Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.

Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule.

Synthesis and Pharmacokinetic Evaluation of Siderophore Biosynthesis Inhibitors for Mycobacterium tuberculosis.

MbtA catalyzes the first committed biosynthetic step of the mycobactins, which are important virulence factors associated with iron acquisition in Mycobacterium tuberculosis. MbtA is a validated therapeutic target for antitubercular drug development. 5'-O-[N-(Salicyl)sulfamoyl]adenosine (1) is a bisubstrate inhibitor of MbtA and exhibits exceptionally potent biochemical and antitubercular activity.

Fluorescent peptide sensors for tyrosylprotein sulfotransferase activity.

Tyrosine sulfurylation is a post-translational modification important for protein-protein interactions in the extracellular space that are instrumental in cell adhesion, cell signaling, immune responses, and pathogen recognition of host cells. Tyrosine sulfurylation is catalyzed by the tyrosylprotein sulfotransferases (TPSTs), and in humans there are two isoforms: hTPST1 and hTPST2. The study of hTPST function and the development of small molecule probes to examine the role of hTPSTs in cell biology have been delayed by the absence of a continuous direct assay for hTPST activity.

Structure-activity relationships of 2-aminothiazoles effective against Mycobacterium tuberculosis.

A series of 2-aminothiazoles was synthesized based on a HTS scaffold from a whole-cell screen against Mycobacterium tuberculosis (Mtb). The SAR shows the central thiazole moiety and the 2-pyridyl moiety at C-4 of the thiazole are intolerant to modification. However, the N-2 position of the aminothiazole exhibits high flexibility and we successfully improved the antitubercular activity of the initial hit by more than 128-fold through introduction of substituted benzoyl groups at this position.

Development of a selective activity-based probe for adenylating enzymes: profiling MbtA Involved in siderophore biosynthesis from Mycobacterium tuberculosis.

MbtA is an adenylating enzyme from Mycobacterium tuberculosis that catalyzes the first step in the biosynthesis of the mycobactins. A bisubstrate inhibitor of MbtA (Sal-AMS) was previously described that displays potent antitubercular activity under iron-replete as well as iron-deficient growth conditions. This finding is surprising since mycobactin biosynthesis is not required under iron-replete conditions and suggests off-target inhibition of additional biochemical pathways.

Adenylating enzymes in Mycobacterium tuberculosis as drug targets.

Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence.

Bisubstrate adenylation inhibitors of biotin protein ligase from Mycobacterium tuberculosis.

The mycobacterial biotin protein ligase (MtBPL) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases involved in lipid biosynthesis that catalyze the first step in fatty acid biosynthesis and pyruvate coenzyme A carboxylase, a gluconeogenic enzyme vital for lipid catabolism. Here we describe the design, development, and evaluation of a rationally designed bisubstrate inhibitor of MtBPL. This inhibitor displays potent subnanomolar enzyme inhibition and antitubercular activity against multidrug resistant and extensively drug resistant Mtb strains.

A continuous fluorescence displacement assay for BioA: an enzyme involved in biotin biosynthesis.

Cofactor biosynthetic pathways represent a rich source of potential antibiotic targets. The second step in biotin biosynthesis is performed by BioA, a pyridoxal 5'-phosphate (PLP)-dependent enzyme. This enzyme has been confirmed as a candidate target in Mycobacterium tuberculosis; however, the current bioassay used to measure BioA activity is cumbersome and low throughput.

Biochemical and structural characterization of bisubstrate inhibitors of BasE, the self-standing nonribosomal peptide synthetase adenylate-forming enzyme of acinetobactin synthesis.

The human pathogen Acinetobacter baumannii produces a siderophore called acinetobactin that is derived from one molecule each of threonine, histidine, and 2,3-dihydroxybenzoic acid (DHB). The activity of several nonribosomal peptide synthetase (NRPS) enzymes is used to combine the building blocks into the final molecule. The acinetobactin synthesis pathway initiates with a self-standing adenylation enzyme, BasE, that activates the DHB molecule and covalently transfers it to the pantetheine cofactor of an aryl-carrier protein of BasF, a strategy that is shared with many siderophore-producing NRPS clusters.

Assigning enzyme function from the metabolic milieu.

In this issue of , Rhee and co-workers develop a metabolic profiling technique using a mycobacterial small molecule extract and use it to correctly assign enzyme function to a previously mis-annotated gene product of (de Carvalho et al., 2010).

Development of a high-throughput fluorescence polarization assay for the discovery of phosphopantetheinyl transferase inhibitors.

An alarming number of clinically relevant bacterial pathogens are becoming resistant to many antibiotics, thereby fueling intense research into the discovery of novel therapeutic targets. Phosphopantetheinyl transferases (PPTases) represent a promising target for antibacterial development because these enzymes are crucial for the biosynthesis of a multitude of a pathogen's collection of essential metabolites and virulence factors biosynthesized via polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways. Here we describe the development of a fluorescence polarization (FP) assay that is amenable for high-throughput screening to identify PPTase inhibitors.

Selective labeling of proteins by using protein farnesyltransferase.

The challenging task of identifying and studying protein function has been greatly aided by labeling proteins with reporter groups. Here, we present a strategy that utilizes an enzyme that labels a four-residue sequence appended onto the C terminus of a protein, with an alkyne-containing substrate. By using a bio-orthogonal cycloaddition reaction, a fluorophore that carried an azide moiety was then covalently coupled to the alkyne appended on the protein.

Synthesis and reactivity of 6,7-dihydrogeranylazides: reagents for primary azide incorporation into peptides and subsequent staudinger ligation.

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide moiety to a peptide substrate, N-dansyl-GCVIA. Because geranylazide is actually a mixture of isomeric, interconverting primary and secondary azides, incorporation of this isoprenoid into peptides can potentially result in a corresponding mixture of prenylated peptides. Here, we first examined the reactivity of geranyl azide in a model Staudinger reaction and determined that a mixture of products is formed.

Site-specific, covalent attachment of proteins to a solid surface.

Immobilized and site-specifically labeled proteins are becoming invaluable tools in proteomics. Here, we describe a strategy to attach a desired protein to a solid surface in a covalent, site-specific manner. This approach employs an enzymatic posttranslational modification method to site-specifically label a target protein with an azide; an alternative substrate for protein farnesyl transferase containing an azide group was developed for this purpose.

Oxathiolene oxides: a novel family of compounds that induce ferritin, glutathione S-transferase, and other proteins of the phase II response.

Compounds that induce the synthesis of cytoprotective phase II enzymes have shown promise as cancer chemopreventive agents. Although chemically diverse, phase II enzyme inducers are capable of participating in Michael reaction chemistry. We have synthesized a novel class of organosulfur compounds, termed oxathiolene oxides (OTEOs).