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
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RESULTS
Enzyme Substrate and Inhibition Studies
The potential role of clopidogrel and 2-oxoclopi-
dogrel as substrates or inhibitors of AKR1D1 was investigated through a set of enzymatic activity assays using spectrofluorometric measurements. The enzymatic activity was monitored by measuring NADPH fluorescence, and no significant changes were observed in the presence of either clopidogrel or 2-oxoclopidogrel, suggesting that neither compound acts as a substrate (Fig. 1) or an inhibitor (Fig. 2) of AKR1D1. The fluorescence remained unchanged across all treatment groups, supporting the conclusion that these compounds do not interact directly with AKR1D1 (p > 0.05).
Expression Studies
The impact of clopidogrel and 2-oxoclopidogrel on
AKR1D1 expression was assessed by conducting qRT-
PCR on HepG2 cells treated with each compound. The
absolute quantification approach, employing a calibration curve generated from linearized AKR1D1, revealed no statistically significant changes in AKR1D1 mRNA levels in response to treatment with either clopidogrel or 2-oxoclopidogrel compared to control samples (p > 0.05; Fig. 3). These results suggest that these compounds do not induce or repress AKR1D1 expression at the transcriptional
level in HepG2 cells.
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