Unveiling the synergistic power of 3-hydrazinoquinoxaline-2-thiol and vancomycin against MRSA: An in vitro and in silico evaluation

Ohood S   Alharbi; Mohanned Talal   Alharbi; Mazen A. Ismail; Ahmad M  Sait; Mohammed  Mufrrih; Wafaa   Alhazmi; Bandar Hasan  Saleh; Manal A.  Zubair; Noha A. Juma; Noof R. Helmi; Hatoon A.  Niyazi; Hanouf A.  Niyazi; Hussam  Daghistani; Taghreed  Shamrani; Waiel S. Halabi; Abdalbagi Alfadil; Hisham N. Altayb; Karem Ibrahem

doi:10.17305/bb.2025.11886

Authors

Ohood S Alharbi Department of Microbiology and Parasitology, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia
Mohanned Talal Alharbi Department of Basic Medical Sciences, College of Medicine, University of Jeddah, Jeddah, Saudi Arabia https://orcid.org/0009-0005-0848-5465
Mazen A. Ismail Department of Medical Education, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia https://orcid.org/0000-0001-6237-1841
Ahmad M Sait Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia; Regenerative Medicine Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia https://orcid.org/0000-0002-1698-5444
Mohammed Mufrrih Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia; Special Infectious Agents Unit BSL-3, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia https://orcid.org/0000-0002-6834-2379
Wafaa Alhazmi Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia https://orcid.org/0000-0001-5322-0953
Bandar Hasan Saleh Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia; Department of Clinical Microbiology Laboratory, King Abdulaziz University Hospital, Jeddah, Saudi Arabia https://orcid.org/0000-0002-3205-2986
Manal A. Zubair Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia https://orcid.org/0009-0002-5937-5336
Noha A. Juma Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
Noof R. Helmi Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
Hatoon A. Niyazi Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
Hanouf A. Niyazi Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
Hussam Daghistani Regenerative Medicine Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia; Department of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University Jeddah, Saudi Arabia https://orcid.org/0000-0001-8491-8350
Taghreed Shamrani Department of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University Jeddah, Saudi Arabia; Food, Nutrition and Lifestyle Unit, King Fahd Medical Research Centre, King Abdulaziz University, Jeddah, Saudi Arabia
Waiel S. Halabi Department of Optometry, Faculty of Applied Medical Sciences, University of Jeddah, Saudi Arabia https://orcid.org/0000-0002-6198-5566
Abdalbagi Alfadil Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia; Centre of Research Excellence for Drug Research and Pharmaceutical Industries, King Abdulaziz University, Jeddah, Saudi Arabia
Hisham N. Altayb Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
Karem Ibrahem Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia; Department of Clinical Microbiology Laboratory, King Abdulaziz University Hospital, Jeddah, Saudi Arabia

DOI:

https://doi.org/10.17305/bb.2025.11886

Keywords:

MRSA, vancomycin, 3-Hydrazinoquinoxaline-2-thiol, 3HL, combination therapy, in silico analysis

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen causing infections ranging from skin disorders to severe conditions like infective endocarditis. Its evolving resistance, including resistance to β-lactams and last-resort antibiotics, such as vancomycin, daptomycin, and linezolid, necessitates alternative therapies. This study investigates the synergistic efficacy of vancomycin and 3-hydrazinoquinoxaline-2-thiol (3HL) against 23 clinical MRSA isolates. Susceptibility testing was performed using broth microdilution and checkerboard assays, while in silico analyses assessed interactions between vancomycin and 3HL. Vancomycin exhibited minimum inhibitory concentrations (MICs) ranging from 0.25 to 1 µg/mL, whereas 3HL showed higher MICs of 16–32 µg/mL. Synergistic interactions were confirmed via checkerboard assays, with fractional inhibitory concentration index (FICI) values between 0.236 and 0.5, indicating enhanced vancomycin efficacy. Notably, vancomycin MICs decreased significantly when combined with 3HL. In silico docking revealed interactions with penicillin-binding protein 2a (PBP2a), suggesting promising therapeutic potential. Vancomycin exhibited superior docking scores (−8.9 kcal/mol) and stabilizing hydrogen bonds, effectively targeting key protein grooves. Both compounds demonstrated potential for overcoming PBP2a’s structural occlusions, suggesting their role in combating β-lactam-resistant strains through targeted protein inhibition and structural stabilization.