Hybrid Pharmacophore- and Structure-Based Virtual Screening Pipeline to Identify Novel EGFR Inhibitors That Suppress Non-Small Cell Lung Cancer Cell Growth

Dysregulated epidermal growth factor receptor (EGFR) expression is frequently observed in non-small cell lung cancer (NSCLC) growth and metastasis. Despite recent successes in the development of tyrosine kinase inhibitors (TKIs), inevitable resistance to TKIs has led to urgent calls for novel EGFR inhibitors. Herein, we report a rational workflow used to identify novel EGFR-TKIs by combining hybrid ligand- and structure-based pharmacophore models. Three types of models were developed in this workflow, including 3D QSAR-, common feature-, and structure-based EGFR-TK domain-containing pharmacophores. A National Cancer Institute (NCI) compound dataset was adopted for multiple-stage pharmacophore-based virtual screening (PBVS) of various pharmacophore models. The six top-scoring compounds were identified through the PBVS pipeline coupled with molecular docking. Among these compounds, NSC609077 exerted a significant inhibitory effect on EGFR activity in gefitinib-resistant H1975 cells, as determined by an enzyme-linked immunosorbent assay (ELISA). Further investigations showed that NSC609077 inhibited the anchorage-dependent growth and migration of lung cancer cells. Furthermore, NSC609077 exerted a suppressive effect on the EGFR/PI3K/AKT pathway in H1975 cells. In conclusion, these findings suggest that hybrid virtual screening may accelerate the development of targeted drugs for lung cancer treatment.     

EGFR(T790M)抑制剂的设计、合成及活性评价

表皮生长因子受体(EGFR)的T790M突变最为频发,也是肺癌临床治疗失败的主要原因之一。鉴于先导化合物B6优良的抗H1975细胞系和异植瘤活性,对其进行了EGFR(T790M)激酶抑制活性的确认,并使用Autodock软件确认了两者的相互作用。以EGFR(T790M)为靶点对B6进行定向结构修饰,所得目标化合物经NMR和MS表征后,联合体外激酶、细胞生物活性与Autodock软件解释它们的构效关系。结果表明,3-(苯并[d][1,3]二氧杂-5-基)-1-(1-(乙烯基磺酰基)哌啶-4-基)-1H-吡唑并[3,4-d]嘧啶-4-胺的抗H1975细胞增殖活性(IC₅₀=(1.16±0.24)μmol/L)与B6(IC₅₀=(0.91±0.36)μmol/L)相似,尽管其对EGFR(T790M)的抑制活性(IC₅₀=(148.2±7.2)nmol/L)不如B6(IC₅₀=(22.0±2.6)nmol/L)。以7H-吡咯并[2,3-d]嘧啶-4-胺为母核,取代基分别为胡椒环基和4-取代哌啶基者可开发活性更优的EGFR(T790M)抑制剂,指导后期研究。     

Targeting an EGFR Water Network with 4-Anilinoquin(az)oline Inhibitors for Chordoma

Quinoline- and quinazoline-based kinase inhibitors of the epidermal growth factor receptor (EGFR) have been used to target non-small cell lung cancer (NSCLC) and chordomas with varying amounts of success. We designed and prepared compounds to probe several key structural features including an interaction with Asp855 within the EGFR DGF motif and interactions with the active site water network. EGFR target engagement was then evaluated in a cellular assay, with the inhibitors then profiled in representative cellular models of NSCLC and chordomas. In addition, structure–activity relationship insight into EGFR inhibitor design with potent dimethoxyquin(az)olines identified compounds 1 [N-(3-ethynylphenyl)-6,7-dimethoxyquinolin-4-amine], 4 [N-(3-ethynylphenyl)-6,7-dimethoxyquinazolin-4-amine], and 7 [4-((3-ethynylphenyl)amino)-6,7-dimethoxyquinoline-3-carbonitrile]. We also identified 6,7-dimethoxy-N-(4-((4-methylbenzyl)oxy)phenyl)quinolin-4-amine (compound 18 ), which is the most potent inhibitor (IC₅₀=310 nm ) of the UCH-2 chordoma cell line to date.     

Synthesis,Molecular Docking and Biological Characterization of Pyrazine Linked 2-Aminobenzamides as New Class I Selective Histone Deacetylase (HDAC) Inhibitors with Anti-Leukemic Activity

Class I histone deacetylases (HDACs) are key regulators of cell proliferation and they are frequently dysregulated in cancer cells. We report here the synthesis of a novel series of class-I selective HDAC inhibitors (HDACi) containing a 2-aminobenzamide moiety as a zinc-binding group connected with a central (piperazin-1-yl)pyrazine or (piperazin-1-yl)pyrimidine moiety. Some of the compounds were additionally substituted with an aromatic capping group. Compounds were tested in vitro against human HDAC1, 2, 3, and 8 enzymes and compared to reference class I HDACi (Entinostat (MS-275), Mocetinostat, CI994 and RGFP-966). The most promising compounds were found to be highly selective against HDAC1, 2 and 3 over the remaining HDAC subtypes from other classes. Molecular docking studies and MD simulations were performed to rationalize the in vitro data and to deduce a complete structure activity relationship (SAR) analysis of this novel series of class-I HDACi. The most potent compounds, including 19f, which blocks HDAC1, HDAC2, and HDAC3, as well as the selective HDAC1/HDAC2 inhibitors 21a and 29b, were selected for further cellular testing against human acute myeloid leukemia (AML) and erythroleukemic cancer (HEL) cells, taking into consideration their low toxicity against human embryonic HEK293 cells. We found that 19f is superior to the clinically tested class-I HDACi Entinostat (MS-275). Thus, 19f is a new and specific HDACi with the potential to eliminate blood cancer cells of various origins.     

The Histone Deacetylase Inhibitor BML-210 Influences Gene and Protein Expression in Human Promyelocytic Leukemia NB4 Cells via Epigenetic Reprogramming

Today, cancer is understood as an epigenetic as well as genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. Proteins such as histone deacetylases (HDACs) that cause modifications of histones and other proteins can be targets for novel anticancer agents. Recently, interest in compounds that can inhibit HDACs increased, and now there are many HDACs inhibitors (HDACIs) available with different chemical structures, biological and biochemical properties; hopefully some of them will succeed, probably in combination with other agents, in cancer therapies. In our study we focused on the novel HDACI–BML-210. We found that BML-210 (N-phenyl-Nʹ-(2-Aminophenyl)hexamethylenediamide) inhibits the growth of NB4 cells in dose- and time-dependent manner. In this study we also examined how expression and activity of HDACs are affected after leukemia cell treatment with BML-210. Using a mass spectrometry method we identified proteins that changed expression after treatment with BML-210. We prepared RT-PCR analysis of these genes and the results correlated with proteomic data. Based on these and other findings from our group, we suggest that HDACIs, like BML-210, can be promising anticancer agents in promyelocytic leukemia treatment.     

Synthesis and Biological Characterization of Amidopropenyl Hydroxamates as HDAC Inhibitors

A series of amidopropenyl hydroxamic acid derivatives were prepared as novel inhibitors of human histone deacetylases (HDACs). Several compounds showed potency at <100 nM in the HDAC inhibition assays, sub‐micromolar IC₅₀ values in tests against three tumor cell lines, and remarkable stability in human and mouse microsomes was observed. Three representative compounds were selected for further characterization and submitted to a selectivity profile against a series of class I and class II HDACs as well as to preliminary in vivo pharmacokinetic (PK) experiments. Despite their high microsomal stability, the compounds showed medium‐to‐high clearance rates in in vivo PK studies as well as in rat and human hepatocytes, indicating that a major metabolic pathway is catalyzed by non‐microsomal enzymes.     

Potent and Selective Non‐hydroxamate Histone Deacetylase 8 Inhibitors

Specific inhibition of histone deacetylase 8 (HDAC8) has been suggested as a promising option for the treatment of neuroblastoma and T‐cell malignancies. A novel class of highly potent and selective HDAC8 inhibitors with a pyrimido[1,2‐c][1,3]benzothiazin‐6‐imine scaffold was studied that is completely different from the traditional concept of HDAC inhibitors comprising a zinc binding group (ZBG), in most cases a hydroxamate group, a spacer, and a capping group that may interact with the surface of the target protein. Although lacking a ZBG, some of the new compounds were shown to have outstanding potency against HDAC8 in the single‐digit nanomolar range. The pyrimido[1,2‐c][1,3]benzothiazin‐6‐imines also inhibited the growth of solid and hematological tumor cells. The small size and beneficial physicochemical properties of the novel HDAC inhibitor class underline the high degree of drug likeness. This and the broad structure–activity relationship suggest great potential for the further development of compounds with the pyrimido[1,2‐c][1,3]benzothiazin‐6‐imine scaffold into innovative and highly effective therapeutic drugs against cancer.     

A Combination Strategy to Inhibit Pim‐1: Synergism between Noncompetitive and ATP‐Competitive Inhibitors

Pim‐1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim‐1 inhibitors, a previously identified ATP‐competitive indolyl‐pyrrolone scaffold was expanded to derive structure–activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP‐competitive inhibitors as well as a series of 2‐aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim‐1. Notably, further evaluation of the 2‐aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP‐competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP‐competitive and ATP‐noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim‐1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim‐1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim‐1 is associated with chemotherapeutic resistance.     

The Role of EREG/EGFR Pathway in Tumor Progression

Aberrant activation of the epidermal growth factor receptor (EGFR/ERBB1) by erythroblastic leukemia viral oncogene homolog (ERBB) ligands contributes to various tumor malignancies, including lung cancer and colorectal cancer (CRC). Epiregulin (EREG) is one of the EGFR ligands and is low expressed in most normal tissues. Elevated EREG in various cancers mainly activates EGFR signaling pathways and promotes cancer progression. Notably, a higher EREG expression level in CRC with wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS) is related to better efficacy of therapeutic treatment. By contrast, the resistance of anti-EGFR therapy in CRC was driven by low EREG expression, aberrant genetic mutation and signal pathway alterations. Additionally, EREG overexpression in non-small cell lung cancer (NSCLC) is anticipated to be a therapeutic target for EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, recent findings indicate that EREG derived from macrophages promotes NSCLC cell resistance to EGFR-TKI treatment. The emerging events of EREG-mediated tumor promotion signals are generated by autocrine and paracrine loops that arise from tumor epithelial cells, fibroblasts, and macrophages in the tumor microenvironment (TME). The TME is a crucial element for the development of various cancer types and drug resistance. The regulation of EREG/EGFR pathways depends on distinct oncogenic driver mutations and cell contexts that allows specific pharmacological targeting alone or combinational treatment for tailored therapy. Novel strategies targeting EREG/EGFR, tumor-associated macrophages, and alternative activation oncoproteins are under development or undergoing clinical trials. In this review, we summarize the clinical outcomes of EREG expression and the interaction of this ligand in the TME. The EREG/EGFR pathway may be a potential target and may be combined with other driver mutation targets to combat specific cancers.     

Identification of HDAC6‐Selective Inhibitors of Low Cancer Cell Cytotoxicity

The histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform‐selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first‐generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell‐cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole‐based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1–3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of class 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.     

Preliminary Evidence on the Diagnostic and Molecular Role of Circulating Soluble EGFR in Non-Small Cell Lung Cancer

Assessment of biological diagnostic factors providing clinically-relevant information to guide physician decision-making are still needed for diseases with poor outcomes, such as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a promising molecule in the clinical management of NSCLC. While the EGFR transmembrane form has been extensively investigated in large clinical trials, the soluble, circulating EGFR isoform (sEGFR), which may have a potential clinical use, has rarely been considered. This study investigates the use of sEGFR as a potential diagnostic biomarker for NSCLC and also characterizes the biological function of sEGFR to clarify the molecular mechanisms involved in the course of action of this protein. Plasma sEGFR levels from a heterogeneous cohort of 37 non-advanced NSCLC patients and 54 healthy subjects were analyzed by using an enzyme-linked immunosorbent assay. The biological function of sEGFR was analyzed in vitro using NSCLC cell lines, investigating effects on cell proliferation and migration. We found that plasma sEGFR was significantly decreased in the NSCLC patient group as compared to the control group (median value: 48.6 vs. 55.6 ng/mL respectively; p = 0.0002). Moreover, we demonstrated that sEGFR inhibits growth and migration of NSCLC cells in vitro through molecular mechanisms that included perturbation of EGF/EGFR cell signaling and holoreceptor internalization. These data show that sEGFR is a potential circulating biomarker with a physiological protective role, providing a first approach to the functional role of the soluble isoform of EGFR. However, the impact of these data on daily clinical practice needs to be further investigated in larger prospective studies.     

Salicylketoximes That Target Glucose Transporter 1 Restrict Energy Supply to Lung Cancer Cells

The glucose transporter GLUT1 is frequently overexpressed in most tumor tissues because rapidly proliferating cancer cells rely primarily on glycolysis, a low‐efficiency metabolic pathway that necessitates a very high rate of glucose consumption. Because blocking GLUT1 is a promising anticancer strategy, we developed a novel class of GLUT1 inhibitors based on the 4‐aryl‐substituted salicylketoxime scaffold. Some of these compounds are efficient inhibitors of glucose uptake in lung cancer cells and have a notable antiproliferative effect. In contrast to their 5‐aryl‐substituted regioisomers, the newly synthesized compounds reported herein do not display significant binding to the estrogen receptors. The inhibition of glucose uptake in cancer cells by these compounds was further observed by fluorescence microscopy imaging using a fluorescent analogue of glucose. Therefore, blocking the ability of tumor cells to take up glucose by means of these small molecules, or by further optimized derivatives, may be a successful approach in the development of novel anticancer drugs.     

Synthesis and cytotoxicity of cyanoborane adducts of n6-benzoyladenine and 6-triphenylphosphonylpurine

N6-Benzoyladenine-cyanoborane (2), and 6-triphenylphosphonylpurine-cyanoborane (3) were selected for investigation of cytotoxicity in murine and human tumor cell lines, effects on human HL-60 leukemic metabolism and DNA strand scission to determine the feasibility of these compounds as clinical antineoplastic agents. Compounds 2 and 3 both showed effective cytotoxicity based on ED(50) values less than 4 mug/ml for L1210, P388, HL-60, Tmolt(3), HUT-78, HeLa-S(3) uterine, ileum HCT-8, and liver Hepe-2. Compound 2 had activity against ovary 1-A9, while compound 3 was only active against prostate PL and glioma UM. Neither compound was active against the growth of lung 549, breast MCF-7, osteosarcoma HSO, melanoma SK2, KB nasopharynx, and THP-1 acute monocytic leukemia. In mode of action studies in human leukemia HL-60 cells, both compounds demonstrated inhibition of DNA and protein syntheses after 60 min at 100 muM. These compounds inhibited RNA synthesis to a lesser extent. The utilization of the DNA template was suppressed by the compounds as determined by inhibition of the activities of DNA polymerase alpha, m-RNA polymerase, r-RNA polymerase and t-RNA polymerase, which would cause adequate inhibition of the synthesis of both DNA and RNA. Both compounds markedly inhibited dihydrofolate reductase activity, especially in compound 2. The compounds appeared to have caused cross-linking of the DNA strands after 24 hr at 100 muM in HL-60 cells, which was consistent with the observed increased in ct-DNA viscosity after 24 hr at 100 muM. The compounds had no inhibitory effects on DNA topoisomerase I and II activities or DNA-protein linked breaks. Neither compound interacted with the DNA molecule itself through alkylation of the nucleotide bases nor caused DNA interculation between base pairs. Overall, these antineoplastic agents caused reduction of DNA and protein replication, which would lead to killing of cancer cells.     

Discovery of Pyridone‐Based Histone Deacetylase Inhibitors: Approaches for Metabolic Stability

Histone deacetylases (HDACs) are important enzymes in epigenetic regulation and are therapeutic targets for cancer. Most zinc‐dependent HDACs induce proliferation, dedifferentiation, and anti‐apoptotic effects in cancer cells. We designed and synthesized a new series of pyridone‐based HDAC inhibitors that have a pyridone ring in the core structure and a conjugated system with an olefin connecting the hydroxamic acid moiety. Consequently, most of the selected pyridone‐based HDAC inhibitors showed similar or higher inhibition profiles in addition to remarkable metabolic stability against hydrolysis relative to the corresponding lactam‐based HDAC inhibitors. Furthermore, the selectivity of the novel pyridine‐based compounds was evaluated across all of the HDAC isoforms. One of these compounds, (E)‐N‐hydroxy‐3‐{1‐[3‐(naphthalen‐2‐yl)propyl]‐2‐oxo‐1,2‐dihydropyridin‐3‐yl}acrylamide, exhibited the highest level of HDAC inhibition (IC₅₀=0.07 μM ), highly selective inhibition of class I HDAC1 and class II HDAC6 enzymes, metabolic stability in mouse liver microsomal studies, and effective growth inhibition of various cancer cell lines. Docking studies indicated that a long alkyl linker and bulky hydrophobic cap groups affect in vitro activities. Overall, the findings reported herein regarding pyridone‐based HDAC inhibitors can be used to guide future research efforts to develop new and effective anticancer therapeutics.     

GZ17-6.02 Inhibits the Growth of EGFRvIII+ Glioblastoma

Epidermal Growth Factor Receptor (EGFR) is amplified in over 50% of glioblastomas and promotes tumor formation and progression. However, attempts to treat glioblastoma with EGFR tyrosine kinase inhibitors have been unsuccessful thus far. The current standard of care is especially poor in patients with a constitutively active form of EGFR, EGFRvIII, which is associated with shorter survival time. This study examined the effect of GZ17-6.02, a novel anti-cancer agent undergoing phase 1 studies, on two EGFRvIII+ glioblastoma stem cells: D10-0171 and D317. In vitro analyses showed that GZ17-6.02 inhibited the growth of both D10-0171 and D317 cells with IC₅₀ values of 24.84 and 28.28 µg/mL respectively. RNA sequencing and reverse phase protein array analyses revealed that GZ17-6.02 downregulates pathways primarily related to steroid synthesis and cell cycle progression. Interestingly, G17-6.02’s mechanism of action involves the downregulation of the recently identified glioblastoma super-enhancer genes WSCD1, EVOL2, and KLHDC8A. Finally, a subcutaneous xenograft model showed that GZ17-6.02 inhibits glioblastoma growth in vivo. We conclude that GZ17-6.02 is a promising combination drug effective at inhibiting the growth of a subset of glioblastomas and our data warrants further preclinical studies utilizing xenograft models to identify patients that may respond to this drug.