FROM HIGH-THROUGHPUT SCREENING TO CELLULAR VALIDATION: DEVELOPMENT OF A NOVEL IDO1 INHIBITOR TARGETING NEUROPATHIC PAIN.

Neuropathic pain is one of the most significant types of chronic pain, affecting about 10% of the global population. This condition may be triggered by damage to the central or peripheral nervous system. Known treatments for neuropathic pain are limited and scarce, so new pharmacological approaches are required. Results show neuropathic pain can be reduced by inhibiting the tryptophan catabolic pathway, especially the kynurenine pathway (kynpath), suggesting enzymes in this pathway could be drug targets. The first enzyme, indoleamine-2,3-dioxygenase 1 (IDO1), converts tryptophan into a product and is a key enzyme in kynpath, making IDO1 the chosen molecular target. Results: We developed a high-throughput screening (HTS) assay using fluorescence readout. This HTS assay yielded a Z’ factor above 0.75 and a signal-to-background ratio (S/N) over 120, demonstrating its suitability for HTS campaigns. The IDO1 HTS campaign involved 14,000 compounds from innovative chemical libraries, sourced from both synthetic and natural origins. We identified 89 candidates showing at least 50% IDO1 inhibition, including 76 isolated compounds (tested at 20 µM) and 13 natural product fractions from plants and deep-sea bacteria (tested at 20 µg/ml). Of these, 46 isolated compounds were confirmed by concentration-response curves with IC₅₀ < 10 µM, and 8 from natural samples had IC₅₀ < 10 µg/ml. Following hit selection, we conducted orthogonal assays (IDO1 product detection via absorbance with an alternative probe) and counter screens (coupled enzyme inhibition). Based on the selection criteria, 39 hits were confirmed as IDO1 inhibitors: 33 isolated compounds and 6 natural products. Further testing with saturation transfer difference (STD) NMR experiments confirmed 10 isolated compounds as IDO1 ligands. So far, 6 of these confirmed IDO1 inhibitors and ligands have been tested in cellular assays, confirming 2 as novel intracellular IDO1 inhibitors with IC₅₀ < 20 µM (from natural plants; three were tested, and two showed activity with IC₅₀ < 20 µg/ml). To date, bioavailability and toxicity profiles have been assessed using in silico models and literature data, leading to the selection of two novel IDO1 ligands with suitable drug profiles for proof-of-efficacy tests in animal pain models.