Helicobacter pylori is a significant human pathogen, discovered by Warren and Marshall in 1982 (Ahmed, 2005). The human stomach is home to the helical, microaerophilic, Gram-negative H. pylori bacteria (Pandey, 2016). Nearly half of the world's population is afflicted by this significant pathogen (Wroblewski et al., 2010). Chronic gastritis brought on by its infections can progress to intestinal metaplasia, peptic ulcers, mucosa-associated lymphoid tissue lymphoma, adenocarcinoma, and gastric atrophy (Alyahawi et al., 2018). Common medication for H. pylori infection includes combinations of antibiotics and proton pump inhibitor (Malfertheiner, 2022). Treatment for individuals with active stomach or duodenal ulcers includes bismuth quadruplex therapy,—which is comprised of bismuth, metronidazole, tetracycline, and a proton pump inhibitor (PPI) or H2-blocker (Hasanuzzaman et al., 2024). Patients who have been exposed to H. pylori strains with 15% resistance rate to clarithromycin may benefit from clarithromycin triple therapy (PPI + clarithromycin + amoxicillin or metronidazole) (Goodarzi, 2024).

However, the recent emergence of antibiotic resistance and multi drug resistance in H. pylori has led to a significant decrease in the efficiency of these treatments. In WHO regions, the combined prevalences of resistance to levofloxacin, amoxicillin, metronidazole, clarithromycin, and tetracycline were 15%, 91%, 14%, 38%, and 13%, respectively (Goodarzi, 2024, Aumpan et al., 2023) In every geographical area, metronidazole showed the highest rate of antibiotic resistance (Savoldi, 2018). We may soon face the possibility of shortage of effective medication alternatives because of the dynamic and evolving nature of antibiotic resistance in H. pylori (Lin, 2023). Thus, it is crucial to develop and implement efficient strategies to combat H. pylori infection. Earlier we have reported b-clamp binding FDA approved repurposed drug diflunisal as inhibitor of H. pylori growth (Pandey, 2017).

In all living forms, kinases are the primary regulators of cellular functions. They mediate the transfer of phosphoryl groups. Since many infectious pathogens genomes have been sequenced, kinases have become more well-known and have shown to be effective therapeutic targets. H. pylori 26,695 have total 30 kinases genes encoded in its genome. In the current work, we have focused on thymidylate kinase, a member of Nucleotide Monophosphate Kinase (NMPK) family (Caillat, 2008). The NMPK family includes Cytidylate kinase (CMK), adenylate kinase (AMK), uridylate kinase (UMK), and guanylate kinase (GMK) (Yan and Tsai, 1999). The base of the nucleoside monophosphate determines the substrate of the NMPK family of enzymes. Every NMPK enzyme has a similar fold made up of the P-loop region, LID (Ligand Induced Degradation) region, and NMP and core domain. All members of the NMPK family share a similar active site architecture in addition to the overall fold (Segura-Pena, 2004).

Thymidylate kinase is a dimeric protein, found in all organisms, including prokaryotes, eukaryotes and viruses (Chaudhary et al., 2018). The thymidylate kinase is crucial enzyme for DNA synthesis. It is responsible for the reversible transfer of the phosphate group from ATP (phosphate donor) to deoxythymidine monophosphate (dTMP). The dTMP gets phosphorylated to give deoxythymidine diphosphate (dTDP). It requires magnesium ions as a cofactor, Mg2+ brings both the substrates nearby for the proper phosphoryl transfer reaction. The overall structure of thymidylate kinase is similar in most of the organisms and is comprised of seven-eleven alpha helices surrounding the central five to seven stranded beta sheets (Kumari and Gourinath, 2025). There are three important loops or regions in the structure which is important for the function of the protein. These are highly conserved P-loop, DR(X) motif, and a LID region (Sinha and Rule, 2017).

The expression of thymidylate kinase is cell cycle regulated and restricted to the S-phase of cell replication, due to its critical role in the synthesis of thymidine nucleotide (Hu and Chang, 2007). Because of its location at the intersection of the salvage and de novo pathways, it is a highly effective target for inhibitors that stop DNA replication, which in turn stops the pathogen from surviving (Kumari and Gourinath, 2025).

This study solves the H. pylori thymidylate kinase (HpTMPK) three-dimensional structure at 2.5 Å. It is evident from the thorough analysis that there are significant differences between human TMPK and HpTMPK, making HpTMPK a viable drug target for inhibitors that target H. pylori. We used the HpTMPK structure for in silico screening of small molecules and shortlisted the top three compounds based on their docking and energy scores.

Using the shortlisted compounds, we also functionally examined this enzyme by looking at in vitro inhibition kinetics. This analysis revealed that two (F725_0025, D430_0467) of the three compounds demonstrated good inhibition of HpTMPK with IC50 values of 84 µM and 134 µM, with compound F725_0025 showing the most effective inhibition of HpTMPK. In order to confirm the F725_0025 and D430_0467 inhibitory function, an in vivo test of the compound's impact on H. pylori proliferation revealed that, after 48 h, the drug reduced H. pylori growth with IC50 value for F725_0025 and D430_0467 equals to 30.14 µM and 53.21 µM respectively.