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. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.

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. However, 1 suffers from suboptimal drug disposition properties resulting in a short half-life (t(1/2)), low exposure (AUC), and low bioavailability (F). Four strategies were pursued to address these liabilities including the synthesis of prodrugs, increasing the pK(a) of the acyl-sulfonyl moiety, modulation of the lipophilicity, and strategic introduction of fluorine into 1. Complete pharmacokinetic (PK) analysis of all compounds was performed. The most successful modifications involved fluorination of the nucleoside that provided substantial improvements in t(1/2) and AUC. Increasing the pK(a) of the acyl-sulfonyl linker yielded incremental enhancements, while modulation of the lipophilicity and prodrug approaches led to substantially poorer PK parameters.

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. We have developed a fluorescent peptide-based assay to directly monitor tyrosine sulfurylation in real time. TPST-mediated tyrosine sulfurylation of the peptides disrupts fluorophore quenching and results in increased fluorescence emission. The assay can be used to study TPST enzymatic activity, and we show that recombinant hTPSTs are active in the absence of divalent metal ions and that optimal activity is at pH 6.0. We further show that the assay can also be used to identify inhibitors of tyrosine sulfurylation. A clear understanding of hTPST function in normal cell biology and in disease states will require the identification of small molecule inhibitors or probes to modulate enzymatic activity, and our results will facilitate that process.

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. N-(3-Chlorobenzoyl)-4-(2-pyridinyl)-1,3-thiazol-2-amine (55) emerged as one of the most promising analogues with a MIC of 0.024µM or 0.008µg/mL in 7H9 media and therapeutic index of nearly ∼300. However, 55 is rapidly metabolized by human liver microsomes (t1/2=28min) with metabolism occurring at the invariant aminothiazole moiety and Mtb develops spontaneous low-level resistance with a frequency of ∼10(-5).

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. As a first step toward a complete understanding of the mechanism of action of Sal-AMS, we have designed and validated an activity-based probe (ABP) for studying Sal-AMS inhibition in M. tuberculosis. This probe labels pure MbtA as well as MbtA in mycobacterial lysate, and labeling can be completely inhibited by preincubation with Sal-AMS. Furthermore, this probe provides a prototypical core scaffold for the creation of ABPs to profile any of the other 66 adenylating enzymes in Mtb or the multitude of adenylating enzymes in other pathogenic bacteria.

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. Several strategies have been used to develop potent and selective AE inhibitors including highthroughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNAsynthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl- AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases.

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. We show that the inhibitor decreases in vivo protein biotinylation of key enzymes involved in fatty acid biosynthesis and that the antibacterial activity is MtBPL dependent. Additionally, the gene encoding BPL was found to be essential in M. smegmatis. Finally, the X-ray cocrystal structure of inhibitor bound MtBPL was solved providing detailed insight for further structure-activity analysis. Collectively, these data suggest that MtBPL is a promising target for further antitubercular therapeutic development.

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. Here we describe the design, development, and optimization of a continuous coupled fluorescence displacement assay to measure BioA activity. In this coupled assay, BioD converts the product of the BioA-catalyzed reaction into dethiobiotin, which is subsequently detected by displacement of a fluorescently labeled dethiobiotin probe from streptavidin. The assay was further adapted to a high-throughput screening format and validated against the LOPAC(1280) library.

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. In this reaction, DHB reacts with ATP to form the aryl adenylate and pyrophosphate. In a second partial reaction, the DHB is transferred to the carrier protein. Inhibitors of BasE and related enzymes have been identified that prevent growth of bacteria on iron-limiting media. Recently, a new inhibitor of BasE has been identified via high-throughput screening using a fluorescence polarization displacement assay. We present here biochemical and structural studies to examine the binding mode of this inhibitor. The kinetics of the wild-type BasE enzyme is shown, and inhibition studies demonstrate that the new compound exhibits competitive inhibition against both ATP and 2,3-dihydroxybenzoate. Structural examination of BasE bound to this inhibitor illustrates a novel binding mode in which the phenyl moiety partially fills the enzyme pantetheine binding tunnel. Structures of rationally designed bisubstrate inhibitors are also presented.

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. The FP assay was validated against a panel of competitive ligands and displayed an excellent Z' score.

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. FRET was used to calculate a Förster (R) distance of 40 A between the eGFP chromophore and the newly appended Texas Red fluorophore. This experimental value is in good agreement with the predicted R value determined by using molecular modeling. The small recognition tag, the high specificity of the enzyme, and the orthogonal nature of the derivatization reaction will make this approach highly useful in protein chemistry.

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. We then describe the synthesis of 6,7-dihydrogeranylazide diphosphate and demonstrate that this compound allows exclusive incorporation of a primary azide into a peptide. The resulting azide-containing peptide was derivatized with a triphenylphosphine-based reagent to generate an O-alkyl imidate-linked product. Finally, we show, using a series of model reactions, that the Staudinger ligation frequently produces small amounts of O-alkyl imidate products in addition to the major amide-linked products. Thus, the alkoxyimidates we have observed as the exclusive products in the reactions of peptides containing prenylated azides also appear to be a common type of product formed using other azide-containing reactants, although at greatly reduced levels. This method for chemical modification of the C-terminus of a protein should be useful for a variety of applications in protein chemistry.

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. A bio-orthogonal Cu(I)-catalyzed cycloaddition reaction is then used to covalently attach the protein to agarose beads bearing an alkyne functional group. We demonstrate that both the azide incorporation and the capture steps can be performed on either a purified protein target or on a protein present within a complex mixture. This approach involves the use of a four-residue tag which is significantly smaller than most other tags reported to date and results in covalent immobilization of the target protein. Hence it should have significant applicability in protein science.

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). Based on their chemical properties, we hypothesized that these compounds could function as phase II enzyme inducers. Northern blot analysis showed that oxathiolene oxides induce the phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), and ferritin H and L mRNA in a concentration-dependent fashion in a normal embryonic mouse liver cell line, BNLCL.2. OTEO-562 (3-cyclohexenyl-4-methyl-1,2-oxathiol-3-ene-2-oxide) was the strongest inducer. Western blot analysis demonstrated that GST-alpha and ferritin H protein levels were also induced in cells treated with OTEO-562, as was total GST and NQO1 enzyme activity. Further, induction of NQO1 activity by OTEO-562 was equivalent in aromatic hydrocarbon (Ah) receptor wild-type and Ah receptor mutant cell lines, suggesting that oxathiolene oxides activate phase II enzymes by an Ah receptor-independent mechanism. Consistent with this observation, OTEO-562 failed to induce cytochrome P450 1A1 mRNA. These results suggest that oxathiolene oxides may merit further investigation as candidate chemopreventive agents.