Interfering with DNA Decondensation as a Strategy Against Mycobacteria

Tuberculosis is once again a major global threat, leading to more than 1 million deaths each year. Treatment options for tuberculosis patients are limited, expensive and characterized by severe side effects, especially in the case of multidrug-resistant forms. Uncovering novel vulnerabilities of the pathogen is crucial to generate new therapeutic strategies. Using high resolution microscopy techniques, we discovered one such vulnerability of Mycobacterium tuberculosis. We demonstrate that the DNA of M. tuberculosis can condense under stressful conditions such as starvation and antibiotic treatment. The DNA condensation is reversible and specific for viable bacteria. Based on these observations, we hypothesized that blocking the recovery from the condensed state could weaken the bacteria. We showed that after inducing DNA condensation, and subsequent blocking of acetylation of DNA binding proteins, the DNA localization in the bacteria is altered. Importantly under these conditions, Mycobacterium smegmatis did not replicate and its survival was significantly reduced. Our work demonstrates that agents that block recovery from the condensed state of the nucleoid can be exploited as antibiotic. The combination of fusidic acid and inhibition of acetylation of DNA binding proteins, via the Eis enzyme, potentiate the efficacy of fusidic acid by 10 and the Eis inhibitor to 1,000-fold. Hence, we propose that successive treatment with antibiotics and drugs interfering with recovery from DNA condensation constitutes a novel approach for treatment of tuberculosis and related bacterial infections

Deciphering Nature’s Intricate Way of <i>N</i>,<i>S</i>-Dimethylating l‑Cysteine: Sequential Action of Two Bifunctional Adenylation Domains

Dimethylation of amino acids consists of an interesting and puzzling series of events that could be achieved, during nonribosomal peptide biosynthesis, either by a single adenylation (A) domain interrupted by a methyltransferase (M) domain or by the sequential action of two of such independent enzymes. Herein, to establish the method by which Nature <i>N</i>,<i>S</i>-dimethylates l-Cys, we studied its formation during thiochondrilline A biosynthesis by evaluating TioS­(A_3aM_3SA_3bT₃) and TioN­(A_aM_NA_b). This study not only led to identification of the exact pathway followed in Nature by these two enzymes for <i>N</i>,<i>S</i>-dimethylation of l-Cys, but also revealed that a single interrupted A domain can <i>N</i>,<i>N</i>-dimethylate amino acids, a novel phenomenon in the nonribosomal peptide field. These findings offer important and useful insights for the development and engineering of novel interrupted A domain enzymes to serve, in the future, as tools for combinatorial biosynthesis

CCDC 890236: Experimental Crystal Structure Determination

Related Article: Atefeh Garzan, Arvind Jaganathan, Nastaran Salehi Marzijarani, Roozbeh Yousefi, Daniel C. Whitehead, James E. Jackson, Babak Borhan|2013|Chem.-Eur.J.|19|9015|doi:10.1002/chem.201300189,An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures

Discovery and Optimization of Two Eis Inhibitor Families as Kanamycin Adjuvants against Drug-Resistant <i>M. tuberculosis</i>

Drug-resistant tuberculosis (TB) is a global threat and innovative approaches such as using adjuvants of anti-TB therapeutics are required to combat it. High-throughput screening yielded two lead scaffolds of inhibitors of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) acetyltransferase Eis, whose upregulation causes resistance to the anti-TB drug kanamycin (KAN). Chemical optimization on these scaffolds resulted in potent Eis inhibitors. One compound restored the activity of KAN in a KAN-resistant <i>Mtb</i> strain. Model structures of Eis-inhibitor complexes explain the structure–activity relationship

Discovery of Allosteric and Selective Inhibitors of Inorganic Pyrophosphatase from <i>Mycobacterium tuberculosis</i>

Inorganic pyrophosphatase (PPiase) is an essential enzyme that hydrolyzes inorganic pyrophosphate (PP_i), driving numerous metabolic processes. We report a discovery of an allosteric inhibitor (2,4-bis­(aziridin-1-yl)-6-(1-phenylpyrrol-2-yl)-<i>s</i>-triazine) of bacterial PPiases. Analogues of this lead compound were synthesized to target specifically <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) PPiase (<i>Mt</i>PPiase). The best analogue (compound <b>16</b>) with a <i>K</i>_i of 11 μM for <i>Mt</i>PPiase is a species-specific inhibitor. Crystal structures of <i>Mt</i>PPiase in complex with the lead compound and one of its analogues (compound <b>6</b>) demonstrate that the inhibitors bind in a nonconserved interface between monomers of the hexameric <i>Mt</i>PPiase in a yet unprecedented pairwise manner, while the remote conserved active site of the enzyme is occupied by a bound PP_i substrate. Consistent with the structural studies, the kinetic analysis of the most potent inhibitor has indicated that it functions uncompetitively, by binding to the enzyme–substrate complex. The inhibitors appear to allosterically lock the active site in a closed state causing its dysfunctionalization and blocking the hydrolysis. These inhibitors are the first examples of allosteric, species-selective inhibitors of PPiases, serving as a proof-of-principle that PPiases can be selectively targeted

Pyrimidone inhibitors targeting Chikungunya Virus nsP3 macrodomain by fragment-based drug design.

The macrodomain of nsP3 (nsP3MD) is highly conserved among the alphaviruses and ADP-ribosylhydrolase activity of Chikungunya Virus (CHIKV) nsP3MD is critical for CHIKV viral replication and virulence. No small molecule drugs targeting CHIKV nsP3 have been identified to date. Here we report small fragments that bind to nsP3MD which were discovered by virtually screening a fragment library and X-ray crystallography. These identified fragments share a similar scaffold, 2-pyrimidone-4-carboxylic acid, and are specifically bound to the ADP-ribose binding site of nsP3MD. Among the fragments, 2-oxo-5,6-benzopyrimidine-4-carboxylic acid showed anti-CHIKV activity with an IC50 of 23 μM. Our fragment-based drug discovery approach provides valuable information to further develop a specific and potent nsP3 inhibitor of CHIKV viral replication based on the 2-pyrimidone-4-carboxylic acid scaffold. In silico studies suggest this pyrimidone scaffold could also bind to the macrodomains of other alphaviruses and coronaviruses and thus, have potential pan-antiviral activity

Activation and Loading of the Starter Unit during Thiocoraline Biosynthesis

The initiation of the nonribosomal peptide synthetase (NRPS) assembly of the bisintercalator natural product thiocoraline involves key enzymatic steps for AMP activation and carrier protein loading of the starter unit 3-hydroxyquinaldic acid (3HQA). Gene cluster data combined with protein sequence homology analysis originally led us to propose that TioJ could be responsible for the AMP activation step, whereas TioO could act as the thiolation (T) domain, facilitating the transfer of 3HQA to the next NRPS module, TioR. Herein, we confirmed the involvement of TioJ in thiocoraline biosynthesis by <i>tioJ</i> knockout and <i>in vitro</i> activation of 3HQA studies. However, we demonstrated that TioJ-activated 3HQA is not loaded onto the T domain TioO, as originally believed, but instead onto a fatty acid synthase (FAS) acyl carrier protein (ACP) domain FabC, which is located outside of the thiocoraline gene cluster. We showed a strong interaction between TioJ and FabC. By generating TioJ point mutants mimicking the active site of highly homologous enzymes activating different molecules, we showed that the identity of the substrate activated by adenylation domains such as TioJ is not determined by only the active site residues that directly interact with the substrate. The insights gained from these enzymatic transformations are valuable in the efforts toward deciphering the complete biosynthetic pathway of thiocoraline and bisintercalators in general

Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis

A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB

Optimization of pyrazole-containing 1,2,4-triazolo-[3,4-b]thiadiazines, a new class of STAT3 pathway inhibitors

Structure-activity relationship studies of a 1,2,4-triazolo-[3,4-b]thiadiazine scaffold, identified in an HTS campaign for selective STAT3 pathway inhibitors, determined that a pyrazole group and specific aryl substitution on the thiadiazine were necessary for activity. Improvements in potency and metabolic stability were accomplished by the introduction of an α-methyl group on the thiadiazine. Optimized compounds exhibited anti-proliferative activity, reduction of phosphorylated STAT3 levels and effects on STAT3 target genes. These compounds represent a starting point for further drug discovery efforts targeting the STAT3 pathway