Changing the antibiotic playbook to catch an ESKAPE artist

Nearly a century ago, a single colony of Staphylococcus aureus (S. aureus) helped launch the antibiotic age. In 1928, Alexander Fleming returned to his London lab to find that a stray mold had wiped out the Staphylococcus growing on one of his plates. The accident gave the world penicillin, ushering in the age of biomedicine and ensuring that an infected cut or scrape would not be fatal.

S. aureus was among the first bacteria that penicillin humbled. It is ironic that the same organism now sits on the World Health Organization’s roster of most-feared superbugs.

Methicillin-resistant S. aureus, or MRSA, represents the “S” in ESKAPE, a group of six pathogens notorious for their ability to escape or evade multiple antibiotics. The bacterium is notorious for causing skin, bloodstream and surgical-implant infections.

Public health officials routinely warn of a “silent pandemic” of antimicrobial resistance (AMR), in which routine medical procedures could once again become life-threatening because of untreatable infections.

According to the 2024 Global Research on Antimicrobial Resistance (GRAM) study published in The Lancet, drug-resistant infections could directly kill more than 39 million people between 2025 and 2050, with resistant Staphylococcus infections directly linked to about 130,000 deaths. South Asia, including India, is expected to bear the heaviest burden, with an estimated 11.8 million deaths.

It is against this backdrop that a collaborative study involving the Indian Institute of Technology Gandhinagar (IITGN) has designed a laboratory-made molecule that kills S. aureus by disabling an enzyme most antibiotics ignore. The study was published in Chemistry & Biodiversity.

“Most antibiotics attack bacteria through a handful of familiar routes, including puncturing their walls and jamming their protein-making machinery. The trouble is that bacteria have spent decades learning to dodge those blows,” explains Professor Bhaskar Datta, corresponding author of the study and affiliated with IITGN’s Departments of Chemistry and Biological Sciences and Engineering.

“Antibiotic resistance is, in many ways, an evolutionary arms race. That is why researchers hunt for fresh points of attack.”

IITGN researchers, working with colleagues at Jamia Millia Islamia, Jamia Hamdard, Xi’an Jiaotong-Liverpool University and the Ahmedabad-based company Sushen Medicamentos, decided to try a new approach to tackling the AMR problem. To survive and multiply, every bacterial cell must replicate its genome.

This process relies on a continuous supply of molecular building blocks, specifically pyrimidine nucleotides. Thymidine kinase (TK) is a small enzyme that helps a bacterium recycle the chemical building blocks it needs to copy and repair its DNA. If TK is disabled, the cell’s genetic engine stalls.

Earlier work had flagged TK as an essential gene. Because today’s antibiotics have not extensively targeted the enzyme, bacteria may not have evolved widespread resistance mechanisms, making it an attractive drug target. The researchers also had a detailed structural map to work with.

Md. Imtaiyaz Hassan’s team recently solved the crystal structure of S. aureus thymidine kinase, giving the researchers an atomic-level view of the enzyme and, crucially, of the pocket they wanted their molecule to target. Hassan is the co-corresponding author of the study and is affiliated with the Center for Interdisciplinary Research in Basic Sciences at Jamia Millia Islamia, New Delhi.

Rather than copy the enzyme’s natural chemistry, the chemists assembled a new molecule from two fragments with long pedigrees in antibacterial research: a thiazole ring and a sulfonamide group, the latter a chemical descendant of the earliest antibiotics. From nine synthesized candidates, one iodo- and trifluoromethyl-substituted compound, DSA3, emerged as the most potent.

Using computer-based molecular docking, the researchers predicted how each compound might fit into the enzyme. DSA3 showed the strongest predicted interaction with thymidine kinase, particularly within the ATP-binding pocket that is essential for enzyme function. This is the pocket where the enzyme normally grabs its chemical fuel, so blocking it stalls the enzyme’s work.

Inside that pocket, DSA3 latches onto several key amino acids (the building blocks that make up the enzyme), including one directly responsible for the enzyme’s activity. Longer computer simulations suggested that the enzyme remained stable while DSA3 stayed within this region.

After computational modeling identified DSA3 as the most promising candidate, the researchers tested whether it worked as predicted. Laboratory experiments showed that DSA3 binds to thymidine kinase and reduces its activity, with 6.996 μM reducing the enzyme’s activity by half. This provided evidence that the molecule could interfere with its target.

Furthermore, the team examined how DSA3 interacts with the enzyme. Using fluorescence measurements, they observed changes in the enzyme’s natural fluorescence upon the addition of DSA3. This indicated that the molecule was interacting with the protein.

A separate technique, called isothermal titration calorimetry, measured the small amounts of heat produced or absorbed when the two came together. The results provided further evidence that DSA3 forms a stable interaction with thymidine kinase.

Finally, the researchers tested whether blocking TK translated into an effect on the bacterium itself. In laboratory tests against Staphylococcus aureus, DSA3 inhibited bacterial growth and, at a higher concentration, killed the bacteria. The researchers also observed a substantial reduction in viable bacterial cells at the concentration that inhibited growth.

“We see DSA3 as a proof of concept and a starting scaffold. It is not a drug,” said Dr. Rajesh K. Hadiya, co-first author and a former Ph.D. scholar at IITGN.

“Its potency needs to improve, and the future steps include testing against resistant clinical isolates, checking selectivity over the human enzyme, and moving into animal models. But that is exactly where medicinal chemistry begins.”

The innovation aligns with national and international initiatives to combat AMR, as drug-resistant bacteria readily spread between people and livestock. The research also contributes to the broader effort to strengthen India’s capacity for antibacterial drug discovery and development.

This approach is consistent with the “One Health” framework, which recognizes the interconnected nature of health across people, animals and the environment and underpins initiatives such as India’s National Action Plan on Antimicrobial Resistance 2.0 (NAP-AMR 2.0) and the National One Health Mission.

Speaking about how this work fits within the aforementioned policies, Datta stated, “We have previously reported on using distinctive strategies and less-explored targets to fulfill the larger goal these frameworks set out. As part of our continuing efforts here, we have shown that a particular chemical class can engage an underused antimicrobial target in a specific, measurable way.”

It is a fitting place for that effort to begin. Nearly a century after a stray mold killed the Staphylococcus on Alexander Fleming’s plate and opened the antibiotic age, the same bug that first proved these drugs work has become a reason to keep searching for vulnerabilities it has not yet learned to guard against.

Who’s behind this story?

Sadie Harley

BSc Life Sciences & Ecology. Microbiology lab background with pharmaceutical news experience in oil, gas, and renewable industries.

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Andrew Zinin

Master’s in physics with research experience. Long-time science news enthusiast. Plays key role in Science X’s editorial success.

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