AXL antibody and AXL-ADC mediate antitumor efficacy via targeting AXL in tumor-intrinsic epithelial-mesenchymal transition and tumor-associated M2-like macrophage.

The receptor tyrosine kinase AXL is an emerging driver of cancer recurrence, while its molecular mechanism remains unclear. In this study we investigated how AXL regulated the disease progression and poor prognosis in non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC). We performed AXL transcriptome analysis from TCGA datasets, and found that AXL expression was significantly elevated in NSCLC and TNBC correlating with poor prognosis, epithelial-mesenchymal transition (EMT) and immune-tolerant tumor microenvironment (TME). Knockdown of AXL or treatment with two independent AXL antibodies (named anti-AXL and AXL02) all diminished cell migration and EMT in AXL-high expressing NSCLC and TNBC cell lines. In a mouse model of 4T1 TNBC, administration of anti-AXL antibody substantially inhibited lung metastases formation and growth, accompanied by reduced downstream signaling activation, EMT and proliferation index, as well as an increased apoptosis and activated anti-tumor immunity. We found that AXL was abundantly activated in tumor nodule-infiltrated M2-macrophages. A specific anti-AXL antibody blocked bone marrow-derived macrophage (BMDM) M2-polarization in vitro. Targeting of AXL in M2-macrophage in addition to tumor cell substantially suppressed CSF-1 production and eliminated M2-macrophage in TME, leading to a coordinated enhancement in both the innate and adaptive immunity reflecting M1-like macrophages, mature dendritic cells, cytotoxic T cells and B cells. We generated a novel and humanized AXL-ADC (AXL02-MMAE) employing a site-specific conjugation platform. AXL02-MMAE exerted potent cytotoxicity against a panel of AXL-high expressing tumor cell lines (IC50 < 0.1 nmol/L) and suppressed in vivo growth of multiple NSCLC and glioma tumors (a minimum efficacy dose<1 mg/kg). Compared to chemotherapy, AXL02-MMAE achieved a superior efficacy in regressing large sized tumors, eliminated AXL-H tumor cell-dependent M2-macrophage infiltration with a robust accumulation of inflammatory macrophages and mature dendritic cells. Our results support AXL-targeted therapy for treatment of advanced NSCLC and TNBC.

Discovery of MTR-106 as a highly potent G-quadruplex stabilizer for treating BRCA-deficient cancers.

G-quadruplexes (G4s) are DNA or RNA structures formed by guanine-rich repeating sequences. Recently, G4s have become a highly attractive therapeutic target for BRCA-deficient cancers. Here, we show that a substituted quinolone amide compound, MTR-106, stabilizes DNA G-quadruplexes in vitro. MTR-106 displayed significant antiproliferative activity in homologous recombination repair (HR)-deficient and PARP inhibitor (PARPi)-resistant cancer cells. Moreover, MTR-106 increased DNA damage and promoted cell cycle arrest and apoptosis to inhibit cell growth. Importantly, its oral and i.v. administration significantly impaired tumor growth in BRCA-deficient xenograft mouse models. However, MTR-106 showed modest activity against talazoparib-resistant xenograft models. In rats, the drug rapidly distributes to tissues within 5 min, and its average concentrations were 12-fold higher in the tissues than in the plasma. Overall, we identified MTR-106 as a novel G-quadruplex stabilizer with high tissue distribution, and it may serve as a potential anticancer agent.

Author Correction: Targeting Hsp90 with FS-108 circumvents gefitinib resistance in EGFR mutant non-small cell lung cancer cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A new BET inhibitor, 171, inhibits tumor growth through cell proliferation inhibition more than apoptosis induction.

The bromodomain and extra-terminal domain (BET) family of proteins, especially bromodomain-containing protein 4 (BRD4), has emerged as exciting anti-tumor targets due to their important roles in epigenetic regulation. Therefore, the discovery of BET inhibitors with promising anti-tumor efficacy will provide a novel approach to epigenetic anticancer therapy. Recently, we discovered the new BET inhibitor compound 171, which is derived from a polo-like kinase 1 (PLK1)-BRD4 dual inhibitor based on our previous research. Compound 171 was found to maintain BET inhibition ability without PLK1 inhibition, and there was no selectivity among BET family members. The in vitro and in vivo results both indicated that the overall anti-tumor activity of compound 171 was improved compared with the (+)-JQ-1 or OTX-015 BET inhibitors. Furthermore, we found that compound 171 could regulate the expression of cell cycle-regulating proteins including c-Myc and p21 and induce cell cycle arrest in the G0/G1 phase. However, compound 171 only has a quite limited effect on apoptosis, in considering that apoptosis was only observed at doses greater than 50 µM. To determine the mechanisms underlying cell death, proliferation activity assay was conducted. The results showed that compound 171 induced clear anti-proliferative effects at doses that no obvious apoptosis was induced, which indicated that the cell cycle arresting effect contributed mostly to its anti-tumor activity. The result of this study revealed the anti-tumor mechanism of compound 171, and laid a foundation for the combination therapy in clinical practice, if compound 171 or its series compounds become drug candidates in the future.

Inhibition of the BET family reduces its new target gene IDO1 expression and the production of L-kynurenine.

The bromodomain and extra terminal domain (BET) family members, including BRD2, BRD3, and BRD4, act as epigenetic readers to regulate gene expression. Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that participates in tumor immune escape primarily by catalyzing tryptophan to L-kynurenine. Here, we report that IDO1 is a new target gene of the BET family. RNA profiling showed that compound 9, a new BET inhibitor, reduced IDO1 mRNA up to seven times in Ty-82 cells. IDO1 differentially expressed in tumor cells and its expression could be induced with interferon gamma (IFN-γ). BET inhibitors (ABBV-075, JQ1, and OTX015) inhibited both constitutive and IFN-γ-inducible expression of IDO1. Similarly, reduction of BRD2, BRD3, or BRD4 decreased IDO1 expression. All these BET family members bound to the IDO1 promoter via the acetylated histone H3. JQ1 led to their release and reduced enrichment of RNA polymerase II (Pol II) on the promoter. IFN-γ increased the binding of BRD2, BRD3, BRD4, and Pol II on the IDO1 promoter by increasing the acetylation of histone H3, which could be prevented by JQ1 partially or even completely. Furthermore, both JQ1 and OTX015 decreased the production of L-kynurenine. The combination of BET inhibitors with the IDO1 inhibitor further reduced L-kynurenine, though only marginally. Importantly, the BET inhibitor ABBV-075 significantly inhibited the growth of human Ty-82 xenografts in nude mice and reduced both protein and mRNA levels of IDO1 in the xenografts. This finding lays a basis for the potential combination of BET inhibitors and IDO1 inhibitors for the treatment of IDO1-expressing cancers.

New microtubulin inhibitor MT189 suppresses angiogenesis via the JNK-VEGF/VEGFR2 signaling axis.

The microtubulin inhibitor MT189 possesses anticancer activity and has been shown to overcome multidrug resistance. Here, we report that MT189 also inhibits angiogenesis. MT189 inhibited the proliferation, migration and differentiation of endothelial cells, with or without VEGF stimulation, and suppressed microvessel formation ex vivo and in vivo. MT189 reduced VEGF expression and secretion in both tumor and endothelial cells, under either hypoxic or normoxic conditions. The activation of VEGFR2 and downstream Src was thus abrogated in the MT189-treated endothelial cells. MT189 subsequently stabilized endothelial cell-cell junctions consist of VE-cadherin, ß-catenin, vinculin, and actin. MT189 also disrupted endothelial cell-matrix junctions by inhibiting the turnover of focal adhesions containing FAK, paxillin, vinculin, and actin. Inhibition of JNK reversed MT189-mediated inhibition of endothelial migration and differentiation, JNK activation, the reduction of VEGF expression and secretion, and the decrease of Src and FAK phosphorylation. These results indicate that MT189 suppresses angiogenesis by reducing endothelial proliferation, migration, and differentiation via the JNK-VEGF/VEGFR2 signaling axis. Together with our previous report showing that MT189 exhibited anticancer activity via the JNK-MCL-1 pathway, these new findings further support MT189-based drug development for cancer therapy.

Targeting Hsp90 with FS-108 circumvents gefitinib resistance in EGFR mutant non-small cell lung cancer cells.

AIM: Inhibition of heat shock protein (Hsp90) has been proven to be effective in overriding primary and acquired resistance of kinase inhibitors. In this study, we investigated the role of FS-108, a newly developed Hsp90 inhibitor, to overcome gefitinib resistance in EGFR mutant non-small cell lung cancer cells. METHODS: Cell proliferation was assessed using the SRB assay. Cell cycle distribution and apoptosis were analyzed by flow cytometry. Protein expression was examined by Western blotting. The in vivo effectiveness of FS-108 was determined in an NCI-H1975 subcutaneous xenograft model. RESULTS: FS-108 triggered obvious growth inhibition in gefitinib-resistant HCC827/GR6, NCI-H1650 and NCI-H1975 cells through inducing G2/M phase arrest and apoptosis. FS-108 treatment resulted in a remarkable degradation of key client proteins involved in gefitinib resistance and further abrogated their downstream signaling pathways. Interestingly, FS-108 alone exerted an identical or superior effect on circumventing gefitinib resistance compared to combined kinase inhibition. Finally, the ability of FS-108 to overcome gefitinib resistance in vivo was validated in an NCI-H1975 xenograft model. CONCLUSION: FS-108 is a powerful agent that impacts the survival of gefitinib-resistant cells in vitro and in vivo through targeting Hsp90.

NMR-based platform for fragment-based lead discovery used in screening BRD4-targeted compounds.

AIM: Fragment-based lead discovery (FBLD) is a complementary approach in drug research and development. In this study, we established an NMR-based FBLD platform that was used to screen novel scaffolds targeting human bromodomain of BRD4, and investigated the binding interactions between hit compounds and the target protein. METHODS: 1D NMR techniques were primarily used to generate the fragment library and to screen compounds. The inhibitory activity of hits on the first bromodomain of BRD4 [BRD4(I)] was examined using fluorescence anisotropy binding assay. 2D NMR and X-ray crystallography were applied to characterize the binding interactions between hit compounds and the target protein. RESULTS: An NMR-based fragment library containing 539 compounds was established, which were clustered into 56 groups (8-10 compounds in each group). Eight hits with new scaffolds were found to inhibit BRD4(I). Four out of the 8 hits (compounds 1, 2, 8 and 9) had IC50 values of 100-260 µmol/L, demonstrating their potential for further BRD4-targeted hit-to-lead optimization. Analysis of the binding interactions revealed that compounds 1 and 2 shared a common quinazolin core structure and bound to BRD4(I) in a non-acetylated lysine mimetic mode. CONCLUSION: An NMR-based platform for FBLD was established and used in discovery of BRD4-targeted compounds. Four potential hit-to-lead optimization candidates have been found, two of them bound to BRD4(I) in a non-acetylated lysine mimetic mode, being selective BRD4(I) inhibitors.

Discovery of a new series of imidazo[1,2-a]pyridine compounds as selective c-Met inhibitors.

AIM: Aberrant c-Met activation plays a critical role in cancer formation, progression and dissemination, as well as in development of resistance to anticancer drugs. Therefore, c-Met has emerged as an attractive target for cancer therapy. The aim of this study was to develop new c-Met inhibitors and elaborate the structure-activity relationships of identified inhibitors. METHODS: Based on the predicted binding modes of Compounds 5 and 14 in docking studies, a new series of c-Met inhibitor-harboring 3-((1H-pyrrolo[3,2-c]pyridin-1-yl)sulfonyl)imidazo[1,2-a]pyridine scaffolds was discovered. Potent inhibitors were identified through extensive optimizations combined with enzymatic and cellular assays. A promising compound was further investigated in regard to its selectivity, its effects on c-Met signaling, cell proliferation and cell scattering in vitro. RESULTS: The most potent Compound 31 inhibited c-Met kinase activity with an IC50 value of 12.8 nmol/L, which was >78-fold higher than those of a panel of 16 different tyrosine kinases. Compound 31 (8, 40, 200 nmol/L) dose-dependently inhibited the phosphorylation of c-Met and its key downstream Akt and ERK signaling cascades in c-Met aberrant human EBC-1 cancer cells. In 12 human cancer cell lines harboring different background levels of c-Met expression/activation, Compound 31 potently inhibited c-Met-driven cell proliferation. Furthermore, Compound 31 dose-dependently impaired c-Met-mediated cell scattering of MDCK cells. CONCLUSION: This series of c-Met inhibitors is a promising lead for development of novel anticancer drugs.

Palladium-Catalyzed Synthesis of α-Iminonitriles from Aryl Halides via Isocyanide Double Insertion Reaction.

An efficient one-pot synthesis of α-iminonitriles from readily available aryl halides via palladium-catalyzed double isocyanide insertion and elimination has been developed, without using various hypertoxic cyanides and excess oxidants. Furthermore, the utility of this reaction was demonstrated by the rapid total synthesis of quinoxaline and the reaction of functional groups exchanged with aryl halides.

[Progress in the study of small molecule inhibitors of HSP90].

HSP90, which is the biomarker of cell stress and endogenous protective protein, functions as a molecular chaperone. Many client proteins of HSP90, including EGFR, Met, Raf-1, IKK and p53, play important roles in the occurrence and development of tumor. Binding of HSP90 inhibitors triggers the deactivation of HSP90, resulting in client protein degradation, and hence inhibits the tumor growth by blocking multiple targets involved in signaling of tumor proliferation. This review summarizes recent development of small molecule inhibitors bound to N-terminal of HSP90.

MCL-1 degradation mediated by JNK activation via MEKK1/TAK1-MKK4 contributes to anticancer activity of new tubulin inhibitor MT189.

Colchicine site-targeted tubulin inhibitors are a promising type of anticancer drugs. MT189 is a new derivative of MT119, a previously reported colchicine site-binding antitubulin agent. In this study, MT189 was demonstrated to retain the property of MT119 in disrupting microtubulin via binding to the colchicine site, causing mitotic arrest and inducing apoptosis, and to display 8.7-fold enhanced proliferative inhibition in a panel of cancer cells. MT189 was shown to elicit in vivo anticancer effects on MDA-MB-231 xenografts in nude mice, and the tumor growth was suppressed by 35.9% over 14 days. MT189 led to degradation of MCL-1, a member of the antiapoptotic BCL-2 protein family. Its overexpression reduced but its silenced expression increased the apoptotic induction followed by the treatment with MT189. Moreover, the treatment with MT189 caused activation of the MEKK1/TAK1-MKK4-JNK signaling pathway. The activated JNK resulted in phosphorylation of MCL-1, which facilitated its ubiquitination-mediated degradation. Our results show that MT189 inhibits microtubulin polymerization by binding to the colchicine site. Relief of apoptotic suppression by MCL-1 degradation together with mitotic arrest contributes to the anticancer activity of MT189.

Synthesis and bioassay of ß-(1,4)-D-mannans as potential agents against Alzheimer's disease.

AIM: Oligomannurarate 971 derived from a marine plant has shown neuroprotective effects. In this study we synthesized a series of truncated derivatives of the oligosaccharide, and investigated the effect of these derivatives against Aß peptide toxicity in vitro. METHODS: The sulfoxide method was applied to synthesize the derivatives. SH-SY5Y human neuroblastoma cells were treated with Aß1-40 (2 µmol/L), and the cell viability was detected using a CCK8 assay. RESULTS: A series of ß-(1,4)-D-mannosyl oligosaccharide, ranging from the disaccharide to the hexasaccharide, were synthesized. Addition of 10 µmol/L ß-(1,4)-D-mannobiose 6, ß-(1,4)-D-mannotriose 9 or ß-(1,4)-D-mannotetraose 12 in SH-SY5Y cells significantly attenuated Aß1-40-induced toxicity. The efficacies were similar to those caused by 10 µmol/L oligomannurarate 971 or alzhemed. Other oligosaccharides including oligomaltoses and oligocelluloses were less active. CONCLUSION: Synthetic homogeneous short chain ß-(1,4)-D-mannans shows neuroprotective effect against Aß peptide toxicity similar to that of heterogeneous oligomannurarate 971 and alzhemed.

Energetic factors determining the binding of type I inhibitors to c-Met kinase: experimental studies and quantum mechanical calculations.

AIM: To decipher the molecular interactions between c-Met and its type I inhibitors and to facilitate the design of novel c-Met inhibitors. METHODS: Based on the prototype model inhibitor 1, four ligands with subtle differences in the fused aromatic rings were synthesized. Quantum chemistry was employed to calculate the binding free energy for each ligand. Symmetry-adapted perturbation theory (SAPT) was used to decompose the binding energy into several fundamental forces to elucidate the determinant factors. RESULTS: Binding free energies calculated from quantum chemistry were correlated well with experimental data. SAPT calculations showed that the predominant driving force for binding was derived from a sandwich π-π interaction with Tyr-1230. Arg-1208 was the differentiating factor, interacting with the 6-position of the fused aromatic ring system through the backbone carbonyl with a force pattern similar to hydrogen bonding. Therefore, a hydrogen atom must be attached at the 6-position, and changing the carbon atom to nitrogen caused unfavorable electrostatic interactions. CONCLUSION: The theoretical studies have elucidated the determinant factors involved in the binding of type I inhibitors to c-Met.

Novel small-molecule AMP-activated protein kinase allosteric activator with beneficial effects in db/db mice.

AMP-activated protein kinase (AMPK) is an energy sensor of metabolism that is an attractive therapeutic target for type 2 diabetes mellitus and metabolic syndrome. Using a homogeneous scintillation proximity assay (SPA), we identified a new small-molecule AMPK activator, ZLN024, which allosterically stimulated active AMPK heterotrimers and the inactive α1 subunit truncations α1 (1-394) and α1 (1-335) but not α1 (1-312). AMPK activation by ZLN024 requires the pre-phosphorylation of Thr-172 by at least one upstream kinase and protects AMPK Thr-172 against dephosphorylation by PP2Cα. ZLN024 activated AMPK in L6 myotubes and stimulated glucose uptake and fatty acid oxidation without increasing the ADP/ATP ratio. ZLN024 also activated AMPK in primary hepatocytes, decreased fatty acid synthesis and glucose output. Treatment of db/db mice with 15 mg/kg/day ZLN024 improved glucose tolerance; liver tissue weight, triacylglycerol and the total cholesterol content were decreased. The hepatic transcriptional level of G6Pase, FAS and mtGPAT were reduced. The transcription of genes involved in fatty acid oxidation and the mitochondrial biogenesis of muscle tissue were elevated. The ACC phosphorylation was increased in muscle and liver. This study provides a novel allosteric AMPK activator for functional study in vitro and in vivo and demonstrates that AMPK allosteric activators could be a promising therapeutic approach for type 2 diabetes mellitus and metabolic syndrome.

[Progress in the fragment-based drug discovery].

As an extension of the structure-based drug discovery, fragment-based drug discovery is matured increasingly, and plays an important role in drug development. Fragments in a small library, with lower molecular mass and high "ligand efficiency", are detected by SPR, MS, NMR, X-ray crystallography technologies and other biophysical methods. Then they are considered as starting points for chemical optimization with the guidance of structural biology methods to get good "drug-like" lead and candidate compounds. In this article, we reviewed the current progress of fragment-based drug discovery and detailed a number of examples to illustrate the novel strategies.

Design, synthesis and biological evaluation of bivalent ligands against A(1)-D(1) receptor heteromers.

AIM: To design and synthesize bivalent ligands for adenosine A1-dopamine D1 receptor heteromers (A1-D1R), and evaluate their pharmacological activities. METHODS: Bivalent ligands and their corresponding A1R monovalent ligands were designed and synthesized. The affinities of the bivalent ligands for A1R and D1R in rat brain membrane preparation were examined using radiolabeled binding assays. To demonstrate the formation of A1-D1R, fluorescence resonance energy transfer (FRET) was conducted in HEK293 cells transfected with D1-CFP and A1-YFP. Molecular modeling was used to analyze the possible mode of protein-protein and protein-ligand interactions. RESULTS: Two bivalent ligands for A1R and D1R (20a, 20b), as well as the corresponding A1R monovalent ligands (21a, 21b) were synthesized. In radiolabeled binding assays, the bivalent ligands showed affinities for A1R 10-100 times higher than those of the corresponding monovalent ligands. In FRET experiments, the bivalent ligands significantly increased the heterodimerization of A1R and D1R compared with the corresponding monovalent ligands. A heterodimer model with the interface of helixes 3, 4, 5 of A1R and helixes 1, 6, 7 from D1R was established with molecular modeling. The distance between the two ligand binding sites in the heterodimer model was approximately 48.4 Å, which was shorter than the length of the bivalent ligands. CONCLUSION: This study demonstrates the existence of A1-D1R in situ and a simultaneous interaction of bivalent ligands with both the receptors.

Novel small-molecule PGC-1α transcriptional regulator with beneficial effects on diabetic db/db mice.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has been shown to influence energy metabolism. Hence, we explored a strategy to target PGC-1α expression to treat metabolic syndromes. We developed a high-throughput screening assay that uses the human PGC-1α promoter to drive expression of luciferase. The effects of lead compound stimulation on PGC-1α expression in muscle cells and hepatocytes were investigated in vitro and in vivo. A novel small molecule, ZLN005, led to changes in PGC-1α mRNA levels, glucose uptake, and fatty acid oxidation in L6 myotubes. Activation of AMP-activated protein kinase was involved in the induction of PGC-1α expression. In diabetic db/db mice, chronic administration of ZLN005 increased PGC-1α and downstream gene transcription in skeletal muscle, whereas hepatic PGC-1α and gluconeogenesis genes were reduced. ZLN005 increased fat oxidation and improved the glucose tolerance, pyruvate tolerance, and insulin sensitivity of diabetic db/db mice. Hyperglycemia and dyslipidemia also were ameliorated after treatment with ZLN005. Our results demonstrated that a novel small molecule selectively elevated the expression of PGC-1α in myotubes and skeletal muscle and exerted promising therapeutic effects for treating type 2 diabetes.

[Research progress of sodium-glucose co-transporter 2 inhibitors for treatment of type 2 diabetes].

Sodium-glucose co-transporters are a family of glucose transporter found in the intestinal mucosa of the small intestine (SGLT-2) and the proximal tubule of the nephron (SGLT-1 and SGLT-2). They contribute to renal glucose reabsorption and most of renal glucose (about 90%) is reabsorbed by SGLT-2 located in the proximal renal tubule. Selectively inhibiting activity of SGLT-2 is an innovative therapeutic strategy for treatment of type 2 diabetes by enhancing urinary glucose excretion from the body. Therefore SGLT-2 inhibitors are considered to be potential antidiabetic drugs with an unique mechanism. This review will highlight some recent advances and structure-activity relationships in the discovery and development of SGLT-2 inhibitors including O-glycoside, C-glycoside, C, O-spiro glycoside and non glycosides.