6-(1-Hydroxyalkyl))penam sulfone derivatives as inhibitors of class A and class C beta-lactamases II

Two stereoselective processes for the synthesis of novel 3,6-disubstituted penam sulfone derivatives were developed. One 6beta-(1-hydroxyethyl) and four 6beta-hydroxymethyl penam sulfone derivatives were synthesized. All four 6beta-(hydroxymethyl)penam sulfone derivatives demonstrated good IC50 against both TEM-1 and AmpC beta-lactamases. Of these, 6beta-hydroxymethyl penam sulfone derivative 25 was the most active inhibitor which was able to restore the activity of piperacillin in vitro and in vivo against both TEM-1 and AmpC beta-lactamases producing organisms.     

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

The chemical synthesis of a series of new penam sulfone derivatives bearing a 2beta-substituted-oxyimino and -hydrazone substituents, their beta-lactamase inhibitory properties against selected enzymes representing class A and C beta-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 2beta-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.     

Penicillin-derived inhibitors that simultaneously target both metallo- and serine-beta-lactamases

The synthesis and beta-lactamase inhibitory activity of four 6-(mercaptomethyl)penicillinates and the four corresponding 6-(hydroxymethyl)penicillinates are described. These penicillins include both C6 stereoisomers as well as the sulfide and sulfone oxidation states of the penam thiazolidine sulfur. All compounds were evaluated as inhibitors of representative metallo- and serine-beta-lactamases enzymes. Selected (mercaptomethyl)penicillinates are shown to inactivate both metallo- and serine-beta-lactamases and to display synergism with piperacillin against beta-lactamase producing strains.     

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

Several alkenyl derivatives were prepared using allyl penam sulfone as the key intermediate. Isomers of these derivatives having beta configuration at C-6 showed potent activity 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.     

6-Acylamino-penam derivatives: synthesis and inhibition of cathepsins B,L, K,and S

The synthesis of a new series of 6-acylamino penam derivatives and their inhibition of cysteine proteases cathepsins B, L, K, and S is described. The 6-acylamino-penam sulfone compounds showed excellent cathepsin L, K, and S inhibition activity with IC(50) values in the nanomolar and subnanomolar range.     

Properties of a class C beta-lactamase from Serratia marcescens.

A beta-lactamase produced by a penicillin-resistant strain of Serratia marcescens was isolated and purified. The kcat. value for benzylpenicillin was about 5% of that observed for the best cephalosporin substrates. However, the low Km of the penam resulted in a high catalytic efficiency (kcat./Km) and the classification of the enzyme as a cephalosporinase might not be completely justified. It also exhibited a low but measurable activity against cefotaxime, cefuroxime, cefoxitin and moxalactam. Substrate-induced inactivation was observed both with a very good (cephalothin) or a very bad (moxalactam) substrate. The active site was labelled by beta-iodopenicillanate. Trypsin digestion produced a 19-residue active-site peptide whose sequence clearly allowed the classification of the enzyme as a class C beta-lactamase.     

Efficient inhibition of class A and class D beta-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.     

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

The synthesis of 6,6-dibromo-3alpha-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3a), 6alpha-chloro-3alpha-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3b), benzyl 6alpha-(diphenyl-phosphate)oxypenicillanate sulfone (4) and 6,6-dibromo-3alpha-(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.     

Evaluation of penicillin-based inhibitors of the class A and B beta-lactamases from Bacillus anthracis

Bacillus anthracis contains a class A (Bla1) and class B (Bla2) beta-lactamase, which confer resistance to beta-lactam antibiotics when expressed in Escherichia coli. In an effort to find new beta-lactamase inhibitors, several penicillin derivatives have been evaluated including experimental compounds incorporating a 6-mercaptomethyl group or a 6-pyridylmethylidene group, along with clavulanate and tazobactam, as inhibitors against Bla1 and Bla2. The 6-mercaptomethyl-substituted penicillins showed much greater activity against the zinc-containing Bla2 than Bla1. The compound that incorporated a 6-pyridylmethylidene substituent and a catecholic substituent at the 2' position was the most effective inhibitor of Bla1 with Ki=0.057 microM. Inhibitors containing iron-chelating functional groups have previously been shown to work in combination with antibiotics to inhibit growth of antibiotic-resistant bacteria expressing beta-lactamase. The development of similar compounds, incorporating these types of substituents, may help overcome resistance to currently used antibiotics.     

Novel Penicillin Analogues as Potential Antimicrobial Agents; Design,Synthesis and Docking Studies

A number of penicillin derivatives (4a-h) were synthesized by the condensation of 6-amino penicillinic acid (6-APA) with non-steroidal anti-inflammatory drugs as antimicrobial agents. In silico docking study of these analogues was performed against Penicillin Binding Protein (PDBID 1CEF) using AutoDock Tools 1.5.6 in order to investigate the antimicrobial data on structural basis. Penicillin binding proteins function as either transpeptidases or carboxypeptidases and in few cases demonstrate transglycosylase activity in bacteria. The excellent antibacterial potential was depicted by compounds 4c and 4e against Escherichia coli, Staphylococcus epidermidus and Staphylococcus aureus compared to the standard amoxicillin. The most potent penicillin derivative 4e exhibited same activity as standard amoxicillin against S. aureus. In the enzyme inhibitory assay the compound 4e inhibited E. coli MurC with an IC₅₀ value of 12.5 μM. The docking scores of these compounds 4c and 4e also verified their greater antibacterial potential. The results verified the importance of side chain functionalities along with the presence of central penam nucleus. The binding affinities calculated from docking results expressed in the form of binding energies ranges from -7.8 to -9.2kcal/mol. The carboxylic group of penam nucleus in all these compounds is responsible for strong binding with receptor protein with the bond length ranges from 3.4 to 4.4 Ǻ. The results of present work ratify that derivatives 4c and 4e may serve as a structural template for the design and development of potent antimicrobial agents.     

Naphthazarin derivatives (II): formation of glutathione conjugate, inhibition of DNA topoisomerase-I and cytotoxicity.

6-(1-Hydroxyalkyl)-5,8-dimethoxy-1,4-naphthoquinones, expressing a higher reactivity in conjugation with glutathione, showed a greater potency in the inhibition of DNA topoisomerase-I and the cytotoxicity against L1210 cells than 2-(1-hydroxyalkyl)-DMNQ derivatives, implying the participation of electrophilic arylation in the bioactivities. In further study 6-(1-Hydroxyalkyl)-5,8-dimethoxy-1,4-naphthoquinones with an alkyl group of shorter chain length (C2-C6) exerted a greater bioactivities than those with longer chain length(>C6).     

Inhibition of class C beta-lactamases: structure of a reaction intermediate with a cephem sulfone

The crystallographic structure of the Enterobacter cloacae GC1 extended-spectrum class C beta-lactamase, inhibited by a new 7-alkylidenecephalosporin sulfone, has been determined by X-ray diffraction at 100 K to a resolution of 1.6 A. The crystal structure was solved by molecular replacement using the unliganded structure [Crichlow et al. (1999) Biochemistry 38, 10256-10261] and refined to a crystallographic R-factor equal to 0.183 (R(free) 0.208). Cryoquenching of the reaction of the sulfone with the enzyme produced an intermediate that is covalently bound via Ser64. After acylation of the beta-lactam ring, the dihydrothiazine dioxide ring opened with departure of the sulfinate. Nucleophilic attack of a side chain pyridine nitrogen atom on the C6 atom of the resultant imine yielded a bicyclic aromatic system which helps to stabilize the acyl enzyme to hydrolysis. A structural assist to this resonance stabilization is the positioning of the anionic sulfinate group between the probable catalytic base (Tyr150) and the acyl ester bond so as to block the approach of a potentially deacylating water molecule. Comparison of the liganded and unliganded protein structures showed that a major movement (up to 7 A) and refolding of part of the Omega-loop (215-224) accompanies the binding of the inhibitor. This conformational flexibility in the Omega-loop may form the basis of an extended-spectrum activity of class C beta-lactamases against modern cephalosporins.     

Strategic design of an effective beta-lactamase inhibitor: LN-1-255, a 6-alkylidene-2'-substituted penicillin sulfone

In an effort to devise strategies for overcoming bacterial beta-lactamases, we studied LN-1-255, a 6-alkylidene-2'-substituted penicillin sulfone inhibitor. By possessing a catecholic functionality that resembles a natural bacterial siderophore, LN-1-255 is unique among beta-lactamase inhibitors. LN-1-255 combined with piperacillin was more potent against Escherichia coli DH10B strains bearing bla(SHV) extended-spectrum and inhibitor-resistant beta-lactamases than an equivalent amount of tazobactam and piperacillin. In addition, LN-1-255 significantly enhanced the activity of ceftazidime and cefpirome against extended-spectrum cephalosporin and Sme-1 containing carbapenem-resistant clinical strains. LN-1-255 inhibited SHV-1 and SHV-2 beta-lactamases with nm affinity (K(I) = 110 +/- 10 and 100 +/- 10 nm, respectively). When LN-1-255 inactivated SHV beta-lactamases, a single intermediate was detected by mass spectrometry. The crystal structure of LN-1-255 in complex with SHV-1 was determined at 1.55A resolution. Interestingly, this novel inhibitor forms a bicyclic aromatic intermediate with its carbonyl oxygen pointing out of the oxyanion hole and forming hydrogen bonds with Lys-234 and Ser-130 in the active site. Electron density for the "tail" of LN-1-255 is less ordered and modeled in two conformations. Both conformations have the LN-1-255 carboxyl group interacting with Arg-244, yet the remaining tails of the two conformations diverge. The observed presence of the bicyclic aromatic intermediate with its carbonyl oxygen positioned outside of the oxyanion hole provides a rationale for the stability of this inhibitory intermediate. The 2'-substituted penicillin sulfone, LN-1-255, is proving to be an important lead compound for novel beta-lactamase inhibitor design.     

Inhibition of alpha-glucosidase by oleanolic acid and its synthetic derivatives

Oleanolic acid (1) and five synthetic derivatives (2-6) were tested spectrophotometrically for inhibition of urease, beta-lactamase, acetyl cholinesterase and alpha-glucosidase. All products showed a positive response only against alpha-glucosidase but not against the other enzymes; IC(50) calculations showed that the dihydroxy-olide derivative (4) was the most potent among all tested samples.     

6-(Methoxymethylene)penicillanic acid: inactivator of RTEM beta-lactamase from Escherichia coli

The Z and E isomers of 6-(methoxymethylene)-penicillanic acid have been synthesized, and their interaction with the RTEM beta-lactamase has been studied. The Z isomer is an inhibitor and an inactivator of the enzyme, and there is some similarity between its behavior and that of other mechanism-based inactivators such as clavulanic acid and the penam sulfones. Kinetic analysis of the interaction of the enzyme with the Z isomer has allowed a detailed evaluation of the factors that are important in the design of anti-beta-lactamase agents. In contrast to the Z compound, the E isomer of 6-(methoxymethylene)penicillanic acid is not a substrate, an inhibitor, or an inactivator of the enzyme.     

Inhibition of class A and class C beta-lactamases by penems: crystallographic structures of a novel 1,4-thiazepine intermediate

A new beta-lactamase inhibitor, a methylidene penem having a 5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazine heterocyclic substituent at the C6 position with a Z configuration, irreversibly inhibits both class A and class C serine beta-lactamases with IC(50) values of 0.4 and 9.0 nM for TEM-1 and SHV-1 (class A), respectively, and 4.8 nM in AmpC (class C) beta-lactamases. The compound also inhibits irreversibly the class C extended-spectrum GC1 beta-lactamase (IC(50) = 6.2 nM). High-resolution crystallographic structures of a reaction intermediate of (5R)-(6Z)-6-(5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazin-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-3-carboxylic acid 1 with the SHV-1 beta-lactamase and with the GC1 beta-lactamase have been determined by X-ray diffraction to resolutions of 1.10 and 1.38 A, respectively. The two complexes were refined to crystallographic R-factors (R(free)) of 0.141 (0.186) and 0.138 (0.202), respectively. Cryoquenching of the reaction of 1 with each beta-lactamase crystal produced a common, covalently bound intermediate. After acylation of the serine, a nucleophilic attack by the departing thiolate on the C6' atom yielded a novel seven-membered 1,4-thiazepine ring having R stereochemistry at the new C7 moiety. The orientation of this ring in each complex differs by a 180 degrees rotation about the bond to the acylated serine. The acyl ester bond is stabilized to hydrolysis through resonance stabilization with the dihydrothiazepine ring and by low occupancy or disorder of hydrolytic water molecules. In the class A complex, the buried water molecule on the alpha-face of the ester bond appears to be loosely bound or absent. In the class C complex, a water molecule on the beta-face is disordered and poorly activated for hydrolysis. Here, the acyl intermediate is unable to assist its own hydrolysis, as is thought to occur with many class C substrates.     

Thiophene-degrading Escherichia coli mutants possess sulfone oxidase activity and show altered resistance to sulfur-containing antibiotics.

We have previously isolated mutants of Escherichia coli which show increased oxidation of heterocyclic furan and thiophene substrates. We have now found that strains carrying the thdA mutation express a novel enzyme activity which oxidizes a variety of substrates containing a sulfone (SO2) moiety. Both heterocyclic sulfones (e.g., tetramethylene sulfone) and simple aliphatic sulfones (e.g., ethyl sulfone) were oxidized. The thdA mutants were more resistant than wild-type strains to aromatic sulfone antibiotics such as dapsone. In contrast they showed increased susceptibility to thiolutin, a cyclic antibiotic containing sulfur at the sulfide level of oxidation. Several new thdA mutant alleles were isolated by selecting for increased oxidation of various aliphatic sulfur compounds. These new thdA mutants showed similar sulfone oxidase activity and the same map location (at 10.7 min) as the original thdA1 mutation. The constitutive fadR mutation was required for the phenotypic expression of thdA-mediated oxidation of sulfur compounds. However, the thdA-directed expression of sulfone oxidase activity was not fadR dependent. The thdC and thdD mutations probably protect against the toxicity of thiophene derivatives rather than conferring improved metabolic capability.     

Thiophene-degrading Escherichia coli mutants possess sulfone oxidase activity and show altered resistance to sulfur-containing antibiotics

We have previously isolated mutants of Escherichia coli which show increased oxidation of heterocyclic furan and thiophene substrates. We have now found that strains carrying the thdA mutation express a novel enzyme activity which oxidizes a variety of substrates containing a sulfone (SO2) moiety. Both heterocyclic sulfones (e.g., tetramethylene sulfone) and simple aliphatic sulfones (e.g., ethyl sulfone) were oxidized. The thdA mutants were more resistant than wild-type strains to aromatic sulfone antibiotics such as dapsone. In contrast they showed increased susceptibility to thiolutin, a cyclic antibiotic containing sulfur at the sulfide level of oxidation. Several new thdA mutant alleles were isolated by selecting for increased oxidation of various aliphatic sulfur compounds. These new thdA mutants showed similar sulfone oxidase activity and the same map location (at 10.7 min) as the original thdA1 mutation. The constitutive fadR mutation was required for the phenotypic expression of thdA-mediated oxidation of sulfur compounds. However, the thdA-directed expression of sulfone oxidase activity was not fadR dependent. The thdC and thdD mutations probably protect against the toxicity of thiophene derivatives rather than conferring improved metabolic capability.     

Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.     

Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

AIMS: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the beta-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. METHODS AND RESULTS: Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and beta-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 microg ml(-1) and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 microg ml(-1). The highest concentration that gave permissible growth rate was 50 microg ml(-1). The isolated survivals were screened on the bases of PGA and beta-lactamase activities. Among the retained mutants, the occurrence of beta-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). CONCLUSIONS: In seven of the mutants, PGA activity was enhanced with considerable decrease in beta-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of beta-lactamase activity and twofold increase in PGA activity [12.7 mg 6-amino-penicillanic acid (6-APA) h(-1) mg(-1) wet cells] compared with that in the wild-type strain (6.3 mg 6-APA h(-1) mg(-1) wet cells). SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of beta-lactamase. These mutants could be used for industrial production of PGA.