RESUMO
The increasing rate of carbapenem-resistant bacteria within healthcare environments is an issue of great concern that needs urgent attention. This resistance is driven by metallo-ß-lactamases (MBLs), which can catalyse the hydrolysis of almost all clinically available ß-lactams and are resistant to all the clinically utilized ß-lactamase inhibitors. In this study, an uncharacterized MBL is identified in a multidrug resistant isolate of the opportunistic pathogen, Chryseobacterium indologenes. Sequence analysis predicts this MBL (CIM-1) to be a lipoprotein with an atypical lipobox. Characterization of CIM-1 reveals it to be a high-affinity carbapenemase with a broad spectrum of activity that includes all cephalosporins and carbapenems. Results also shown that CIM-1 is potentially a membrane-associated MBL with an uncharacterized lipobox. Using prediction tools, we also identify more potentially lipidated MBLs with non-canonical lipoboxes highlighting the necessity of further investigation of lipidated MBLs.
Assuntos
Antibacterianos , Fatores R , Antibacterianos/farmacologia , beta-Lactamases/genética , Proteínas de Bactérias/genéticaRESUMO
Recently, several ß-lactam (BL)/ß-lactamase inhibitor (BLI) combinations have entered clinical testing or have been marketed for use, but limited direct comparative studies of their in vitro activity exist. Xeruborbactam (XER, also known as QPX7728), which is undergoing clinical development, is a cyclic boronate BLI with potent inhibitory activity against serine (serine ß-lactamase) and metallo-ß-lactamases (MBLs). The objectives of this study were (i) to compare the potency and spectrum of ß-lactamase inhibition by various BLIs in biochemical assays using purified ß-lactamases and in microbiological assays using the panel of laboratory strains expressing diverse serine and metallo-ß-lactamases and (ii) to compare the in vitro potency of XER in combination with multiple ß-lactam antibiotics to that of other BL/BLI combinations in head-to-head testing against recent isolates of carbapenem-resistant Enterobacterales (CRE). Minimal inhibitory concentrations (MICs) of XER combinations were tested with XER at fixed 4 or 8 µg/mL, and MIC testing was conducted in a blinded fashion using Clinical and Laboratory Standards Institute reference methods. Xeruborbactam and taniborbactam (TAN) were the only BLIs that inhibited clinically important MBLs. The spectrum of activity of xeruborbactam included several MBLs identified in Enterobacterales, e.g., and various IMP enzymes and NDM-9 that were not inhibited by taniborbactam. Xeruborbactam potency against the majority of purified ß-lactamases was the highest in comparison with other BLIs. Meropenem-xeruborbactam (MEM-XER, fixed 8 µg/mL) was the most potent combination against MBL-negative CRE with MIC90 values of 0.125 µg/mL. MEM-XER and cefepime-taniborbactam (FEP-TAN) were the only BL/BLIs with activity against MBL-producing CREs; with MEM-XER (MIC90 of 1 µg/mL) being at least 16-fold more potent than FEP-TAN (MIC90 of 16 µg/mL). MEM-XER MIC values were ≤8 µg/mL for >90% of CRE, including both MBL-negative and MBL-positive isolates, with FEP-TAN MIC of >8 µg/mL. Xeruborbactam also significantly enhanced potency of other ß-lactam antibiotics, including cefepime, ceftolozane, ceftriaxone, aztreonam, piperacillin, and ertapenem, against clinical isolates of Enterobacterales that carried various class A, class C, and class D extended-spectrum ß-lactamases and carbapenem-resistant Enterobacterales, including metallo-ß-lactamase-producing isolates. These results strongly support further clinical development of xeruborbactam combinations.
Assuntos
Antibacterianos , Inibidores de beta-Lactamases , Inibidores de beta-Lactamases/farmacologia , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Antibióticos beta Lactam , Cefepima , Lactamas , beta-Lactamases , Serina , Testes de Sensibilidade Microbiana , Compostos Azabicíclicos/farmacologiaRESUMO
Taniborbactam and xeruborbactam are dual serine-/metallo-beta-lactamase inhibitors (BLIs) based on a cyclic boronic acid pharmacophore that undergo clinical development. Recent report demonstrated that New Delhi metallo-beta-lactamase (NDM)-9 (differs from NDM-1 by a single amino acid substitution, E152K, evolved to overcome Zn (II) deprivation) is resistant to inhibition by taniborbactam constituting pre-existing taniborbactam resistance mechanism. Using microbiological and biochemical experiments, we show that xeruborbactam is capable of inhibiting NDM-9 and propose the structural basis for differences between two BLIs.
Assuntos
Ácidos Borínicos , Substituição de Aminoácidos , Ácidos Borônicos/farmacologia , Resistência beta-Lactâmica/genética , Inibidores de beta-Lactamases/farmacologiaRESUMO
Transporters of the resistance-nodulation-cell division (RND) superfamily of proteins are the dominant multidrug efflux power of Gram-negative bacteria. The major RND efflux pump of Pseudomonas aeruginosa is MexAB-OprM, in which the inner membrane transporter MexB is responsible for the recognition and binding of compounds. The high importance of this pump in clinical antibiotic resistance made it a subject of intense investigations and a promising target for the discovery of efflux pump inhibitors. This study is focused on a series of peptidomimetic compounds developed as effective inhibitors of MexAB-OprM. We performed multi-copy molecular dynamics simulations, machine-learning (ML) analyses, and site-directed mutagenesis of MexB to investigate interactions of MexB with representatives of efflux avoiders, substrates, and inhibitors. The analysis of both direct and water-mediated protein-ligand interactions revealed characteristic patterns for each class, highlighting significant differences between them. We found that efflux avoiders poorly interact with the access binding site of MexB, and inhibition engages amino acid residues that are not directly involved in binding and transport of substrates. In agreement, machine-learning models selected different residues predictive of MexB substrates and inhibitors. The differences in interactions were further validated by site-directed mutagenesis. We conclude that the substrate translocation and inhibition pathways of MexB split at the interface (between the main putative binding sites) and at the deep binding pocket and that interactions outside of the hydrophobic patch contribute to the inhibition of MexB. This molecular-level information could help in the rational design of new inhibitors and antibiotics less susceptible to the efflux mechanism. IMPORTANCE Multidrug transporters recognize and expel from cells a broad range of ligands including their own inhibitors. The difference between the substrate translocation and inhibition routes remains unclear. In this study, machine learning and computational and experimental approaches were used to understand dynamics of MexB interactions with its ligands. Our results show that some ligands engage a certain combination of polar and charged residues in MexB binding sites to be effectively expelled into the exit funnel, whereas others engage aromatic and hydrophobic residues that slow down or hinder the next step in the transporter cycle. These findings suggest that all MexB ligands fit into this substrate-inhibitor spectrum depending on their physico-chemical structures and properties.
Assuntos
Proteínas da Membrana Bacteriana Externa , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Ligantes , Testes de Sensibilidade Microbiana , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Xeruborbactam (formerly QPX7728) is a cyclic boronate inhibitor of numerous serine and metallo-beta-lactamases. At concentrations generally higher than those required for beta-lactamase inhibition, xeruborbactam has direct antibacterial activity against some Gram-negative bacteria, with MIC50/MIC90 values of 16/32 µg/mL and 16/64 µg/mL against carbapenem-resistant Enterobacterales and carbapenem-resistant Acinetobacter baumannii, respectively (the MIC50/MIC90 values against Pseudomonas aeruginosa are >64 µg/mL). In Klebsiella pneumoniae, inactivation of OmpK36 alone or in combination with OmpK35 resulted in 2- to 4-fold increases in the xeruborbactam MIC. In A. baumannii and P. aeruginosa, AdeIJK and MexAB-OprM, respectively, affected xeruborbactam's antibacterial potency (the MICs were 4- to 16-fold higher in efflux-proficient strains). In Escherichia coli and K. pneumoniae, the 50% inhibitory concentrations (IC50s) of xeruborbactam's binding to penicillin-binding proteins (PBPs) PBP1a/PBP1b, PBP2, and PBP3 were in the 40 to 70 µM range; in A. baumannii, xeruborbactam bound to PBP1a, PBP2, and PBP3 with IC50s of 1.4 µM, 23 µM, and 140 µM, respectively. Treating K. pneumoniae and P. aeruginosa with xeruborbactam at 1× and 2× MIC resulted in changes of cellular morphology similar to those observed with meropenem; the morphological changes observed after treatment of A. baumannii were consistent with inhibition of multiple PBPs but were unique to xeruborbactam compared to the results for control beta-lactams. No single-step xeruborbactam resistance mutants were obtained after selection at 4× MIC of xeruborbactam using wild-type strains of E. coli, K. pneumoniae, and A. baumannii; mutations selected at 2× MIC in K. pneumoniae did not affect antibiotic potentiation by xeruborbactam through beta-lactamase inhibition. Consistent with inhibition of PBPs, xeruborbactam enhanced the potencies of beta-lactam antibiotics even against strains that lacked beta-lactamase. In a large panel of KPC-producing clinical isolates, the MIC90 values of meropenem tested with xeruborbactam (8 µg/mL) were at least 4-fold lower than those in combination with vaborbactam at 64 µg/mL, the concentration of vaborbactam that is associated with complete inhibition of KPC. The additional enhancement of the potency of beta-lactam antibiotics beyond beta-lactamase inhibition may contribute to the potentiation of beta-lactam antibiotics by xeruborbactam.
Assuntos
Antibacterianos , Escherichia coli , Meropeném/farmacologia , Meropeném/metabolismo , Proteínas de Ligação às Penicilinas/genética , Escherichia coli/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , beta-Lactamases/genética , beta-Lactamas/farmacologia , Testes de Sensibilidade Microbiana , Klebsiella pneumoniae/genética , Carbapenêmicos/farmacologia , Carbapenêmicos/metabolismo , Monobactamas/metabolismo , Pseudomonas aeruginosa/metabolismo , Serina/metabolismoRESUMO
Early efforts to broaden the spectrum and potency of cyclic boronic acid ß-lactamase inhibitor vaborbactam included a series of 7-membered ring boronates. Exploration of stereoisomers and incorporation of heteroatoms allowed identification of the all-carbon cyclic boronate with substituents trans as the preferred core structure, showing inhibition of Class A and C enzymes. Crystal structures of one analog bound to important ß-lactamase enzymes were obtained. When isolated under acidic conditions, these compounds spontaneously formed a neutral cyclic anhydride (intramolecular prodrug) which was shown to have much-improved oral bioavailability (52-69%) compared to the ring-opened carboxylate salt (9%).
Assuntos
Pró-Fármacos , Inibidores de beta-Lactamases , Antibacterianos/química , Antibacterianos/farmacologia , Disponibilidade Biológica , Pró-Fármacos/farmacologia , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismoRESUMO
The emergence of multidrug-resistant (MDR) Gram-negative pathogens is an urgent global medical challenge. The old polymyxin lipopeptide antibiotics (polymyxin B and colistin) are often the only therapeutic option due to resistance to all other classes of antibiotics and the lean antibiotic drug development pipeline. However, polymyxin B and colistin suffer from major issues in safety (dose-limiting nephrotoxicity, acute toxicity), pharmacokinetics (poor exposure in the lungs) and efficacy (negligible activity against pulmonary infections) that have severely limited their clinical utility. Here we employ chemical biology to systematically optimize multiple non-conserved positions in the polymyxin scaffold, and successfully disconnect the therapeutic efficacy from the toxicity to develop a new synthetic lipopeptide, structurally and pharmacologically distinct from polymyxin B and colistin. This resulted in the clinical candidate F365 (QPX9003) with superior safety and efficacy against lung infections caused by top-priority MDR pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.
Assuntos
Colistina , Polimixina B , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla , Lipopeptídeos/farmacologia , Lipopeptídeos/uso terapêutico , Testes de Sensibilidade Microbiana , Polimixinas/farmacologia , Polimixinas/uso terapêutico , Pseudomonas aeruginosaRESUMO
QPX7728 is a cyclic boronate ultrabroad-spectrum beta-lactamase inhibitor, with potent activity against both serine beta-lactamases and metallo-beta-lactamases. QPX7728 can be delivered systemically by the intravenous (i.v.) or oral route of administration. Oral beta-lactam antibiotics alone or in combination with QPX7728 were evaluated for (i) sensitivity to hydrolysis by various common beta-lactamases and inhibition of hydrolysis by QPX7728, (ii) the impact of non-beta-lactamase-mediated resistance mechanisms on potency of beta-lactams, and (iii) in vitro activity against a panel of clinical strains producing diverse beta-lactamases. The carbapenem tebipenem had stability for many serine beta-lactamases from all molecular classes, followed by the cephalosporin ceftibuten. Addition of QPX7728 to tebipenem, ceftibuten, and amdinocillin completely reversed beta-lactamase-mediated resistance in cloned beta-lactamases from serine enzyme and metalloenzyme classes; the degree of potentiation of other beta-lactams varied according to the beta-lactamase produced. Tebipenem, ceftibuten, and cefixime had the lowest MICs against laboratory strains with various combinations of beta-lactamases and the intrinsic drug resistance mechanisms of porin and efflux mutations. There was a high degree of correlation between potency of various combinations against cloned beta-lactamases and efflux/porin mutants and the activity against clinical isolates, showing the importance of inhibition of beta-lactamase along with minimal impact of general intrinsic resistance mechanisms affecting the beta-lactam. Tebipenem and ceftibuten appeared to be the best beta-lactam antibiotics when combined with QPX7728 for activity against Enterobacterales that produce serine beta-lactamases or metallo-beta-lactamases.
Assuntos
Inibidores de beta-Lactamases , beta-Lactamases , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ácidos Borínicos , Ácidos Carboxílicos , Testes de Sensibilidade Microbiana , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/uso terapêutico , beta-Lactamases/genética , beta-Lactamas/farmacologiaRESUMO
In recognition of the need for effective oral therapies to treat Gram-negative bacterial infections, efforts were directed toward identifying an oral prodrug of ß-lactamase inhibitor clinical candidate QPX7728. Seventeen prodrugs were synthesized; key properties investigated were rates of cleavage to the active form in vitro, pharmacokinetics across species, and crystallinity. Compound 5-Na (QPX7831 Sodium) emerged with optimal properties across all key attributes.
Assuntos
Ácidos Borínicos/farmacologia , Ácidos Carboxílicos/farmacologia , Pró-Fármacos/farmacologia , Inibidores de beta-Lactamases/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , HumanosRESUMO
QPX7728 is a novel ß-lactamase inhibitor (BLI) that belongs to a class of cyclic boronates. The first member of this class, vaborbactam, is a BLI in the recently approved Vabomere (meropenem-vaborbactam). In this paper we provide the overview of the biochemical, structural and microbiological studies that were recently conducted with QPX7728. We show that QPX7728 is an ultra-broad-spectrum ß-lactamase inhibitor with the broadest spectrum of inhibition reported to date in a single BLI molecule; in addition to potent inhibition of clinically important serine ß-lactamases, including Class A and D carbapenemases from Enterobacterales and notably, diverse Class D carbapenemases from Acinetobacter, it also inhibits many metallo ß-lactamases. Importantly, it is minimally affected by general intrinsic resistance mechanisms such as efflux and porin mutations that impede entry of drugs into gram-negative bacteria. QPX7728 combinations with several intravenous (IV) ß-lactam antibiotics shows broad coverage of Enterobacterales, Acinetobacter baumannii and Pseudomonas aeruginosa, including strains that are resistant to other IV ß-lactam-BLI combinations, e.g., ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam and imipenem-relebactam that were recently approved for clinical use. Based on studies with P. aeruginosa, different partner ß-lactams in combination with QPX7728 may be optimal for the coverage of susceptible organisms. This provides microbiological justification for a stand-alone BLI product for co-administration with different ß-lactams. QPX7728 can also be delivered orally; thus, its ultra-broad ß-lactamase inhibition spectrum and other features could be also applied to oral QPX7728-based combination products. Clinical development of QPX7728 has been initiated.
RESUMO
QPX7728 is an ultrabroad-spectrum beta-lactamase inhibitor with potent inhibition of key serine and metallo beta-lactamases. QPX7728 enhances the potency of multiple beta-lactams in beta-lactamase-producing Enterobacterales and Acinetobacter spp. In this study, we evaluated the in vitro activity of QPX7728 (QPX; 8 µg/ml) combined with multiple beta-lactams against clinical isolates of Pseudomonas aeruginosa with various beta-lactam resistance mechanisms. Seven hundred ninety clinical isolates were included in this study; 500 isolates, termed a "representative panel," were selected to be representative of the MIC distribution of meropenem (MEM), ceftazidime-avibactam (CAZ-AVI), and ceftolozane-tazobactam (TOL-TAZ) resistance for clinical isolates according to 2017 SENTRY surveillance data. An additional 290 selected isolates ("challenge panel") that were either nonsusceptible to MEM or were resistant to TOL-TAZ or CAZ-AVI were also tested; 61 strains carried metallo-beta-lactamases (MBLs), 211 strains were defective in the carbapenem porin OprD, and 185 strains had the MexAB-OprM efflux pump overproduced based on a phenotypic test. Against the representative panel, susceptibility for all QPX7728/beta-lactam combinations was >90%. For the challenge panel, QPX-ceftolozane (TOL) was the most active combination (78.6% susceptible) followed by equipotent QPX-piperacillin (PIP) and QPX-cefepime (FEP), restoring susceptibility in 70.3% of strains (CLSI breakpoints for the beta-lactam compound alone). For MBL-negative strains, QPX-TOL and QPX-FEP restored the MIC values to susceptibility rates in â¼90% and â¼80% of strains, respectively, versus 68% to 70% for QPX-MEM and QPX-PIP and 63% to 65% for TOL-TAZ and CAZ-AVI, respectively. For MBL-positive strains, QPX-PIP restored the MIC to susceptibility values for â¼70% of strains versus 2% to 40% for other combinations. Increased efflux and impaired OprD had various effect on QPX7728 combination depending on the partner beta-lactam tested. QPX7728 enhanced the potency of multiple beta-lactams against P. aeruginosa, with varied results according to beta-lactamase production and other intrinsic resistance mechanisms.
Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/tratamento farmacológico , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
Antibiotic-resistant bacteria rapidly spread in clinical and natural environments and challenge our modern lifestyle. A major component of defense against antibiotics in Gram-negative bacteria is a drug permeation barrier created by active efflux across the outer membrane. We identified molecular determinants defining the propensity of small peptidomimetic molecules to avoid and inhibit efflux pumps in Pseudomonas aeruginosa, a human pathogen notorious for its antibiotic resistance. Combining experimental and computational protocols, we mapped the fate of the compounds from structure-activity relationships through their dynamic behavior in solution, permeation across both the inner and outer membranes, and interaction with MexB, the major efflux transporter of P. aeruginosa We identified predictors of efflux avoidance and inhibition and demonstrated their power by using a library of traditional antibiotics and compound series and by generating new inhibitors of MexB. The identified predictors will enable the discovery and optimization of antibacterial agents suitable for treatment of P. aeruginosa infections.IMPORTANCE Efflux pump avoidance and inhibition are desired properties for the optimization of antibacterial activities against Gram-negative bacteria. However, molecular and physicochemical interactions defining the interface between compounds and efflux pumps remain poorly understood. We identified properties that correlate with efflux avoidance and inhibition, are predictive of similar features in structurally diverse compounds, and allow researchers to distinguish between efflux substrates, inhibitors, and avoiders in P. aeruginosa The developed predictive models are based on the descriptors representative of different clusters comprising a physically intuitive combination of properties. Molecular shape (represented by acylindricity), amphiphilicity (anisotropic polarizability), aromaticity (number of aromatic rings), and the partition coefficient (LogD) are physicochemical predictors of efflux inhibitors, whereas interactions with Pro668 and Leu674 residues of MexB distinguish between inhibitors/substrates and efflux avoiders. The predictive models and efflux rules are applicable to compounds with unrelated chemical scaffolds and pave the way for development of compounds with the desired efflux interface properties.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Modelos Biológicos , Peptidomiméticos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Expressão Gênica , Cinética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Análise de Componente Principal , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Relação Estrutura-Atividade , TermodinâmicaRESUMO
QPX7728 is a recently discovered ultra-broad-spectrum beta-lactamase inhibitor (BLI) with potent inhibition of key serine and metallo-beta-lactamases. QPX7728 enhances the potency of many beta-lactams, including carbapenems, in beta-lactamase-producing Gram-negative bacteria, including Acinetobacter spp. The potency of meropenem alone and in combination with QPX7728 (1 to 16 µg/ml) was tested against 275 clinical isolates of Acinetobacter baumannii (carbapenem-resistant A. baumannii [CRAB]) collected worldwide that were highly resistant to carbapenems (MIC50 and MIC90 for meropenem, 64 and >64 µg/ml). Addition of QPX7728 resulted in a marked concentration-dependent increase in meropenem potency, with the MIC90 of meropenem alone decreasing from >64 µg/ml to 8 and 4 µg/ml when tested with fixed concentrations of QPX7728 at 4 and 8 µg/ml, respectively. In order to identify the mechanisms that modulate the meropenem-QPX7728 MIC, the whole-genome sequences were determined for 135 isolates with a wide distribution of meropenem-QPX7728 MICs. This panel of strains included 116 strains producing OXA carbapenemases (71 OXA-23, 16 OXA-72, 16 OXA-24, 9 OXA-58, and 4 OXA-239), 5 strains producing NDM-1, one KPC-producing strain, and 13 strains that did not carry any known carbapenemases but were resistant to meropenem (MIC ≥ 4 µg/ml). Our analysis indicated that mutated PBP3 (with mutations localized in the vicinity of the substrate/inhibitor binding site) is the main factor that contributes to the reduction of meropenem-QPX7728 potency. Still, >90% of isolates that carried PBP3 mutations remained susceptible to ≤8 µg/ml of meropenem when tested with a fixed 4 to 8 µg/ml of QPX7728. In the absence of PBP3 mutations, the MICs of meropenem tested in combination with 4 to 8 µg/ml of QPX7728 did not exceed 8 µg/ml. In the presence of both PBP3 and efflux mutations, 84.6% of isolates were susceptible to ≤8 µg/ml of meropenem with 4 or 8 µg/ml of QPX7728. The combination of QPX7728 with meropenem against CRAB isolates with multiple resistance mechanisms has an attractive microbiological profile.
Assuntos
Acinetobacter baumannii , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Meropeném/farmacologia , Testes de Sensibilidade Microbiana , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
Class A ß-lactamases are a major cause of ß-lactam resistance in Gram-negative bacteria. The recently FDA-approved cyclic boronate vaborbactam is a reversible covalent inhibitor of class A ß-lactamases, including CTX-M extended-spectrum ß-lactamase and KPC carbapenemase, both frequently observed in the clinic. Intriguingly, vaborbactam displayed different binding kinetics and cell-based activity for these two enzymes, despite their similarity. A 1.0-Å crystal structure of CTX-M-14 demonstrated that two catalytic residues, K73 and E166, are positively charged and neutral, respectively. Meanwhile, a 1.25-Å crystal structure of KPC-2 revealed a more compact binding mode of vaborbactam versus CTX-M-14, as well as alternative conformations of W105. Together with kinetic analysis of W105 mutants, the structures demonstrate the influence of this residue and the unusual conformation of the ß3 strand on the inactivation rate, as well as the stability of the reversible covalent bond with S70. Furthermore, studies of KPC-2 S130G mutant shed light on the different impacts of S130 in the binding of vaborbactam versus avibactam, another recently approved ß-lactamase inhibitor. Taken together, these new data provide valuable insights into the inhibition mechanism of vaborbactam and future development of cyclic boronate inhibitors.
Assuntos
Antibacterianos , beta-Lactamases , Antibacterianos/farmacologia , Ácidos Borônicos , Cinética , Inibidores de beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
QPX7728 is an investigational ultrabroad-spectrum-beta-lactamase inhibitor (BLI) with potent inhibition of key serine and metallo-beta-lactamases. QPX7728 enhances the potency of many beta-lactams, including carbapenems, in isogenic strains of Gram-negative bacteria producing various beta-lactamases. The potency of meropenem alone and in combination with QPX7728 (tested at fixed concentrations of 1 to 16 µg/ml) was tested against 598 clinical isolates of carbapenem-resistant Enterobacterales (CRE). The panel included 363 strains producing serine carbapenemases, 224 strains producing metallo-beta-lactamases (151 NDM, 53 VIM, and 20 IMP), and 50 strains that did not carry any known carbapenemases but were resistant to meropenem (MIC ≥ 4 µg/ml). The panel was also enriched in strains that had various defects in the major porins OmpK35/OmpF and OmpK36/OmpC. Increasing concentrations of QPX7728 restored the potency of meropenem against CRE, with the meropenem MIC90 decreasing from >64 µg/ml to 0.5 µg/ml for QPX7728 (8 µg/ml). QPX7728 significantly increased the potency of meropenem against CRE with multiple resistance mechanisms; the reduction in the meropenem MIC90 with QPX7728 (8 µg/ml) ranged from 32- to >256-fold. Compared with other beta-lactamase inhibitor combinations, meropenem-vaborbactam, ceftazidime-avibactam, and imipenem-relebactam, meropenem with QPX7728 was the most potent beta-lactam-BLI combination tested against all groups of CRE with multiple resistance mechanisms. Defects in OmpK36 in KPC-producing strains markedly decreased the potency of meropenem with vaborbactam (128-fold increase in the MIC90), whereas only an 8- to 16-fold change was observed with QPX7728 plus meropenem. More than 90% of various CRE subsets (including those with reduced permeability) were susceptible to ≤8 µg/ml of meropenem with QPX7728 at 8 µg/ml or lower. The combination of QPX7728 with meropenem against CRE has an attractive microbiological profile in CRE with multiple resistance mechanisms.
Assuntos
Carbapenêmicos , Enterobacteriaceae/efeitos dos fármacos , Inibidores de beta-Lactamases , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Combinação de Medicamentos , Testes de Sensibilidade Microbiana , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
QPX7728 is an ultrabroad-spectrum boronic acid beta-lactamase inhibitor, with potent inhibition of key serine and metallo-beta-lactamases being observed in biochemical assays. Microbiological studies using characterized strains were used to provide a comprehensive characterization of the spectrum of beta-lactamase inhibition by QPX7728. The MICs of multiple antibiotics administered intravenously only (ceftazidime, piperacillin, cefepime, ceftolozane, and meropenem) and orally bioavailable antibiotics (ceftibuten, cefpodoxime, tebipenem) alone and in combination with QPX7728 (4 µg/ml), as well as comparator agents, were determined against panels of laboratory strains of Pseudomonas aeruginosa and Klebsiella pneumoniae expressing over 55 diverse serine and metallo-beta-lactamases. QPX7728 significantly enhanced the potency of antibiotics against strains expressing class A extended-spectrum beta-lactamases (CTX-M, SHV, TEM, VEB, PER) and carbapenemases (KPC, SME, NMC-A, BKC-1), consistent with the beta-lactamase inhibition demonstrated in biochemical assays. It also inhibited both plasmidic (CMY, FOX, MIR, DHA) and chromosomally encoded (P99, PDC, ADC) class C beta-lactamases and class D enzymes, including carbapenemases, such as OXA-48 from Enterobacteriaceae and OXA enzymes from Acinetobacter baumannii (OXA-23/24/72/58). QPX7728 is also a potent inhibitor of many class B metallo-beta-lactamases (NDM, VIM, CcrA, IMP, and GIM but not SPM or L1). Addition of QPX7728 (4 µg/ml) reduced the MICs for a majority of the strains to the level observed for the control with the vector alone, indicative of complete beta-lactamase inhibition. The ultrabroad-spectrum beta-lactamase inhibition profile makes QPX7728 a viable candidate for further development.
Assuntos
Antibacterianos , Inibidores de beta-Lactamases , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Monobactamas , Serina , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
QPX7728 is an ultrabroad-spectrum boronic acid beta-lactamase inhibitor that demonstrates inhibition of key serine and metallo-beta-lactamases at a nanomolar concentration range in biochemical assays with purified enzymes. The broad-spectrum inhibitory activity of QPX7728 observed in biochemical experiments translates into enhancement of the potency of many beta-lactams against strains of target pathogens producing beta-lactamases. The impacts of bacterial efflux and permeability on inhibitory potency were determined using isogenic panels of KPC-3-producing isogenic strains of Klebsiella pneumoniae and Pseudomonas aeruginosa and OXA-23-producing strains of Acinetobacter baumannii with various combinations of efflux and porin mutations. QPX7728 was minimally affected by multidrug resistance efflux pumps either in Enterobacteriaceae or in nonfermenters, such as P. aeruginosa or A. baumannii Against P. aeruginosa, the potency of QPX7728 was further enhanced when the outer membrane was permeabilized. The potency of QPX7728 against P. aeruginosa was not affected by inactivation of the carbapenem porin OprD. While changes in OmpK36 (but not OmpK35) reduced the potency of QPX7728 (8- to 16-fold), QPX7728 (4 µg/ml) nevertheless completely reversed the KPC-mediated meropenem resistance in strains with porin mutations, consistent with the lesser effect of these mutations on the potency of QPX7728 compared to that of other agents. The ultrabroad-spectrum beta-lactamase inhibition profile, combined with enhancement of the activity of multiple beta-lactam antibiotics with various sensitivities to the intrinsic resistance mechanisms of efflux and permeability, indicates that QPX7728 is a useful inhibitor for use with multiple beta-lactam antibiotics.
Assuntos
Acinetobacter baumannii , Inibidores de beta-Lactamases , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Enterobacteriaceae , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Serina , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
Despite major advances in the ß-lactamase inhibitor field, certain enzymes remain refractory to inhibition by agents recently introduced. Most important among these are the class B (metallo) enzyme NDM-1 of Enterobacteriaceae and the class D (OXA) enzymes of Acinetobacter baumannii. Continuing the boronic acid program that led to vaborbactam, efforts were directed toward expanding the spectrum to allow treatment of a wider range of organisms. Through key structural modifications of a bicyclic lead, stepwise gains in spectrum of inhibition were achieved, ultimately resulting in QPX7728 (35). This compound displays a remarkably broad spectrum of inhibition, including class B and class D enzymes, and is little affected by porin modifications and efflux. Compound 35 is a promising agent for use in combination with a ß-lactam antibiotic for the treatment of a wide range of multidrug resistant Gram-negative bacterial infections, by both intravenous and oral administration.
Assuntos
Ácidos Borínicos/farmacologia , Ácidos Borônicos/farmacologia , Ácidos Carboxílicos/farmacologia , Inibidores de beta-Lactamases/farmacologia , Animais , Bactérias/efeitos dos fármacos , Ácidos Borínicos/química , Ácidos Borínicos/farmacocinética , Ácidos Borínicos/uso terapêutico , Ácidos Borônicos/química , Ácidos Borônicos/farmacocinética , Ácidos Borônicos/uso terapêutico , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacocinética , Ácidos Carboxílicos/uso terapêutico , Descoberta de Drogas , Infecções por Klebsiella/tratamento farmacológico , Camundongos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacocinética , Inibidores de beta-Lactamases/uso terapêuticoRESUMO
QPX7728 is a new ultrabroad-spectrum inhibitor of serine and metallo-beta-lactamases (MBLs) from a class of cyclic boronates that gave rise to vaborbactam. The spectrum and mechanism of beta-lactamase inhibition by QPX7728 were assessed using purified enzymes from all molecular classes. QPX7728 inhibits class A extended-spectrum beta-lactamases (ESBLs) (50% inhibitory concentration [IC50] range, 1 to 3 nM) and carbapenemases such as KPC (IC50, 2.9 ± 0.4 nM) as well as class C P99 (IC50 of 22 ± 8 nM) with a potency that is comparable to or higher than recently FDA-approved beta-lactamase inhibitors (BLIs) avibactam, relebactam, and vaborbactam. Unlike those other BLIs, QPX7728 is also a potent inhibitor of class D carbapenemases such as OXA-48 from Enterobacteriaceae and OXA enzymes from Acinetobacter baumannii (OXA-23/24/58, IC50 range, 1 to 2 nM) as well as MBLs such as NDM-1 (IC50, 55 ± 25 nM), VIM-1 (IC50, 14 ± 4 nM), and IMP-1 (IC50, 610 ± 70 nM). Inhibition of serine enzymes by QPX7728 is associated with progressive inactivation with a high-efficiency k2/K ranging from 6.3 × 104 (for P99) to 9.9 × 105 M-1 s-1 (for OXA-23). This inhibition is reversible with variable stability of the QPX7728-beta-lactamase complexes with target residence time ranging from minutes to several hours: 5 to 20 min for OXA carbapenemases from A. baumannii, â¼50 min for OXA-48, and 2 to 3 h for KPC and CTX-M-15. QPX7728 inhibited all tested serine enzymes at a 1:1 molar ratio. Metallo-beta-lactamases NDM, VIM, and IMP were inhibited by a competitive mechanism with fast-on-fast-off kinetics, with Ki s of 7.5 ± 2.1 nM, 32 ± 14 nM, and 240 ± 30 nM for VIM-1, NDM-1, and IMP-1, respectively. QPX7728's ultrabroad spectrum of BLI inhibition combined with its high potency enables combinations with multiple different beta-lactam antibiotics.
Assuntos
Serina , Inibidores de beta-Lactamases , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Monobactamas , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
Resistance to ceftazidime-avibactam due to mutations in KPC genes has been reported both in vitro and in clinical settings. The most frequently reported mutation leads to the amino acid substitution D179Y in the Ω loop of the enzyme. Bacterial cells that carry mutant KPC acquire a higher level of ceftazidime resistance, become more sensitive to other cephalosporins, and almost completely lose resistance to carbapenems. In this study, we demonstrated that two substitutions in KPC-2, D179Y and L169P, reduce the ability of avibactam to enhance the activity of ceftazidime, cefepime, or piperacillin against isogenic efflux-deficient strains of Pseudomonas aeruginosa, 8- to 32-fold and 4- to 16-fold for the D179Y and L169P variants, respectively, depending on the antibiotic. In contrast, the potency of vaborbactam, the structurally unrelated ß-lactamase inhibitor that was recently approved by the FDA in combination with meropenem, is reduced no more than 2-fold. Experiments with purified enzymes demonstrate that the D179Y substitution causes an â¼20-fold increase in the 50% inhibitory concentration (IC50) for inhibition of ceftazidime hydrolysis by avibactam, versus 2-fold for vaborbactam, and that the L169P substitution has an â¼4.5-fold-stronger effect on the affinity for avibactam than for vaborbactam. In addition, the D179Y and L169P variants hydrolyze ceftazidime with 10-fold and 4-fold-higher efficiencies, respectively, than that of wild-type KPC-2. Thus, microbiological and biochemical experiments implicate both decreased ability of avibactam to interact with KPC-2 variants and an increase in the efficiency of ceftazidime hydrolysis in resistance to ceftazidime-avibactam. These substitutions have a considerably lesser effect on interactions with vaborbactam, making the meropenem-vaborbactam combination a valuable agent in managing infections due to KPC-producing carbapenem-resistant Enterobacteriaceae.