Penam Sulfones Research Articles

How Clavulanic Acid Inhibits Serine β-Lactamases.

Clavulanic acid is a medicinally important inhibitor of serine β-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC‑MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest.

Studies on enmetazobactam clarify mechanisms of widely used β-lactamase inhibitors

β-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine β-lactamase (SBL)-catalyzed hydrolysis. The scope of β-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone–derived acyl–enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl–enzyme complexes.

Penam sulfones and β-lactamase inhibition: SA2-13 and the importance of the C2 side chain length and composition.

β-Lactamases are the major reason β-lactam resistance is seen in Gram-negative bacteria. To combat this resistance mechanism, β-lactamase inhibitors are currently being developed. Presently, there are only three that are in clinical use (clavulanate, sulbactam and tazobactam). In order to address this important medical need, we explored a new inhibition strategy that takes advantage of a long-lived inhibitory trans-enamine intermediate. SA2-13 was previously synthesized and shown to have a lower k react than tazobactam. We investigated here the importance of the carboxyl linker length and composition by synthesizing three analogs of SA2-13 (PSR-4-157, PSR-4-155, and PSR-3-226). All SA2-13 analogs yielded higher turnover numbers and k react compared to SA2-13. We next demonstrated using protein crystallography that increasing the linker length by one carbon allowed for better capture of a trans-enamine intermediate; in contrast, this trans-enamine intermediate did not occur when the C2 linker length was decreased by one carbon. If the linker was altered by both shortening it and changing the carboxyl moiety into a neutral amide moiety, the stable trans-enamine intermediate in wt SHV-1 did not form; this intermediate could only be observed when a deacylation deficient E166A variant was studied. We subsequently studied SA2-13 against a relatively recently discovered inhibitor-resistant (IR) variant of SHV-1, SHV K234R. Despite the alteration in the mechanism of resistance due to the K→R change in this variant, SA2-13 was effective at inhibiting this IR enzyme and formed a trans-enamine inhibitory intermediate similar to the intermediate seen in the wt SHV-1 structure. Taken together, our data reveals that the C2 side chain linker length and composition profoundly affect the formation of the trans-enamine intermediate of penam sulfones. We also show that the design of SA2-13 derivatives offers promise against IR SHV β-lactamases that possess the K234R substitution.

Structures of SHV-1 β-Lactamase with Penem and Penam Sulfone Inhibitors That Form Cyclic Intermediates Stabilized by Carbonyl Conjugation

Bacterial β-lactamase enzymes are in large part responsible for the decreased ability of β-lactam antibiotics to combat infections. The inability to overcome β-lactamase mediated resistance spurred the development of inhibitors with penems and penam sulfones being amongst the most potent and broad spectrum mechanism-based inactivators. These inhibitors form covalent, “suicide-type” inhibitory intermediates that are attached to the catalytic S70 residue. To further probe the details of the mechanism of β-lactamase inhibition by these novel compounds, we determined the crystal structures of SHV-1 bound with penem 1, and penam sulfones SA1-204 and SA3-53. Comparison with each other and with previously determined crystal structures of members of these classes of inhibitors suggests that the final conformation of the covalent adduct can vary greatly amongst the complex structures. In contrast, a common theme of carbonyl conjugation as a mechanism to avoid deacylation emerges despite that the penem and penam sulfone inhibitors form different types of intermediates. The detailed insights gained from this study could be used to further improve new mechanism-based inhibitors of these common class A serine β-lactamases.

Carboxylation and Decarboxylation of Active Site Lys 84 Controls the Activity of OXA-24 β-Lactamase of Acinetobacter baumannii: Raman Crystallographic and Solution Evidence

The class D β-lactamases are characterized by the presence of a carboxylated lysine in the active site that participates in catalysis. Found in Acinetobacter baumannii, OXA-24 is a class D carbapenem hydrolyzing enzyme that exhibits resistance to most available β-lactamase inhibitors. In this study, the reaction between a 6-alkylidiene penam sulfone inhibitor, SA-1-204, in single crystals of OXA-24 is followed by Raman microscopy. Details of its reaction with SA-1-204 provide insight into the enzyme's mode of action and help define the mechanism of inhibition. When the crystal is maintained in HEPES buffer, the reaction is fast, shorter than the time scale of the Raman experiment. However, when the crystal holding solution contains 28% PEG 2000, the reaction is slower and can be recorded by Raman microscopy in real time; the inhibitor's Raman bands quickly disappear, transient features are seen due to an early intermediate, and, at approximately 2-11 min, new bands appear that are assigned to the late intermediate species. At about 50 min, bands due to all intermediates are replaced by Raman signals of the unreacted inhibitor. The new population remains unchanged indicating (i) that the OXA-24 is no longer active and (ii) that the decarboxylation of Lys84 occurred during the first reaction cycle. Using absorbance spectroscopy, a one-cycle reaction could be carried out in aqueous solution producing inactive OXA-24 as assayed by the chromogenic substrate nitrocefin. However, activity could be restored by reacting aqueous OXA-24 with a large excess of NaHCO(3), which recarboxylates Lys84. In contrast, the addition of NaHCO(3) was not successful in reactivating OXA-24 in the crystalline state; this is ascribed to the inability to create a concentration of NaHCO(3) in large excess over the OXA-24 that is present in the crystal. The finding that inhibitor compounds can inactivate a class D enzyme by promoting decarboxylation of an active site lysine suggests a novel function that could be exploited in inhibitor design.

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

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 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 μM 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 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.

β-Lactamase Inhibition: Mechanistic Details and Novel Inhibitors

Bacteria that are resistant to β-lactam antibiotics by producing β-lactamases are a significant public health threat. These β-lactamases hydrolyze β-lactams and render them inactive. To combat these resistant strains, β-lactams are often administered with β-lactamase inhibitors. Unfortunately, β-lactamases are beginning to acquire mutations which confer resistance to these inhibitors as well. Therefore, a clear need exists to identify novel inhibitors to ensure continued antibiotic efficacy.We used a synergistic X-ray and Raman crystallographic approach to investigate the mechanisms of inhibitor binding and to further the development of new inhibitors of the β-lactamase SHV-1. To this end, we first engineered an acylation deficient mutant to capture the pre-acylation complex of β-lactamase and inhibitor. Our 1.45 A structure, as well Raman measurements, reveals an unreacted sulbactam species in the active site of the mutant SHV enzyme and represents the first pre-acylation structure between inhibitor and β-lactamase. The structure identifies key interactions that are made immediately before acylation.The second project involves a series of derivative compounds designed to improve upon the novel C2 penam sulfone inhibitor, SA2-13, which forms a stabilized trans-enamine conformation in the active site. Structures of the derivative compounds suggest that the C2 chain length is important in SA2-13 stabilization. Exploring a series of C2 derivatives by X-ray and Raman spectroscopy permitted deeper insight into more favorable inhibitor conformations.Understanding the interactions which occur in the steps immediately prior to and during covalent complex formation will allow for design of better β-lactamase inhibitors against these important drug targets. Using Raman and protein crystallography allow us to track and trap reaction intermediates inside protein crystals.

Efficient Inhibition of Class A and Class D β-Lactamases by Michaelis Complexes

A 6-alkylidiene penam sulfone, SA-1-204, is an efficient inhibitor of both SHV-1 and OXA-1 beta-lactamases with K(I) = 42 +/- 4 nm and 1.0 +/- 0.1 microm, respectively. To gain insight into the reaction chemistry of SA-1-204, the reactions between this inhibitor and SHV-1 and OXA-1 were studied by Raman spectroscopy in single crystals and in solution. Raman signatures characteristic of the unreacted beta-lactam ring show that in both phases the inhibitor binds as a noncovalent Michaelis-like complex. This complex is present as the major population for periods of up to an hour. On longer time scales, the Raman data show that beta-lactam ring opening eventually leads to a complex mixture of reaction products. However, the data clearly demonstrate that the key species for inhibition on the time scale of bacterial half-lives is the noncovalent complex preceding acylation.

Rational Design of a β-Lactamase Inhibitor Achieved via Stabilization of the trans-Enamine Intermediate: 1.28 Å Crystal Structure of wt SHV-1 Complex with a Penam Sulfone

beta-Lactamases are one of the major causes of antibiotic resistance in Gram negative bacteria. The continuing evolution of beta-lactamases that are capable of hydrolyzing our most potent beta-lactams presents a vexing clinical problem, in particular since a number of them are resistant to inhibitors. The efficient inhibition of these enzymes is therefore of great clinical importance. Building upon our previous structural studies that examined tazobactam trapped as a trans-enamine intermediate in a deacylation deficient SHV variant, we designed a novel penam sulfone derivative that forms a more stable trans-enamine intermediate. We report here the 1.28 A resolution crystal structure of wt SHV-1 in complex with a rationally designed penam sulfone, SA2-13. The compound is covalently bound to the active site of wt SHV-1 similar to tazobactam yet forms an additional salt-bridge with K234 and hydrogen bonds with S130 and T235 to stabilize the trans-enamine intermediate. Kinetic measurements show that SA2-13, once reacted with SHV-1 beta-lactamase, is about 10-fold slower at being released from the enzyme compared to tazobactam. Stabilizing the trans-enamine intermediate represents a novel strategy for the rational design of mechanism-based class A beta-lactamase inhibitors.

Synthesis and biological evaluation of penam sulfones as inhibitors of β-lactamases

The chemical synthesis of a series of new penam sulfone derivatives bearing a 2β-substituted-oxyimino and -hydrazone substituents, their β-lactamase inhibitory properties against selected enzymes representing class A and C β-lactamases are reported. The oxime containing penam sulfones strongly inhibited the Escherichia coli TEM-1 and Klebsiella pneumoniae cefotaximase (CTX-1) enzymes, but moderately inhibited the Pseudomonas aeruginosa 46012 cephalosporinase; while the 2β-substituted-hydrazone derivatives were generally less active against these enzymes. Furthermore, most of the inhibitors enhanced the antibacterial activities of piperacillin (PIP) and ceftazidime (CAZ) particularly against TEM-1 and CTX-1 producing bacterial strains.

ChemInform Abstract: Allyl and Propargyl Substituted Penam Sulfones as Versatile Intermediates Toward the Syntheses of New β‐Lactamase Inhibitors.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis and human leukocyte elastase inhibitory evaluation of phosphate triesters and acyl phosphates of penam sulfides and sulfones

The synthesis of 6,6-dibromo-3α-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone ( 3a), 6α-chloro-3α-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone ( 3b), benzyl 6α-(diphenyl-phosphate)oxypenicillanate sulfone ( 4) and 6,6-dibromo-3α-(methylphosphate)carbonyl-2,2-dimethylpenam sulfone ( 12) are reported. When tested as inhibitors of human leukocyte elastase, the compound 4 proved to be the most active.

Allyl and propargyl substituted penam sulfones as versatile intermediates toward the syntheses of new β-lactamase inhibitors

Several alkenyl derivatives were prepared using allyl penam sulfone as the key intermediate. Isomers of these derivatives having β configuration at C-6 showed potent acitivity against CcrA enzyme. A new method was developed to prepare propargyl penam sulfone. The majority of the triazoles prepared by this route exhibited good activity against all three representative enzymes used for the inhibition assay.

Dipolar cycloaddition of novel 6-(nitrileoxidomethyl) penam sulfone: an efficient route to a new class of beta-lactamase inhibitors.

6-(Nitrileoxidomethyl) penam sulfone intermediate was prepared in a few steps starting from commercially available (+)-6-aminopenicillanic acid. This intermediate underwent smooth 1, 3-dipolar cycloaddition reactions with various alkenes and alkynes to give cycloadducts in moderate to good yields. By this new method, several potent beta-lactamase inhibitors were synthesized. The regio- and stereoselectivity outcomes of the cycloaddition process are also discussed.

Synthesis and Elastase Inhibitory Evaluation of Phosphate Esters and Mixed Phosphate Anhydride of Penam Sulfones

first_page settings Order Article Reprints Font Type: Arial Georgia Verdana Font Size: Aa Aa Aa Line Spacing:    Column Width:    Background: Open AccessArticle Synthesis and Elastase Inhibitory Evaluation of Phosphate Esters and Mixed Phosphate Anhydride of Penam Sulfones by María A. Laborde * and O. A. Mascaretti Instituto de Química Orgánica de Síntesis (IQUIOS-UNR), Casilla de Correo 991, 2000 Rosario, Argentina * Author to whom correspondence should be addressed. Molecules 2000, 5(4), 655-656; https://doi.org/10.3390/50400655 Received: 1 April 2000 / Accepted: 3 April 2000 / Published: 5 April 2000 Download Download PDF Download PDF with Cover Download XML Versions Notes Abstract: The evaluation of human leucocite elastase inhibition by phosphate esters (1-2) and phosphate mixed anhydride (3) of penicillin sulfones and their precursor sulfides is described. Keywords: Elastase Inhibition; Phosphate Esters; Phosphate Anhydride; Penam Sulfones IntroductionHuman Leukocyte Elastase [1] (HLE, EC 3.4.21.37) is a serine proteinase found in the azurophilic granules of polymorphonuclear leukocytes. This enzyme has been the subject of extensive studies, both in terms of its biological role in numerous diseases [2] and in the development of suitable inhibitors to be used as potential therapeutic agents. This interest has led over the past fifteen years to the synthesis of a wide variety of inhibitors based on the β-lactam nucleus. Results and DiscussionWe have recently reported the structure-activity relationship (S.A.R) of several benzyl and methyl 6-α-substituted penicillanate sulfones [3]. In this communication we describe the synthesis and our initial structure-activity relationship study of a series of phosphate triesters (1-2) and phosphatecarboxylate mixed anhydride (3) of penicillin sulfones. These new penicillin derivatives were evaluated as elastase inhibitors using H.L.E. AcknowledgementsUNR, CONICET, Agencia Nacional de Promoción Científica.References and Notesa) Edwards, P.D.; Bernstein, P.R. Med. Res. Rev. 1994, 14, 127. b) Hlasta, D.J.; Pagani, E.D. Ann. Rep. Med. Chem. 1994, 29, 195. c) Durette, P.L.; Chabin, R.M. Bioorg. Med. Chem. Lett. 1995, 5, 271. Weinbaum, G.; Giles, R.E.; Krell, R.D. Ann. N.Y. Acad. Sci. 1991, 624, 1–370. Boschetti, C.E.; Mata, E.G.; Mascaretti, O.A.; Cricco, J.A.; Coux, G.; Roveri, O.A. Bioorg. Med. Chem. Lett. 1995, 5, 2033. b) Boschetti, C.E.; Mascaretti, O.A.; Cricco, J.A.; Roveri, O.A. Bioorg. Med. Chem. 1995, 3, 95. c) Boschetti, C.E.; Mata, E.G.; Mascaretti, O.A.; Cricco, J.A.; Coux, G.; Lodeyro, A.; Roveri, O.A. J. Braz. Chem. Soc. 1996, 7, 285. © 2000 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes. Share and Cite MDPI and ACS Style Laborde, M.A.; Mascaretti, O.A. Synthesis and Elastase Inhibitory Evaluation of Phosphate Esters and Mixed Phosphate Anhydride of Penam Sulfones. Molecules 2000, 5, 655-656. https://doi.org/10.3390/50400655 AMA Style Laborde MA, Mascaretti OA. Synthesis and Elastase Inhibitory Evaluation of Phosphate Esters and Mixed Phosphate Anhydride of Penam Sulfones. Molecules. 2000; 5(4):655-656. https://doi.org/10.3390/50400655 Chicago/Turabian Style Laborde, María A., and O. A. Mascaretti. 2000. "Synthesis and Elastase Inhibitory Evaluation of Phosphate Esters and Mixed Phosphate Anhydride of Penam Sulfones" Molecules 5, no. 4: 655-656. https://doi.org/10.3390/50400655 Find Other Styles Article Metrics No No Article Access Statistics For more information on the journal statistics, click here. Multiple requests from the same IP address are counted as one view.

B-Lactamase inhibitors.

The use of beta-lactamase inhibitors in combination with a beta-lactamase-susceptible antibiotic is a useful strategy to rescue otherwise good antibiotics from failure. However, recent years have seen a rise in the numbers of beta-lactamases that are insensitive to the available beta-lactamase inhibitors. This review summarizes of the mechanisms of action of the principal types of inhibitors and the ways in which beta-lactamase are thought to develop resistance towards them. Ten general classes of inhibitors are reviewed, especially those of therapeutic importance (clavulanic acid, penam sulfones and carbapenems). Copyright 2000 Harcourt Publishers Ltd.

ChemInform Abstract: The Synthesis and Evaluation of 6‐Alkylidene‐2′β‐substituted Penam Sulfones as β‐Lactamase Inhibitors.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Catalytic single-chain antibodies possessing β-lactamase activity selected from a phage displayed combinatorial library using a mechanism-based inhibitor

Catalytic single-chain antibodies (scFvs) possessing β-lactamase activity were selected from a phage displayed combinatorial antibody library using a penam sulfone mechanism-based inhibitor of β-lactamase. The scFvs FT6 and FT12 catalyzed the hydrolysis of ampicillin with rate accelerations ( k cat k uncat ) of 5200 and 320.

ChemInform Abstract: 6‐(1‐Hydroxyalkyl)penam Sulfone Derivatives as Inhibitors of Class A and Class C β‐Lactamases. Part 2.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: 6‐(1‐Hydroxyalkyl)penam Sulfone Derivatives as Inhibitors of Class A and Class C β‐Lactamases. Part 1.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.