HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND PHOTOAFFINITY CROSS-LINKING TO EXPLORE THE BINDING ENVIRONMENT OF NEVIRAPINE TO REVERSE-TRANSCRIPTASE OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activit y of reverse transcriptase of human immunodeficiency virus type-1. Nev irapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 1 81 and 188 of the p66 subunit of reverse transcriptase. The chromatogr aphy which was utilized to explore this binding site is described. BI- RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of ne virapine, are each crosslinked to reverse transcriptase. The use of se veral HPLC-based techniques employing different modes of detection mak es it possible to demonstrate a dramatic difference between the two az ido analogues in crosslinking behavior. In particular, by comparing HP LC tryptic peptide maps of the photoadducts formed between reverse tra nscriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and he nce exploitable for the identification of the specifically bonded amin o acid residue(s). In addition, comparison of the tryptic maps also ma tes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectros copy to identify the crosslinked amino acid residue.