Maintaining the efficacy of β-lactam antibiotics against Staphylococcus aureus is a clinical priority given the prevalence of methicillin-resistant S. aureus (MRSA). We previously showed that the pyrimidine analogs 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUrd) synergize with β-lactams. Here, we extended this by evaluating additional nucleotide metabolism-targeting agents. Gemcitabine (Gem) and mitomycin C (Mito), such as 5-FU and 5-FUrd, exhibited intrinsic anti-MRSA activity and potentiated β-lactams, whereas the purine analog 6-thioguanine (6-TG) showed distinct, often antagonistic effects. Transcriptomic analysis revealed that pyrimidine-targeting agents repress lysine and glutamate biosynthesis, while 6-TG induced these pathways, implicating amino acid metabolism in β-lactam potentiation. Consistent with this, pyrimidine analogs also suppressed GlmS expression, potentially limiting UDP-GlcNAc production required for cell wall synthesis, and synergized with fosfomycin. Fluorescence microscopy confirmed that the potentiation of oxacillin activity by pyrimidine-targeting agents, but not 6-TG, was accompanied by impaired peptidoglycan synthesis. Additionally, glutathione-mediated attenuation of killing implicated reactive oxygen species in the bactericidal activity of cloxacillin combinations. Finally, these agents displayed strong antibiofilm activity, further enhanced in combination with daptomycin and rifampicin. Together, these findings highlight the potential of pyrimidine analogs to potentiate cell wall-targeting antibiotics and identify an important role for modulation of cell wall precursor pathways in this anti-MRSA activity.

Thiopurine immunosuppressive drugs are indicated in children with acute lymphoblastic leukemia and in both children and adults with chronic inflammatory diseases. Therapeutic drug monitoring (TDM) of its DNA-incorporated metabolites 6-thioguanine nucleotides (6TGN) and 6-methylmercaptopurine nucleotides (6MMPN) is of high interest to optimize efficacy and prevent toxicity. The objective of this study was to develop and validate a sensitive and simple LC-MS/MS method for the simultaneous quantification of 6TGN and 6MMPN in whole blood for TDM. Following red blood cell count and whole blood extraction with dithiothreitol plus acidic hydrolysis at 100°C, quantification was performed using an LC-MS/MS in positive ionization mode. Calibration curves were linear over the range of 20-1000 ng/mL for 6TGN and 20-20,000 ng/mL for 6MMPN, with correlation coefficients above 0.99 for all analytes. Intra- and inter-day precisions were below 9.45%. Extraction recovery and matrix effects reached 78.9%-83.2% and 121% for 6TGN and 92.2%-95.2% and 112%-118% for 6MMPN, respectively. The method was applied to 749 clinical samples from patients with acute lymphoblastic leukemia or chronic inflammatory diseases. A sensitive and simple LC-MS/MS method was validated for 6TGN and 6MMPN in whole blood and was successfully applied to clinical samples for TDM.

Motivated by their potential use as a photosensitizer in photodynamic therapy, we report an electrostatic embedding quantum mechanics/molecular mechanics (QM/MM) study of 6-selenoguanine and 6-thioguanine in water. We analyzed triplet-state nonradiative decay within quasi-Marcus theory and assessed the influence of solvation by comparing QM/MM with previous microsolvation and PCM models. Both molecules follow similar deactivation pathways, involving torsional distortion around the chalcogen center and decay through a T1/S0 crossing point. Experimental studies show that triplet decay of 6-selenoguanine (6SeGua) is 835 times faster than 6-thioguanine (6tGua) in water. The shorter T1 lifetime of 6SeGua is due to its higher spin-orbit coupling (SOC) and lower activation barrier to reach the T1/S0 crossing point. Stronger hydrogen bonding in 6tGua stabilizes the T1 minimum relative to the T1/S0 crossing point, thereby increasing the activation barrier for triplet decay. The decay rates obtained from QM/MM are in good agreement with the previous microsolvation model, indicating that both approaches capture the essential solvent effects. The findings underscore the role of solvent effects and hydrogen bonding in modulating the photophysical properties of chalcogen-substituted nucleobases.

Genome integrity relies on DNA mismatch repair (MMR) to correct replication errors, yet whether non-protein cofactors regulate this pathway remains unexplored. Here, we identify nuclear phosphatidylinositol-3-phosphate (PI3P) as a lipid regulator of MMR. Using biosensors, lipid pulldown, and proximity ligation assays, we show that PI3P forms discrete nuclear puncta in close proximity to the MutSα (MSH2:MSH6) and MutSβ (MSH2:MSH3) MMR recognition complexes. Pharmacological or genetic depletion of the class III PI3-kinase Vps34 impaired MutSα and MutSβ heterodimer assembly without altering MMR protein nuclear abundance, compromised DNA substrate association of MMR components in nuclear extracts, and elevated microsatellite instability at mononucleotide repeats. Exogenous PI3P enhanced MMR recognition complex assembly and DNA association in PI3P-deficient nuclear extracts, supporting a role for PI3P in promoting MMR. We further show that a nuclear Beclin-1/Vps34 complex produces this PI3P pool through an autophagy-independent mechanism. Functionally, loss of nuclear PI3P blunts MMR-dependent DNA damage signaling and confers 6-thioguanine resistance in cultured cells and in Beclin-1-deficient zebrafish in vivo. These findings reveal an autophagy-independent nuclear function for the Beclin-1/Vps34 complex in genome maintenance and identify PI3P as a lipid mediator of MMR, thereby expanding the functional repertoire of nuclear phosphoinositide signaling.

The immunosuppressive prodrugs azathioprine and 6-mercaptopurine necessitate therapeutic drug monitoring of their active metabolite, 6-thioguanine (6-TG), due to their narrow therapeutic window and substantial risk of severe adverse effects. Conventional chromatographic techniques are accurate but impractical for point-of-care (POC) applications. Conversely, existing electrochemical sensors, despite their widespread use, suffer from an over-reliance on nanomaterial modifications to enhance analytical performance and face persistent challenges associated with calibration. This work resolves a long-standing knowledge gap by providing the first experimental validation of the 6-TG redox mechanism on a Nafion-modified screen-printed carbon electrode (SPCE). Through dithiothreitol intervention and electrochemical kinetic analysis, we confirm that 6-TG oxidation proceeds via a disulfide bond-mediated, four-electron, four-proton multi-step pathway. Critically, elucidation of this mechanism reveals a consistent kinetic framework, which directly inspires a universal-slope (US) calibration-free strategy wherein the calibration slope becomes a stable parameter, obviating frequent recalibration for POC applications. Leveraging this mechanistic insight, we constructed a smartphone-assisted electrochemical sensor on the same Nafion/SPCE platform for on-site detection of 6-TG in erythrocyte lysate and serum. The sensor exhibits a linear response from 0.1 to 15 μM in erythrocyte lysate, effectively covering the clinically relevant therapeutic window, alongside excellent selectivity and reproducibility. Clinical validation using 50 patient samples demonstrates strong agreement with high-performance liquid chromatography results, and the US method yields outcomes statistically equivalent to those obtained via the standard addition approach. By resolving the redox mechanism and translating this insight into a calibration-free POC sensor, this work offers a clinically deployable solution for personalized thiopurine therapy.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. Genetic polymorphisms in TPMT can reduce enzyme activity and increase the risk of adverse drug reactions. One such variant, TPMT2 (A80P), has been reported to impair TPMT function. In this study, we used molecular dynamics simulations, molecular docking, and umbrella sampling to investigate how the A80P mutation affects TPMT's structure and its interaction with the cofactor S-adenosylmethionine (SAM). Our results indicate that the mutation increases structural flexibility, reduces stability, and weakens SAM binding, suggesting compromised enzymatic function. Although TPMT2 is not directly linked to acute myeloid leukemia (AML), identifying its prevalence in AML patients has clinical value due to the potential use of thiopurines in treatment. We screened 50 AML patients and 50 healthy controls from northern India for the TPMT2 variant using PCR-based genotyping. The variant was absent in all individuals tested. These findings support the importance of population-specific pharmacogenetic screening. Tailoring genetic tests to local variant frequencies can improve cost-effectiveness, reduce unnecessary testing, and contribute to safer, more personalized cancer therapy.

6-Thioguanine (6-TG) is an FDA-approved antimetabolite drug that is widely used clinically, including for the treatment of leukemia. Its cellular effects require metabolic activation and are regulated through interactions with various proteins such as NUDT15, which catalyzes the hydrolysis of the active 6-TG metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP. Recent genome-wide CRISPR loss-of-function studies have identified another NUDIX hydrolase, NUDT5, as a crucial mediator of 6-TG toxicity. Here, we develop and validate a selective, cell-active NUDT5 degrader toolkit and orthogonally characterize target engagement, ternary complex formation, degradation kinetics, and proteome-wide selectivity. These degraders, in conjunction with orthogonal CRISPR knock-out and reconstitution experiments, support a non-enzymatic role for NUDT5 in modulating the cellular response to 6-TG. Depletion of NUDT5 protein is antagonistic to NUDT15 inhibition, suggesting a distinct mode-of-action with potential implications for patient therapy.

Thiopurines are efficient triplet-state photosensitisers; however, the practical application of canonical derivatives such as 6-thioguanine (6TG) and 6-thioguanosine (6TGuo) is limited by competing deactivation pathways that reduce the fraction of triplet states available for productive interaction with molecular oxygen. In this work, we investigated how structural modification of the thiopurine scaffold through introducing of an additional five-membered etheno ring affects triplet-state energetics, deactivation pathways, and singlet oxygen sensitisation. The photophysical properties of four tricyclic thiopurine analogues-9-thio-1,N2-ethenoguanine (TEGua), 9-thio-1,N2-ethenoguanosine (TEGuo), 6-methyl-9-thio-1,N2-ethenoguanine (6MeTEGua), and 6-methyl-9-thio-1,N2-ethenoguanosine (6MeTEGuo)-were investigated using steady-state spectroscopy, low-temperature phosphorescence, nanosecond transient absorption spectroscopy, and direct detection of singlet oxygen phosphorescence. All investigated compounds exhibited efficient intersystem crossing and microsecond-lived triplet states. Compared with canonical thiopurines, the tricyclic analogues displayed lower triplet-state energies and significantly enhanced singlet oxygen generation. Quantum yields of singlet oxygen sensitisation reached ~0.56 in acetonitrile, approximately twofold higher than those observed for 6TG and 6TGuo under identical conditions. Analysis of triplet-state deactivation pathways showed that the enhanced photosensitising efficiency does not result from increased triplet formation, but from more effective use of the triplet-state population for energy transfer to molecular oxygen leading to singlet oxygen formation. These findings demonstrate that structural modification of the thiopurine scaffold enables control over triplet-state reactivity and provides a strategy for designing improved thiopurine-based photosensitisers for photodynamic therapy applications (PDT).

Variants in TPMT and NUDT15 genes that affect thiopurine metabolism can guide personalized dosing to minimize toxicity. Decreased or no appreciable NUDT15 activity demonstrates impaired breakdown of active thiopurine metabolites which can lead to severe adverse events including potentially life-threatening myelosuppression. Recently, the NUDT15*6 allele was re-classified from having uncertain function to no function by the Clinical Pharmacogenetics Implementation Consortium. Here, we present a pediatric patient with a NUDT15*1/*6 genotype who experienced significant thiopurine-induced myelosuppression. The patient is a 4-year-old female with standard risk-average precursor B-cell acute lymphoblastic leukemia treated per AALL1731. Exome sequencing determined TPMT*1/*1 (normal metabolizer) and NUDT15*1/*6 (indeterminate metabolizer). After 75 mg/m[2]/day mercaptopurine, myelosuppression necessitated a three-week delay prior to the next phase of therapy. With 60 mg/m[2]/day thioguanine in a subsequent phase of therapy, she was admitted twice for fever and neutropenia. In the final phase of therapy, mercaptopurine at standard dosing of 75 mg/m[2]/day caused severe neutropenia and thrombocytopenia with elevated metabolite levels that required stopping mercaptopurine. After neutrophil recovery, a trial of 50% standard dosing was not tolerated and her dose was reduced to 27%, which was tolerated. Among 339 patients with cancer, sequencing revealed an allele frequency of 0.88% for NUDT15*6. In a larger cohort sequenced for suspicion of rare genetic disease, the frequency of NUDT15*6 was 0.15% among 1011 unrelated individuals. This brief report supports the recent update that patients with the NUDT15*1/*6 genotype should be classified as intermediate metabolizers.

6-Thioguanosine 5'-monophosphate (6-TGMP) is the biologically active nucleotide form of the antimetabolite 6-thioguanine produced by Erwinia amylovorans. The YcfA-YcfC enzymatic pair catalyzes the incorporation of the thioamide functional group in GMP via an oxygen-to-sulfur replacement process. YcfA is a member of the adenine nucleotide alpha hydrolase-like (AANH-like) superfamily and YcfC is a pyridoxal 5'-phosphate (PLP)-dependent enzyme. Two mechanistic models exist, depending on whether YcfC is a C-S lyase or a cysteine desulfurase, to explain the trans-sulfuration reaction. Herein, we investigate the YcfA-YcfC trans-sulfuration reaction using anaerobically purified YfcA and YcfC enzymes in the absence of reductants. Steady state and pre-steady state kinetic studies suggest the mechanism involves two distinct intermediates and differs from the common trans-sulfuration pathway used in, for example, tRNA thionucleotide biosynthesis.

Ebola virus (EBOV) is among the most lethal human pathogens, yet effective treatment options remain limited. While extensive efforts have elucidated the functions of viral proteins, the temporal orchestration of host transcriptional responses-and their exploitation by EBOV during infection-remains poorly defined. Here, we performed integrated time-series transcriptomic profiling using both RNA-seq and microarray analyses to characterize dynamic host and viral gene expression. Through the combination of co-expression network construction and causal inference modeling, we reconstructed the sequential activation of viral and host genes across distinct phases of infection. Our findings show that EBOV induces only minimal transcriptional perturbations during early infection but extensively reprograms host gene expression at later stages, forming infection-specific co-expression modules enriched for antiviral signaling, immune regulation, and stress responses. To prioritize functional host factors, we integrated transcriptional modules with virus-host protein-protein interaction networks and gene-drug databases. This approach identified early-induced host genes with known or predicted interactions with EBOV proteins and pharmacologically actionable targets. RNA interference-mediated silencing of three key regulatory genes (RELB, LDLR, and MYC) significantly impaired EBOV RNA replication and progeny virus production. Furthermore, pharmacological screening identified sorafenib and thioguanine as effective inhibitors of EBOV replication, with EC50 values of 1.529 μM and 2.469 μM, respectively. Collectively, our study uncovers temporally resolved host regulatory programs hijacked by EBOV and demonstrates the utility of integrating dynamic transcriptomics with systems biology, functional validation, and drug screening to identify host-targeted antivirals. These findings provide a conceptual and methodological framework for developing host-targeted therapies against highly pathogenic viruses.IMPORTANCEEbola virus is a devastating pathogen with limited treatment options. A major challenge in developing therapies is understanding how the virus dynamically hijacks our cells over time. This study provides a time-resolved map of the host transcriptional landscape during Ebola virus infection. We reveal that the virus causes minimal early changes but extensively reprograms human gene expression later, creating specific co-expression networks that are essential for viral replication. By integrating these networks with virus-host interaction data, we identified key human genes and demonstrated that silencing them impairs viral replication. Furthermore, we repurposed existing drugs, identifying sorafenib and thioguanine as effective inhibitors. Our work uncovers the temporal strategy of Ebola virus and establishes a framework for discovering host-directed therapies against this and other highly pathogenic viruses.