Nanomolar inhibitors of AmpC beta-lactamase

beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and the cephalosporins. In an effort to combat these enzymes, a combination of stereoselective organic synthesis, enzymology, microbiology, and X-ray crystallography was used to design and evaluate new carboxyphenyl-glycylboronic acid transition-state analogue inhibitors of the class C beta-lactamase AmpC. The new compounds improve inhibition by over 2 orders of magnitude compared to analogous glycylboronic acids, with K(i) values as low as 1 nM. On the basis of the differential binding of different analogues, the introduced carboxylate alone contributes about 2.1 kcal/mol in affinity. This carboxylate corresponds to the ubiquitous C3(4)' carboxylate of beta-lactams, and this energy represents the first thermodynamic measurement of the importance of this group in molecular recognition by class C beta-lactamases. The structures of AmpC in complex with two of these inhibitors were determined by X-ray crystallography at 1.72 and 1.83 A resolution. These structures suggest a structural basis for the high affinity of the new compounds and provide templates for further design. The highest affinity inhibitor was 5 orders of magnitude more selective for AmpC than for characteristic serine proteases, such as chymotrypsin. This inhibitor reversed the resistance of clinical pathogens to the third generation cephalosporin ceftazidime; it may serve as a lead compound for drug discovery to combat bacterial resistance to beta-lactam antibiotics.     

Using steric hindrance to design new inhibitors of class C beta-lactamases

beta-lactamases confer resistance to beta-lactam antibiotics such as penicillins and cephalosporins. However, beta-lactams that form an acyl-intermediate with the enzyme but subsequently are hindered from forming a catalytically competent conformation seem to be inhibitors of beta-lactamases. This inhibition may be imparted by specific groups on the ubiquitous R(1) side chain of beta-lactams, such as the 2-amino-4-thiazolyl methoxyimino (ATMO) group common among third-generation cephalosporins. Using steric hindrance of deacylation as a design guide, penicillin and carbacephem substrates were converted into effective beta-lactamase inhibitors and antiresistance antibiotics. To investigate the structural bases of inhibition, the crystal structures of the acyl-adducts of the penicillin substrate amoxicillin and the new analogous inhibitor ATMO-penicillin were determined. ATMO-penicillin binds in a catalytically incompetent conformation resembling that adopted by third-generation cephalosporins, demonstrating the transferability of such sterically hindered groups in inhibitor design.     

Structure-based design and in-parallel synthesis of inhibitors of AmpC beta-lactamase.

BACKGROUND: Group I beta-lactamases are a major cause of antibiotic resistance to beta-lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic beta-lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I beta-lactamase AmpC; this cleft binds the ubiquitous R1 side chain of beta-lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. RESULTS: To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K(i) 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with K(i) values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 A resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. CONCLUSIONS: Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from beta-lactams but nevertheless inhibit the enzyme well. The crystal structure of 2 (K(i) 83 nM) in complex with AmpC may guide exploration of a highly conserved, largely unexplored cleft, providing a template for further design against AmpC beta-lactamase.     

Structure-based optimization of a non-beta-lactam lead results in inhibitors that do not up-regulate beta-lactamase expression in cell culture

Bacterial expression of beta-lactamases is the most widespread resistance mechanism to beta-lactam antibiotics, such as penicillins and cephalosporins. There is a pressing need for novel, non-beta-lactam inhibitors of these enzymes. One previously discovered novel inhibitor of the beta-lactamase AmpC, compound 1, has several favorable properties: it is chemically dissimilar to beta-lactams and is a noncovalent, competitive inhibitor of the enzyme. However, at 26 microM its activity is modest. Using the X-ray structure of the AmpC/1 complex as a template, 14 analogues were designed and synthesized. The most active of these, compound 10, had a K(i) of 1 microM, 26-fold better than the lead. To understand the origins of this improved activity, the structures of AmpC in complex with compound 10 and an analogue, compound 11, were determined by X-ray crystallography to 1.97 and 1.96 A, respectively. Compound 10 was active in cell culture, reversing resistance to the third generation cephalosporin ceftazidime in bacterial pathogens expressing AmpC. In contrast to beta-lactam-based inhibitors clavulanate and cefoxitin, compound 10 did not up-regulate beta-lactamase expression in cell culture but simply inhibited the enzyme expressed by the resistant bacteria. Its escape from this resistance mechanism derives from its dissimilarity to beta-lactam antibiotics.     

Communication between the active site and the allosteric site in class A beta-lactamases

Bacterial production of beta-lactamases, which hydrolyze beta-lactam type antibiotics, is a common antibiotic resistance mechanism. Antibiotic resistance is a high priority intervention area and one strategy to overcome resistance is to administer antibiotics with beta-lactamase inhibitors in the treatment of infectious diseases. Unfortunately, beta-lactamases are evolving at a rapid pace with new inhibitor resistant mutants emerging every day, driving the design and development of novel beta-lactamase inhibitors. Here, we examined the inhibitor recognition mechanism of two common beta-lactamases using molecular dynamics simulations. Binding of beta-lactamase inhibitor protein (BLIP) caused changes in the flexibility of regions away from the binding site. One of these regions was the H10 helix, which was previously identified to form a lid over an allosteric inhibitor binding site. Closer examination of the H10 helix using sequence and structure comparisons with other beta-lactamases revealed the presence of a highly conserved Trp229 residue, which forms a stacking interaction with two conserved proline residues. Molecular dynamics simulations on the Trp229Ala mutants of TEM-1 and SHV-1 resulted in decreased stability in the apo form, possibly due to loss of the stacking interaction as a result of the mutation. The mutant TEM-1 beta-lactamase had higher H10 fluctuations in the presence of BLIP, higher affinity to BLIP and higher cross-correlations with BLIP. Our results suggest that the H10 helix and specifically W229 are important modulators of the allosteric communication between the active site and the allosteric site.     

Molecular dynamics at the root of expansion of function in the M69L inhibitor-resistant TEM beta-lactamase from Escherichia coli

Clavulanate, an inhibitor for beta-lactamases, was the very first inhibitor for an antibiotic resistance enzyme that found clinical utility in 1985. The clinical use of clavulanate and that of sulbactam and tazobactam, which were introduced to the clinic subsequently, has facilitated evolution of a set of beta-lactamases that not only retain their original function as resistance enzymes but also are refractory to inhibition by the inhibitors. This article characterizes the properties of the clinically identified M69L mutant variant of the TEM-1 beta-lactamase from Escherichia coli, an inhibitor-resistant beta-lactamase, and compares it to the wild-type enzyme. The enzyme is as active as the wild-type in turnover of typical beta-lactam antibiotics. Furthermore, many of the parameters for interactions of the inhibitors with the mutant enzyme are largely unaffected. The significant effect of the inhibitor-resistant trait was a relatively modest elevation of the dissociation constant for the formation of the pre-acylation complex. The high-resolution X-ray crystal structure for the M69L mutant variant revealed essentially no alteration of the three-dimensional structure, both for the protein backbone and for the positions of the side chains of the amino acids. It was surmised that the difference in the two enzymes must reside with the dynamic motions of the two proteins. Molecular dynamics simulations of the mutant and wild-type proteins were carried out for 2 ns each. Dynamic cross-correlated maps revealed the collective motions of the two proteins to be very similar, yet the two proteins did not behave identically. Differences in behavior of the two proteins existed in the regions between residues 145-179 and 155-162. Additional calculations revealed that kinetic effects measured experimentally for the dissociation constant for the pre-acylation complex could be mostly attributed to the electrostatic and van der Waals components of the binding free energy. The effects of the mutation on the behavior of the beta-lactamase were subtle, including the differences in the measured dissociation constants that account for the inhibitor-resistant trait. It would appear that nature has selected for incorporation of the most benign alteration in the structure of the wild-type TEM-1 beta-lactamase that is sufficient to give the inhibitor-resistant trait.     

Screening of bacterial genes responsible for resistance to beta-lactam antibiotics using microarrays with enzymatic detection

The method of hybridization analysis on microarrays with enzymatic detection based on horseradish peroxidase is applied to screen infectious agents of nosocomial and community-acquired infections for beta-lactamase genes causing resistance to beta-lactam antibiotics. The advantages of using this method for the rapid identification of genes are demonstrated. Similarities and differences in the distribution of beta-lactamase genes identified in the infectious agents of nosocomial and community-acquired infections are revealed. The most common type of extended-spectrum beta-lactamases (ESBLs) is CTX-M. The high prevalence of extended-spectrum beta-lactamases, particularly of the TEM-1 beta-lactamase, is demonstrated. Individually or in combination with genes of TEM-1 and SVH-1 beta-lactamases, the genes of subgroup CTX-M-1 beta-lactamases were the most frequently identified in community-acquired infectious agents. There were no cases of the simultaneous detection of multiple ESBLs in community-acquired infectious agents. Much more varied combinations of beta-lactamases were identified in nosocomial infectious agents: a combination of extended-spectrum beta-lactamases and broad-spectrum beta-lactamases was identified in 62% of strains and the simultaneous presence of two different types of ESBLs was identified in 18% of strains.     

An ultrahigh resolution structure of TEM-1 beta-lactamase suggests a role for Glu166 as the general base in acylation

Although TEM-1 beta-lactamase is among the best studied enzymes, its acylation mechanism remains controversial. To investigate this problem, the structure of TEM-1 in complex with an acylation transition-state analogue was determined at ultrahigh resolution (0.85 A) by X-ray crystallography. The quality of the data was such as to allow for refinement to an R-factor of 9.1% and an R(free) of 11.2%. In the resulting structure, the electron density features were clear enough to differentiate between single and double bonds in carboxylate groups, to identify multiple conformations that are occupied by residues and loops, and to assign 70% of the protons in the protein. Unexpectedly, even at pH 8.0 where the protein was crystallized, the active site residue Glu166 is clearly protonated. This supports the hypothesis that Glu166 is the general base in the acylation half of the reaction cycle. This structure suggests that Glu166 acts through the catalytic water to activate Ser70 for nucleophilic attack on the beta-lactam ring of the substrate. The hydrolytic mechanism of class A beta-lactamases, such as TEM-1, appears to be symmetrical, as are the serine proteases. Apart from its mechanistic implications, this atomic resolution structure affords an unusually detailed view of the structure, dynamics, and hydrogen-bonding networks of TEM-1, which may be useful for the design of inhibitors against this key antibiotic resistance target.     

Molecular Dynamics Study of the Complex Formation of TEM-Type β-Lactamases with Substrates and Inhibitors

The complex formation of TEM-1 β-lactamase and its three mutant forms TEM-32, TEM-37, and TEM-39 with substrates cephalothin and CENTA and serine beta-lactamase inhibitors sulbactam, tazobactam, and clavulanic acid is studied using the methods of molecular dynamics. It is found that the stability of the complexes is caused by the electrostatic attraction between the deprotonated carboxyl group of the β-lactam ring of the substrate (inhibitor) and the positively charged amino groups of the lysine 234 and 73 residues, located in the active site of the enzymes. The formation of a hydrogen bond between this substrate group or its carbonyl oxygen with the hydroxyl group of the catalytic serine 70 residue and also between the negatively charged substituent groups and the positive charge region formed by the arginine 244 guanidine group and the asparagine 276 amino group is observed for some complexes. The binding energy of CENTA with TEM-1 β-lactamase is below the analogous binding energy of cephalothin, which is confirmed by the values of the Michaelis constants, determined experimentally. It is also found that the inhibitors bind to the mutant forms of β-lactamases related to the inhibitor-resistant phenotype, with higher affinity than TEM-1 β-lactamase.     

Ab initio QM/MM study of class A beta-lactamase acylation: dual participation of Glu166 and Lys73 in a concerted base promotion of Ser70

Beta-lactamase acquisition is the most prevalent basis for Gram-negative bacteria resistance to the beta-lactam antibiotics. The mechanism used by the most common class A Gram-negative beta-lactamases is serine acylation followed by hydrolytic deacylation, destroying the beta-lactam. The ab initio quantum mechanical/molecular mechanical (QM/MM) calculations, augmented by extensive molecular dynamics simulations reported herein, describe the serine acylation mechanism for the class A TEM-1 beta-lactamase with penicillanic acid as substrate. Potential energy surfaces (based on approximately 350 MP2/6-31+G calculations) reveal the proton movements that govern Ser70 tetrahedral formation and then collapse to the acyl-enzyme. A remarkable duality of mechanism for tetrahedral formation is implicated. Following substrate binding, the pathway initiates by a low energy barrier (5 kcal mol(-1)) and an energetically favorable transfer of a proton from Lys73 to Glu166, through the catalytic water molecule and Ser70. This gives unprotonated Lys73 and protonated Glu166. Tetrahedral formation ensues in a concerted general base process, with Lys73 promoting Ser70 addition to the beta-lactam carbonyl. Moreover, the three-dimensional potential energy surface also shows that the previously proposed pathway, involving Glu166 as the general base promoting Ser70 through a conserved water molecule, exists in competition with the Lys73 process. The existence of two routes to the tetrahedral species is fully consistent with experimental data for mutant variants of the TEM beta-lactamase.     

Stereoselective synthesis of optically active bicyclic beta-lactam carboxylic acids that target pilus biogenesis in pathogenic bacteria

Optically active bicyclic beta-lactams were synthesized, starting from 2-H-delta 2-thiazolines and Meldrum's acid derivatives. Several methods to accomplish an ester hydrolysis without damaging the beta-lactam framework were investigated. A rapid CsOH saponification of the beta-lactam methyl esters was developed and protonation of the Cs-carboxylates by Amberlite (IR-120 H+) afforded a series of bicyclic beta-lactam carboxylic acids. Moreover, a convenient method for the synthesis of 2-H-delta 2-thiazolinecarboxylic acid methyl ester 2 was developed. Bicyclic beta-lactam carboxylic acids 7a-g and aldehydes 4a-d were screened for their affinity to the bacterial periplasmic chaperone PapD using a surface plasmon resonance technique. beta-Lactams substituted with large acyl substitutents showed better binding to the chaperone than the native C-terminal peptide PapG 8, demonstrating that bicyclic beta-lactams constitute a new class of potential bacterial chaperone inhibitors.     

Different transition-state structures for the reactions of beta-lactams and analogous beta-sultams with serine beta-lactamases

Beta-sultams are the sulfonyl analogues of beta-lactams, and N-acyl beta-sultams are novel inactivators of the class C beta-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C-O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl beta-lactams are substrates for beta-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation-deacylation process. The rates of acylation of the enzyme by the beta-lactams, measured by the second-order rate constant for hydrolysis, kcat/K(m), and those of sulfonylation by the beta-sultams, measured by the second-order rate constant for inactivation, k(i), both show a similar pH dependence to that exhibited by the beta-lactamase-catalyzed hydrolysis of beta-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl beta-lactams increase the rate of alkaline hydrolysis and give a Bronsted beta(lg) of -0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted beta(lg) for the beta-lactamase-catalyzed hydrolysis of the same substrates is -0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted beta(lg) for the alkaline hydrolysis of N-aroyl beta-sultams is -0.73, similar to that for the beta-lactams, that for the sulfonylation of beta-lactamase by these compounds is -1.46, compatible with significant amide anion expulsion/S-N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the beta-sultam.     

Structure-based design and in-parallel synthesis of inhibitors of AmpC β-lactamase

Background: Group I β-lactamases are a major cause of antibiotic resistance to β-lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic β-lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I β-lactamase AmpC; this cleft binds the ubiquitous R1 side chain of β-lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids.Results: To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K_i 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with K_i values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 Å resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored.Conclusions: Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from β-lactams but nevertheless inhibit the enzyme well. The crystal structure of 2 (K_i 83 nM) in complex with AmpC may guide exploration of a highly conserved, largely unexplored cleft, providing a template for further design against AmpC β-lactamase.     

Structural aspects for evolution of beta-lactamases from penicillin-binding proteins

Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and beta-lactamases, resistance enzymes to beta-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for beta-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7beta-[N-Acetyl-L-alanyl-gamma-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporin bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC beta-lactamase from Escherichia coli was solved at 1.71 A resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the -lactamase active site. Furthermore, insertion of a peptide in the beta-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the beta-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.     

Molecular modeling and chemical reactivity of sanfetrinem and derivatives

The indiscriminate use of beta-lactams has considerably diminished their efficiency as a result of bacteria developing effective defense mechanisms against them. Recent pharmaceutical research has led to the synthesis of tricyclic beta-lactam antibiotics known as "tricyclic carbapenems" or "trinems". In this work, we studied the chemical reactivity, an essential property for antibiotic action, of trinems and found it to be similar to that of cephalosporins. Also, we elucidated the interaction pattern for sanfetrinem and 4beta-methoxy trinem and compared it to that for classical beta-lactams. The behavior of both trinems was found to be similar to that of penicillin G toward Staphylococcus aureus PC1, and that of cephalothin and imipenem toward Enterobacter cloacae P99.     

Reactions of pyrazolylborate-zinc-hydroxide complexes related to beta-lactamase activity

Simple beta-lactams and their hydrolysis products, the beta-amino acids, react with TpZn-OH under deprotonation. The latter become semibidentate carboxylate ligands with a NH...O hydrogen bond, and the former become N-bound beta-lactamide ligands. Likewise the antibiotic derivatives 6-aminopenicillanic acid and 7-aminocephalosporanic acid are incorporated as carboxylate ligands. beta-Lactams bearing nitrophenyl or acyl substituents at the nitrogen atoms are opened hydrolytically by TpZn-OH, and the resulting N-substituted beta-amino acids are attached to zinc by their carboxylate functions. Only with trifluoroacetyl as the N-substituent does the hydrolytic cleavage occur at the external amide bond, yielding the free beta-lactam and TpZn-trifluoroacetate. The kinetic investigation of the opening reactions has shown them to be of second order like all other TpZn-OH-induced hydrolytic cleavages, thereby supporting the four-center mechanism for the monozinc beta-lactamases.     

pKa,MM, and QM studies of mechanisms of beta-lactamases and penicillin-binding proteins: acylation step

The acylation step of the catalytic mechanism of beta-lactamases and penicillin-binding proteins (PBPs) has been studied with various approaches. The methods applied range from molecular dynamics (MD) simulations to multiple titration calculations using the Poisson-Boltzmann approach to quantum mechanical (QM) methods. The mechanism of class A beta-lactamases was investigated in the greatest detail. Most approaches support the critical role of Glu-166 and hydrolytic water in the acylation step of the enzymatic catalysis in class A beta-lactamases. The details of the catalytic mechanism have been revealed by the QM approach, which clearly pointed out the critical role of Glu-166 acting as a general base in the acylation step with preferred substrates. Lys-73 shuffles a proton abstracted by Glu-166 O(epsilon ) to the beta-lactam nitrogen through Ser-130 hydroxyl. This proton is transferred from O(gamma) of the catalytic Ser-70 through the bridging hydrolytic water to Glu-166 O(epsilon ). Then the hydrogen is simultaneously passed through S(N)2 inversion mechanism at Lys-73 N(zeta) to Ser-130 O(gamma), which loses its proton to the beta-lactam nitrogen. The protonation of beta-lactam nitrogen proceeds with an immediate ring opening and collapse of the first tetrahedral species into an acyl-enzyme intermediate. However, the studies that considered the effect of solvation lower the barrier for the pathway, which utilizes Lys-73 as a general base, thus creating a possibility of multiple mechanisms for the acylation step in the class A beta-lactamases. These findings help explain the exceptional efficiency of these enzymes. They emphasize an important role of Glu-166, Lys-73, and Ser-130 for enzymatic catalysis and shed light on details of the acylation step of class A beta-lactamase mechanism. The acylation step for class C beta-lactamases and six classes of PBPs were also considered with continuum solvent models and MD simulations.     

Synthesis and evaluation of ketophosph(on)ates as beta-lactamase inhibitors

A series of amidoketophosph(on)ates of general structure PhCH2OCONHCH(R)COCHR'(CH2)n(O)P(O2-)(O)R' (R = H, CH3; R' = H, CH3; n = 0, 1; R' = H, CH3, Et, Ph) have been prepared as a potential source of beta-lactamase inhibitors. The phosphonates (n = 0) were obtained by means of the Arbuzov reaction while most of the phosphates were achieved from reaction of phosph(or/on)ic acids with the appropriate diazoketone PhCH2OCONHCH(R)COCR'N2. The electrophilicity of the carbonyl group in the resulting phosph(on)ates was assessed by the degree of hydration in aqueous solution, determined from NMR spectra. These compounds inhibited typical class C and class D beta-lactamases, particularly the latter group, but showed no activity against class A enzymes. To enhance the carbonyl electrophilicity, an alpha-difluorinated analogue (R = H, CHR' = CF2, n = 0, R' = Et) was also prepared, but no enhanced inhibitory activity was observed. All evidence suggested that these compounds inhibited in the carbonyl form rather than by formation of tetrahedral adducts at the beta-lactamase active site. They show promise as leads to specific class D beta-lactamase inhibitors.     

Crystal structure of BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, in complex with Enterobacter cloacae 908R beta-lactamase: evidence for a stereoselective mechanism from docking studies

BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, is an active-site-directed inactivator of bacterial beta-lactamases. The crystal structure of Enterobacter cloacae 908R class C beta-lactamase in complex with BRL 42715, docking, and energy minimization studies explain stereoselectivity of the binding of C6-(heterocyclic methylene)penems against class C beta-lactamase.