Exploring the Biochemical Mechanisms of Drug Resistance: A Prospective Study on Target Enzyme Inhibition

Abstract

Background: Drug resistance mediated by target enzyme alteration remains a global challenge, undermining therapeutic efficacy and increasing morbidity and mortality in diverse clinical conditions.  Objective: This study aims to investigate the biochemical mechanisms underlying drug resistance by evaluating structural, kinetic, and regulatory variations in enzyme inhibition across resistant patient samples.  Methods: A prospective observational study was conducted in the Department of Biochemistry, Naogaon Medical College, Bangladesh, from January 2023 to June 2023, involving 88 patients. Enzyme activity assays, spectrophotometry, molecular docking, and statistical analyses (ANOVA, Student’s t-test, chi-square) were applied to assess inhibitor binding affinity, catalytic turnover, and mutation frequency. Data were analyzed using SPSS v26.0.  Results: Of 88 patients, 61 (69.3%) exhibited significant resistance-associated enzyme mutations. Mean inhibitory constant (Ki) increased from 12.5 ± 2.1 μM in sensitive cases to 38.7 ± 3.4 μM in resistant cases (p < 0.001). Enzyme turnover (kcat) rose by 41.2%, with resistant samples showing 76.8 ± 5.6 s⁻¹ versus 54.4 ± 4.9 s⁻¹ in controls (p = 0.002). Binding free energy shifted from –8.6 ± 0.4 kcal/mol in sensitive to –5.2 ± 0.3 kcal/mol in resistant enzymes, reflecting reduced affinity. Standard deviation analyses confirmed low variability in resistant subgroups, strengthening statistical reliability. Stratified data indicated resistance prevalence of 72.1% in males versus 65.1% in females, though not statistically significant (p = 0.278). Mutation clustering correlated with a 27% decline in inhibition efficiency, confirming structural adaptation as a primary resistance determinant.  Conclusion: Resistance to enzyme-targeted therapy arises from increased catalytic turnover, reduced inhibitor binding affinity, and mutation-driven conformational changes, emphasizing the need for advanced drug design strategies.