부산대학교 도서관

Widespread resistance was already detected when penicillin was first used clinically in 1946 to treat infections brought on by gram-positive bacteria. After the discovery of β-lactamase inhibitors that inhibit class A β-lactamase, many inhibitors have been developed through screening of natural products. As a representative inhibitor, avibactam formerly NXL104) was approved by the FDA as a combination of ceftazidime-avibactam (antibacterial-inhibitor) and sold in 2015. Avibactam is a diazabicyclooctane (DBO, non-lactam class) that has been reported to inhibit class A and C serine β-lactamases. However, inhibitors for metallo-β-lactamase (MBL), a class B carbapenemase, which is the biggest problem due to antimicrobials resistance in clinical practice, have not been developed. Therefore, it was necessary to design a novel inhibitor for the inhibition of class B carbapenemases recognized as the source of multidrug resistance based on this and to establish an optimal inhibitor selection system based on the class B carbapenemase structure. First, tertiary structure-based inhibitor screening of class B carbapenemases (GIM-1, IMP-1, NDM-1, VIM-2, CphA and GOB-18) was performed using ICM-VLS software (Molsoft, LLC) was determined using virtual library screening (VLS). The MolCart compound and building block database (Molsoft, LLC) consists of a library of 10,661,544 compounds, and as a result of docking to 6 targets using this, 50 compounds of the most effective docking scores were selected. Of the 50 compounds, 26 compounds that can be synthesized were synthesized. A checkerboard assay was performed with a combination of 13 antibacterial agents and 26 compounds (a total of 338) targeting 8 types of class B carbapenemase-producing multidrug-resistant strains. 8 compounds having an inhibitory effect (MS-1, MS-2, MS-3, MS-5, MS-6, MS-7, MS-9 and MS-10) were selected. In particular, MS-1, which showed the best effect on class B carbapenemase-producing multidrug-resistant strains in the checkerboard assay result, was named compound 1. In order to increase the inhibitory effect of compound 1, a fragment of OCL-4 (2,4-dichloro- 1-nitrobenzene) was added to compound 1 to synthesize a derivative (named compound 2). In order to increase the outer membrane permeability and inhibitory effect of Gram-negative bacteria, ethylamine was added to compound 2 to synthesize a derivative (named compound 3). LC/MS/MS system was used to confirm bacterial outer membrane permeability. As a result of the experiment, it was confirmed that compound 3 was present in cells at a higher concentration than compound 1, and from these results, it was confirmed that the intracellular permeability of compound 3 was improved. In order to perform the time-kill assay, an optimal combination of novel inhibitors (compound 1, compound 2 and compound 3) and antibacterial agent (meropenem) selected through checkerboard assay was applied. A time-kill assay was performed on four clinical strains [Klebsiella pneumoniae (producing NDM-1), Acinetobacter baumannii (producing IMP-1), Citrobacter freundii (VIM-2), and E. coli (GIM-1)] and two recombinant strains [E. coli TOP10 (CphA) and E. coli TOP10 (GOM-1)]. As a result, the three novel inhibitors (compound 1, compound 2, and compound 3) showed a synergistic effect against the three strains [K. pneumoniae (NDM-1), A. baumannii (IMP-1), and E. coli TOP10 (GOB-1)], and -3.6 ~ -5.6 log10 CFU/ml showed a reduction effect. In the case of a novel inhibitor (compound 3) with improved penetration ability, it was confirmed that there was a bactericidal effect because reduction effects of -3.5 to -5.6 log10 CFU/ml were obtained in all MIC combinations of the time-kill assay.Finally, IC50 analysis of novel inhibitors (compound 1, compound 2, and compound 3) against 6 class B carbapenemase (NDM-1, VIM-2, IMP-1, GIM-1, CphA, and GOB-1) were performed. As a result, compound 3 showed the best inhibitory effect (IC50). In order to predict the inhibitory mechanism of class B carbapenemases, the interaction (docking) analysis of compound 3 and class B carbapenemase in the substrate binding pocket was performed using the structure of the class B carbapenemase, which had the highest inhibitory effect through IC50. In this study, a novel inhibitor selection system was established based on the tertiary structure of class B carbapenemases, and a novel inhibitor (compound 3) with improved inhibitory effect and cell permeability against multidrug-resistant strains producing class B carbapenemases was developed. Through the analysis of the interaction (docking) of compound 3 and class B carbapenemase in the substrate binding pocket, the inhibition mechanism of class B carbapenemase could be predicted.
1946년 페니실린이 그람 양성 박테리아에 의해 유발된 감염을 치료하기 위해 임상적으로 처음 사용되었을 때 광범위한 내성이 이미 감지되었다. 클래스 A β-락타마제를 억제하는 β-락타마제 억제제가 발견된 후 천연물 스크리닝을 통해 많은 억제제가 개발되었다. 대표적인 저해제로 avibactam (formerly NXL104)이 2015년에 ceftazidime-avibactam(항균제-저해제) 조합으로 FDA에 승인되어 판매되고 있으며, Avibactam은 diazabicyclooctane (DBO, non-lactam class)으로 class A, C serine β-lactamase를 저해는 것이 보고되었다. 하지만, 임상에서 항균제 내성으로 가장 큰 문제가 되고 있는 class B carbapenemase인 GIM-type, IMP-type, NDM-type, VIM-type, CphA-type 및 GOB-type에 대한 저해제는 개발이 되어 있지 않았다. 따라서, class B carbapenemase 구조를 기반으로 최적의 저해제 선별 시스템을 구축하고, 이를 기반으로 다제내성의 원인으로 규명된 class B carbapenemase들의 억제를 위한 신규 저해제의 개발이 필요하였다. 첫번째, class B carbapenemases (GIM-1, IMP-1, NDM-1, VIM-2, CphA 및 GOB-18)의 3차 구조 기반의 저해제 스크리닝은 ICM-VLS software (Molsoft, LLC)를 사용한 VLS (virtual library screening)를 이용하여 수행하였다. MolCart compound and building block database (Molsoft, LLC)는 10,661,544 화합물의 라이브러리로 구성되어 있으며 이를 사용하여 6개의 타겟에 도킹한 결과 도킹스코어가 가장 효과적인 50개의 화합물들을 선별하였다. 50개의 화합물 중에 합성 가능한 26개의 화합물을 합성하였다. 8종의 class B carbapenemase 생성 다제내성균을 대상으로 13개의 항균제와 26개의 화합물(총 338개)들의 조합으로 checkerboard assay를 수행하였다. 그 결과 억제 효과가 있는 8개의 화합물들(MS-1, MS-2, MS-3, MS-5, MS-6, MS-7, MS-9 그리고 MS-10)을 선별하였다. 특히, checkerboard assay에서 MBLs에 대하여 가장 좋은 효과를 보인 MS-1을 compound 1로 명명하였다. Compound 1의 저해 효과를 높이기 위해서, Our Chemical Library (OCL)의 화합물 6,600개를 대상으로 checkerboard assay를 수행한 결과로부터 선별된 OCL-4의 단편(2,4-dichloro-1-nitrobenzene)을 compound 1에 추가하여 유도체(compound 2로 명명)를 합성하였다. 그람 음성 세균의 외막 투과력과 저해 효능을 높이기 위해, compound 2에 에틸아민(ethylamine)을 추가하여 유도체(compound 3로 명명)을 합성하였다.두번째로, 세균 외막 투과성을 확인하기 위해서 LC/MS/MS 시스템을 사용하였다. 실험 결과 compound 3이 compound 1 보다 높은 농도로 세포 내에 존재하는 것을 확인하였고, 이러한 결과로부터 compound 3의 세포 내의 투과성이 향상되었음을 확인하였다. Checkerboard assay를 통하여 선별한 신규 저해제(compound 1, compound 2 및 compound 3)들과 항균제(meropenem)의 최적 조합을 적용하여 time-kill assay를 수행하였다. 4종의 임상 균주들[Klebsiella pneumoniae (NDM-1 생산), Acinetobacter baumannii (IMP-1 생산), Citrobacter freundii (VIM-2), E. coli (GIM-1)] 및 2종의 재조합 균주들[E. coli TOP10 (CphA) 및 E. coli TOP10 (GOM-1)]을 사용하여 time-kill assay를 수행하였다. 그 결과 3종의 신규 저해제들(compound 1, compound 2 및 compound 3)은 K. pneumoniae (NDM-1), A. baumannii (IMP-1) 및 E. coli TOP10 (GOB-1)에 대하여 시너지 효과(synergy effect)를 나타냈었고, –3.6 ∼ –5.6 log10CFU/ml의 감소 효과를 나타냈었다. 특히, 침투 능력이 향상된 신규 저해제(compound 3)의 경우 time-kill assay의 모든 MIC 조합에서 –3.5 ∼ –5.6 log10CFU/ml의 감소 효과를 얻었기 때문에 살균 효과(bactericidal effect)가 있는 것을 확인하였다. 마지막으로, 6종의 class B carbapenemase (NDM-1, VIM-2, IMP-1, GIM-1, CphA 및 GOB-1)에 대한 신규 저해제들(compound 1, compound 2 및 compound 3)의 저해 효능(IC50) 분석하였다. 그 결과 compound 3이 가장 좋은 저해 효능(IC50)를 보였다. Class B carbapenemases의 저해 메커니즘을 예측하기 위해서 class B carbapenemase 구조를 이용하여 compound 3과 class B carbapenemase의 substrate binding pocket에서의 상호작용(docking)을 분석하였다.본 연구는 class B carbapenemase들의 3차 구조를 기반으로 신규 저해제를 선별 시스템을 구축 및 class B carbapenemase를 생성하는 다제내성균주들에 대한 저해 효과 및 세포 투과성이 향상된 신규 저해제(compound 3)를 개발하였고, compound 3과 class B carbapenemase의 substrate binding pocket에서의 상호작용(docking) 분석을 통하여 저해 메커니즘을 분석할 수 있었다.