Penicillin-binding Protein Target Research Articles

Discovery of beta-lactamase CMY-10 inhibitors for combination therapy against multi-drug resistant Enterobacteriaceae.

β-lactam antibiotics are the most widely used antimicrobial agents since the discovery of benzylpenicillin in the 1920s. Unfortunately, these life-saving antibiotics are vulnerable to inactivation by continuously evolving β-lactamase enzymes that are primary resistance determinants in multi-drug resistant pathogens. The current study exploits the strategy of combination therapeutics and aims at identifying novel β-lactamase inhibitors that can inactivate the β-lactamase enzyme of the pathogen while allowing the β-lactam antibiotic to act against its penicillin-binding protein target. Inhibitor discovery applied the Site-Identification by Ligand Competitive Saturation (SILCS) technology to map the functional group requirements of the β-lactamase CMY-10 and generate pharmacophore models of active site. SILCS-MC, Ligand-grid Free Energy (LGFE) analysis and Machine-learning based random-forest (RF) scoring methods were then used to screen and filter a library of 700,000 compounds. From the computational screens 74 compounds were subjected to experimental validation in which β-lactamase activity assay, in vitro susceptibility testing, and Scanning Electron Microscope (SEM) analysis were conducted to explore their antibacterial potential. Eleven compounds were identified as enhancers while 7 compounds were recognized as inhibitors of CMY-10. Of these, compound 11 showed promising activity in β-lactamase activity assay, in vitro susceptibility testing against ATCC strains (E. coli, E. cloacae, E. agglomerans, E. alvei) and MDR clinical isolates (E. cloacae, E. alvei and E. agglomerans), with synergistic assay indicating its potential as a β-lactam enhancer and β-lactamase inhibitor. Structural similarity search against the active compound 11 yielded 28 more compounds. The majority of these compounds also exhibited β-lactamase inhibition potential and antibacterial activity. The non-β-lactam-based β-lactamase inhibitors identified in the current study have the potential to be used in combination therapy with lactam-based antibiotics against MDR clinical isolates that have been found resistant against last-line antibiotics.

Pharmacokinetics and Pharmacodynamics of β-Lactamase Inhibitors.

Novel β-lactamase inhibitors have extended the reach of new and existing β-lactams against multidrug-resistant bacteria expressing β-lactamases. The efficacy of these combination therapeutics relies on a complex two-component pharmacodynamic (PD) system where the β-lactamase inhibitor inactivates the bacterial β-lactamase enzyme and frees the companion β-lactam to act against its penicillin-binding protein target. Despite considerable investigation into the pharmacokinetics (PK) and pharmacodynamics of β-lactams, the pharmacology of their companion β-lactamase inhibitors has only recently been rigorously explored. This review describes the diversity of β-lactamase enzymes, mechanisms of enzyme inhibition, and factors impacting the efficacy of clinically available β-lactamase inhibitors. Relevant PK differences among available inhibitors and the PK/PD properties of these agents are described independently of their companion β-lactams. In the modern era of antibiotic resistance, a comprehensive understanding of the pharmacology, PK, and PD of β-lactamase inhibitors is paramount to maximizing the therapeutic efficacy of existing β-lactam/β-lactamase inhibitor combinations and protecting novel agents in the drug development pipeline.

Sideromimic Modification of Lactivicin Dramatically Increases Potency against Extensively Drug-Resistant Stenotrophomonas maltophilia Clinical Isolates.

Acetamido derivatives of the naturally antibacterial non-β-lactam lactivicin (LTV) have improved activity against their penicillin binding protein targets and reduced hydrolysis by β-lactamases, but penetration into Gram-negative bacteria is still relatively poor. Here we report that modification of the LTV lactone with a catechol-type siderophore increases potency 1,000-fold against Stenotrophomonas maltophilia, a species renowned for its insusceptibility to antimicrobials. The MIC90 of modified lactone compound 17 (LTV17) against a global collection of extensively drug-resistant clinical S. maltophilia isolates was 0.063 μg · ml(-1) Sideromimic modification does not reduce the ability of LTVs to induce production of the L1 and L2 β-lactamases in S. maltophilia and does not reduce the rate at which LTVs are hydrolyzed by L1 or L2. We conclude, therefore, that lactivicin modification with a siderophore known to be preferentially used by S. maltophilia substantially increases penetration via siderophore uptake. LTV17 has the potential to be developed as a novel antimicrobial for treatment of infections by S. maltophilia More generally, our work shows that sideromimic modification in a species-targeted manner might prove useful for the development of narrow-spectrum antimicrobials that have reduced collateral effects.

A peptidoglycan fragment triggers β-lactam resistance in Bacillus licheniformis.

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation.

Reduction of Beta-Lactam Antimicrobial Activity in <i>Staphylococcus aureus</i> Abscesses by Neutrophil Alteration of Penicillin-Binding Protein 2

We previously demonstrated that brief nonkilling neutrophil exposure diminishes the binding affinity of S. aureus penicillin-binding protein (PBP) 2. We sought to investigate further the role of the neutrophil in the alteration of antimicrobial activity and its interaction with PBP-2 by studying the activity of cefotaxime, which highly binds to PBP 2, and cephalexin, which minimally binds to PBP 2. Using S. aureus, cultured in vitro in sterile-filtered normal and neutrophil depleted abscess fluid, we sought to demonstrate an in vivo significance of the neutrophil effect upon the activity of antimicrobials that target PBP-2 by studying the same antimicrobials in an experimental S. aureus abscess. Rats were implanted with perforated tissue cages and infected with S. aureus; some rats were neutrophil depleted by mechlorethamine. Abscess fluids from normal and neutropenic abscesses were harvested, pooled, sterile-filtered and stored for the time-kill studies. Treatment studies were performed by administering either 300 μg/kg/d cefotaxime or cephalexin for 7 days in other rats with 24 hour-old tissue-cage S. aureus abscesses. In time-kill studies, cefotaxime was highly active against stationary phase S. aureus in MHB and in neutropenic abscess fluid, but less active in the non-neutropenic abscess fluid (p 10 kill, p = 0.029 vs. 0.81 ± 2.5, p = NS). These data suggest that neutrophil exposure, which diminishes S. aureus PBP-2 binding affinity [or total quantity], also adversely affects the antimicrobial activity of cefotaxime, which binds to PBP-2, as compared to cephalexin. Altered PBP targets from neutrophil exposure may be a mechanism of antimicrobial resistance within abscesses.

A7.12 The efficacy and safety of tigecycline in the treatment of complicated intra-abdominal infections: Results of pooled clinical trial data

Cefditoren, a broad-spectrum orally administered cephalosporin ester, has documented in vitro efficacy against many Gram-positive and -negative pathogens and stability against clinically important β-lactamases. We have reviewed the microbiology and the pharmacokinetic/pharmacodynamic literature regarding the spectrum and potency of this newer agent against the major etiologic agents of community-acquired respiratory infection, (Streptococcus pneumoniae, Hemophilus influenzae and Moraxella catarrhalis), as well as the Enterobacteriaceae and non-enteric Gram-negative bacilli, staphylococci, and other anerobic and anaerobic Gram-positive cocci. The level of cefditoren activity against S. pneumoniae (MIC90, 0.5 μg/mL) was superior to all marketed oral cephalosporins and at least equal to amoxicillin ± clavulanate. H. influenzae (MIC90, 0.016–0.03 μg/mL) and M. catarrhalis (MIC90, 0.06–0.5 μg/mL) were also very susceptible to cefditoren. In contrast to cefixime and ceftibuten, cefditoren was active against oxacillin-susceptible staphylococci (MIC90, ≤ 1 μg/mL) at a level comparable to cefuroxime axetil, cefaclor or cefprozil. Enterococci, Pseudomonas aeruginosa and most anaerobes (Gram-negative) were not cefditoren-susceptible, but most Enterobacteriaceae, β-haemolytic and viridans group streptococci were highly susceptible. Furthermore, an overview of key in vitro susceptibility testing methods and issues including disk diffusion testing and Etest (AB BIODISK, Solna, Sweden) method accuracy, interpretive criteria, and pharmacodynamic considerations for the selection of a breakpoint concentration are provided. The rapid bactericidal nature of the antibacterial activity of cefditoren, its post antibiotic effect, penicillin binding protein targets, and extent of β-lactamase stability are all favorable qualities. In conclusion, this orally administered (BID) β-lactam possesses promise for use against commonly isolated problematic respiratory tract pathogens such as penicillin-non-susceptible pneumococci and β-lactamase-positive M. catarrhalis or H. influenzae. Success in the clinical trials will further define the role of cefditoren in this era of emerging resistant bacterial pathogens.

Cefditoren in vitro activity and spectrum: a review of international studies using reference methods

Cefditoren, a broad-spectrum orally administered cephalosporin ester, has documented in vitro efficacy against many Gram-positive and -negative pathogens and stability against clinically important β-lactamases. We have reviewed the microbiology and the pharmacokinetic/pharmacodynamic literature regarding the spectrum and potency of this newer agent against the major etiologic agents of community-acquired respiratory infection, ( Streptococcus pneumoniae, Hemophilus influenzae and Moraxella catarrhalis), as well as the Enterobacteriaceae and non-enteric Gram-negative bacilli, staphylococci, and other anerobic and anaerobic Gram-positive cocci. The level of cefditoren activity against S. pneumoniae (MIC 90, 0.5 μg/mL) was superior to all marketed oral cephalosporins and at least equal to amoxicillin ± clavulanate. H. influenzae (MIC 90, 0.016–0.03 μg/mL) and M. catarrhalis (MIC 90, 0.06–0.5 μg/mL) were also very susceptible to cefditoren. In contrast to cefixime and ceftibuten, cefditoren was active against oxacillin-susceptible staphylococci (MIC 90, ≤ 1 μg/mL) at a level comparable to cefuroxime axetil, cefaclor or cefprozil. Enterococci, Pseudomonas aeruginosa and most anaerobes (Gram-negative) were not cefditoren-susceptible, but most Enterobacteriaceae, β-haemolytic and viridans group streptococci were highly susceptible. Furthermore, an overview of key in vitro susceptibility testing methods and issues including disk diffusion testing and Etest (AB BIODISK, Solna, Sweden) method accuracy, interpretive criteria, and pharmacodynamic considerations for the selection of a breakpoint concentration are provided. The rapid bactericidal nature of the antibacterial activity of cefditoren, its post antibiotic effect, penicillin binding protein targets, and extent of β-lactamase stability are all favorable qualities. In conclusion, this orally administered (BID) β-lactam possesses promise for use against commonly isolated problematic respiratory tract pathogens such as penicillin-non-susceptible pneumococci and β-lactamase-positive M. catarrhalis or H. influenzae. Success in the clinical trials will further define the role of cefditoren in this era of emerging resistant bacterial pathogens.

In vitro evaluation of contemporary β-lactam drugs tested against viridans group and β-haemolytic streptococci

Streptococci continue to be prevalent causes of mild as well as of serious, life-threatening infections. Furthermore, some species harbor antimicrobial resistances (pneumococcus, viridans gr. streptococci) that compromise effective chemotherapy with β-lactam drugs or other antimicrobial classes. In this study, the potency and spectrum of 12 β-lactams was assessed versus 274 contemporary isolates of viridans group and β-haemolytic streptococci using reference methods. Cefotaxime and ceftriaxone (MIC 90s, 0.015 to 2 μg/ml, 84 to 100% susceptible) were consistently most potent among the agents tested. Ceftazidime (MIC 90s, 0.25 to 8 μg/ml) and ticarcillin (MIC 90s, 0.5 to >32 μg/ml) were least active among the cephalosporins and penicillins, respectively. When 25% pooled serum was added to the reference test medium, ceftriaxone activity decreased fourfold, and cefotaxime remained highly active. As penicillin/β-lactam-resistant streptococci with altered penicillin-binding protein target sites become more prevalent, only a few “third-generation” cephems seem to have sustained activity when used alone or possibly with a carefully selected co-drug. Routine testing of these species against β-lactams and alternative drugs should be encouraged to detect emerging resistance patterns.

Target for bacteriostatic and bactericidal activities of beta-lactam antibiotics against Escherichia coli resides in different penicillin-binding proteins

The relationship between cell-killing kinetics and penicillin-binding protein (PBP) saturation has been evaluated in the permeability mutant Escherichia coli DC2 in which the antimicrobial activity of beta-lactams has been described as being directly related to the extent of saturation of the PBP target(s). Saturation of a single PBP by cefsulodin (PBP 1s), mecillinam (PBP 2), and aztreonam (PBP 3) resulted in a slow rate of killing (2.5-, 1.5-, and 0.8-log-unit decreases in the number of CFU per milliliter, respectively, in 6 h). Saturation of two of the three essential PBPs resulted in a marked increase in the rate of killing, which reached the maximum value when PBPs 1s and 2 were simultaneously saturated by a combination of cefsulodin and mecillinam (4.7-log-unit decrease in the number of CFU per milliliter in 6 h). Inactivation of all three essential PBPs by the combination of cefsulodin, mecillinam, and aztreonam further increased the killing kinetics (5.5-log-unit decrease in the number of CFU per milliliter), and this was not significantly changed upon additional saturation of the nonessential PBPs 5 and 6 by cefoxitin. Similar relationships between PBP saturation and killing kinetics were obtained with imipenem and meropenem at concentrations which inhibited only one PBP (PBP 2), only two PBPs (PBP 1s and 2), or all three essential PBPs. Saturation of one or more PBPs also resulted in a different rate of bacteriolysis, the highest rate being obtained by the cefsulodin-mecillinam combination and by 5 micrograms of either imipenem or meropenem per ml. All of these conditions caused saturation of PBP 2 and saturation or extensive binding of PBP 1s. However, none of these conditions caused determined the fastest possible rate of killing, which occurred only when all three essential PBPs were saturated. It was concluded that the actual killing effect of beta-lactams is reflected by killing rates that approach the fastest possible rate for the given microorganism and that the targets for the bactericidal activity are precisely those PBPs whose saturation or binding occurs under conditions.

Iron-regulated outer membrane proteins of Escherichia coli K-12 and mechanism of action of catechol-substituted cephalosporins.

Selected aminothiazolyl-oxime cephalosporin congeners substituted at C-3' with a catechol moiety were used to probe the basis of the enhanced antibacterial activity against Escherichia coli K-12 often associated with chemical modifications of this type. Evidence is presented for a tonB-dependent illicit transport of the compounds across the outer membrane of E. coli K-12, the process involving jointly and specifically the Fiu and Cir iron-regulated outer membrane proteins. Thus, both tonB and fiu cir mutants showed a comparably reduced susceptibility to the probe compounds, whereas mutants singularly lacking any one of the six iron-regulated outer membrane proteins (Fiu, FepA, FecA, FhuA, FhuE, and Cir) or lacking any combination of any two of these proteins (except Fiu plus Cir) did not show this resistance. Mutants devoid of all six iron-regulated outer membrane proteins were no more resistant to the probe compounds than fiu cir or tonB strains. In addition to the latter genes, the products of the exbB and possibly the exbC loci were necessary for maximal antibacterial potency. A dependence of antibacterial activity on the level of expression of the uptake system components was noted. Comparison of penicillin-binding protein target affinity with antibacterial activity suggested a possible periplasmic accumulation of active compounds by E. coli K-12. Free vicinal hydroxyl groups of the catechol residue were a primary chemical requirement for recognition by the uptake pathway and thus for high antibacterial activity.

Affinities of penicillins and cephalosporins for the penicillin-binding proteins of Escherichia coli K-12 and their antibacterial activity

The affinities of a range of penicillins and cephalosporins for ther penicillin-binding proteins of Escherichia coli K-12 have been studied, and the results were compared with the antibacterial activity of the compounds against E. coli K-12 and an isogenic permeability mutant. Different penicillins and cephalosporins exhibited different affinities for the "essential" penicillin-binding proteins of E. coli K-12, in a manner which directly correlated with their observed effects upon bacterial morphology. Furthermore, the affinities of the compounds for their "primary" lethal penicillin-binding protein targets showed close agreement with their antibacterial activities against the permeability mutant.