Enzymology and medicinal chemistry of N5-carboxyaminoimidazole ribonucleotide synthetase: A novel antibacterial target

N5-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 structural and functional studies on the Aspergillus clavatus N5-CAIR synthetase and identification of inhibitors for the enzyme. In collaboration with Dr. Hazel Holden of the University of Wisconsin, the three-dimensional structure of Aspergillus clavatus N5-CAIR synthetase was solved in the presence of either Mg2ATP or MgADP and AIR. These structures, determined to 2.1 and 2.0 Å resolution, 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 Asp153 and Lys353 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 Mg2ATP and bicarbonate first react to form the unstable intermediate carboxyphosphate. This intermediate subsequently decarboxylates to CO2 and inorganic phosphate, and the amino group of AIR, through general base assistance by Asp153, attacks CO2 to form N5-CAIR. To identify the inhibitors for this enzyme, we have conducted high-throughput screening (HTS) against Escherichia coli N5-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 N5-CAIR synthetase by reacting with the substrate AIR. Kinetic analysis indicates that the class II families 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 indicating that these agents are selective inhibitors of N5-CAIR synthetase. Given the importance of the class II, non-competitive inhibitors, we developed a diazirine-based photocrosslinking agent to identify the binding site of these inhibitors. These studies revealed that the isatin core of class II inhibitors is capable of undergoing photochemical conversion to isatoic anhydride. Once formed, the anhydride is capable of reacting with the protein. Treatment of N5-CAIR synthetase with the photoreactive agent lead to the dimerization of two monomers of the synthetase. Proteomic analysis of the crosslinked protein identified serine 227 as a possible site of modification. These studies also revealed two peptides that were missing in the dimerized protein sample. These two peptides were located near serine 227. While compelling, the location of the missing peptides and serine 227 is 20 Å away from the dimerization interface observed in the crystal structure. Thus, our photocrosslinking studies suggest that N5-CAIR synthetase may exist in multiple dimer conformations

Design, Synthesis and Evaluation of Fe-S Targeted Adenosine 5′-Phosphosulfate Reductase Inhibitors

<div><p>Adenosine 5′-phosphosulfate reductase (APR) is an iron-sulfur enzyme that is vital for survival of <i>Mycobacterium tuberculosis</i> 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 <i>N</i>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.</p></div

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