Discovery of Strong 3-Nitro-2-Phenyl- 2H-Chromene Analogues as Antitrypanosomal Agents and Inhibitors of Trypanosoma cruzi Glucokinase

Abstract

Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC₅₀ values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC₅₀ determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.

Keywords: 3-nitro-2-phenyl-2H-chromene analogues; Chagas disease; drug discovery; glucokinase; glucose kinases; hexokinase; human African trypanosomiasis; inhibitors; neglected tropical diseases.

Figure 1

Library of 3-nitro-2-phenyl-2H-chromene analogues (1–13). For systematic names and SMILES codes of the compounds, please refer to Tables S1 and S2 in the Supplementary Information section, respectively.

Figure 2

Flowchart diagrams with assay checkpoints (trapezoids) and compound filters (dashed rectangles) to narrow down top-performing compounds from a 13-compound library of 3-nitro-2-phenyl-2H-chromene analogues (cylinders). Compounds were screened against (a) TcGlcK/T. cruzi parasites and (b) T. brucei parasites. N indicates the number of times an assay was performed; N = 1 for the TcGlcK primary screen, N = 4 for the confirmatory assay, and N = 3 for the anti-T. cruzi and anti-T. brucei activity assays.

Figure 3

Enzyme-inhibitor kinetics for TcGlcK and compounds 1 (GLK2-003), 9 (GLK2-004), 4, and 11 in order to determine K_i values. Panels (a,c,e,g) represent Dixon plots of 1/V as a function of inhibitor concentration. Values calculated from the extrapolation of the intersection of the three lines with the X-axis were as follows: K_i = 6.2 ± 1.2 μM for compound 1, K_i = 12.3 ± 5.5 μM for compound 9, K_i = 5.5 ± 1.8 μM for compound 4, and K_i = 20.0 ± 4.3 μM for compound 11. Panels (b,d,f,h) represent a second set of Dixon plots characterized as [d-Glc]/V as a function of inhibitor concentration, which were used to determine the mode of inhibition. The modes of inhibition were determined as uncompetitive inhibition for compound 1, mixed-mode inhibition for compounds 9 and 4, and noncompetitive inhibition for compound 11. The inhibitor concentrations ranged from 0.0–10.0 μM at three different substrate concentrations, 0.4 mM d-Glc (red circles), 0.6 mM d-Glc (blue squares), and 0.8 mM d-Glc (black triangles). In all panels, one representative experiment is displayed but three independent experiments were performed. Dixon plot graphs corresponding to an individual compound are from the same experiment.

Figure 4

The in vitro dose-response curves of compound activity on T. cruzi (Tulahuen strain) infective form co-cultured in NIH-3T3 fibroblasts (blue) in comparison to NIH-3T3 fibroblast cytotoxicity (black) for compounds (a) 1, (b) 4, (c) 9, and (d) 11.

Figure 5

The in vitro dose-response curves of compound activity on T. brucei brucei (427 strain) bloodstream form (blue) in comparison to NIH-3T3 fibroblast cytotoxicity (black) for the high-performing compounds (a) 3, (b) 4, (c) 5, and (d) 6.

Figure 6

Structure–activity relationships observed from the 3-nitro-2-phenyl-2H-chromene scaffold representing the thirteen analogues. Substituent effects are highlighted for (a) TcGlcK inhibition and (b) in vitro growth inhibition of trypanosomatid parasites T. cruzi and T. brucei. An atom numbering system is provided for the scaffold in blue.