Functional Site Discovery in a Sulfur Metabolism Enzyme by Using Directed Evolution.

In human pathogens, the sulfate assimilation pathway provides reduced sulfur for biosynthesis of essential metabolites, including cysteine and low-molecular-weight thiol compounds. Sulfonucleotide reductases (SRs) catalyze the first committed step of sulfate reduction. In this reaction, activated sulfate in the form of adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is reduced to sulfite. Gene knockout, transcriptomic and proteomic data have established the importance of SRs in oxidative stress-inducible antimicrobial resistance mechanisms. In previous work, we focused on rational and high-throughput design of small-molecule inhibitors that target the active site of SRs. However, another critical goal is to discover functionally important regions in SRs beyond the traditional active site. As an alternative to conservation analysis, we used directed evolution to rapidly identify functional sites in PAPS reductase (PAPR). Four new regions were discovered that are essential to PAPR function and lie outside the substrate binding pocket. Our results highlight the use of directed evolution as a tool to rapidly discover functionally important sites in proteins.

Reactivity, Selectivity, and Stability in Sulfenic Acid Detection: A Comparative Study of Nucleophilic and Electrophilic Probes.

The comparative reaction efficiencies of currently used nucleophilic and electrophilic probes toward cysteine sulfenic acid have been thoroughly evaluated in two different settings-(i) a small molecule dipeptide based model and (ii) a recombinant protein model. We further evaluated the stability of corresponding thioether and sulfoxide adducts under reducing conditions which are commonly encountered during proteomic protocols and in cell analysis. Powered by the development of new cyclic and linear C-nucleophiles, the unsurpassed efficiency in the capture of sulfenic acid under competitive conditions is achieved and thus holds great promise as highly potent tools for activity-based sulfenome profiling.

Design, synthesis and evaluation of Fe-S targeted adenosine 5'-phosphosulfate reductase inhibitors.

Adenosine 5'-phosphosulfate reductase (APR) is an iron-sulfur enzyme that is vital for survival of Mycobacterium tuberculosis during dormancy and is an attractive target for the treatment of latent tuberculosis (TB) infection. The 4Fe-4S cluster is coordinated to APR by sulfur atoms of four cysteine residues, is proximal to substrate, adenosine 5'-phopsphosulfate (APS), and is essential for catalytic activity. Herein, we present an approach for the development of a new class of APR inhibitors. As an initial step, we have employed an improved solid-phase chemistry method to prepare a series of N(6)-substituted adenosine analogues and their 5'-phosphates as well as adenosine 5'-phosphate diesters bearing different Fe and S binding groups, such as thiols or carboxylic and hydroxamic acid moieties. Evaluation of the resulting compounds indicates a clearly defined spacing requirement between the Fe-S targeting group and adenosine scaffold and that smaller Fe-S targeting groups are better tolerated. Molecular docking analysis suggests that the S atom of the most potent inhibitor may establish a favorable interaction with an S atom in the cluster. In summary, this study showcases an improved solid-phase method that expedites the preparation of adenosine and related 5'-phosphate derivatives and presents a unique Fe-S targeting strategy for the development of APR inhibitors.

New targets and inhibitors of mycobacterial sulfur metabolism.

The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress made in the development of inhibitors of sulfur metabolism enzymes.

Efficient microwave-assisted solid phase coupling of nucleosides, small library generation and mild conditions for release of nucleoside derivatives.

Nucleosides are essential bio-molecules that participate in a wide array of biological processes involved in maintaining physiologic homeostasis. Recent efforts geared towards the synthesis of nucleoside analogues and development of nucleoside combinatorial libraries using solid phase synthesis has contributed invaluable information towards drug design and development. These studies have provided information concerning the structural requirements of substrate binding pockets of enzymes and evaluation of enzyme kinetics. However, the synthesis of nucleosides and its corresponding analogues remains a challenging and time consuming process. Herein, we report an efficient, microwave assisted solid phase coupling of nucleosides, combinatorial chemistry on the coupled nucleosides to generate small library and mild cleavage conditions to release nucleoside derivatives from its solid support. We anticipate these findings will accelerate the development of synthetic methods or combinatorial library design of nucleoside analogues in similar settings.

A continuous spectrophotometric assay for adenosine 5'-phosphosulfate reductase activity with sulfite-selective probes.

Mycobacterium tuberculosis (Mtb) adenosine 5'-phosphosulfate (APS) reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of essential reduced sulfur-containing biomolecules, such as cysteine, and is essential for survival in the latent phase of tuberculosis (TB) infection. Despite the importance of APR to Mtb and other bacterial pathogens, current assay methods depend on the use of (35)S-labeled APS or shunt adenosine 5'-monophosphate (AMP) to a coupled-enzyme system. Both methods are cumbersome and require the use of expensive reagents. Here, we report the development of a continuous spectrophotometric method for measuring APR activity by using novel sulfite-selective colorimetric or "off-on" fluorescent levulinate-based probes. Thus, the APR activity can be followed by monitoring the increase in absorbance or fluorescence of the resulting phenolate product. Using this assay, we determined Michaelis-Menten kinetic constants (K(m), k(cat), and k(cat)/K(m)) and the apparent inhibition constant (Ki) for adenosine 5'-diphosphate (ADP), which compared favorably with values obtained in the "gold standard" radioactive assay. The newly developed assay is robust and easy to perform with a simple spectrophotometer.

Characterization of insulin ILPR sequences for their ability to adopt a G-quadruplex structure.

A major genetic factor linked to the progression of type 1 diabetes occurs in the insulin-linked polymorphic repeat region (ILPR) located 363 bp upstream of the human insulin gene. Genetic studies have shown that individuals with class I repeats (30-60) are predisposed to the development of type 1 diabetes while individuals with longer repeats are protected. Previous research has suggested that some sequences found within the ILPR can adopt a G-quadruplex structure, and this finding has lead to speculation that G-quadruplexes may control insulin expression in certain circumstances. Unfortunately, relatively little study has been done on whether sequences found in the ILPR can adopt a quadruplex fold. In this study, we have utilized circular dichroism, thermal difference spectroscopy and ultraviolet (UV) melting studies to examine the first seven common repeat sequences (A-G) found in the ILPR. We find that sequences A-E adopt a quadruplex fold while sequences F and G likely do not. Examination of sequence B and a single nucleotide variant, B2, revealed that both folded into a G-quadruplex. This result casts doubt on previous studies suggesting that the formation of a quadruplex was related to the ability of ILPR sequences to regulate transcription.

Structural and functional studies of Aspergillus clavatus N(5)-carboxyaminoimidazole ribonucleotide synthetase .

N(5)-Carboxyaminoimidazole ribonucleotide synthetase (N(5)-CAIR synthetase), a key enzyme in microbial de novo purine biosynthesis, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to N(5)-CAIR. To date, this enzyme has been observed only in microorganisms, and thus, it represents an ideal target for antimicrobial drug development. Here we report the cloning, crystallization, and three-dimensional structural analysis of Aspergillus clavatus N(5)-CAIR synthetase solved in the presence of either Mg(2)ATP or MgADP and AIR. These structures, determined to 2.1 and 2.0 A, respectively, revealed that AIR binds in a pocket analogous to that observed for other ATP-grasp enzymes involved in purine metabolism. On the basis of these models, a site-directed mutagenesis study was subsequently conducted that focused on five amino acid residues located in the active site region of the enzyme. These investigations demonstrated that Asp 153 and Lys 353 play critical roles in catalysis without affecting substrate binding. All other mutations affected substrate binding and, in some instances, catalysis as well. Taken together, the structural and kinetic data presented here suggest a catalytic mechanism whereby Mg(2)ATP and bicarbonate first react to form the unstable intermediate carboxyphosphate. This intermediate subsequently decarboxylates to CO(2) and inorganic phosphate, and the amino group of AIR, through general base assistance by Asp 153, attacks CO(2) to form N(5)-CAIR.

Identification of inhibitors of N5-carboxyaminoimidazole ribonucleotide synthetase by high-throughput screening.

The increasing risk of drug-resistant bacterial infections indicates that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis in microbes is different from that in humans. The differences in the pathways are centered around the synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR) which requires the enzyme N(5)-carboxyaminoimidazole ribonucleotide (N(5)-CAIR) synthetase. Humans do not require and have no homologs of this enzyme. Unfortunately, no studies aimed at identifying small-molecule inhibitors of N(5)-CAIR synthetase have been published. To remedy this problem, we have conducted high-throughput screening (HTS) against Escherichia coliN(5)-CAIR synthetase using a highly reproducible phosphate assay. HTS of 48,000 compounds identified 14 compounds that inhibited the enzyme. The hits identified could be classified into three classes based on chemical structure. Class I contains compounds with an indenedione core. Class II contains an indolinedione group, and Class III contains compounds that are structurally unrelated to other inhibitors in the group. We determined the Michaelis-Menten kinetics for five compounds representing each of the classes. Examination of compounds belonging to Class I indicates that these compounds do not follow normal Michaelis-Menten kinetics. Instead, these compounds inhibit N(5)-CAIR synthetase by reacting with the substrate AIR. Kinetic analysis indicates that the Class II family of compounds are non-competitive with both AIR and ATP. One compound in Class III is competitive with AIR but uncompetitive with ATP, whereas the other is non-competitive with both substrates. Finally, these compounds display no inhibition of human AIR carboxylase:SAICAR synthetase indicating that these agents are selective inhibitors of N(5)-CAIR synthetase.

Benzo(h)quinoline derivatives as G-quadruplex binding agents.

G-quadruplexes are unusual structures formed from guanine-rich sequences of nucleic acids. G-quadruplexes have been postulated to play important roles in a number of biological systems including gene regulation and the inhibition of enzyme function. Recently, our laboratory reported on the synthesis and evaluation of a triaza-cyclopentaphenanthrene compound which bound to G-quadruplexes with good affinity and selectivity. This compound contains a 4-pyridone group which has not been previously utilized in other quadruplex binding agents. In this Letter, we describe the synthesis and evaluation of 4-pyridone containing 2- and 3-carboxy-benzoquinolines as G-quadruplex binding agents. We find that these compounds are capable of binding G-quadruplexes with a K(a) in the range of 3 x 10(5)M(-1) and with a 10-fold selectivity for quadruplex over duplex DNA.