Tiki1 is required for head formation via Wnt cleavage-oxidation and inactivation.

Secreted Wnt morphogens are signaling molecules essential for embryogenesis, pathogenesis, and regeneration and require distinct modifications for secretion, gradient formation, and activity. Whether Wnt proteins can be posttranslationally inactivated during development and homeostasis is unknown. Here we identify, through functional cDNA screening, a transmembrane protein Tiki1 that is expressed specifically in the dorsal Spemann-Mangold Organizer and is required for anterior development during Xenopus embryogenesis. Tiki1 antagonizes Wnt function in embryos and human cells via a TIKI homology domain that is conserved from bacteria to mammals and acts likely as a protease to cleave eight amino-terminal residues of a Wnt protein, resulting in oxidized Wnt oligomers that exhibit normal secretion but minimized receptor-binding capability. Our findings identify a Wnt-specific protease that controls head formation, reveal a mechanism for morphogen inactivation through proteolysis-induced oxidation-oligomerization, and suggest a role of the Wnt amino terminus in evasion of oxidizing inactivation. TIKI proteins may represent potential therapeutic targets.

Self-derived structure-disrupting peptides targeting methionine aminopeptidase in pathogenic bacteria: a new strategy to generate antimicrobial peptides.

Bacterial infection is one of the leading causes of death in young, elderly, and immune-compromised patients. The rapid spread of multi-drug-resistant (MDR) bacteria is a global health emergency and there is a lack of new drugs to control MDR pathogens. We describe a heretofore-unexplored discovery pathway for novel antibiotics that is based on self-targeting, structure-disrupting peptides. We show that a helical peptide, KFF- EcH3, derived from the Escherichia coli methionine aminopeptidase can disrupt secondary and tertiary structure of this essential enzyme, thereby killing the bacterium (including MDR strains). Significantly, no detectable resistance developed against this peptide. Based on a computational analysis, our study predicted that peptide KFF- EcH3 has the strongest interaction with the structural core of the methionine aminopeptidase. We further used our approach to identify peptide KFF- NgH1 to target the same enzyme from Neisseria gonorrhoeae. This peptide inhibited bacterial growth and was able to treat a gonococcal infection in a human cervical epithelial cell model. These findings present an exciting new paradigm in antibiotic discovery using self-derived peptides that can be developed to target the structures of any essential bacterial proteins.-Zhan, J., Jia, H., Semchenko, E. A., Bian, Y., Zhou, A. M., Li, Z., Yang, Y., Wang, J., Sarkar, S., Totsika, M., Blanchard, H., Jen, F. E.-C., Ye, Q., Haselhorst, T., Jennings, M. P., Seib, K. L., Zhou, Y. Self-derived structure-disrupting peptides targeting methionine aminopeptidase in pathogenic bacteria: a new strategy to generate antimicrobial peptides.

Synthesis and biological evaluation of salicylate-based compounds as a novel class of methionine aminopeptidase inhibitors.

A series of salicylate-based compounds were designed and synthesized based on the simple function group replacement from our previously reported catechol-containing inhibitors of methionine aminopeptidase (MetAP). Some of these salicylate derivatives showed similar potency and metalloform selectivity, and some showed considerable antibacterial activity. These findings are consistent with our previous conclusion that Fe(II) is the likely metal used by MetAP in bacterial cells and provide new lead structures that can be further developed as novel antibacterial agents.

Two methionine aminopeptidases from Acinetobacter baumannii are functional enzymes.

Drug resistance in gram-negative bacteria, such as Acinetobacter baumannii, is emerging as a significant healthcare problem. New antibiotics with a novel mechanism of action are urgently needed to overcome the drug resistance. Methionine aminopeptidase (MetAP) carries out an essential cotranslational methionine excision in many bacteria and is a potential target to develop such novel antibiotics. Two putative MetAP genes were identified in A. baumannii genome, but whether they actually function as MetAP enzymes was not known. Therefore, we established an efficient E. coli expression system for their production as soluble and metal-free proteins for biochemical characterization. We demonstrated that both could carry out the metal-dependent catalysis and could be activated by divalent metal ions with the order Fe(II) ≈ Ni(II) > Co(II) > Mn(II) for both. By using a set of metalloform-selective inhibitors discovered on other MetAP enzymes, potency and metalloform selectivity on the A. baumannii MetAP proteins were observed. The similarity of their catalysis and inhibition to other MetAP enzymes confirmed that both may function as competent MetAP enzymes in A. baumannii and either or both may serve as the potential drug target.

Repurposing celecoxib analogues as leads for antibiotics.

There is an urgent need for new antibiotics and alternative strategies to combat bacterial pathogens. Molecular docking, antibacterial evaluation in vitro and in vivo, cytotoxicity assessment and enzyme inhibition analyses were performed. Compound 12 exhibited antimicrobial activity against Staphylococcus aureus (MIC: 4 µg/ml), various clinically isolated strains of MRSA (MIC: 4-16 µg/ml) and Acinetobacter baumannii (MIC: 4 µg/ml) when combined with subinhibitory concentrations of colistin B. Compound 12 (20 mg/kg) yielded mild improvement in survival of methicillin-resistant Staphylococcus aureus (MRSA)-infected mice. Additionally, enzyme inhibition tests showed that compound 12 exhibited inhibitory effects against S. aureus dihydrofolate reductase (105.1 µg/ml) and DNA gyrase (122.8 µg/ml). Compound 12 is a promising antibacterial candidate for further development.

A cell-based assay that targets methionine aminopeptidase in a physiologically relevant environment.

Methionine aminopeptidase (MetAP) is a promising target for the development of novel antibiotics. However, many potent inhibitors of the purified enzyme failed to show significant antibacterial activity. It is uncertain which divalent metal MetAP uses as its native cofactor in bacterial cells. Herein, we describe a cell-based assay that monitors the hydrolysis of a fluorogenic substrate by overexpressed MetAP in permeabilized Escherichia coli cells and its validation with a set of MetAP inhibitors. This cell-based assay is applicable to those cellular targets with poorly defined native cofactor, increasing the chances of identifying inhibitors that can inhibit the cellular target.

Expression and characterization of Mycobacterium tuberculosis methionine aminopeptidase type 1a.

Methionine aminopeptidase (MetAP) carries out the cotranslational N-terminal methionine excision and is essential for bacterial survival. Mycobacterium tuberculosis expresses two MetAPs, MtMetAP1a and MtMetAP1c, at different levels in growing and stationary phases, and both are potential targets to develop novel antitubercular therapeutics. Recombinant MtMetAP1a was purified as an apoenzyme, and metal binding and activation were characterized with an activity assay using a fluorogenic substrate. Ni(II), Co(II) and Fe(II) bound tightly at micromolar concentrations, and Ni(II) was the most efficient activator for the MetAP-catalyzed substrate hydrolysis. Although the characteristics of metal binding and activation are similar to MtMetAP1c we characterized before, MtMetAP1a was significantly more active, and more importantly, a set of inhibitors displayed completely different inhibitory profiles on the two mycobacterial MetAPs in both potency and metalloform selectivity. The differences in catalysis and inhibition predicted the significant differences in active site structure.

Determination of binding affinity of metal cofactor to the active site of methionine aminopeptidase based on quantitation of functional enzyme.

Determination of metal affinity to the active site of metalloenzymes constitutes an integral part in the understanding of enzyme catalysis and regulation. Nonlinear curve fitting of metal titration curves using the multiple independent binding sites (MIBS) model was adapted to determine K(D) values based on functional enzyme concentrations. This approach provides a more accurate evaluation of K(D) compared with existing methods that are based on total protein concentrations. We applied this concept to methionine aminopeptidase from Mycobacterium tuberculosis and showed that it is a monometalated enzyme with a K(D) of 0.13 microM for Co(2+).

Analysis of the stoichiometric metal activation of methionine aminopeptidase.

BACKGROUND: Methionine aminopeptidase (MetAP) is a ubiquitous enzyme required for cell survival and an attractive target for antibacterial and anticancer drug development. The number of a divalent metal required for catalysis is under intense debate. E. coli MetAP was shown to be fully active with one equivalent of metal by graphical analysis, but it was inferred to require at least two metals by a Hill equation model. Herein, we report a mathematical model and detailed analysis of the stoichiometric activation of MetAP by metal cofactors. RESULTS: Because of diverging results with significant implications in drug discovery, the experimental titration curve for Co2+ activating MetAP was analyzed by fitting with a multiple independent binding sites (MIBS) model, and the quality of the fitting was compared to that of the Hill equation. The fitting by the MIBS model was clearly superior and indicated that complete activity is observed at a one metal to one protein ratio. The shape of the titration curve was also examined for activation of metalloenzymes in general by one or two metals. CONCLUSIONS: Considering different scenarios of MetAP activation by one or two metal ions, it is concluded that E. coli MetAP is fully active as a monometalated enzyme. Our approach can be of value in proper determination of the number of cations needed for catalysis by metalloenzymes.

Discovery and development of a small molecule library with lumazine synthase inhibitory activity.

(E)-5-Nitro-6-(2-hydroxystyryl)pyrimidine-2,4(1H,3H)-dione (9) was identified as a novel inhibitor of Schizosaccharomyces pombe lumazine synthase by high-throughput screening of a 100000 compound library. The K(i) of 9 vs Mycobacterium tuberculosis lumazine synthase was 95 microM. Compound 9 is a structural analogue of the lumazine synthase substrate 5-amino-6-(d-ribitylamino)-2,4-(1H,3H)pyrimidinedione (1). This indicates that the ribitylamino side chain of the substrate is not essential for binding to the enzyme. Optimization of the enzyme inhibitory activity through systematic structure modification of the lead compound 9 led to (E)-5-nitro-6-(4-nitrostyryl)pyrimidine-2,4(1H,3H)-dione (26), which has a K(i) of 3.7 microM vs M. tuberculosis lumazine synthase.

Discovery and development of the covalent hydrates of trifluoromethylated pyrazoles as riboflavin synthase inhibitors with antibiotic activity against Mycobacterium tuberculosis.

A high-throughput screening (HTS) hit compound displayed moderate inhibition of Mycobacterium tuberculosis and Escherichia coli riboflavin synthases. The structure of the hit compound provided by the commercial vendor was reassigned as [3-(4-chlorophenyl)-5-hydroxy-5-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](o-tolyl)methanone (18). The hit compound had a k(is) of 8.7 microM vs. M. tuberculosis riboflavin synthase and moderate antibiotic activity against both M. tuberculosis replicating phenotype and nonreplicating persistent phenotype. Molecular modeling studies suggest that two inhibitor molecules bind in the active site of the enzyme, and that the binding is stabilized by stacking between the benzene rings of two adjacent ligands. The most potent antibiotic in the series proved to be [5-(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](m-tolyl)methanone (16), which displayed a minimum inhibitory concentration (MIC) of 36.6 microM vs. M. tuberculosis replicating phenotype and 48.9 microM vs. M. tuberculosis nonreplicating phenotype. The HTS hit compound and its analogues provide the first examples of riboflavin synthase inhibitors with antibiotic activity.

Metal-mediated inhibition is a viable approach for inhibiting cellular methionine aminopeptidase.

Methionine aminopeptidase (MetAP) plays an essential role for cell survival. Hence, MetAP is a promising target for developing broad spectrum antibacterial agents. MetAP can be activated in vitro by a number of divalent metals, and X-ray structures show that the active site can accommodate two cations. Herein, we demonstrate bacterial growth inhibition by a compound that targets MetAP by recruitment of a third auxiliary metal. Contrary to previous beliefs, this shows that metal-mediated inhibition is a viable approach for discovering MetAP inhibitors that are effective for therapeutic application.

Synthesis and structure-function analysis of Fe(II)-form-selective antibacterial inhibitors of Escherichia coli methionine aminopeptidase.

Methionine aminopeptidase (MetAP) is a promising target for the development of novel antibacterial, antifungal and anticancer therapy. Based on our previous results, catechol derivatives coupled with a thiazole or thiophene moiety showed high potency and selectivity toward the Fe(II)-form of Escherichia coli MetAP, and some of them clearly showed antibacterial activity, indicating that Fe(II) is likely the physiologically relevant metal for MetAP in E. coli and other bacterial cells. To further understand the structure-function relationship of these Fe(II)-form selective MetAP inhibitors, a series of catechol derivatives was designed and synthesized by replacement of the thiazole or thiophene moiety with different five-membered and six-membered heterocycles. Inhibitory activities of these newly synthesized MetAP inhibitors indicate that many five- and six-membered rings can be accommodated by MetAP and potency on the Fe(II)-form can be improved by introducing substitutions on the heterocyles to explore additional interactions with the enzyme. The furan-containing catechols 11-13 showed the highest potency at 1muM on the Fe(II)-form MetAP, and they were also among the best inhibitors for growth inhibition against E. coli AS19 strain. These findings provide useful information for the design and discovery of more effective MetAP inhibitors for therapeutic applications.

Identification and characterization of novel isothiazolones with potent bactericidal activity against multi-drug resistant Acinetobacter baumannii clinical isolates.

Acinetobacter baumannii has emerged as a globally important nosocomial pathogen characterized by an increased multi-drug resistance (MDR), leaving limited options for treating its infection. To identify novel antibacterial compounds with activity against clinical isolates of A. baumannii, we performed high-throughput screening against a chemical library of 42,944 compounds using nonpathogenic Escherichia coli MG1655 and identified 55 hit compounds. The antibacterial activities of 30 pure compounds were determined against MDR clinical isolates of A. baumannii obtained from Los Angeles County hospitals. Two isothiazolones identified, 5-chloro-2-(4-chloro-3-methylphenyl)-4-methyl-3(2H)-isothiazolone (Compound 6) and 5-chloro-2-(4-chlorophenyl)-4-methyl-3(2H)-isothiazolone (Compound 7), possess novel structure and exhibited consistent, potent and cidal activity against all 46 MDR A. baumannii clinical isolates and reference strains. Additionally, structure-activity relationship analysis involving several additional isothiazolones supports the link between a chloro-group on the heterocyclic ring or a fused benzene ring and the cidal activity. Attempts to obtain isothiazolone resistant mutants failed, consistent with the rapid cidal action and indicative of a complex mechanism of action. While cytotoxicity was observed with Compound 7, it had a therapeutic index value of 28 suggesting future therapeutic potential. Our results indicate that high-throughput screening of compound libraries followed by in vitro biological evaluations is a viable approach for the discovery of novel antibacterial agents to contribute in the fight against MDR bacterial pathogens.

Studies towards the synthesis of methionine aminopeptidase inhibitors: diversification utilizing a ROMP-derived coupling reagent.

Efforts to synthesize potential methionine aminopeptidase inhibitors is described. Preliminary SAR and docking studies served as a guide to design the compound libraries. "Chromatography-free" synthesis of various heterocyclic amides was realized by using a high-load, soluble coupling reagent derived via ring-opening metathesis polymerization (ROMP). Subsequent microwave-assisted Suzuki reactions with ortho-substituted arylboronic acids, followed by chromatographic purification afforded a 55-member library in high yields and purities. While the biological testing was not satisfactory, concurrent X-ray crystallography studies revealed key structural features essential for inhibition of methionine aminopeptidase, which directed fruitful results reported in the accompanying manuscript. In addition, in silico Lipinksi profiles and ADME properties of the library are also reported.

Ionic immobilization, diversification, and release: application to the generation of a library of methionine aminopeptidase inhibitors.

Development of an ionic immobilization, diversification, and release method for the generation of methionine aminopeptidase inhibitors is reported. This method involves the immobilization of 5-bromofuran-2-carboxylic acid and 5-bromothiophene-2-carboxylic acid onto PS-BEMP, followed by Suzuki reaction on a resin-bound intermediate and subsequent release to provide products in moderate yields and excellent purities. Compound potencies were evaluated on the Co(II), Mn(II), Ni(II), and Fe(II) forms of Escherichia coli MetAP1. The furoic-acid analogs were found to be Mn(II) selective with IC 50 values in the low micromolar range. Qualitative SAR analysis, supplemented by molecular modeling studies, provides valuable information on structural elements responsible for potency and selectivity.

Structural analysis of inhibition of E. coli methionine aminopeptidase: implication of loop adaptability in selective inhibition of bacterial enzymes.

BACKGROUND: Methionine aminopeptidase is a potential target of future antibacterial and anticancer drugs. Structural analysis of complexes of the enzyme with its inhibitors provides valuable information for structure-based drug design efforts. RESULTS: Five new X-ray structures of such enzyme-inhibitor complexes were obtained. Analysis of these and other three similar structures reveals the adaptability of a surface-exposed loop bearing Y62, H63, G64 and Y65 (the YHGY loop) that is an integral part of the substrate and inhibitor binding pocket. This adaptability is important for accommodating inhibitors with variations in size. When compared with the human isozymes, this loop either becomes buried in the human type I enzyme due to an N-terminal extension that covers its position or is replaced by a unique insert in the human type II enzyme. CONCLUSION: The adaptability of the YHGY loop in E. coli methionine aminopeptidase, and likely in other bacterial methionine aminopeptidases, enables the enzyme active pocket to accommodate inhibitors of differing size. The differences in this adaptable loop between the bacterial and human methionine aminopeptidases is a structural feature that can be exploited to design inhibitors of bacterial methionine aminopeptidases as therapeutic agents with minimal inhibition of the corresponding human enzymes.

Inhibition of monometalated methionine aminopeptidase: inhibitor discovery and crystallographic analysis.

Two divalent metal ions are commonly seen in the active-site cavity of methionine aminopeptidase, and at least one of the metal ions is directly involved in catalysis. Although ample structural and functional information is available for dimetalated enzyme, methionine aminopeptidase likely functions as a monometalated enzyme under physiological conditions. Information on structure, as well as catalysis and inhibition, of the monometalated enzyme is lacking. By improving conditions of high-throughput screening, we identified a unique inhibitor with specificity toward the monometalated enzyme. Kinetic characterization indicates a mutual exclusivity in binding between the inhibitor and the second metal ion at the active site. This is confirmed by X-ray structure, and this inhibitor coordinates with the first metal ion and occupies the space normally occupied by the second metal ion. Kinetic and structural analyses of the inhibition by this and other inhibitors provide insight in designing effective inhibitors of methionine aminopeptidase.

Selective inhibition of matrix metalloproteinase isozymes and in vivo protection against emphysema by substituted gamma-keto carboxylic acids.

The synthesis and matrix metalloproteinase (MMP) inhibitory activity of a series of gamma-keto carboxylic acids are described. Among nine MMP isozymes tested, compound 1j displays selective inhibition of MMP-2, -9, and -12 with IC(50) values between 0.20 and 1.51 microuM, and in male golden Syrian hamsters, it shows protection against PPE-induced emphysema.

Support vector machines in HTS data mining: Type I MetAPs inhibition study.

This article reports a successful application of support vector machines (SVMs) in mining high-throughput screening (HTS) data of a type I methionine aminopeptidases (MetAPs) inhibition study. A library with 43,736 small organic molecules was used in the study, and 1355 compounds in the library with 40% or higher inhibition activity were considered as active. The data set was randomly split into a training set and a test set (3:1 ratio). The authors were able to rank compounds in the test set using their decision values predicted by SVM models that were built on the training set. They defined a novel score PT50, the percentage of the test set needed to be screened to recover 50% of the actives, to measure the performance of the models. With carefully selected parameters, SVM models increased the hit rates significantly, and 50% of the active compounds could be recovered by screening just 7% of the test set. The authors found that the size of the training set played a significant role in the performance of the models. A training set with 10,000 member compounds is likely the minimum size required to build a model with reasonable predictive power.