Identification of mycobacterial Thymidylate kinase inhibitors: a comprehensive pharmacophore, machine learning, molecular docking, and molecular dynamics simulation studies.

Thymidylate kinase (TMK) is a pivotal enzyme in Mycobacterium tuberculosis (Mtb), crucial for phosphorylating thymidine monophosphate (dTMP) to thymidine diphosphate (dTDP), thereby playing a critical role in DNA biosynthesis. Dysregulation or inhibition of TMK activity disrupts DNA replication and cell division, making it an attractive target for anti-tuberculosis drug development. In this study, the statistically validated pharmacophore mode was developed from a set of known TMK inhibitors. Further, the robust pharmacophore was considered for screening the Enamine database. The chemical space was reduced through multiple molecular docking approaches, pharmacokinetics, and absolute binding energy estimation. Two different molecular docking algorithms favor the strong binding affinity of the proposed molecules towards TMK. Machine learning-based absolute binding energy also showed the potentiality of the proposed molecules. The binding interactions analysis exposed the strong binding affinity between the proposed molecules and active site amino residues of TMK. Several statistical parameters from all atoms MD simulation explained the stability between proposed molecules and TMK in the dynamic states. The MM-GBSA approach also found a strong binding affinity for each proposed molecule. Therefore, the proposed molecules might be crucial TMK inhibitors for managing Mtb inhibition subjected to in vitro/in vivo validations.

Fate control engagement augments NK cell responses in LV/hu-IL-12 transduced sarcoma.

INTRODUCTION: NK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. "suicide mechanisms" regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. OBJECTIVES: We sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. METHODS: Primary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. RESULTS: AZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. CONCLUSIONS: mTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.

Molecular characterization of Drosophila melanogaster thymidylate kinase.

Drosophila has been used as an animal model to study pathogenic mechanism of neurological disorders. Thymidylate kinase (TMPK) is an essential enzyme in dTTP synthesis catalyzing the phosphorylation of dTMP to dTDP. Loss of function mutations in the DTYMK gene, coding for TMPK, cause severe microcephaly in human patients. In this study, Drosophila melanogaster TMPK (DmTMPK) was cloned, expressed, purified and characterized. Unlike human TMPK, DmTMPK phosphorylated not only dTMP and dUMP but also dGMP and dIMP although with low efficiency. ATP and dATP are the most efficient phosphate donor but at higher concentration (>1 mM) ATP inhibited DmTMPK activity. Sequence and structural model analysis explain why DmTMPK could phosphorylate purine nucleoside monophosphates. This study has laid a solid foundation for future study of TMPK function in Drosophila.

Identification of the myxobacterial secondary metabolites Aurachin A and Soraphinol A as promising inhibitors of thymidylate kinase of the Monkeypox virus.

Thymidylate kinase (TMPK) of monkeypox virus (MPXV) has emerged as a promising target for potential therapeutics due to its significant role in pyrimidine metabolism. While smallpox drugs are advised for treating monkeypox, the European Medicine Agency has sanctioned Tecovirimat due to its potent nanomolar activity. Nonetheless, there is a need for monkeypox-specific therapeutic options. In this work, we employed docking-based virtual screening and molecular dynamics (MD) simulations to identify myxobacterial secondary metabolites as promising anti-viral natural compounds capable of inhibiting thymidylate kinase. The computational pharmacokinetics and manual curation of top-scoring compounds identified six lead compounds that were compared in terms of protein-ligand contacts and protein-essential dynamics. The study shows that among the six candidates, Aurachin A and the Soraphinol analogues such as Soraphinol A and Soraphinol C remain very stable compared to other compounds, enabling the active site integrity via a stable dynamics pattern. We also show that other compounds such as Phenoxan, Phenylnannolone C, and 8E-Aurafuron B remain unstable and have a negative impact on the active site integrity and may not be suitable binders for TMPK protein. Analyzing the Aurachin A and Soraphinol A binding, the established hydrogen bonds with Arg93 and the conserved hydrophobic interaction with Tyr101 are consistent with previous experimental interactions. Additionally, a deeper insight into the indole and the aromatic ring interaction through π-π stacking and π-cation interactions, as well as the background of Aurachin A and Soraphinol A as a bioactive compound, has significant implications not only for its potential as a promising drug but also for directing future drug discovery efforts targeting the TMPK protein.

Molecular level investigation for identifying potential inhibitors against thymidylate kinase of monkeypox through in silico approaches.

The need for more advanced and effective monkeypox (Mpox) treatments has become evident with numerous Mpox virus (MPXV) outbreaks. Over the years, interest has increased in developing targeted medicines that are efficient, safe, and precise while avoiding adverse effects. Here, we screened 32409 compounds against thymidylate kinase (TMPK), an emerging target for Mpox treatment. We studied their pharmacological characteristics and analyzed those through all-atom molecular dynamics simulations followed by molecular mechanics Poisson Boltzmann surface area (MM-PBSA) based free energy calculations. According to our findings, the leads CID40777874 and CID28960001 had the highest binding affinities towards TMPK with ΔGbind of -8.04 and -5.58 kcal/mol, respectively, which outperformed our control drug cidofovir (ΔGbind = -2.92 kcal/mol) in terms of binding favourability. Additionally, we observed crucial TMPK dynamics brought on by ligand-binding and identified key residues such as Phe68 and Tyr101 as the critical points of the protein-ligand interaction. The DCCM analysis revealed the role of ligand binding in stabilizing TMPK's binding region, as indicated by residual correlation motions. Moreover, the PSN analysis revealed that the interaction with ligand induces changes in residual network properties, enhancing the stability of complexes. We successfully identified novel compounds that may serve as potential building blocks for constructing contemporary antivirals against MPXV and highlighted the molecular mechanisms underlying their binding with TMPK. Overall, our findings will play a significant role in advancing the development of new therapies against Mpox and facilitating a comprehensive understanding of their interaction patterns.Communicated by Ramaswamy H. Sarma.

Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440.

IMPORTANCE: Investigating fundamental aspects of metabolism is vital for advancing our understanding of the diverse biochemical capabilities and biotechnological applications of bacteria. The origin of the essential thymidylate kinase function in the model bacterium Pseudomonas putida KT2440, seemingly interrupted due to the presence of a large genomic island that disrupts the cognate gene, eluded a satisfactory explanation thus far. This is a first-case example of an essential metabolic function, likely acquired by horizontal gene transfer, which "landed" in a locus encoding the same activity. As such, foreign DNA encoding an essential dNMPK could immediately adjust to the recipient host-instead of long-term accommodation and adaptation. Understanding how these functions evolve is a major biological question, and the work presented here is a decisive step toward this direction. Furthermore, identifying essential and accessory genes facilitates removing those deemed irrelevant in industrial settings-yielding genome-reduced cell factories with enhanced properties and genetic stability.

Cheminformatics and machine learning approaches for repurposing anti-viral compounds against monkeypox virus thymidylate kinase.

One of the emerging epidemic concerns is Monkeypox disease which is spreading globally. This disease is caused by the monkeypox virus (MPXV), with an increasing global incidence with an outbreak in 2022. One of the novel targets for monkeypox disease is thymidylate kinase, which is involved in pyrimidine metabolism. In this study, docking-based virtual screening and molecular dynamics techniques were employed in addition to the machine learning (ML) model to investigate the potential anti-viral natural small compounds to inhibit thymidylate kinase of MPXV. Several potential hits were identified through high-throughput virtual screening, and further top three candidates were selected, which ranked using the ML model. These three compounds were then examined under molecular dynamics simulation and MM/GBSA-binding free energy analysis. Among these, Chlorhexidine HCl showed high potential for binding to the thymidylate kinase with stable and consistent conformation with RMSD < 0.3 nm. The MM/GBSA analysis also showed the minimum binding free energy (ΔGTOTAL) of -62.41 kcal/mol for this compound. Overall, this study used structure-based drug design complemented by machine learning-guided ligand-based drug design to screen potential hit compounds from the anti-viral natural compound database.

The evolving landscape of involvement of DTYMK enzymes in cancer.

Cancer cells require continuous synthesis of nucleotides for their uncontrolled proliferation. Deoxy thymidylate kinase (DTYMK) belongs to the thymidylate kinase family and is concerned with pyrimidine metabolism. DTYMK catalyzes the ATP-based conversion of deoxy-TMP to deoxy-TDP in both de novo and salvage pathways. Different studies demonstrated that DTYMK was increased in various types of cancers such as hepatocellular carcinoma, colon cancer, lung cancer, etc. Increased level of DTYMK was associated with poorer survival and prognosis, stage, grade and size of tumor, cell proliferation, colony formation, enhanced sensitivity to chemotherapy drugs, migration. Some studies were showed that knockdown of DTYMK reduced the signaling pathway of PI3K/AKT and downregulated expression of CART, MAPKAPK2, AKT1 and NRF1. Moreover, some microRNAs could suppress DTYMK expressions. On the other hand based on the TIMER database, the infiltration of macrophages, dendritic cells, neutrophils, B cells, CD4+ T cell and CD8+ T cell is affected by DTYMK. In the present review, we describe the genomic location, protein structure and isoforms of DTYMK and focus on its role in cancer development.

Machine learning-based drug design for identification of thymidylate kinase inhibitors as a potential anti-Mycobacterium tuberculosis.

The rise of antibiotic-resistant Mycobacterium tuberculosis (Mtb) has reduced the availability of medications for tuberculosis therapy, resulting in increased morbidity and mortality globally. Tuberculosis spreads from the lungs to other parts of the body, including the brain and spine. Developing a single drug can take several decades, making drug discovery costly and time-consuming. Machine learning algorithms like support vector machines (SVM), k-nearest neighbor (k-NN), random forest (RF) and Gaussian naive base (GNB) are fast and effective and are commonly used in drug discovery. These algorithms are ideal for the virtual screening of large compound libraries to classify molecules as active or inactive. For the training of the models, a dataset of 307 was downloaded from BindingDB. Among 307 compounds, 85 compounds were labeled as active, having an IC50 below 58 mM, while 222 compounds were labeled inactive against thymidylate kinase, with 87.2% accuracy. The developed models were subjected to an external ZINC dataset of 136,564 compounds. Furthermore, we performed the 100-ns dynamic simulation and post trajectories analysis of compounds having good interaction and score in molecular docking. As compared to the standard reference compound, the top three hits revealed greater stability and compactness. In conclusion, our predicted hits can inhibit thymidylate kinase overexpression to combat Mycobacterium tuberculosis.Communicated by Ramaswamy H. Sarma.

Identification of novel potential inhibitor of thymidylate kinase from Variola virus.

A hit compound was designed using Fragment Based Drug Designing (FBDD) approach, density functional theory (DFT) calculations were performed to find the structural and electronic properties. Additionally, pharmacokinetic properties were studied to understand the biological response of the compound. Docking studies were carried out with the protein structure of VrTMPK and HssTMPK with the reported hit compound. The favored docked complex was further carried to perform MD simulations; the RMSD plot and H-bond analysis was done for 200 ns. Also, MM-PBSA was done to understand the binding energy constituents and stability of the complex. A comparative study of the designed hit compound was done with FDA approved Tecovirimat. As a result, it was found that the reported compound (POX-A)is a potential selective inhibitor for Variola virus. Hence, it can be used to study further in vivo and in vitro behavior of the compound.Communicated by Ramaswamy H. Sarma.

In-silico targeting TMPK from monkey pox virus: Molecular docking analysis, density functional theory studies and molecular dynamic simulation analysis.

The World Health Organization (WHO) proclaimed the monkeypox epidemic a "public health emergency of worldwide significance" recently. The monkeypox virus is a member of the same Orthopoxvirus genus as the smallpox virus. Although smallpox medications are advised against monkeypox, no monkeypox-specific drugs are currently available. In the event of such an outbreak, in-silico medication identification is a practical and efficient strategy. As a result, we report a computational drug repurposing analysis to discover medicines that may be potential inhibitors of thymidylate kinase, a critical monkeypox viral enzyme. The target protein structure of the monkeypox virus was modeled using the vaccinia virus's homologous protein structure. Using molecular docking and density functional theory, we found 11 possible inhibitors of the monkeypox virus from an Asinex library of 261120 chemicals. The primary purpose of this in silico work is to find possible inhibitors of monkeypox viral proteins that can then be experimentally tested in order to develop innovative therapeutic medicines for monkeypox infection.Communicated by Ramaswamy H. Sarma.

Promising Antibacterial and Antifungal Agents Based on Thiolated Vitamin K3 Analogs: Synthesis, Bioevaluation, Molecular Docking.

In the present study, we designed and synthesized thiolated VK3 analogs (VK3a-g) along with an extensive antimicrobial study. After the evaluation of the antibacterial and antifungal activity against various bacterial and fungal strains, we presented an initial structure-activity relationship study on these VK3 analogs. In particular, four thiolated VK3 analogs exhibited superior biological potency against some Gram-positive bacterial strains, including Staphylococcus aureus (ATCC® 29213) and Enterococcus faecalis (ATCC® 29212). Next, all thiolated VK3 analogs were evaluated for their potential of cell growth inhibition on the NCI-60 cancer cell lines panel. This screening underlined that the thiolated VK3 analogs have no visible cytotoxicity on different cancer cell lines. The selected two thiolated VK3 analogs (VK3a and VK3b), having minimal hemolytic activity, which also have the lowest MIC values on S. aureus and E. faecalis, were further evaluated for their inhibition capacities on biofilm formation after evaluating their potential in vitro antimicrobial activity against each of the 20 clinically obtained resistant strains of Staphylococcus aureus. VK3b showed excellent antimicrobial activity against clinically resistant S. aureus isolates. Furthermore, the tested molecules showed nearly two log10 reduction in the viable cell count at six hours according to the time kill curve studies. Although these molecules decreased biofilm attachment about 50%, when sub-MIC concentrations were used these molecules increased the percentage of biofilm formation. The molecular docking of VK3a and VK3b in S. aureus thymidylate kinase was conducted in order to predict their molecular interactions. VK3a and VK3b exhibited excellent lead-likeness properties and pharmacokinetic profiles that qualify them for further optimization and development. In conclusion, since investigating efficient novel antimicrobial molecules is quite difficult, these studies are of high importance, especially in the present era of antimicrobial resistance.

A Role for the Chicken Interferon-Stimulated Gene CMPK2 in the Host Response Against Virus Infection.

Virus infection can lead to the production of interferon, which activates the JAK/STAT pathway and induces the expression of multiple downstream interferon-stimulated genes (ISGs) to achieve their antiviral function. Cytidine/uridine monophosphate kinase 2 (CMPK2) gene has been identified as an ISG in human and fish, and is also known as a rate-limiting enzyme in mitochondria to maintain intracellular UTP/CTP levels, which is necessary for de novo mitochondrial DNA synthesis. By mining previous microarray data, it was found that both Avian Influenza Virus (AIV) and Newcastle Disease Virus (NDV) infection can lead to the significant upregulation of chicken CMPK2 gene. However, little is known about the function of CMPK2 gene in chickens. In the present study, the open reading frame (ORF) of chicken CMPK2 (chCMPK2) was cloned from DF-1, a chicken embryo fibroblasts cell line, and subjected to further analysis. Sequence analysis showed that chCMPK2 shared high similarity in amino acid with CMPK2 sequences from all the other species, especially reptiles. A thymidylate kinase (TMK) domain was identified in the C-terminus of chCMPK2, which is highly conserved among all species. In vitro, AIV infection induced significant increases in chCMPK2 expression in DF-1, HD11, and the chicken embryonic fibroblasts (CEF), while obvious increase only detected in DF-1 cells and CEF cells after NDV infection. In vivo, the expression levels of chCMPK2 were up-regulated in several tissues from AIV infected chickens, especially the brain, spleen, bursa, kidney, intestine, heart and thymus, and notable increase of chCMPK2 was detected in the bursa, kidney, duodenum, lung, heart, and thymus during NDV infection. Here, using MDA5 and IFN-ß knockdown cells, we demonstrated that as a novel ISG, chCMPK2 could be regulated by the MDA5/IFN-ß pathway. The high expression level of exogenous chCMPK2 displayed inhibitory effects on AIV and NDV as well as reduced viral RNA in infected cells. We further demonstrated that Asp135, a key site on the TMK catalytic domain, was identified as critical for the antiviral activities of chCMPK2. Taken together, these data demonstrated that chCMPK2 is involved in the chicken immune system and may play important roles in host anti-viral responses.

Structure-Based Discovery of Potent Staphylococcus aureus Thymidylate Kinase Inhibitors by Virtual Screening.

INTRODUCTION: Multidrug-resistant bacteria are rapidly increasing worldwide, increasing antibiotic resistance. The exploitation, misuse, overuse, and decrease of the therapeutic potential of currently available antibiotics have resulted in the development of resistance against bacteria. As the most common bacterial pathogen in humans, Staphylococcus aureus can cause many adverse health effects. In fighting multidrug-resistant Staphylococcus aureus, scientists have identified an extremely relevant target - SaTMPK. SaTMPK is essential for DNA synthesis, which, in turn, is necessary for the replication and cell division of bacteria. OBJECTIVE: To perform multi-stage screening using the ZINC database, followed by molecular docking, ADMET profiling, molecular dynamics simulations, and energy calculations. METHODS: Based on the similar pharmacophoric characteristics of existing SaTMPK crystal structures, a model of interaction-based pharmacophores was developed. We then performed molecular docking studies on the positive hits obtained from the pharmacophore screening. Compounds that exhibited good molecular interactions within the SaTMPK binding sites were further evaluated using in-silico ADMET profiling. RESULTS: In a multi-stage screening campaign, three compounds were shortlisted that exhibited physicochemical characteristics suitable for human administration. CONCLUSION: The findings from this study should contribute to in vitro and in vivo studies for clinical applications.

Heavy metal tolerance of Mesorhizobium delmotii thymidylate kinase.

Metal ions play an important role in many metabolic processes in all living organisms. At low concentrations, heavy metals such as Fe2+, Cu2+ and Zn2+ are essential cofactors for many enzymes. However, at high concentrations they are toxic. Mesorhizobium species belong to the class α-proteobacteria and have high tolerance to soil acidity, salinity, temperature extremes, and metallicolous conditions. To identify factors responsible for this tolerance we have studied the effects of metal ions on Mesorhizobium delmotii thymidylate kinase (MdTMPK), an essential enzyme in the synthesis of dTTP, thus being vital for cell growth. We show that Mg2+ and Mn2+ are the divalent metal ions required for catalysis and that Mn2+ gives the highest catalytic efficiency. MdTMPK activity in the presence of Mg2+ was strongly inhibited by the co-presence of Zn2+, Ni2+ and Co2+. However, the addition of Cs+ caused >2-fold enhanced MdTMPK activity. For TMPK from Bacilus anthracis and humans, the effects of Mg2+ and Mn2+ were similar, whereas the effects of other divalent metal ions were different, and no stimulatory effect of Cs+ was observed. Together, our results demonstrate that MdTMPK and BaTMPK function well in the presence of high concentrations of heavy metal ions, introducing a potential contribution of these enzymes to the heavy metal tolerance of Mesorhizobium delmotii and Bacillus anthracis.

Differential expression of enzymes in thymidylate biosynthesis in zebrafish at different developmental stages: implications for dtymk mutation-caused neurodegenerative disorders.

BACKGROUND: Deoxythymidine triphosphate (dTTP) is an essential building block of DNA, and defects in enzymes involved in dTTP synthesis cause neurodegenerative disorders. For instance, mutations in DTYMK, the gene coding for thymidylate kinase (TMPK), cause severe microcephaly in human. However, the mechanism behind this is not well-understood. Here we used the zebrafish model and studied (i) TMPK, an enzyme required for both the de novo and the salvage pathways of dTTP synthesis, and (ii) thymidine kinases (TK) of the salvage pathway in order to understand their role in neuropathology. RESULTS: Our findings reveal that maternal-stored dNTPs are only sufficient for 6 cell division cycles, and the levels of dNTPs are inversely correlated to cell cycle length during early embryogenesis. TMPK and TK activities are prominent in the cytosol of embryos, larvae and adult fish and brain contains the highest TMPK activity. During early development, TMPK activity increased gradually from 6 hpf and a profound increase was observed at 72 hpf, and TMPK activity reached its maximal level at 96 hpf, and remained at high level until 144 hpf. The expression of dtymk encoded Dtymk protein correlated to its mRNA expression and neuronal development but not to the TMPK activity detected. However, despite the high TMPK activity detected at later stages of development, the Dtymk protein was undetectable. Furthermore, the TMPK enzyme detected at later stages showed similar biochemical properties as the Dtymk enzyme but was not recognized by the Dtymk specific antibody. CONCLUSIONS: Our results suggest that active dNTP synthesis in early embryogenesis is vital and that Dtymk is essential for neurodevelopment, which is supported by a recent study of dtymk knockout zebrafish with neurological disorder and lethal outcomes. Furthermore, there is a novel TMPK-like enzyme expressed at later stages of development.

First-in-class pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones against leishmaniasis and tuberculosis: Rationale, in vitro, ex vivo studies and mechanistic insights.

Pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones were synthesized, for the first time, from indole chalcones and 6-aminouracil, and their ability to inhibit leishmaniasis and tuberculosis (Tb) infections was evaluated. The in vitro antileishmanial activity against promastigotes of Leishmania donovani revealed exceptional activities of compounds 3, 12 and 13, with IC50 values ranging from 10.23 ± 1.50 to 15.58 ± 1.67 µg/ml, which is better than the IC50 value of the standard drug pentostam of 500 µg/ml. The selectivity of the compounds towards Leishmania parasites was evaluated via ex vivo studies in Swiss albino mice. The efficiency of these compounds against Tb infection was then evaluated using the in vitro anti-Tb microplate Alamar Blue assay. Five compounds, 3, 7, 8, 9 and 12, showed MIC100 values against the Mycobacterium tuberculosis H37 Rv strain at 25 µg/ml, and compound 20 yielded an MIC100 value of 50 µg/ml. Molecular modelling of these compounds highlighted interactions with binding sites of dihydrofolate reductase, pteridine reductase and thymidylate kinase, thus establishing the rationale of their pharmacological activity against both pathogens, which is consistent with the in vitro results. From the above results, it is clear that compounds 3 and 12 are promising lead candidates for Leishmania and Mycobacterium infections and may be promising for coinfections.

Structural and functional analysis of human thymidylate kinase isoforms.

Thymidylate kinase (TMPK) phosphorylates deoxythymidine monophosphate (dTMP) and plays an important role in genome stability. Deficiency in TMPK activity due to genetic alterations of DTYMK, i.e., the gene coding for TMPK, causes severe microcephaly in humans. However, no defects were observed in other tissues, suggesting the existence of a compensatory enzyme for dTTP synthesis. In search for this compensatory enzyme we analyzed 6 isoforms of TMPK mRNA deposited in the GenBank. Of these, only isoform 1 has been characterized and represents the known human TMPK. Our results reveal that isoform 2, 3, 4 and 5 lack essential structural elements for substrate binding and, thus, they are considered as nonfunctional isoforms. Isoform 6, however, has intact catalytic centers, i.e., dTMP-binding, DRX motif, ATP-binding p-loop and lid region, which are the key structural elements of an active TMPK, suggesting that isoform 6 may function as TMPK. When isoform 6 was expressed and purified, it showed only minimal activity (<0.1%) as compared with isoform 1. A putative isoform 6 was detected in a cancer cell line, in addition to the dominant isoform 1. However, because of its low activity, isoform 6 is unlikely be able to compensate for the loss of TMPK activity caused by deletions and/or point mutations of the DTYMK gene. Thereby, future studies to identify and characterize the compensatory TMPK enzyme found in patients with DTYMK mutations may contribute to the understanding of dTTP synthesis and of the pathophysiological role of DTYMK mutations in neurodegenerative disorders.

In Silico Studies of Potential Selective Inhibitors of Thymidylate Kinase from Variola virus.

Continuing the work developed by our research group, in the present manuscript, we performed a theoretical study of 10 new structures derived from the antivirals cidofovir and ribavirin, as inhibitor prototypes for the enzyme thymidylate kinase from Variola virus (VarTMPK). The proposed structures were subjected to docking calculations, molecular dynamics simulations, and free energy calculations, using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, inside the active sites of VarTMPK and human TMPK (HssTMPK). The docking and molecular dynamic studies pointed to structures 2, 3, 4, 6, and 9 as more selective towards VarTMPK. In addition, the free energy data calculated through the MM-PBSA method, corroborated these results. This suggests that these compounds are potential selective inhibitors of VarTMPK and, thus, can be considered as template molecules to be synthesized and experimentally evaluated against smallpox.

Structure-aided optimization of non-nucleoside M. tuberculosis thymidylate kinase inhibitors.

Mycobacterium tuberculosis thymidylate kinase (MtTMPK) has emerged as an attractive target for rational drug design. We recently investigated new families of non-nucleoside MtTMPK inhibitors in an effort to diversify MtTMPK inhibitor chemical space. We here report a new series of MtTMPK inhibitors by combining the Topliss scheme with rational drug design approaches, fueled by two co-crystal structures of MtTMPK in complex with developed inhibitors. These efforts furnished the most potent MtTMPK inhibitors in our assay, with two analogues displaying low micromolar MIC values against H37Rv Mtb. Prepared inhibitors address new sub-sites in the MtTMPK nucleotide binding pocket, thereby offering new insights into its druggability. We studied the role of efflux pumps as well as the impact of cell wall permeabilizers for selected compounds to potentially provide an explanation for the lack of correlation between potent enzyme inhibition and whole-cell activity.