The Role of a Second-Shell Residue in Modifying Substrate and Inhibitor Interactions in the SHV β-Lactamase: A Study of Ambler Position Asn276

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g Boronic acid transition state inhibitors (BATSIs) are competitive, reversible ␤-lactamase inhibitors (BLIs). In this study, a series of BATSIs with selectively modified regions (R1, R2, and amide group) were strategically designed and tested against representative class A ␤-lactamases of Klebsiella pneumoniae, KPC-2 and SHV-1. Firstly, the R1 group of compounds 1a to 1c and 2a to 2e mimicked the side chain of cephalothin, whereas for compounds 3a to 3c, 4a, and 4b, the thiophene ring was replaced by a phenyl, typical of benzylpenicillin. Secondly, variations in the R2 groups which included substituted aryl side chains (compounds 1a, 1b, 1c, 3a, 3b, and 3c) and triazole groups (compounds 2a to 2e) were chosen to mimic the thiazolidine and dihydrothiazine ring of penicillins and cephalosporins, respectively. Thirdly, the amide backbone of the BATSI, which corresponds to the amide at C-6 or C-7 of ␤-lactams, was also changed to the following bioisosteric groups: urea (compound 3b), thiourea (compound 3c), and sulfonamide (compounds 4a and 4b). Among the compounds that inhibited KPC-2 and SHV-1 ␤-lactamases, nine possessed 50% inhibitory concentrations (IC 50 s) of <600 nM. The most active compounds contained the thiopheneacetyl group at R1 and for the chiral BATSIs, a carboxy-or hydroxy-substituted aryl group at R2. The most active sulfonamido derivative, compound 4b, lacked an R2 group. Compound 2b (S02030) was the most active, with acylation rates (k 2 /K) of 1.2 ؎ 0.2 ؋ 10 4 M ؊1 s ؊1 for KPC-2 and 4.7 ؎ 0.6 ؋ 10 3 M ؊1 s ؊1 for SHV-1, and demonstrated antimicrobial activity against Escherichia coli DH10B carrying bla SHV variants and bla KPC-2 or bla KPC-3 and against clinical strains of Klebsiella pneumoniae and E. coli producing different class A ␤-lactamase genes. At most, MICs decreased from 16 to 0.5 mg/liter.

Section: Resultssupporting

confidence: 85%

Boronic Acid Transition State Inhibitors Active against KPC and Other Class A β-Lactamases: Structure-Activity Relationships as a Guide to Inhibitor Design

Rojas

Taracila

Papp-Wallace

et al. 2016

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“…MICs for various bacterial isolates were determined by the Mueller-Hinton agar dilution method according to the Clinical and Laboratory Standards Institute guidelines (17). The MIC measurements were performed using a Steers replicator that delivered 10 l of a diluted overnight culture containing 10 4 CFU. Avibactam was tested at 4 mg/liter in combination with increasing concentrations of ampicillin or ceftazidime (18).…”

Section: Methodsmentioning

confidence: 99%

“…Based upon this knowledge and results of previous studies on the mechanism of inactivation by clavulanic acid of other class A ␤-lactamases (e.g., SHV-1), we hypothesized that the structural and kinetic determinants that mediate resistance to clavulanic acid, sulbactam, and tazobactam in class A ␤-lactamases will also result in resistance to avibactam. In this work, we determined the amino acid sequence requirements of the KPC-2 ␤-lactamase as they affect ␤-lactam-avibactam resistance, focusing on residues (i.e., 69, 130, 234, 220, and 276) that were previously shown to be important for inhibitor resistance in TEM and SHV class A ␤-lactamases (6,(10)(11)(12)(13)(14)(15).…”

mentioning

confidence: 99%

Variants of β-Lactamase KPC-2 That Are Resistant to Inhibition by Avibactam

Papp-Wallace

Winkler

Taracila

et al. 2015

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KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the ␤-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 ␤-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-␤-lactam ␤-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 ␤-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 ␤-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV ␤-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 ␤-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k 2 /K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M ؊1 s ؊1 to 1.2 M ؊1 s ؊1 ). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent against E. coli DH10B strains possessing all of these variants. In 2013, the Centers for Disease Control and Prevention (CDC) in the United States announced that carbapenem-resistant Enterobacteriaceae (CRE; e.g., Klebsiella pneumoniae possessing bla KPC-2 ) were one of the highest-priority target pathogens for the identification and development of novel antibacterials (1). CREs are often resistant to all antibiotics; thus, treatment options are limited to toxic agents, such as polymyxins B and E. Every year in the United States, 9,000 people become infected with CREs, and 600 of these individuals die (1). To combat these trends, the Infectious Diseases Society of America (IDSA) presented a "call to arms," known as the 10 by '20 Initiative, which demands the development of 10 new antibiotics by 2020 (2). Regr...

“…The steady-state kinetic parameters V max and K m were obtained by fitting reaction curves to the Michaelis-Menten equation using Origin 8.0 (OriginLab, Northampton, MA). When hydrolysis could not be measured directly, a competition assay using NCF as a reporter substrate was performed; an apparent K m is reported for the compound, correcting for the NCF K m (22). We initiated the reaction with the addition of enzyme to a mixture of NCF and the poor substrate and took initial velocity measurements during the first 5 to 10 s of the progress curves to better approximate formation of the MichaelisMenten complex (23).…”

Section: Mutagenesismentioning

confidence: 99%

N152G, -S, and -T Substitutions in CMY-2 β-Lactamase Increase Catalytic Efficiency for Cefoxitin and Inactivation Rates for Tazobactam

Skalweit

Conklin

et al. 2013

Class C cephalosporinases are a growing threat, and clinical inhibitors of these enzymes are currently unavailable. Previous studies have explored the role of Asn152 in the Escherichia coli AmpC and P99 enzymes and have suggested that interactions between C-6= or C-7= substituents on penicillins or cephalosporins and Asn152 are important in determining substrate specificity and enzymatic stability. We sought to characterize the role of Asn152 in the clinically important CMY-2 cephalosporinase with substrates and inhibitors. Mutagenesis of CMY-2 at position 152 yields functional mutants (N152G, -S, and -T) that exhibit improved penicillinase activity and retain cephamycinase activity. We also tested whether the position 152 substitutions would affect the inactivation kinetics of tazobactam, a class A ␤-lactamase inhibitor with in vitro activity against CMY-2. Using standard assays, we showed that the N152G, -S, and -T variants possessed increased catalytic activity against cefoxitin compared to the wild type. The 50% inhibitory concentration (IC 50 ) for tazobactam improved dramatically, with an 18-fold reduction for the N152S mutant due to higher rates of enzyme inactivation. Modeling studies have shown active-site expansion due to interactions between Y150 and S152 in the apoenzyme and the Michaelis-Menten complex with tazobactam. Substitutions at N152 might become clinically important as new class C ␤-lactamase inhibitors are developed. C lass C ␤-lactamases such as CMY-2, found in Gram-negative pathogens, confer resistance to a wide variety of ␤-lactam antibiotics, including narrow-and extended-spectrum cephalosporins and penicillins (1). When combined with other resistance mechanisms, such as porin loss or efflux (2-5), or when increased expression occurs in derepressed strains (1, 6), organisms expressing class C ␤-lactamases become resistant to cefepime and carbapenems. Point mutations and deletions in the omega loop or helix H2 or H10 and near the C terminus of the AmpC ␤-lactamases that cause an extended-spectrum AmpC (ESAC) phenotype have been described (1, 7). Of the CMY enzymes, 97 unique types have been described to date (see http://www.lahey.org/Studies), including enzymes such as , with alterations of the omega loop or the H10 helix. Recently, Dahyot and Mammeri described a ceftazidime-and cefepime-hydrolyzing CMY-2 laboratory variant based on a clinical mutant in which a Y-X-N loop mutation (R148H) accounted for the ESAC phenotype (13). Little has been written about the behavior of the ESAC variants in regard to ␤-lactamase inhibitors, although a tazobactam-susceptible H-10 helix variant of Escherichia coli AmpC has been described (14).Previous studies on the Y-X-N loop of class C ␤-lactamases explored the role of N152 in the E. coli AmpC (15) and P99 (16) enzymes and suggested that interactions between C-6= or C-7= substituents of penicillins or cephalosporins and N152 are important in determining substrate specificity and enzymatic stability. We sought to characterize the role of N152 in the cli...