Nuno T. Antunes

Discovery of a New Class of Non-β-lactam Inhibitors of Penicillin-Binding Proteins with Gram-Positive Antibacterial Activity

Infections caused by hard-to-treat methicillin-resistant Staphylococcus aureus (MRSA) are a serious global public-health concern, as MRSA has become broadly resistant to many classes of antibiotics. We disclose herein the discovery of a new class of non-β-lactam antibiotics, the oxadiazoles, which inhibit penicillin-binding protein 2a (PBP2a) of MRSA. The oxadiazoles show bactericidal activity against vancomycin- and linezolid-resistant MRSA and other Gram-positive bacterial strains, in vivo efficacy in a mouse model of infection, and have 100% oral bioavailability.

Class D β-Lactamases: Are They All Carbapenemases?

ABSTRACT Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are enzymes of the utmost clinical importance due to their ability to produce resistance to carbapenems, the antibiotics of last resort for the treatment of various life-threatening infections. The vast majority of these enzymes have been identified in Acinetobacter spp., notably in Acinetobacter baumannii. The OXA-2 and OXA-10 enzymes predominantly occur in Pseudomonas aeruginosa and are currently classified as narrow-spectrum class D β-lactamases. Here we demonstrate that when OXA-2 and OXA-10 are expressed in Escherichia coli strain JM83, they produce a narrow-spectrum antibiotic resistance pattern. When the enzymes are expressed in A. baumannii ATCC 17978, however, they behave as extended-spectrum β-lactamases and confer resistance to carbapenem antibiotics. Kinetic studies of OXA-2 and OXA-10 with four carbapenems have demonstrated that their catalytic efficiencies with these antibiotics are in the same range as those of some recognized class D carbapenemases. These results are in disagreement with the classification of the OXA-2 and OXA-10 enzymes as narrow-spectrum β-lactamases, and they suggest that other class D enzymes that are currently regarded as noncarbapenemases may in fact be CHDLs.

Structure−Activity Relationship for the Oxadiazole Class of Antibiotics

The structure-activity relationship (SAR) for the newly discovered oxadiazole class of antibiotics is described with evaluation of 120 derivatives of the lead structure. This class of antibiotics was discovered by in silico docking and scoring against the crystal structure of a penicillin-binding protein. They impair cell-wall biosynthesis and exhibit activities against the Gram-positive bacterium Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant and linezolid-resistant S. aureus. 5-(1H-Indol-5-yl)-3-(4-(4-(trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadiazole (antibiotic 75b) was efficacious in a mouse model of MRSA infection, exhibiting a long half-life, a high volume of distribution, and low clearance. This antibiotic is bactericidal and is orally bioavailable in mice. This class of antibiotics holds great promise in recourse against infections by MRSA.

Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase‐2″‐IVa

Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside‐modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2″‐IVa (APH(2″)‐IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi‐Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2″)‐IVa enzyme phosphorylates various 4,6‐disubstituted aminoglycoside antibiotics with catalytic efficiencies (kcat/Km) of 1.5 × 103 to 1.2 × 106 (M−1 s−1). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2″)‐IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP‐utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP‐based enzyme inhibitors which would not be expected to interfere with crucial ATP‐dependent enzymes.

The Class A β-Lactamase FTU-1 Is Native to Francisella tularensis

ABSTRACT The class A β-lactamase FTU-1 produces resistance to penicillins and ceftazidime but not to any other β-lactam antibiotics tested. FTU-1 hydrolyzes penicillin antibiotics with catalytic efficiencies of 105 to 106 M−1 s−1 and cephalosporins and carbapenems with catalytic efficiencies of 102 to 103 M−1 s−1, but the monobactam aztreonam and the cephamycin cefoxitin are not substrates for the enzyme. FTU-1 shares 21 to 34% amino acid sequence identity with other class A β-lactamases and harbors two cysteine residues conserved in all class A carbapenemases. FTU-1 is the first weak class A carbapenemase that is native to Francisella tularensis.

Resistance to the Third-Generation Cephalosporin Ceftazidime by a Deacylation-Deficient Mutant of the TEM β-Lactamase by the Uncommon Covalent-Trapping Mechanism

The Glu166Arg/Met182Thr mutant of Escherichia coli TEM(pTZ19-3) β-lactamase produces a 128-fold increase in the level of resistance to the antibiotic ceftazidime in comparison to that of the parental wild-type enzyme. The single Glu166Arg mutation resulted in a dramatic decrease in both the level of enzyme expression in bacteria and the resistance to penicillins, with a concomitant 4-fold increase in the resistance to ceftazidime, a third-generation cephalosporin. Introduction of the second amino acid substitution, Met182Thr, restored enzyme expression to a level comparable to that of the wild-type enzyme and resulted in an additional 32-fold increase in the minimal inhibitory concentration of ceftazidime to 64 μg/mL. The double mutant formed a stable covalent complex with ceftazidime that remained intact for the entire duration of the monitoring, which exceeded a time period of 40 bacterial generations. Compared to those of the wild-type enzyme, the affinity of the TEM(pTZ19-3) Glu166Arg/Met182Thr mutant for ceftazidime increased by at least 110-fold and the acylation rate constant was augmented by at least 16-fold. The collective experimental data and computer modeling indicate that the deacylation-deficient Glu166Arg/Met182Thr mutant of TEM(pTZ19-3) produces resistance to the third-generation cephalosporin ceftazidime by an uncommon covalent-trapping mechanism. This is the first documentation of such a mechanism by a class A β-lactamase in a manifestation of resistance.

Crystal Structure of Carbapenemase OXA-58 from Acinetobacter baumannii

ABSTRACT Class D β-lactamases capable of hydrolyzing last-resort carbapenem antibiotics represent a major challenge for treatment of bacterial infections. Wide dissemination of these enzymes in Acinetobacter baumannii elevated this pathogen to the category of most deadly and difficult to treat. We present here the structure of the OXA-58 β-lactamase, a major class D carbapenemase of A. baumannii, determined to 1.30-Å resolution. Unlike two other Acinetobacter carbapenemases, OXA23 and OXA-24, the OXA-58 enzyme lacks the characteristic hydrophobic bridge over the active site, despite conservation of the residues which participate in its formation. The active-site residues in OXA-58 are spatially conserved in comparison to those in other class D β-lactamases. Lys86, which activates water molecules during the acylation and deacylation steps, is fully carboxylated in the OXA-58 structure. In the absence of a substrate, a water molecule is observed in the active site of the enzyme and is positioned in the pocket that is usually occupied by the 6α-hydroxyethyl moiety of carbapenems. A water molecule in this location would efficiently deacylate good substrates, such as the penicillins, but in the case of carbapenems, it would be expelled by the 6α-hydroxyethyl moiety of the antibiotics and a water from the surrounding medium would find its way to the vicinity of the carboxylated Lys86 to perform deacylation. Subtle differences in the position of this water in the acyl-enzyme complexes of class D β-lactamases could ultimately be responsible for differences in the catalytic efficiencies of these enzymes against last-resort carbapenem antibiotics.

Novel Aminoglycoside 2″-Phosphotransferase Identified in a Gram-Negative Pathogen

ABSTRACT Aminoglycoside 2″-phosphotransferases are the major aminoglycoside-modifying enzymes in clinical isolates of enterococci and staphylococci. We describe a novel aminoglycoside 2″-phosphotransferase from the Gram-negative pathogen Campylobacter jejuni, which shares 78% amino acid sequence identity with the APH(2″)-Ia domain of the bifunctional aminoglycoside-modifying enzyme aminoglycoside (6′) acetyltransferase-Ie/aminoglycoside 2″-phosphotransferase-Ia or AAC(6′)-Ie/APH(2″)-Ia from Gram-positive cocci, which we called APH(2″)-If. This enzyme confers resistance to the 4,6-disubstituted aminoglycosides kanamycin, tobramycin, dibekacin, gentamicin, and sisomicin, but not to arbekacin, amikacin, isepamicin, or netilmicin, but not to any of the 4,5-disubstituted antibiotics tested. Steady-state kinetic studies demonstrated that GTP, and not ATP, is the preferred cosubstrate for APH(2″)-If. The enzyme phosphorylates the majority of 4,6-disubstituted aminoglycosides with high catalytic efficiencies (kcat/Km = 105 to 107 M−1 s−1), while the catalytic efficiencies against the 4,6-disubstituted antibiotics amikacin and isepamicin are 1 to 2 orders of magnitude lower, due mainly to the low apparent affinities of these substrates for the enzyme. Both 4,5-disubstituted antibiotics and the atypical aminoglycoside neamine are not substrates of APH(2″)-If, but are inhibitors. The antibiotic susceptibility and substrate profiles of APH(2″)-If are very similar to those of the APH(2″)-Ia phosphotransferase domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme.

Importance of Position 170 in the Inhibition of GES-Type β-Lactamases by Clavulanic Acid

ABSTRACT Bacterial resistance to β-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is commonly the result of the production of β-lactamases. The emergence of β-lactamases capable of turning over carbapenem antibiotics is of great concern, since these are often considered the last resort antibiotics in the treatment of life-threatening infections. β-Lactamases of the GES family are extended-spectrum enzymes that include members that have acquired carbapenemase activity through a single amino acid substitution at position 170. We investigated inhibition of the GES-1, -2, and -5 β-lactamases by the clinically important β-lactamase inhibitor clavulanic acid. While GES-1 and -5 are susceptible to inhibition by clavulanic acid, GES-2 shows the greatest susceptibility. This is the only variant to possess the canonical asparagine at position 170. The enzyme with asparagine, as opposed to glycine (GES-1) or serine (GES-5), then leads to a higher affinity for clavulanic acid (Ki = 5 μM), a higher rate constant for inhibition, and a lower partition ratio (r ≈ 20). Asparagine at position 170 also results in the formation of stable complexes, such as a cross-linked species and a hydrated aldehyde. In contrast, serine at position 170 leads to formation of a long-lived trans-enamine species. These studies provide new insight into the importance of the residue at position 170 in determining the susceptibility of GES enzymes to clavulanic acid.

Application of flow cytometry for the determination of minimal inhibitory concentration of several antibacterial agents on Mycoplasma hyopneumoniae.

Aim:  In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MICs) of nine antibacterial agents (enrofloxacin, ciprofloxacin, oxytetracycline, chloramphenicol, tylosin, lincomycin, gentamycin, spectinomycin and streptomycin) against M. hyopneumoniae.