Cemtirestat, 2-(3-thioxo-2H-[1,2,4]triazino[5,6-b]indol-5(3H)-yl) acetic acid, was recently designed and patented as a highly selective and efficient aldose reductase inhibitor endowed with antioxidant activity (Stefek et al. 2015; Šoltesova Prnova et al. 2020). Presently the compound is under complex preclinical evaluation as a promising drug to treat diabetic complications. The aim of the present study was to compare two methods of determination of cemtirestat inhibition constant (Ki).
At the very beginning, Michaelis-Menten constant (Km) value for aldose reductase mediated reduction of the glyceraldehyde substrate, was determined. Then cemtirestat was subjected to inhibition assessment by two approaches. In the first way, concentrations of inhibitor for 50% inhibition (IC50) of rat lens aldose reductase by cemtirestat were determined for three different concentration of the glyceraldehyde substrate, S<>Km. In the second way, inhibition constant Ki was determined by applying enzyme kinetics approach with Lineweaver-Burk plot. To relate the IC50 values to the inhibition constant Ki, the method based on modified Cheng and Prusoff equations (Brandt et al., 1987), was used. To conclude, both approaches revealed an uncompetitive type of aldose reductase inhibition by cemtirestat and both methods yielded comparable Ki values.
Cemtirestat, kyselina 2-(3-tioxo-2H-[1,2,4]triazino[5,6-b]indol-5(3H)-yl) octová, bol nedávno navrhnutý a patentovaný ako vysoko selektívny a efektívny inhibítor aldózareduktázy s antioxidačnou aktivitou (Štefek et al. 2015; Šoltésová Prnová et al. 2020). V súčasnosti sa zlúčenina nachádza v štádiu komplexného predklinického hodnotenia ako potencionálne liečivo voči diabetickým komplikáciám. Cieľom tejto štúdie bolo porovnať dve metódy stanovenia inhibičnej konštanty (Ki) cemtirestatu.
Na začiatku bola stanovená Michaelis-Mentenovej konštanta (Km) pre aldózareduktázu, ktorá ako substrát využíva glyceraldehyd. Následne sa pomocou dvoch rôznych prístupov hodnotila inhibícia aldózareduktázy cemtirestatom. V prvom prípade sa koncentrácia inhibítora pre 50% inhibíciu (IC50) aldózareduktázy pochádzajúcej z potkaních šošoviek stanovila pre tri rôzne koncentrácie substrátu, glyceraldehydu, a to pre S<>Km. V druhom prípade bola inhibičná konštanta Ki určená aplikovaním metód enzýmovej kinetiky a grafu Lineweaver-Burka. Stanovené hodnoty IC50 a inhibičnej konštanty Ki boli uvedené do vzťahu pomocou modifikovaných Chengových a Prusoffových rovníc (Brandt et al. 1987). Prostredníctvom oboch prístupov sa určil akompetitívny typ inhibície aldózareduktázy cemtirestatom a obe metódy poskytli porovnateľné hodnoty Ki.
This work was supported by VEGA 2/0005/2018 and APVV-15-0455.
Táto práca bola podporená projektami VEGA 2/0005/2018 a APVV-15-0455.
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