspecific mechanism of inhibition.

A tetrodotoxin-resistant voltage-gated sodium channel from human dorsal root ganglia, hPN3/SCN10A.

Neuropathic pain may be produced, at least in part, by the increased activity of primary afferent neurons. Studies have suggested that an accumulation of voltage-gated sodium channels at the site of peripheral nerve injury is a primary precursory event for subsequent afferent hyperexcitability. In this study, a human sodium channel (hPN3, SCN10A) has been cloned from the lumbar 4/5 dorsal root ganglia (DRG). Expression of hPN3 in Xenopus oocytes showed that this clone is a functional voltage-gated sodium channel. The amino acid sequence of hPN3 is most closely related to the rat PN3/SNS sodium channels which are expressed primarily in the small neurons of rat DRGs. The homologous relationship between rPN3 and hPN3 is defined by (i) a high level of sequence identity (ii) sodium currents that are highly resistant to tetrodotoxin (TTX) (iii) similar tissue distribution profiles and (iv) orthologous chromosomal map positions. Since rPN3/SNS has been implicated in nociceptive transmission, hPN3 may prove to be a valuable target for therapeutic agents against neuropathic pain.