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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To front emergence of antibiotic resistance there is an urgent need for new therapeutics, and one seemingly relevant target is thymidylate monophosphate kinase (TMPK). Serendipitously, we discovered a naphthyl substituted pyrrolopyrimidine possessing activity towards E. coli TMPK. Based on this hit, synthesis, and screening of 61 fused pyrimidines were undertaken. The most potent derivatives were also counter assayed towards the human variant of the enzyme. Two of the inhibitors possessed promising drug‐like properties and selectivity for E. coli TMPK. Although the initial pyrrolopyrimidine hit failed to have cellular activity, two alternative scaffolds were discovered providing starting points for further work. [ABSTRACT FROM AUTHOR]

Assam is a state with different ethnic groups which are known for their distinct identity and colourful culture. If we look after the Independence then we find that in the nation building process the Indian state framed policies which aims at homogenization and integration based on a dominant language. Such policies by the state opened up doors for ethnic communities to assert their identity. The Mising autonomy movement is one such example in which the Mising tribe demanded for the autonomy of their community. The implementation of the Assam Official Language Act of 1960 and various other reasons augmented the struggle for autonomy. After struggling for many years, the Mising Autonomous Council was established in 2005 fulfilling the demand and expectations of the Mising people. However, the movement did not end there and sporadic movements have been going on for various other reasons. This paper is an attempt to study the autonomy movement and the creation of the Mising Autonomous Council and the recent developments thereafter. [ABSTRACT FROM AUTHOR]

Cellular supply of deoxythymidine triphosphate (dTTP) is crucial for DNA replication and repair. Thymidylate kinase (TMPK) catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dTTP synthesis. Despite their major cellular localization in cytosol, TMPK and ribonucleotide reductase (RNR) are detected at DNA damage sites for local dNDP formation. Because deoxyuridine diphosphate is synthesized by RNR, the simultaneous recruitment of TMPK and RNR to DNA damage sites is critical for preventing deoxyuridine triphosphate-mediated toxic repair. This study investigates the mechanism responsible for the recruitment of TMPK to DNA damage sites. Our data demonstrate the requirement of ataxia telangiectasia mutated (ATM) kinase activity for TMPK recruitment to DNA lesion sites. Moreover, we find that TMPK is able to form the complex with histone acetyltransferase Tip60 and RNR. Inhibition of ATM kinase reduces the complex formation and TMPK phosphorylation. Our analysis further shows the presence of TMPK phosphorylation at serine 88, which is an ATM kinase consensus site. A phosphorylation-defective mutation at this site suppresses TMPK recruitment to DNA damage sites and the complex formation with Tip60. Finally, we provide evidence that this site is critical for the function of TMPK in DNA repair but not for catalytic activity. Together, these findings suggest that Tip60-ATM signaling has a functional contribution to the recruitment of TMPK to DNA damage sites, thereby increasing local dTTP synthesis for DNA repair. [ABSTRACT FROM AUTHOR]

Monkeypox (MPXV) is a globally growing public health concern with 80,328 active cases and 53 deaths have been reported. No specific vaccine or drug is available for the treatment of MPXV. Hence, the current study also employed structure-based drug designing, molecular simulation, and free energy calculation methods to identify potential hit molecules against the TMPK of MPXV, which is a replicatory protein that helps the virus to replicate its DNA and increase the number of DNAs in the host cell. The 3D structure of TMPK was modeled with AlphaFold and screening of multiple natural products libraries (4,71,470 compounds) identified TCM26463, TCM2079, and TCM29893 from traditional Chinese medicines database (TCM), SANC00240, SANC00984, and SANC00986 South African natural compounds database (SANCDB), NPC474409, NPC278434 and NPC158847 from NPASS (natural product activity and species source database) while CNP0404204, CNP0262936, and CNP0289137 were shortlisted from coconut database (collection of open natural products) as the best hits. These compounds interact with the key active site residues through hydrogen bonds, salt bridges, and pie-pie interactions. The structural dynamics and binding free energy results further revealed that these compounds possess stable dynamics with excellent binding free energy scores. Moreover, the dissociation constant (K D ) and bioactivity analysis revealed stronger activity of these compounds exhibit stronger biological activity against MPXV and may inhibit it in in vitro conditions. All the results demonstrated that the designed novel compounds possess stronger inhibitory activity than the control complex (TPD-TMPK) from the vaccinia virus. The current study is the first to design small molecule inhibitors for the replication protein of MPXV which may help in controlling the current epidemic and also overcome the challenge of vaccine evasion., Competing Interests: Declaration of competing interest Declared none., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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.

Tuberculosis (TB) is a chronic infectious disease with high epidemiological rates. The emergence of multidrug and extensively drug-resistant TB strains, as well as the long-term treatment and the side effects, become urgent the need for the development of new anti-tuberculosis drugs. Furthermore, the search for new targets is also necessary; as the current antimycobacterial drugs have just a small number of enzymes related to essential functions of the mycobacteria as targets. TMPKmt is an attractive target for the design of new antituberculosis agents since this enzyme is essential for DNA replication. In this work, we used the 3D-QSAR, by applying the Comparative Molecular Field Analysis (CoMFA), in order to elucidate the structural requirements relevant to the biological activity and to generate 3D-QSAR models for predicting the biological activity of compounds not yet synthesized, considering quantitative biological data and the interaction with TMPKmt of 106 thymidine analogues obtained from the literature. Robust and significant statistical models were obtained, confirming the importance of groups with higher electronic density and less bulky near ring 2. The studies herein developed with thymidine analogues through rational drug design lead to important data towards the search of new candidates as new anti-tuberculosis drugs. [ABSTRACT FROM AUTHOR]

Bacillus anthracis is well known in connection with biological warfare. The search for new drug targets and antibiotics is highly motivated because of upcoming multiresistant strains. Thymidylate kinase is an ideal target since this enzyme is at the junction of the de novo and salvage synthesis of dTTP, an essential precursor for DNA synthesis. Here the expression and characterization of thymidylate kinase from B. anthracis ( Ba-TMPK) is presented. The enzyme phosphorylated deoxythymidine-5′-monophosphate (dTMP) efficiently with K m and V max values of 33 μM and 48 μmol mg−1 min−1, respectively. The efficiency of deoxyuridine-5′-monophosphate phosphorylation was ∼10% of that of dTMP. Several dTMP analogs were tested, and D-FMAUMP (2′-fluoroarabinosyl-5-methyldeoxyuridine-5′-monophosphate) was selectively phosphorylated with an efficiency of 172% of that of D-dTMP, but l-FMAUMP was a poor substrate as were 5-fluorodeoxyuridine-5′-monophosphate (5FdUMP) and 2′,3′-dideoxy-2′,3′-didehydrothymidine-5′-monophosphate (d4TMP). No activity could be detected with 3′-azidothymidine-5′-monophosphate (AZTMP). The corresponding nucleosides known as efficient anticancer and antiviral compounds were also tested, and d-FMAU was a strong inhibitor with an IC50 value of 10 μM, while other nucleosides- l-FMAU, dThd, 5-FdUrd, d4T, and AZT, and 2′-arabinosylthymidine-were poor inhibitors. A structure model was built for Ba-TMPK based on the Staphylococcus aureus TMPK structure. Docking with various substrates suggested mechanisms explaining the differences in substrate selectivity of the human and the bacterial TMPKs. These results may serve as a start point for development of new antibacterial agents. [ABSTRACT FROM AUTHOR]

Cellular supply of deoxythymidine triphosphate (dTTP) is crucial for DNA replication and repair. Thymidylate kinase (TMPK) catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dTTP synthesis. Despite their major cellular localization in cytosol, TMPK and ribonucleotide reductase (RNR) are detected at DNA damage sites for local dNDP formation. Because deoxyuridine diphosphate is synthesized by RNR, the simultaneous recruitment of TMPK and RNR to DNA damage sites is critical for preventing deoxyuridine triphosphate-mediated toxic repair. This study investigates the mechanism responsible for the recruitment of TMPK to DNA damage sites. Our data demonstrate the requirement of ataxia telangiectasia mutated (ATM) kinase activity for TMPK recruitment to DNA lesion sites. Moreover, we find that TMPK is able to form the complex with histone acetyltransferase Tip60 and RNR. Inhibition of ATM kinase reduces the complex formation and TMPK phosphorylation. Our analysis further shows the presence of TMPK phosphorylation at serine 88, which is an ATM kinase consensus site. A phosphorylation-defective mutation at this site suppresses TMPK recruitment to DNA damage sites and the complex formation with Tip60. Finally, we provide evidence that this site is critical for the function of TMPK in DNA repair but not for catalytic activity. Together, these findings suggest that Tip60-ATM signaling has a functional contribution to the recruitment of TMPK to DNA damage sites, thereby increasing local dTTP synthesis for DNA repair.-Hu, C.-M., Tsao, N., Wang, Y.-T., Chen, Y.-J., Chang, Z.-F. Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.