Flexible docking of dihydrofolate reductase and beta-lactamase inhibitors into target enzymes using QXP

Abstract

PART I In this study, an automatic method with computer docking program, QXP (Quick eXPlore) was used for docking novel dihydrofolate reductase inhibitors into target enzymes. Dihydrofolate reductase (DHFR) has been used as a target protein for anticancer, antibacterial and antifungal drugs. From Protein Data Bank (PDB), non-mutated human, Escherichia coli and Candida albicans DHFR X-ray crystal structures were selected. The original ligand was extracted from the ternary complex resulting DHFR-NADP(H) binary complex. The extracted ligand was docked back into the complex to check the reproducibility by docking. The result gave an average ligand RMSD of 0.87 Å with the positions and orientations almost overlapping with each other. By extracting coenzyme of each X-ray crystal structure, DHFR-substrate binary complex was made and extracted coenzyme was docked back into this complex. The average coenzyme RMSD was 0.93 Å with also almost overlapping conformation. These results demonstrated excellent reproducibility of X-ray crystal structure by QXP docking. The derivatives of thiosemicarbazone which are known to inhibit the growth of microorganisms and inhibit bovine DHFR were docked into human, Escherichia coli and Candida albicans DHFR-NADP(H) binary complex, DHFR-substrate binary complex and DHFR alone to study their binding positions and interactions with three species of DHFRs. The three derivatives used were all docked into the substrate site of DHFR-NADP(H) binary complex with reasonable RMSD, energy and interactions. These derivatives were also docked well into the coenzyme site of DHFR-substrate binary complex. When these derivatives were docked into DHFR alone, due to structural differences of different species of DHFR, these ligands were docked into either the coenzyme site or both substrate and coenzyme sites. The results indicate that these derivatives can bind to either the substrate site or the coenzyme site, supporting the experimental data of uncompetitive inhibition kinetics reported for these compounds. The results also show the species differences in binding modes, suggesting the possibility of designing species selective inhibitors of the enzyme. PART II Two inhibitors of β-lactamase with (E)- and (Z)- isomers were docked into the Enterobacter cloacae β-lactamase with computer docking program, QXP. In order to test the compatibility of the program, reproducibility test of the original ligand of X-ray crystal structure was done. The results show the rmsd of 0.84 Å, indicating close overlap of the original ligand with the docked ligand in the active site of the enzyme. The docking results of (E)- and (Z)- isomers of 1,1-Dioxo-6-bromo-6-[(2-bromo, 3-phenyl) allylidene] penicillanic acid (1) showed that more prevalent (Z)-isomer docked better into the active site than the (E)-isomer. They were oriented in the direction of 180°degree with each other. When (E)- and (Z)- isomers of 2β-(3-methyl-1,3-butadaenyl)-2-α-methyl penam 3α carboxylate 1,1-dioxide (2) was studied, both isomers were docked into the enzyme with similar position and orientation. Their rmsd and energy values were also very similar as they showed the same degree of inhibition in enzyme assays. The results seem to demonstrate the correlation between the activity of the isomer nature of interactions formed by docking. This study may provide not only the binding modes of new inhibitors with the target enzymes but also the means to distinguish stereoisomers to select biologically active forms.;part I Dihydrofolate reductase (DHFR)은 dihydrofolate를 tetrahydrofolate로 환원하는 과정에 과여하는 enzyme으로서, DNA 합성에 중요한 역할을 한다. 따라서, 특정 species의 DHFR에 유효한 저해제는 DNA 합성을 저해하며 세포 사멸을 초래함으로서 항암제, 항생제, 항진균제 등으로 개발될 수 있다. 본 연구에서는 human, Escherichia coli 및 Candida albicans의 DHFR X-ray crystal structures를 Protein Data Bank (PDB)로부터 얻었다. Original ligand를 ternary complex으로부터 extract하여 DHFR-NADP(H) binary complex를 얻은 후, 여기에 extract한 original ligand를 다시 이 complex로 docking함으로써 X-ray crystal structure의 재현성을 확인했다. 그 결과, Escherichia coli, human, 및 Candida albicans의 DHFR의 ligand RMSD가 평균 0.87 Å으로 나타났으며 position이나 orientation이 서로 겹쳐졌다. 또한, 각 X-ray crystal structure의 coenzyme을 extract하여 DHFR-substrate binary complex를 얻은 후 여기에 extract한 coenzyme을 다시 이 complex으로 docking 하였다. 그 결과, 세가지 종의 DHFRs의 coenzyme RMSD가 평균 0.93 Å으로 나타났으며 구조가 거의 겹쳐졌다. 이 결과들은 QXP의 X-ray crystal structure 재현성의 탁월함을 입증하였다. 다음으로 microorganisms의 성장을 억제하고 bovine DHFR에 대한 저해 작용이 있다고 보고된 바 있는 thiosemicarbazone 유도체들을 Escherichia coli, human 및 Candida albicans의 DHFR-NADP(H) binary complex, DHFR-coenzyme binary complex 및 DHFR free enzyme에 docking하여 세 가지 종의 DHFRs에 대한 binding position과 interactions를 조사하였다. 그 결과 docking에 사용한 세 가지 유도체들은 모두 DHFR-NADP(H) binary complex의 substrate site에 만족스러운 RMSD와 energy 값 및 interaction을 가짐으로써 안정하게 결합되었다. 이 유도체들은 DHFR-substrate binary complex의 coenzyme site에도 좋은 RMSD와 energy 값 및 interaction으로 안정하게 결합되었다. 이 유도체들을 DHFR alone에 docking하였을 때에는 ligand와 species에 의한 단백질의 구조적인 차이 때문에 이 유도체들은 coenzyme site에 결합하거나 substrate site와 coenzyme site에 걸쳐서 docking되었다. 이러한 결과들은 thiosemicarbazone 유도체들이 substrate site나 coenzyme site에 결합할 수 있다는 것을 제시함으로써 이러한 유도체들의 uncompetitive inhibition kinetics 실험 data를 뒷받침한다. 이 결과들은 또한 binding modes에서의 species differences를 보여줌으로써 단백질의 species selective한 저해제의 design에 대한 가능성을 제시한다. part II Computer docking program QXP를 사용하여 새로운 penam sulfone계 β-lactamase 저해제들의 (E)- and (Z)- isomers를 Enterobacter cloacae β -lactamase 단백질에 docking하는 연구를 진행하였다. Docking하기 이전에, program의 효율성을 확인하기 위해서 X-ray crystal 구조의 original ligand를 docking하는 재현성 실험을 하였다. Rmsd 결과는 0.84 Å였고 original ligand와 docked ligand가 잘 겹쳐졌다. 1,1-Dioxo-6-bromo-6-[(2-bromo, 3-phenyl) allylidene] penicillanic acid (1)의 (E)- and (Z)- isomer들을 docking한 결과, 더 많은 비율로 존재하는 (Z)-isomer가 단백질의 활성 부위에 잘 docking되었으나, 적은 비율로 존재하는 (E)-isomer의 결과는 상대적으로 좋지 않았다. 2β -(3-methyl-1,3-butadaenyl)-2-α-methyl penam 3α carboxylate 1,1-dioxide (2)의 (E)- and (Z)- isomer들을 docking한 결과, 두 isomer들은 실험적으로 비슷한 저해도를 나타내었기 때문에 각각은 유사한 binding mode와 energy 결과들을 나타내었다. 이러한 docking 결과들은 약물의 활성과 docking에 의한 interaction의 상관관계를 제시해 주고 있다. 또한, 이 연구는 docking study가 새로운 저해제들과 target 단백질에 대한 binding modes에 대한 정보를 제공할 뿐만 아니라 biologically active한 stereoisomers를 선택하는데 사용될 수 있음을 제시하였다.