STUDIES DIRECTED TOWARD THE DEVELOPMENT OF A TRANSITION STATE ANALOG INHIBITOR FOR TRYPTOPHAN DIOXYGENASE

The enzyme tryptophan dioxygenase (E.C.1.13.11.11), found in mammalian liver, catalyzes the initial, irreversible step in the metabolism of the amino acid L-tryptophan i.e. that of addition of molecular oxygen. The enzyme has been extensively studied over the last 20 years because it provided a good model for both the mechanism of an oxygenase enzyme and for regulatory control. Recently, overactivity of this enzyme has been implicated in the genesis of spontaneous bladder cancer in humans. An inhibitor of the catalytic activity of this enzyme would be useful as an effective metabolic regulator in those patients exhibiting abnormally elevated urinary tryptophan metabolites. A most effective enzyme inhibitor is that of the transition state analog type and a structure-activity study was undertaken for the development of such an inhibitor. The sulfur, sulfone, dihydrosulfone, and dihydro-tryptophan analogs of tryptophan were synthesized and tested as tryptophan dioxygenase inhibitors. The mechanism of the extremely potent inhibitor of tryptophan dioxygenase, 5-hydroxy-L-tryptophan (5-HTP) was also investigated. Due to apparent tight fit of the pyrrole ring of tryptophan into the enzyme\u27s active site the bulky sulfone and dihydrosulfone analogs were very poor inhibitors, K(,i) \u3e 25mM; K(,m) = 1mM. However, the sulfur analog of tryptophan was bound by the enzyme almost as well as the substrate; K(,i) = 1.4mM, indicating the lack of a necessity of a nitrogen atom in the pyrrole ring for substrate-like recognition. The dihydro-L-tryptophan analog was found to bind like a transition state, K(,i) = .13mM, due to its structural resemblance to the initial oxygen-tryptophan adduct, a hydroperoxyindolenine, predicted by the published photochemical model for the enzyme. The powerful inhibition of tryptophan dioxygenase by 5-HTP was due, as we theorize, to the ability of the enzyme to initially oxygenate 5-HTP as substrate followed by rapid rearrangement of the initially formed indolenine intermediate to its quinoneimine tautomer. This quinoneimine functions as a mechanistic poison of tryptophan dioxygenase since it cannot rearrange to product nor can it readily diffuse form the enzyme\u27s active site due to the enzyme\u27s high affinity for the L-alanyl and hydro-peroxy groups present. The ideal, stable transition state analog inhibitor of tryptophan dioxygenase would therefore possess the ring system and L-alanyl side chain of dihydro-L-tryptophan and a stable geometric analog to the hydroperoxy group present in the oxygen-5-HTP adduct