The Chlamydiota phylum consists of obligate intracellular bacteria, including well-known pathogens and emerging environmental species, with diverse host ranges and metabolic capabilities. Among these bacteria, the gene which encodes nucleoside diphosphate kinase, ndk is present in variable numbers. While most chlamydial species carry a single paralog of ndk, some species have two paralogs. In Waddlia chondrophila, the two Ndk proteins encoded by ndk paralogs retain conserved kinase motifs but differ in subcellular localization, suggesting divergent functional roles. According to localization studies performed in heterologous expression systems, WcNdk1 is confined to the bacteria-containing vacuole and probably supports nucleotide metabolism, while WcNdk2 localizes to the host nucleus, perinuclear space, and Golgi apparatus, suggesting involvement in host interaction. Azidothymidine (AZT), a known Ndk inhibitor, impaired W. chondrophila growth, potentially through inhibition of WcNdk2. However, the lack of genetic tools currently limits definitive functional conclusions. Our data suggests potential functions for Ndks in W. chondrophila, providing a foundation for future studies on Ndk-mediated interactions between this pathogen and its host, which might possibly be translated into a new therapeutic approach.

Background/Objectives: Despite the presumption of bioequivalence for BCS Class I drugs due to their high solubility and permeability, recent evidence indicates that those with rapid systemic elimination exhibit heightened vulnerability to Cmax non-equivalence, primarily attributable to intrasubject variability in gastrointestinal transit and absorption kinetics. It is well known that gastric emptying is a significant physiological-dependent factor. But, does the formulation affect gastric emptying? Methods: Using zidovudine as a model drug, formulations containing sodium carboxymethyl starch (CMS-Na), pregelatinized starch, hydroxypropyl methylcellulose (HPMC), and lactose were investigated for their effects on gastric emptying kinetics, and the impact of excipient-mediated gastric emptying prolongation on pharmacokinetic parameters was also evaluated. Results: Relative to AZT alone (Cmax = 13,350 ng/mL; gastric %ID = 11.3%), co-administration with CMS-Na, pregelatinized starch, or HPMC significantly prolonged gastric retention (%ID: 23.4%, 30.5%, and 40.8% at 22.5 min) and reduced Cmax in rats by 47.8%, 34.4%, and 35.1%, respectively, with no effect on intestinal permeability. Viscosity positively correlated with gastric emptying delay. Conclusions: Our rat findings provide new possible mechanistic evidence that certain viscosity-modifying excipients can delay gastric emptying and reduce Cmax of zidovudine, a rapidly eliminated BCS Class I drug, with potential implications for biowaiver risk assessment. Gastric emptying is not only a physiological-dependent variation but also, in cases where common excipients may significantly delay gastric emptying, a formulation-dependent rate-limiting step. For such drugs, excipient-induced gastric emptying delay poses an underappreciated risk to the biowaiver approach, necessitating more prudent regulatory assessment that encompasses the dynamic interplay among sequential rate processes governing drug disposition.

Colorectal cancer is the third most commonly diagnosed cancer worldwide and the second leading cause of cancer-related deaths, while its resistance to treatment continues to represent a major therapeutic challenge. In the present study, a series of phenothiazine derivatives, including hybrids containing a 1,2,3-triazole linker and the zidovudine (AZT) fragment, were synthesized and evaluated for their anticancer activity against colorectal cancer cell lines HCT116 and HT-29 as well as non-cancerous BEAS-2B cells. Cytotoxic activity was determined using the Alamar Blue assay, while the mechanisms of action were investigated by flow cytometric analysis of apoptosis, cell cycle progression, and reactive oxygen species (ROS) generation. Additionally, changes in the expression of genes associated with apoptosis, oxidative stress, and DNA damage response were analyzed by RT-qPCR. The obtained results demonstrated that AZT-containing derivatives exhibited stronger anticancer activity than non-conjugated phenothiazine analogs. Compounds A9-A12 induced pronounced apoptosis and significant disturbances in cell cycle progression, particularly in HCT116 cells. Among the analyzed derivatives, compound A9 displayed the most favorable overall biological profile, combining strong proapoptotic and cytotoxic activity with relatively high selectivity toward cancer cells and moderate effects on non-cancerous cells. The results indicate that molecular hybridization of phenothiazine derivatives with the AZT scaffold represents a promising strategy for the development of novel anticancer agents targeting colorectal cancer.

Adult T cell leukemia/lymphoma (ATLL), caused by human T cell leukemia virus type 1, is an aggressive malignancy with limited treatment options. Although the combination therapy of azidothymidine (AZT) and interferon-α (IFN-α) has demonstrated clinical efficacy in ATLL, its underlying mechanism remains unclear. In this study, through comprehensive analyses, we reveal that the combination therapy can enhance CD8+ T cell function both in vitro and in vivo: it promotes cytotoxic T lymphocyte (CTL)-mediated killing of ATLL cell lines, and exhibits antitumor activities in immunocompetent but not immunodeficient mouse xenograft models (MC38 and EL4 were used due to the lack of immunocompetent mouse model for ATLL). Granzyme B, which is downregulated in ATLL patients, was found to be key to the effectiveness of AZT/IFN-α therapy. In addition, AZT promotes IFN-α-mediated antigen processing and presentation, and it further transcriptionally upregulates the expression of the ATLL antigen HBZ. Collectively, AZT could potentiate the immunomodulatory function of IFN-α in ATLL likely via enhancing tumor antigen expression and processing and boosting CTL activities. This study suggests a key role of immunostimulation in AZT/IFN-α-treated ATLL and may provide mechanistic insights into the development of novel therapies.

The NS3 protein of dengue virus (DENV) is highly conserved and plays a vital role in replication, and thus it is considered as a potential antiviral target. The current study was carried out to observe interactions between the NS3 protein of DENV-4 and 3'-azido-3'-deoxythymidine (AZT-TP) using biophysical studies and ATPase inhibition assays, and further complemented it with in silico approaches. Fluorescence and time-resolved studies showed significant static quenching of NS3 fluorophores. Surface plasmon resonance (SPR) studies showed strong binding affinity with a significant pharmacodynamics rate, i.e., higher kinetic rate of association (k a) and lower dissociation rate constant (Kd). Isothermal titration calorimetry (ITC) measurements revealed strong and spontaneous binding between NS3 and AZT-TP with feasible thermodynamic parameters. The ATPase inhibition assay showed a decrease in NS3 ATPase activities in a dose-dependent manner, indicating the inhibitory potential of AZT-TP. Molecular docking showed significant binding (-8.0 kcal/mol) with the conserved residues involved in NS3 helicase/ATPase activities. Molecular dynamics (MD) simulation studies showed no major conformational changes in the NS3 and AZT-TP complex, and it was well-anchored at its binding position with nearly a linear trajectory up to 200 ns. Total Gibbs free energy and contribution of different interactions calculated using MMPB-(GB)-SA complemented the biophysical and docking results. The study suggests that there might be a direct binding between AZT-TP and residues involved in the ATPase activities and thus provides promising data to explore the future prospects of AZT-TP as an NS3 inhibitor for antiviral development.

Faithful DNA replication in Escherichia coli requires the DNA polymerase III holoenzyme (DNA pol III HE) and its clamp loader complex, which couples processive DNA synthesis with β-clamp loading. The clamp loader accessory subunits χ and ψ link single-stranded DNA-binding protein (SSB) to the replisome, stabilizing replication on SSB-coated templates through interactions with the SSB C-terminal tail. Chi has also been implicated in tolerance to the chain-terminating nucleoside analog azidothymidine (AZT), though whether this function depends on χ within DNA pol III HE or on its independent interaction with the YoaA helicase remains unclear. To address this, we engineered ψ-χ fusion proteins with flexible glycine-serine linkers to tether the two subunits while preserving folding and activity. Both fusions were biochemically competent, supporting ATP hydrolysis and clamp loading on SSB-coated DNA. In vivo, however, neither the ψ-GS12-χ fusion nor expression of a ψχ operon restored AZT tolerance in ΔholC cells, whereas expression of χ alone was sufficient. Fusion expression impaired growth in both wild-type (WT) and ΔholC backgrounds, a phenotype alleviated by disrupting χ-SSB binding. These findings support a model in which χ must dynamically engage SSB and YoaA outside of the clamp loader to promote AZT tolerance, highlighting the importance of regulated χ-SSB interactions in genome maintenance.

The widespread use of pharmaceuticals has led to their frequent detection in aquatic environments, raising concerns about adverse effects on non-target organisms. This study examined behavioral and endocrine responses to acute repetitive exposure (4 days, 15 min day-1) to environmentally relevant concentrations of azithromycin (AZT, 12.5 μg mL-1), nimesulide (NIME, 0.4 μg mL-1), and their combination in zebrafish (Danio rerio). Behavior was assessed using the Novel Tank Test, Social Preference Test, and Light/Dark Test. Whole-body cortisol, acetylcholinesterase (AChE) and catalase (CAT) activities, lipid peroxidation (TBARS), and non-protein thiols (NTP) were measured as stress biomarkers. NIME significantly reduced locomotion (∼30%) and time spent in upper zones (p < 0.05), indicating hypolocomotion and anxiety-like behavior. AZT elevated cortisol (p < 0.05) but caused minimal behavioral changes. Co-exposure intensified behavioral impairments and further increased cortisol (p < 0.01), suggesting synergistic effects. NIME increased TBARS, indicating oxidative damage. AChE activity was inhibited in the brain by NIME, while AZT and the combination increased systemic AChE. CAT activity rose in the combination group, possibly reflecting a compensatory response to reactive oxygen species. These findings demonstrate that short-term, repeated exposure to mixtures of commonly detected pharmaceuticals, even at environmentally relevant concentrations, can disrupt behavioral, biochemical, and endocrine homeostasis in aquatic organisms. This highlights the ecological risks posed by multi-compound contamination in aquatic environments.

It was first hypothesized in 2009 that endogenous host reverse transcriptase (eRT) arising from retrotransposon elements in arthropod genomes would be critical for defense against viral pathogens in crustaceans and insects. This was not proven until 2016 in insects. In the succeeding 20 years, insect studies have confirmed the eRT role in antiviral defenses that result in tolerated, persistent infections via viral accommodation mechanisms (VAM). In insects, eRT transcribes viral RNA fragments into viral copy DNA (vcDNA) in both linear (lvcDNA) and circular (cvcDNA) forms. These, in turn, give rise to small interfering RNA (siRNA) fragments to feed the host RNA interference (RNAi) mechanism. At the same time, some vcDNA fragments are integrated into the host genome as endogenous viral elements (EVE), that can also give rise to siRNA. Studies with shrimp have progressed more slowly but results so far have mimicked those revealed with insects. Here we describe the presence and functional activity of a 5383 bp-RT-related gene from a retrotransposon in the black tiger shrimp Penaeus monodon upon challenge with white spot syndrome virus (WSSV). It encompasses four conserved domains of protease (retropepsin), reverse transcriptase (RT-RNase H), transposase, and integrase as a single ORF of 1248 deduced amino acids. The recombinant protein of a conserved 408-amino acid RT-RNase H domain, designated as Pm-eRT was expressed and found to possess reverse transcriptase activity. During WSSV infection, Pm-eRT was upregulated in shrimp hemocytes and pleopods and DNA/RNA hybrids in hemocytes were detected. The DNA/RNA hybrid signals were translocated from the plasma membrane to the nuclear membrane in the shrimp that survived WSSV infection with low viral loads. Pharmacological inhibition of Pm-eRT activity using azidothymidine (AZT) resulted in enhanced viral replication and accelerated shrimp mortality. Collectively, these findings support those described for insects, paving the way to further work on details of VAM in shrimp with the ultimate aim of developing genetically viral-tolerant breeding stocks for shrimp cultivation.

Currently, the increasing isolation rates of New Delhi metallo-beta-lactamase (NDM) positive Klebsiella pneumoniae and the lack of a standard algorithm for the treatment of infections caused by these isolates necessitate the search for effective treatment options. This study aimed to investigate the efficacy of the combination of ceftazidime-avibactam (CZA), frequently used in the treatment of carbapenem-resistant Enterobacterales (CRE) infections in recent years with aztreonam (AZT), which is expected to become available in our country soon and the combination of meropenem (MEM) with colistin (COL), which is frequently preferred in our hospital against NDM-positive K.pneumoniae isolates using in vitro synergy tests and to determine the susceptibility of these isolates to cefiderocol (FDC), which is recommended as an alternative treatment option in CRE infections. A total of 63 K.pneumoniae isolates, isolated from clinical samples in the Bacteriology Laboratory of the Department of Medical Microbiology at Süleyman Demirel University Faculty of Medicine and identified as NDM positive by real-time polymerase chain reaction, were included in the study. The susceptibility of the isolates to CZA, AZT, MEM and COL was tested by broth microdilution method and FDC susceptibility was tested by Kirby-Bauer disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing criteria. The efficacy of CZA-AZT and MEM-COL combinations against NDM positive K.pneumoniae isolates was tested using the checkerboard method. Time-kill assays were performed to investigate the bactericidal activity of CZA-AZT and MEM-COL combinations at different concentrations and time points in five isolates harboring different carbapenemase genes (two NDM+OXA-48, one NDM, one NDM+OXA-48+KPC, and one NDM+OXA-48/VIM/IMP). All isolates included in our study were found as resistant to CZA, AZT and MEM, while 77.8% were resistant to FDC and 54% were resistant to COL. According to the checkerboard test results, the CZA-AZT combination showed synergistic interaction against all isolates, while the MEM-COL combination exhibited synergistic interaction against 57.1% of the isolates, additive interaction against 25.4% and undefined interaction against 17.5%. In time-kill assays, synergistic activity was observed at various time points in all isolates with the CZA-AZT combination and in four isolates with the MEM-COL combination, while bactericidal activity was detected at certain time points. Although an undefined interaction was determined in the checkerboard test against one isolate (NDM+OXA-48 positive) with the MEM-COL combination, synergistic activity and bactericidal effect were observed at the 12th and 24th hours with the 1xMIC MEM + 1xMIC COL combination in the time-kill assay against the same isolate. In conclusion, the combination of CZA-AZT seems to be a promising treatment option in infections caused by NDM positive K.pneumoniae isolates. In cases where CZA-AZT combination therapy cannot be applied, MEM-COL combination therapy may be considered as an alternative treatment option. In situations where both treatment options cannot be used, cefiderocol monotherapy may be considered as an alternative in selected cases, given the low and heterogeneous susceptibility rates among NDM-positive isolates, provided that close clinical and microbiological monitoring is ensured. However, careful monitoring is required during cefiderocol therapy and treatment modification should be considered in cases of clinical non-response.

Faithful DNA replication in Escherichia coli requires the DNA polymerase III holoenzyme (DNA pol III HE) and its clamp loader complex, which couples processive DNA synthesis with β -clamp loading. The clamp loader accessory subunits χ and ψ link single-stranded DNA-binding protein (SSB) to the replisome, stabilizing replication on SSB-coated templates through interactions with the SSB C-terminal tail. Chi has also been implicated in tolerance to the chain-terminating nucleoside analog azidothymidine (AZT), though whether this function depends on χ within DNA pol III HE or on its independent interaction with the YoaA helicase remains unclear. To address this, we engineered ψ - χ fusion proteins with flexible glycine-serine linkers to tether the two subunits while preserving folding and activity. Both fusions were biochemically competent, supporting ATP hydrolysis and clamp loading on SSB-coated DNA. In vivo, however, neither the ψ -GS12- χ fusion nor expression of a ψχ operon restored AZT tolerance in ΔholC cells, whereas expression of χ alone was sufficient. Fusion expression impaired growth in both wild-type (WT) and ΔholC backgrounds, a phenotype alleviated by disrupting χ -SSB binding. These findings support a model in which χ must dynamically engage SSB and YoaA outside of the clamp loader to promote AZT tolerance, highlighting the importance of regulated χ -SSB interactions in genome maintenance.

Zidovudine (AZT), a nucleoside reverse transcriptase inhibitor, exerts its antiviral effect through intracellular conversion to AZT-triphosphate (AZT-TP), with therapeutic efficacy and toxicity closely linked to intracellular AZT-TP levels rather than plasma AZT concentrations. However, research on AZT transport and phosphorylation in the Chinese population is scarce, and the relationship between transporter protein expression and AZT intracellular metabolism remains unclear. This study aimed to characterize the cellular transport and phosphorylation kinetics of AZT in Chinese individuals, develop a pharmacokinetic model linking plasma AZT to intracellular AZT-TP, and explore the impact of transporter expression, to support personalized antiretroviral therapy. In vitro experiments used Molt-4 and HUVEC cells. Cytotoxicity was assessed via CCK-8 assay. Concentration-dependent studies incubated cells with 0–200 µM AZT for 12 h, while time-course studies exposed cells to 100 µM AZT for 0.5–36 h, quantifying intracellular AZT and metabolites via HPLC-MS/MS. In clinical study, six healthy Chinese volunteers received a single 600 mg oral AZT dose, with blood samples collected over 10 h to measure plasma AZT and intracellular metabolites in peripheral blood mononuclear cells (PBMCs). A two-compartment pharmacokinetic model was developed, and ABC transporter expression was quantified using HPLC-MS/MS. In vitro, AZT showed negligible cytotoxicity at 50 µM for 36 h. Intracellular AZT, zidovudine monophosphate (AZT-MP), zidovudine diphosphat (AZT-DP), and zidovudine triphosphate (AZT-TP) increased linearly with extracellular AZT (0–200 µM), with time-course studies showing rapid initial accumulation followed by plateauing, and cell-type differences in kinetics. In clinical study, plasma AZT had a Tmax of 0.62 ± 0.20 h and T1/2 of 1.95 ± 0.20 h. Intracellular AZT-TP in PBMCs exhibited marked inter-individual variability. The pharmacokinetic model parameters differed from non-Chinese cohorts. ABCB1 and ABCG2 expression strongly correlated with AZT efflux, while ABCC1 did not. This study clarifies AZT’s transport and phosphorylation kinetics in Chinese subjects, showing that intracellular AZT-TP levels, linked to efficacy and toxicity, are influenced by plasma concentrations, transporter activity, and protein expression. The findings highlight the role of ABCB1 and ABCG2 in AZT efflux and support the need for individualized dosing based on intracellular metabolite levels and transporter expression, advancing personalized antiretroviral therapy in the Chinese population.