Insights into inhibition of heme-dependent dioxygenases

Abstract

Tryptophan 2,3-dioxygenase (TDO), along with indoleamine 2,3-dioxygenase (IDO) and indoleamine 2,3-dioxygenase-2 (IDO2) are the three enzymes that catalyse oxidation of L-tryptophan (L-Trp) in the first step of the kynurenine pathway. Despite the fact that all three catalyse the same reaction, they were detected and characterized in different chronological periods; TDO, IDO and IDO2 were discovered in 1936, 1967 and 2007 respectively. Years of studies showed that abnormal regulation of L-Trp, in the first step of kynurenine pathway, is related with several disorders, including cancer. Regardless of their distinct dissimilarities, TDO, IDO and IDO2 were all detected in various cancers, supporting tumour escape and survival. The early identification of IDO immunomodulatory action (1990s) led to intense research for the development of IDO inhibitors, but not TDO. Despite this effort, the most pharmacologically suitable IDO inhibitor, 1-methyltryptophan (1- MT), appears to be ineffective as monotherapeutic drug. Discovery of IDO2 showed that 1-MT action is not fully understood, raising questions about the biological significance of IDO2.

The ultimate goal of the current study was to address the problems outlined above. Because TDO and IDO are two druggable molecular targets, the discovery of a new class of effective inhibitors was pursued. Plate screening of ~2800 potential inhibitor compounds obtained from National Cancer Institute (NCI), USA, indicated seven promising compounds that inhibit both TDO and IDO in either nanomolar or low micromolar range. Interestingly, of these seven inhibitors, six have been identified to have cytotoxic action against several types of tumour cell lines (NCI data). NSC 26326, known as ß-lapachone, is a natural occurring quinone and the strongest inhibitor of all seven. This NCI compound inhibits both TDO and IDO with inhibition constants of ~30-70 nM and 97 ± 14 nM respectively. Like NSC 26326, NSC 36398 is another natural occurring product and the only compound that showed selectivity against TDO with inhibition constant of 16.3 ± 3.8 μM. Among the seven compounds that displayed promise as inhibitors of TDO and IDO was mitomycin C. Mitomycin C, which is an approved oncology drug and a known inhibitor of IDO (Kᵢ = 24.2 ± 1.2 μM), is also inhibitor of TDO with inhibition constant of 2.86 ± 0.03 μM. Another major goal of the current work was the discovery of isatin derivatives as inhibitors of TDO and IDO. Using the tryptophan-like structure of isatin as starting point, a number of structural modifications were carried out (structureactivity relationship (SAR)) succeeding the optimization of their inhibition activity. This new family of TDO and IDO inhibitors demonstrated inhibition potencies in the low micromolar range with 5,7-dicholoisatin to reach the nanomolar range (in the case of TDO). Halogenation of isatin and its derivatives was found to increase noticeably the inhibition potencies of these molecules by 12fold and 6fold for TDO and IDO respectively while breakdown of isatin’s pyrrolidine ring had a disastrous result on the inhibition of both enzymes. Combinations of 1-MT with either the newly-identified NCI inhibitors or the isatin derivatives were also examined. The in vitro combinations of 1-MT with either the NCI inhibitors or the isatin derivatives revealed an additive effect without though excluding the possibility of synergistic effect in vivo.

The specificity of TDO, IDO and IDO2 against the two stereoisomers of 1- MT was also investigated, with interesting results. While IDO is inhibited only by the L-isoform of 1-MT (Kᵢ= 18.0 ± 3.4 μM), IDO2 is inhibited by both 1-Me-L-Trp and 1-Me-D-Trp with inhibition constants of 306 ± 17 μM and 3419 ± 259 μM respectively. Biochemical characterization of human IDO2 was another goal of the current thesis, which completed successfully. Kinetic, redox and inhibition study of human IDO2 indicated significant differences in comparison with human IDO something which suggests the potential implication of IDO2 in an identified biological pathway (other than tryptophan catabolism function).The findings presented herein help to solve the mystery of 1-MT action, at least in vitro, give answers in regards to IDO2 function, and provide a number of new, promising inhibitors for TDO and IDO.