Research Case Study 1
研究成果案例分享
Title: Combatting Antibiotic-Resistant Staphylococcus aureus: Discovery of TST1N-224, a Potent Inhibitor Targeting Response Regulator VraRC, through Pharmacophore-Based Screening and Molecular Characterizations
題目: 對抗抗生素耐藥性金黃色葡萄球菌：通過基於藥效團的篩選和分子特徵分析發現TST1N-224，一種針對響應調節因子VraRC的強效抑制劑。
ABSTRACT:
Staphylococcus aureus (S. aureus) is a major global health concern, causing various infections and presenting challenges due to antibiotic resistance. In particular, MRSA, VISA, and VRSA, pose significant obstacles in treating S. aureus infections. Therefore, a critical need for novel drugs to counter these resistant forms is pressing. Two-component systems (TCS), integral to bacterial regulation, offer promising targets for disruption. In this study a comprehensive approach, involving pharmacophore-based inhibitor screening, along with biochemical and biophysical analyses were conducted to identify, characterize, and validate potential inhibitors targeting the response regulator VraRC of S. aureus. The constructed pharmacophore model, Phar-VRPR-N3, demonstrated effectiveness to identify a potent inhibitor, TST1N-224 (IC50 = 55.9 ± 2.7 µM), against the formation of the VraRC-DNA complex. Notably, TST1N-224 exhibited strong binding to VraRC (KD = 23.4 ± 1.2 mM) by using a fast-on-fast-off binding mechanism. Additionally, NMR-based molecular modeling revealed that TST1N-224 predominantly interact with the a9- and a10-helixes of the DNA-binding domain (DBD) of VraR, where the interactive and functionally essential residues (N165, K180, S184, and R195) act as hotspots for structure-based inhibitor optimization. Furthermore, TST1N-224 evidently enhanced the susceptibility of VISA to both vancomycin and methicillin. Importantly, TST1N-224 distinguished by a 1,2,5,6-tetrathiocane with the 3 and 8 positions modified with ethanesulfonates, hold significant potential as a lead compound for the development of new antimicrobial agents.
摘要:
金黃色葡萄球菌（S. aureus）是全球主要的健康問題之一，它能引發各種感染，並因抗生素耐藥性而帶來挑戰。特別是耐甲氧西林金黃色葡萄球菌（MRSA）、耐萬古黴素中介金黃色葡萄球菌（VISA）和耐萬古黴素金黃色葡萄球菌（VRSA）在治療S. aureus感染時構成了重大障礙。因此，迫切需要新型藥物來對抗這些耐藥性形式。雙組分系統（TCS）是細菌調節的重要組成部分，為藥物干擾提供了有希望的靶點。在本研究中，通過藥效團基的抑制劑篩選，結合生化和生物物理分析，識別、特徵化並驗證了針對S. aureus響應調節因子VraRC的潛在抑制劑。構建的藥效團模型Phar-VRPR-N3展示了識別強效抑制劑TST1N-224（IC50 = 55.9 ± 2.7 µM）對VraRC-DNA複合物形成的有效性。值得注意的是，TST1N-224通過一種快進快出的結合機制表現出對VraRC的強結合力（KD = 23.4 ± 1.2 uM）。此外，基於NMR的分子建模顯示，TST1N-224主要與VraR的DNA結合域（DBD）中的a9-和a10-螺旋相互作用，其中的交互和功能必需殘基（N165、K180、S184和R195）成為基於結構的抑制劑優化的熱點。此外，TST1N-224顯著增強了VISA對萬古黴素和甲氧西林的敏感性。重要的是，TST1N-224以3位和8位修飾為乙磺酸鹽的1,2,5,6-四硫烷為特色，作為新型抗菌劑開發的潛在先導化合物具有重要意義。
Results 1
Receptor-ligand pharmacophore generation
To efficiently identify potent inhibitors against VraR, it is crucial to consider the functionally essential features that play key roles in the interactions between VraR and its target DNA. Pharmacophore modelling is a powerful technique for identifying and characterizing crucial elements within a ligand, ensuring precise and effective binding to a receptor 33, 34. Receptor-ligand pharmacophore generation involves the translation of protein properties into corresponding ligand features 35. This method can be utilized to investigate the essential functional features in DNA and target protein interactions. Nowadays, the structure of the VraRC-DNA complex has been determined and is available for analysis. Therefore, we used the complex structure of VraRC-DNA (PDB ID: 7VE5) (Figure 1A) to construct the pharmacophore model by using receptor-ligand pharmacophore generation. In this process, VraRC was employed as the receptor, while the DNA structure was used as the ligand to construct the pharmacophore model. Subsequently, two distinct clusters of pharmacophore features were successfully generated and designated as Phar-VRPL and Phar-VRPR (Figure 1B). Phar-VRPL comprises 5 hydrogen-bond acceptors (depicted as green spheres), 3 negative-charged features (represented by blue spheres), and 1 hydrophobic feature (illustrated as a cyan sphere) (Figure 1B). Phar-VRPR encompasses 7 hydrogen-bond acceptors, 1 hydrogen-bond donor (depicted as magenta spheres), 1 hydrophobic feature, and 7 negative-charged features (Figure 1B). These pharmacophore models offer a comprehensive representation of the essential features necessary for DNA binding and interaction with VraRC and useful for identifying and designing potential inhibitors.
結果一
受體-配體藥效團生成
為了有效識別針對VraR的強效抑制劑，考慮在VraR與其目標DNA之間相互作用中起關鍵作用的功能要素至關重要。藥效團建模是一種強大的技術，可識別和表徵配體中的關鍵元素，確保精確且有效地與受體結合。受體-配體藥效團生成涉及將蛋白質的特性轉化為相應的配體特徵。此方法可用於研究DNA與目標蛋白質相互作用中的必要功能特徵。如今，VraRC-DNA複合物的結構已經確定，並可供分析。因此，我們使用VraRC-DNA複合物結構（PDB ID: 7VE5）（圖1A）通過受體-配體藥效團生成來構建藥效團模型。在此過程中，VraRC被用作受體，而DNA結構則被用作配體來構建藥效團模型。隨後，成功生成了兩個不同的藥效團特徵簇，分別命名為Phar-VRPL和Phar-VRPR（圖1B）。Phar-VRPL包含5個氫鍵受體（以綠色球體表示）、3個負電荷特徵（以藍色球體表示）和1個疏水特徵（以青色球體表示）（圖1B）。Phar-VRPR包含7個氫鍵受體、1個氫鍵供體（以洋紅色球體表示）、1個疏水特徵和7個負電荷特徵（圖1B）。這些藥效團模型全面展現了DNA結合與VraRC相互作用所需的關鍵特徵，並可用於識別和設計潛在抑制劑。
Figure 1. The receptor-ligand pharmacophore generation based on the structure of VraRC-DNA complex. (A) Construction of pharmacophore models using the complex structure of VraRC-DNA (PDB ID: 7VE5). The protein structure is represented in ribbons, and the DNA molecule is displayed in sticks. (B) Depiction of generated pharmacophore features in conjunction with the VraRC-DNA complex structure. Pharmacophore features are color-coded: green for hydrogen bond acceptor, magenta for hydrogen-bond donor, and deep-blue for negatively charged features. (C) Detailed view of Phar-VRPR. (D) Features at specific distances corresponding to the pharmacophore model, Phar-VRPR-N3.
圖1. 基於VraRC-DNA複合物結構的受體-配體藥效團生成
(A) 使用VraRC-DNA複合物結構（PDB ID: 7VE5）構建藥效團模型。蛋白質結構以絲帶表示，DNA分子以棒狀顯示。(B) 與VraRC-DNA複合物結構結合生成的藥效團特徵的圖示。藥效團特徵以顏色編碼：綠色代表氫鍵受體，洋紅色代表氫鍵供體，深藍色代表負電荷特徵。(C) Phar-VRPR的詳細視圖。(D) 與藥效團模型Phar-VRPR-N3相對應的特徵在特定距離的分佈。
Result 2
Pharmacophore‐based inhibitor screening
Efficiently screening inhibitors through pharmacophore modelling necessitates the careful selection of a pharmacophore scaffold for ligand-pharmacophore mapping. Consequently, we undertook a comprehensive examination of the pharmacophore properties associated with Phar-VRPL and Phar-VRPR. Our analysis unveiled that a DNA bioactive scaffold, which interacts with residues N165, K180, S184 and R195 (Figure 2A and 2B), can be represented by three negatively charged features (n1, n2, and n3) and 3 hydrogen-bond acceptors (HA1, HA2 and HA3) (Figure 2). The features of Phar-VRPR were further consolidated and organized into a pharmacophore scaffold called Phar-VRPR-N3 (Figure 1D and 2). This scaffold was then utilized to screen a compound library consisting of 68,000 molecules obtained from the IBS database. The ligand-pharmacophore mapping process was carried out to screen and align these compounds onto the Phar-VRPR-N3 scaffold. In the process of ligand-pharmacophore mapping, the 3D coordinates of the ligands are aligned with the pharmacophore features of Phar-VRPR-N3. This alignment allows for the assessment of the fit between the ligand and the pharmacophore. Fit values are assigned to indicate the quality of the match between the ligand and the pharmacophore, with higher scores indicating a stronger and more favorable fit. Consequently, the top 9 ranked hits from the screening process were chosen as potential candidates (Figure 3). The fit values follow the hierarchy: TST1S-887 > TST1N-224 > TST1S-251 > TST1S-545 > TST1N-494 > TST1N-691 > TST1N-440 > TST1S-012 > TST1N-218. The detailed chemical structures of these identified candidates are displayed in Figure S1.
結果二
基於藥效團的抑制劑篩選
通過藥效團建模來高效篩選抑制劑，需要謹慎選擇藥效團骨架進行配體-藥效團映射。因此，我們對與Phar-VRPL和Phar-VRPR相關的藥效團特徵進行了全面分析。我們的分析揭示了一個與殘基N165、K180、S184和R195相互作用的DNA生物活性骨架（圖2A和2B），該骨架可以用三個負電荷特徵（n1、n2和n3）和三個氫鍵受體（HA1、HA2和HA3）來表示（圖2）。Phar-VRPR的特徵進一步被整合和組織成一個稱為Phar-VRPR-N3的藥效團骨架（圖1D和2）。隨後，我們使用該骨架對來自IBS數據庫的68,000種分子組成的化合物庫進行篩選。配體-藥效團映射過程中，這些化合物被篩選並對齊至Phar-VRPR-N3骨架上。在配體-藥效團映射過程中，配體的3D坐標與Phar-VRPR-N3的藥效團特徵對齊。這種對齊允許評估配體與藥效團的契合度，並根據契合度給予分數，較高的分數表示更強且更有利的契合度。因此，篩選過程中排名前9位的命中結果被選為潛在候選者（圖3）。契合值的等級如下：TST1S-887 > TST1N-224 > TST1S-251 > TST1S-545 > TST1N-494 > TST1N-691 > TST1N-440 > TST1S-012 > TST1N-218。這些候選者的詳細化學結構顯示於圖S1。
Figure 2. Schematic representations of pharmacophore model, Phar-VRPR-N3. (A) The amplified view of pharmacophore model, Phar-VRPR-N3. The protein is presented in ribbon and the interactive residues are shown in sticks (orange) and labeled. Pharmacophore features are color-coded: green for hydrogen bond acceptor, magenta for hydrogen-bond donor, and deep-blue for negatively charged features. (B) The molecular interactions of the functional residues of VraRC binding to DNA. The DNA molecule is shown in thin stick (gray).
圖2. 藥效團模型Phar-VRPR-N3的示意圖
(A) 藥效團模型Phar-VRPR-N3的放大視圖。蛋白質以絲帶表示，交互殘基以橙色棒狀顯示並標註。藥效團特徵以顏色編碼：綠色代表氫鍵受體，洋紅色代表氫鍵供體，深藍色代表負電荷特徵。(B) VraRC與DNA結合的功能殘基的分子相互作用。DNA分子以細棒狀（灰色）顯示。
Figure 3. Pharmacophore-based inhibitor screening. Illustration of the results from ligand pharmacophore (Phar-VRPR-N3) mapping for hits screened from the IBS database. The top 9 ranked hits are aligned with the pharmacophore model, Phar-VRPR-N3. Pharmacophore features are color-coded: hydrogen-bond acceptor in green and negative charge in deep-blue.
圖3. 基於藥效團的抑制劑篩選
顯示從IBS數據庫篩選出的配體藥效團（Phar-VRPR-N3）映射結果。排名前9位的命中結果與藥效團模型Phar-VRPR-N3對齊。藥效團特徵以顏色編碼：綠色代表氫鍵受體，深藍色代表負電荷。
Result 3
The disruptive ability of inhibitors to the formation of VraRC-DNA complex
To assess compounds' ability to inhibit VraR binding to DNA, we initially expressed and purified full-length VraR for assays. However, due to strict regulatory controls on BeCl2 (required to generate BeF3- (a phosphate analog for proteins phosphorylated on aspartate)) in our country, we cannot experimentally activate VraR. Alternatively, VraRC showed greater stability during protein preparation, facilitating subsequent experiments. Moreover, the availability of the VraRC-DNA complex structure allows for pharmacophore modeling and inhibitor screening focused on the DNA-binding domain, simplifying the identification of inhibitors that disrupt VraR-DNA binding. Therefore, we evaluated the inhibitory activity of identified compounds against VraRC binding to DNA using fluorescence polarization experiments. Prior to these experiments, the interaction of VraRC with DNA was initially characterized to provide a basis for the inhibition assay. The results demonstrated an increase in polarization intensity as the protein concentration of VraRC ascended, as depicted in Figure 4. The result indicated that VraRC strongly binds to DNA, with a KD value of 4.1 uM (Figure 4). Notably, the polarization intensity reached a plateau when the concentration of VraRC is around 36 uM. Therefore, a concentration of 36 uM VraRC was employed for the inhibition assay. Subsequently, the inhibitory abilities of top10 ranked hits screened from the ligand-pharmacophore mapping were evaluated at a compound concentration of 100 uM. The results showed that the compounds TST1N-224, and TST1N-619 exhibited inhibitions of over 50% (Figure 5). Conversely, TST1N-218 displayed approximately 40% inhibition. On the other hand, TST1S-887, TST1N-251, TST1S-545, TST1S-012, TST1S-494, and TST1S-938 demonstrated lower or no inhibition against the binding of VraRC to DNA (Figure 5). Moreover, compounds that demonstrated inhibitions exceeding 50% were subjected to further inhibitory experiments, varying the compound concentrations, to determine their IC50. Regarding the inhibition of VraRC binding to DNA, TST1N-224, and TST1N-691 displayed dose-dependent inhibitions, as shown in Figure 6. The IC50 values for TST1N-224 and TST1N-691 were determined to be 60.2 and 75.2 uM, respectively.
結果三
抑制劑對VraRC-DNA複合物形成的干擾能力
為了評估化合物抑制VraR結合DNA的能力，我們首先表達並純化了全長VraR以進行測試。然而，由於我們國家對BeCl2的嚴格管制（這是生成BeF3-（一種磷酸鹽類似物，用於天冬氨酸磷酸化蛋白質）所必需的），我們無法在實驗中激活VraR。作為替代，VraRC在蛋白質製備過程中顯示出更高的穩定性，從而促進了後續實驗。此外，VraRC-DNA複合物結構的可用性使得藥效團建模和針對DNA結合域的抑制劑篩選成為可能，簡化了識別能夠干擾VraR-DNA結合的抑制劑的過程。因此，我們使用螢光偏振實驗評估了已識別化合物對VraRC結合DNA的抑制活性。在這些實驗之前，首先表徵了VraRC與DNA的相互作用，為抑制測定提供了依據。結果顯示，隨著VraRC蛋白濃度的增加，偏振強度也有所增強，如圖4所示。結果表明，VraRC與DNA具有很強的結合力，其KD值為4.1 µM（圖4）。值得注意的是，當VraRC濃度達到約36 µM時，偏振強度達到平臺。因此，在抑制測定中使用了36 µM的VraRC濃度。隨後，評估了從配體-藥效團映射中篩選出的排名前10位的命中化合物在100 µM濃度下的抑制能力。結果顯示，化合物TST1N-224和TST1N-619的抑制率超過50%（圖5）。相反，TST1N-218的抑制率約為40%。另一方面，TST1S-887、TST1N-251、TST1S-545、TST1S-012、TST1S-494和TST1S-938對VraRC與DNA的結合顯示出較低或無抑制效果（圖5）。此外，抑制率超過50%的化合物進一步進行了抑制實驗，通過調整化合物濃度來確定其IC50值。關於VraRC結合DNA的抑制，TST1N-224和TST1N-691顯示出劑量依賴性的抑制，如圖6所示。TST1N-224和TST1N-691的IC50值分別為60.2 µM和 75.2 µM。
Figure 4. The DNA binding property of VraRC. The DNA binding ability of VraRC is observed by FP experiments as a function of protein concentration. The determined KD value is 4.1 uM.
圖4. VraRC的DNA結合特性
通過FP實驗觀察VraRC的DNA結合能力，並隨著蛋白質濃度的變化進行測定。確定的KD值為4.1 µM。
Figure 5. Inhibitory potency of Top 9 ranked hits against the complex formation of VraRC-DNA. Evaluation of the inhibitory capacity of the top 9 ranked hits against the formation of the VraRC-DNA complex at 100 uM concentration is depicted.
圖5. 前9名排名命中化合物對VraRC-DNA複合物形成的抑制效力
顯示了在100 µM濃度下，前9名排名命中化合物對VraRC-DNA複合物形成的抑制能力的評估。
Figure 6. The inhibitory potencies of TST1N-224 and TST1N-691 as a function of compound concentration. The dose‐dependent inhibition curves of TST1N-224 and TST1N-691 against the formation of VraRC-DNA complex are shown.
圖6. TST1N-224和TST1N-691的抑制效力與化合物濃度的關係
顯示了TST1N-224和TST1N-691對VraRC-DNA複合物形成的劑量依賴性抑制曲線。
Result 4
The binding affinity of TST1N-224 towards VraRC
In our pharmacophore-based inhibitor screening, we successfully identified TST1N -166 and TST1N-224 as potent inhibitors against VraRC. To confirm the binding between the inhibitors and VraRC, the Localized Surface Plasmon Resonance (LSPR) experiments were performed. During the experiments, TST1N-224 was tested at concentrations of 6.25, 12.5, 25, and 50 uM against VraRC. The sensorgrams revealed a fast association and fast dissociation binding pattern of TST1N-224 with VraRC, resulting in a KD value of 23.4 uM (Figure 7). Similarly, the binding of TST1N-691 to VraRC was investigated at concentrations of 6.25, 12.5, 25, and 50uM. However, the sensorgrams showed very minor and weak binding signal, compared to that of buffer blank (data not shown). The association signal of TST1N-691 to VraRC was observed not significantly ascended even if the compound concentration increased to 100-200 uM (data not shown).
結果4
TST1N-224對VraRC的結合親和力
在我們基於藥效團的抑制劑篩選中，我們成功地識別了TST1N-166和TST1N-224作為對VraRC的強效抑制劑。為了確認這些抑制劑與VraRC的結合，進行了局部表面等離子體共振（LSPR）實驗。在實驗中，對TST1N-224在6.25、12.5、25和50 µM濃度下進行了測試。感應圖顯示，TST1N-224與VraRC的結合模式為快速聯合和快速解離，導致KD值為23.4 µM（圖7）。同樣，對TST1N-691在6.25、12.5、25和50 µM濃度下對VraRC的結合進行了調查。然而，與緩衝液空白對照相比，感應圖顯示TST1N-691的結合信號非常微弱且較弱（數據未顯示）。即使將化合物濃度提高至100-200 µM，TST1N-691與VraRC的結合信號也未顯著上升（數據未顯示）。
Figure 7. The LSPR sensorgrams of TST1N-224 binding to VraRC. The binding affinity of TST1N-224 (KD = 23.4 uM) to VraRC.
圖7. TST1N-224 與 VraRC 結合的 LSPR 感應圖
顯示了TST1N-224對VraRC的結合親和力 (KD = 23.4 µM）。
Result 5
Exploring inhibitor binding site by NMR spectroscopy
To explore the inhibitor binding site, we characterized the molecular interactions of TST1N-224 with VraRC by NMR-HSQC. Firstly, the chemical structure and purity of TST1N-224 were verified and confirmed by high-resolution electrospray ionization mass spectrometry, high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy (Figures S2–S4). Subsequently, the titrations of TST1N-224 towards VraRC were carried out at the molar ratios of protein to inhibitor = 1 : 0, 1 : 2, 1 : 4, and 1 : 6 to acquire the HSQC spectra, respectively. The results showed that with the additions of TST1N-224, the HQSC spectra of VraRC all showed chemical shift perturbations (CSP) (Figure 8A). The determined average CSP of residues of VraRC upon TST1N-224 titration (molar ratio of protein: inhibitor = 1: 4) was determined to be 0.022. Therefore, residue with CPS value ≥ 0.022 is defined to be most perturbed. The most perturbed residues are M147, E152, E154, L158, I159, K161, G162, S164, T175, K177, T181, S184, I186, L187, K189, L190, Q193, D194, T196 and A198 (Figure 8B). These interactive residues of VraRC pointed out the possible binding site of TST1N-224 (Figure 8C).
結果 5
通過NMR光譜探索抑制劑結合位點
為了探索抑制劑的結合位點，我們使用NMR-HSQC表徵了TST1N-224與VraRC的分子相互作用。首先，通過高解析度電噴霧離子化質譜、高效液相色譜和核磁共振光譜確認了TST1N-224的化學結構和純度（圖S2–S4）。隨後，對 TST1N-224 進行了不同摩爾比（蛋白質對抑制劑 = 1:0, 1:2, 1:4, 1:6）的滴定以獲取HSQC光譜。結果顯示，隨著 TST1N-224 添加，VraRC 的 HSQC 光譜顯示出化學位移擾動（CSP）（圖8A）。TST1N-224滴定後（蛋白質對抑制劑的摩爾比 = 1:4）確定的VraRC殘基的平均CSP值為0.022。因此，CSP值 ≥ 0.022的殘基被定義為擾動最顯著。最顯著擾動的殘基包括M147、E152、E154、L158、I159、K161、G162、S164、T175、K177、T181、S184、I186、L187、K189、L190、Q193、D194、T196和A198（圖8B）。這些VraRC的交互作用胺基酸指出了 TST1N-224 的可能結合位點（圖8C）。
Figure 8. Determination of binding site of TST1N-224 toward VraRC by NMR titrations. (A) The acquired 2D 1H-15N HSQC spectra of VraRC with the addition of district concentrations of TST1N-224 are overlapped and shown. The molar ratios of VraRC to TST1N-224 were set to 1 : 0, 1 : 2, 1 : 4, and 1 : 6. The perturbed residues of VraRC upon TST1N-224 binding are shown and labeled (highlighted with green and cyan colors). (B) The chemical shift perturbation (CSP) values for backbone amide resonances of VraRC on titration with TST1N-224 (1: 4). Green and cyan bars indicate that residues with CSP values more than the average (0.022). (C) Cartoon structure of VraRC showing chemical shift-perturbed residues upon titration of TST1N-224 highlighted in green and cyan, respectively. The protein structure of VraRC is presented as ribbon and the Ca atoms of each perturbed residues are shown in spheres.
圖 8. 通過NMR滴定確定TST1N-224對VraRC的結合位點
(A) 顯示了隨著不同濃度的TST1N-224添加而獲得的VraRC 2D 1H-15N HSQC光譜的重疊圖。VraRC與TST1N-224的摩爾比設置為1:0、1:2、1:4和1:6。顯示並標註了TST1N-224結合後VraRC的擾動殘基（以綠色和青色突出顯示）。
(B) TST1N-224（1:4）滴定後VraRC骨架酰胺共振的化學位移擾動（CSP）值。綠色和青色條表示CSP值超過平均值（0.022）的殘基。(C) VraRC的卡通結構顯示了在TST1N-224滴定後化學位移擾動的殘基，分別以綠色和青色突出顯示。VraRC的蛋白質結構以絲帶表示，每個擾動殘基的Cα原子以球狀顯示。
Result 6
The complex structure of VraRC-TST1N-224
To gain a more comprehensive understanding of the atomic-level interactions between VraRC and the potent inhibitor, TST1N-224, we utilized molecular modeling techniques to construct the complex structure. The residues (M147, E152, T175, K177, T181, S184, I186, L187, K189, L190, Q193, D194, and T196) in VraRC that displayed perturbations during the NMR titration with TST1N-224 were identified as the binding site for the subsequent protein-ligand flexible docking. During the protein-ligand flexible docking, we allowed for the flexibility of side chains in the binding site residues to explore various rotamers. Eventually, we selected the model with the lowest energy, where TST1N-224 conformed closely to the characteristics of Phar-VRPR-N3, as the final complex structure of VraRC-TST1N-224. The built complex structure was further analyzed by non-bond interaction analysis (Discovery Studio 2021) to unveil the detail molecular interactions. The results showed that TST1N-224 was positioned between the α9- and α10-helixes of VraRC. The specific molecular interactions between TST1N-224 and VraRC are visualized in Figure 9. Notably, in this binding orientation, the terminal sulfonic groups of TST1N-224, which correspond to n1 and n3 of Phar-VRPR-N3, engaged in charge-charge interactions with residues R195 and K180. Remarkably, residue R195 demonstrated interactions with TST1N-224 through a carbon-hydrogen bond and an additional hydrogen bond. Additionally, the S1 atom on the 1,2,5,6-tetrathiocane and the O7 atom of TST1N-224 formed hydrogen bonds with the side chain of residue S184. Furthermore, the C22 atom of TST1N-224 interacted with Q193 through a carbon-hydrogen bond (Figure 9).
結果6
VraRC-TST1N-224複合物結構
為了更全面地了解VraRC與強效抑制劑TST1N-224之間的原子級相互作用，我們使用分子建模技術構建了複合物結構。在NMR滴定中顯示出擾動的VraRC殘基（M147、E152、T175、K177、T181、S184、I186、L187、K189、L190、Q193、D194和T196）被確定為後續蛋白質-配體柔性對接的結合位點。在蛋白質-配體柔性對接過程中，我們允許結合位點殘基的側鏈具有柔性，以探索各種旋轉異構體。最終，我們選擇了能夠與Phar-VRPR-N3特徵最為匹配的最低能量模型，作為VraRC-TST1N-224的最終複合物結構。構建的複合物結構進一步通過非鍵合相互作用分析進行了詳細的分子相互作用分析。結果顯示，TST1N-224定位在VraRC的α9-和α10-螺旋之間。TST1N-224與VraRC之間的具體分子相互作用如圖9所示。值得注意的是，在這種結合取向中，TST1N-224的末端磺酸基團（對應於Phar-VRPR-N3中的n1和n3）與殘基R195和K180發生了電荷-電荷相互作用。特別地，殘基R195通過一個碳-氫鍵和額外的氫鍵與TST1N-224互動。此外，1,2,5,6-四硫環上的S1原子與TST1N-224的O7原子形成了與殘基S184側鏈的氫鍵。此外，TST1N-224的C22原子通過碳-氫鍵與Q193相互作用（圖9）。
Figure 9. The complex structure of VraRC-TST1N-224. (A) The complex structure of VraRC-TST1N-224 was built by NMR-based molecular modelling. TST1N-224 binds between a9 and a10-helix of VraRC. The residues of VraRC interacting with TST1N-224 are shown in sticks (white) and labeled (black). VraRC is presented in ribbon and TST1N-224 is shown in stick (magenta). (B) The complex structure of VraRC-TST1N-224 is aligned with the pharmacophore model, Phar-VRPR-N3.
圖 9. VraRC-TST1N-224複合物結構
(A) VraRC-TST1N-224複合物的結構是通過基於NMR的分子建模構建的。TST1N-224結合在VraRC的α9和α10螺旋之間。與TST1N-224互動的VraRC殘基顯示為白色棒狀並標註為黑色。VraRC以絲帶表示，TST1N-224顯示為品紅色棒狀。(B) VraRC-TST1N-224複合物結構與藥效團模型Phar-VRPR-N3對齊。
Result 7
In vitro inhibition of TST1N-224 against MRSA and VISA
To test the biological activity of TST1N-224, the growth of Staphylococcus aureus subsp. aureus Z172 (VISA) was observed with the addition of inhibitor to determine the MIC. Meanwhile, the susceptibility of standard strains of Staphylococcus aureus (SA) and Methicillin-resistant Staphylococcus aureus (MRSA) to TST1N-224 were also tested. The experiments were performed with a final bacterial count of 5 × 104 CFU/ml and followed the guideline of CLSI (M100 and M07). The results showed that TST1N-224 can inhibit the growths of SA (MIC > 126 µM), MRSA (MIC > 126 µM), and VISA (MIC = 63 µM) (Table 1). In addition, the MICs of vancomycin complied with the concentrations specified in CLSI (M100). These results revealed that TST1N-224 exerted better antibacterial effects on VISA.
結果7
TST1N-224對MRSA和VISA的體外抑制效果
為了測試TST1N-224的生物活性，我們觀察了Staphylococcus aureus subsp. aureus Z172（VISA）的生長情況，並通過添加抑制劑來確定最小抑菌濃度（MIC）。同時，我們還測試了標準菌株Staphylococcus aureus（SA）和抗甲氧西林的Staphylococcus aureus（MRSA）對TST1N-224的敏感性。實驗以5 × 10^4 CFU/ml的最終菌數進行，並遵循了CLSI（M100和M07）的指導原則。結果顯示，TST1N-224能夠抑制SA（MIC > 126 µM）、MRSA（MIC > 126 µM）和VISA（MIC = 63 µM）的生長（表1）。此外，萬古霉素的MIC符合CLSI（M100）中規定的濃度。這些結果顯示，TST1N-224對VISA表現出更好的抗菌效果。
Result 8
The synergetic effects of TST1N-224 combined with methicillin or vancomycin against VISA
The Fractional Inhibitory Concentration Index (FICI) is an experimental method based on the MIC to investigate the synergistic effects of drugs in inhibiting bacteria. By using this, we can explore the biological function of TST1N-224 in combination with vancomycin and/or methicillin against Staphylococcus aureus subsp. aureus Z172 (VISA). The results showed that the combination of TST1N-224 and vancomycin led to the FICI > 1.0, indicating no synergistic effect on the growth of bacteria (Figure S5). In contrast, the growth of VISA was significantly inhibited, when treated with the combination of TST1N-224 and methicillin (FICI = 0.675) (Figure S6 and Table 2). This indicates an additive effect on bacterial growth, implying that TST1N-224 has a better synergistic effect when combined with β-lactam antibiotics, such as methicillin.
結果 8
TST1N-224與甲氧西林或萬古霉素聯合使用對VISA的協同效應
分數抑菌濃度指數（FICI）是一種基於MIC的實驗方法，用於研究藥物抑制細菌的協同效應。通過使用這種方法，我們可以探討TST1N-224與萬古霉素和/或甲氧西林聯合使用對 Staphylococcus aureus subsp. aureus Z172（VISA）的生物學功能。結果顯示，TST1N-224 與萬古霉素聯合使用時，FICI > 1.0，表明對細菌生長沒有協同效應（圖S5）。相比之下，當 TST1N-224 與甲氧西林聯合使用時，VISA的生長顯著被抑制（FICI = 0.675）（圖S6和表2）。這表明對抑制細菌生長具有加成效應，暗示TST1N-224在與β-內酰胺抗生素（如甲氧西林）聯合使用時具有更好的協同抑菌殺菌效果。
Result 9
The cytotoxicity of TST1N-224
To reveal the possibility of TST1N-224 as a drug for the treatment of Staphylococcus species, its safety profile was investigated. Oral cancer cell line OECM-1 was treated with 0, 50, or 100 μM of TST1N-224 for 0, 2, 4 days. The results showed that the cell viability of the condition treated with TST1N-224 (100 μM) showed no significant change, compared to that of the mock control (Figure 10). This result indicated that TST1N-224 have no apparent cytotoxicity.
結果9
TST1N-224的細胞毒性
為了評估TST1N-224作為治療葡萄球菌類的藥物的可能性，我們調查了其安全性。對OECM-1哺乳動物細胞進行了0、50或100 μM的TST1N-224處理，持續時間為0、2、4天。結果顯示，TST1N-224（100 μM）處理的細胞存活率與對照組相比沒有顯著變化（圖10）。這表明TST1N-224沒有明顯的細胞毒性。
Figure 10. The cell viability of oral cancer cell, OECM-1, under the treatments of TST1N-224.
圖10. 口腔癌細胞OECM-1在TST1N-224處理下的細胞存活率。
This study has been published in J. Chem. Inf. Model. 2024, 64, 15, 6132–6146
本研究成果已發表於國際知名期刊 (J. Chem. Inf. Model. 2024, 64, 15, 6132–6146)
