RESUMEN
Nitroxides are stable organic radicals with exceptionally long lifetimes, which render them uniquely suitable as observable probes or polarising agents for spectroscopic investigation of biomolecular structure and dynamics. Radical-based probes for biological applications are ideally characterized by both robustness towards reductive degradation and beneficial electron spin relaxation parameters. These properties are largely influenced by the molecular structure of the nitroxide scaffold, and also by the conformations it prefers to adopt. In this study we present the synthesis of the first nitroxides based on a spirocyclic pyrrolidine scaffold with an exocyclic methylene substituent. The conformations adopted by these nitroxides were evaluated by X-ray crystallography, both with single nitroxide crystals and by inclusion of nitroxides in a microporous crystalline sponge. The kinetic and thermodynamic stability of the new nitroxides towards reduction was investigated by electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry (CV). In combination with EPR measurements of electron spin relaxation properties, these results suggest that this new family of nitroxides can provide access to multifunctionalized probes and polarising agents suitable for use in biological environments at elevated temperatures.
RESUMEN
The upswing of antibiotic resistance is an escalating threat to human health. Resistance mediated by bacterial metallo-ß-lactamases is of particular concern as these enzymes degrade ß-lactams, our most frequently prescribed class of antibiotics. Inhibition of metallo-ß-lactamases could allow the continued use of existing ß-lactam antibiotics, such as penicillins, cephalosporins and carbapenems, whose applicability is becoming ever more limited. The design, synthesis, and NDM-1, VIM-2, and GIM-1 inhibitory activities (IC50 4.1-506 µM) of a series of novel non-cytotoxic α-aminophosphonate-based inhibitor candidates are presented herein. We disclose the solution NMR spectroscopic and computational investigation of their NDM-1 and VIM-2 binding sites and binding modes. Whereas the binding modes of the inhibitors are similar, VIM-2 showed a somewhat higher conformational flexibility, and complexed a larger number of inhibitor candidates in more varying binding modes than NDM-1. Phosphonate-type inhibitors may be potential candidates for development into therapeutics to combat metallo-ß-lactamase resistant bacteria.
RESUMEN
Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted, compared to mesophilic orthologs. In several cases, the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation. In the psychrophilic α-amylase from an Antarctic bacterium, the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature. Here, we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward. A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations of the catalytic reaction at different temperatures. The predictions were verified by kinetic experiments and crystal structures of the redesigned α-amylase, showing that the temperature optimum is indeed markedly shifted upward and that the critical surface loop controlling the temperature dependence approaches the target conformation observed in a mesophilic ortholog.
Asunto(s)
Frío , Proteínas , Temperatura , Conformación Molecular , alfa-Amilasas/química , alfa-Amilasas/metabolismoRESUMEN
Being the second leading cause of death and the leading cause of disability-adjusted life years worldwide, infectious diseases remain-contrary to earlier predictions-a major consideration for the public health of the 21st century. Resistance development of microbes to antimicrobial drugs constitutes a large part of this devastating problem. The most widely spread mechanism of bacterial resistance operates through the degradation of existing ß-lactam antibiotics. Inhibition of metallo-ß-lactamases is expected to allow the continued use of existing antibiotics, whose applicability is becoming ever more limited. Herein, we describe the synthesis, the metallo-ß-lactamase inhibition activity, the cytotoxicity studies, and the NMR spectroscopic determination of the protein binding site of phosphonamidate monoesters. The expression of single- and double-labeled NDM-1 and its backbone NMR assignment are also disclosed, providing helpful information for future development of NDM-1 inhibitors. We show phosphonamidates to have the potential to become a new generation of antibiotic therapeutics to combat metallo-ß-lactamase-resistant bacteria.
RESUMEN
Metallo-ß-lactamases (MBLs) are an emerging cause of bacterial antibiotic resistance by hydrolysing all classes of ß-lactams except monobactams, and the MBLs are not inhibited by clinically available serine-ß-lactamase inhibitors. Two of the most commonly encountered MBLs in clinical isolates worldwide - the New Delhi metallo-ß-lactamase (NDM-1) and the Verona integron-encoded metallo-ß-lactamase (VIM-2) - are included in this study. A series of several NH-1,2,3-triazoles was prepared by a three-step protocol utilizing Banert cascade reaction as the key step. The inhibitor properties were evaluated in biochemical assays against the MBLs VIM-2, NDM-1 and GIM-1, and VIM-2 showed IC50 values down to nanomolar range. High-resolution crystal structures of four inhibitors in complex with VIM-2 revealed hydrogen bonds from the triazole inhibitors to Arg228 and to the backbone of Ala231 or Asn233, along with hydrophobic interactions to Trp87, Phe61 and Tyr67. The inhibitors show reduced MIC in synergy assays with Pseudomonas aeruginosa and Escherichia coli strains harbouring VIM enzymes. The obtained results will be useful for further structural guided design of MBL inhibitors.
Asunto(s)
Triazoles/farmacología , Resistencia betalactámica/efectos de los fármacos , Inhibidores de beta-Lactamasas/farmacología , Antibacterianos/farmacología , Dominio Catalítico , Cristalografía por Rayos X , Sinergismo Farmacológico , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , Klebsiella pneumoniae/efectos de los fármacos , Meropenem/farmacología , Estructura Molecular , Unión Proteica , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/metabolismo , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/metabolismo , beta-Lactamasas/metabolismoRESUMEN
Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (ß-lactamases able to inactivate carbapenems) have been identified in both serine ß-lactamase (SBL) and metallo-ß-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 µM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by â¼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.
Asunto(s)
Inhibidores de beta-Lactamasas , beta-Lactamasas , Animales , Antibacterianos/farmacología , Carbapenémicos/farmacología , Meropenem/farmacología , Ratones , Resistencia betalactámica , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/genéticaRESUMEN
Metallo-ß-lactamases (MBLs) threaten the effectiveness of ß-lactam antibiotics, including carbapenems, and are a concern for global public health. ß-Lactam/ß-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-ß-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC50) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC50 levels of the new thiol-based inhibitors were 0.66 µM (inhibitor 2a) and 0.62 µM (inhibitor 2b), and the equilibrium dissociation constant (KD ) of inhibitor 2a was 1.6 µM; thus, both were more potent inhibitors than l-captopril (IC50 = 47 µM; KD = 25 µM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than l-captopril.
Asunto(s)
Achromobacter denitrificans , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , beta-Lactamasas/genética , Achromobacter denitrificans/efectos de los fármacos , Achromobacter denitrificans/enzimología , Achromobacter denitrificans/genética , Proteínas Bacterianas/metabolismo , Captopril/farmacología , Carbapenémicos/uso terapéutico , Pruebas de Sensibilidad Microbiana , Resonancia por Plasmón de Superficie , Inhibidores de beta-Lactamasas/uso terapéutico , beta-Lactamasas/metabolismoRESUMEN
Bacterial resistance is compromising the use of ß-lactam antibiotics including carbapenems. The main resistance mechanism against ß-lactams is hydrolysis of the ß-lactam ring mediated by serine- or metallo-ß-lactamases (MBLs). Although several inhibitors of MBLs have been reported, none has been developed into a clinically useful inhibitor. Mercaptocarboxylic acids are among the most prominent scaffolds reported as MBL inhibitors. In this study, the carboxylate group of mercaptocarboxylic acids was replaced with bioisosteric groups like phosphonate esters, phosphonic acids and NH-tetrazoles. The influence of the replacement on the bioactivity and inhibitor binding was evaluated. A series of bioisosteres of previously reported inhibitors was synthesized and evaluated against the MBLs VIM-2, NDM-1 and GIM-1. The most active inhibitors combined a mercapto group and a phosphonate ester or acid, with two/three carbon chains connecting a phenyl group. Surprisingly, also compounds containing thioacetate groups instead of thiols showed low IC50 values. High-resolution crystal structures of three inhibitors in complex with VIM-2 revealed hydrophobic interactions for the diethyl groups in the phosphonate ester (inhibitor 2b), the mercapto bridging the two active site zinc ions, and tight stacking of the benzene ring to the inhibitor between Phe62, Tyr67, Arg228 and His263. The inhibitors show reduced enzyme activity in Escherichia coli cells harboring MBL. The obtained results will be useful for further structural guided design of MBL inhibitors.
Asunto(s)
Ácidos Carboxílicos/farmacología , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/metabolismo , Sitios de Unión/efectos de los fármacos , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Relación Dosis-Respuesta a Droga , Estructura Molecular , Pseudomonas aeruginosa/enzimología , Relación Estructura-Actividad , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/químicaRESUMEN
Metallo-ß-lactamases (MBLs) hydrolyze virtually all ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative ß-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with ß-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the ß-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.
Asunto(s)
beta-Lactamasas/química , beta-Lactamasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Ampicilina/metabolismo , Dominio Catalítico , Ceftazidima/metabolismo , Cristalografía por Rayos X , Estabilidad de Enzimas , Hidrólisis , Imipenem/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , beta-Lactamasas/genéticaRESUMEN
Metallo-ß-lactamases (MBLs) are the causative mechanism for resistance to ß-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (T(m)) of 48.5°C, compared to 55.3 °C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higher Tm (57.1 °C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (T(m), 62.9 °C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.