ABCB1-dependent collateral sensitivity of multidrug-resistant colorectal cancer cells to the survivin inhibitor MX106-4C.

AIMS: To investigate the collateral sensitivity (CS) of ABCB1-positive multidrug resistant (MDR) colorectal cancer cells to the survivin inhibitor MX106-4C and the mechanism. METHODS: Biochemical assays (MTT, ATPase, drug accumulation/efflux, Western blot, RT-qPCR, immunofluorescence, flow cytometry) and bioinformatic analyses (mRNA-sequencing, reversed-phase protein array) were performed to investigate the hypersensitivity of ABCB1 overexpressing colorectal cancer cells to MX106-4C and the mechanisms. Synergism assay, long-term selection, and 3D tumor spheroid test were used to evaluate the anti-cancer efficacy of MX106-4C. RESULTS: MX106-4C selectively killed ABCB1-positive colorectal cancer cells, which could be reversed by an ABCB1 inhibitor, knockout of ABCB1, or loss-of-function ABCB1 mutation, indicating an ABCB1 expression and function-dependent mechanism. MX106-4C's selective toxicity was associated with cell cycle arrest and apoptosis through ABCB1-dependent survivin inhibition and activation on caspases-3/7 as well as modulation on p21-CDK4/6-pRb pathway. MX106-4C had good selectivity against ABCB1-positive colorectal cancer cells and retained this in multicellular tumor spheroids. In addition, MX106-4C could exert a synergistic anti-cancer effect with doxorubicin or re-sensitize ABCB1-positive cancer cells to doxorubicin by reducing ABCB1 expression in the cell population via long-term exposure. CONCLUSIONS: MX106-4C selectively kills ABCB1-positive MDR colorectal cancer cells via a novel ABCB1-dependent survivin inhibition mechanism, providing a clue for designing CS compound as an alternative strategy to overcome ABCB1-mediated colorectal cancer MDR.

Inhibition of TRPC3 channels by a novel pyrazole compound confers antiseizure effects.

OBJECTIVE: As a key member of the transient receptor potential (TRP) superfamily, TRP canonical 3 (TRPC3) regulates calcium homeostasis and contributes to neuronal excitability. Ablation of TRPC3 lessens pilocarpine-induced seizures in mice, suggesting that TRPC3 inhibition might represent a novel antiseizure strategy. Among current TRPC3 inhibitors, pyrazole 3 (Pyr3) is most selective and potent. However, Pyr3 only provides limited benefits in pilocarpine-treated mice, likely due to its low metabolic stability and potential toxicity. We recently reported a modified pyrazole compound 20 (or JW-65) that has improved stability and safety. The objective of this study was to explore the effects of TRPC3 inhibition by our current lead compound JW-65 on seizure susceptibility. METHODS: We first examined the pharmacokinetic properties including plasma half-life and brain to plasma ratio of JW-65 after systemic administration in mice. We then investigated the effects of TRPC3 inhibition by JW-65 on behavioral and electrographic seizures in mice treated with pilocarpine. To ensure our findings are not model specific, we assessed the susceptibility of JW-65-treated mice to pentylenetetrazole (PTZ)-induced seizures with phenytoin as a comparator. RESULTS: JW-65 showed adequate half-life and brain penetration in mice, justifying its use for central nervous system conditions. Systemic treatment with JW-65 before pilocarpine injection in mice markedly impaired the initiation of behavioral seizures. This antiseizure action was recapitulated when JW-65 was administered after pilocarpine-induced behavioral seizures were well established and was confirmed by time-locked electroencephalographic monitoring and synchronized video. Moreover, JW-65-treated mice showed substantially decreased susceptibility to PTZ-induced seizures in a dose-dependent manner. SIGNIFICANCE: These results suggest that pharmacological inhibition of the TRPC3 channels by our novel compound JW-65 might represent a new antiseizure strategy engaging a previously undrugged mechanism of action. Hence, this proof-of-concept study establishes TRPC3 as a novel feasible therapeutic target for the treatment of some forms of epilepsy.

Survivin Inhibitors Mitigate Chemotherapeutic Resistance in Breast Cancer Cells by Suppressing Genotoxic Nuclear Factor-κB Activation.

Therapeutic resistance developed after chemotherapy and aggressive metastasis are the major causes of cancer-related death in patients with triple-negative breast cancer (TNBC). Survivin is the smallest member of the inhibitor-of-apoptosis proteins (IAPs) family, which plays critical roles in cell division and cell survival. High expression levels of survivin have been associated with therapeutic resistance in various cancers. We recently developed a novel small-molecule survivin inhibitor mimicking the IAP-binding motif of second mitochondria-derived activator of caspase, which showed high potency in promoting survivin degradation. Here, we show that survivin inhibitor MX106/MX107 suppresses TNBC cell proliferation. Moreover, MX106/MX107 synergized with chemotherapeutic drugs or radiation and significantly enhanced tumoricidal efficacy of genotoxic treatments. Mechanistically, MX106/MX107 induced degradation of XIAP and/or cIAP1, which inhibited nuclear factor κB (NF-κB) activation by genotoxic agents. Treatment with MX106/MX107 alone did not activate alternative NF-κB signaling in breast cancer cells, which is likely attributable to their selective potency in degrading survivin in these cells. In addition, survivin degradation by MX106/MX107 dramatically increased abnormal mitotic spindle formation and cell division failure, which led to cell cycle arrest in breast cancer cells. Overall, our study suggests that combination treatment of TNBC using survivin inhibitors MX106/MX107 with cytotoxic chemotherapeutic drugs can achieve significantly improved therapeutic efficacy, which depends on MX106/MX107-mediated inhibition of genotoxic NF-κB activation.

An MDM2 degrader shows potent cytotoxicity to MDM2-overexpressing acute lymphoblastic leukemia cells with minimal toxicity to normal cells/tissues.

The MDM2 oncogene is amplified and/or overexpressed in various human cancers and elevated expression of MDM2 protein acts as a survival factor promoting cancer progression through both p53-dependent and -independent pathways. Here, we report a novel small-molecule chemical compound (MX69-102) that we identified to induce MDM2 protein degradation, resulting in reactivation of p53, inhibition of XIAP, and potent cell growth inhibition and apoptosis in MDM2-overexpressing acute lymphoblastic leukemia (ALL) in vitro and in vivo. We have previously identified a compound (MX69) that binds to the MDM2 C-terminal RING domain and induces MDM2 protein degradation. In the present study, we performed structural modifications of MX69 and selected analog MX69-102, showing increased MDM2-targeting activity. MX69-102 exhibited significantly enhanced inhibitory and apoptotic effects on a group of MDM2-overexpressing ALL cell lines in vitro with IC50 values of about 0.2 µM, representing an approximately 38-fold increase in activity compared to MX69. MX69-102 also showed effective inhibition on xenografted human MDM2-overexpressing ALL in SCID mice. Importantly, MX69-102 had minimal or no inhibitory effect on normal human hematopoiesis in vitro and was very well tolerated in vivo in animal models. Based on the strong inhibitory and apoptotic activity against MDM2-overexpressing ALL, along with minimal or no toxicity to normal cells/tissues, MX69-102 is a candidate for further development as a novel MDM2-targeted therapeutic drug for refractory/MDM2-overexpressing ALL.

Small molecule conjugates with selective estrogen receptor ß agonism promote anti-aging benefits in metabolism and skin recovery.

Estrogen is imperative to mammalian reproductivity, metabolism, and aging. However, the hormone activating estrogen receptor (ERs) α can cause major safety concerns due to the enrichment of ERα in female tissues and certain malignancies. In contrast, ERß is more broadly expressed in metabolic tissues and the skin. Thus, it is desirable to generate selective ERß agonist conjugates for maximizing the therapeutic effects of ERs while minimizing the risks of ERα activation. Here, we report the design and production of small molecule conjugates containing selective non-steroid ERß agonists Gtx878 or genistein. Treatment of aged mice with our synthesized conjugates improved aging-associated declines in insulin sensitivity, visceral adipose integrity, skeletal muscle function, and skin health, with validation in vitro. We further uncovered the benefits of ERß conjugates in the skin using two inducible skin injury mouse models, showing increased skin basal cell proliferation, epidermal thickness, and wound healing. Therefore, our ERß-selective agonist conjugates offer novel therapeutic potential to improve aging-associated conditions and aid in rejuvenating skin health.

TRPC3 suppression ameliorates synaptic dysfunctions and memory deficits in Alzheimer's disease.

Transient receptor potential canonical (TRPC) channels are widely expressed in the brain; however, their precise roles in neurodegeneration, such as Alzheimer's disease (AD) remain elusive. Bioinformatic analysis of the published single-cell RNA-seq data collected from AD patient cohorts indicates that the Trpc3 gene is uniquely upregulated in excitatory neurons. TRPC3 expression is also upregulated in post-mortem AD brains, and in both acute and chronic mouse models of AD. Functional screening of TRPC3 antagonists resulted in a lead inhibitor JW-65, which completely rescued Aß-induced neurotoxicity, impaired synaptic plasticity (e.g., LTP), and learning memory in acute and chronic experimental AD models. In cultured rat hippocampal neurons, we found that treatment with soluble ß-amyloid oligomers (AßOs) induces rapid and sustained upregulation of the TRPC3 expression selectively in excitatory neurons. This aberrantly upregulated TRPC3 contributes to AßOs-induced Ca 2+ overload through the calcium entry and store-release mechanisms. The neuroprotective action of JW-65 is primarily mediated via restoring AßOs-impaired Ca 2+ /calmodulin-mediated signaling pathways, including calmodulin kinases CaMKII/IV and calcineurin (CaN). The synaptic protective mechanism via TRPC3 inhibition was further supported by hippocampal RNA-seq data from the symptomatic 5xFAD mice after chronic treatment with JW-65. Overall, these findings not only validate TRPC3 as a novel therapeutic target for treating synaptic dysfunction of AD but most importantly, disclose a distinct role of upregulated TRPC3 in AD pathogenesis in mediating Ca 2+ dyshomeostasis.

Synthesis and biological evaluation of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold.

Overexpression of both human murine double minute 2 (MDM2) and X-linked inhibitor of apoptosis protein (XIAP) is detected in tumor cells from several cancer types, including childhood acute leukemia lymphoma (ALL), neuroblastoma (NB), and prostate cancer, and is associated with disease progression and treatment resistance. In this report, we described the design and syntheses of a series of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold from our previously reported lead compound JW-2-107 and tested their cytotoxicity in a panel of human cancer cell lines. The best compound identified in this study is compound 3e. Western blot analyses demonstrated that treatments with 3e decreased MDM2 and XIAP protein levels and increased expression of p53, resulting in cancer cell growth inhibition and cell death. Furthermore, compound 3e effectively inhibited tumor growth in vivo when tested using a human 22Rv1 prostate cancer xenograft model. Collectively, results in this study strongly suggest that the tetrahydroquinoline scaffold, represented by 3e and our earlier lead compound JW-2-107, has abilities to dual target MDM2 and XIAP and is promising for further preclinical development.

SB226, an inhibitor of tubulin polymerization, inhibits paclitaxel-resistant melanoma growth and spontaneous metastasis.

Extensive preclinical studies have shown that colchicine-binding site inhibitors (CBSIs) are promising drug candidates for cancer therapy. Although numerous CBSIs were generated and evaluated, but so far the FDA has not approved any of them due to undesired adverse events or insufficient efficacies. We previously reported two very potent CBSIs, the dihydroquinoxalinone compounds 5 m and 5t. In this study, we further optimized the structures of compounds 5 m and 5t and integrated them to generate a new analog, SB226. X-ray crystal structure studies and a tubulin polymerization assay confirmed that SB226 is a CBSI that could disrupt the microtubule dynamics and interfere with microtubule assembly. Biophysical measurements using surface plasmon resonance (SPR) spectroscopy verified the high binding affinity of SB226 to tubulin dimers. The in vitro studies showed that SB226 possessed sub-nanomolar anti-proliferative activities with an average IC50 of 0.76 nM against a panel of cancer cell lines, some of which are paclitaxel-resistant, including melanoma, breast cancer and prostate cancer cells. SB226 inhibited the colony formation and migration of Taxol-resistant A375/TxR cells, and induced their G2/M phase arrest and apoptosis. Our subsequent in vivo studies confirmed that 4 mg/kg SB226 strongly inhibited the tumor growth of A375/TxR melanoma xenografts in mice and induced necrosis, anti-angiogenesis, and apoptosis in tumors. Moreover, SB226 treatment significantly inhibited spontaneous axillary lymph node, lung, and liver metastases originating from subcutaneous tumors in mice without any obvious toxicity to the animals' major organs, demonstrating the therapeutic potential of SB226 as a novel anticancer agent for cancer therapy.

MYCN mRNA degradation and cancer suppression by a selective small-molecule inhibitor in MYCN-amplified neuroblastoma.

Amplification of the MYCN gene leads to its overexpression at both the mRNA and protein levels. Overexpression of MYCN mRNA may also have an important role in promoting neuroblastoma (NB) beyond the translation of MYCN protein. In the present study, we report a small molecule compound (MX25-1) that was able to bind to the 3'UTR of MYCN mRNA and induce MYCN mRNA degradation; this resulted in potent cell-growth inhibition and cell death specifically in MYCN-amplified or MYCN 3'UTR overexpressing NB cells. To evaluate the role of MYCN 3'UTR-mediated signals in contributing to the anticancer activity of MX25-1, we examined the status and activation of the tumor suppressor microRNA (miRNA) let-7, which is a target of MYCN 3'UTR in MYCN-amplified NB. We first observed that overexpression of MYCN mRNA was associated with high-level expression of the let-7 oncogenic targets DICER1, ARID3B and HMGA2. Following MYCN mRNA degradation, the expression of DICER1, ARID3B and HMGA2 was downregulated in MX25-1-treated cells. Inhibition of let-7 reversed the downregulation of these oncogenic mRNAs and significantly increased resistance of NB cells to MX25-1. Our results from this study supported the notion that overexpression of MYCN mRNA due to gene amplification has an independent function in NB cell growth and disease progression and suggest that targeting MYCN mRNA may represent an attractive strategy for therapy of MYCN amplified NB, both by inhibiting MYCN's cell-survival effects and activating the tumor-suppressor effect of let-7.

Synthesis and biological evaluation of selective survivin inhibitors derived from the MX-106 hydroxyquinoline scaffold.

The survivin (BIRC5) expression is very low in normal differentiated adult tissues, but it is one of the most widely upregulated genes in tumor cells. The overexpression of survivin in many cancer types has been positively correlated with resistance to chemotherapy, tumor metastasis, and poor patient survival. Survivin is considered to be a cancer specific biomarker and serves as a potential cancer drug target. In this report, we describe the design and syntheses of a series of novel selective survivin inhibitors based on the hydroxyquinoline scaffold from our previously reported lead compound MX-106. The best compound identified in this study is compound 12b. In vitro, 12b inhibited cancer cell proliferation with an average IC50 value of 1.4 µM, using a panel of melanoma, breast, and ovarian cancer cell lines. The metabolic stability of 12b improved over MX-106 by 1.7-fold (88 vs 51 min in human microsomes). Western blot analyses demonstrated that treatments with 12b selectively decreased survivin protein levels, but negligibly affected other closely related members in the IAP family proteins, and strongly induced cancer cell apoptosis. In vivo, compound 12b effectively inhibited melanoma tumor growth when tested using a human A375 melanoma xenograft model. Further evaluation using an aggressive, orthotopic ovarian cancer mouse model showed that 12b was highly efficacious in suppressing both primary tumor growth in ovaries and tumor metastasis to multiple peritoneal organs. Collectively, results in this study strongly suggest that the hydroxyquinoline scaffold, represented by 12b and our earlier lead compound MX-106, has abilities to selectively target survivin and is promising for further preclinical development.

Discovery of N-(3,4-Dimethylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide as a Potent Dual MDM2/XIAP Inhibitor.

Murine double minute 2 (MDM2) and X-linked inhibitor of apoptosis protein (XIAP) are important cell survival proteins in tumor cells. As a dual MDM2/XIAP inhibitor reported previously, compound MX69 has low potency with an IC50 value of 7.5 µM against an acute lymphoblastic leukemia cell line EU-1. Herein, we report the structural optimization based on the MX69 scaffold, leading to the discovery of a 25-fold more potent analogue 14 (IC50 = 0.3 µM against EU-1). We demonstrate that 14 maintains its mode of action by dual targeting of MDM2 and XIAP through inducing MDM2 protein degradation and inhibiting XIAP mRNA translation, respectively, which resulted in cancer cell growth inhibition and cell death. The results strongly suggest that the scaffold based on 14 is promising for further optimization to develop a new therapeutic agent for leukemia and possibly other cancers where MDM2 and XIAP are dysregulated.

Discovery of a Highly Selective and Potent TRPC3 Inhibitor with High Metabolic Stability and Low Toxicity.

The overactivation of transient receptor potential canonical 3 (TRPC3) is associated with neurodegenerative diseases and hypertension. Pyrazole 3 (Pyr3) is reported as the most selective TRPC3 inhibitor, but it has two inherent structural limitations: (1) the labile ester moiety leads to its rapid hydrolysis to the inactive Pyr8 in vivo, and (2) the alkylating trichloroacrylic amide moiety is known to be toxic. To circumvent these limitations, we designed a series of conformationally restricted Pyr3 analogues and reported that compound 20 maintains high potency and selectivity for human TRPC3 over its closely related TRP channels. It has significantly improved metabolic stability compared with Pyr3 and has a good safety profile. Preliminary evaluation of 20 demonstrated its ability to rescue Aß-induced neuron damage with similar potency to that of Pyr3 in vitro. Collectively, these results suggest that 20 represents a promising scaffold to potentially ameliorate the symptoms associated with TRPC3-mediated neurological and cardiovascular disorders.

A Luminacin D Analog HL142 Inhibits Ovarian Tumor Growth and Metastasis by Reversing EMT and Attenuating the TGFß and FAK Pathways.

Epithelial to mesenchymal transition (EMT) is known to contribute to tumor metastasis and chemoresistance. Reversing EMT using small molecule inhibitors to target EMT associated gene expression represents an effective strategy for cancer treatment. The purpose of this study is to test whether a new luminacin D analog HL142 reverses EMT in ovarian cancer (OC) and has the therapeutic potential for OC. We chemically synthesized HL142 and tested its functions in OC cells in vitro and its efficacy in inhibiting ovarian tumor growth and metastasis in vivo using orthotopic OC mouse models. We first demonstrate that ASAP1 is co-amplified and interacts with the focal adhesion kinase (FAK) protein in serous ovarian carcinoma. HL142 inhibits ASAP1 and its interaction protein FAK in highly invasive OVCAR8 and moderately invasive OVCAR3 cells. HL142 inhibits EMT phenotypic switch, accompanied by upregulating epithelial marker E-cadherin and cytokeratin-7 and downregulating mesenchymal markers vimentin, ß-catenin, and snail2 in both cell lines. Functionally, HL142 inhibits proliferation, colony formation, migration, and invasion. HL142 also sensitizes cell responses to chemotherapy drug paclitaxel treatment and inhibits ovarian tumor growth and metastasis in orthotopic OC mouse models. We further show that HL142 attenuates the TGFß and FAK pathways in vitro using OC cells and in vivo using orthotopic mouse models.

X-ray Crystallography-Guided Design, Antitumor Efficacy, and QSAR Analysis of Metabolically Stable Cyclopenta-Pyrimidinyl Dihydroquinoxalinone as a Potent Tubulin Polymerization Inhibitor.

Small molecules that interact with the colchicine binding site in tubulin have demonstrated therapeutic efficacy in treating cancers. We report the design, syntheses, and antitumor efficacies of new analogues of pyridopyrimidine and hydroquinoxalinone compounds with improved drug-like characteristics. Eight analogues, 5j, 5k, 5l, 5m, 5n, 5r, 5t, and 5u, showed significant improvement in metabolic stability and demonstrated strong antiproliferative potency in a panel of human cancer cell lines, including melanoma, lung cancer, and breast cancer. We report crystal structures of tubulin in complex with five representative compounds, 5j, 5k, 5l, 5m, and 5t, providing direct confirmation for their binding to the colchicine site in tubulin. A quantitative structure-activity relationship analysis of the synthesized analogues showed strong ability to predict potency. In vivo, 5m (4 mg/kg) and 5t (5 mg/kg) significantly inhibited tumor growth as well as melanoma spontaneous metastasis into the lung and liver against a highly paclitaxel-resistant A375/TxR xenograft model.

Recent and emerging anti-diabetes targets.

Diabetes is a disease that affects over 150 million people worldwide for which there are multiple oral and injectable medications. Because of trends in obesity and sedentary lifestyles, diabetes rates in both developed and developing countries are increasing at an alarming rate. Current medications are not adequately effective in maintaining long-term glycemic control in most patients, even when used in combination, leaving diabetics susceptible to developing life threatening and debilitating complications such as cardiovascular disease, blindness, kidney complications, and amputations. Consequently, there is a critical need for more potent pharmacotherapies with novel mechanisms of action. A panel of 20 emerging diabetes targets is presented, and small molecule modulators for each target will be discussed.

Design, synthesis and biological evaluation of selective survivin inhibitors.

The differential distribution between cancer cells and normal adult tissues makes survivin a very attractive cancer drug target. We have previously reported a series of novel selective survivin inhibitors with the most potent compound MX106 reaching nanomolar activity in several cancer cell lines. Further optimization of the MX106 scaffold leads to the discovery of more potent and more selective survivin inhibitors. Various structural modifications were synthesized and their anticancer activities were evaluated to determine the structure activity relationships for this MX106 scaffold. In vitro anti-proliferative assays using two human melanoma cell lines showed that several new analogs have improved potency compared to MX106. Very interestingly, these new analogs generally showed significantly higher potency against P-glycoprotein overexpressed cells compared with the corresponding parental cells, suggesting that these compounds may strongly sensitize tumors that have high expressions of the P-glycoprotein drug efflux pumps. Western blotting analysis confirmed that the new MX106 analogs maintained their mechanism of actions by selectively suppressing survivin expression level among major inhibitors of apoptotic proteins and induced strong apoptosis in melanoma tumor cells.

Ovarian Primary and Metastatic Tumors Suppressed by Survivin Knockout or a Novel Survivin Inhibitor.

Survivin, a member of the inhibitor of apoptosis family, is upregulated in multiple cancers including ovarian cancer, but is rarely detectable in normal tissues. We previously reported that survivin promoted epithelial-to-mesenchymal transition (EMT) in ovarian cancer cells, suggesting that survivin may contribute to ovarian tumor metastasis and chemoresistance. In this study, we tested whether knockout or pharmacologic inhibition of survivin overcomes chemoresistance and suppresses tumor metastasis. The genetic loss of survivin suppressed tumor metastasis in an orthotopic ovarian cancer mouse model. To pharmacologically test the role of survivin on ovarian tumor metastasis, we treated chemo-resistant ovarian cancer cells with a selective survivin inhibitor, MX106, and found that MX106 effectively overcame chemoresistance in vitro MX106 inhibited cell migration and invasion by attenuating the TGFß pathway and inhibiting EMT in ovarian cancer cells. To evaluate the efficacy of MX106 in inhibiting ovarian tumor metastasis, we treated an orthotopic ovarian cancer mouse model with MX106, and found that MX106 efficiently inhibited primary tumor growth in ovaries and metastasis in multiple peritoneal organs as compared with vehicle-treated control mice. Our data demonstrate that inhibition of survivin using either genetic knockout or a novel inhibitor MX106 suppresses primary ovarian tumor growth and metastasis, supporting that targeting survivin could be an effective therapeutic approach in ovarian cancer.

Structural Modification of the 3,4,5-Trimethoxyphenyl Moiety in the Tubulin Inhibitor VERU-111 Leads to Improved Antiproliferative Activities.

Colchicine binding site inhibitors (CBSIs) hold great potential in developing new generations of antimitotic drugs. Unlike existing tubulin inhibitors such as paclitaxel, they are generally much less susceptible to resistance caused by the overexpression of drug efflux pumps. The 3,4,5-trimethoxyphenyl (TMP) moiety is a critical component present in many CBSIs, playing an important role in maintaining suitable molecular conformations of CBSIs and contributing to their high binding affinities to tubulin. Previously reported modifications to the TMP moiety in a variety of scaffolds of CBSIs have usually resulted in reduced antiproliferative potency. We previously reported a potent CBSI, VERU-111, that also contains the TMP moiety. Herein, we report the discovery of a VERU-111 analogue 13f that is significantly more potent than VERU-111. The X-ray crystal structure of 13f in complex with tubulin confirms its direct binding to the colchicine site. In addition, 13f exhibited a strong inhibitory effect on tumor growth in vivo.