Restricted Rotational Flexibility of the C5α-Methyl-Substituted Carbapenem NA-1-157 Leads to Potent Inhibition of the GES-5 Carbapenemase

Carbapenem antibiotics are used as a last-resort treatment for infections caused by multidrug-resistant bacteria. The wide spread of carbapenemases in Gram-negative bacteria has severely compromised the utility of these drugs and represents a serious public health threat. To combat carbapenemase-mediated resistance, new antimicrobials and inhibitors of these enzymes are urgently needed. Here, we describe the interaction of the atypically C5α-methyl-substituted carbapenem, NA-1-157, with the GES-5 carbapenemase. MICs of this compound against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii producing the enzyme were reduced 4–16-fold when compared to MICs of the commercial carbapenems, reaching clinically sensitive breakpoints. When NA-1-157 was combined with meropenem, a strong synergistic effect was observed. Kinetic and ESI-LC/MS studies demonstrated that NA-1-157 is a potent inhibitor of GES-5, with a high inactivation efficiency of (2.9 ± 0.9) × 105 M–1 s–1. Acylation of GES-5 by NA-1-157 was biphasic, with the fast phase completing within seconds, and the slow phase taking several hours and likely proceeding through a reversible tetrahedral intermediate. Deacylation was extremely slow (k3 = (2.4 ± 0.3) × 10–7 s–1), resulting in a residence time of 48 ± 6 days. MD simulation of the GES-5-meropenem and GES-5-NA-1-157 acyl-enzyme complexes revealed that the C5α-methyl group in NA-1-157 sterically restricts rotation of the 6α-hydroxyethyl group preventing ingress of the deacylating water into the vicinity of the scissile bond of the acyl-enzyme intermediate. These data demonstrate that NA-1-157 is a potent irreversible inhibitor of the GES-5 carbapenemase

QM/MM Simulations Reveal the Determinants of Carbapenemase Activity in Class A β-lactamases

[Image: see text] β-lactam antibiotic resistance in Gram-negative bacteria, primarily caused by β-lactamase enzymes that hydrolyze the β-lactam ring, has become a serious clinical problem. Carbapenems were formerly considered “last resort” antibiotics because they escaped breakdown by most β-lactamases, due to slow deacylation of the acyl-enzyme intermediate. However, an increasing number of Gram-negative bacteria now produce β-lactamases with carbapenemase activity: these efficiently hydrolyze the carbapenem β-lactam ring, severely limiting the treatment of some bacterial infections. Here, we use quantum mechanics/molecular mechanics (QM/MM) simulations of the deacylation reactions of acyl-enzyme complexes of eight β-lactamases of class A (the most widely distributed β-lactamase group) with the carbapenem meropenem to investigate differences between those inhibited by carbapenems (TEM-1, SHV-1, BlaC, and CTX-M-16) and those that hydrolyze them (SFC-1, KPC-2, NMC-A, and SME-1). QM/MM molecular dynamics simulations confirm the two enzyme groups to differ in the preferred acyl-enzyme orientation: carbapenem-inhibited enzymes favor hydrogen bonding of the carbapenem hydroxyethyl group to deacylating water (DW). QM/MM simulations of deacylation give activation free energies in good agreement with experimental hydrolysis rates, correctly distinguishing carbapenemases. For the carbapenem-inhibited enzymes, free energies for deacylation are significantly higher than for the carbapenemases, even when the hydroxyethyl group was restrained to prevent interaction with the DW. Analysis of these simulations, and additional simulations of mutant enzymes, shows how factors including the hydroxyethyl orientation, the active site volume, and architecture (conformations of Asn170 and Asn132; organization of the oxyanion hole; and the Cys69-Cys238 disulfide bond) collectively determine catalytic efficiency toward carbapenems

The Importance of a Critical Protonation State and the Fate of the Catalytic Steps in Class A β-Lactamases and Penicillin-binding Proteins

b-Lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to b-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active-site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins and crystal structures demonstrate that the side chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys73) in the TEM-1 b-lactamase from Escherichia coli. These techniques included perturbation of the pKa of Lys73 by the study of the g-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance and by computational treatment using the thermodynamic integration method. All three methods indicated that the pKa of Lys73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu166 and Lys73 of class A b-lactamases has actually raised the pKa of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot definitively rule out that Glu166 activates the active site water, which in turn promotes Ser70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu166 and unprotonated Lys73. As such, unprotonated Lys73 could promote serine for acylation, a process that should be shared among all active-site-serine b-lactamases and penicillin-binding proteins

Exploring The Role of A Conserved Class A Residue in The Ω-Loop of KPC-2 β-Lactamase: A Mechanism For Ceftazidime Hydrolysis

Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative β-lactamase for more detailed analysis, we observed only a modest 25% increase in kcat/Km for ceftazidime (0.015→0.019 μm−1 s−1). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of Ω-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through “covalent trapping” of the substrate by a deacylation impaired enzyme with a lower Km. Future antibiotic design must consider the distinctive behavior of the Ω-loop of KPC-2

Exploring The Role of A Conserved Class A Residue in The Ω-Loop of KPC-2 β-Lactamase: A Mechanism For Ceftazidime Hydrolysis

Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative β-lactamase for more detailed analysis, we observed only a modest 25% increase in kcat/Km for ceftazidime (0.015→0.019 μm−1 s−1). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of Ω-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through “covalent trapping” of the substrate by a deacylation impaired enzyme with a lower Km. Future antibiotic design must consider the distinctive behavior of the Ω-loop of KPC-2

Antimicrobial resistance conferred by OXA-48 β-lactamases:towards a detailed mechanistic understanding

OXA-48-type β-lactamases are now routinely encountered in bacterial infections caused by carbapenem-resistant Enterobacterales. These enzymes are of high and growing clinical significance due to the importance of carbapenems in treatment of health care-associated infections by Gram-negative bacteria, the wide and increasing dissemination of OXA-48 enzymes on plasmids, and the challenges posed by their detection. OXA-48 confers resistance to penicillin (which is efficiently hydrolyzed) and carbapenem antibiotics (which is more slowly broken down). In addition to the parent enzyme, a growing array of variants of OXA-48 is now emerging. The spectrum of activity of these variants varies, with some hydrolyzing expanded-spectrum oxyimino-cephalosporins. The growth in importance and diversity of the OXA-48 group has motivated increasing numbers of studies that aim to elucidate the relationship between structure and specificity and establish the mechanistic basis for β-lactam turnover in this enzyme family. In this review, we collate recently published structural, kinetic, and mechanistic information on the interactions between clinically relevant β-lactam antibiotics and inhibitors and OXA-48 β-lactamases. Collectively, these studies are starting to form a detailed picture of the underlying bases for the differences in β-lactam specificity between OXA-48 variants and the consequent differences in resistance phenotype. We focus specifically on aspects of carbapenemase and cephalosporinase activities of OXA-48 β-lactamases and discuss β-lactamase inhibitor development in this context. Throughout the review, we also outline key open research questions for future investigation

Transduction as the method of horizontal gene transfer of the Staphylococcal Chromosomal Cassette mec (SCCmec)

Methicillin-resistant Staphylococcus aureus (MRSA) gains resistance to β-lactam antibiotics through a mutated penicillin binding protein (PBP2a) encoded on the SCCmec element. In combination with the recombinase encoded by ccr, these two genes are used as markers of the mobile genetic element (SCCmec). Due to recent increases in community acquired MRSA infections, the mechanisms of antibiotic resistance gene transfer have gained attention. Transduction, a method of horizontal gene transfer mediated by bacteriophage, is believed to be responsible for the movement of the SCCmec element. Recent studies have shown the transduction of the SCCmec element in clinical isolates; however, this study is more concerned with transduction in the environment. The preliminary study presented here was based on two studies demonstrating the presence of the mecA gene in viral fractions from environmental sources by polymerase chain reaction (PCR). This study aimed to confirm the presence of the SCCmec element in environmental bacteriophage populations through PCR analysis and sequencing. Approximately 22% of the environmental samples collected contain mecA and/or ccr. One positive sample was sequenced, confirming the presence of the mecA gene and defining it as Type 1. Samples from non-fecal sources were more likely to contain one or both genes, and compost samples have the greatest percent (65%) positive. This preliminary study left many questions unanswered, spurring a second study with goals to determine the frequency of transduction and the allotype of the SCCmec element most frequently transduced. A number of bacterial isolates were collected and characterized. This works sets the stage for isolation of phages and transduction experiments in the future. The results of this work will lead to a better understanding of how antibiotic resistance genes are transferred in the environment, which could lead to preventative applications

Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography

Ligand binding can change the pKa of protein residues and influence enzyme catalysis. Herein, we report three sub-Angstrom resolution X-ray crystal structures of CTX-M \u3b2-lactamase, representing three stages of the enzymatic pathway, apo protein (0.79 \uc5), pre-covalent complex (0.89 \uc5), and acylation transition state analog (0.84 \uc5). The binding of a non-covalent ligand induces a proton transfer from the catalytic Ser70 to the general base Glu166, and the formation of a low-barrier hydrogen bond (LBHB) between Ser70 and Lys73. QM/MM reaction path calculations determined the proton transfer barrier between Ser70 and Lys73 to be 1.53 kcal/mol, further confirming the presence of a LBHB. This LBHB is absent in the other two structures. Our data represents the first evidence of a direct and transient LBHB stabilizing a nucleophilic serine, as hypothesized by Cleland and Kreevoy. These results have important implications for the study of enzyme mechanisms as well as protein-inhibitor interactions

An experiment-informed signal transduction model for the role of the Staphylococcus aureus MecR1 protein in β-lactam resistance

The treatment of hospital- and community-associated infections by methicillin-resistant Staphylococcus aureus (MRSA) is a perpetual challenge. This Gram-positive bacterium is resistant specifically to β-lactam antibiotics, and generally to many other antibacterial agents. Its resistance mechanisms to β-lactam antibiotics are activated only when the bacterium encounters a β-lactam. This activation is regulated by the transmembrane sensor/signal transducer proteins BlaR1 and MecR1. Neither the transmembrane/metalloprotease domain, nor the complete MecR1 and BlaR1 proteins, are isolatable for mechanistic study. Here we propose a model for full-length MecR1 based on homology modeling, residue coevolution data, a new extensive experimental mapping of transmembrane topology, partial structures, molecular simulations, and available NMR data. Our model defines the metalloprotease domain as a hydrophilic transmembrane chamber effectively sealed by the apo-sensor domain. It proposes that the amphipathic helices inserted into the gluzincin domain constitute the route for transmission of the β-lactam-binding event in the extracellular sensor domain, to the intracellular and membrane-embedded zinc-containing active site. From here, we discuss possible routes for subsequent activation of proteolytic action. This study provides the first coherent model of the structure of MecR1, opening routes for future functional investigations on how β-lactam binding culminates in the proteolytic degradation of MecI.Fil: Belluzo, Bruno Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Abriata, Luciano Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. École Polytechnique Fédérale de Lausanne; SuizaFil: Giannini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mihovilcevic, Damila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Dal Peraro, Matteo. École Polytechnique Fédérale de Lausanne; SuizaFil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

DISCOVERY OF NEW ANTIMICROBIAL OPTIONS AND EVALUATION OF AMINOGLYCOSIDE RESISTANCE ENZYME-ASSOCIATED RESISTANCE EPIDEMIC

The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6\u27) domain and its mutant enzymes from a bifunctional AME, AAC(6\u27)-Ie/APH(2 )-Ia as well as another common AME, APH(3\u27)-IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi