Thymoquinone–3HQ Combo Targets ESBL Antibiotic Resistance

Researchers report the first evidence that two existing compounds work together to hit hard-to-treat ESBL-producing bacteria and the CTX-M-15 enzyme.

Why ESBL-producing bacteria are so difficult to treat

Bacterial infections remain a major cause of death worldwide and are increasingly complicated by antibiotic resistance. In 2019, antimicrobial resistance was linked to an estimated 1.27 million deaths globally, with high mortality in sub-Saharan Africa and key pathogens such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. Projections suggest that infections caused by drug-resistant organisms could cause 10 million deaths annually by 2050, together with treatment failures, longer hospital stays, and rising healthcare costs.

Extended-spectrum β-lactamases (ESBLs) are central to this problem. These enzymes are produced by members of the Enterobacteriaceae family and P. aeruginosa and can break down several β-lactam antibiotics, including aztreonam and third-generation cephalosporins. Their spread severely restricts treatment options. CTX-M-type ESBLs, including CTX-M-15, have been found in Klebsiella, Proteus mirabilis, Enterobacter, Salmonella, Shigella, Morganella morganii, A. baumannii and P. aeruginosa.

Because the discovery of entirely new antibiotics is slow and expensive, combining and repurposing existing agents is an increasingly attractive strategy. The authors note that using known drugs in new combinations can save more than $1 billion and cut the time to regulatory approval by about half.

Two familiar compounds with new potential

Thymoquinone (TQ) is a natural compound extracted from the seeds of Nigella sativa (black cumin or black seed). It has been used traditionally and is now recognized for diverse biological activities. In vitro, TQ shows antibacterial effects against Gram-negative bacteria and can inhibit biofilm formation in E. coli and P. aeruginosa.

3-Hydrazinoquinoxaline-2-thiol (3HQ), a quinoxaline derivative, has previously shown significant activity against a range of clinical strains associated with methicillin-resistant S. aureus (MRSA). The authors also report that 3HQ has activity against ESBL-producing Gram-negative strains (unpublished data).

Based on this background, they hypothesized that combining TQ and 3HQ could enhance activity against ESBL-producing clinical isolates and might inhibit the CTX-M-15 enzyme.

Study design in brief

The team tested TQ and 3HQ against 18 ESBL-producing clinical isolates obtained from a hospital in Jeddah, Saudi Arabia. The panel included E. coli (the majority of strains), K. pneumoniae, P. aeruginosa and A. baumannii.

They first determined the minimum inhibitory concentrations (MICs) of each compound alone by broth microdilution. Both TQ and 3HQ showed MIC values between 16 and 128 μg/mL across the isolates.

To assess synergy, they used a checkerboard assay and calculated the fractional inhibitory concentration index (FICI). Synergy was defined as FICI ≤ 0.5.

In parallel, the authors conducted molecular docking and 50-ns molecular dynamics simulations using the CTX-M-15 structure (PDB 7TI0). RPX-7063, a co-crystallized inhibitor, served as a control.

Key findings: consistent synergy and CTX-M-15 targeting

The core result is clear: every one of the 18 ESBL clinical isolates showed synergy between TQ and 3HQ, with FICI values from 0.178 to 0.491 (all <0.5).

• When combined, the MICs of 3HQ decreased 4- to 16-fold.
• The MICs of TQ decreased 4- to 8-fold.

For example, in E. coli strain 4, the MIC of 3HQ dropped from 32 μg/mL to 4 μg/mL when combined with TQ, and TQ dropped from 64 μg/mL to 8 μg/mL in the presence of 3HQ.

As the authors conclude, “Our study offers the first evidence of synergy between 3HQ and TQ against a variety of ESBL strains.

Docking results showed:

• 3HQ binds within the canonical CTX-M-15 active site and displays an interaction pattern similar to RPX-7063, with a docking score of –3.56.

• TQ binds at a distinct site, spatially separated from the catalytic center, with a docking score of –3.80, suggesting an allosteric pocket.

Molecular dynamics simulations supported stable binding of both ligands and indicated that TQ’s binding might alter protein conformation in a way that enhances 3HQ binding and stability. This mechanistic hypothesis aligns with the observed reduction in MICs and FICI values.

How might the combination work?

The discussion links the synergy to complementary mechanisms:

• Thymoquinone (TQ) damages bacterial cell morphology, compromises membrane integrity and causes leakage of essential proteins. It also penetrates the cell and disrupts critical intracellular proteins.

• 3HQ inhibits DNA synthesis in bacterial cells, interfering with replication and proliferation.

The authors also highlight the potential role of increased reactive oxygen species (ROS). TQ is known to induce ROS formation, and when combined with 3HQ this oxidative stress may be further enhanced, adding another layer to the antimicrobial effect against ESBL strains.

In addition, dual binding to CTX-M-15—3HQ at the active site and TQ at a possible allosteric pocket—could help suppress β-lactamase activity and contribute to the observed synergy.

Importantly, the study notes that synergy may allow lower doses of each compound, which is relevant given the potential toxicity of high concentrations of TQ, 3HQ and other drugs.

Implications and next steps for the field

Taken together, the findings suggest that combining TQ and 3HQ could be a promising strategy for tackling ESBL-mediated resistance, especially in E. coli and related Gram-negative pathogens. The work also illustrates how drug repurposing, combination therapy, and structure-based design can be integrated to address antimicrobial resistance.

However, the authors are careful to outline limitations. Most isolates in this study were E. coli, with only single isolates of K. pneumoniae, P. aeruginosa and A. baumannii, and all experiments were in vitro. They emphasize the need for:

• Time-kill and resistance assays

• Proteomic studies to map bacterial responses to the combination

• Expanded strain panels

• In vivo studies to evaluate toxicity, pharmacokinetics and therapeutic potential

For researchers working on antimicrobial resistance, this study offers a concrete example of how existing compounds—one natural product and one synthetic derivative—can be combined and mechanistically characterized to generate new options against ESBL-producing bacteria and CTX-M-type enzymes.

 

The translation of the preceding English text in Arabic: