scholarly journals Insights Into the Inhibition of MOX-1 β-Lactamase by S02030, a Boronic Acid Transition State Inhibitor

2021 ◽  
Vol 12 ◽  
Author(s):  
Tatsuya Ishikawa ◽  
Nayuta Furukawa ◽  
Emilia Caselli ◽  
Fabio Prati ◽  
Magdalena A. Taracila ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Boronic Acid ◽  
Laboratory Strain ◽  
Multidrug Resistant ◽  
Mechanisms Of Action ◽  
Gram Negative Bacteria ◽  
Class A ◽  
Fixed Concentration ◽  
Active Site Cavity

The rise of multidrug resistant (MDR) Gram-negative bacteria has accelerated the development of novel inhibitors of class A and C β-lactamases. Presently, the search for novel compounds with new mechanisms of action is a clinical and scientific priority. To this end, we determined the 2.13-Å resolution crystal structure of S02030, a boronic acid transition state inhibitor (BATSI), bound to MOX-1 β-lactamase, a plasmid-borne, expanded-spectrum AmpC β-lactamase (ESAC) and compared this to the previously reported aztreonam (ATM)-bound MOX-1 structure. Superposition of these two complexes shows that S02030 binds in the active-site cavity more deeply than ATM. In contrast, the SO3 interactions and the positional change of the β-strand amino acids from Lys315 to Asn320 were more prominent in the ATM-bound structure. MICs were performed using a fixed concentration of S02030 (4 μg/ml) as a proof of principle. Microbiological evaluation against a laboratory strain of Escherichia coli expressing MOX-1 revealed that MICs against ceftazidime are reduced from 2.0 to 0.12 μg/ml when S02030 is added at a concentration of 4 μg/ml. The IC50 and Ki of S02030 vs. MOX-1 were 1.25 ± 0.34 and 0.56 ± 0.03 μM, respectively. Monobactams such as ATM can serve as informative templates for design of mechanism-based inhibitors such as S02030 against ESAC β-lactamases.

scholarly journals Novel Insights into the Mode of Inhibition of Class A SHV-1 β-Lactamases Revealed by Boronic Acid Transition State Inhibitors

2010 ◽  
Vol 55 (1) ◽  
pp. 174-183 ◽  
Author(s):  
Wei Ke ◽  
Jared M. Sampson ◽  
Claudia Ori ◽  
Fabio Prati ◽  
Sarah M. Drawz ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Boronic Acid ◽  
Kinetic Data ◽  
Binding Pocket ◽  
Stacking Interactions ◽  
Relative Resistance ◽  
Class A ◽  
Important Enzyme ◽  
Phenol Moiety

ABSTRACTBoronic acid transition state inhibitors (BATSIs) are potent class A and C β-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 β-lactamase, a clinically important enzyme found inKlebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 β-lactamase with micromolar affinity that is considerably weaker than their inhibition of other β-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other β-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.

scholarly journals Inhibition of pancreatic lipase in vitro by the covalent inhibitor tetrahydrolipstatin

1988 ◽  
Vol 256 (2) ◽  
pp. 357-361 ◽  
Author(s):  
P Hadváry ◽  
H Lengsfeld ◽  
H Wolfer
Keyword(s):  
Transition State ◽  
Acid Derivative ◽  
Pancreatic Lipase ◽  
Active Site ◽  
Boronic Acid ◽  
Selective Inhibitor ◽  
Covalent Intermediate ◽  
State Form ◽  
Covalent Inhibitor

Tetrahydrolipstatin inhibits pancreatic lipase from several species, including man, with comparable potency. The lipase is progressively inactivated through the formation of a long-lived covalent intermediate, probably with a 1:1 stoichiometry. The lipase substrate triolein and also a boronic acid derivative, which is presumed to be a transition-state-form inhibitor, retard the rate of inactivation. Therefore, in all probability, tetrahydrolipstatin reacts with pancreatic lipase at, or near, the substrate binding or active site. Tetrahydrolipstatin is a selective inhibitor of lipase; other hydrolases tested were at least a thousand times less potently inhibited.

scholarly journals Crystal Structures of KPC-2 β-Lactamase in Complex with 3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226

2012 ◽  
Vol 56 (5) ◽  
pp. 2713-2718 ◽  
Author(s):  
Wei Ke ◽  
Christopher R. Bethel ◽  
Krisztina M. Papp-Wallace ◽  
Sundar Ram Reddy Pagadala ◽  
Micheal Nottingham ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Boronic Acid ◽  
Carbonyl Oxygen ◽  
Oxyanion Hole ◽  
Covalent Interaction ◽  
Class A ◽  
Inactivation Rate Constant ◽  
Reversible Covalent ◽  
Acid Compound

ABSTRACTClass 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 aKmvalue 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 penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed aKmvalue of 3.8 ± 0.4 μM for KPC-2, and the inactivation rate constant (kinact) was 0.034 ± 0.003 s−1. When covalently bound to S70, PSR-3-226 forms atrans-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 β-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.

scholarly journals Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

2004 ◽  
Vol 78 (6) ◽  
pp. 3123-3132 ◽  
Author(s):  
Bradley C. Logsdon ◽  
John F. Vickrey ◽  
Philip Martin ◽  
Gheorghe Proteasa ◽  
Jay I. Koepke ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Surface Plasmon Resonance ◽  
Protease Inhibitors ◽  
Surface Plasmon ◽  
Active Site ◽  
Multidrug Resistant ◽  
Immunodeficiency Virus ◽  
Active Site Cavity ◽  
Hiv 1

ABSTRACT The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (Å) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease “flaps” stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 Å. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1′, S3, and S3′ pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k off rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k on and k off data (Kd = k off/k on) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

scholarly journals Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Melina Ruggiero ◽  
Krisztina M. Papp-Wallace ◽  
Magdalena A. Taracila ◽  
Maria F. Mojica ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Option ◽  
Structural Features ◽  
Atomic Mass ◽  
Stable Complex ◽  
Water Molecules ◽  
Gram Negative Bacteria ◽  
Biophysical Properties ◽  
Content Type ◽  
Class A

ABSTRACT PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2/K = 2 × 103 ± 0.1 × 103 M−1 s−1, where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k 2/K range of ≈103 M−1 s−1) and not class A β-lactamases (i.e., k 2/K range, 104 to 105 M−1 s−1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k 2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam–AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing bla PER-2. Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

scholarly journals Site-saturation Mutagenesis of Tyr-105 Reveals Its Importance in Substrate Stabilization and Discrimination in TEM-1 β-Lactamase

2004 ◽  
Vol 279 (44) ◽  
pp. 46295-46303 ◽  
Author(s):  
Nicolas Doucet ◽  
Pierre-Yves De Wals ◽  
Joelle N. Pelletier
Keyword(s):  
Active Site ◽  
Survival Rates ◽  
Saturation Mutagenesis ◽  
Structural Requirement ◽  
Class A ◽  
Active Site Cavity ◽  
Cephalosporin Antibiotics ◽  
The Difference ◽  
Substrate Stabilization

The conserved Class A β-lactamase active site residue Tyr-105 was substituted by saturation mutagenesis in TEM-1 β-lactamase fromEscherichia coliin order to clarify its role in enzyme activity and in substrate stabilization and discrimination. Minimum inhibitory concentrations were calculated forE. colicells harboring each Y105Xmutant in the presence of various penicillin and cephalosporin antibiotics. We found that only aromatic residues as well as asparagine replacements conferred highin vivosurvival rates for all substrates tested. At position 105, the small residues alanine and glycine provide weak substrate discrimination as evidenced by the difference in benzylpenicillin hydrolysis relative to cephalothin, two typical penicillin and cephalosporin antibiotics. Kinetic analyses of mutants of interest revealed that the Y105Xreplacements have a greater effect onKmthankcat, highlighting the importance of Tyr-105 in substrate recognition. Finally, by performing a short molecular dynamics study on a restricted set of Y105Xmutants of TEM-1, we found that the strong aromatic bias observed at position 105 in Class A β-lactamases is primarily defined by a structural requirement, selecting planar residues that form a stabilizing wall to the active site. The adopted conformation of residue 105 prevents detrimental steric interactions with the substrate molecule in the active site cavity and provides a rationalization for the strong aromatic bias found in nature at this position among Class A β-lactamases.

Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity

2013 ◽  
Vol 451 (2) ◽  
pp. 217-226 ◽  
Author(s):  
Emily Golden ◽  
Rachel Paterson ◽  
Wan Jun Tie ◽  
Anandhi Anandan ◽  
Gavin Flematti ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Cephalosporin C ◽  
Class Iii ◽  
Wild Type ◽  
Serine Residue ◽  
Active Site Cavity ◽  
Β Chain ◽  
Enzyme Variant

The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57βS/H70βS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a β-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser1β, is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.

scholarly journals Structural basis of the complementary activity of two ketosynthases in aryl polyene biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Cheol Lee ◽  
Sungjae Choi ◽  
Ahjin Jang ◽  
Jiwon Yeon ◽  
Eunha Hwang ◽  
...  
Keyword(s):  
Active Site ◽  
Acyl Carrier Protein ◽  
Gene Clusters ◽  
Structural Basis ◽  
Gram Negative Bacteria ◽  
Aryl Group ◽  
Vitro Assay ◽  
Active Site Cavity

AbstractAryl polyenes (APE) are one of the most widespread secondary metabolites among gram-negative bacteria. In Acinetobacter baumannii, strains belonging to the virulent global clone 2 (GC2) mostly contain APE biosynthesis genes; its relevance in elevated pathogenicity is of great interest. APE biosynthesis gene clusters harbor two ketosynthases (KSs): the heterodimeric KS-chain length factor complex, ApeO-ApeC, and the homodimeric ketoacyl-acyl carrier protein synthase I (FabB)-like KS, ApeR. The role of the two KSs in APE biosynthesis is unclear. We determined the crystal structures of the two KSs from a pathogenic A. baumannii strain. ApeO-ApeC and ApeR have similar cavity volumes; however, ApeR has a narrow cavity near the entrance. In vitro assay based on the absorption characteristics of polyene species indicated the generation of fully elongated polyene with only ApeO-ApeC, probably because of the funnel shaped active site cavity. However, adding ApeR to the reaction increases the throughput of APE biosynthesis. Mutagenesis at Tyr135 in the active site cavity of ApeR reduces the activity significantly, which suggests that the stacking of the aryl group between Tyr135 and Phe202 is important for substrate recognition. Therefore, the two KSs function complementarily in the generation of APE to enhance its production.

scholarly journals 1372. In Vitro Activity of Novel Ceftazidime–Avibactam and Aztreonam–Avibactam Combinations Against Carbapenem-Nonsusceptible Enterobacteriaceae Isolates by Phenotype Collected in Latin America From 2014 to 2017 as Part of the INFORM Surveillance Program

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S420-S420
Author(s):  
Krystyna Kazmierczak ◽  
Boudewijn De Jonge ◽  
Gregory G Stone ◽  
Dan Sahm
Keyword(s):  
Latin America ◽  
Susceptibility Testing ◽  
Multidrug Resistant ◽  
Vitro Activity ◽  
Surveillance Program ◽  
In Vitro Activity ◽  
Class A ◽  
Fixed Concentration ◽  
Clinically Significant

Abstract Background Carbapenem-nonsusceptible Enterobacteriaceae (CRE) are often multidrug-resistant and infections caused by these organisms are associated with increased morbidity and mortality. The combination of avibactam (AVI), a non-β-lactam/β-lactamase inhibitor of Class A, C, and some D serine β-lactamases, with ceftazidime (CAZ) and aztreonam (ATM) is being developed to treat infections caused by CRE. CAZ-AVI reveals potent in vitro activity against CRE, except those producing metallo-β-lactamases (MBLs), whereas ATM-AVI inhibits growth of both MBL-positive and MBL-negative CRE. We evaluated the in vitro activity of CAZ-AVI and ATM-AVI against Enterobacteriaceae isolates nonsusceptible to meropenem (MEM-NS) collected in 2014–2017 in Latin America through the INFORM global surveillance program. Methods Nonduplicate clinically significant isolates were collected from 29 hospital laboratories located in Argentina, Brazil, Chile, Colombia, Mexico, and Venezuela. Susceptibility testing was performed by CLSI broth microdilution. AVI was tested at a fixed concentration of 4 µg/mL in combination with CAZ and ATM. MEM-NS Eba (MIC >1 µg/mL) were screened for the presence of β-lactamase genes by PCR and sequencing. Results Five hundred fifty-seven MEM-NS isolates were identified (440 Klebsiella pneumoniae and 117 isolates of 13 other species). Of these, 441 (79.2%) carried carbapenemases (Cpase) (KPC only, n = 383; MBL only, n = 48; OXA-48-like only, n = 5; KPC and OXA-48-like, n = 2; MBL and GES, n = 2; MBL and KPC, n = 1). CAZ-AVI showed potent in vitro activity against Cpase-positive MBL-negative and Cpase-negative Eba and against all MEM-NS Eba, but was not active against MBL-positive Eba. 100% of MEM-NS Eba were inhibited by ≤8 µg/mL of ATM-AVI. Conclusion CAZ-AVI and ATM-AVI displayed potent in vitro activity against MEM-NS Eba collected in LA. These agents could serve as promising options for treatment of infections caused by CRE. Disclosures K. Kazmierczak, Pfizer Inc.: Consultant, Consulting fee. IHMA, Inc.: Employee, Salary. B. De Jonge, AstraZeneca: Shareholder, Dividends. Pfizer Inc: Employee, Salary. G. G. Stone, Pfizer Inc.: Employee, Salary. AstraZeneca: Former Employee and Shareholder, Salary. D. Sahm, Pfizer Inc.: Consultant, Consulting fee. IHMA, Inc.: Employee, Salary.

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

2016 ◽  
Vol 60 (3) ◽  
pp. 1751-1759 ◽  
Author(s):  
Laura J. Rojas ◽  
Magdalena A. Taracila ◽  
Krisztina M. Papp-Wallace ◽  
Christopher R. Bethel ◽  
Emilia Caselli ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Transition State ◽  
Boronic Acid ◽  
Thiophene Ring ◽  
Side Chain ◽  
Aryl Group ◽  
Content Type ◽  
E Coli ◽  
Class A ◽  
Structure Activity

Boronic acid transition state inhibitors (BATSIs) are competitive, reversible β-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 β-lactamases ofKlebsiella 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 β-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 β-lactamases, nine possessed 50% inhibitory concentrations (IC50s) 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 (k2/K) of 1.2 ± 0.2 × 104M−1s−1for KPC-2 and 4.7 ± 0.6 × 103M−1s−1for SHV-1, and demonstrated antimicrobial activity againstEscherichia coliDH10B carryingblaSHVvariants andblaKPC-2orblaKPC-3and against clinical strains ofKlebsiella pneumoniaeandE. coliproducing different class A β-lactamase genes. At most, MICs decreased from 16 to 0.5 mg/liter.

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