Synthesis of 3-indolylmethyl substituted (pyrazolo/benzo)triazinone derivatives under Pd/Cu-catalysis: Identification of potent inhibitors of chorismate mutase (CM).

The chorismate mutase (CM) is considered as an attractive target for the identification of potential antitubercular agents due to its absence in animals but not in bacteria. A series of 3-indolylmethyl substituted pyrazolotriazinone derivatives were designed and docked into CM in silico as potential inhibitors. These compounds were efficiently synthesized using the Pd/Cu-catalyzed coupling-cyclization in a single pot involving the construction of indole ring. The methodology was later extended to the preparation of corresponding benzo analogs of pyrazolotriazinones i.e. 3-indolylmethyl substituted benzotriazinone derivatives. Several of these novel compounds showed significant inhibition of CM when tested in vitro at 30 µM. The SAR (Structure-Activity-Relationship) studies suggested that benzotriazinone moiety was more favorable over the pyrazolotriazinone ring. The two best active compounds showed IC50 ∼ 0.4-0.9 µM (better than the reference/known compounds used) and no toxicity till 30 µM in vitro.

A kiwellin disarms the metabolic activity of a secreted fungal virulence factor.

Fungi-induced plant diseases affect global food security and plant ecology. The biotrophic fungus Ustilago maydis causes smut disease in maize (Zea mays) plants by secreting numerous virulence effectors that reprogram plant metabolism and immune responses1,2. The secreted fungal chorismate mutase Cmu1 presumably affects biosynthesis of the plant immune signal salicylic acid by channelling chorismate into the phenylpropanoid pathway3. Here we show that one of the 20 maize-encoded kiwellins (ZmKWL1) specifically blocks the catalytic activity of Cmu1. ZmKWL1 hinders substrate access to the active site of Cmu1 through intimate interactions involving structural features that are specific to fungal Cmu1 orthologues. Phylogenetic analysis suggests that plant kiwellins have a versatile scaffold that can specifically counteract pathogen effectors such as Cmu1. We reveal the biological activity of a member of the kiwellin family, a widely conserved group of proteins that have previously been recognized only as important human allergens.

Rational Discovery of (+) (S) Abscisic Acid as a Potential Antifungal Agent: a Repurposing Approach.

Fungal infections are spreading widely worldwide, and the types of treatment are limited due to the lack of diverse therapeutic agents and their associated side effects and toxicity. The discovery of new antifungal classes is vital and critical. We discovered the antifungal activity of abscisic acid through a rational drug design methodology that included the building of homology models for fungal chorismate mutases and a pharmacophore model derived from a transition state inhibitor. Ligand-based virtual screening resulted in some hits that were filtered using molecular docking and molecular dynamic simulations studies. Both in silico methods and in vitro antifungal assays were used as tools to select and validate the abscisic acid repurposing. Abscisic acid inhibition assays confirmed the inhibitory effect of abscisic acid on chorismate mutase through the inhibition of phenylpyruvate production. The repositioning of abscisic acid, the well-known and naturally occurring plant growth regulator, as a potential antifungal agent because of its suggested action as an inhibitor to several fungal chorismate mutases was the main result of this work.

Mycobacterium tuberculosis chorismate mutase: A potential target for TB.

Mycobacterium tuberculosis chorismate mutase (MtbCM) catalyzes the rearrangement of chorismate to prephenate in the shikimate biosynthetic pathway to form the essential amino acids, phenylalanine and tyrosine. Two genes encoding chorismate mutase have been identified in Mtb. The secretory form,∗MtbCM (encoded by Rv1885c) is assumed to play a key role in pathogenesis of tuberculosis. Also, the inhibition of MtbCM may hinder the supply of nutrients to the organism. Indeed, the existence of chorismate mutase (CM) in bacteria, fungi and higher plants but not in human and low sequence homology among known CM makes it an interesting target for the discovery of anti-tubercular agents. The present article mainly focuses on the recent developments in the structure, function and inhibition of MtbCM. The understanding of various aspects of MtbCM as presented in the current article may facilitate the design and subsequent chemical synthesis of new inhibitors against ∗MtbCM, that could lead to the discovery and development of novel and potent anti-tubercular agents in future.

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.

Discovery and structure optimization of a series of isatin derivatives as Mycobacterium tuberculosis chorismate mutase inhibitors.

In this study, the crystal structure of the Mycobacterium tuberculosis (MTB) enzyme chorismate mutase (CM) bound to transition state analogue (PDB: 2FP2) was used as a framework for virtual screening of the BITS-Pilani in-house database (2500 compounds) to identify new scaffold. We identified isatin as novel small molecule MTB CM inhibitors; further twenty-four isatin derivatives were synthesized and evaluated in vitro for their ability to inhibit MTB CM, and activity against M. tuberculosis as steps towards the derivation of structure-activity relationships (SAR) and lead optimization. Compound 3-(4-nitrobenzylidene)indolin-2-one, 24 emerged as the most promising lead with an IC50 of 1.01 ± 0.22 µm for purified CM and MIC of 23.5 µm for M. tuberculosis, with little or no cytotoxicity.

Identification and structure-activity relationship study of carvacrol derivatives as Mycobacterium tuberculosis chorismate mutase inhibitors.

In the present study, we identified carvacrol, a major phenolic component of oregano oil as a novel small molecule inhibitor of Mycobacterium tuberculosis (MTB) chorismate mutase (CM) enzyme with IC50 of 1.06 ± 0.4 µM. Virtual screening of the BITS-Pilani in-house database using the crystal structure of the MTB CM bound transition state intermediate (PDB: 2FP2) as framework identified carvacrol as a potential lead. Further various carvacrol derivatives were evaluated in vitro for their ability to inhibit MTB CM enzyme, whole cell MTB and cytotoxicity as steps toward the derivation of structure-activity relationships (SAR) and lead optimization.

Novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one derivatives as potential inhibitors of chorismate mutase.

A series of novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one (DHPM) derivatives were designed, synthesized and evaluated in vitro as potential inhibitors of chorismate mutase (CM). All these compounds were prepared via a multi-component reaction (MCR) involving sequential I2-mediated Biginelli reaction followed by Cu-free Sonogashira coupling. Some of them showed promising inhibitory activities when tested at 30µM. One compound showed dose dependent inhibition of CM with IC50 value of 14.76±0.54µM indicating o-alkynylphenyl substituted DHPM as a new scaffold for the discovery of promising inhibitors of CM.

When inhibitors do not inhibit: critical evaluation of rational drug design targeting chorismate mutase from Mycobacterium tuberculosis.

Tuberculosis (TB) is a devastating disease that claims millions of lives every year. Hindered access or non-compliance to medication, especially in developing countries, led to drug resistance, further aggravating the situation. With current standard therapies in use for over 50 years and only few new candidates in clinical trials, there is an urgent call for new TB drugs. A powerful tool for the development of new medication is structure-guided design, combined with virtual screening or docking studies. Here, we report the results of a drug-design project, which we based on a publication that claimed the structure-guided discovery of several promising and highly active inhibitors targeting the secreted chorismate mutase (*MtCM) from Mycobacterium tuberculosis. We set out to further improve on these compounds and synthesized a series of new derivatives. Thorough evaluation of these molecules in enzymatic assays revealed, to our dismay, that neither the claimed lead compounds, nor any of the synthesized derivatives, show any inhibitory effects against *MtCM.

AlCl3 mediated unexpected migration of sulfonyl groups: regioselective synthesis of 7-sulfonyl indoles of potential pharmacological interest.

A conceptually new and straightforward introduction of sulfonyl groups at the C-7 position of an indole ring has been achieved via AlCl(3) mediated unexpected regioselective sulfonyl group migration for N-alkyl/aryl/heteroarylsulfonyl indoles affording potential inhibitors of Mycobacterium tuberculosis H37Rv chorismate mutase.

C-N bond formation under Cu-catalysis: synthesis and in vitro evaluation of N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones against chorismate mutase.

A series of novel N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones were designed and synthesized as potential inhibitors of chorismate mutase. Synthesis of this class of compounds was carried out by using Cu-mediated C-N bond forming reaction between thieno[2,3-d]pyrimidin-4(3H)-ones and aryl boronic acids. The reaction can be performed in an open flask as the conversion was found to be not sensitive to the presence of air or atmospheric moisture. A range of compounds were prepared by using this method and single crystal X-ray diffraction study was performed using a representative compound. In vitro pharmacological data of some of the compounds synthesized along with dose response studies using active molecules are presented. In silico interactions of these molecules with chorismate mutase are also presented.

Cu-mediated N-arylation of 1,2,3-triazin-4-ones: synthesis of fused triazinone derivatives as potential inhibitors of chorismate mutase.

A rapid and direct access to N-aryl substituted fused triazinone derivatives has been accomplished via N-arylation of 1,2,3-triazin-4-one ring involving a Cu-mediated coupling between triazinone derivatives and aryl boronic acids. A combination of Cu(OAc)(2)-Et(3)N in 1,2-dichloroethane was found to be effective and various fused triazinone derivatives have been prepared by using this methodology. Molecular structure of a representative compound was confirmed by single crystal X-ray diffraction study. The scope and limitations of this reaction is discussed. Some of the compounds synthesized were tested for chorismate mutase inhibitory properties in vitro. The in vitro dose response study of an active compound is presented.

Ligand based virtual screening and biological evaluation of inhibitors of chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv.

We have identified new lead candidates that possess inhibitory activity against Mycobacterium tuberculosis H37Rv chorismate mutase by a ligand-based virtual screening optimized for lead evaluation in combination with in vitro enzymatic assay. The initial virtual screening using a ligand-based pharmacophore model identified 95 compounds from an in-house small molecule database of 15,452 compounds. The obtained hits were further evaluated by molecular docking and 15 compounds were short listed based on docking scores and the other scoring functions and subjected to biological assay. Chorismate mutase activity assays identified four compounds as inhibitors of M. tuberculosis chorismate mutase (MtCM) with low K(i) values. The structural models for these ligands in the chorismate mutase binding site will facilitate medicinal chemistry efforts for lead optimization against this protein.

Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved.

Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQ(gamma) subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5-15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000), Protein Sci. 9, 755-757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 A resolution. The crystals belong to space group P2(1), with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 angstroms, beta = 104.5 degrees. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.

Feedback inhibition of chorismate mutase/prephenate dehydrogenase (TyrA) of Escherichia coli: generation and characterization of tyrosine-insensitive mutants.

In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA(fbr) mutants were selected by virtue of their resistance toward m-fluoro-D,L-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrA(WT), but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrA(fbr) mutants were similar to those of the TyrA(WT) protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA(fbr) contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.

Design, synthesis, and evaluation of aza inhibitors of chorismate mutase.

A series of aza inhibitors (4-9) of chorismate mutase (E.C. 5.4.99.5) was designed, prepared, and evaluated against the enzyme by monitoring the direct inhibition of the chorismate, 1, to prephenate, 2, conversion. None of these aza inhibitors displayed tighter binding to the enzyme than the native substrate chorismate or greater inhibitory action than the previously reported ether analogue, 3. Furthermore, no time-dependent loss of enzyme activity was observed in the presence of the two potentially reactive aza inhibitors (7 and 9). These results in conjunction with inhibition data from a broader series of chorismate mutase inhibitors allowed a novel proposal for the mechanistic role of chorismate mutase to be developed. This proposed mechanism was computationally verified and correlated with crystallographic studies of various chorismate mutases.

Chorismate mutase of Thermus thermophilus is a monofunctional AroH class enzyme inhibited by tyrosine.

aroG, encoding the monofunctional chorismate mutase (TtCM) of the thermophilic gram-negative bacterium Thermus thermophilus, was cloned and its gene product characterized. TtCM was purified to homogeneity on an SDS polyacrylamide gel as a His-fusion protein with a deduced molecular mass of 15.8 kDa. The enzyme belongs to the rare group of AroH-type chorismate mutases which are mainly found in gram-positive bacteria of the Bacillus/ Clostridia group and have recently also been described for gram-negative organisms. The native molecular mass is consistent with a pseudo-alpha/beta barrel enzyme that is organized as a trimer. Comparison of the enzyme's structure with that of its mesophilic counterpart from Bacillus revealed an increase in hydrophilicity on the protein's surface, greater hydrophobicity in cavities within the protein, and greater restriction of conformational freedom, features that contribute to the thermal stability of this chorismate mutase. The kinetic data show Michaelis-Menten substrate saturation with a Km of 290 microM, and a kcat/ Km value of 180 s(-1) mM(-1). TtCM was inhibited by tyrosine with a Ki =34 microM, possibly in a competitive manner.

Quantitative evaluation of noncovalent chorismate mutase-inhibitor binding by ESI-MS.

Electrospray time-of-flight mass spectrometry was used to quantitatively determine the dissociation constant of chorismate mutase and a transition state analogue inhibitor. This system presents a fairly complex stoichiometry because the native protein is a homotrimer with three equal and independent substrate binding sites. We can detect the chorismate mutase trimer as well as chorismate mutase-inhibitor complexes by choosing appropriate conditions in the ESI source. To verify that the protein-inhibitor complexes are specific, titration experiments with different enzyme variants and different inhibitors were performed. A plot of the number of bound inhibitors versus added inhibitor concentration revealed saturation behavior with 3:1 (inhibitor:functional trimer) stoichiometry for the TSA. The soft ESI conditions, the relatively high protein mass of 43.5 kDa, and the low charge state (high m/z) result in broad peaks, a typical problem in analyzing noncovalent protein complexes. Due to the low molecular weight of the TSA (226 Da) the peaks of the free protein and the protein with one, two or three inhibitors bound cannot be clearly resolved. For data analysis, relative peak areas of the deconvoluted spectra of chorismate mutase-inhibitor complexes were obtained by fitting appropriate peak shapes to the signals corresponding to the free enzyme and its complexes with one, two, or three inhibitor molecules. From the relative peak areas we were able to calculate a dissociation constant that agreed well with known solution-phase data. This method may be generally useful for interpreting mass spectra of noncovalent complexes that exhibit broad peaks in the high m/z range.

Charge optimization increases the potency and selectivity of a chorismate mutase inhibitor.

The highest affinity inhibitor for chorismate mutases, a conformationally constrained oxabicyclic dicarboxylate transition state analogue, was modified as suggested by computational charge optimization methods. As predicted, replacement of the C10 carboxylate in this molecule with a nitro group yields an even more potent inhibitor of a chorismate mutase from Bacillus subtilis (BsCM), but the magnitude of the improvement (roughly 3-fold, corresponding to a DeltaDeltaG of -0.7 kcal/mol) is substantially lower than the gain of 2-3 kcal/mol binding free energy anticipated for the reduced desolvation penalty upon binding. Experiments with a truncated version of the enzyme show that the flexible C terminus, which was only partially resolved in the crystal structure and hence omitted from the calculations, provides favorable interactions with the C10 group that partially compensate for its desolvation. Although truncation diminishes the affinity of the enzyme for both inhibitors, the nitro derivative binds 1.7 kcal/mol more tightly than the dicarboxylate, in reasonable agreement with the calculations. Significantly, substitution of the C10 carboxylate with a nitro group also enhances the selectivity of inhibition of BsCM relative to a chorismate mutase from Escherichia coli (EcCM), which has a completely different fold and binding pocket, by 10-fold. These results experimentally verify the utility of charge optimization methods for improving interactions between proteins and low-molecular weight ligands.

Studies on 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase using chorismate mutase inhibitors.

The proposed cyclic mechanism of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase and the mechanism of chorismate mutase share certain structural and electronic similarities. In this report, we examine several inhibitors of chorismate mutase for their efficacy against KDO 8-P synthase.