Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: Effects on catalytic activity

ABSTRACT Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) may confer resistance to acyclovir (ACV). Because of the high genetic polymorphism of this gene, discriminating between mutations related to resistance and mutations related to gene polymorphism can be difficult, especially when no sensitive strain has been previously isolated from the same patient. To assess the role of the mutations located at codons 51, 77, 83, and 175, previously detected in HSV-1 clinical isolates (F. Morfin, G. Souillet, K. Bilger, T. Ooka, M. Aymard, and D. Thouvenot, J. Infect. Dis. 182:290-293, 2000), in the acquisition of resistance to ACV, four mutants with site-directed mutations at these respective codons were constructed. The enzymatic activity of the proteins, produced using both a reticulocyte lysate system and a bacterial system, was evaluated using [3H]thymidine as substrate. This site-directed mutagenesis revealed that mutations at codons 51, 83, and 175 induce a loss of HSV-1 TK activity and are thus clearly involved in the acquisition of resistance to ACV. On the other hand, the mutation at codon 77 does not affect enzyme activity.

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Critical Residues in HSP70 Nucleotide Binding Domain for Challenges in Drug Design

Current Proteomics ◽

10.2174/1570164618666210413111223 ◽

2021 ◽

Vol 18 ◽

Author(s):

Mustafa Ergul ◽

Fugen Aktan ◽

Yusuf Tutar

Keyword(s):

Drug Design ◽

Nucleotide Binding ◽

Competitive Inhibitor ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Inhibition Mechanism ◽

Loss Of Function ◽

Nucleotide Binding Domain ◽

Critical Residues ◽

The Impact

Background: The association of a drug with its target protein correlates to its medicinal activity and the microenvironment plays a key role in this association. The key challenge is to identify mutations which unlikely to respond to designed drugs. Objective: Hsp70 is an anti-apoptotic factor and tumor cells overexpress Hsp70 to survive against anti-cancer agents. The impact of pathogenic mutations on Hsp70 is unknown. Elucidation of these alterations is essential to understand the molecular switch mechanism. Thus, critical spots on Hsp70 Nucleotide Binding Domain (NBD) are important since mutation-driven sensitivity may be useful in designing innovative inhibitors. Methods: ATP, AMP-PNP (non-hydrolyzable analog of ATP) along with commercially available compounds VER-155008 (ATP analog and competitive inhibitor) and MKT-077 (allosteric inhibitor of ADP bound form) were docked to Hsp70 NBD structure in silico to identify critical amino acids of inhibition mechanism. Site-directed mutagenesis of the determined critical residues along with ATP hydrolysis and luciferase refolding were performed. Wild-type and mutant Hsp70s were compared to determine the effect on protein functions in the presence or the absence of inhibitors. Results: This study identified three mutants that have a loss of function for Hsp70, which may alter the drug inhibition activity as oncogenic cells have multiple mutations. Conclusion: Two commercial inhibitors employed here that mimic ATP and ADP states respectively are not affected by these mutational perturbations and displayed effective interference for Hsp70 functions. Designing inhibitors by considering these critical residues may improve drug design and increase drug efficiency.

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