At the outer part of the active site in Trypanosoma cruzi glucokinase: The role of phenylalanine 337

Carey, Shane M.; Kearns, Sean P.; Millington, Matthew E.; Buechner, Gregory S.; Alvarez, Beda E.; Daneshian, Leily; Abiskaroon, Brendan; Chruszcz, Maksymilian; D'Antonio, Edward L.

doi:10.1016/j.biochi.2023.09.014

Cited by 1 publication

(3 citation statements)

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“…We cannot explain the difference in the enzyme kinetics to the computational study at the present time, but we can suggest an alternative experimental method to study the 3-nitro-2-phenyl-2H-chromene binding site interaction of TcGlcK with compounds 1 and/or 9 (and others of the class). It appears that the best method would be X-ray crystallography since there are many published structures available in the protein databank for TcGlcK, including structures of TcGlcK-inhibitor complexes [25,27,33]. Circling back to the key residues of inhibitor interactions proposed by Omolabi et al (vide supra), we recently reported a structural superposition between the X-ray crystal structure of TcGlcK (PDB entry 7S2H) and the AlphaFold computed structure model of TcHxK (AlphaFold DB entry AF-Q8ST54-F1) [25].…”

Section: T Brucei Biological Assaymentioning

confidence: 99%

“…It appears that the best method would be X-ray crystallography since there are many published structures available in the protein databank for TcGlcK, including structures of TcGlcK-inhibitor complexes [25,27,33]. Circling back to the key residues of inhibitor interactions proposed by Omolabi et al (vide supra), we recently reported a structural superposition between the X-ray crystal structure of TcGlcK (PDB entry 7S2H) and the AlphaFold computed structure model of TcHxK (AlphaFold DB entry AF-Q8ST54-F1) [25]. This allowed for the development of a starting framework to identify the location of these residues; our laboratory is pursuing testing to determine whether they have a key role in the proposed inhibitor binding sites.…”

Section: T Brucei Biological Assaymentioning

confidence: 99%

“…The concept of limiting the flux of G6P by inhibiting either TcHxK and/or TcGlcK to cause T. cruzi cell death appears to be a good tactic and it is one on which our laboratory has focused for the past decade. TcGlcK has been recognized as a potential drug target based on various studies involving small-molecule inhibitors and natural products [17,[25][26][27]. We recently discovered that the 3-nitro-2-phenyl-2H-chromene compound class, via a high-throughput screening (HTS) campaign of 13,040 compounds, includes two antichagasic lead compounds [17].…”

Section: Introductionmentioning

confidence: 99%

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

Carey,

O’Neill,

Conner

et al. 2024

IJMS

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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.

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