Search

The emergence of drug-resistant Mycobacterium tuberculosis strains highlights the need to discover anti-tuberculosis drugs with novel mechanisms of action. Here we discovered a mycobactericidal strategy based on the prodrug activation of selected chemical derivatives classified as nitronaphthofurans (nNFs) mediated by the coordinated action of the sigH and mrx2 genes. The transcription factor SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in M. tuberculosis. The nNF action induced the SigH stress response which in turn induced the mrx2 overexpression. The nitroreductase Mrx2 was found to activate nNF prodrugs, killing replicating, non-replicating and intracellular forms of M. tuberculosis. Analysis of SigH DNA sequences obtained from spontaneous nNF-resistant M. tuberculosis mutants suggests disruption of SigH binding to the mrx2 promoter site and/or RNA polymerase core, likely promoting the observed loss of transcriptional control over Mrx2. Mutations found in mrx2 lead to structural defects in the thioredoxin fold of the Mrx2 protein, significantly impairing the activity of the Mrx2 enzyme against nNFs. Altogether, our work brings out the SigH/Mrx2 stress response pathway as a promising target for future drug discovery programs., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Swapping the substituents in positions 2 and 4 of the previously synthesized but yet undisclosed 5-cyano-4-(methylthio)-2-arylpyrimidin-6-ones 4 , ring closure, and further optimization led to the identification of the potent antitubercular 2-thio-substituted quinazolinone 26 . Structure-activity relationship (SAR) studies indicated a crucial role for both meta -nitro substituents for antitubercular activity, while the introduction of polar substituents on the quinazolinone core allowed reduction of bovine serum albumin (BSA) binding ( 63c , 63d ). While most of the tested quinazolinones exhibited no cytotoxicity against MRC-5, the most potent compound 26 was found to be mutagenic via the Ames test. This analogue exhibited moderate inhibitory potency against Mycobacterium tuberculosis thymidylate kinase, the target of the 3-cyanopyridones that lies at the basis of the current analogues, indicating that the whole-cell antimycobacterial activity of the present S -substituted thioquinazolinones is likely due to modulation of alternative or additional targets. Diminished antimycobacterial activity was observed against mutants affected in cofactor F 420 biosynthesis ( fbiC ), cofactor reduction ( fgd ), or deazaflavin-dependent nitroreductase activity ( rv3547 ), indicating that reductive activation of the 3,5-dinitrobenzyl analogues is key to antimycobacterial activity.

As the last enzyme in nucleotide synthesis as precursors for DNA replication, thymidylate kinase of M. tuberculosis (MtbTMPK) attracts significant interest as a target in the discovery of new anti-tuberculosis agents. Earlier, we discovered potent MtbTMPK inhibitors, but these generally suffered from poor antimycobacterial activity, which we hypothesize is due to poor bacterial uptake. To address this, we herein describe our efforts to equip previously reported MtbTMPK inhibitors with targeting moieties to increase the whole cell activity of the hybrid analogues. Introduction of a simplified Fe-chelating siderophore motif gave rise to analogue 17 that combined favorable enzyme inhibitory activity with significant activity against M. tuberculosis (MIC of 12.5 μM). Conjugation of MtbTMPK inhibitors with an imidazo[1,2-a]pyridine or 3,5-dinitrobenzamide scaffold afforded analogues 26, 27 and 28, with moderate MtbTMPK enzyme inhibitory potency, but sub-micromolar activity against mycobacteria without significant cytotoxicity. These results indicate that conjugation with structural motifs known to favor mycobacterial uptake may be a valid approach for discovering new antimycobacterial agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

A series of Mycobacterium tuberculosis TMPK ( Mtb TMPK) inhibitors based on a reported compound 3 were synthesized and evaluated for their capacity to inhibit Mtb TMPK catalytic activity and the growth of a virulent M. tuberculosis strain (H37Rv). Modifications of the scaffold of 3 failed to afford substantial improvements in Mtb TMPK inhibitory activity and antimycobacterial activity. Optimization of the substitution pattern of the D ring of 3 resulted in compound 21j with improved Mtb TMPK inhibitory potency (three-fold) and H37Rv growth inhibitory activity (two-fold). Moving the 3-chloro substituent of 21j to the para -position afforded isomer 21h , which, despite a 10-fold increase in IC 50 -value, displayed promising whole cell activity (minimum inhibitory concentration (MIC) = 12.5 μM).

A series of readily accessible 1-(piperidin-3-yl)thymine amides was designed, synthesised and evaluated as Mycobacterium tuberculosis TMPK ( Mtb TMPK) inhibitors. In line with the modelling results, most inhibitors showed reasonable Mtb TMPK inhibitory activity. Compounds 4b and 4i were slightly more potent than the parent compound 3 . Moreover, contrary to the latter, amide analogue 4g was active against the avirulent M. tuberculosis H37Ra strain (MIC 50 =35 µM). This finding opens avenues for future modifications.

In recent years, thymidylate kinase (TMPK), an enzyme indispensable for bacterial DNA biosynthesis, has been pursued for the development of new antibacterial agents including against Mycobacterium tuberculosis, the causative agent for the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous efforts toward the exploration of novel and potent Mycobacterium tuberculosis TMPK ( MtTMPK) inhibitors, and reported here the design of a novel series of non-nucleoside inhibitors of MtTMPK. The inhibitors display hitherto unexplored interactions in the active site of MtTMPK, offering new insights into structure-activity relationships. To investigate the discrepancy between enzyme inhibitory activity and the whole-cell activity, experiments with efflux pump inhibitors and efflux pump knockout mutants were performed. The minimum inhibitory concentrations of particular inhibitors increased significantly when determined for the efflux pump mmr knockout mutant, which partly explains the observed dissonance.

We report the design and synthesis of a series of non-nucleoside MtbTMPK inhibitors (1-14) based on the gram-positive bacterial TMPK inhibitor hit compound 1. A practical synthesis was developed to access these analogues. Several compounds show promising MtbTMPK inhibitory potency and allow the establishment of a structure-activity relationship, which is helpful for further optimization., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We report the synthesis of 5'-modified thymidines (16, 18, 21, 23) and 5,5'-bis-substituted 2'-deoxyuridine analogues (30, 47) as inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). These analogues were evaluated for their capacity to inhibit TMPKmt and solely two 5'-modified thymidines were found to possess moderate inhibitory activity. In addition, a feasibility study of protecting groups for the 5-CH(2)OH moiety of 2'-deoxyuridines is described that enables to introduce the desired 5'-modification., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A series of 5-substituted analogs of 6-aza-2'-deoxyuridine 5'-monophosphate, 6-aza-dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin-dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N-atom in 6-aza-dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 μM). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3-(octanamido)prop-1-ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 μM). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N-atom furnished 6-aza-dTMP (1b), which still was recognized as a substrate by TMPKmt., (Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.)

We report on Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) inhibitory activities of a series of new 3'- and 5'-modified thymidine analogues including α- and β-derivatives. In addition, several analogues were synthesized in which the 4-oxygen was replaced by a more lipophilic sulfur atom to probe the influence of this modification on TMPKmt inhibitory activity. Several compounds showed an inhibitory potency in the low micromolar range, with the 5'-arylthiourea 4-thio-α-thymidine analogue being the most active one (K(i)=0.17 μM). This compound was capable of inhibiting mycobacteria growth at a concentration of 25 μg/mL., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Nucleoside Monophosphate Kinases (NMPKs) family are key enzymes in nucleotide metabolism. Bacterial UMPKs depart from the main superfamily of NMPKs. Having no eukaryotic counterparts they represent attractive therapeutic targets. They are regulated by GTP and UTP, while showing different mechanisms in Gram(+), Gram(-) and archaeal bacteria. In this work, we have characterized the mycobacterial UMPK (UMPKmt) combining enzymatic and structural investigations with site-directed mutagenesis. UMPKmt exhibits cooperativity toward ATP and an allosteric regulation by GTP and UTP. The crystal structure of the complex of UMPKmt with GTP solved at 2.5 Å, was merely identical to the modelled apo-form, in agreement with SAXS experiments. Only a small stretch of residues was affected upon nucleotide binding, pointing out the role of macromolecular dynamics rather than major structural changes in the allosteric regulation of bacterial UMPKs. We further probe allosteric regulation by site-directed mutagenesis. In particular, a key residue involved in the allosteric regulation of this enzyme was identified.

Thymidine monophosphate kinase from Mycobacterium tuberculosis (TMPKmt) has been proposed as an attractive target in the search of new agents to fight against tuberculosis. We recently reported that thymine derivatives carrying a naphtholactam or naphthosultam moiety at position 4 of a (Z)-butenyl chain inhibit TMPKmt in the subμM range. Here we describe the replacement of the planar naphtholactam and naphthosultam rings in our identified hits by 5,6-dihydro-1H-imidazo[4,5,1-ij]quinolinones and a 5,6-dihydro-1H,4H-1,2,5-thiadiazolo[4,3,2-ij]quinoline-2,2-dioxide where the planarity has been broken. Interestingly, these non-planar compounds were similarly potent against the target enzyme than their aromatic analogues, suggesting a bioisosteric behavior that may also be applied to other biologically active compounds. The synthesis of the different targeted imidazoquinolinones has been successfully performed via a hypervalent iodide mediated oxidative cyclization of N-methoxyureas catalized by bis(trifluoroacetoxy)iodobenzene (PIFA) expanding the reported use of this reagent for the synthesis of differently substituted imidazoquinolinones., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

In the search for novel inhibitors of the enzyme thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt), an attractive target for novel antituberculosis agents, we report herein the discovery of the first acyclic nucleoside analogues that potently and selectively inhibit TMPKmt. The most potent compounds in this series are (Z)-butenylthymines carrying a naphtholactam or naphthosultam moiety at position 4, which display K(i) values of 0.42 and 0.27 microM, respectively. Docking studies followed by molecular dynamics simulations performed to rationalize the interaction of this new family of inhibitors with the target enzyme revealed a key interaction between the distal substituent and Arg 95 in the target enzyme. The fact that these inhibitors are more easily synthesizable than previously identified TMPKmt inhibitors, together with their potency against the target enzyme, makes them attractive lead compounds for further optimization.

A series of N(1)-(4-substituted-benzyl)-pyrimidines were synthesized as potential inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). Key SAR parameters included the chain length substitution in para position of the benzyl ring, the functional group terminating the alkyl chain, and the substituent on the C-5 pyrimidine ring. Synthesized molecules were assayed against both recombinant enzyme and mycobacteria cultures. The most potent compounds have K(i) values in the micromolar range and an MIC(50) of 50microg/mL against Mycobacterium bovis. These results will guide the design of a new generation of lead compounds.

Herein we describe the synthesis and conformational analysis of a series of bicyclic thymidine derivatives and their evaluation as inhibitors of thymidine monophosphate kinase from Mycobacterium tuberculosis (TMPKmt), based on previously discovered bicyclic sugar nucleosides. With a K(i) value of 2.3 microm, 1-[3-aminomethyl-3,5-dideoxy-2-O,6-N-(thiocarbonyl)-beta-D-ribofuranosyl]thymine emerged as the most potent TMPK inhibitor of this series. Moreover, this promising compound displays inhibitory potency against Mycobacteria cultures with an IC(99) value of 100 microg mL(-1), thus promoting TMPKmt for the first time as a validated target for further inhibitory design. Attempts to rationalise the observed structure-activity relationship (SAR) involving molecular modelling and conformational analysis are described.

The crystal structure of Mycobacterium tuberculosis adenylate kinase (MtAK) in complex with two ADP molecules and Mg2+ has been determined at 1.9 A resolution. Comparison with the solution structure of the enzyme, obtained in the absence of substrates, shows significant conformational changes of the LID and NMP-binding domains upon substrate binding. The ternary complex represents the state of the enzyme at the start of the backward reaction (ATP synthesis). The structure is consistent with a direct nucleophilic attack of a terminal oxygen from the acceptor ADP molecule on the beta-phosphate from the donor substrate, and both the geometry and the distribution of positive charge in the active site support the hypothesis of an associative mechanism for phosphoryl transfer.

Bacterial nucleoside monophosphate (NMP) kinases, which convert NMPs to nucleoside diphosphates (NDP), are investigated as potential antibacterial targets against pathogenic bacteria. Herein, we report the biochemical and structural characterization of GMP kinase from Mycobacterium tuberculosis (GMPKMt). GMPKMt is a monomer with an unusual specificity for ATP as a phosphate donor, a lower catalytic efficiency compared with eukaryotic GMPKs, and it carries two redox-sensitive cysteines in the central CORE domain. These properties were analyzed in the light of the high-resolution crystal structures of unbound, GMP-bound, and GDP-bound GMPKMt. The latter structure was obtained in both an oxidized form, in which the cysteines form a disulfide bridge, and a reduced form which is expected to correspond to the physiological enzyme. GMPKMt has a modular domain structure as most NMP kinases. However, it departs from eukaryotic GMPKs by the unusual conformation of its CORE domain, and by its partially open LID and GMP-binding domains which are the same in the apo-, GMP-bound, and GDP-bound forms. GMPKMt also features a unique GMP binding site which is less close-packed than that of mammalian GMPKs, and in which the replacement of a critical tyrosine by a serine removes a catalytic interaction. In contrast, the specificity of GMPKMt for ATP may be a general feature of GMPKs because of an invariant structural motif that recognizes the adenine base. Altogether, differences in domain dynamics and GMP binding between GMPKMt and mammalian GMPKs should reveal clues for the design of GMPKMt-specific inhibitors., (2005 Wiley-Liss, Inc.)

Tuberculosis (TB) is the primary cause of mortality among infectious diseases. Mycobacterium tuberculosis thymidylate kinase (TMPK(Mtub)) catalyzes the ATP-dependent phosphorylation of deoxythymidine 5'-monophosphate (dTMP). Essential to DNA replication, this enzyme represents a promising target for developing new drugs against TB, because the configuration of its active site is unique within the TMPK family. Indeed, it has been proposed that, as opposed to other TMPKs, catalysis by TMPK(Mtub) necessitates the transient binding of a magnesium ion coordinating the phosphate acceptor. Moreover, 3'-azidodeoxythymidine monophosphate (AZTMP) is a competitive inhibitor of TMPK(Mtub), whereas it is a substrate for human and other TMPKs. Here, the crystal structures of TMPK(Mtub) in complex with deoxythymidine (dT) and AZTMP were determined to 2.1 and 2.0 A resolution, respectively, and suggest a mechanism for inhibition. The azido group of AZTMP perturbs the induced-fit mechanism normally adopted by the enzyme. Magnesium is prevented from binding, and the resulting electrostatic environment precludes phosphoryl transfer from occurring. Our data provide a model for drug development against tuberculosis.

Thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt) represents an attractive target for selectively blocking bacterial DNA synthesis. Hereby, we report on the discovery of a novel class of bicyclic nucleosides (10 and 11) and one dinucleoside (12), belonging to the most selective inhibitors of TMPKmt discovered so far.

Tuberculosis is the leading cause of death worldwide from a single infectious disease. Search of new therapeutic tools requires the discovery and biochemical characterization of new potential targets among the bacterial proteins essential for the survival and virulence. Among them are the nucleoside monophosphate kinases, involved in the nucleotide biosynthesis. In this work, we determined the solution structure of adenylate kinase (AK) from Mycobacterium tuberculosis (AKmt), a protein of 181 residues that was found to be essential for bacterial survival. The structure was calculated by a simulated annealing protocol and energy minimization using experimental restraints, collected by nuclear magnetic resonance spectroscopy. The final, well-defined 20 NMR structures show an average root-mean-square deviation of 0.77 A for the backbone atoms in regular secondary structure segments. The protein has a central CORE domain, composed of a five-stranded parallel beta-sheet surrounded by seven alpha-helices, and two peripheral domains, AMPbd and LID. As compared to other crystallographic structures of free form AKs, AKmt is more compact, with the AMP(bd) domain closer to the CORE of the protein. Analysis of the (15)N relaxation data enabled us to obtain the global rotational correlation time (9.19 ns) and the generalized order parameters (S(2)) of amide vectors along the polypeptide sequence. The protein exhibits restricted movements on a picosecond to nanosecond time scale in the secondary structural regions with amplitudes characterized by an average S(2)() value of 0.87. The loops beta1/alpha1, beta2/alpha2, alpha2/alpha3, alpha3/alpha4, alpha4/beta3, beta3/alpha5, alpha6/alpha7 (LID), alpha7/alpha8, and beta5/alpha9 exhibit rapid fluctuations with enhanced amplitudes. These structural and dynamic features of AKmt may be related to its low catalytic activity that is 10-fold lower than in their eukaryote counterparts.

The affinity of a series of 2', 3'- and 5-modified thymidine analogues for Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) was evaluated. The affinities of several non-phosphorylated analogues are in the same order of magnitude as those of their phosphorylated congeners. In view of drug delivery problems associated with phosphorylated compounds, these 'free' nucleosides seem more promising leads in the search of TMPKmt inhibitors as novel anti-tuberculosis agents.

Thymidine monophosphate kinase (TMPK) of Mycobacterium tuberculosis (TMPKmt) represents an attractive target for blocking the bacterial DNA synthesis. In an attempt to find high-affinity inhibitors of TMPKmt, a cavity in the enzyme at the 3'-position was explored via the introduction of various substituents at the 3'-position of the thymidine monophosphate (dTMP) scaffold. Various 3'-C-branched chain substituted nucleotides in the 2'-deoxyribo (3-6) and ribo series (7, 8) were synthesized from one key intermediate (23). 2'-Deoxy analogues proved to be potent inhibitors of TMPKmt: 3'-CH(2)NH(2) (4), 3'-CH(2)N(3) (3), and 3'-CH(2)F (5) nucleotides exhibit the highest affinities within this series, with K(i) values of 10.5, 12, and 15 microM, respectively. These results show that TMPKmt tolerates the introduction of sterically demanding substituents at the 3'-position. Ribo analogues experience a significant affinity decrease, which is probably due to steric hindrance of Tyr103 in close vicinity of the 2'-position. Although the 5'-O-phosphorylated compounds have somewhat higher affinities for the enzyme, the parent nucleosides generally exhibit affinities for TMPKmt in the same order of magnitude and display a superior selectivity profile versus human TMPK. This series of inhibitors holds promise for the development of a new class of antituberculosis agents.

Thymidine monophosphate kinase (TMPK) from Mycobacterium tuberculosis (TMPKmt) is an attractive target for the design of specific inhibitors. This fact is the result of its key role in the thymidine pathway and of unique structural features in the active site observed by X-ray crystallography, especially in comparison to its human counterpart (TMPKh). Different 5-modified thymidine derivatives, as well as purine and pyrimidine analogues or C-nucleosides were tested on TMPKmt and TMPKh, and the results were rationalized by docking studies. 5-Halogenated 2'-deoxyuridines are the best inhibitors of TMPKmt found and present the highest selectivity indexes in favor of TMPKmt.

The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg(2+) ion. The crystallographic structure of the complex of one of the compounds, 5-CH(2)OH-dUMP, with TMPK has been determined at 2.0 A. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser(99). Looking for a role for Ser(99), we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu(6), Ser(99), Arg(95), and Asp(9)) that probably serves to protonate the transferred PO(3) group. The crystallographic structure of the commercially available bisubstrate analogue P(1)-(adenosine-5')-P(5)-(thymidine-5')-pentaphosphate bound to TMPK is also reported at 2.45 A and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors.

A number of 2'- and 3'-modified thymidine 5'-O-monophosphate analogues were synthesized as potential leads for new anti-mycobacterial drugs. Evaluation of their affinity for Mycobacterium tuberculosis thymidine monophosphate kinase showed that a 2'-halogeno substituent and a 3'-azido function are the most favorable leads for further development of potent inhibitors of this enzyme.

The crystal structure of Mycobacterium tuberculosis adenylate kinase (MtAK) in complex with two ADP molecules and Mg2+ has been determined at 1.9 Å resolution. Comparison with the solution structure of the enzyme, obtained in the absence of substrates, shows significant conformational changes of the LID and NMP-binding domains upon substrate binding. The ternary complex represents the state of the enzyme at the start of the backward reaction (ATP synthesis). The structure is consistent with a direct nucleophilic attack of a terminal oxygen from the acceptor ADP molecule on the β-phosphate from the donor substrate, and both the geometry and the distribution of positive charge in the active site support the hypothesis of an associative mechanism for phosphoryl transfer. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about molecular structure of Mycobacterium tuberculosis adenylate kinase.

Mycobacterium tuberculosis , the causative agent of tuberculosis, relies on thymidylate kinase (Mtb TMPK) for the synthesis of thymidine triphosphates and thus also DNA synthesis. Therefore, this enzyme constitutes a potential Achilles heel of the pathogen. Based on a previously reported Mtb TMPK 6-aryl-substituted pyridone inhibitor and guided by two co-crystal structures of Mtb TMPK with pyridone- and thymine-based inhibitors, we report the synthesis of a series of aryl-shifted cyanopyridone analogues. These compounds generally lacked significant Mtb TMPK inhibitory potency, but some analogues did exhibit promising antitubercular activity. Analogue 11i demonstrated a 10-fold increased antitubercular activity (MIC H37Rv, 1.2 μM) compared to literature compound 5. Many analogues with whole-cell antimycobacterial activity were devoid of significant cytotoxicity. Image 1 • 5-aryl-substituted cyanopyridone analogues were prepared and evaluated as anti- M. tuberculosis agents. • Biphenyl analogue 11i demonstrated promising antitubercular activity (MIC H37Rv, 1.2 μM) and reasonable selectivity. • The lack of correlation between Mtb TMPK inhibitory potency and whole-cell activity points towards a new mode of action. [ABSTRACT FROM AUTHOR]