Identification of New EGFR Inhibitors by Structure-Based Virtual Screening and Biological Evaluation.

Epidermal growth factor receptor (EGFR) inhibitors have been used in clinical for the treatment of non-small-cell lung cancer for years. However, the emergence of drug resistance continues to be a major problem. To identify potential inhibitors, molecular docking-based virtual screening was conducted on ChemDiv and Enamine commercial databases using the Glide program. After multi-step VS and visual inspection, a total of 23 compounds with novel and varied structures were selected, and the predicted ADMET properties were within the satisfactory range. Further molecular dynamics simulations revealed that the reprehensive compound ZINC49691377 formed a stable complex with the allosteric pocket of EGFR and exhibited conserved hydrogen bond interactions with Lys 745 and Asp855 of EGFR over the course of simulation. All compounds were further tested in experiments. Among them, the most promising hit ZINC49691377 demonstrated excellent anti-proliferation activity against H1975 and PC-9 cells, while showing no significant anti-proliferation activity against A549 cells. Meanwhile, apoptosis analysis indicated that the compound ZINC49691377 can effectively induce apoptosis of H1975 and PC-9 cells in a dose-dependent manner, while having no significant effect on the apoptosis of A549 cells. The results indicate that ZINC49691377 exhibits good selectivity. Based on virtual screening and bioassays, ZINC4961377 can be considered as an excellent starting point for the development of new EGFR inhibitors.

Discovery and Computational Studies of Potent Covalent Kinase Inhibitors with α-Substituent Electrophiles Targeting Cysteine.

Both reversible noncovalent inhibitors and irreversible covalent inhibitors targeting tyrosine kinases have their disadvantages. The reversible covalent inhibitors with electrophilic group cyanoacrylamide as warheads reacting with cysteine residues could solve the dilemmas. However, there are still several unresolved issues regarding the electrophilic groups. In this manuscript, a series of EGFR inhibitors with double electron-withdrawing substituents introduced into the Cα position on the olefin bond were designed and synthesized. The binding structures and characteristics of inhibitors with the kinase in both the first noncovalent binding phase and the second covalent binding step were explored and combined with molecular docking and molecular dynamics simulations. Then, the reverse ß-elimination reactions of the thiol-Michael adducts were investigated by applying density functional theory calculations. In addition, the effects of different electrophilic substituents of Cα on the binding between the inhibitors and kinase were elucidated. The results suggested that the electrophilicity and size of the electron-withdrawing groups play an important role in the specific interactions during the reaction. The compounds with the electron-withdrawing groups that had medium electrostatic and steric complementarity to the kinase active site could cooperatively stabilize the complexes and showed relatively good potent activities in the kinase assay experiment. The mechanical and structural information in this study could enhance our understanding of the functioning of the electron-withdrawing groups in the covalent inhibitors. The results might help to design efficient cysteine targeting inhibitors in the future.

Discovery of novel 2-aminopyridine derivatives as ROS1 and ALK dual inhibitors to combat drug-resistant mutants including ROS1G2032R and ALKG1202R.

Clinical treatment by FDA-approved ROS1/ALK inhibitor Crizotinib significantly improved the therapeutic outcomes. However, the emergence of drug resistance, especially driven by acquired mutations, have become an inevitable problem and worsened the clinical effects of Crizotinib. To combat drug resistance, some novel 2-aminopyridine derivatives were designed rationally based on molecular simulation, then synthesised and subjected to biological test. The preferred spiro derivative C01 exhibited remarkable activity against CD74-ROS1G2032R cell with an IC50 value of 42.3 nM, which was about 30-fold more potent than Crizotinib. Moreover, C01 also potently inhibited enzymatic activity against clinically Crizotinib-resistant ALKG1202R, harbouring a 10-fold potency superior to Crizotinib. Furthermore, molecular dynamic disclosed that introducing the spiro group could reduce the steric hindrance with bulky side chain (Arginine) in solvent region of ROS1G2032R, which explained the sensitivity of C01 to drug-resistant mutant. These results indicated a path forward for the generation of anti Crizotinib-resistant ROS1/ALK dual inhibitors.

Discovery of 4-methyl-3-(pyridin-2-ylamino)benzamide derivatives as C-Abl inhibitors with potential neuroprotective effect.

C-Abl is involved in various biological processes and plays an important role in neurodegenerative diseases, especially Parkinson's disease (PD). Previous studies have found that nilotinib shows a neuroprotective effect in cell and animal models of PD by inhibiting the activation of c-Abl. But the low blood-brain barrier permeability and potential toxicity limit the further use of nilotinib in PD. Based on molecular modeling studies, a series of 4-methyl-3-(pyridin-2-ylamino)benzamide derivatives were designed and synthesized. In particular, compound 9a exhibited significant inhibitory activity against c-Abl and a potent neuroprotective effect against MPP+-induced SH-SY5Y cell death. Moreover, 9a not only displayed lower cell toxicity compared with nilotinib, but also showed higher oral bioavailability and proper permeability of the blood-brain barrier. This paper provides 4-methyl-3-(pyridin-2-ylamino)benzamide derivatives as a new scaffold for c-Abl inhibitor with potential neuroprotective effect.

Novel natural scaffold as hURAT1 inhibitor identified by 3D-shape-based, docking-based virtual screening approach and biological evaluation.

As a promising therapeutic target for gout, hURAT1 has attracted increasing attention. In this work, we identified a novel scaffold of hURAT1 inhibitors from a personal natural product database of verified herb-treated gout. First, we constructed more than 800 natural compounds from Chinese medicine that were verified to treat gout. Following the application of both shape-based and docking-based virtual screening (VS) methods, taking into account the shape similarity and flexibility of the target, we identified isopentenyl dihydroflavones that might inhibit hURAT1. Specifically, 9 compounds with commercial availability were tested with biochemical assays for the inhibition of 14C-uric acid uptake in high-expression hURAT1 cells (HEK293-hURAT1), and their structure-activity relationship was evaluated. As a result, 8-isopentenyl dihydroflavone was identified as a novel scaffold of hURAT1 inhibitors since isobavachin (DHF3) inhibited hURAT1 with an IC50 value of 0.39 ± 0.17 µM, which was comparable to verinurad with an IC50 value of 0.32 ± 0.23 µM. Remarkably, isobavachin also displayed an eminent effect in the decline of serum uric acid in vivo experiments. Taken together, isobavachin is a promising candidate for the treatment of hyperuricemia and gout.

Identification of imidazo[4,5-c]pyridin-2-one derivatives as novel Src family kinase inhibitors against glioblastoma.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour in the central nervous system (CNS). As the ideal targets for GBM treatment, Src family kinases (SFKs) have attracted much attention. Herein, a new series of imidazo[4,5-c]pyridin-2-one derivatives were designed and synthesised as SFK inhibitors. Compounds 1d, 1e, 1q, 1s exhibited potential Src and Fyn kinase inhibition in the submicromolar range, of which were next tested for their antiproliferative potency on four GBM cell lines. Compound 1s showed effective activity against U87, U251, T98G, and U87-EGFRvIII GBM cell lines, comparable to that of lead compound PP2. Molecular dynamics (MDs) simulation revealed the possible binding patterns of the most active compound 1s in ATP binding site of SFKs. ADME prediction suggested that 1s accord with the criteria of CNS drugs. These results led us to identify a novel SFK inhibitor as candidate for GBM treatment.

Mibefradil and Flunarizine, Two T-Type Calcium Channel Inhibitors, Protect Mice against Lipopolysaccharide-Induced Acute Lung Injury.

Recent studies have illuminated that blocking Ca2+ influx into effector cells is an attractive therapeutic strategy for lung injury. We hypothesize that T-type calcium channel may be a potential therapeutic target for acute lung injury (ALI). In this study, the pharmacological activity of mibefradil (a classical T-type calcium channel inhibitor) was assessed in a mouse model of lipopolysaccharide- (LPS-) induced ALI. In LPS challenged mice, mibefradil (20 and 40 mg/kg) dramatically decreased the total cell number, as well as the productions of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF). Mibefradil also suppressed total protein concentration in BALF, attenuated Evans blue extravasation, MPO activity, and NF-κB activation in lung tissue. Furthermore, flunarizine, a widely prescripted antimigraine agent with potent inhibition on T-type channel, was also found to protect mice against lung injury. These data demonstrated that T-type calcium channel inhibitors may be beneficial for treating acute lung injury. The important role of T-type calcium channel in the acute lung injury is encouraged to be further investigated.

Protective effects of S-carvedilol on doxorubicin-induced damages to human umbilical vein endothelial cells and rats.

Doxorubicin (DOX) is a highly active anticancer drug with severe cytotoxicity, which is strongly associated with oxidative stress. Carvedilol (CAR), used as its racemate with S-CAR and R-CAR (1:1), has been previously reported to ameliorate the DOX-induced cytotoxicity. However, the main contributor from CAR of its protective effects has not been clear. Therefore, in this study, we aimed to investigate further the different effects of CAR enantiomers on DOX-induced cytotoxicity in human umbilical vein endothelial cells and rats, respectively. Results indicated that S-CAR could significantly attenuate DOX-induced cell death, apoptotic morphological changes, decrease the mitochondrial membrane potential and oxidative stress responses by increasing the superoxide dismutase and catalase activities, and decreasing malondialdehyde contents and reactive oxygen species levels via the phosphoinositide 3-kinase/AKT/endothelial nitric oxide synthase pathway in vitro. Consistent with the in vitro study, the protective effects of S-CAR on the myocardial tissues and hemodynamics were also detected in rats suffering because of DOX treatment. With the obtained results, we can first conclude that S-CAR provides superior protection to injury induced by DOX relative to that of racemic CAR and R-CAR.

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.

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.

Standardized myrtol attenuates lipopolysaccharide induced acute lung injury in mice.

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.

Discovery of Novel Small-Molecule-Based Potential PD-L1/EGFR Dual Inhibitors with High Druggability for Glioblastoma Immunotherapy.

Based on the close relationship between programmed death protein ligand 1 (PD-L1) and epidermal growth factor receptor (EGFR) in glioblastoma (GBM), we designed and synthesized a series of small molecules as potential dual inhibitors of EGFR and PD-L1. Among them, compound EP26 exhibited the highest inhibitory activity against EGFR (IC50 = 37.5 nM) and PD-1/PD-L1 interaction (IC50 = 1.77 µM). In addition, EP26 displayed superior in vitro antiproliferative activities and in vitro immunomodulatory effects by promoting U87MG cell death in a U87MG/Jurkat cell coculture model. Furthermore, EP26 possessed favorable pharmacokinetic properties (F = 22%) and inhibited tumor growth (TGI = 92.0%) in a GBM mouse model more effectively than Gefitinib (77.2%) and NP19 (82.8%). Moreover, EP26 increased CD4+ cells and CD8+ cells in tumor microenvironment. Collectively, these results suggest that EP26 represents the first small-molecule-based PD-L1/EGFR dual inhibitor deserving further investigation as an immunomodulating agent for cancer treatment.

Development and validation of a liquid chromatography-tandem mass spectrometry method for topotecan determination in beagle dog plasma and its application in a bioequivalence study.

Topotecan (TPT) is an important anti-cancer drug that inhibits topoisomerase I. A sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that potentially determines TPT in beagle dog plasma is needed for a bioequivalence study of TPT formulations. We developed and validated LC-MS/MS to evaluate TPT in beagle dog plasma in terms of specificity, linearity, precision, accuracy, stability, extraction recovery and matrix effect. Plasma samples were treated with an Ostro(TM) sorbent plate (a robust and effective tool) to eliminate phospholipids and proteins before analysis. TPT and camptothecin (internal standard) were separated on an Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 50 mm) with 0.1% formic acid and methanol as the mobile phase at a flow rate of 0.25 mL/min. TPT was analyzed using positive ion electrospray ionization in multiple-reaction monitoring mode. The obtained lower limit of quantitation was 1 ng/mL (signal-to-noise ratio > 10). The standard calibration curve for TPT was linear (correlation coefficient > 0.99) at the concentration range of 1-400 ng/mL. The intra-day and inter-day precision, accuracy, stability, extraction recovery and matrix effect of TPT were within the acceptable limits. The validated method was successfully applied in a bioequivalence study of TPT in healthy beagle dogs.

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.

A Side-Effect-Free Interventional Therapy for Precisely Eliminating Unresectable Cancer Pain.

Of patients bearing unresectable tumors at advanced stages, most undergo serious pain. For unresectable tumors adjacent to vital organs or nerves, eliminating local cancer pain without adverse effects remains a formidable challenge. Interventional ablative therapies (IATs), such as radio frequency ablation (RFA), microwave ablation, and irreversible electroporation, have been clinically adopted to treat various carcinomas. In this study, we established another palliative interventional therapy to eliminate local cancer pain, instead of relieving nociception temporarily. Here, we developed another interventional ablative therapy (termed nanoparticle-mediated microknife ablation) to locoregionally eliminate cancer pain and tumors. The IAT system was composed of self-assembled nanodrugs, infusion catheters, puncture needles, injection pump, and an empirical tumor ablation formula. Notably, the ablation formula established in the IAT system enables us to predict the essential nanoparticle (NP) numbers used for completely destroying tumors. In a mouse model of cancer pain, tumor-targeted nanodrugs made of Paclitaxel and Hematoporphyrin, which have an extremely high drug-loading efficiency (more than 60%), were infused into tumors through injection pumps under imaging guidance. In conclusion, when compared to classic chemotherapeutic agents, IAT showed significantly higher effectiveness in cancer pain removal. It also presented no damage to the nervous, sensory, and motor capabilities of the treated mice. All of these merits resulted from NPs' long-lasting retention, targeted ablation, and confined diffusion in tumor stroma. Therefore, this safe treatment modality has great potential to eradicate local cancer pain in the clinic.

Structure Modification of Ginsenoside Rh2 and Cytostatic Activity on Cancer Cells.

Ginsenoside Rh2 (Rh2) is one of the most effective anticancer components extracted from red ginseng, but the poor solubility limits its clinical application. In this paper, ginsenoside Rh2 was modified with maleimidocaproic acid or maleimidoundecanoic acid with functional groups at both ends. The structures of derivatives were determined by analysis of 1D and 2D nuclear magnetic resonance, Fourier transform infrared, and high-resolution mass spectrometry. Antiproliferative cell experiments showed that Rh2 modified with maleimidocaproic acid (C-Rh2) displayed higher cytostatic activity against different tumor cells compared with Rh2, while Rh2 modified with maleimidoundecanoic acid (U-Rh2) did not exhibit obvious cytotoxicity. The results suggest that the length of the spacer arm may play an important role in the cytostatic activity of the Rh2 derivatives.

Mechanism of anti-hyperuricemia of isobavachin based on network pharmacology and molecular docking.

BACKGROUND: Hyperuricemia is a more popular metabolic disease caused by a disorder of purine metabolism. Our previous study firstly screened out a natural product Isobavachin as anti-hyperuricemia targeted hURAT1 from a Chinese medicine Haitongpi (Cortex Erythrinae). In view of Isobavachin's diverse pharmacological activities, similar to the Tranilast (as another hURAT1 inhibitor), our study focused on its potential targets and molecular mechanisms of Isobavachin anti-hyperuricemia based on network pharmacology and molecular docking. METHODS: First of all, the putative target genes of compounds were screen out based on the public databases with different methods, such as SwissTargetPerdiction, PharmMapper and TargetNet,etc. Then the compound-pathways were obtained by the compounds' targets gene from David database for Gene Ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis. The cross pathways of compound-pathways and the diseases pathways of hyperuricemia from Comparative Toxicogenomics Database were be considered as the compound-disease pathways. Next, based on the compound-disease pathways and the PPI network, the core targets were identified based on the retrieved disease-genes. Finally, the compound-target-pathway-disease network was constructed by Cytoscape and the mechanism of isobavachin anti-hyperuricemia was discussed based on the network analysis. RESULTS: Our study demonstrated that there were five pathways involved in Isobavachin against hyperuricemia, including Drug metabolism-other enzymes, Metabolic pathways, Bile secretion, Renin-angiotensin system and Renin secretion. Among the proteins involved in these pathways, HPRT1, REN and ABCG2 were identified as the core targets associated with hyperuricemia, which regulated the five pathways mentioned above. It is quite different from that of Tranilast, which involved in the same pathways except Bile secretion instead of purine metabolism. CONCLUSION: This study revealed Isobavachin could regulate the pathways including Drug metabolism-other enzymes, Metabolic pathways, Bile secretion, Renin-angiotensin system, Renin secretion by core targets HPRT1, REN and ABCG2, in the treatment of hyperuricemia effect. Among them, the Bile secretion regulated by ABCG2 probably would be a novel pathway. Our work provided a theoretical basis for the pharmacological study of Isobavachin in lowering uric acid and further basic research.

A Novel and Highly Selective Epidermal Growth Factor Receptor Inhibitor, SMUZ106, for the Treatment of Glioblastoma.

Targeting the epidermal growth factor receptor (EGFR) is one of the potential ways to treat glioblastoma (GBM). In this study, we investigate the anti-GBM tumor effects of the EGFR inhibitor SMUZ106 in both in vitro and in vivo conditions. The effects of SMUZ106 on the growth and proliferation of GBM cells were explored through MTT and clone formation experiments. Additionally, flow cytometry experiments were conducted to study the effects of SMUZ106 on the cell cycle and apoptosis of GBM cells. The inhibitory activity and selectivity of SMUZ106 to the EGFR protein were proved by Western blotting, molecular docking, and kinase spectrum screening methods. We also conducted a pharmacokinetic analysis of SMUZ106 hydrochloride following i.v. or p.o. administration to mice and assessed the acute toxicity level of SMUZ106 hydrochloride following p.o. administration to mice. Subcutaneous and orthotopic xenograft models of U87MG-EGFRvIII cells were established to assess the antitumor activity of SMUZ106 hydrochloride in vivo. SMUZ106 could inhibit the growth and proliferation of GBM cells, especially for the U87MG-EGFRvIII cells with a mean IC50 value of 4.36 µM. Western blotting analyses showed that compound SMUZ106 inhibits the level of EGFR phosphorylation in GBM cells. It was also shown that SMUZ106 targets EGFR and presents high selectivity. In vivo, the absolute bioavailability of SMUZ106 hydrochloride was 51.97%, and its LD50 exceeded 5000 mg/kg. SMUZ106 hydrochloride significantly inhibited GBM growth in vivo. Furthermore, SMUZ106 inhibited the activity of U87MG-resistant cells induced by temozolomide (TMZ) (IC50: 7.86 µM). These results suggest that SMUZ106 hydrochloride has the potential to be used as a treatment method for GBM as an EGFR inhibitor.

Design and synthesis of proteolysis targeting chimeras (PROTACs) as an EGFR degrader based on CO-1686.

Epidermal growth factor receptor (EGFR) inhibitors represent the first-line treatment of non-small-cell lung cancer (NSCLC). However, the emergence of acquired drug resistance and side effects largely encumbered their application in clinic. The emerging technology proteolysis targeting chimera (PROTAC) could be an alternative strategy to overcome these problems. Herein, we reported the discovery of EGFRL858R/T790M degraders based on CO-1686. Promising PROTAC 1q could effectively and selectively inhibit the growth of PC-9 (EGFRDel 19) and H1975 (EGFRL858R/T790M) cells, but not that of A549 (EGFRWT) cells. In addition, 1q could time- and dose-dependently induce degradation of EGFRL858R/T790M in H1975 cells with a DC50 value of 355.9 nM, while did not show obvious effect on the EGFRDel 19 and EGFRWT protein. Preliminary mechanism study demonstrated that the protein degradation was mediated through ubiquitin-proteasome system (UPS). Furthermore, 1q could significantly induce the apoptosis of H1975 cells and arrest the cells in G0/G1 phase. These findings demonstrated that compound 1q could be used as initial lead compound for the development of new EGFRL858R/T790M degraders based therapy.

Molecular mechanism study of EGFR allosteric inhibitors using molecular dynamics simulations and free energy calculations.

ABTRACTThe epidermal growth factor receptor (EGFR) kinase inhibitors Gefitinib, Erlotinib, Afatinib and Osimertinib have been approved for the treatments of non-small cell lung cancer patients harboring sensitive EGFR mutations, but resistance arises rapidly. To date all approved EGFR inhibitors are ATP-competitive inhibitors, highlighting the need for therapeutic agents with alternative mechanisms of action. Allosteric kinase inhibitors offer a promising new therapeutic strategy to ATP-competitive inhibitors. The mutant-selective allosteric EGFR inhibitors EAI045 exhibited higher potency for EGFRL858R&T790M compared to WT, which was also effective in EGFR-mutant models including those harboring the C797S mutation. However, it was not effective as a single-agent inhibitor, and require the co-administration of the anti-EGFR antibody Cetuximab. Further efforts produced a more potent analog JBJ-04-125-02, which can inhibit cell proliferation as a single-agent inhibitor. In the present study, molecular dynamics simulations and free energy calculations were performed and revealed the detailed inhibitory mechanism of JBJ-04-125-02 as more potent EGFR inhibitor. Moreover, the energy difference between HOMO and LUMO calculated by DFT implied the higher interaction of JBJ-04-125-02 than EAI045 in the active site of the EGFR. The identified key features obtained from the molecular modeling enabled us to design novel EGFR allosteric inhibitors.Communicated by Ramaswamy H. Sarma.