Shanhe Wan

Molecular dynamics simulation and free energy calculation studies of kinase inhibitors binding to active and inactive conformations of VEGFR-2

Vascular endothelial growth factors receptor-2 (VEGFR-2) inhibitors have been proved as very effective anticancer agents. Structurally similar ligands 1 and 2 show almost the same inhibitory activities against VEGFR-2, but they bind to the enzyme in distinct binding mode. Ligand 1 targets DFG-in active conformation of VEGFR-2, known as Type I inhibitor. On the other hand, ligand 2 targets DFG-out inactive conformation of VEGFR-2, known as Type II inhibitor or allosteric kinase inhibitor. Ligand 2 shows high inhibitory activity, while the compound 3, a close analog of 2 with the cyclopropylamide replaced by tert-butylamide, exhibits drastically diminished potency. In this work, molecular dynamics simulations and free energy calculations were performed on inhibitors 1-3 binding to active and inactive conformation of VEGFR-2. Molecular dynamics simulations find that the active conformation binding to Type I inhibitor 1 appears more flexible when compared to the unbound form. In contrast, binding of Type II inhibitor 2 to the inactive conformation helps to stabilize the inactive conformation of the protein. Binding free energy calculations verify that inhibitors 1 and 2 have almost the same activities against VEGFR-2, and that ligand 1 binds to and stabilizes the DFG-in conformation of VEGFR-2, which is in agree with the experimental observation. Molecular dynamics simulations and binding free energy calculations of 3 binding to VEGFR-2 can give a good explanation of the drastically diminished potency. Free energy analysis revealed that van der Waals interactions provided the substantial driving force for the binding process. The important hydrophobic property of the terminal 4-Cl phenyl was required to be Type II inhibitors. Furthermore, per-residue free energy decomposition analysis revealed that the most favorable contribution came from Leu840, Val848, Ala866, Lys868, Leu889, Val899, Thr916, Phe918, Cys919, Leu1035, Cys1045, Asp1046, and Phe1047. These results are expected to be useful for future rational design of novel potent VEGFR-2 inhibitors.

Molecular Simulation Studies on the Binding Selectivity of Type-I Inhibitors in the Complexes with ROS1 versus ALK

ROS1 and ALK are promising targets of anticancer drugs for non-small-cell lung cancer. Since they have 49% amide acid sequence homology in the kinases domain and 77% identity at the ATP binding area, some ALK inhibitors also showed some significant responses for ROS1 in the clinical trial, such as the type-I binding inhibitor crizotinib and PF-06463922. As a newly therapeutic target, the selective ROS1 inhibitor is relatively rare. Moreover, the molecular basis for the selectivity of ROS1 versus ALK still remains unclear. In order to disclose the binding preference toward ROS1 over ALK and to aid the design of selective ROS1 inhibitors, the specific interactions and difference of conformational changes in the dual and selective ROS1/ALK inhibitors systems were investigated by molecular dynamics (MD) simulation and principle component analysis (PCA) in our work. Afterward, binding free energies (MM/GBSA) and binding free energies decomposition analysis indicated that the dominating effect of Van der Waals interaction drives the specific binding process of the type-I inhibitor, and residues of the P-loop and the DFG motif would play an important role in selectivity. On the basis of the modeling results, the new designed compound 14c was verified as a selective ROS1 inhibitor versus ALK, and SMU-B was a dual ROS1/ALK inhibitor by the kinase inhibitory study. These results are expected to facilitate the discovery and rational design of novel and specific ROS1 inhibitors.

3D-QSAR study on 2,3-dihydroimidazo[4,5]-pyridin-2-one derivatives with a meta substitution pattern as V600EBRAF inhibitors

V600EBRAF have been identified as new promising targets for the design of novel anticancer agents. It is reported that the inhibitors based on 2,3-dihydroimidazo[4,5]pyridin-2-one scaffold and a meta-substituted middle ring exhibit potent inhibitory activities toward V600EBRAF. To investigate how their chemical structures relate to the inhibitory activities and to identify the key structural elements that are required in the rational design of potential drug candidates of this class, molecular docking simulations and three-dimensional quantitative structure–activity relationship (3D-QSAR) methods were performed. The bioactive conformation was explored by docking one potent compound 1 into the active site of BRAF in its DFG-out inactive conformation. The constructed CoMFA and CoMSIA models produced statistically significant results with the cross-validated correlation coefficients q2 of 0.563 and 0.624, non-cross-validated correlation coefficients r2 of 0.982 and 0.962, and predicted correction coefficients rpred2 of 0.822 and 0.875, respectively. In addition, the CoMFA and CoMSIA models were used to guide the design of a series of new inhibitors of this class with predicted excellent activities. Thus, these models may be used as an efficient tool to predict the inhibitory activities and to guide the future rational design of 2,3-dihydroimidazo[4,5]pyridin-2-one derivatives with a meta substitution pattern as BRAF inhibitors with potent activities.

Bisarylureas Based on 1H-Pyrazolo[3,4-d]pyrimidine Scaffold as Novel Pan-RAF Inhibitors with Potent Anti-Proliferative Activities: Structure-Based Design, Synthesis, Biological Evaluation and Molecular Modelling Studies

RAF (Ras activating factor) kinases are important and attractive targets for cancer therapy. With the aim of discovering RAF inhibitors that bind to DFG-out inactive conformation created by the movement of Asp-Phe-Gly (DFG), we conducted structure-based drug design using the X-ray cocrystal structures of BRAF (v-raf murine sarcoma viral oncogene homolog B1), starting from bisarylurea derivative based on 1H-pyrazolo[3,4-d]pyrimidine scaffold 1a. Most of the synthesized compounds showed good to excellent inhibitory activities against BRAFV600E kinase, possessed moderate to potent anti-proliferative activities against four tumor cell lines (A375, HT-29, PC-3 and A549) and good selectivity towards cancer cells rather normal cells (Madin-Darby canine kidney, MDCK). The most promising compound, 1v, exhibited potent inhibitory activity against not only BRAFV600E (half maximal inhibitory concentration, IC50 = 23.6 nM) but also wild-type BRAF (IC50 = 51.5 nM) and C-RAF (IC50 = 8.5 nM), and effective cellular anti-proliferative activities against A375, HT-29, PC-3 and A549 cell lines as well as a very good selectivity profile. Moreover, compound 1v mainly arrested the A375 cell line in the G0/G1 stage, and showed significant suppression of MEK (mitogen-activated protein kinase kinase) phosphorylation in A375 and HT-29 cell lines. Taken together, the optimal compound 1v showed excellent in vitro potency as a pan-RAF inhibitor. In addition, the promise of compound 1v was further confirmed by molecular dynamics simulation and binding free energy calculations.

Computational investigation on inhibition mechanism of BRAFV600E by Vemurafenib (PLX4032) and its analogue PLX4720

BRAFV600E small-molecule inhibitors have recently received increasing attention as very effective agents for the therapy of cancer, especially melanoma. In this current work, a computational investigation was performed to investigate the interaction details of BRAFV600E kinase with inhibitors Vemurafenib (PLX4032) and its analog PLX4720. Binding free energy calculations based on molecular dynamics simulations reveal that ligands PLX4032 and PLX4720 bind to and stabilize the DFG-in conformation of BRAFV600E. Component analysis of binding free energy revealed that vdWaals interactions play a dominating effect on BRAFV600E inhibition. Furthermore, the per-residue binding free energy decomposition revealed that the most favorable contribution came from Ile463, Val471, Ala481, Lys483, Leu514, Ile527, Thr529, Gln530, Trp531, Cys532, Phe583, Gly593, and Asp594. These results agree well with experimental data, which provide valuable resources for understanding the inhibition mechanism of BRAFV600E by PLX4032 and PLX4720 and clues for the design of novel potent BRAFV600E inhibitors.

Standardized myrtol attenuates lipopolysaccharide induced acute lung injury in mice

Abstract Context: Standardized myrtol, an essential oil containing primarily cineole, limonene and α-pinene, has been used for treating nasosinusitis, bronchitis and chronic obstructive pulmonary disease (COPD). Objective: To investigate the effects of standardized myrtol in a model of acute lung injury (ALI) induced by lipopolysaccharides (LPS). Materials and methods: Male BALB/c mice were treated with standardized myrtol for 1.5 h prior to exposure of atomized LPS. Six hours after LPS challenge, lung injury was determined by the neutrophil recruitment, cytokine levels and total protein concentration in the bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity in the lung tissue. Additionally, pathological changes and NF-κB activation in the lung were examined by haematoxylin and eosin staining and western blot, respectively. Results: In LPS-challenged mice, standardized myrtol at a dose of 1200 mg/kg significantly inhibited the neutrophile counts (from 820.97 ± 142.44 to 280.42 ± 65.45, 103/mL), protein concentration (from 0.331 ± 0.02 to 0.183 ± 0.01, mg/mL) and inflammatory cytokines level (TNF-α: from 6072.70 ± 748.40 to 2317.70 ± 500.14, ng/mL; IL-6: from 1184.85 ± 143.58 to 509.57 ± 133.03, ng/mL) in BALF. Standardized myrtol also attenuated LPS-induced MPO activity (from 0.82 ± 0.04 to 0.48 ± 0.06, U/g) and pathological changes (lung injury score: from 11.67 ± 0.33 to 7.83 ± 0.79) in the lung. Further study demonstrated that standardized myrtol prevented LPS-induced NF-κB activation in lung tissues. Discussion and conclusion: Together, these data suggest that standardized myrtol has the potential to protect against LPS-induced airway inflammation in a model of ALI.

Design, synthesis, biological evaluation and molecular modeling of novel 1 H -pyrazolo[3,4-d]pyrimidine derivatives as BRAF V600E and VEGFR-2 dual inhibitors

Aiming to explore novel BRAFV600E and VEGFR-2 dual inhibitors, a series of 1H-pyrazolo[3,4-d]pyrimidine derivatives were designed, synthesized and biologically evaluated in this study. Most of the synthesized 1H-pyrazolo[3,4-d]pyrimidine compounds displayed moderate to high potent activity in both enzymatic and cellular proliferation assays. Among these compounds, 9e, 9g, 9m and 9u showed remarkably high inhibitory activities against both BRAFV600E and VEGFR-2 kinase comparable to positive control Sorafenib. Particularly, compound 9u also showed potent anti-proliferative activity against BRAFV600E-expressing A375 (IC50 = 1.74 μM) and H-29 (IC50 = 6.92 μM) as well as VEGFR-2-expressing HUVEC (IC50 = 5.89 μM), which was also comparable to Sorafenib. Furthermore, kinase selectivity profile showed that 9u had almost poor or no significant inhibitory activity against wild-type BRAF and 15 other tested protein kinases. Flow cytometric analysis showed that compound 9u mainly arrested the A375 and HUVEC cell lines in the G0/G1 stage with a concentration-dependent effect. In addition, the molecular docking and molecular dynamics simulations suggested that 9u adopted a similar binding pattern with Sorafenib at the ATP-binding sites of BRAFV600E and VEGFR-2. Taken together, these results indicated that compound 9u may serve as novel lead compound in research on more effective BRAFV600E and VEGFR-2 dual inhibitors.

Investigation on the binding mechanism of loratinib with the c-ros oncogene 1 (ROS1) receptor tyrosine kinase via molecular dynamics simulation and binding free energy calculations

The c-ros oncogene 1 (ROS1) has proven to be an important cancer target for the treatment of various human cancers. The anaplastic lymphoma kinase inhibitor crizotinib has been granted approval for the treatment of patients with ROS1 positive metastatic non-small-cell lung cancer by the Food and Drug Administration on 2016. However, serious resistance due to the secondary mutation of glycine 2032 to arginine (G2032R) was developed in clinical studies. Loratinib (PF-06463922), a macrocyclic analog of crizotinib, showed significantly improved inhibitory activity against wild–type (WT) ROS1 and ROS1G2032R mutant. To provide insights into the inhibition mechanism, molecular dynamics simulations and free energy calculations were carried out for the complexes of loratinib with WT and G2032R mutated ROS1. The apo-ROS1WT and apo-ROS1G2032R systems showed similar RMSF distributions, while ROS1G2032R-loratinib showed significantly higher than that of WT ROS1-loratinib, which revealed that the binding of loratinib to ROS1G2032R significantly interfered the fluctuation of protein. Calculations of binding free energies indicate that G2032R mutation significantly reduces the binding affinity of loratinib for ROS1, which arose mostly from the increase of conformation entropy and the decrease of solvation energy. Furthermore, detailed per-residue binding free energies highlighted the increased and decreased contributions of some residues in the G2032R mutated systems. The present study revealed the detailed inhibitory mechanism of loratinib as potent WT and G2032R mutated ROS1 inhibitor, which was expected to provide a basis for rational drug design.

Three Dimensional Quantitative Structure-Activity Relationship of 5H-Pyrido[4,3-b]indol-4-carboxamide JAK2 Inhibitors

Janus kinase 2 (JAK2) is an intracellular nonreceptor tyrosine kinase that belongs to the JAK family of kinases, which play an important role in survival, proliferation, and differentiation of a variety of cells. JAK2 inhibitors are potential drugs for the treatment of myeloproliferative neoplasms. The three dimensional quantitative structure-activity relationships have been studied on a series of JAK2 inhibitors by comparative molecular field analysis (CoMFA), and comparative molecular similarity indices analysis (CoMSIA). The CoMFA model had a cross-validated coefficient q2 of 0.633, and the relation non-cross-validated coefficient r2 of 0.976. The F value is 225.030. The contributions of steric and electrostatic fields to the activity are 55.2% and 44.8%, respectively. For the CoMSIA study, the q2, r2, and F values of the model are 0.614, 0.929, and 88.771, respectively. The contributions of steric, electrostatic, hydrophobic, hydrogen bond donor, and hydrogen bond donor fields to the activity are 27.3%, 23.9%, 16.4%, 21.7%, and 10.7%, respectively. The CoMFA and CoMSIA models showed strong predictive ability, and the 3D contour plots give the basis on the structure modification of JAK2 inhibitors.