京都大学 / 新制・論文博士 / 博士(工学) / 乙第13581号 / 論工博第4212号 / 新制||工||1990(附属図書館) / (主査)教授 松原 誠二郎, 教授 中尾 佳亮, 教授 浦山 健治 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

The emergence and proliferation of Gram-negative bacteria expressing β-lactamases is a significant threat to human health. β-Lactamases are enzymes that degrade the β-lactam antibiotics (e.g., penicillins, cephalosporins, monobactams, and carbapenems) that we use to treat a diverse range of bacterial infections. Specifically, β-lactamases catalyze a hydrolysis reaction where the β-lactam ring common to all β-lactam antibiotics and responsible for their antibacterial activity, is opened, leaving an inactive drug. There are two groups of β-lactamases: serine enzymes that use an active site serine residue for β-lactam hydrolysis and metalloenzymes that use either one or two zinc ions for catalysis. Serine enzymes are divided into three classes (A, C, and D), while there is only one class of metalloenzymes, class B. Clavulanic acid, sulbactam, and tazobactam are β-lactam-based BLIs that demonstrate activity against class A and C β-lactamases; however, they have no activity against the class A KPC and MBLs, NDM and VIM. Avibactam and vaborbactam are novel BLIs approved in the last two years that have activity against serine carbapenemases (e.g., KPC), but do not inhibit MBLs. The overall goals of this project were to use X-ray crystallography to study the catalytic mechanism of serine β-lactamases with β-lactam antibiotics and to understand the mechanisms behind the broad-spectrum inhibition of class A β-lactamases by avibactam and vaborbactam. This project also set out to find novel inhibitors using molecular docking and FBDD that would simultaneously inactivate serine β-lactamases and MBLs commonly expressed in Gram-negative pathogenic bacteria. The first project involved examining the structural basis for the class A KPC-2 β-lactamase broad-spectrum of activity that includes cephalosporins and carbapenems. Three crystal structures were solved of KPC-2: (1) an apo-structure at 1.15 Å; (2) a complex structure with the hydrolyzed cephalosporin, cefotaxime at 1.45 Å; and (3) a complex structure with the hydrolyzed penem, faropenem at 1.40 Å. These complex structures show how alternative conformations of Ser70 and Lys73 play a role in the product release step. The large and shallow active site of KPC-2 can accommodate a wide variety of β-lactams, including the bulky oxyimino side chain of cefotaxime and also permits the rotation of faropenem’s 6-alpha-1R-hydroxyethyl group to promote carbapenem hydrolysis. Lastly, the complex structures highlight that the catalytic versatility of KPC-2 may expose a potential opportunity for drug discovery. The second project focused on understanding the stability of the BLI, avibactam, against hydrolysis by serine β-lactamases. A 0.83 Å crystal structure of CTX-M-14 bound by avibactam revealed that binding of the inhibitor impedes a critical proton transfer between Glu166 and Lys73. This results in a neutral Glu166 and neutral Lys73. A neutral Glu166 is unable to serve as a general base to activate the catalytic water for the hydrolysis reaction. Overall, this structure suggests that avibactam can influence the protonation state of catalytic residues. The third project centered on vaborbactam, a cyclic boronic acid inhibitor of class A and C β-lactamases, including the serine class A carbapenemase KPC-2. To characterize vaborbactam inhibition, binding kinetic experiments, MIC assays, and mutagenesis studies were performed. A crystal structure of the inhibitor bound to KPC-2 was solved to 1.25 Å. These data revealed that vaborbactam achieves nanomolar potency against KPC-2 due to its covalent and extensive non-covalent interactions with conserved active site residues. Also, a slow off-rate and long drug-target residence time of vaborbactam with KPC-2 strongly correlates with in vitro and in vivo activity. The final project focused on discovering dual action inhibitors targeting serine carbapenemases and MBLs. Performing molecular docking against KPC-2 led to the identification of a compound with a phosphonate-based scaffold. Testing this compound using a nitrocefin assay confirmed that it had micromolar potency against KPC-2. SAR studies were performed on this scaffold, which led to a nanomolar inhibitor against KPC-2. Crystal structures of the inhibitors complexed with KPC-2 revealed interactions with active site residues such as Trp105, Ser130, Thr235, and Thr237, which are all important in ligand binding and catalysis. Interestingly, the phosphonate inhibitors that displayed activity against KPC-2, also displayed activity against the MBLs NDM-1 and VIM-2. Crystal structures of the inhibitors complexed with NDM-1 and VIM-2 showed that the phosphonate group displaces a catalytic hydroxide ion located between the two zinc ions in the active site. Additionally, the compounds form extensive hydrophobic interactions that contribute to their activity against NDM-1 and VIM-2. MIC assays were performed on select inhibitors against clinical isolates of Gram-negative bacteria expressing KPC-2, NDM-1, and VIM-2. One phosphonate inhibitor was able to reduce the MIC of the carbapenem, imipenem 64-fold against a K. pneumoniae strain producing KPC-2. The same phosphonate inhibitor also reduced the MIC of imipenem 4-fold against an E. coli strain producing NDM-1. Unfortunately, no cell-based activity was observed for any of the phosphonate inhibitors when tested against a P. aeruginosa strain producing VIM-2. Ultimately, this project demonstrated the feasibility of developing cross-class BLIs using molecular docking, FBDD, and SAR studies.

Antibiotic resistance is one of the most serious medical problems in the world. To counteract the increase in antibiotic resistance, new rapid and effective analytical methods are needed. To effectively treat infections in critically ill patients, optimal antibiotic dosages are required. DrugLog® is an instrument that uses a spectrophotometric method to analyze antibiotics in plasma in the wavelength range 200-800 nm. The aim of this study was to do a method validation of the instrument DrugLog®.     The study material that was used was whole blood from healthy donor and routine citrate plasma samples from the laboratory. The precision of the method and stability of plasma, the best way to filtrate lipids from plasma and four antibiotics (meropenem, cefotaxime, vancomycin, piperacillin/tazobactam) were investigated.     The precision of the method, measured as CV% was less than 0.62 and stability plasma showed a CV% of 135.74 after 24 h in room temperature. The stability for the different antibiotics after 24 h in room temperature showed a CV% of 8.11 for meropenem, 40.80 for vancomycin, 16.55 for cefotaxime and 2.92 for the combination antibiotic piperacillin/tazobactam. It was also determined that bacterial filter was the best way to remove lipids from plasma.     In conclusion DrugLog® is a suitable instrument to analyze concentration of antibiotics in patients during antibiotic treatment, however further validations are needed.

Pseudomonas aeruginosa, a notorious opportunistic pathogen, is a leading cause of hospital-acquired infections. The newest generation of β-lactam antibiotics, the carbapenems, are often used to treat multi-drug resistant (MDR) P. aeruginosa infections. Treatment of P. aeruginosa has become increasingly difficult due to its remarkable ability to resist antibiotics through various intrinsic and acquired mechanisms. Physicians rely on a limited group of antibiotics to treat these infections, but many P. aeruginosa isolates are evolving to become resistant to all available antibiotics, including carbapenems. The multifaceted mechanisms underlying P. aeruginosa antibiotic resistance include β -lactamases, efflux pumps, altered membrane porins, and antibiotic binding site mutations of the penicillin binding proteins. There is an urgent need for continued research to better understand the resistance mechanisms used by P. aeruginosa, in order to develop novel therapeutic strategies. The purpose of this project was to investigate the effect of β-lactam antibiotics used in combination with the known efflux pump inhibitor mefloquine, on the growth of MDR P. aeruginosa. The effect of the combination of mefloquine andβ-lactams was investigated in vitro using the checkerboard method. In vitro assays showed that mefloquine when combined with certain β-lactam antibiotics produced no significant additional inhibition than the β-lactams antibiotics alone on MDR P. aeruginosa. Mefloquine, in combination with various β-lactams, did not restore clinically relevant sensitivity, even in those isolates where resistance is thought to be mediated by efflux pumps.

Infectious diseases cause substantial morbidity and mortality, exacerbated by increasing antibiotic resistance. In critically ill patients, recent studies indicate a substantial variability in β-lactam antibiotic levels when standardized dosing is applied. New methods for characterizing nosocomial outbreaks of bacterial infections are needed to limit transmission. The goals of this thesis were to investigate new strategies towards individualized treatment and control of bacterial infections.  In Paper I we confirmed high variability in β-lactam antibiotic levels among intensive care unit (ICU) patients from southeastern Sweden, where 45 % failed to reach treatment targets (100 % fT>MIC). Augmented renal clearance and establishing the minimum inhibitory concentration of the bacteria were important for evaluating the risk of not attaining adequate drug levels. In Paper II a rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of 11 commonly used antibiotics was developed and tested in clinical samples. Performance goals (CV<15%) were reached. A microbiological method for quantification of β-lactam antibiotics in serum was developed in Paper III. The method could be important for hospitals without access to an LC-MS method. Paper IV and Paper V investigated ligation-mediated qPCR with high resolution melt analysis (LMqPCR HRMA), for transmission investigation of extended spectrum β-lactamase (ESBL)-producing E. coli and other common bacterial pathogens. Results comparable to the reference method (PFGE) could be achieved within one day in a closed system and confirmed a nosocomial outbreak in Kalmar County. In Paper VI whole genome sequencing followed by bioinformatic analysis resolved transmission links within a nosocomial outbreak due to improved discriminatory power compared to LMqPCR HRMA. The high proportion of ICU patients with insufficient β-lactam drug levels emphasizes the need for individualized treatment by therapeutic drug monitoring (TDM). TDM is enabled by a highly sensitive method, such as UPLC-MS/MS, but if unavailable, also by a microbial method. Molecular typing methods used for transmission investigation can detect nosocomial outbreaks. LMqPCR HRMA can be used for screening purposes. For enhanced resolution, whole genome sequencing should be used, but always together with a rigorous epidemiological investigation.

Different catalytic approaches have been applied to new -lactams preparations. olefin metathesis has been successfully performed with 3--methylidene--lactams. It was verified that 3--methylidene--lactams olefin metathesis is applicable to preparation of biologically active -lactam of Ezetimibe-type.

The Turos laboratory has been working with N-thiolated β-lactams for years trying to understand the mode of action and structural features it needs to have biological activity. Over the years new data has shown promising inhibitory activity against various microbes. In this dissertation, a review of the vast amount of work carried out on N-thiolated β-lactams in Turos laboratory has been done and their novelty, in terms of structure and mechanism has been discussed. A complete outline of our work in the discovery and ongoing development of these compounds, starting from our initial, unexpected finding of antimicrobial activity for one of the lead compounds, to a more complete understanding of their chemical and biological mode of action and potential utility as antibacterial compounds, has been provided. Previous researches by graduate students in the Turos laboratory have shown that N-thiolated β-lactams targets Type II Fatty Acid Synthesis (FAS). In process of understanding this further, other FAS inhibiting antibiotics like Triclosan were compared to our lactams by adding excess of exogenous fatty acids. Results revealed vast differences in the MIC value of triclosan and N-thiolated β-lactams, giving an idea that there might be a different mode of action or a different target altogether. The third chapter discusses the study of attaching hydrophilic C3 side chains like amino acids and carbohydrates on N-thiolated β-lactams while studying the influence of microbiological activity. From the study it was found that the lengthening of the side chain halts the inhibitory activity regardless of whether the side chain contains unsaturation or branching. Results showed that polar groups were not well tolerated and the inhibitory activity goes down regardless of polarity. Finally, research on dual-action antibiotics was discussed. Antibiotics cause continuous bacterial resistance and in this aspect use of two drugs with different mode of action can call for reduction of the resistance. Herein, N-acyl ciprofloxacin and N-thiolated β-lactams were connected together via an ester linkage. Six new hybrid compounds have been synthesized successfully and tested against E. faecium, K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae.

Pyrrolo [2,1-c] [1,4] benzodiazepine (PBD) is a class of natural products obtained from various actinomycetes which have both anti-tumor and antibiotic activities and can bind to specific sequences of DNA. PBD-dilactam was initially produced using isatoic anhydride and (L)-proline which was then converted to the PBD-thiolactam using Lawesson's reagent. Reaction of thiolactam with hydrazine in ethanol afforded PBD-11-hydrazinyl. Condensation of 11-hydrazinyl PBD with aldehydes possessing various substitutions was performed to obtain (S,E)-11-[2-(arylmethylene) hydrazono] pyrrolo [2,1-c] [1,4] benzodiazepine derivatives. 1HNMR, 13C-NMR, DEPT, IR, GC-MS and X-ray crystallography were used for the characterization. Inhibition activity of the products were carried out using TEM-1, AmpC and P99 β-lactamases. A minimal inhibition growth of 25% was observed for one of the selected PBDs on cancer cell line. A promising result was observed on preliminary cannabinoid binding activity test on one of the compounds.

<p>Antibiotic treatment causes an ecological disturbance on the human microflora. Four commensal bacteria: E. coli, enterococci, a-streptococci and coagulase-negative staphylococci, from patients with extensive, high antibiotic usage were investigated with regard to resistance pattern and mutation frequency. Among 193 investigated strains it was found that high antibiotic usage selected for resistant bacteria and enriched for bacteria with a small but significantly increased mutation frequency. </p><p>The relative biological fitness cost of resistance in <i>Staphylococcus epidermidis</i> was assessed in a human in vivo model where the indigenous flora was present. In vitro data of the bacterial growth rate correlated well to in vivo fitness assayed in the competition experiments on skin. </p><p>An in vitro kinetic model was shown to be a useful tool to establish the pharmacokinetic and pharmacodynamic (PK/PD) indices for efficacy of antibiotics. It was confirmed that the time, when the concentration exceeds the minimal inhibitory concentration (MIC), correlates with efficacy for b-lactam antibiotics. To achieve maximal killing for penicillin-resistant pneumococci, with an MIC of 2 mg/L, the peak concentration was also of importance. </p><p>Suboptimal dosing regimen facilitates selection of resistance. Penicillin-resistant pneumococci were easily selected in a mixed population with penicillin-sensitive, -intermediate and -resistant pneumococci in an in vitro kinetic model. The selection of the resistant strain was prevented when the benzylpenicillin concentration exceeded the MIC for approximately 50% of 24 h.</p>

<p>Toxic products, such as endotoxin from the gram-negative and exotoxin from the gram-positive bacteria, are the most important initiators of the inflammatory host response in sepsis. In addition to antibacterial treatment, numerous attempts have been made to interfere with the exaggerated proinflammatory cascade initiated by the toxins. As most antitoxic and anti-inflammatory agents have shown no clear efficacy, an attractive alternative has been to prevent or minimise their release. Therefore, it was of interest to further study the antibiotic-induced release of toxins after exposure to antibiotics used for the treatment of the most severe infections, especially if protein synthesis inhibitors could reduce the release induced by PBP 3-specific β-lactam antibiotics.</p><p>There were significant reductions in endotoxin release from gram-negative bacteria when the combination of the PBP 3-specific β-lactam antibiotic, cefuroxime, and the protein synthesis inhibitor, tobramycin, was compared with cefuroxime alone. Increasing doses of tobramycin reduced endotoxin release and increased the killing rate. In a kinetic <i>in vitro</i> model the endotoxin release from <i>E.coli</i> was higher after the second dose of cefuroxime. Nevertheless, it was reduced after addition of tobramycin.</p><p>No binding of tobramycin to endotoxin was observed, either <i>in vivo</i> or <i>in vitro</i>. In a porcine sepsis model, a possible anti-inflammatory effect of ceftazidime and tobramycin, expressed as late cytokine inhibition, was seen.</p><p>The protein synthesis inhibitor, clindamycin, released less streptococcal pyrogenic exotoxin A (SpeA) from a group A streptococcus strain than penicillin, and addition of clindamycin to penicillin resulted in less toxin production than penicillin alone. The SpeA production was dependent on the bacterial number at the start of treatment. Higher doses of penicillin also led to less SpeA. </p><p>The choice of antibiotic class and dose may be important in the severely ill septic patient in whom an additional toxin release could be deleterious. A combination of a β-lactam antibiotic and a protein synthesis inhibitor seems beneficial but further investigations are needed.</p>