Parallel discovery of selective and dual inhibitors of tryptophan dioxygenases IDO1 and TDO2 with a newly-modified enzymatic assay

Abstract

The expression of tryptophan catabolising enzyme indoleamine 2,3-dioxygenase 1 (IDO1) or tryptophan 2,3-dioxygenase 2 (TDO2) in cancers is associated with suppressed immunity and poor patient prognosis. Results from human clinical trials of IDO1 inhibitors have been disappointing. There is now a strong interest in the development of TDO2-selective or dual IDO1/TDO2 inhibitors that may surpass IDO1 inhibitors by providing broader efficacy and blocking constitutively-expressed hepatic TDO2. To expedite the discovery of novel TDO2-specific and dual inhibitors, an assay that enabled the efficient and accurate measurement of the inhibitory activity of compounds against both IDO1 and TDO2 enzymes, concurrently in the same experiment was established to screen 5,682 compounds that included the National Cancer Institute Diversity set 5, for inhibition of IDO1 and TDO2 activity. This screen identified 82 compounds that inhibited either IDO1, TDO2 or both enzymes > 50% at 20 µM. Thirty Pan Assay Interference compounds were removed from the list and the IC₅₀ of the remaining 52 compounds against IDO1 and TDO2 was subsequently determined using the newly-developed concurrent assay. Ten compounds were confirmed as dual IDO1/TDO2 inhibitors having IC₅₀ values under 50 µM against both enzymes and within 2-fold of each other. Six compounds with IC₅₀ values between 1.39 and 8.41 µM were identified as potential TDO2-selective leads. The use of this concurrent protocol is anticipated to expedite the discovery of novel leads for dual and selective inhibitors against IDO1 and or TDO2 and speed the evaluation of novel analogues that will ensue.

Keywords: Cancer-immunotherapy; Drug discovery; IDO1; Inhibitor-screening; TDO2; Tryptophan dioxygenases.