STRUCTURAL REQUIREMENTS OF THE COMPETITIVE-BINDING SITE OF RECOMBINANT HUMAN INDOLEAMINE 2,3-DIOXYGENASE

The structural requirements for substrate/inhibitor binding to the act ive site of recombinant human IDO are reported in this paper. Tryptoph an analogues with substituents at the 5 or 6 positions were found to b ind to the tryptophan binding site of IDO and serve as either substrat es or inhibitors. Analogues that were more effective as substrates tha n inhibitors include 5-methyl-D,L-tryptophan 18, 5-methoxy-D,L-tryptop han 19, 5-hydroxy-L-tryptophan 21 and 6-methyl-D,L-tryptophan 24. Inte restingly, 5-methyl-D,L-tryptophan appeared to be a better substrate t han L-tryptophan. Compounds which were more active as inhibitors than substrates include 5-bromo-D,L-tryptophan 22 and 6-fluoro-D,L-tryptoph an 25. 5-Fluoro-D,L-tryptophan 23 was slightly more active as a substr ate. The most effective competitive inhibitor of recombinant human IDO was 6-nitro-L-tryptophan 26 which inhibited enzyme activity 52% at 1 mM concentrations (K-i = 180 mu M) and was not active as a substrate. The optical isomer, 6-nitro-D-tryptophan 27 did not inhibit IDO activi ty indicating that binding to the active site is stereoselective. An a nalogue with a large substituent at the 5 position, 5-benzyloxy-D,L-tr yptophan 20, was excluded from the active site. Compounds with substit uents at other positions around the indole ring or the amino acid port ion of tryptophan generally have low activity as substrates or inhibit ors although the benzofuran analogue 5 gave moderate (43%) inhibition.