Inhibitor Resistance in the KPC-2 -Lactamase: A. Pre-eminent Property of This Class A -Lactamase

Transcription

1 AAC Accepts, published online ahead of print on 1 December 00 Antimicrob. Agents Chemother. doi:./aac.00-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Inhibitor Resistance in the KPC- -Lactamase: A Pre-eminent Property of This Class A -Lactamase Krisztina M. Papp-Wallace 1,, Christopher R. Bethel 1, Anne M. Distler, Courtney Kasuboski 1, Magdalena Taracila 1, and Robert A. Bonomo 1-,* 1 Research Service, Louis Stokes Cleveland Department of Veteran Affairs Medical Center, Cleveland, Ohio, and Department of Medicine, Pharmacology, and Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio. * Corresponding author: Robert A. Bonomo, MD, Infectious Diseases Section, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, 01 East Blvd., Cleveland, Ohio, Ph: 10 x, Fax:, robert.bonomo@med.va.gov Downloaded from on October 1, 01 by guest

2 ABSTRACT As resistance determinants, KPC -lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC type -lactamases often test resistant to standard -lactam- -lactamase inhibitor combinations in susceptibility testing. The KPC- carbapenemase presents a significant clinical challenge as the mechanistic bases for KPC- s phenotypes remain elusive. Here, we show that KPC- is resistant to -lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC- with partition ratios (k cat /k inact ) of,00, 1,000, and 00, respectively. Methylidene penems that contain a sp hybridized C carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,-c][1,]thiazole (penem 1) or dihydropyrazolo[,1- c][1,]thiazine (penem )) are potent inhibitors : K m of penem 1= 0.0 ± 0.01 M; K m of penem = 0.00 ± M). We also demonstrate that penems 1 and are mechanism-based inactivators having partition ratios (k cat /k inact ) of 0 and 0, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-. These models suggest that: i) penem 1 and penem interact differently with active site residues, with the carbonyl of penem being positioned outside of the oxyanion hole and in a less favorable position for hydrolysis; ii) support the kinetic observations that penem is the better inhibitor (k inact /K m =. ± 0. M -1 s -1 ). We conclude that KPC- is unique among class A -lactamases by being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem type -lactamase inhibitors by exhibiting unique active site chemistry may serve as an important scaffold for future development and offer an attractive alternative to our current -lactamase inhibitors. Downloaded from on October 1, 01 by guest

3 INTRODUCTION In Klebsiella pneumoniae, -lactam resistance is predominantly mediated by class A SHV, TEM, and CTX-M -lactamases (, ). Single amino acid substitutions in the SHV and TEM -lactamases can drastically alter the substrate profile of enzymes and confer resistance to extended-spectrum cephalosporins and -lactamase inhibitors (, 1,, ). -lactamases with altered substrate profiles (i.e., extended-spectrum or inhibitor-resistant -lactamases) have significantly challenged the clinician s approach to the treatment of serious infectious diseases (). Thus, the search for effective mechanism-based inhibitors against novel -lactamases merits significant effort (,, ). First identified in Klebsiella pneumoniae, KPC class A -lactamases threaten the use of all current -lactam antibiotics (). These -lactamase enzymes are present in an increasing number of bacterial genera, becoming the major carbapenemase expressed by Gram negative pathogens (e.g., Enterobacter spp., Escherichia coli, Citrobacter freundii, Pseudomonas spp., Serratia marcescens, Proteus mirabilis, and Salmonella enterica) in the U.S. (,,, 1, 1,,,,,, ). Moreover, KPC -lactamases are becoming geographically widespread (e.g., U.S., China, France, Colombia, Greece, Sweden, Norway, Argentina, United Kingdom, Israel, Brazil, Puerto Rico, Canada, Ireland, Trinidad and Tobago, Poland, Italy, and Finland) (1,, 1,,, -1,,,,, 0, 1,,, ). Evidence suggests that many K. pneumoniae strains in the United States harboring KPCs are genetically related (1). Why are KPC -lactamases so problematic? KPC- has an overall structure similar to other class A enzymes, and, interestingly, this -lactamase has only 0% protein sequence conservation compared to CTX-M-1, % to SHV-1, and % to TEM-1. KPC- is more like other class A carbapenemases having % identity to NmcA and Imi-1, % to Sfc-1, and % Downloaded from on October 1, 01 by guest

4 to Sme-1. The KPC- -lactamase possesses a large and shallow active site, allowing it to accommodate bulkier -lactams (). As a result of these structural changes, KPC- is regarded as a versatile -lactamase (); it is a penicillinase, carbapenemase, cephamycinase, and an extended-spectrum -lactamase, ESBL (, ). Microbiologists and clinicians observed that many bla KPC- containing strains are resistant to -lactam- -lactamase inhibitor combinations (, 1, 0,,, ). According to Clinical and Laboratory Standards Institute (CLSI) breakpoints, clinical strains carrying bla KPC- with a MIC of /1 mg/l for amoxicillinclavulanic acid and 1/ mg/l for piperacillin-tazobactam are resistant (1, ). These observations led us to examine the kinetic properties of the KPC- -lactamase against commercially available and novel inhibitors. A -lactamase inhibitor demonstrating an affinity in the nm range against for KPC- and other class A carbapenemases would be an important addition to our therapeutic armamentarium. Thus, we wondered if penem inhibitors that possess a sp hybridized C carboxylate (a property resembling carbapenems), a complex and reactive R1 side chain, and a different inactivation chemistry than clavulanic acid could be exploited to inhibit KPC enzymes (1). The methylidene inhibitors, penem 1 and penem, have either a dihydropyrazolo[1,-c][1,]thiazole or dihydropyrazolo[,1-c][1,]thiazine moiety, respectively (Figure 1). These penems demonstrate similar in vivo efficacy in mice and are shown to be effective inhibitors of several class A, C, and D -lactamases (,, -, ). In this paper we show why K. pneumoniae containing bla KPC- and an E. coli laboratory strain harboring bla KPC- are not susceptible to the commercially available -lactamase inhibitors. Our results demonstrated that clavulanic acid, sulbactam, tazobactam, are hydrolyzed by the KPC- -lactamase. In contrast, -methylidene penems with complex fused bicyclic R 1 side Downloaded from on October 1, 01 by guest

5 chains are better inhibitors by possessing greater affinity for the active site, low K m s, and acting as mechanism-based inactivators. Downloaded from on October 1, 01 by guest

6 MATERIALS AND METHODS Bacterial strains and plasmids. K. pneumoniae possessing bla KPC- and E. coli containing bla KPC- in pbr-cati vector were a kind gifts of Dr. Fred Tenover from the Centers for Disease Control and Prevention (Atlanta, GA) (). We verified the sequence of bla KPC- in the pbr-cati vector. All DNA sequencing reactions were conducted by the Genomics Core Facility at Case Western Reserve University (Cleveland, OH). E. coli DHB cells (Invitrogen, Carlsbad, CA) were used as a host strain for the pbr-cati-bla KPC- plasmid. Antibiotic susceptibility. K. pneumoniae possessing bla KPC-, E. coli DHB, E. coli DHB expressing bla KPC- were phenotypically characterized using lysogeny broth (LB) agar dilution MICs according to CLSI (1). MICs for antibiotics were determined using a Steers Replicator that delivered L containing CFU per spot. For the -lactamase inhibitor MICs, ampicillin was maintained at a constant concentration of 0 mg/l and clavulanic acid and sulbactam concentrations were increased (). In testing piperacillin/tazobactam, the combination used was an :1 ratio. Finally, penem 1 and penem were maintained at mg/l while the concentrations of piperacillin, imipenem, and cefotaxime were increased. Sulbactam was a gift from Dr. Thomas Gootz formerly from Pfizer (Groton, CT). Clavulanic acid was obtained from Glaxo Smith Kline (Brentford, UK). Tazobactam was given to us by Wyeth Pharmaceuticals (Pearl River, NY). Penem 1 and penem were kindly provided by Dr. Tarek Mansour of Wyeth Pharmaceuticals (Pearl River, NY). The details of the chemical synthesis of penems 1 and are summarized in Venkatesan et al. (). Imipenem was purchased from U. S. Pharmacopeia (Rockville, MD). Piperacillin and cefotaxime were purchased from Sigma (St. Louis, MO). The chemical structures of compounds used in this study are shown in Figure 1. Downloaded from on October 1, 01 by guest

7 Lactamase purification. The KPC- -lactamase was purified from E. coli DHB cells carrying the pbr-cati-bla KPC- plasmid. The cells were grown for 1 h in 00 ml of SOB broth containing 0 mg/l chloramphenicol (Sigma) at C shaking. Cells were pelleted by centrifugation at 000g for 1 min. Cell pellets were frozen for 1 h at -0 C., resuspended in 0 mm Tris-chloride (Cl) at ph., and periplasmic proteins were released using stringent periplasmic fractionation with 0 mg/l lysozyme followed by addition of.0 mm EDTA. The supernatant was further enriched for -lactamase using preparative isoelectric focusing as previously described (, ). A second purification step was performed using fast protein liquid chromatography (FPLC) with a size exclusion HiLoad 1/0 Superdex- column (GE Healthcare Life Sciences, Uppsalla, Sweden) on the ÄKTA P-00 System (GE Healthcare Life Sciences). Proteins were eluted with mm phosphate buffered saline (PBS) at ph., and fractions were analyzed for purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples were resolved on a % stacking, 1% separating SDS-PAGE gel and stained with Coomassie Brilliant Blue R0. A final purification step was needed using anion exchange chromatography with a HiTrap Q HP column (GE Healthcare Life Sciences). Proteins were eluted with a salt gradient using a mixture of 0 mm Tris-Cl at ph. and 0 mm Tris-Cl 1.0 M sodium chloride at ph.. The purity of each preparation was again assessed by SDS-PAGE. Protein concentrations were determined with Bio-Rad s Protein Assay (Biorad, Hercules, CA) using bovine serum albumin as a protein standard according to the manufacturer s protocol. Electrospray ionization (ESI) mass spectrometry (MS). ESI-MS of the intact KPC- lactamase, purified from E. coli DHB cells carrying pbr-cati-bla KPC- plasmid, was performed on an Applied Biosystems (Foster City, CA) Q-STAR XL quadrupole-time-of-flight Downloaded from on October 1, 01 by guest

8 mass spectrometer equipped with a nanospray source as described previously (). - Lactamases (E) were incubated with inhibitor (I) for 1 min at room temperature in mm PBS, ph.. The I:E ratios were greater than the turnover number at 1 min (see below). Reactions were terminated by equilibration to 0.1% trifluoroacetic acid (TFA). All samples were desalted and concentrated using a C 1 ZipTip (Millipore, Billerica, MA) according to the manufacturer s protocol. Eluted protein samples were diluted with 0% acetonitrile and 0.1% TFA to a concentration of M, infused at a rate of 0. L per min, and data were collected for min. Spectra were deconvoluted using the Applied Biosystems Analyst program. Kinetics. Steady state kinetic parameters were determined using an Agilent (Santa Clara, CA) Diode Array spectrophotometer. Each assay was performed in mm PBS at ph. at room temperature. The enzyme was maintained at nm while substrate concentrations were varied from M to 00 M. To measure hydrolysis rates, we used the following extinction coefficients: nitrocefin (NCF) = 1,00 M -1 cm -1 at nm; piperacillin = -0 M -1 cm -1 at nm; cefotaxime = -,0 M -1 cm -1 at nm; imipenem = -,000 M -1 cm -1 at nm; and penem 1 = -,00 M -1 cm -1 at 0 nm. We also observed that in mm PBS at room temperature imipenem undergoes spontaneous hydrolysis (personal communication, Dr. Karen Bush). We subtracted this rate from our measured velocity to determine the true velocity. The kinetic parameters, V max and K m, were obtained with non-linear least squares fit of the data (Henri Michaelis-Menten equation) using Enzfitter (Biosoft Corporation, Ferguson, MO): v = (V max x [S]) / (K m + [S]) Eq. 1 Kinetic assays were performed under steady state conditions to determine the K m of inhibitors for the enzyme according to a previously established model represented in Eq. (1, Downloaded from on October 1, 01 by guest

9 ). In every case, the enzyme concentration was maintained at nm while inhibitor concentrations varied from M to 00 M for clavulanic acid, 1 M to 1.0 mm for sulbactam, 0 M to. mm for tazobactam, 0 nm to M for penem 1, and nm to 0 nm for penem. A final concentration of 0 M NCF was used as the reporter substrate. The branched pathway model can be represented by the following rate equation: k 1 k k E + I EI EI* E + P' k -1 k EI** k E + P'' Eq. EI*** In this model, formation of the non-covalent complex, EI, is represented by the dissociation constant, K, which is equivalent to k -1 /k 1. k is the first order rate constant for the acylation step, or formation of EI*. k is the rate constant for the hydrolysis of the EI* acylenzyme. The rearrangement of EI* to EI** is represented by the rate constant k. The rate constant for the hydrolysis of the EI** acyl-enzyme corresponds to rate constant k. Finally, the formation of a terminally inactivated acyl-enzyme species, EI*** is represented by rate constant k. Inverse initial steady state velocities (1/v 0 ) were plotted against inhibitor concentration (I) to obtain a straight line. As previously established, for brief periods of observation, the k step of Eq. can be neglected (1, ). Under these conditions, measuring the initial steady state velocity immediately after mixing yielded a K m for the hydrolysis reaction, or the Michaelis constant for the inhibitors. The velocity (v 0 ) measured after mixing is represented by Eq.. k Downloaded from on October 1, 01 by guest

10 v 0 = (V max *[S]) / (K m (ncf) * (1 + I/K m ) + [S]) Eq. K m (observed) was determined by dividing the value for the y-intercept by the slope of line. The data were corrected to account for the affinity of NCF for the -lactamase: K m (corrected) = K m (observed)/ [1 + ([S] / K m (ncf)] Eq. The k inact was measured directly by time dependent inactivation of the enzyme in the presence of inhibitor. To perform this determination, a fixed concentration of enzyme ( nm), 0 M NCF, and increasing concentrations of inactivator (0 M to 1,0 M for clavulanic acid, 00 M to.0 mm for sulbactam, 0 M to. mm for tazobactam, 00 nm to 0 M for penem 1, and 0 nm to 1. M for penem ) were used in each assay. The k obs for inactivation was determined using a non-linear least squares fit of the data using Origin.0 (Northampton, MA). Here, A 0 is initial absorbance, v f is final velocity, and t is time: A = A 0 + v f x t + (v 0 v f ) x [1 exp(-k obs x t)]/k obs Eq. Each k obs was plotted versus inhibitor concentration and fit to determine k inact. The kinetic parameter, k inact was obtained with non-linear least squares fit of the data using Enzfitter (Biosoft Corporation) and the hyperbolic equation below: k obs = (k inact x [I]) / (K m + [I]) Eq. The k inact as presented in Eq. corresponds to (k *k /(k +k +k ). The inhibitor efficiency of k inact /K m is equivalent to k *k /K*k. Partition ratios or turnover numbers (t n = k cat /k inact or k /k ) for KPC- were obtained by incubating 1.0 M KPC- with increasing concentrations of inhibitor (1.0 mm to.0 mm for clavulanic acid, 00 M to.0 mm for sulbactam, 0 M to 1.0 mm for tazobactam, 0 M to Downloaded from on October 1, 01 by guest

11 mm for penem 1, and M to 0 M for penem ) at room temperature for 1 min in mm PBS, ph.. The ratio of inhibitor to enzyme (I:E) necessary to inhibit the hydrolysis of NCF by greater than 0% was determined. Recovery from inhibition by penem 1 was measured by incubating 1.0 M KPC- with 1.0 mm penem 1 for 1h at room temperature. A spin concentrator with a,000 MWCO was used to remove the unbound inhibitor/product from the enzyme inhibitor (E, I) mixture. NCF (0 M) hydrolysis by KPC- was followed for 0 min. Hydrolysis of M penem 1 by 00, 0, and nm KPC- was measured at Abs 0nm at room temperature over 0 min ( max of penem 1 = 0nm). Ultraviolet difference (UVD) spectra. UVD spectra were obtained for M penem 1, M penem 1 reacted with 0. M KPC-, and M penem 1 reacted with 0 mm sodium hydroxide (NaOH) in mm PBS, ph.. UVD spectra were measured from wavelengths 00 to 00 nm on an Agilent Diode Array spectrophotometer for 1 min at room temperature. Molecular modeling. The crystal structure of KPC- (OV, Protein Data Bank) was used to generate molecular representations of the -lactamase interactions with the two penem inhibitors. The applications, Proteins Reports and Utility Tools of Discovery Studio.1 molecular modeling software (Accelrys, San Diego, CA), were used to correct crystallographic disorder and prepare the protein for molecular modeling. Hydrogen atoms were added, the ph was set at., and the crystallographic waters were removed. The KPC- -lactamase was immersed in a water box and was centered.0 Å away from any face of the box. Then, the molecule was energy minimized in several steps using Steepest Descendant and Conjugate Gradient algorithms to reach the minimum convergence (0.0 after,000 iterations and a final potential energy - 0, kj/mole). Energy minimizations and molecular dynamics simulations of the enzyme Downloaded from on October 1, 01 by guest

12 and complexes were determined using force-field parameters of CHARMm and a dielectric constant of 1.0. The Particle Mesh Ewald (PME) method was used to treat long-range electrostatics. Bonds that involved hydrogen atoms were constrained with the SHAKE algorithm. The penem structures were constructed using Fragment Builder tools. The CHARMm force field was applied. The protein was solvated and minimized using a Standard Dynamics Cascade protocol of Discovery Studio.1. The hydrolyzed penems were automatically docked into the active site of the -lactamase using the Flexible Docking module of Discovery Studio.1. The protocol allowed docking of penems into the flexible active site of KPC-. Flexible Docking using the ChiFlex algorithm (0) created side-chains and penem conformations while LibDock algorithm (1) docked the low energy penem conformations into the active site of KPC-. In the presence of penems, the side-chains of selected active site residues were refined using the ChiRotor algorithm (0), followed by a final simulated annealing and energy minimization of each ligand conformation using CDOCKER (). Downloaded from on October 1, 01 by guest 1

13 RESULTS and DISCUSSION Susceptibility testing. In Table 1 we summarize the results of our susceptibility testing of the K. pneumoniae clinical isolate containing KPC-, an E. coli transformant with bla KPC-, and an E. coli DHB control for -lactams. As expected the K. pneumoniae clinical strain and E. coli strain containing bla KPC- showed elevated MICs to all -lactams tested ( mg/l for imipenem to,1 mg/l for ampicillin). bla KPC- expressing strains also demonstrated high MICs to the lactam- -lactamase inhibitor combinations (ampicillin-clavulanic acid, 0/ mg/l; ampicillinsulbactam, 0/1 mg/l; and piperacillin-tazobactam, 1/ mg/l). These observations are consistent with current reports of MICs performed against clinical strains (, 1, 0,,, ). We next assayed the activities of piperacillin, cefotaxime, and imipenem in combination with penem 1 or penem. The lowest MICs were obtained when either penem 1 or is combined with piperacillin, cefotaxime, or imipenem. Notably, penem 1 and penem lower the MICs from mg/l to mg/l for imipenem in bla KPC- bearing strains. Interestingly, the most significant reduction for bla KPC- carrying strains was seen when cefotaxime was combined with penem 1 and penem. MICs were lowered from /1 mg/l to 0./0. mg/l, a six doubling dilution difference. Kinetics of KPC- with substrates and inhibitors. KPC- was purified and steady state kinetic parameters were measured (Table ). As demonstrated previously, KPC- hydrolyzes piperacillin, NCF, cefotaxime, and imipenem. Consistent with the MIC data, the catalytic efficiency (k cat/ K m ) is lowest for cefotaxime at 0.1 M -1 s -1 and highest for NCF at. M -1 s -1 compared to other substrates (). To measure the parameters of inactivation, we used NCF as the reporter substrate (Table ). In our assays, clavulanic acid, tazobactam, and sulbactam were unable to effectively inhibit Downloaded from on October 1, 01 by guest 1

14 NCF hydrolysis by KPC- (k inact /K m ) < 0.00 M -1 s -1 for all three inhibitors). More importantly, we also showed that KPC- hydrolyzes over,00 molecules of clavulanic acid, 1,000 molecules of sulbactam, and 00 molecules of tazobactam in 1 min. After hours, there is at least 0% further recovery of KPC- activity from inhibition by sulbactam and tazobactam at inhibitor concentrations equal to its t n measured at 1 min. By hours, KPC- partially recovers (~%) from inhibition by clavulanic acid. In contrast, we observed that the k inact /K m for penem 1 was 0.1 M -1 s -1 and penem was. M -1 s -1 for the KPC- -lactamase; these values were in accordance with the lower MIC determinations. Despite significantly lower MICs compared to combinations with other lactamase inhibitors, KPC- also hydrolyzed 0 molecules of penem 1 and molecules of penem in 1 min. In the case of penem 1, we were able to determine a k cat =. s -1. After hours, KPC- can recover from inhibition by penem 1 (~0%) or penem (~0%) at inhibitor concentrations equal to its t n measured at 1 min. Recovery after hours at a 1,000:1 penem 1 to KPC- ratio is represented in Figure D. KPC-, inhibitor inactivation products, and mechanism of inhibition. Timed mass spectrometry was used to assess the formation of KPC- -lactamase-inhibitor complexes. A molecular weight of, ± Daltons (amu) was obtained for KPC- alone. In preparation for mass spectrometry, KPC- was incubated for 1 min with clavulanic acid, sulbactam, tazobactam, penem 1, or penem at a concentration greater than its t n. Notwithstanding the lower partition ratios (t n ), we were not able to trap penem 1 or penem inactivating KPC-. We next employed timed UVD spectra to gain further insight into the inhibition process of penem 1 and penem with KPC-. The UVD spectra at 0 s for penem 1 alone, penem 1 reacted with KPC-, and penem 1 reacted with NaOH are represented in Figure A. The Downloaded from on October 1, 01 by guest 1

15 significant change at Abs 0nm when KPC- was incubated with penem 1 suggests that penem 1 is hydrolyzed by KPC- (Figure B). This interpretation is supported by the UVD changes that are observed after the base hydrolysis of penem 1 (Abs 0nm also decreases with time). We next studied the hydrolysis of penem 1 by KPC- at Abs 0nm (Figure C). Our results show that when the I/E ratio is > t n (i.e. > 0:1) a new steady state is reached. We observed that the hydrolysis of penem 1 is biphasic with a rapid initial hydrolysis (EI* E + P, rate constant= k )) followed by a slower steady state rate (EI** E + P, rate constant = k ) (Eq.) after about 00 s. After hours at high inhibitor to enzyme ratios (1,000:1), not all of the penem 1 was hydrolyzed (data not shown). Remarkably, if excess penem 1 is removed, most of KPC- s activity rapidly recovers from inhibition at 1,000:1 ratio with a slight lag (Figure D). We also observed that there is an initial rate of hydrolysis which may be due to free enzyme (either enzyme that has not acylated or enzyme that has acylated and deacylated (Figure D). In addition, the slope of the line after the lag is lower than the no penem 1 control, which is indicative of terminally inactivated enzyme-inhibitor complex (EI***) (Eq. ). To begin to understand how penem 1 and penem are hydrolyzed and yet inhibit KPC-, we modeled the penems in the active site of KPC-. We focused upon the penems as they represented the best inhibitors when compared to clavulanate, sulbactam and tazobactam. Based upon our work with SHV-1 and OXA-1, we conceptualized a mechanism in which the acyl enzyme proceeds to the linear imine that ultimately undergoes -endo-trig cyclization to yield a cyclic enamine, the 1,-thiazepine derivative (, ). Here, we focus on the deacylated form of penems 1 and before formation of the postulated seven-membered 1,-thiazepine ring. In Figure, the molecular representation of penem 1 (orange) within the active site of Downloaded from on October 1, 01 by guest KPC- is superimposed with the representation of penem (purple) in the active site. When 1

16 comparing the models of the major active site interactions between the penem 1 and penem, we note several major differences. To begin with, the carbonyl oxygen atom of penem 1 is pointing towards the oxyanion hole, whereas the carbonyl oxygen atom of penem is flipped and pointing away from the oxyanion hole. Then, we note that residues T and R0 make hydrogen bond interactions with the C carboxylate of penem 1, whereas neither is close enough to the C carboxylate of penem to make hydrogen bonding interactions. Instead, the C carboxylate of penem is close enough to hydrogen bond with either K or T. Lastly, we observe hydrophobic interactions with a potential for - stacking between the W ring and the bicyclic ring of penem 1. However in the penem model, W shifts away about 0 or. Å from the penem molecule. Overall, our model supports why the penems form interactions leading to a lower K m and a higher k inact /K m ratio. Conclusions. Herein, we summarize the kinetic and biochemical correlates of resistance to inhibition of KPC- by clavulanic acid, sulbactam, and tazobactam and we explore the turnover of two novel penems. Three important conclusions arise from our study. Firstly, we show why the commercially available -lactamase inhibitors are ineffective against KPC-. To our knowledge this ability to readily hydrolyze clavulanic acid, sulbactam, and tazobactam is very uncommon in class A enzymes (). This unprecedented observation partly explains why MICs are so high against -lactam -lactamase inhibitor combinations. In clinical isolates, this is compounded by the presence of multiple -lactamases (e.g., TEM, SHV, etc). Although penem 1 and penem are hydrolyzed by KPC- by acting as mechanism-based inactivators, they potentially offer a better alternative for inhibition of KPC producing strains compared to the commercial inhibitors. We suspect unraveling the chemistry that drives the hydrolysis of the Downloaded from on October 1, 01 by guest 1

17 commercially available inhibitors and penem 1 and through a branched kinetic mechanism (0, 1, ), may serve to offer new approaches to inhibiting carbapenemases. Secondly, we were intrigued by the synergy between cefotaxime and penems 1 or. We predict this synergy is due to the low catalytic efficiency k cat /K m of 0. ± 0.1 M -1 s -1 for cefotaxime by the KPC- -lactamase, as compared to the other substrate which have higher catalytic efficiencies. One wonders if combinations like this may potentially be exploited to inhibit KPC producing bacteria in the future. Thirdly, penem 1 and penem only differ by one carbon atom, thus it was surprising that their turnovers were different. Using our model, we illustrate why these inhibitors are effective and why they behave so differently. We close with the thought that the future challenge in medicinal chemistry will be to anticipate the different catalytic pathways these versatile enzymes (i.e., -lactamases) will follow as we continuously stress them with each new generation of -lactam. We recall that both imipenem (thienamycin) and clavulanic acid were discovered in the late s. It is sobering to contemplate that it has taken only 0 years to evolve a versatile -lactamase whose carbapenem resistant phenotype is matched by a very robust inhibitor resistant profile. In fact, the latter may be the pre-eminent property of the KPC -lactamase. Downloaded from on October 1, 01 by guest 1

18 Acknowledgements The Veterans Affairs Merit Review Program, the National Institutes of Health (RO1 AI01-01), and the Geriatric Research Education and Clinical Care VISN supported these studies. The authors also thank Dr. Andrea Endimiani and Ms. Andrea M. Hujer for careful review of the manuscript. Downloaded from on October 1, 01 by guest 1

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28 oxytoca harboring carbapenem-hydrolyzing -lactamase KPC-. Antimicrob Agents Chemother :1-.. Zhang, R., H. W. Zhou, J. C. Cai, and G. X. Chen. 00. Plasmid-mediated carbapenem-hydrolysing -lactamase KPC- in carbapenem-resistant Serratia marcescens isolates from Hangzhou, China. J Antimicrob Chemother :-. Downloaded from on October 1, 01 by guest

29 TABLE 1. Minimal Inhibitory Concentrations of -Lactams (mg/l), -Lactam and Commercially Available -Lactamase Inhibitors (mg/l) and Penems 1 and (mg/l) K. pneumoniae bla KPC- E. coli DHB pbr bla KPC- E. coli DHB Ampicillin,1,0 1 Ampicillin 0/ 0/ 0/1 clavulanic acid a Ampicillin 0/1 0/1 0/1 sulbactam a Piperacillin 1,0 1,0 Piperacillin 1/ 1/ /0. tazobactam b Piperacillin penem 1 c 1/ / 1/ Piperacillin / / / penem c Cefotaxime Cefotaxime 0./ 0./ 0.0/ penem 1 c Cefotaxime 0./ 0./ 0.0/ penem c Imipenem 0.1 Imipenem / / 0.1/ penem 1 c Imipenem / / 0./ penem c a Ampicillin was maintained at a constant concentration of 0 mg/l and clavulanic acid and sulbactam concentrations were increased. b For piperacillin/tazobactam, both were increased at a ratio :1. c Penem 1 and penem were held at a constant mg/l while the concentrations of piperacillin, imipenem, and cefotaxime were increased. Downloaded from on October 1, 01 by guest

30 TABLE. KPC- Kinetic Parameters K m (µm) k cat (s -1 ) k cat /K m ( M -1 s -1 ) Piperacillin 1 ± 1 ± 1.1 ± 0. NCF ± 1 0 ± 1. ± 0. Cefotaxime ± 1 ± 0. ± 0.1 Imipenem 1 ± 1 1 ± ± 0.1 Downloaded from on October 1, 01 by guest 0

31 TABLE. KPC- Inhibitor Kinetics with the -Lactamase Inhibitors K m ( M) k inact (s -1 ) k inact /K m ( M -1 s -1 ) t n (k cat /k inact ) Clavulanate. ± ± ± ~,00 Sulbactam 1 ± ± ± ~1,000 Tazobactam. ±. 0.0 ± ± ~00 Penem ± ± ± 0.0 ~0 Penem 0.00 ± ± ± 0. ~ Downloaded from on October 1, 01 by guest 1

32 FIGURE LEGENDS FIGURE 1. Chemical structures of the classical -lactamase inhibitors, the novel penem - lactamase inhibitors, cefotaxime, and imipenem FIGURE. A) UVD spectra of M penem 1 ( ), M penem 1 and 0. M KPC- ( ), and M penem 1 and 0 mm NaOH ( ) after 0 sec incubation. B) UVD spectra for M penem 1 and 0. M KPC- monitored over time s ( ), s ( ), and 0 sec ( )(bottom). C) Penem 1 hydrolysis by KPC- monitored at Abs 0nm using ratios of 1:1 ( M:00nM) ( ), 00:1 ( M:0nM) ( ), and 00:1 ( M:nM) ( ). D) NCF hydrolysis (0 M) by 1.0 M KPC- (gray) after inhibition by 1.0 mm penem 1 at a 00:1 inhibitor to enzyme ratio after hr incubation at room temperature compared to KPC- alone (black). FIGURE. Superimposition of molecular representations of penem 1 (orange) and penem (purple) within the KPC- active site. Downloaded from on October 1, 01 by guest

33 FIGURE 1. O O OH H N O CH OH H N O Clavulanic acid CO O CO O CO Sulbactam Tazobactam H N Imipenem N N S N S S O O CO O - Penem 1 - Penem CO S CO - HN NH NH S N S N N N H N N O S S OCH H N O O H N O Cefotaxime N N S - CO N O CH O Downloaded from on October 1, 01 by guest

34 Absorbance Absorbance 1 FIGURE. 0.0 mm penem 0 nm KPC- and 0.0 mm penem 1 0 mm NaOH and 0.0 mm penem 1 B. A. sec sec 0 sec Wavelength Wavelength C. D. 1 to 1 00 to 1 00 to no drug hours Absorbance Absorbance Time (seconds) Time (seconds) Downloaded from on October 1, 01 by guest

35 FIGURE. E1 S0 S W penem 1 penem T R0 Downloaded from on October 1, 01 by guest