Crystal Structures of KPC-2 -Lactamase in Complex with 3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226 (original) (raw)
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Antimicrobial Agents and Chemotherapy, 2012
Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated a K m value of 1.0 ± 0.1 μM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the ...
Antimicrob Agents Chemother, 2012
Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by -lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two -lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated a K m value of 1.0 ؎ 0.1 M (mean ؎ standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed a K m value of 3.8 ؎ 0.4 M for KPC-2, and the inactivation rate constant (k inact) was 0.034 ؎ 0.003 s ؊1. When covalently bound to S70, PSR-3-226 forms a trans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first -lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.
Antimicrobial agents and chemotherapy, 2016
Resistance to expanded-spectrum cephalosporins and carbapenems has rendered certain strains of Klebsiella pneumoniae as the most problematic pathogens infecting patients in the hospital and community. This broad spectrum resistance to β-lactamas emerges in part via the expression of KPC-2 and SHV-1 β-lactamases, and variants thereof, KPC-2 carbapenemase is particularly worrisome as the genetic determinant encoding this β-lactamase is rapidly spread via plasmids. Moreover, KPC-2, a class A enzyme, is difficult to inhibit with mechanism based inactivators (i.e. clavulanate). In order to develop new β-lactamase inhibitors (BLIs) to add to the limited available armamentarium that can inhibit KPC-2, we have structurally probed the boronic acid transition state analog S02030 for its inhibition of KPC-2 and SHV-1. S02030 contains a boronic acid, a thiophene, and a carboxyl triazole moiety. We present here the 1.54 and 1.87 Å resolution crystal structures of S02030 bound to SHV-1 and KPC-2 ...
Journal of Medicinal Chemistry, 2015
The increasing dissemination of carbapenemases in Gram-negative bacteria has threatened the clinical usefulness of the β-lactam class of antimicrobials. A program was initiated to discover a new series of serine β-lactamase inhibitors containing a boronic acid pharmacophore, with the goal of finding a potent inhibitor of serine carbapenemase enzymes that are currently compromising the utility of the carbapenem class of antibacterials. Potential lead structures were screened in silico by modeling into the active sites of key serine β-lactamases. Promising candidate molecules were synthesized and evaluated in biochemical and whole-cell assays. Inhibitors were identified with potent inhibition of serine carbapenemases, particularly the Klebsiella pneumoniae carbapenemase (KPC), with no inhibition of mammalian serine proteases. Studies in vitro and in vivo show that RPX7009 (9f) is a broad-spectrum inhibitor, notably restoring the activity of carbapenems against KPCproducing strains. Combined with a carbapenem, 9f is a promising product for the treatment of multidrug resistant Gramnegative bacteria.
Crystal Structure of KPC-2: Insights into Carbapenemase Activity in Class A β-Lactamases
Biochemistry, 2007
β-lactamases inactivate β-lactam antibiotics and are a major cause of antibiotic resistance. The recent outbreaks of Klebsiella pneumoniae carbapenem-resistant (KPC) infections mediated by KPC type β-lactamases are creating a serious threat to our "last resort" antibiotics, the carbapenems. KPC βlactamases are thus carbapenemases and are a subclass of Class A β-lactamases that have evolved to efficiently hydrolyze carbapenems and cephamycins which contain substitutions at the α position proximal to the carbonyl group that normally render these β-lactams resistant to hydrolysis. To investigate the molecular basis of this carbapenemase activity, we have determined the structure of KPC-2 at 1.85Å resolution. The active site of KPC-2 reveals the presence of a bicine buffer molecule which interacts via its carboxyl group with conserved active site residues S130, K234, T235, and T237; this likely resembles the interactions the β-lactam carboxyl moiety makes in the Michaelis-Menten complex. Comparison of the KPC-2 structure with non-carbapenemases and previously determined NMC-A and SME-1 carbapenemase structures shows several active site alterations that are unique among carbapenemases. An outward shift of the catalytic S70 residue renders the active sites of the carbapenemases more shallow likely allowing easier access of the bulkier substrates. Further space for the α-substituents is likely provided by shifts in N132 and N170 in addition to concerted movements in the postulated carboxyl binding pocket that might allow the substrates to bind in a slightly different angle to accommodate these α-substituents. The structure of KPC-2 thus provides key insights into the carbapenemase activity of emerging Class A β-lactamases. Carbapenems (imipenem, meropenem, and ertapenem) are the "last resort" β-lactam antibiotics for treating serious infections caused by multi-drug resistant gram-negative bacteria. Unfortunately, the acquisition of carbapenem-hydrolyzing β-lactamases by bacteria has resulted in a major threat to the clinical utility of these compounds (1;2). Currently the Klebsiella pneumoniae carbapenemases (KPC-type β-lactamases), are rapidly emerging as a major threat in the New York area (3-5), Pennsylvania (6), and internationally (7;8). Three KPC variants have been found so far (KPC-1,-2, and-3) of which KPC-1 and KPC-2 are almost indistinguishable, whereas KPC-3 exhibits different hydrolytic properties (9). KPC carbapenemases belong to the Ambler class A β-lactamases (E.C. 3.5.2.6) (10;11). In common with other class A β-lactamase, these highly proficient class A enzymes have an efficient hydrolysis 'machinery' involving a catalytic serine residue (S70), which acylates β
PloS one, 2014
The use of three classical β-lactamase inhibitors (Clavulanic acid, tazobactam and sulbactam) in combination with β-lactam antibiotics is presently the mainstay of antibiotic therapy against Gram-negative bacterial infections. However these inhibitors are unable to inhibit carbapenemase KPC-2 effectively. They being β-lactam derivatives behave as substrates for this enzyme instead of inactivating it. We have initiated our study to check the in vitro inhibition activity of the two novel screened inhibitors (ZINC01807204 and ZINC02318494) in combination with carbapenems against KPC-2 expressing bacterial strain and their effect on purified enzyme KPC-2. The MIC values of meropenem and ertapenem showed maximum reduction (8 folds) in combination with screened compounds (ZINC01807204 and ZINC02318494). CLSM images also depicted their strong antibacterial activity in comparison to conventional β-lactamase inhibitors. Moreover no toxic effect has been shown on HeLa cell line. Though the IC...
Journal of the American Chemical Society, 1998
Bacteria resistant to antibiotics are being selected in a relatively short time, and cases of infections resistant to treatment by all known antibiotics are being identified at alarming rates. The primary mechanism for resistance to-lactam antibiotics is the catalytic function of-lactamases. However, imipenem (a-lactam) resists the action of most-lactamases and is virtually the last effective agent against the vancomycin-resistant Gram-positive bacteria, as well as against multiple antibiotic-resistant Gram-negative organisms. Here, we report the crystal structure, to 1.8 Å resolution, of an acyl-enzyme intermediate for imipenem bound to the TEM-1-lactamase from Escherichia coli, the parent enzyme of 67 clinical variants. The structure indicates an unprecedented conformational change for the complex which accounts for the ability of this antibiotic to resist hydrolytic deactivation by-lactamases. Computational molecular dynamics underscored the importance of the motion of the acyl-enzyme intermediate, which may be a general feature for catalysis by these enzymes.
2014
The genome of Mycobacterium tuberculosis contains a gene, blaC, which encodes a highly active β-lactamase (BlaC). We have previously shown that BlaC has an extremely broad spectrum of activity against penicillins and cephalosporins but weak activity against newer carbapenems. We have shown that carbapenems such as meropenem, doripenem, and ertapenem react with the enzyme to form enzyme−drug covalent complexes that are hydrolyzed extremely slowly. In the current study, we have determined apparent K m and k cat values of 0.8 μM and 0.03 min −1 , respectively, for tebipenem, a novel carbapenem whose prodrug form, the pivalyl ester, is orally available. Tebipenem exhibits slow tightbinding inhibition at low micromolar concentrations versus the chromogenic substrate nitrocefin. FT-ICR mass spectrometry demonstrated that the tebipenem acyl−enzyme complex remains stable for greater than 90 min and exists as mixture of the covalently bound drug and the bound retro-aldol cleavage product. We have also determined the high-resolution crystal structures of the BlaC−tebipenem covalent acylated adduct (1.9 Å) with wild-type BlaC and the BlaC−tebipenem Michaelis− Menten complex (1.75 Å) with the K73A BlaC variant. These structures are compared to each other and to other carbapenem− BlaC structures.
Antimicrobial agents and chemotherapy, 2016
Boronic acid transition state inhibitors (BATSIs) are competitive, reversible β-lactamase inhibitors (BLIs). Herein, a series of BATSIs with selectively modified regions (R1, R2 and amide group) were strategically designed and tested against representative class A β-lactamases of Klebsiella pneumoniae, KPC-2 and SHV-1. Firstly, the R1 group of compounds 1A: , 1B: , 1C: , 2A -2E: mimicked the side chain of cephalothin whereas for compounds 3A: , 3B: , 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 -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 C6/C7 of β-lactams, was also changed to the following bioisosteric groups: urea (com...
Carbapenems (imipenem, meropenem, biapenem, ertapenem, and doripenem) are β-lactam antimicrobial agents. Because carbapenems have the broadest spectra among all β-lactams and are primarily used to treat infections by multi-resistant Gram-negative bacteria, the emergence and spread of carbapenemases became a major public health concern. Carbapenemases are the most versatile family of β-lactamases that are able to hydrolyze carbapenems and many other β-lactams. According to the dependency of divalent cations for enzyme activation, carbapenemases can be divided into metallo-carbapenemases (zinc-dependent class B) and non-metallo-carbapenemases (zinc-independent classes A, C, and D). Many studies have provided various carbapenemase structures. Here we present a comprehensive and systematic review of three-dimensional structures of carbapenemase-carbapenem complexes as well as those of carbapenemases. We update recent studies in understanding the enzymatic mechanism of each class of carbapenemase, and summarize structural insights about regions and residues that are important in acquiring the carbapenemase activity.