Toxicological Profiling of Potential Shikimate Kinase Inhibitors Against Mycobacterium tuberculosis.

Over the last decade, Mycobacterium tuberculosis has mutated into a putative 'superbug', as treatments against it have failed due to increasing antimicrobial resistance. As a result, the rising incidence of multidrug-resistant tuberculosis (MDR-TB) is posing a significant public health threat, thus, the need to develop effective drugs for MDR-TB has become an urgent priority. To identify new drug candidates for the treatment of MDR-TB, the present study was based on mycobacterial shikimate kinase (MtSK) as the pharmacological target. One hundred potential MtSK inhibitors were identified from literature and database searches to identify compounds that were designed to specifically function as MtSK antagonists. The ADME properties of these compounds were evaluated by using the SwissADME web tool. ProTox-II software was also used to investigate any potential endocrine disrupting effects, mediated through their interaction with oestrogenic and/or androgenic receptors. This study also aimed to predict LD50 values of potential drug candidates that would be active against the standard H37Rv strain of M. tuberculosis, by using the ProTox-II in silico tool. The molecules for which no structural hazard alerts were identified with these software tools were further subjected to molecular docking analyses and molecular dynamic simulations to estimate their ability to interact with the MtSK enzyme. Preliminary results from SwissADME indicated that 30 molecules were drug-like, due to their physicochemical and pharmacokinetic properties. However, subsequent analysis with ToxTree and ProTox-II indicated that only three of these 30 drug-like molecules were suitable for taking forward into further in vitro experiments. This study, which is based on the use of commonly used open-source in silico tools, identified new MtSK ligands for potential use in the development of new drugs for the therapeutic management of tuberculosis. An initial prediction of their safety profile was also generated.

Inhibition of Shikimate Kinase from Methicillin-Resistant Staphylococcus aureus by Benzimidazole Derivatives. Kinetic, Computational, Toxicological, and Biological Activity Studies.

Antimicrobial resistance (AMR) is one of the biggest threats in modern times. It was estimated that in 2019, 1.27 million deaths occurred around the globe due to AMR. Methicillin-resistant Staphylococcus aureus (MRSA) strains, a pathogen considered of high priority by the World Health Organization, have proven to be resistant to most of the actual antimicrobial treatments. Therefore, new treatments are required to be able to manage this increasing threat. Under this perspective, an important metabolic pathway for MRSA survival, and absent in mammals, is the shikimate pathway, which is involved in the biosynthesis of chorismate, an intermediate for the synthesis of aromatic amino acids, folates, and ubiquinone. Therefore, the enzymes of this route have been considered good targets to design novel antibiotics. The fifth step of the route is performed by shikimate kinase (SK). In this study, an in-house chemical library of 170 benzimidazole derivatives was screened against MRSA shikimate kinase (SaSK). This effort led to the identification of the first SaSK inhibitors, and the two inhibitors with the greatest inhibition activity (C1 and C2) were characterized. Kinetic studies showed that both compounds were competitive inhibitors with respect to ATP and non-competitive for shikimate. Structural analysis through molecular docking and molecular dynamics simulations indicated that both inhibitors interacted with ARG113, an important residue involved in ATP binding, and formed stable complexes during the simulation period. Biological activity evaluation showed that both compounds were able to inhibit the growth of a MRSA strain. Mitochondrial assays showed that both compounds modify the activity of electron transport chain complexes. Finally, ADMETox predictions suggested that, in general, C1 and C2 can be considered as potential drug candidates. Therefore, the benzimidazole derivatives reported here are the first SaSK inhibitors, representing a promising scaffold and a guide to design new drugs against MRSA.

Identification of potential inhibitors of Mycobacterium tuberculosis shikimate kinase: molecular docking, in silico toxicity and in vitro experiments.

Tuberculosis (TB) is one of the main causes of death from a single pathological agent, Mycobacterium tuberculosis (Mtb). In addition, the emergence of drug-resistant TB strains has exacerbated even further the treatment outcome of TB patients. It is thus needed the search for new therapeutic strategies to improve the current treatment and to circumvent the resistance mechanisms of Mtb. The shikimate kinase (SK) is the fifth enzyme of the shikimate pathway, which is essential for the survival of Mtb. The shikimate pathway is absent in humans, thereby indicating SK as an attractive target for the development of anti-TB drugs. In this work, a combination of in silico and in vitro techniques was used to identify potential inhibitors for SK from Mtb (MtSK). All compounds of our in-house database (Centro de Pesquisas em Biologia Molecular e Funcional, CPBMF) were submitted to in silico toxicity analysis to evaluate the risk of hepatotoxicity. Docking experiments were performed to identify the potential inhibitors of MtSK according to the predicted binding energy. In vitro inhibitory activity of MtSK-catalyzed chemical reaction at a single compound concentration was assessed. Minimum inhibitory concentration values for in vitro growth of pan-sensitive Mtb H37Rv strain were also determined. The mixed approach implemented in this work was able to identify five compounds that inhibit both MtSK and the in vitro growth of Mtb.

Finding the First Potential Inhibitors of Shikimate Kinase from Methicillin Resistant Staphylococcus aureus through Computer-Assisted Drug Design.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important threat as it causes serious hospital and community acquired infections with deathly outcomes oftentimes, therefore, development of new treatments against this bacterium is a priority. Shikimate kinase, an enzyme in the shikimate pathway, is considered a good target for developing antimicrobial drugs; this is given because of its pathway, which is essential in bacteria whereas it is absent in mammals. In this work, a computer-assisted drug design strategy was used to report the first potentials inhibitors for Shikimate kinase from methicillin-resistant Staphylococcus aureus (SaSK), employing approximately 5 million compounds from ZINC15 database. Diverse filtering criteria, related to druglike characteristics and virtual docking screening in the shikimate binding site, were performed to select structurally diverse potential inhibitors from SaSK. Molecular dynamics simulations were performed to elucidate the dynamic behavior of each SaSK-ligand complex. The potential inhibitors formed important interactions with residues that are crucial for enzyme catalysis, such as Asp37, Arg61, Gly82, and Arg138. Therefore, the compounds reported provide valuable information and can be seen as the first step toward developing SaSK inhibitors in the search of new drugs against MRSA.

Identification of novel scaffold using ligand and structure based approach targeting shikimate kinase.

Tuberculosis (TB) remains a major global health problem. It causes ill-health among millions of people each year and rank as the second leading cause of death from an infectious disease worldwide, after the human immunodeficiency virus (HIV). Shikimate kinase is one of the major enzymes targeted for TB. Most approaches to overcome TB were based on synthesis and screening of a known compounds to obtain a few representatives with desired potency. In this study, we have applied a virtual screening approach which combines ligand- and structure-based approaches to screen a large library of compounds as a starting point for the identification of new scaffolds for the development of shikimate kinase inhibitors. The combined approach has identified 2 new scaffolds as potential inhibitors of shikimate kinase. To prove the approach, few of the molecules and their derivatives, a total of 17 compounds, were synthesized. The compounds were tested for biological activity and shows moderate activity against shikimate kinase. The shikimate kinase enzyme inhibition study reveals that the compounds showed inhibition (IC50) at concentrations of 50 µg/mL (Compounds 21, 22, 24, 25, 26, 27, 30, 32, 34) and 25 µg/mL (14, 19, 23, 31, 33).

Mechanism of irreversible inhibition of Mycobacterium tuberculosis shikimate kinase by ilimaquinone.

Ilimaquinone (IQ), a marine sponge metabolite, has been considered as a potential therapeutic agent for various diseases due to its broad range of biological activities. We show that IQ irreversibly inactivates Mycobacterium tuberculosis shikimate kinase (MtSK) through covalent modification of the protein. Inactivation occurred with an apparent second-order rate constant of about 60 M-1 s-1. Following reaction with IQ, LC-MS analyses of intact MtSK revealed covalent modification of MtSK by IQ, with the concomitant loss of a methoxy group, suggesting a Michael-addition mechanism. Evaluation of tryptic fragments of IQ-derivatized MtSK by MS/MS demonstrated that Ser and Thr residues were most frequently modified with lesser involvement of Lys and Tyr. In or near the MtSK active site, three residues of the P-loop (K15, S16, and T17) as well as S77, T111, and S44 showed evidence of IQ-dependent derivatization. Accordingly, inclusion of ATP in IQ reactions with MtSK partially protected the enzyme from inactivation and limited IQ-based derivatization of K15 and S16. Additionally, molecular docking models for MtSK-IQ were generated for IQ-derivatized S77 and T111. In the latter, ATP was observed to sterically clash with the IQ moiety. Out of three other enzymes evaluated, lactate dehydrogenase was derivatized and inactivated by IQ, but pyruvate kinase and catalase-peroxidase (KatG) were unaffected. Together, these data suggest that IQ is promiscuous (though not entirely indiscriminant) in its reactivity. As such, the potential of IQ as a lead in the development of antitubercular agents directed against MtSK or other targets is questionable.

Understanding the Catalytic Mechanism and the Nature of the Transition State of an Attractive Drug-Target Enzyme (Shikimate Kinase) by Quantum Mechanical/Molecular Mechanical (QM/MM) Studies.

Shikimate kinase (SK) is the fifth bacterial enzyme involved in the shikimate pathway for biosynthesis of life-indispensable components, such as aromatic amino acids. The absence of the shikimate pathway in humans makes SK an attractive target for the rational design of drugs aimed at pathogenesis bacteria, such as Mycobacterium tuberculosis and Helicobacter pylori. However, an effective inhibitor of SK (e.g., a transition-state analogue) is still not available on the market due, at least in part, to a lack of knowledge on the catalytic mechanism and the nature of the rate-limiting transition state. Herein, quantum mechanical/molecular mechanical (QM/MM) reaction coordinate, molecular dynamics (MD), and free-energy simulations have been performed to answer these questions. The results presented herein demonstrate that the phosphoryl-transfer process, which is the rate-limiting step of SK-catalyzed phosphorylation of shikimic acid (SKM), is a concerted one-step reaction proceeding through a loose transition state. The computational results agree well with those of experimental studies, specifically NMR results, X-ray crystal structure observation, and activation free-energy barrier.

Protein kinase C-δ inhibitor, Rottlerin inhibits growth and survival of mycobacteria exclusively through Shikimate kinase.

The molecular bases of disease provide exceptional prospect to translate research findings into new drugs. Nevertheless, to develop new and novel chemical entities takes huge amount of time and efforts, mainly due to the stringent processes. Therefore, drug repurposing is one of such strategies which is being used in recent times to identify new pharmacophores. The essential first step in discovery of the specific inhibitor with low toxicity is the identification and elucidation of pathways exclusive to target pathogen. One such target is the shikimate pathway, which is essential for algae, higher plants, bacteria and fungi. Since, this enzyme system is absent in higher eukaryotes and in mammals, the enzymes involved in the pathway provide an attractive target for the development of potentially selective and non toxic antimicrobial agents. Since, so far there is no specific inhibitor which is able to restrain mycobacterial shikimate pathway; we expanded the use of a known kinase inhibitor; Rottlerin, in order to predict the prototype in discovering the specific molecules against this enzyme. For the first time we have shown that Rottlerin inhibits extracellular mycobacteria by affecting Shikimate Kinase (SK) and this effect is further enhanced during the intracellular infection due to the added effect of PKC- δ down-regulation. The molecular docking of Rottlerin with both the mycobacterial SKs, corroborated the inhibition data, and revealed that the effects of SK, in slow and in fast grower mycobacteria are due to the changes in affinity of binding with the drug.

Screening of antitubercular compound library identifies novel shikimate kinase inhibitors of Mycobacterium tuberculosis.

Shikimate kinase of Mycobacterium tuberculosis is involved in the biosynthesis of aromatic amino acids through shikimate pathway. The enzyme is essential for the survival of M. tuberculosis and is absent from mammals, thus providing an excellent opportunity for identifying new chemical entities to combat tuberculosis with a novel mechanism of action. In this study, an antitubercular library of 1000 compounds was screened against M. tuberculosis shikimate kinase (MtSK). This effort led to the identification of 20 inhibitors, among which five promising leads exhibited half maximal inhibitory concentration (IC50) values below 10 µM. The most potent inhibitor ("5631296") showed an IC50 value of 5.10 µM ± 0.6. The leads were further evaluated for the activity against multidrug-resistant (MDR)-TB, Gram-positive and Gram-negative bacterial strains, mode of action, docking simulations, and combinatorial study with three frontline anti-TB drugs. Compound "5491210" displayed a nearly synergistic activity with rifampicin, isoniazid, and ethambutol while compound "5631296" was synergistic with rifampicin. In vitro cytotoxicity against HepG2 cell line was evaluated and barring one compound; all were found to be non-toxic (SI > 10). In order to rule out mitochondrial toxicity, the promising inhibitors were also evaluated for cell cytotoxicity using galactose medium where compounds "5631296" and "5122752" appeared non-toxic. Upon comprehensive analysis, compound "5631296" was found to be the most promising MtSK inhibitor that was safe, synergistic with rifampicin, and bactericidal against M. tuberculosis.

Isolation of alpha-linolenic acid from Sutherlandia frutescens and its inhibition of Mycobacterium tuberculosis' shikimate kinase enzyme.

BACKGROUND: Sutherlandia frutescens (L) R.Br. is one of traditional herbal medicines that formed the basis of primary health care systems since the earliest days and is still widely used. Sutherlandia is prescribed for people with tuberculosis (TB), but is still not known which compound(s) acts against M. tuberculosis and its mode of action. The aim of this study was to identify and isolate antimycobacterial compounds from S. frutescens extracts against shikimate kinase, a drug target for M. tuberculosis. METHODS: S. frutescens were dried, ground and extracted with ethanol, dichloromethane: methanol and water. Fractionation and separation of compounds was done with column chromatography. Chromatograms were developed in butanol/acetic acid/water (BAW) [21:6:3]; chloroform/methanol/water/formic acid (CMWF1) [60:15:2:1] and (CMWF2) [21:9:1:0.3]. Separation and isolation of active compounds were done using preparative HPLC. The activity of the plant extracts were also screened against shikimate kinase enzyme (MtbSK) using the MtbSK inhibition assay. RESULTS: The DCM: MeOH (1:1) extract showed a high percentage inhibition (with an IC50 of 0.1 µg/ml) of MtbSK and the purified inhibitor was an Alpha-Linolenic Acid (ALA) compound and it had a significant IC50 of 3.7 µg/ml. CONCLUSIONS: This study demonstrated that ALA from S. frustescens is an inhibitor of shikimate kinase a good drug target for M. tuberculosis.

Design, Synthesis, and Characterization of Some Hybridized Pyrazolone Pharmacophore Analogs against Mycobacterium tuberculosis.

Twenty-seven hybridized pyrazolone analogs were designed, docked, synthesized in two series and evaluated for their in vitro antimycobacterial properties. In the first series, four Schiff base derivatives, 6b, 7b, 7h, and 7i, show good antitubercular activity with minimum inhibition concentration (MIC) values in the range of 32.56-42.55 µM. In the second series, two compounds, 8b and 8c, possessed significant antitubercular activity with MIC <0.37 and <0.44 µM, respectively; they were even more potent than the standards pyrazinamide (12.99 µM), ciprofloxacin (4.82 µM), and streptomycin (5.36 µM), with a selectivity index of >630. Compounds 8b and 8c showed shikimate kinase inhibition activity at 5.84 and 6.93 µM, respectively. The activity and docking results lead to the conclusion that the compounds without double bond in the imine side chain and hydrophobic clashes at the pyrazolone end are necessary for good accommodation in the binding pocket and for imparting flexibility. All the compounds were also tested for antimicrobial activity (antibacterial and antifungal) and show highly significant activities against all the microorganisms tested.

Studies on the production of shikimic acid using the aroK knockout strain of Bacillus megaterium.

Shikimic acid has various pharmaceutical and industrial applications. It is the sole chemical building block for the antiviral drug oseltamivir (Tamiflu(®)) and one of the potent pharmaceutical intermediates with three chiral centres. Here we report a modified strain of Bacillus megaterium with aroK (shikimate kinase) knock out to block the aromatic biosynthetic pathway downstream of shikimic acid. Homologous recombination based gene disruption approach was used for generating aroK knock out mutant of B. megaterium. Shake flask cultivation showed shikimic acid yield of 2.98 g/L which is ~6 times more than the wild type (0.53 g/L). Furthermore, the shikimate kinase activity was assayed and it was 32 % of the wild type. Effect of various carbon sources on the production of shikimic acid was studied and fructose (4 %, w/v) was found to yield maximum shikimic acid (4.94 g/L). The kinetics of growth and shikimic acid production by aroK knockout mutant was studied in 10 L bioreactor and the yield of shikimic acid had increased to 6 g/L which is ~12 fold higher over the wild type. It is evident from the results that aroK gene disruption had an immense effect in enhancing the shikimic acid production.

Structure of shikimate kinase, an in vivo essential metabolic enzyme in the nosocomial pathogen Acinetobacter baumannii, in complex with shikimate.

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that is an important cause of healthcare-associated infections exhibiting high mortality rates. Clinical isolates of multidrug-resistant (MDR) and extremely drug-resistant (XDR) A. baumannii strains are increasingly being observed. Compounding this concern is the dearth of new antibacterial agents in late-stage development that are effective against MDR and XDR A. baumannii. As part of an effort to address these concerns, two genes (aroA and aroC) of the shikimate pathway have previously been determined to be essential for the growth and survival of A. baumannii during host infection (i.e. to be essential in vivo). This study expands upon these results by demonstrating that the A. baumannii aroK gene, encoding shikimate kinase (SK), is also essential in vivo in a rat soft-tissue infection model. The crystal structure of A. baumannii SK in complex with the substrate shikimate and a sulfate ion that mimics the binding interactions expected for the ß-phosphate of ATP was then determined to 1.91 Å resolution and the enzyme kinetics were characterized. The flexible shikimate-binding domain and LID region are compared with the analogous regions in other SK crystal structures. The impact of structural differences and sequence divergence between SKs from pathogenic bacteria that may influence antibiotic-development efforts is discussed.

Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis.

Aromatic amino acids are precursors of numerous plant secondary metabolites with diverse biological functions. Many of these secondary metabolites are already being used as active pharmaceutical or nutraceutical ingredients, and there are numerous exploratory studies of other compounds with promising applications. p-Coumaric acid is derived from aromatic amino acids and, besides being a valuable chemical building block, it serves as precursor for biosynthesis of many secondary metabolites, such as polyphenols, flavonoids, and some polyketides. Here we developed a p-coumaric acid-overproducing Saccharomyces cerevisiae platform strain. First, we reduced by-product formation by knocking out phenylpyruvate decarboxylase ARO10 and pyruvate decarboxylase PDC5. Second, different versions of feedback-resistant DAHP synthase and chorismate mutase were overexpressed. Finally, we identified shikimate kinase as another important flux-controlling step in the aromatic amino acid pathway by overexpressing enzymes from Escherichia coli, homologous to the pentafunctional enzyme Aro1p and to the bifunctional chorismate synthase-flavin reductase Aro2p. The highest titer of p-coumaric acid of 1.93 ± 0.26 g L(-1) was obtained, when overexpressing tyrosine ammonia-lyase TAL from Flavobacterium johnsoniaeu, DAHP synthase ARO4(K229L), chorismate mutase ARO7(G141S) and E. coli shikimate kinase II (aroL) in Δpdc5Δaro10 strain background. To our knowledge this is the highest reported titer of an aromatic compound produced by yeast. The developed S. cerevisiae strain represents an attractive platform host for production of p-coumaric-acid derived secondary metabolites, such as flavonoids, polyphenols, and polyketides.

Exploring Indonesian actinomycete extracts for anti-tubercular compounds: Integrating inhibition assessment, genomic analysis, and prediction of its target by molecular docking.

Tuberculosis (TB) is the foremost cause of infectious fatality globally. The primary global challenge in combatting TB lies in addressing the emergence of drug-resistant variants of the disease. However, the number of newly approved agents for treating TB has remained remarkably low over recent decades. Hence, research endeavors for discovering novel anti-TB agents are always needed. In the present study, we screened over 1,500 culture extracts from actinomycetes isolated in Indonesia for their inhibitory activity against Mycobacterium smegmatis used as a surrogate in the primary screening. The initial screening yielded approximately 6.2 % hit extracts, with a selection criterion of >80 % growth inhibition. The confirmed hit extracts were subsequently subjected to growth inhibition assay against Mycobacterium bovis and Mycobacterium tuberculosis. Approximately 20 % of the hit extracts that showed growth inhibition also exhibited efficacy against M. bovis BCG and M. tuberculosis H37Rv pathogenic strain. An active compound was successfully purified from a large-scale culture of the most potent representative extract by high-performance liquid chromatography and thin-layer chromatography. The structure of the active compound was elucidated by mass spectrometry and nuclear magnetic resonance. This compound displayed structural similarities to actinomycin group and exhibited robust inhibition, with IC50 values of 0.74, 0.02, and 0.07 µg/mL against M. smegmatis, M. bovis, and M. tuberculosis, respectively. The Actinomycetes strain A612, which produced the active compound, was taxonomically classified by phylogenetic analysis of 16s rRNA gene and whole genome sequencing data as Streptomyces parvus. Computational genome analysis utilizing anti-SMASH 7.0 unveiled that S. parvus A612 strain harbors 40 biosynthetic gene clusters with the potential to produce 16 known (with >70 % similarity) and 24 unknown compounds. A non-ribosomal peptide synthesis (NRPS) gene cluster associated with actinomycin D biosynthesis was also identified, boasting an 85 % similarity. Molecular docking analysis of actinomycin D and 21 potential M. tuberculosis targets revealed possible interactions with multiple targets. The purified active compound inhibited recombinant M. tuberculosis shikimate kinase (MtSK), which validated the results obtained from the docking analysis.

Shikimate Kinase Inhibitors: An Update on Promising Strategy against Mycobacterium tuberculosis.

Humanity has been battling with tuberculosis (TB) for a long period, and despite the availability of drugs well-known to act against the deadly microbe, the menace is still very far from reaching its end. Moreover, problems related to TB chemotherapy, such as lengthy treatment periods leading to poor patient compliance, increasing drug resistance, and association with another deadlier disease HIV-AIDS, make the situation alarming, thereby pressing the need for the discovery of new potent drugs urgently. Therefore, a drug target that is essential for survival and exclusive to M. tuberculosis presents a promising platform to explore novel molecules against the microorganism for better pathogen clearance with minimal toxicity. The shikimate pathway that leads to the synthesis of essential aromatic amino acids is one such attractive target. Shikimate kinase, the fifth enzyme of this pathway, converts shikimate to shikimate-3-phosphate by using ATP as a cosubstrate. Targeting shikimate kinase could be an effective strategy in light of its essentiality and absence of any homologue in mammals. This review discusses different strategies adopted for discovering novel compounds or scaffolds targeting M. tuberculosis shikimate kinase (MtSK) in vitro. The application of substrate analogues, their structure, and ligand-based approach for screening a library of anti-mycobacterial compounds, marine-derived molecules, and commercially available libraries have yielded promising MtSK inhibitors exhibiting micro-molar activities. To develop these leads into future drugs with minimum off-target effects on the host microenvironment, the molecules need to be structurally optimized for improved activities against enzymes and whole-cell organisms.

An overview of mechanism and chemical inhibitors of shikimate kinase.

Tuberculosis is a highly infectious disease other than HIV/AIDS and it is one of the top ten causes of death worldwide. Resistance development in the bacteria occurs because of genetic alterations, and the molecular insights suggest that the accumulation of mutation in the individual drug target genes is the primary mechanism of multi-drug resistant tuberculosis. Chorismate is an essential structural fragment for the synthesis of aromatic amino acids and synthesized biochemically by a number of bacteria, including Mycobacterium tuberculosis, utilizing the shikimate pathway. This shikimate kinase is the newer possible target for the generation of novel antitubercular drug because this pathway is expressed only in mycobacterium and not in Mammals. The discovery and development of shikimate kinase inhibitors provide an opportunity for the development of novel selective medications. Multiple shikimate kinase inhibitors have been identified via insilico virtual screening and related protein-ligand interactions along with their in-vitro studies. These inhibitors bind to the active site in a similar fashion to shikimate. In the current review, we present an overview of the biology and chemistry of the shikimate kinase protein and its inhibitors, with special emphasis on the various active scaffold against the enzyme. A variety of chemically diversified synthetic scaffolds including Benzothiazoles, Oxadiazoles, Thiobarbiturates, Naphthoquinones, Thiazoleacetonitriles, Hybridized Pyrazolone derivatives, Orthologous biological macromolecule derivatives, Manzamine Alkaloids derivatives, Dipeptide inhibitor, and Chalcones are discussed in detail. These derivatives bind to the specific target appropriately proving their potential ability through different binding interactions and effectively explored as an effective and selective Sk inhibitor.Communicated by Ramaswamy H. Sarma.

Multiple Metabolic Engineering Strategies to Improve Shikimate Titer in Escherichia coli.

Shikimate is a valuable chiral precursor for synthesizing oseltamivir (Tamiflu®) and other chemicals. High production of shikimate via microbial fermentation has attracted increasing attention to overcome the unstable and expensive supply of shikimate extracted from plant resources. The current cost of microbial production of shikimate via engineered strains is still unsatisfactory, and thus more metabolic strategies need to be investigated to further increase the production efficiency. In this study, we first constructed a shikimate E. coli producer through the application of the non-phosphoenolpyruvate: carbohydrate phosphotransferase system (non-PTS) glucose uptake pathway, the attenuation of the shikimate degradation metabolism, and the introduction of a mutant of feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase. Inspired by the natural presence of bifunctional 3-dehydroquinate dehydratase (DHD)-shikimate dehydrogenase (SDH) enzyme in plants, we then designed an artificial fusion protein of DHD-SDH to decrease the accumulation of the byproduct 3-dehydroshikimate (DHS). Subsequently, a repressed shikimate kinase (SK) mutant was selected to promote shikimate accumulation without the supplementation of expensive aromatic substances. Furthermore, EsaR-based quorum sensing (QS) circuits were employed to regulate the metabolic flux distribution between cell growth and product synthesis. The final engineered strain dSA10 produced 60.31 g/L shikimate with a yield of 0.30 g/g glucose in a 5 L bioreactor.

Identification of novel antimicrobial compounds targeting Mycobacterium tuberculosis shikimate kinase using in silico hierarchical structure-based drug screening.

The development of new anti-TB drugs to prevent the spread of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains is imperative. Mtb shikimate kinase (MtSK) was selected as the target protein to screen for new anti-TB drugs. We performed hierarchical in silico screening using a library of 154,118 compounds to search for novel compounds that could bind to the active site of MtSK. The growth-inhibitory effects of the candidate compounds on Mycobacterium smegmatis were evaluated in vitro. Nine of the 11 candidate compounds exhibited inhibitory effects against mycobacteria in vitro. The inhibitory activity of Compound 2 (IC50 = 1.39 µM) was higher than that of isoniazid, the first-line drug for TB treatment. Moreover, Compound 2 did not exhibit toxicity against mammalian cells and Escherichia coli. Molecular dynamics simulations using the MtSK-Compound 2 complex structure in a timeframe of 100 ns suggested that Compound 2 could stably bind to MtSK. The binding free energy of Compound 2 was estimated to be -37.96 kcal/mol using the MM/PBSA method, demonstrating that Compound 2 can stably bind to MtSK. These in silico and in vitro results indicated that Compound 2 is a promising hit compound for the development of novel anti-TB drugs.

Synthesis, molecular docking, and biological evaluation of novel 1,2,4-triazole-isatin derivatives as potential Mycobacterium tuberculosis shikimate kinase inhibitors.

The issue of emerging resistance to antitubercular drugs has created a formidable barrier in the effective prevention and cure of tuberculosis globally. In an effort to search for new antimycobacterial agents, possibly comprising new pharmacophore, novel triazole-isatin derivatives were designed as Mycobacterium tuberculosis shikimate kinase inhibitors and synthesized by microwave-assisted method. The synthesized molecules were evaluated for their antimycobacterial activity by MABA assay against M. tuberculosis H37Rv. The molecule 5h demonstrated MIC of 0.8 µg/ml and good safety profile with higher selectivity index with HEK293 cell line. The antimycobacterial activity was further substantiated with molecular docking studies. The triazole-isatin derivatives showed significant binding interactions with amino acid residues in the active site of the enzyme. These studies revealed that molecule 5h could act as a potential lead molecule for further studies to find new target-directed molecules.