초분자 단백질 복합체 및 NMR 분광법을 이용한 리간드 스크리닝 방법 개발

엄윤진 세종대학교 대학원 2020 국내석사

신약 개발 시 필수적인 후보 물질의 탐색에 주로 이용되는 리간드 스크리닝 방법은 타겟 단백질과 리간드를 하나씩 섞어 리간드의 신호 변화를 관찰한다. 본 연구를 통해 제안하는 방법은 2차원 NMR을 이용하여 다수의 후보 물질과 타겟 단백질을 한 번에 스크리닝하며, ELP를 이용하여 기존보다 민감하게 신호 변화를 관찰하고 이를 통해 타겟 단백질과 결합하는 리 간드를 찾는다. 이와 같은 새로운 리간드 스크리닝 방법 개발을 목표로 한다. 본 연구를 위해 사용한 ELP는 VPGXG의 5 가지 아미노산이 반복되는 서열을 가지며 온도나 염 농도에 따라 가역적인 불용성 상태로 상전이가 유도된다. 이와 같은 성질을 이용하여 ELP를 타겟 단백질에 융합하여 퓨전 단백질 상태에서도 가역적인 상전이가 일어나도록 하였다. 또한 타겟 단백질와 ELP가 결합하여 초분자 복합체가 만들어지면 작은 신호 변화라도 그 세기를 증폭시켜 기존의 방법보다 신호 변화가 민감하게 관찰되었다. 타겟 단백질과 리간드가 약하게 결합하는 경우에는 실험 온도가 ELP의 전이 온도 이상이 되면 타겟 단백질에 결합된 리간드의 텀블링 속도가 매우 느려지면서 리간드의 신호가 빠른 시간 내에 사라졌다. 타겟 단백질과 리간드가 강하게 결합하는 경우에도 pull-down 형태의 과정이 일어나 그에 상응하는 신호 변화가 스펙트럼에서 관찰되었다. 타겟 단백질이 생물체 내에서 특정 역할을 담당하기 때문에 생체 내 특정 물질과 결합하므로 후보 물질은 대장균의 대사물질을 추출하여 이용하였다. 정상 배지에는 영양요소가 많이 함유되어 있어 대사 물질 추출을 위한 대장균을 배양할 때는 M9 최소 배지를 사용하였다. M9 최소 배지의 탄소원으로 C-13 글루코오스를 사용하여 대사 물질을 C-13으로 치환 후 추출하여 사용하였다. 여러 종류의 ELP를 타겟 유전자 뒤에 합성하여 융합 단백질을 발현하였고 말토오스와 결합한다고 알려진 MBP를 테스트 타겟 단백질로 설정하였다. 최종적으로 MBP-I48을 다양한 리간드와 함께 1차원 NMR을 측정하였고, 타겟 단백질과 리간드의 결합 여부에 따른 스펙트럼의 신호 변화를 확인하였다. 타겟 단백질과 리간드가 결합할 경우, 온도를 전이 온도 이상으로 올리면 단백질의 신호와 리간드의 신호가 같이 줄어들었다. 반면에 타겟 단백질과 리간드가 결합하지 않았을 경우, 단백질의 신호만 줄어들고 리간드의 신호에는 변화가 없는 것을 확인하였다. 1차원 NMR의 결과를 통해 본 연구에서 제시하는 리간드 스크리닝 방법의 정당성을 확인하였고, 이를 2차원 NMR에 응용하여 다수의 후보 물질 풀에서 타겟 단백질과 리간드의 결합을 확인하는 데 적용하였다. The ligand screening method, which is mainly used for screening candidates, an essential part of drug development, observes the signal change of ligand by mixing target protein and ligand one by one. In this study, the proposed method screened a large number of candidates and target proteins at one time using two-dimensional NMR and observes the signal change of ligand more sensitively using the ELPs. It was aimed at developing such a new ligand screening method. The ELP used for this study had a repeat sequence of the five amino acids of VPGXG, which induces phase transition into a reversible insoluble state depending on temperature or salt concentration. Using this property, the ELPs were fused to a target protein to allow reversible phase transition even in a fusion protein state. In addition, the target protein and ELP combined to form a supermolecular complex, so even small signal variations can amplify their intensity to observe signal changes more sensitively than conventional methods. In the case where the target protein and the ligand are weakly bound, when the experimental temperature is higher than the transition temperature of the ELPs, the tumbling speed of the ligand bound to the target protein was slowed, and the signal of the ligand was disappeared in a short time. Also when the target protein and ligand are strongly bonded, a process in the form of a pull-down was occured and corresponding signal changes were observed in the spectrum. Since the target protein plays a specific role in the organism, it would bind to a specific substance in vivo. So metabolites extracted from E. coli was used as a candidate material pool. As the normal medium contains a lot of nutrients, M9 minimal medium was used when culturing E. coli for metabolite extraction. The metabolites were replaced with C-13 using C-13 glucose as the carbon source of M9 minimal medium, followed by extraction. Various kinds of ELPs were synthesized behind the target gene and the fusion proteins were expressed. MBP, known to bind maltose, was set as the test target protein. Finally, one-dimensional NMR was measured by binding the MBP-I48 with various ligands. Depending on whether they bonded or not, it was able to determine the signal variation in the spectrum. After binding the protein and ligand, the temperature was set up above the transition temperature, and as a result, it showed different outcomes. When the target protein and ligand are bound, if raising the temperature above the transition temperature reduced the signal of the protein and the ligand. On the other hand, when they did not bind on each other, only the signal of protein was reduced and the signal of ligand remained as same. The results of one-dimensional NMR confirmed the validity of the ligand screening method presented in this study. By applying this method to two-dimensional NMR, it enabled the experiment to identify the binding of target proteins and ligands in a number of candidate material pools.

Ligand Supported Homology Modeling of Chemokine Receptor and Its Application to Structure-Based Discovery

김종훈 연세대학교 대학원 2012 국내박사

Homology modeling is the most successful technique predicting the three-dimensional protein structure. However, the conventional homology modeling for G-protein coupled receptors (GPCRs) is very challenging because of several reasons such as limited number of the solved crystal structures (templates), lower sequence identity between the target and the template, and diversity of binding site according to ligands. To obtain a binding site model of GPCR, ligand supported homology modeling is strongly recommended. We developed a new ligand supported homology modeling protocol in which the ligand was incorporated throughout the modeling process and the binding site selection by consensus scoring of docking scores was employed. We have developed the protocol from the application to Chemokine Receptor 2 (CCR2), a GPCR and crucial target for various inflammatory and autoimmune diseases. The structure based ligands design for many GPCRs, including CCR2, is restricted by the lack of an experimental three dimensional structure. In addition, there is substantial diversity for the ligand binding pocket and binding modes among GPCRs. Thus the receptor-ligand binding mode predictions should be derived from homology modeling with supported ligand information. We modeled the binding of our proprietary CCR2 antagonist using ligand supported homology modeling followed by consensus scoring the docking evaluation based on all modeled binding sites. The protein-ligand model was then validated by visual inspection of receptor-ligand interaction for consistency of published site-directed mutagenesis data and virtual screening a decoy compound database. This model was able to successfully identify active compounds within the decoy database. Finally, additional hit compounds were identified through a docking-based virtual screening of a commercial database, followed by a biological assay to validate CCR2 inhibitory activity. Thus, this procedure can be employed to screen large database of compounds to identify new CCR2 antagonists. The protocol developed from CCR2 and our proprietary antagonists was extendedly applied and verified to known CCR2 antagonist, CC chemokine receptor (CCR) 1 antagonist, and CXC chemokine receptor (CXCR) 4 antagonist. The principal features of our ligand supported homology modeling protocol are the incorporation of ligand throughout the homology modeling, the implementation of flexible docking by rather simple energy minimization, the concise selection of best binding site model by consensus scoring and the validation of enrichment by small scale virtual screening.

Identification of Interaction Sites of Ginsenoside and Flavonoid in Ligand-gated Ion Channels

이병환 건국대학교 대학원 2007 국내박사

ABSTRACTTreatment of ginsenosides, major active ingredients of Panax ginseng, and quercetin, one of flavonoids, produce a variety of physiological effects in central and peripheral nervous systems. Recent reports showed that ginsenosides and quercetin inhibit various types of ligand-gated ion channel activity. This study is to identify the interacting site of ginsenoside and quercetin in ligand-gated ion channel in a Xenopus lavies oocyte gene expression system. We expressed various ligand-gated ion channels (human and mouse 5-HT3A receptor, nicotinic acetylcholine receptor α3β4, cGMP-gated ion channel, glycine α1 receptor) by intraoocyte injection of cRNAs. We also used the various mutants for identification of interaction site of ginsenoside (PART Ⅰ) and quercetion (PART Ⅱ) on ligand-gated ion channels. PART ⅠIn the present study, we investigated the action side of ginsenoside Rg3 or M4, a ginsenoside metabolite, in the regulation of ligand-gated ion channel activity using Xenopus oocyte gene expression system. The ligand-gated ion currents were measured using two-electrode voltage clamp techniques. Extracellular treatment but not intacellular injection of ginsenoside Rg3 or M4 inhibited 5-HT3A and α3β4 nACh receptor-mediated ion currents. In oocytes expressing gustatory cGMP-gated ion channel, which is known to have cGMP binding site at intracellular side of plasma membrane and is only activated by cytosolic cGMP, extracellular treatment/excised outside-out patch clamp but not cytosolic injection/excised inside-out patch clamp of ginsenoside Rg3 inhibited cGMP-gated ion currents. (Section Ⅰ)In oocytes expressing wild-type 5-HT3A receptors, Rg3 dose-dependently inhibited peak I5-HT with an IC50 of 27.6 ± 4.3 μM. Mutations V291A, F292A and I295A in TM2 greatly attenuated or abolished the Rg3-induced inhibition of peak I5-HT. Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg3 accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg3 and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC50 values of constitutively active ion currents in V291A mutant receptor were 72.4 ? 23.1 and 6.5 ± 0.7 μM for Rg3 and TMB-8, respectively. Also TMB-8 and diltiazem did not prevent Rg3-induced inhibition of constitutively active ion currents in occlusion experiments. (Section Ⅱ)Rg3 exist as stereoisomers depending on the position of the hydroxyl group on carbon-20; i.e. 20(R)-Rg3 and 20(S)-Rg3 are epimers. In the present study, we further investigated the effects of 20(R)-Rg3 and 20(S)-Rg3 on mouse 5-HT3A receptor channel activity after site-directed mutations of 5-HT3A receptor facilitation site, which is located at pre-transmembrane domain I (pre-TM1). In wild-type, both 20(R)-Rg3 and 20(S)-Rg3 inhibited I5-HT with concentration-dependent and reversible manner. Induction of 5-HT3A receptor facilitation by point mutations of pre-TM1 amino acid residue R222 to R222A, R222D, R222E or R222T not only decreased EC50 values for I5-HT compared to wild-type but also abolished 20(R)-Rg3-induced inhibition of I5-HT. Those mutations also shifted the IC50 values by 20(S)-Rg3 into right direction by 2- to 4-folds compared with wild-type. (Section Ⅲ)PART ⅡIn the present study, we investigated the effect of quercetin on mouse 5-HT3A receptor channel activity, which is involved in pain transmission, analgesia, vomiting, and mood disorders in nervous system. 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured using two-electrode voltage clamp technique. Treatment of quercetin itself had no effect on the oocytes injected with H2O as well as on the oocytes injected with 5-HT3A receptor cRNA. In the oocytes injected with 5-HT3A receptor cRNA, co-treatment of quercetin with 5-HT inhibited 5-HT-induced inward peak current (I5-HT) with dose-dependent and reversible manner. The half-inhibitory concentrations (IC50) of quercetin was 64.7 ± 2.2 ?M. The inhibitions of I5-HT by quercetin were competitive and voltage-independent. Point mutations of pre-transmembraine domain 1 (pre-TM1) such as R222T and R222A but not R222D, R222E and R222K abolished quercetin-induced inhibition of I5-HT, indicating that quercetin interacts pre-TM1 of 5-HT3A receptor. (Section Ⅰ)We investigated the effect of quercetin on the human glycine α1 receptor channel expressed in Xenopus oocytes using a two-electrode voltage clamp technique. Application of quercetin reversibly inhibited glycine-induced current (IGly). Quercetin’s inhibition depends on its dose, with an IC50 of 21.5 ± 0.2 ?M. The inhibition was sensitive to membrane voltages. Site-directed mutations of S267 to S267Y but not S267A, S267F, S267G, S267K, S267L and S267T at transmembrane domain 2 (TM2) nearly abolished quercetin-induced inhibition of IGly. In contrast, in site-directed mutant receptors such as S267 to S267I, S267R and S267V, quercetin enhanced IGly compared to the wild-type receptor. The EC50 was 22.6 ± 1.4, 25.5 ± 4.2, and 14.5 ± 3.1 ?M for S267I, S267R and S267V, respectively. (Section Ⅱ)Conclusively, these results indicate that ginsenoside Rg3 and M4 regulate ligand-gated ion channel activity at extracellular side. Furthermore, Rg3 inhibits 5-HT3A receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2. Rg3 regulates 5-HT3A receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem. 5-HT3A receptor facilitation differentially affects 20(R)-Rg3- and 20(S)-Rg3-mediated 5-HT3A receptor channel regulation. (PART Ⅰ)Quercetin might regulate 5-HT3A receptor channel activity via interaction with N-terminal domain. It is shown that this regulation of 5-HT3A receptor channel activity by quercetin might be one of pharmacological actions of flavonoids. And quercet 초 록Panax ginseng의 주요 성분인 진세노사이드와 플라보노이드의 한 종류인 쿼세틴은 중추신경계와 말초신경계에서 여러 가지 다양한 생리학적인 효과를 나타낸다. 최근의 보고에서 진세노사이드와 쿼세틴은 여러 가지 유형의 ligand-gated ion channel의 활동상태를 억제한다고 보여주었다.이 연구의 목적은 제노퍼스 개구리 알에서 발현시킨 ligand-gated ion channel 에서 진세노사이드와 쿼세틴이 작용하는 자리를 동정하는 것이고 그 자리를 동정하기 위해 이 논문에서는 여러가지 ligand-Gated Ion Channels (human and mouse 5-HT3A, Nicotinic Acetylcholine α3β4, CNG, glycine α1)들을 cRNA의 개구리 알에 주입하여 발현시켰다. 그리고 ligand-gated ion channel들에서 진세노사이드 (PART Ⅰ)와 플라보노이드 (PART Ⅱ)의 상호작용 자리를 동정을 위해 이들 ion channel의 여러 가지 돌연변이체를 이용하였다.PART ⅠPart I에서는 제노퍼스 개구리알 유전자 발현 시스템을 이용하여 ligand-gated ion channel에 대한 진세노사이드 Rg3와 진세노사이드의 대사산물인 M4의 작용자리를 연구하였다. Ligand에 의해 매개된 이온 흐름은 two-electrode voltage clamp 기술을 이용하여 측정하였다. 세포외에서 Rg3와 M4의 처리는 5-HT3A와 α3β4 nACh 수용체를 통한 이온 흐름을 억제하였지만 세포내에서 처리는 억제하지 못하였다. 또한 세포내 원형질막의 안쪽에 cGMP 결합자리를 가지고 있고 세포질내 cGMP에 의해서 활성화되는 gustatory cGMP-gated ion channel를 발현시킨 개구리알에서 outside-out patch clamp 법을 이용하여 세포 바깥에서 Rg3를 처리하면 cGMP에 의한 이온흐름을 억제하였으나 그와 반대로 inside-out patch clamp 법을 이용한 세포내 Rg3의 주입은 그러하지 못했다. (Section Ⅰ)5-HT3A 수용체를 발현시킨 개구리 알에서 Rg3는 5-HT에 의해 유발된 이온 흐름(I5-HT)을 27.6 ± 4.3 μM의 IC50값을 가지며 농도의존적으로 억제하였다. Transmembrane2(TM2)에서 V291A, F292A, I295A의 돌연변이체들에서는 Rg3에 의한 I5-HT의 억제가 크게 약화되거나 소멸되었다. V291A 돌연변이체에서는 F292A와 I295A와는 확연히 다른 current의 감쇠율이 감소되고 자발적으로 활성된 ion current를 일으켰다. Rg3는 5-HT 존재하에서 current 감쇠율의 속도를 농도의존적으로 가속화시켰다. Rg3 와 오픈 채널 차단제인 TMB-8은 자발적으로 활성화된 ion current를 농도의존적으로 억제하였다. V291A 돌연변이 수용체에서 자발적으로 활성화된 ion current를 억제하는 Rg3와 TMB-8의 IC50값은 72.4 ? 23.1과 6.5 ± 0.7 μM이었다. 또한 TMB-8 와 diltiazem은 폐색 실험에서 Rg3에 의해 자발적으로 활성화된 ion current의 억제를 방해하지는 못했다. (Section Ⅱ)Rg3는 20번 탄소에 수산기의 위치에 따른 입체이성질체가 존재한다. 다시 말하면 20(R)-Rg3와 20(S)-Rg3는 서로 epimer이다. 이 연구에서 pre-transmembrane domain I (pre-TM1)에 존재하는 5-HT3A 수용체 반응을 촉진하는 돌연변이체를 만든 후에 20(R)-Rg3와 20(S)-Rg3가 5-HT3A 수용체 통로에서의 효과를 조사하였다. Wild-type 수용체에서 20(R)-Rg3와 20(S)-Rg3는 농도의존적이고 가역적인 방식으로 I5-HT를 억제하였다. pre-TM1에 존재하는 아미노산인 R222(Arginine)을 R222A, R222D, R222E 또는 R22T로 변화시키면 이들 돌연변이체에 의한 5-HT3A 수용체의 반응 촉진 정도는 wild-type과 비교하여 I5-HT에 필요한 EC50값이 감소하였을 뿐만 아니라 20(R)-Rg3에 의한 억제효과도 소멸 되었다. 결국 이 돌연변이체들은 wild-type과 비교하면 20(S)-Rg3의 IC50값을 오른쪽 방향으로 2-4배 이동시켰다. (Section Ⅲ)PART Ⅱ이 연구에서 신경계에서 통증 매개, 통각상실, 구토와 감정 장애에 관여하는 mouse 5-HT3A 수용체에 대한 쿼세틴의 효과를 조사하였다. 5-HT3A 수용체는 제노퍼스 개구리 알에 발현시켰고 current는 two-electrode voltage clamp 방법을 이용하여 측정하였다. H2O를 주입한 대조구와 5-HT3A 수용체 cRNA를 주입한 알에서 쿼세틴 그 자체의 처리는 효과가 없었지만 5-HT와 함께 쿼세틴을 처리하면 5-HT에 의해 유발되는 내향성 전류 흐름(I5-HT)을 농도의존적이고 가역적인 방식으로 억제하였다. 쿼세틴의 IC50농도는 64.7 ± 2.2 μM이었다. 쿼세틴에 의한 I5-HT의 억제는 경쟁적이고 전압 비의존적이었다. pre-TM1에 위치한 R222를 R222T, R222A의 돌연변이체들은 쿼세틴이 유발하는 I5-HT의 억제를 소멸시켰으나 R222D, R222E, R222K는 그러하지 못하였고 이 사실은 쿼세틴이 5-HT3A 수용체의 pre-TM1과 상호작용함을 나타낸다. (Section Ⅰ)이 연구에서는 제노퍼스 개구리 알에 발현된 glycine α1 수용체에 대한 쿼세틴의 효과를 two-electrode voltage clamp법을 이용하여 조사하였다. 쿼세틴의 처리는 glycine에 의해 유발되는 전류 흐름(IGly)을 가역적으로 억제하였다. 쿼세틴의 억제 효과는 농도에 의존적이었고 IC50값은 21.5 ± 0.2 ?M이며 막 전압에 민감하였다. TM2에 위치한 S267의 돌연변이체인 S267Y에서는 쿼세틴에 의한 IGly.의 억제 효과가 거의 없어졌으나 S267A, S267F, S267G, S267K, S267L, S267T에서는 그렇지 않았다. 이와는 대조적으로 S267의 돌연변이인 S267I, S267R, S267V를 wild-type과 비교하면 쿼세틴은 glycine에 의해 유발되는 전류 흐름(IGly)을 더 증가시켰다. S267I, S267R, S267V에 대한 EC50는 각각 22.6 ± 1.4, 25.5 ± 4.2, 14.5 ± 3.1 ?M이었다. (Section Ⅱ)결론적으로 이러한 결과들은

살아있는 세포의 세포막에서 단일 분자의 확산을 통한 리간드와 수용체의 결합 분석 : Analyzing Interactions between Ligand and Receptor Using Single-Molecule Diffusivity in a Living Cell Membrane

김도현 포항공과대학교 일반대학원 2015 국내박사

Ligand-receptor interactions are the fundamental biochemical reactions that generate cellular functions. The investigation of membrane-protein interactions with ligands has been challenging, despite of their biological and therapeutic importance, because the integrity of a membrane protein is highly dependent on a lipid membrane; the biochemical properties of their interactions identified using purified membrane proteins have not often appeared under real physiological conditions. Although recent attempts to analyze these interactions utilizing membrane proteins embedded in lipid vesicles have expanded my understanding of the membrane-protein interactions, these methods still cannot reflect the genuine properties produced only in an intact plasma membrane of a live cell. I develop a novel method that analyze interactions between membrane and soluble proteins in an intact living cell membrane utilizing single particle tracking with super-resolution microscopy. My method (termed single-molecule diffusional mobility shift assay, smDIMSA) overcomes the fundamental limitations of current methods for the analysis of ligand-receptor interactions by providing unprecedented biochemical information on the ligand-receptor interactions occurred in the interface between the solution and crowded cell membrane at the single-molecule population level. smDIMSA is developed based on the novel finding on membrane protein’s diffusivity in a living cell membrane. It has been believed, over 40 years, that the effect of the water-soluble regions of a membrane protein on its diffusivity is negligible due to the orders of magnitude higher viscosity of a plasma membrane than aqueous solution surrounding a cell. This inherent property of membrane protein’s diffusivity fundamentally restrains the diffusion-based imaging assays from measuring the interactions involving water-soluble regions of membrane proteins. However, I unexpectedly observed that the diffusion-coefficient of EGFR decreased up to 40% by the direct binding of its antibody. Because the detection limit of the diffusion-coefficient changes in a living cell membrane using the current methods including FRAP and FCS is typically larger than 400%, this phenomenon can be detected only by single-molecule population analysis that is capable to probe less than 5% of diffusion-coefficient changes. This remarkable finding was extensively validated with various membrane proteins including ErbB receptor family (receptor tyrosine kinases) and adrenergic receptor family (G-protein coupled receptors) with different soluble ligands. Analysis of the ligand-receptor interactions using smDIMSA is extraordinary because it provides novel features that traditional methods, such as using radioactive-isotope labeled ligand, surface plasmon resonance, and co-immunoprecipitation, cannot provide. First, smDIMSA is ligand-label free, eliminating the difficulties associated with ligand labeling and enabling its application to a broad range of ligand concentrations or types. Second, smDIMSA is sensitive to the size of binding partners, generating an unprecedented level of information that can be used to reduce the complexity of a pool of interacting proteins; it can unravel the multiple steps of complex formation on a plasma membrane. Third, it provides quantitative biochemical information to assess binding constants, interaction modes, and kinetics, which is critical to understand the detailed mechanisms of biomolecular reactions. Fmyth, compared with popular label-free assays utilizing SPR, which requires labor-intensive membrane protein purification and expensive gold surfaces, smDISMA directly utilize a single cell seeded on an inexpensive glass surface, greatly reducing both the experimental labor and cost. Last but not least, smDIMSA provides genuine interaction information on a living cell membrane by significantly reducing the false-positives and -negatives derived from chemical purification of membrane proteins for in vitro assays. An immediate biochemical application of smDIMSA was further demonstrated. I revealed, for the first time, the positive cooperativity of cetuximab binding to EGFR L858R on a living cell membrane. Furthermore, based on information about the binding cooperativity of various antibodies targeting different epitopes of the extracellular domain of EGFR L858R, I unraveled the conformation of the extracellular domain of dimeric EGFR L858R, which was controversial in previous studies. By measuring interaction cooperativity and deducing reaction mechanism, my results provide a novel biochemical insight on how dimeric EGFR L858R can be constitutively active without natural ligands on a cell membrane. In conclusion, I developed a novel method that analyze interactions between membrane and soluble proteins in an intact living cell membrane utilizing single particle tracking with super-resolution microscopy. My method overcomes the fundamental limitations of current methods for the analysis of ligand-receptor interactions by providing unprecedented biochemical information on the ligand-receptor interactions occurred in the interface between the solution and crowded cell membrane at the single-molecule population level, which allows detecting a new biochemical aspect of ligand-receptor interactions that appeared only in a crowded living cell membrane, not a solution or a vesicle, as shown in this study. 리간드-수용체간 상호작용은 세포 안과 밖의 신호전달을 매개함으로써 생명유지에 필수적인 역할을 하는 것뿐만이 아니라, 수많은 질병의 원인 인자로서 현재 개발된 50% 이상의 신약들이 리간드-수용체간 상호작용을 대상으로 하고 있다. 이러한 중요성에도 불구하고, 세포막 위에서 일어나고 있는 리간드-수용체간 상호작용에 대한 이해가 많이 부족한 상황이다. 이는 전체 유전자의 15% 이상 되는 다양한 단백질들이 세포막 위에서 매우 높은 농도로 존재함으로써 나타나는 복잡성과 친수성 및 소수성을 모두 가지는 세포막의 생화학적 특이성이 만들어 내는 특수한 환경 때문인데, 현재까지는 기술적인 제약으로 인해 리간드-수용체 간의 상호작용의 연구가 대부분 시험관 수준에서 이루어졌지만 실제 세포막 위에서 나타나는 이들 상호작용의 본질적인 성질을 밝혀내는데 큰 한계가 있었다. 하지만 본 연구에서 최초로 살아있는 세포의 세포막 위에서 리간드-수용체간 상호작용을 연구할 수 있는 기술을 개발함으로써 그 한계를 근본적으로 극복하였다. 이 기술의 핵심은 세포막 위에서 끊임없이 움직이는 수용체가 리간드와 결합하였을 때 단일 분자 수준에서 수용체의 움직임이 둔화된다는 원리에 있다. 40여 년간 세포막 단백질 유동의 표준모델로서 받아들여져 왔던 샤프만-델브룩 (Saffman-Delbrück) 모델에 따르면 세포막의 높은 점성도를 가지는 지질 특성으로 인해 수용성인 리간드가 수용체에 결합하더라도 수용체의 움직임이 변화하지 않는다고 믿어져 왔다. 그러나 본인은 최근에 급속도로 발전하고 있는 초해상도 현미경과 단일 분자 영상 기술들은 응용하여 기존에 측정이 불가능했던 단일 분자 수준에서의 세포막 수용체의 움직임을 직접 관찰하였고, 우연히 리간드가 수용체에 결합하였을 때 그 움직임이 변화한다는 결과를 얻어내었다. 세포막 단백질 유동에 대한 새로운 원리를 토대로 개발된 기술인 smDIMSA (single-molecule DIffusional Mobility Shift Assay, 본 논문에서 명명함)는 리간드-비표지적 방법으로 수용체에 결합하는 리간드의 크기를 정량적으로 인식하기 때문에 대상 수용체에 대한 신약 리간드의 후보 선별에 매우 적합하며, 기존의 방법들과 비교하였을 때 혁명적인 장점들을 가지고 있다. 현재 상용화되어 주로 사용되고 있는 표면 플라즈몬 공명 (Surface Plasmon Resonance)을 이용한 방법은 복잡하고 힘들게 정제한 대상 단백질을 값비싼 금표면에 부착시켜 리간드와의 결합을 측정하게 된다. 특히, 수용체의 경우 높은 세포막 의존도로 인해 정제가 어려워 그 측정이 더욱 힘들었다. 그러나 smDIMSA 기술은 살아있는 세포에서 리간드-수용체간 상호작용을 직접 측정함으로써 시험관 실험에서 오는 양성 및 음성 오류들을 현저히 줄일 수 있고, 까다로운 세포막 단백질 정제 과정을 완전히 없애고, 비용적인 측면에서 기존대비 수천 배 이상 절약할 수 있다.

Development of a protein-ligand docking program based on global optimization

신웅희 서울대학교 대학원 2014 국내박사

Protein-ligand docking has become an essential tool for computer-aided drug discovery since docking programs were first developed in 1980’s. The goals of docking are to predict 1) the binding mode and 2) the binding affinity of a given protein-ligand complex accurately. Accurate prediction of binding mode requires appropriate sampling of both protein and ligand conformations. Many available docking programs sample ligand structures successfully because ligand has a relatively small number of degrees of freedom. However, a lot of current docking programs treat receptor as a rigid molecule although receptor often adapts its shape to bound ligand because treating receptor flexibility is a very complicated problem. First of all, the large conformational space of receptor is a challenge for typical sampling methods. In addition, current energy functions such as empirical docking score functions or force field-based energy functions do not accurately describe flexible receptor-flexible ligand interactions yet. In this thesis, the development process of an efficient docking program that treats receptor flexible, called GalaxyDock, is described. A powerful global optimization technique, called conformational space annealing, was employed for simultaneous sampling of the conformational space of protein and ligand. In addition, a new energy function for flexible-receptor docking was designed by combining the AutoDock energy function and a knowledge-based ROTA potential. With these components for sampling and scoring, GalaxyDock shows high performances in the binding pose prediction and virtual screening benchmark tests when compared to other state-of-art docking programs. This result suggests that the GalaxyDock program can provide a firm basis for further method developments and for practical applications to in sillico drug discovery processes.

Synthesis, structure, and photoluminescence properties of mercury(II) complexes containing tetradentate ligand with pyridine and hydroxy groups

조민지 경북대학교 교육대학원 2019 국내석사

d오비탈에 전자가 모두 점유된 수은(II) 금속과 한 개의 피리딘기와 두 개의 피리딘기를 가지는 네 자리 리간드를 각각 합성하여 배위화합물 [Hg(H2pmide)I2] (1), [Hg(bapeOH)HgI2]2 (2)를 얻었다(H2pmide=N-(2-pyridylmethyl)iminodiethanol, bpaeOH= N,N-bis(2-pyridylmethyl)-2-aminoethanol). 근적외선 분광법, 원소 분석법, 자외-가시 광선 분광법, X-선 회절 분광법, 광루미네선스를 통해 각 화합물의 성질을 확인할 수 있었다. H2pmide리간드와 수은(II) 금속을 합성한 착화합물 1의 배위수는 5로 중심금속인 수은(II)에 H2pmide리간드에 속하는 피리딘의 질소 1개와 하이드록시기의 산소 1개, 3차 질소 1개와 두개의 요오드 음이온이 배위되어 있는 사각뿔 형태의 배위구조를 가진다. 자유 H2pmide리간드와 착화합물 1은 각각 454 nm와 410 nm에서 발광띠가 나타났다. 따라서 착화합물 1의 발광은 파장이 짧은 방향으로 벗어난 청색편이가 일어난 것을 확인할 수 있다. 청색편이는 리간드에서 금속으로 전하가 이동하는 LMCT(ligand to metal charge trasnsfer)에 의해 일어나게 된다. 즉, 방향족 리간드 내에서 일어나는 π → π* 전이보다 LMCT에 의해 리간드의 채워진 π궤도함수에서 금속의 비어있는 σ*로 일어나는 전이의 에너지가 더 크기 때문이다. 또한 화합물 1의 경우 양자수율을 측정하였을 때 로다민-6G에 비해 약 1.5%정도의 양자수율을 가지므로 효율성 면에서는 떨어진다. 화합물 1의 형광수명은 T1, T2, T3의 평균 값인 1.95 ns이었으며 형광 수명에 대한 기여도는 T2가 약 70%로 가장 큰 것으로 확인되었다. bpaeOH리간드와 수은(II) 금속으로 합성된 착화합물 2는 사합체로 네 개의 수은 금속이 아이오딘 음이온에 의해 연결된 형태를 보인다. 착화합물 2는 착화합물 1에 비해 굉장히 약한 발광띠를 나타내며, 따라서 효과적이지는 못하다. 자유 bpaeOH리간드는 자외-가시 분광법에서 나타난 흡수띠를 기반으로 280 nm에서 여기되어졌을 때 450 nm에서 최대 발광띠가 나타났다. 이와 비교하여 착화합물 2는 발광띠가 280 nm에서 여기될 때 467 nm에서 최대 발광띠를 나타낸다. 따라서 파장이 길어진 적색편이가 일어난 것을 확인할 수 있으며 이것은 리간드 내에서 일어나는 전이에 의한 것으로 예측된다. The title coordination compounds [Hg(H2pmide)I2] (1) and [Hg(bpaeOH)HgI2]2 (2) were obtained by mixing mercury(II) metal of d10 system and ligand H2pmide or bpaeOH. The properties of each compound were confirmed by infrared spectroscopy, elemental analysis, ultraviolet-visible spectroscopy, X-ray diffraction spectroscopy and optical luminescence. The coordination number of complex 1 is 5. The mercury(II) ion has a square pyramidal structure in which one nitrogen atom of pyridine belonging to the H2pmide ligand, one oxygen atom of the hydroxyl group, one tertiary nitrogen atom and two iodine anions are coordinated. The emission spectrum of the free H2pmide ligand was found at 454 nm, whereas complex 1 exhibited a strong emission band at 410 nm. Therefore, it was observed the blue shift. The blue shift is caused by the ligand to metal charge trasnsfer (LMCT), which transfers charge from the ligand to the metal. This is because the electron transition caused by LMCT in the compound is larger than the transition energy from π to π* in the aromatic ring. Furthermore, the quantum yield of compound 1 was found to be 1.5% as compared with rhodamine 6G, which is inferior in efficiency. The fluorescence lifetime of compound 1 was 1.95 ns, which is the average value of T1, T2, and T3, and the contribution to fluorescence lifetime was found to be the largest at about 70% of T2. Complex 2 was synthesized from bpaeOH ligand and mercury(II) metal, in which four mercury metals are bridged by iodine anion. Complex 2 exhibits a very weak luminescent band compared to complex 1 and thus is not effective. The maximum emission band of the free bpaeOH ligand was found at 450 nm and the maximum emission band at 467 nm when excited at 280 nm. Thus, it can be seen that an increase in wavelength of red shift occurs, which is presumably due to the transition occurring in the ligand. Therefore, it can be seen that compound 2 has an increased red shift compared to the free ligand, which can be presumed to be caused by a transition occurring in the ligand.

Metal halide-based ligand exchange of Cd-free quantum dots for cell imaging

Sung Han Park Ulsan National Institute of Science and Technology 2023 국내석사

PART A. 새로운 Pd-H 촉매를 이용한 Hydroamination의 개발 PART B. Palladium 촉매를 위한 Hemilabile Ligand의 설계 및 합성

김보은 과학기술연합대학교대학원 2019 국내석사

<Palladium–Hydride Catalyzed Hydroamination> In this study, we tried to develop intermolecular hydroamination of vinylarene using stable palladium hydride (Pd-H) catalyst. Pd-H complex is generated by reacting LPdCl2 (L = ligand) with silver salt, silyl hydride. The reaction conditions of hydroamination using this catalyst were optimized. Under optimized reaction conditions, the hydroamination of various styrenes with anilines, sulfonamides, and indoles were proceeded, showing generally good yields. When 2,2'-bis (diphenylphosphino) -1,1'-binaphthyl (BINAP) type chiral ligands were used, enantioselective products were obtained. Among them, (R) - SEGPHOS gave the best enantioselectivity. <Design and Synthesis of New Hemilabile Ligand for Palladium catalyst> In this study, we tried to develop a new ligand for palladium. We aimed to make a ligand that increases the stability of palladium and does not decrease its reactivity. Therefore, we decided to make hemilabile ligands. For hemilability, seven ligands with controlled electron properties of labile donor were designed. Designed ligands were synthesized through two synthetic routes. And palladium complexes were synthesized by reacting the ligand with several palladium sources. The structure of one palladium complex was confirmed by 1H NMR, MALDI-TOF-MS and X-ray. Currently, studies to reveal structure of palladium complexes and to confirm improvement of stability and hemilability of palladium complex were in progress. <새로운 Pd-H 촉매를 이용한 Hydroamination의 개발> 본 연구에서는 선행 연구를 통해 개발한 안정한 palladium hydride (Pd-H) 촉매를 intermolecular hydroamination에 적용시켜 보고자 하였다. 안정한 palladium hydride 촉매는 LPdCl2 (L=ligand)와 silver salt, silyl hydride를 반응시켜 insitu로 생성되었다. 이러한 촉매를 hydroamination에 적용시키기 위해 반응 조건을 최적화하였다. 최적화된 반응 조건 하에서 다양한 styrene과 aniline, sulfonamide, indole의 hydroamination이 진행되었으며 대체로 좋은 수율을 보였다. 2,2’-bis(diphenyl -phosphino) -1,1’-binaphthyl (BINAP) 계열의 카이랄 리간드를 사용했을 때는 거울상 이성질 선택성이 있는 hydroami -nation 생성물을 얻었다. 그 중에서도 (R)-SEGPHOS를 사용했을 때 가장 좋은 거울상 이성질 선택성을 보였다. <Palladium 촉매를 위한 Hemilabile 리간드의 설계 및 합성> 본 연구에서는 palladium을 위한 새로운 리간드의 개발을 시도하였다. 우리는 palladium의 안정성은 높이면서 반응성을 감소시키지 않을 리간드를 만드는 것을 목표로 하였다. 따라서 hemilability를 가지는 리간드를 만들고자 하였다. hemilability를 위해 labile donor의 전자적 특성을 조절하여 7가지의 리간드를 설계하였다. 설계된 리간드는 두 가지 합성법을 통해 합성되었다. 그리고 리간드를 여러 palladium source와 반응시켜 palladium complex를 합성하였다. 일부 palladium complex의 구조는 1H NMR, MALDI-TOF-MS, X-ray로 확인하였다. 현재 나머지 palladium complex의 구조 규명을 위한 연구 및 palladium complex의 안정성 향상과 hemilability를 확인하기 위한 연구가 진행 중이다.

The Dadi Ligand and Beyond! Benzimidazole-Diamide (Bide) Complexes of Chromium and Titanium and Homoleptic Complexes Featuring Nitroxyl Ligands

Kayser, Ann Kateri Cornell University ProQuest Dissertations & Theses 2023 해외박사(DDOD)

Transition metal catalysis has proven to be vital in organic synthesis and in the production of commodity chemicals. 2nd and 3 rd -row metal species are typical catalysts due to their stability, low-spin electron configurations, and ready engagement in 2- electron chemistry. Unfortunately, they are less abundant, more expensive, and more toxic than their 1st -row analogues, which have been underutilized due to weak ligand fields and high-spin electron configurations. This makes characterization difficult and typically leads to unproductive 1-electron chemistry, thereby spoiling catalytic applications. One approach to circumvent unproductive 1-electron chemistry in 3d metals involves the use of redox non-innocent ligands. Redox non-innocence (RNI) mediates reactivity of metal complexes through a synergistic relationship between the metal center and ligand framework, typically acting as electron reservoirs or participating in bond functionalization events. Consequently, RNI ligands are excellent candidates for expanding chemistry in 1st-row metals.The RNI of a four-coordinate diamide-diimine (dadi) ligand was responsible for undesired reactivity at its C-C backbone, and a methylated version of the ligand was synthesized to attempt to block reactivity at the backbone. (medadi)M complexes were prepared and compared to the previously reported system.A benzimidazole-diamide pincer ligand (bida) that features potential RNI behavior, was coordinated to Cr and Ti to yield (bida)CrCl(THF) and (bida)TiCl2. For Cr, (bida)CrCl(THF) was successfully oxidized with organic azides, generating a collection of Cr(V) imido compounds, and resulting in a nitrene insertion. Alkylation of (bida)CrCl(THF) with alkyllithiums resulted in the clean formation of (bida)CrR(THF) compounds, and the discovery of a single component system for the polymerization of ethylene. For Ti, attempts were made to generate metal-ligand multiple bonds. Simple salt metathesis successfully yielded (bida)TiR2 compounds which thermally rearrange to produce a tuck-in species. Mechanistic investigations were initiated to explore the formation of the cyclometallated compound.TEMPO may also be considered as a redox active ligand with its ability to access three redox states. As such, examination of the coordination of TEMPO to first row metals was also investigated, leading to the isolation of chromium-TEMPO complexes featuring a rare-coordination geometry and unique iron-oxide clusters.

Structural understanding of ligand recognition and selectivity of purinergic P1 and P2Y receptors through the systematic analysis of sequence conservation and ligand interaction patterns

한리 중앙대학교 대학원 2023 국내석사

퓨린성 수용체는 거의 모든 포유동물 조직에서 발견되는 막 단백질이며 퓨린 뉴클레오티드 또는 뉴클레오사이드의 반응을 촉매함으로써 수많은 세포 기능을 조절합니다. 퓨린성 수용체는 내인성 리간드, 분자 구조 및 조직 분포에 따라 P1, P2X 및 P2Y의 세 가지 패밀리로 분류할 수 있고 모든 수용체는 퓨린 기반 물질에 결합을 합니다. 그러나, 퓨린성 수용체들은 진화적으로 보았을 때 긴 진화거리를 갖고 있습니다. P1 및 P2Y 수용체는 G 단백질 결합 수용체(GPCR) 패밀리에 속하는 반면 P2X 수용체는 리간드 개폐 이온 채널로 알려져 있습니다. 더 나아가 P1 및 P2Y 수용체가 클래스 A GPCR의 공통 구조적 특징을 공유하지만, 포괄적인 서열 및 구조 분석은 P1 및 P2Y 수용체가 클래스 A GPCR(각각 α- 및 σ-그룹)에서 두 개의 별개의 그룹에 속하는 것으로 밝혀졌습니다. 추가적으로 퓨린성 수용체들은 특정 활성제를 구별할 수 있는 다른 리간드 결합 부위 기능을 가지고 있습니다. 이러한 결합 공동의 특정 아미노산 잔기는 각 하위 유형의 선택성과 독특한 약리학적 거동에 관여할 수 있습니다. 이 연구에서는 리간드 인식 및 선택성에 대한 구조적 통찰력과 진화적 특징을 확인하기 위해 퓨린성 P1 및 P2Y 수용체의 구조 및 아미노산 서열 기반 분석을 수행했습니다. P1 및 P2Y 수용체의 가장 높게 보존된 잔기를 찾기 위해 상동 단백질 서열을 수집한 후 특정 데이터는 데이터 처리 단계를 수행했습니다. 그 다음 서열을 정렬하여 MSA(다중 서열 정렬) 테이블을 생성하고 섀넌 엔트로피를 기반으로 보존 점수를 계산했습니다. 리간드 상호작용 패턴을 분석하기 위해 ChEMBL 데이터베이스의 리간드 정보를 사용하여 P1 및 P2Y 수용체에 대한 도킹 연구를 수행했습니다. P1과 P2Y 수용체의 보존된 잔기가 리간드 결합 부위와 어떻게 관련되는지, 두 수용체 사이의 유사점과 차이점을 추출하여 이들이 어떻게 달랐는지에 대하여 분석했습니다. Purinergic receptors are membrane proteins found in almost all mammalian tissues and regulate numerous cellular functions by catalyzing the reaction of purine nucleotides or nucleosides. Receptors can be classified into three families according to their endogenous ligands, molecular structures, and tissue distribution: P1, P2X, and P2Y, and all receptors bind to purine-based substances. However, purinergic receptors have a long evolutionary distance. P1 and P2Y receptors belong to the G protein-coupled receptor (GPCR) family, whereas P2X receptors are known as ligand-gated ion channels. Furthermore, although P1 and P2Y receptors share common structural features of class A GPCRs, comprehensive sequence and structural analysis showed that P1 and P2Y receptors belong to two distinct groups in class A GPCRs (α- and σ-groups, respectively). Additionally, purinergic receptors have different ligand binding site functions that can differentiate specific activators. Specific amino acid residues in these binding cavities may be involved in the selectivity and unique pharmacological behavior of each subtype. In this study, sequence- and structure-based analyses of purinergic P1 and P2Y receptors were performed to obtain structural insights and evolutionary features into ligand recognition and selectivity. Homologous protein sequences were collected to find conserved residues of the P1 and P2Y receptors. They were then aligned to generate a multiple sequence alignment (MSA) table, and the conservation score was calculated based on entropy. To analyze the ligand interaction pattern, I conducted a docking study on the P1 and P2Y receptors using the ligand information from the ChEMBL database. I analyzed how the conserved residues of the P1 and P2Y receptors relate to ligand binding sites and how they differ by extracting the similarities and differences between the two receptors.