Antiangiogenic Small Molecule Research Articles

Anlotinib reduces the suppressive capacity of monocytic myeloid-derived suppressor cells and potentiates the immune microenvironment normalization window in a mouse lung cancer model.

By exploring the effects of an antiangiogenic small molecule drug named anlotinib on the levels of myeloid-derived suppressor cells (MDSCs) in a mouse xenograft model of lung cancer, the role of anti-angiogenesis in remodeling the immune microenvironment was discussed. In addition, the impact of anlotinib on the normalization of the immune microenvironment and time window was examined, providing a theoretical basis for the optimization of clinical strategies applying anlotinib combined with PD-1 inhibitors. On the basis of the LLC mouse xenograft model, MDSCs and MDSCs + immune microenvironment were examined in tissues, respectively, according to different samples. The former observation included the control (group A) and anlotinib monotherapy (group B) groups; the latter also included the control (group C) and anlotinib monotherapy (group D) groups. The levels of MDSCs in peripheral blood at different time points were analyzed by flow cytometry, and the levels of MDSCs in tissue samples at different time points were evaluated by immunofluorescence and immunohistochemistry. The volumes of subcutaneous xenografts were significantly smaller in the anlotinib treatment group compared with the control group ( P < 0.005). Flow cytometry showed that compared with the control group, the intratumoral percentages of total MDSCs ( P < 0.01) and mononuclear-MDSCs ( P < 0.05) were significantly decreased on days 3 and 17 after anlotinib treatment in peripheral blood samples; however, there was no significant difference in granulocytic-MDSCs changes between the experimental and control groups. Immunofluorescence showed that the levels of MDSCs in both the experimental and control groups reached the lowest points 10 days after drug administration, and were significantly lower in the experimental group than in the control group ( P < 0.05). Anlotinib reduces the levels of MDSCs in the mouse xenograft model of lung cancer, with the characteristics of time window. This study provides a basis for further exploring strategies for anti-angiogenic treatment combined with immunotherapy in lung cancer based on time-window dosing.

Current status and prospects of anti-angiogenic small-molecule tyrosine kinase inhibitors in the therapy of soft tissue sarcoma

Chemotherapy is the main systemic treatment for patients with advanced soft tissue sarcoma, and immunotherapy is only effective for some special subtypes. Anti-angiogenic small molecule tyrosine kinase inhibitors represented by pazopanib and anlotinib are the main drugs of targeted therapy. They have been clearly recommended as the second-line treatment of non-specific soft tissue sarcoma in guidelines. In recent years, in addition to second-line monotherapy in patients with advanced sarcoma, some studies have been carried out in second-line combination therapy, maintenance therapy, first-line therapy and neoadjuvant therapy. This article briefly reviews the application status and prospect of anti-angiogenic small molecule tyrosine kinase inhibitors in advanced soft tissue sarcoma.

A patent review on efficient strategies for the total synthesis of pazopanib, regorafenib and lenvatinib as novel anti-angiogenesis receptor tyrosine kinase inhibitors for cancer therapy.

Angiogenesis is an important and interesting scientific subject in the area of malignant tumours. Current research importance and interest are directed in connection to blood microvessels in cancer cell proliferation, tumour growth, and metastasis. Tyrosine kinases have been intensely implicated as therapeutic targets that affect the angiogenic process in tumour growth. In the last decades, targeting angiogenesis has led to achievements in the therapy of different carcinomas by different mechanisms, such as the utilization of anti-angiogenic small molecule receptor tyrosine kinase inhibitors. In the current review, we aim to track the advancements in the total synthesis of three receptor tyrosine kinase inhibitors (pazopanib, regorafenib and lenvatinib). This review surveys different synthetic routes for these three approved drugs (pazopanib, regorafenib and lenvatinib) which were previously published as patents (2014-2021). The purity of medicines is a very important factor during manufacturing so we have decided to review the purification process of these anticancer medicines as well. It should be noted that the different patents may have reported some procedures with different yields and purities for the synthesis of desired drug and their intermediates. In order to simplify the understanding of the contents of this review article, only the best results reported in each of these patents are reported for the synthesis of desired drug and their intermediates.

A phase Ib study of TQB2450 plus anlotinib in patients with advanced triple-negative breast cancer.

1074 Background: TQB2450 is a humanized monoclonal antibody targeting programmed death-ligand 1 (PD-L1). Anlotinib is an antiangiogenic small molecule, multi-target tyrosine kinase inhibitor that has improved clinical outcomes in various solid tumors. This phase 1b study aims to evaluate the safety and efficacy of TQB2450 plus anlotinib for patients with advanced triple-negative breast cancer (TNBC) after the failure of standard therapy. Methods: This ongoing study included a dose-escalation phase and an expansion phase. Advanced TNBC patients with prior anthracyclines and/or taxanes treatment and failed at least first-line therapy were enrolled. In the dose-escalation phase, eligible patients received anlotinib (8mg, 10mg, and 12mg, qd, days 1-14; 21 days per cycle) plus TQB2450 (1200mg, day 1; 21 days per cycle) following the conventional 3+3 design. If the starting dose of 10mg anlotinib led to ≥2 dose-limiting toxicities (DLTs), 8mg anlotinib would be administered. After the dose-escalating phase, eligible patients were enrolled into the expansion cohort. The primary endpoint was objective response rate (ORR), and the secondary endpoints were overall survival (OS), disease control rate (DCR), progression-free survival (PFS), and safety. Results: Between May 29, 2019, and December 31, 2020, in the dose-escalation phase, three patients receiving 10mg anlotinib plus 1200mg TQB2450 had no DLTs in the first cycle, neither did three patients with 12mg anlotinib plus TQB2450. Next, 28 patients with advanced TNBC received 12 mg anlotinib plus TQB2450 in the expansion phase. Finally, a total of 34 patients were included with median age of 49.5 (32-70) and median prior lines of 2 (1-6). Numbers of patients with prior platinum therapy: 16, prior anthracycline therapy: 32. The ORR was 26.47% (9/34) and DCR was 82.35% (28/34). The median PFS was 8.57 months. Seventeen patients experienced grade 3 treatment-related AEs (TRAEs). Most frequently occurring (&gt;5%) grade 3 TRAEs were QT interval prolongation (17.65%), hypertension (14.71%), diarrhea (8.82%), hand-foot syndrome (HFS) (8.82%), and hypertriglyceridemia (5.88%). Conclusions: TQB2450 plus anlotinib showed an acceptable safety profile with promising activity for previously anthracyclines and/or taxanes-treated advanced TNBC patients. Clinical trial information: NCT03855358 .[Table: see text]

Combining 1,4-dihydroxy quininib with Bevacizumab/FOLFOX alters angiogenic and inflammatory secretions in ex vivo colorectal tumors

BackgroundColorectal cancer (CRC) is the second most common cause of cancer-related mortality worldwide with one in every five patients diagnosed with metastatic CRC (mCRC). In mCRC cases, the 5-year survival rate remains at approximately 14%, reflecting the lack of effectiveness of currently available treatments such as the anti-VEGF targeting antibody Bevacizumab combined with the chemotherapy folinic acid, fluorouracil and oxaliplatin (FOLFOX). Approximately 60% of patients do not respond to this combined treatment. Furthermore, Bevacizumab inhibits dendritic cell (DC) maturation in poor responders, a key process for tumor eradication.MethodFollowing drug treatment, secreted expression levels of angiogenic and inflammatory markers in tumor conditioned media generated from human ex vivo colorectal tumors were measured by ELISA. Dendritic cell phenotypic and maturation markers were assessed by flow cytometry.ResultsOur novel compound, 1,4-dihydroxy quininib, acts in an alternative pathway compared to the approved therapy Bevacizumab. 1,4-dihydroxy quininib alone, and in combination with Bevacizumab or FOLFOX significantly reduced TIE-2 expression which is involved in the promotion of tumor vascularization. Combination treatment with 1,4-dihydroxy quininib significantly increased the expression level of DC phenotypic and maturation markers.ConclusionOur results indicate the anti-angiogenic small molecule 1,4-dihydroxy quininib could be an alternative novel treatment in combination therapy for CRC patients.

Combination of anlotinib and irinotecan in the second-line treatment of patients with advanced colorectal cancer: An open-label, multicenter phase I/II study.

e16036 Background: Efficacious therapeutic options for advanced colorectal cancer (CRC) are limited, and treatment outcomes remain to be improved in the second-line setting. Anlotinib is a multi-target antiangiogenic small molecule that primarily inhibits VEGFR1/2/3. We conducted this open-label, multi-center phase 1/2 study to evaluate the safety and efficacy of anlotinib plus irinotecan in patients with previously treated advanced CRC. Methods: We enrolled patients with advanced CRC whose disease had progressed after first-line systemic therapy and had not previously received irinotecan. In the 3+3 dose escalation (phase 1) part of the trial, patients received anlotinib (8mg, 10mg and 12mg, po, 2 weeks on/1 week off) in combination with fixed-dose irinotecan (180mg/m2, iv, q2w) to define the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). In the dose-expansion (phase 2) part, patients were treated with the RP2D of anlotinib and irinotecan. The primary endpoints were MTD and objective response rate (ORR). Secondary endpoints included progression-free survival (PFS) and disease control rate (DCR). Results: A total of 31 patients were enrolled from 2 centers between May 2018 and Jan 2020. The median follow-up duration was 12.48m (95% CI 7.43-15.24). Anlotinib was well tolerated in combination with irinotecan with no MTD identified in the phase 1 part, and the RP2D was therefore 12mg. Twenty-eight patients were evaluable for efficacy analysis. Eight patients achieved partial response (PR), in which 6 had confirmed PR and 2 were unconfirmed. The ORR was 21.4% (95%CI 8.3%-41.0%) and the DCR was 100% (95%CI 87.7%-100%). The median PFS and OS were 6.97m (95%CI 3.71-11.20) and 13.87m (95%CI 11.47-NR), respectively. All patients were included in the safety analysis. The most common grade 3 treatment-related adverse events (AEs) (≥5%) were diarrhea (16.1%), neutropenia (12.9%), fatigue (6.5%) and hypertension (6.5%). Grade 4 AEs included neutropenia (6.5%) and thrombocytopenia (3.2%). There were no treatment-related deaths. Conclusions: The combination of anlotinib and irinotecan had promising anti-tumor activity in the second-line treatment of advanced CRC with a manageable safety profile. Clinical trial information: NCT03545711 . [Table: see text]

Enantioselective Synthesis of Homoisoflavanones by Asymmetric Transfer Hydrogenation and Their Biological Evaluation for Antiangiogenic Activity.

Neovascular eye diseases are a major cause of blindness. Excessive angiogenesis is a feature of several conditions, including wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity. Development of novel antiangiogenic small molecules for the treatment of neovascular eye disease is essential to provide new therapeutic leads for these diseases. We have previously reported the therapeutic potential of anti-angiogenic homoisoflavanone derivatives with efficacy in retinal and choroidal neovascularization models, although these are racemic compounds due to the C3-stereogenic center in the molecules. This work presents asymmetric synthesis and structural determination of anti-angiogenic homoisoflavanones and pharmacological characterization of the stereoisomers. We describe an enantioselective synthesis of homoisoflavanones by virtue of ruthenium-catalyzed asymmetric transfer hydrogenation accompanying dynamic kinetic resolution, providing a basis for the further development of these compounds into novel experimental therapeutics for neovascular eye diseases.

Clinical observation of low-dose apatinib in the treatment of small cell lung cancer with antidiuretic hormone secretion.

e20087 Background: The treatment of extensive small cell lung cancer (ED-SCLC) is still based on radiotherapy and chemotherapy, but the overall survival is short and the prognosis is poor. The prognosis of ED-SCLC with hyponatremia is worse. At present, the anti-angiogenic small molecule targeted drug apatinib has achieved satisfactory clinical efficacy in advanced non-squamous non-small cell lung cancer. Although there are reports of a small number of apatinib in the treatment of small cell lung cancer, there is still a lack of low integration. In the study of sodiumemia, in this study, we evaluated the efficacy and safety of low-dose abatatinib in the treatment of ED-SCLC with hyponatremia. Methods: Retrospective analysis of 8 patients with hyponatremia at least twice during hospitalization between January 2016 and September 1818, after low-dose apafitini (125 mg or 250 mg, QD, orally; 28 days for one observation period) Case data for patients with SCLC who progressed after first-line or second-line treatment. Efficacy was assessed using the RECIST criteria and adverse reactions (AEs) were graded using the NCI CTC criteria. Results: The efficacy was evaluated as 4 PR, 2 SD, and 2 PD. ORR and DCR are 50% (4/8) and 100% (75%), respectively. The median progression-free survival (PFS) was 91 days (95% CI, 30-167) and the median overall survival (OS) was 150 days (95% CI, 110-197). Up to the follow-up date, 3 patients survived and 5 died; 1 grade 3/4 hypertension, 1 case of neutropenia, and grade 3/4 toxicity were relieved by symptomatic treatment. Did not affect the use of apatinib. Conclusions: The efficacy and tolerability of patients with LD-SCLC with hyponatremia after low-dose atentinil treatment for first- and second-line treatment were good. However, the conclusion still requires a large sample of prospective studies to further evaluate. [Table: see text]

A Novel Small Molecule Therapy for Corneal Neovascularization

Background and Hypothesis: Corneal neovascularization (CNV) is characterized by abnormal blood vessel growth in the avascular cornea. CNV can be caused by multiple insults such as trauma, infection, and immunological diseases. Current treatments have partial efficacy and associated side effects, revealing a need for novel treatments. &#x0D; We identified the enzyme soluble epoxide hydrolase (sEH) as a target of an antiangiogenic small molecule we developed, SH-11037. SH-11037 was antiangiogenic in mouse models for retinal and choroidal neovascularization. However, its effect on CNV has not been explored. We hypothesize that inhibition of sEH with SH-11037 will block CNV. &#x0D; Experimental Design or Project Methods: To validate sEH’s relevance in CNV, we assessed the effects of inhibition of sEH in an alkali burn CNV mouse model, treating the eyes with either eyedrops of SH-11037 or another known sEH inhibitor, t-AUCB. Neovascularization was determined by clinical assessment and immunohistochemistry. Toxicity in the retina was assessed by optical coherence tomography (OCT). Eyedrops of dexamethasone (0.1% and 0.03%) were positive controls. &#x0D; Results: Clinical assessment indicated that there was a strong trend towards a decrease in CNV in the 0.1% dexamethasone control group (p = 0.0509). Further data generation is underway, but we expect to see decreased CNV in the SH-11037 and t-AUCB experimental groups, compared to the vehicle (PBS) control group. &#x0D; Conclusion and Potential Impact: Upon completion of this study there is potential for a new therapeutic target for reducing CNV. This study provides the assessment of sEH’s involvement in CNV, which opens future areas of study.

Characterization of a Novel Inhibitor of Soluble Epoxide Hydrolase and Role in Ocular Neovascularization

ObjectiveOcular neovascularization causes blindness in diseases such as proliferative diabetic retinopathy (PDR) and wet age‐related macular degeneration (AMD). Today, the effort to treat wet AMD is hampered by patients who are resistant and refractory to the current standard of care, anti‐vascular endothelial growth factor (VEGF) therapies. Therefore, identification of novel therapeutic targets and inhibitors is desirable to address the unmet needs in antiangiogenic treatment. Previously, we have identified soluble epoxide hydrolase (sEH) as a binding target of an antiangiogenic small molecule, SH‐11037. Here, we aimed to further investigate sEH expression in the eye and the inhibition of its activity by SH‐11037.MethodsThe expression and localization of sEH in retinal sections of laser‐induced choroidal neovascularization (L‐CNV) mice were evaluated by immunohistochemistry. The sEH enzymatic activity in ocular tissue homogenates of L‐CNV and control mice was assayed using trans‐stilbene oxide (t‐SO) as substrate. The sEH inhibition by SH‐11037 and mode of inhibition was assessed by recombinant sEH activity assay using a fluorogenic substrate, PHOME. Docking and molecular dynamics simulations were performed to predict the binding mode of SH‐11037 within the active site of sEH. In addition, intravitreal injections of sEH substrate 19,20‐epoxy docosapentaenoic acid (EDP) and product 19,20‐dihydroxy docosapentaenoic acids (DHDP) were given to L‐CNV mice, and effects were assessed by imaging and choroidal flatmount immunofluorescence.ResultssEH was upregulated in the rod photoreceptors in L‐CNV mice compared to control mice and it did not co‐localize with other retinal cell type markers. Correspondingly, sEH activity was increased in L‐CNV eyes and was normalized by SH‐11037 or a known sEH inhibitor. In a recombinant enzyme assay, SH‐11037 inhibited sEH activity in a dose‐dependent manner, with IC50 = 0.15 μM. Enzyme kinetics analysis demonstrated that increasing concentrations of SH‐11037 decreased Vmax and increased KM, revealing SH‐11037 as a mixed‐type inhibitor of sEH, with Ki = 1.73 ± 0.45 μM. Docking of SH‐11037 to sEH showed an energetically favorable binding mode with the ligand spanning the active site of the enzyme and interacting with key catalytic residues. Intravitreal 19,20‐EDP reduced CNV lesion volume compared to vehicle and 19,20‐DHDP, confirming antiangiogenic properties of this sEH substrate.ConclusionOur studies confirm the relevance of sEH for ocular neovascularization and reveal the mechanism for SH‐11037's inhibition of sEH. Further optimization of SH‐11037's structure could yield more potent, novel sEH inhibitors.Support or Funding InformationNIH/NEI R01EY025641, Retina Research Foundation, Research to Prevent Blindness, Inc.

Disruption of FGF2-Heparin-FGFR1 Complex Formation by Antiangiogenic Small Molecule Found In Silico

Background: Basic fibroblast growth factor (FGF2) is one of the most potent proangiogenic proteins involving in tumor angiogenesis. Interaction of heparin with FGF2 and FGF2 receptor (FGFR1) form a ternary complex that is prerequisite for FGF2 proangiogenic activity. Therefore, this interaction can be an important target for inhibition of angiogenesis. Material and Method: In this study, we performed screening studies by computer-aided techniques to find a small molecule interfering with this interaction. Based on ionic interactions, we found seventeen small molecules which had the capability of angiogenic inhibition. According to important negative charge distances, benzene-1, 2, 4-tricarboxylic acids, known as Trimellitic acid (TMLA), was chosen. MTT viability test, real time PCR, tube formation assay, and Flowcytometry technique were used to evaluate TMLA effect. Results: Here, the viability of HUVECs was decreased following exposure to TMLA only at high concentrations. According to real time PCR, gene expression was dramatically decreased in comparison to negative control, indicating that TMLA is an effective agent on reduction of CD31 expression. At half maximal inhibitory concentration (13mM), TMLA inhibited HUVEC tube formation process. Annexin V-FITC / PI flow cytometry technique revealed that TMLA inhibitory effect was via apoptosis. Conclusion: With respect to findings of the present study, it is possible to extend FGF2 inhibitors with much specificity based on ionic interactions strategy.&#x0D;

Targeting of the breast cancer microenvironment with a potent and linkable oxindole based antiangiogenic small molecule.

The clinical efficacy of antiangiogenic small molecules (e.g., sunitinib) in breast carcinoma has largely failed with substantial off-target toxicity. We rationally designed and evaluated preclinically a novel sunitinib analogue, SAP, with favourable pharmacological properties and the ability to be readily conjugated to a targeting peptide or antibody for active tumour targeting.SAP was evaluated in silico and in vitro in order to verify target engagement (e.g., VEGFR2). Pharmacokinetic and biodistribution parameters were determined in mice using LC-MS/MS. SAP efficacy was tested in two breast cancer xenograft and two syngeneic animal models and pharmacodynamic evaluation was accomplished using phosphokinase assays and immunohistochemistry. Cardiac and blood toxicity of SAP were also monitored.SAP retained the antiangiogenic and cytotoxic properties of the parental molecule with an increased blood exposure and tumor accumulation compared to sunitinib. SAP proved efficacious in all animal models. Tumors from SAP treated animals had significantly decreased Ki-67 and CD31 markers and reduced levels of phosphorylated AKT, ERK and S6 compared to vehicle treated animals. In mice dosed with SAP there was negligible hematotoxicity, while cardiac function measurements showed a reduction in the percentage left ventricular fractional shortening compared to vehicle treated animals.In conclusion, SAP is a novel rationally designed conjugatable small antiangiogenic molecule, efficacious in preclinical models of breast cancer.

A Quininib Analogue and Cysteinyl Leukotriene Receptor Antagonist Inhibits Vascular Endothelial Growth Factor (VEGF)-independent Angiogenesis and Exerts an Additive Antiangiogenic Response with Bevacizumab

Excess blood vessel growth contributes to the pathology of metastatic cancers and age-related retinopathies. Despite development of improved treatments, these conditions are associated with high economic costs and drug resistance. Bevacizumab (Avastin®), a monoclonal antibody against vascular endothelial growth factor (VEGF), is used clinically to treat certain types of metastatic cancers. Unfortunately, many patients do not respond or inevitably become resistant to bevacizumab, highlighting the need for more effective antiangiogenic drugs with novel mechanisms of action. Previous studies discovered quininib, an antiangiogenic small molecule antagonist of cysteinyl leukotriene receptors 1 and 2 (CysLT1 and CysLT2). Here, we screened a series of quininib analogues and identified a more potent antiangiogenic novel chemical entity (IUPAC name (E)-2-(2-quinolin-2-yl-vinyl)-benzene-1,4-diol HCl) hereafter designated Q8. Q8 inhibits developmental angiogenesis in Tg(fli1:EGFP) zebrafish and inhibits human microvascular endothelial cell (HMEC-1) proliferation, tubule formation, and migration. Q8 elicits antiangiogenic effects in a VEGF-independent in vitro model of angiogenesis and exerts an additive antiangiogenic response with the anti-VEGF biologic bevacizumab. Cell-based receptor binding assays confirm that Q8 is a CysLT1 antagonist and is sufficient to reduce cellular levels of NF-κB and calpain-2 and secreted levels of the proangiogenic proteins intercellular adhesion molecule-1, vascular cell adhesion protein-1, and VEGF. Distinct reductions of VEGF by bevacizumab explain the additive antiangiogenic effects observed in combination with Q8. In summary, Q8 is a more effective antiangiogenic drug compared with quininib. The VEGF-independent activity coupled with the additive antiangiogenic response observed in combination with bevacizumab demonstrates that Q8 offers an alternative therapeutic strategy to combat resistance associated with conventional anti-VEGF therapies.

Predicting drug-induced liver injury by normalising liver microtissue high content screening data to in vivo tissue concentrations

Mitochondrial reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. In particular, high levels of mitochondrial ROS in hypoxic cells regulate many angiogenesis-related diseases, including cancer and ischemic disorders. Here we report a new angiogenesis inhibitor, YCG063, which suppressed mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library with an ArrayScan HCS reader. YCG063 suppressed mitochondrial ROS generation under a hypoxic condition in a dose-dependent manner, leading to the inhibition of in vitro angiogenic tube formation and chemoinvasion as well as in vivo angiogenesis of the chorioallantoic membrane (CAM) at non-toxic doses. In addition, YCG063 decreased the expression levels of HIF-1α and its target gene, VEGF. Collectively, a new antiangiogenic small molecule that suppresses mitochondrial ROS was identified. This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions.

Targeting Angiogenesis in Colorectal Cancer: Tyrosine Kinase Inhibitors.

Colorectal cancer is commonly diagnosed throughout the world, and treatment options have greatly expanded over the last 2 decades. Targeting angiogenesis has been a major focus of study in a variety of malignancy types. Targeting angiogenesis has been achieved by several mechanisms in colorectal cancer, including use of antiangiogenic small molecule tyrosine kinase inhibitors (TKIs). There have been many attempts and failures to prove efficacy of TKIs in the treatment of colorectal cancer including sorafenib, sunitinib, vatalanib, and tivozanib. Regorafenib was the first TKI to demonstrate efficacy and is an orally active inhibitor of angiogenic (including the vascular endothelial growth factor receptors 1, 2, and 3), stromal, and oncogenic receptor tyrosine kinases. There are ongoing investigations of both regorafenib and ninetanib; however, there remains a critical need to better understand novel combinations with TKIs that could prove more efficacious than available options.

PO-15 - Antiangiogenic small molecule ligands of FGF2 derived from the endogenous inhibitor thrombospondin-1

Platelet thrombospondin-1 (TSP-1) is a major endogenous regulator of growth factor activity in physiological and pathological processes, including tumor onset, progression and angiogenesis. We previously demonstrated that TSP-1 binds to FGF-2, sequestering the growth factor and inhibiting its angiogenic activity. We also identified a non-peptidic antiangiogenic compound (SM27) that retains the structural and functional properties of the FGF2-binding sequence of TSP-1. To identify new small molecule inhibitors of FGF2 that recapitulate the structure and functional properties of the FGF-2-binding site of TSP-1, by investigating the chemical space around SM27. A similarity-based screening of small molecule libraries has been used to identify candidates, followed by docking calculations, and evaluation of the activity of the resulting compounds in biochemical and biophysical assays, to assess interaction with FGF2, and in experimental models of angiogenesis, to assess biological activity. The used integrated approach allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting FGF2 binding to both heparan sulfate proteoglycans and FGFR1. The compounds inhibited FGF2-induced endothelial cell proliferation, vessel sprouting from aortic rings and angiogenesis in the chorioallantoic membrane assay, with improved potency over SM27. We have identified new compounds that are valuable as FGF inhibitors for potential therapeutic purposes. Moreover, these compounds are useful chemical tools to identify the minimal stereochemical requirements for FGF2 binding and activity to improve the design of new agents for antineoplastic therapy. Supported by AIRC (Associazione Italiana per la Ricerca sul Cancro).

Integrating computational and chemical biology tools in the discovery of antiangiogenic small molecule ligands of FGF2 derived from endogenous inhibitors.

The FGFs/FGFRs system is a recognized actionable target for therapeutic approaches aimed at inhibiting tumor growth, angiogenesis, metastasis, and resistance to therapy. We previously identified a non-peptidic compound (SM27) that retains the structural and functional properties of the FGF2-binding sequence of thrombospondin-1 (TSP-1), a major endogenous inhibitor of angiogenesis. Here we identified new small molecule inhibitors of FGF2 based on the initial lead. A similarity-based screening of small molecule libraries, followed by docking calculations and experimental studies, allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting its binding to both heparan sulfate proteoglycans and FGFR-1. The compounds inhibit FGF2 activity in in vitro and ex vivo models of angiogenesis, with improved potency over SM27. Comparative analysis of the selected hits, complemented by NMR and biochemical analysis of 4 newly synthesized functionalized phenylamino-substituted naphthalenes, allowed identifying the minimal stereochemical requirements to improve the design of naphthalene sulfonates as FGF2 inhibitors.

Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma.

Antiangiogenic therapy resistance occurs frequently in patients with metastatic renal cell carcinoma (RCC). The purpose of this study was to understand the mechanism of resistance to sunitinib, an antiangiogenic small molecule, and to exploit this mechanism therapeutically. We hypothesized that sunitinib-induced upregulation of the prometastatic MET and AXL receptors is associated with resistance to sunitinib and with more aggressive tumor behavior. In the present study, tissue microarrays containing sunitinib-treated and untreated RCC tissues were stained with MET and AXL antibodies. The low malignant RCC cell line 786-O was chronically treated with sunitinib and assayed for AXL, MET, epithelial-mesenchymal transition (EMT) protein expression and activation. Co-culture experiments were used to examine the effect of sunitinib pretreatment on endothelial cell growth. The effects of AXL and MET were evaluated in various cell-based models by short hairpin RNA or inhibition by cabozantinib, the multi-tyrosine kinases inhibitor that targets vascular endothelial growth factor receptor, MET and AXL. Xenograft mouse models tested the ability of cabozantinib to rescue sunitinib resistance. We demonstrated that increased AXL and MET expression was associated with inferior clinical outcome in patients. Chronic sunitinib treatment of RCC cell lines activated both AXL and MET, induced EMT-associated gene expression changes, including upregulation of Snail and β-catenin, and increased cell migration and invasion. Pretreatment with sunitinib enhanced angiogenesis in 786-0/human umbilical vein endothelial cell co-culture models. The suppression of AXL or MET expression and the inhibition of AXL and MET activation using cabozantinib both impaired chronic sunitinib treatment-induced prometastatic behavior in cell culture and rescued acquired resistance to sunitinib in xenograft models. In summary, chronic sunitinib treatment induces the activation of AXL and MET signaling and promotes prometastatic behavior and angiogenesis. The inhibition of AXL and MET activity may overcome resistance induced by prolonged sunitinib therapy in metastatic RCC.

A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species

Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identification and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders.

A new anti-angiogenic small molecule, G0811, inhibits angiogenesis via targeting hypoxia inducible factor (HIF)-1α signal transduction

Regulation of hypoxia inducible factor (HIF)-1α stabilization, which in turn contributes to adaptation of tumor cells to hypoxia has been highlighted as a promising therapeutic target in angiogenesis-related diseases. We have identified a new small molecule, G0811, as a potent angiogenesis inhibitor that targets HIF-1α signal transduction. G0811 suppressed HIF-1α stability in cancer cells and inhibited in vitro and in vivo angiogenesis, as validated by tube formation, chemoinvasion, and chorioallantoic membrane (CAM) assays. In addition, G0811 effectively decreased the expression of vascular endothelial growth factor (VEGF), which is one of target genes of HIF-1α. However, G0811 did not exhibit anti-proliferative activities or toxicity in human umbilical vein endothelial cells (HUVECs) at effective doses. These results demonstrate that G0811 could be a new angiogenesis inhibitor that acts by targeting HIF-1α signal transduction pathway.