Balkan Journal of Medical Genetics

IN VITRO ANALYSIS OF AKR1D1 INTERACTIONS WITH CLOPIDOGREL: EFFECTS ON ENZYME ACTIVITY AND GENE EXPRESSION
Shutevska K1*, Kadifkova Panovska T1, Zhivikj Z1, Kapedanovska Nestorovska A2
*Corresponding Author: *Corresponding Author: Kristina Shutevska, Majka Tereza 47, 1000 Skopje, Republic of North Macedonia, +389 2 1326032 (142), k.sutevska@ff.ukim.edu.mk
page: 69

METHODS

Cloning, Expression, and Purification of AKR1D1 The coding sequence for the wild-type AKR1D1 (AKR1D1*1) was amplified and subcloned into the pET28b+ vector using standard molecular cloning tech- niques (23,24). E. coli BL21 (D3) cells were transformed with the prepared pET28b+_AKR1D1 vector, and over- expression of the enzyme was induced using isopropyl β-D-1-thiogalactopyranoside (IPTG). The enzyme was subsequently purified using the NGC™ Discover™ 100 Pro chromatography system (Bio Rad, USA). The clon- ing, expression, and purification methods for AKR1D1, including optimization steps, have been comprehensively described in a recent publication (25). Standard Spectrofluorometric Assay and Enzyme Studies Substrate Evaluation: The enzymatic reduction of testosterone to 5β-dihydrotestosterone (5β-DHT) was monitored using a spectrofluorometric assay. The reac- tion mixture contained AKR1D1 (7.8 µM), NADPH (15 µM), and testosterone (10 µM) in 100 mM potassium phosphate buffer (pH 6.0). The reduction was followed by measuring NADPH fluorescence, with an excitation wavelength of 340 nm and an emission wavelength of 460 nm, on an Infinite M200 Tecan spectrophotometer (Tecan, Switzerland) over a 45-minute period. For substrate evalu- ation, testosterone was replaced with either clopidogrel or 2-oxoclopidogrel at a final concentration of 50 µM. Inhibition Studies: For inhibition studies, AKR1D1 (7.8 µM) was pre-incubated with clopidogrel or 2-oxoclopidogrel (10 µM or 50 µM) in the same reaction conditions as de- scribed above. Reactions were initiated by the addition of NADPH, and fluorescence changes were monitored over 45 minutes. The concentrations of clopidogrel and 2-oxoclopidogrel (10 µM and 50 µM) were chosen based on established practices in enzymatic studies involving related AKR1C enzymes (26). The 50 µM concentration was selected to ensure sufficient substrate availability for detecting poten- tial enzymatic activity, while 10 µM and 50 µM were used in inhibition studies to observe potential dose-dependent effects. This approach allows for exploratory assessment of AKR1D1 interactions with these compounds under condi- tions aligned with prior enzymatic studies. Cell Culture Preparation and Viability Assessment HepG2 cells, chosen for their similarity to AKR1D1 expression in primary human liver cells (27), were maintained in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ atmosphere. An MTT assay was performed to determine non-toxic concentrations of clopidogrel and 2-oxoclopidogrel for expression studies. HepG2 cells were treated with con- centrations ranging from 0.1 µM to 50 µM for 48 hours, and cell viability remained >90% at all tested concentra- tions. Based on these findings 5 µM was selected as a safe and physiologically relevant concentration for further experiments. Expression Studies HepG2 cells were seeded into 12-well plates (1,600,000 cells per well) and allowed to adhere for 24 hours. Cells were then treated with 5 µM clopidogrel or 2-oxoclopidogrel for 24 hours. Following treatment, total RNA was extracted using TRI Reagent®, and RNA quality and concentration were assessed spectrophotometrically. Complementary DNA (cDNA) was synthesized using re- verse transcriptase, and 80 ng of cDNA was utilized for quantitative real-time PCR (qRT-PCR) analysis. qRT-PCR was conducted in triplicate to quantify AKR1D1 expression, with amplification specificity vali- dated by dissociation curve analysis. Absolute quantifica- tion was achieved using a calibration curve generated from serial dilutions of linearized AKR1D1 cDNA. To prepare the plasmid for the calibration curve, the AKR1D1 plasmid was linearized using the HpaI restriction enzyme and subsequently dephosphorylated with shrimp alkaline phosphatase to prevent self-ligation. The concentration of 5 µM aligns with previous studies involving AKR1D1-related compounds, such as anabolic steroids, where 5 µM approximates intracellular levels after cellular uptake (28). While plasma concentra- tions of clopidogrel and its metabolites are in the nanomo- lar range due to rapid metabolism (12), the chosen con- centration ensures sufficient exposure to detect potential regulatory effects on AKR1D1 expression in vitro (29). Statistical Analysis Statistical comparisons between treated and control groups were conducted using a two-tailed Student’s t-test. Data are presented as mean ± standard deviation (SD), with significance set at p < 0.05. All experiments were performed in triplicate unless otherwise stated.

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