RESUMO
ERAP1 is a zinc-dependent M1-aminopeptidase that trims lipophilic amino acids from the N-terminus of peptides. Owing to its importance in the processing of antigens and regulation of the adaptive immune response, dysregulation of the highly polymorphic ERAP1 has been implicated in autoimmune disease and cancer. To test this hypothesis and establish the role of ERAP1 in these disease areas, high affinity, cell permeable and selective chemical probes are essential. DG013A 1, is a phosphinic acid tripeptide mimetic inhibitor with reported low nanomolar affinity for ERAP1. However, this chemotype is a privileged structure for binding to various metal-dependent peptidases and contains a highly charged phosphinic acid moiety, so it was unclear whether it would display the high selectivity and passive permeability required for a chemical probe. Therefore, we designed a new stereoselective route to synthesize a library of DG013A 1 analogues to determine the suitability of this compound as a cellular chemical probe to validate ERAP1 as a drug discovery target.
Assuntos
Aminopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Oligopeptídeos/farmacologia , Ácidos Fosfínicos/farmacologia , Aminopeptidases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Antígenos de Histocompatibilidade Menor/metabolismo , Modelos Moleculares , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Ácidos Fosfínicos/síntese química , Ácidos Fosfínicos/química , Relação Estrutura-AtividadeRESUMO
ß-Lactamase production increasingly threatens the effectiveness of ß-lactams, which remain a mainstay of antimicrobial chemotherapy. New activities emerge through both mutation of previously known ß-lactamases and mobilization from environmental reservoirs. The spread of metallo-ß-lactamases (MBLs) represents a particular challenge because of their typically broad-spectrum activities encompassing carbapenems, in addition to other ß-lactam classes. Increasingly, genomic and metagenomic studies have revealed the distribution of putative MBLs in the environment, but in most cases their activity against clinically relevant ß-lactams and, hence, the extent to which they can be considered a resistance reservoir remain uncharacterized. Here we characterize the product of one such gene, blaRm3, identified through functional metagenomic sampling of an environment with high levels of biocide exposure. blaRm3 encodes a subclass B3 MBL that, when expressed in a recombinant Escherichia coli strain, is exported to the bacterial periplasm and hydrolyzes clinically used penicillins, cephalosporins, and carbapenems with an efficiency limited by high Km values. An Rm3 crystal structure reveals the MBL superfamily αß/ßα fold, which more closely resembles that in mobilized B3 MBLs (AIM-1 and SMB-1) than other chromosomal enzymes (L1 or FEZ-1). A binuclear zinc site sits in a deep channel that is in part defined by a relatively extended N terminus. Structural comparisons suggest that the steric constraints imposed by the N terminus may limit its affinity for ß-lactams. Sequence comparisons identify Rm3-like MBLs in numerous other environmental samples and species. Our data suggest that Rm3-like enzymes represent a distinct group of B3 MBLs with a wide distribution and can be considered an environmental reservoir of determinants of ß-lactam resistance.
Assuntos
Metagenômica/métodos , beta-Lactamases/química , beta-Lactamases/genética , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Filogenia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Lactamases/metabolismoRESUMO
Metallo-ß-Lactamases (MBLs) protect bacteria from almost all ß-lactam antibiotics. Verona integron-encoded MBL (VIM) enzymes are among the most clinically important MBLs, with VIM-1 increasing in carbapenem-resistant Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) that are among the hardest bacterial pathogens to treat. VIM enzymes display sequence variation at residues (224 and 228) that in related MBLs are conserved and participate in substrate binding. How they accommodate this variability, while retaining catalytic efficiency against a broad substrate range, has remained unclear. Here, we present crystal structures of VIM-1 and its complexes with a substrate-mimicking thioenolate inhibitor, ML302F, that restores meropenem activity against a range of VIM-1 producing clinical strains, and the hydrolysed product of the carbapenem meropenem. Comparison of these two structures identifies a water-mediated hydrogen bond, between the carboxylate group of substrate/inhibitor and the backbone carbonyl of the active site zinc ligand Cys221, that is common to both complexes. Structural comparisons show that the responsible Cys221-bound water is observed in all known VIM structures, participates in carboxylate binding with other inhibitor classes, and thus effectively replicates the role of the conserved Lys224 in analogous complexes with other MBLs. These results provide a mechanism for substrate binding that permits the variation at positions 224 and 228 that is a hallmark of VIM MBLs. ENZYMES: EC 3.5.2.6 DATABASES: Co-ordinates and structure factors for protein structures described in this manuscript have been deposited in the Protein Data Bank (www.rcsb.org/pdb) with accession codes 5N5G (VIM-1), 5N5H (VIM-1:ML302F complex) and 5N5I (VIM-1-hydrolysed meropenem complex).
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Enterobacteriaceae/enzimologia , Meropeném/química , Meropeném/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high-resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than 2 orders of magnitude compared to their human homologue, demonstrating a route to the selective chemical inhibition of these bacterial targets.
Assuntos
Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Sondas Moleculares/química , Serina-tRNA Ligase/antagonistas & inibidores , Staphylococcus aureus/enzimologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Estrutura Molecular , Serina-tRNA Ligase/químicaRESUMO
Serine- and metallo-ß-lactamases present a threat to the clinical use of nearly all ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. Efforts to develop metallo-ß-lactamase (MBL) inhibitors require suitable screening platforms to allow the rapid determination of ß-lactamase activity and efficient inhibition. Unfortunately, the platforms currently available are not ideal for this purpose. Further progress in MBL inhibitor identification requires inexpensive and widely applicable assays. Herein the identification of an inexpensive and stable chromogenic substrate suitable for use in assays of clinically relevant MBLs is described. (6R,7R)-3-((4-Nitrophenoxy)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5,5-dioxide (CLS405) was synthesised in a three-step protocol. CLS405 was then characterised spectroscopically, and its stability and kinetic properties evaluated. With a Δλmax value of 100â nm between the parent and hydrolysis product, a higher analytical accuracy is possible with CLS405 than with commonly used chromogenic substrates. The use of CLS405 in assays was validated by MBL activity measurements and inhibitor screening that resulted in the identification of N-hydroxythiazoles as new inhibitor scaffolds for MBLs. Further evaluation of the identified N-hydroxythiazoles against a panel of clinically relevant MBLs showed that they possess inhibitory activities in the mid- to low-micromolar range. The findings of this study provide both a useful tool compound for further inhibitor identification, and novel scaffolds for the design of improved MBL inhibitors with potential as antibiotics against resistant strains of bacteria.
Assuntos
Cefalosporinas/química , Inibidores Enzimáticos/química , Inibidores de beta-Lactamases , Antibacterianos/farmacologia , Cefalosporinas/síntese química , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Cinética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato , Tiazóis/síntese química , Tiazóis/química , Tiazóis/farmacologia , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Metallo-ß-lactamases (MBLs) are a growing threat to the use of almost all clinically used ß-lactam antibiotics. The identification of broad-spectrum MBL inhibitors is hampered by the lack of a suitable screening platform, consisting of appropriate substrates and a set of clinically relevant MBLs. We report procedures for the preparation of a set of clinically relevant metallo-ß-lactamases (i.e., NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (São Paulo MBL), and VIM-2 (Verona integron-encoded MBL)) and the identification of suitable fluorogenic substrates (umbelliferone-derived cephalosporins). The fluorogenic substrates were compared to chromogenic substrates (CENTA, nitrocefin, and imipenem), showing improved sensitivity and kinetic parameters. The efficiency of the fluorogenic substrates was exemplified by inhibitor screening, identifying 4-chloroisoquinolinols as potential pan MBL inhibitors.