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OBJECTIVE: As a key member of 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 merely 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 uses for CNS 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 electroencephalography (EEG) 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 the TRPC3 as a novel feasible therapeutic target for the treatment of some forms of epilepsy.

Supplementary Figure S1. Survivin and EMT markers were stained in sections of ovarian tumor of survivin KO and control mice; Supplementary Figure S2. Ovarian cancer cell migration and invasion following inhibition of apoptosis; Supplementary Figure S3. Cell viability in the upper chamber following treatment of MX106 in the migration assay; Supplementary Figure S4. Cell viability in the upper chamber following treatment of MX106 in the invasion assay; Supplementary Figure S5. SMAD dependent reporter gene luciferase activity; Supplementary Figure S6. TGFβ did not activate non-SMAD pathway in ovarian cancer cells; Supplementary Figure S7. Immunostaining for survivin and EMT markers in sections of ovarian tumor tissue following MX106 or vehicle treatment.

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 TGFβ 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.

Supplementary Figures and Tables - PDF file 898K, Supplementary Table ST1: AKR1C3 over-expression in HEK-293 cells reduces IC50 of androgens. Supplementary Table ST2: Finasteride increases the testosterone formation. Supplementary Figure S1: Over-expression of AKR1C3 increases LNCaP xenograft growth in intact mice. Supplementary Figure S2: AR target FKBP51, but not AR, protein expression is increased in LNCaP-AKR1C3 xenograft tumors. Supplementary Figure S3: AKR1C3 translocation to nucleus requires AR. Top panel. NIH3T3-AKR1C3 cells infected with adenovirus LacZ. Bottom panel. NIH3T3-AKR1C3 cells infected with adenovirus AR.Supplementary Figure S4: AKR1C3 migrates with AR. Supplementary Figure S5: Duolink assay demonstrates interaction between AR and AKR1C3 in LNCaP-AKR1C3 cells. Supplementary Figure S6: AKR1C3 synergizes with SRC-2 in AR transactivation assay. Supplementary Figure S7: AKR1C3 dependent- androgen induced- AR transactivation is not cell type dependent. Transient transactivation studies conducted in COS-1 cells Supplementary Figure S8: AKR1C3-dependent increase in transactivation is selective to AR. Supplementary Figure S9: R1881 induced- AKR1C3 dependent- AR transactivation is not observed with other AKR1C. Supplementary Figure-S10: Different domains mediate the enzymatic and activator functions of AKR1C3. Supplementary Figure S11: GTx-560 is specific for AKR1C3. Supplementary Figure S12: HEK-293-AKR1C3 enzyme activity. Supplementary Figure S14: GTx-560 inhibits AKR1C3-dependent A�dione-induced AR transactivation at all concentration of AKR1C3. Supplementary Figure S15: Expression of steroidogenic enzymes in VCaP cells

Supplementary Methods - PDF file 62K, Additional methods to support the manuscript 1. Cloning and protein purification 2. AKR1C3 enzyme activity and thin layer chromatography (TLC)

Purpose: Castration-resistant prostate cancer (CRPC) may occur by several mechanisms including the upregulation of androgen receptor (AR), coactivators, and steroidogenic enzymes, including aldo keto reductase 1C3 (AKR1C3). AKR1C3 converts weaker 17-keto androgenic precursors to more potent 17-hydroxy androgens and is consistently the major upregulated gene in CRPC. The studies in the manuscript were undertaken to examine the role of AKR1C3 in AR function and CRPC.Experimental Design: LNCaP cells stably transfected with AKR1C3 and VCaP cells endogenously expressing AKR1C3 were used to understand the effect of AKR1C3 on prostate cancer cell and tumor growth in nude mice. Chromatin immunoprecipitation, confocal microscopy, and co-immunoprecipitation studies were used to understand the recruitment of AKR1C3, intracellular localization of AKR1C3 and its interaction with AR in cells, tumor xenograft, and in Gleason sum 7 CRPC tissues. Cells were transiently transfected for AR transactivation. Novel small-molecule AKR1C3-selective inhibitors were synthesized and characterized in androgen-dependent prostate cancer and CRPC models.Results: We identified unique AR-selective coactivator- and prostate cancer growth-promoting roles for AKR1C3. AKR1C3 overexpression promotes the growth of both androgen-dependent prostate cancer and CRPC xenografts, with concomitant reactivation of androgen signaling. AKR1C3 interacted with AR in prostate cancer cells, xenografts, and in human CRPC samples and was recruited to the promoter of an androgen-responsive gene. The coactivator and growth-promoting functions of AKR1C3 were inhibited by an AKR1C3-selective competitive inhibitor.Conclusions: AKR1C3 is a novel AR-selective enzymatic coactivator and may represent the first of more than 200 known nuclear hormone receptor coactivators that can be pharmacologically targeted. Clin Cancer Res; 19(20); 5613–25. ©2013 AACR.

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.

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.

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 5m and 5t. In this study, we further optimized the structures of compounds 5m and 5t and integrated them to generate an 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 IC

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, β-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 TGFβ and FAK pathways in vitro using OC cells and in vivousing orthotopic mouse models.

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 IC(50) 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 (IC(50) = 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.

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 mainly through inhibition of the tumor suppressor p53. Here, we report a novel small-molecule chemical compound (MX69-114b) that we identified to induce MDM2 protein degradation resulting in reactivation of p53 and potent cell growth inhibition and apoptosis in MDM2-overexpressing acute lymphoblastic leukemia (ALL). 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 modification of MX69 and selected analog MX69-114b showing increased MDM2-targeting activity. MX69-114b exhibited significantly enhanced inhibitory and apoptotic effects on a group of MDM2-overexpressing ALL cell lines in vitro with IC50 values of 0.08-0.14 µM, representing a >80-100-fold increase in activity compared to MX69. MX69-114b also showed inhibitory effects on xenografted human MDM2-overexpressing ALL in SCID mice at a much lower dose than did MX69. Importantly, MX69-114b 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, MX69-114b is a candidate for further development as a novel MDM2-targeted therapeutic drug for refractory ALL. Citation Format: tao Liu, Lubing Gu, Anna Mui, Zhongzhi Wu, Wei Li, Muxiang Zhou. A MDM2 degrader inhibits cell proliferation and growth of MDM2-overexpressing acute lymphoblastic leukemia in SCID mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 470.

Melanoma is the most lethal skin cancer, with the increasing incidence in the past decade. Despite the significant advancements in immune therapy and targeted therapy, the overall prognosis for metastatic melanoma remains unsatisfactory. TP53 is a tumor suppressor, but its wildtype is inactivated in about 90% of melanoma. Since activated p53 can promote cancer cell apoptosis and thus effectively inhibit tumor growth and tumor metastasis, reactivation of p53 is considered a promising strategy for cancer therapy. Herein, we report a recently developed small molecule compound, JW-1-283, targeting the interactions between MDM2 E3 ubiquitin-protein ligase and p53 protein. In vitro, JW-1-283 showed potent cytotoxicity against A375 melanoma cell line (IC50 2.2±0.3 μM) which has wildtype p53, but its potency is substantially reduced in two other melanoma cell lines with either p53 mutation (M14) or p53 null (RPMI-7951) status. Treatment with JW-1-283 significantly dose-dependently reduced the stemness in A375 melanoma cells, as indicated by low numbers of colony formation, impaired tumor sphere formation abilities, and inhibition of cancer cell migration. Mechanistically, JW-1-283 disrupts the p53-MDM2 interaction, prevents p53 and MDM2 degradation in the existence of cycloheximide, and induces p38 and p53 phosphorylation in the apoptotic pathway. Genetic knockdown of p38 by siRNA further confirmed that the stabilized p53 could be subsequently phosphorylated by p38, leading to induced apoptosis as well as S phase cell cycle arrest. In vivo, when A375 tumors grown in NSG mice were treated with 7.5 mg/kg of JW-1-283 intraperitoneally every other day for a consecutive of 16 days, significant inhibition of tumor growth was observed, along with decreased tumor cell proliferation, tumor angiogenesis, and enhanced tumor cell apoptosis. In summary, this work provides the initial proof-of-concept of developing the JW-1-283 scaffold as a potential strategy for advanced melanoma, which inhibits tumor growth by activating the p53 signaling pathway. Citation Format: Yang Xie, Kelli Hartman, Zhongzhi Wu, Wei Li. JW-1-283 inhibits melanoma tumor growth via the stabilization of p53 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2784.

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 TGFβ 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.

[Image: see text] 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 Aβ-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.

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.

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.

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.

One of the major challenges in colon cancer chemotherapy is multidrug resistance (MDR), which is typically mediated by the overexpression of ATP-binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp, ABCB1, MDR1). A number of P-gp inhibitors have been developed, however, none of these compounds have improved chemotherapeutic efficacy due to undesirable pharmacokinetic profiles or adverse effects, resulting in limited clinical success. Therefore, alternative approaches are urgently needed to circumvent MDR cancer. In previous study, a series of synthesized analogs of MX-106, as anti-cancer drugs targeting survivin, exhibited collateral sensitivity (CS) effect to P-gp overexpressing MDR colon cancer cells as well as ABCB1 gene transfected cells, reflected by more than 10-fold cytotoxic effect in P-gp positive MDR cell lines compared to drug sensitive cell lines. Among the analogs, MX-106-4C was identified as the leading compound with the most potent selective toxicity to P-gp overexpressing cells. MX-106-4C-induced CS effect was observed in both intrinsic and acquired P-gp overexpressing colon cancer cells, which was only partially reversed with the presence of a P-gp inhibitor. Nevertheless, this CS effect was abolished in ABCB1-knockout cells, indicating that the selective cytotoxicity was P-gp expression dependent, but only partially related to P-gp function. Furthermore, we found that MX-106-4C did not significantly affect P-gp ATPase activity or drug accumulation and efflux in P-gp-overexpressing cells. In P-gp overexpressing colon cancer cells, short-term (up to 72 h) incubation of MX-106-4C significantly down regulated P-gp expression at transcriptional level but not protein level, whereas long-term (14 d) incubation of MX-106-4C significantly down regulated P-gp protein expression and re-sensitized MDR colon cancer cells to doxorubicin. These findings suggested an indirect interaction and regulation between MX-106-4C and P-gp. Further study revealed that the selective cytotoxic effects of MX-106-4C were associated with cell cycle arrest at G1 phase and apoptosis through the downregulation of CDK4. Overall, this study demonstrates that MX-106-4C selectively kills P-gp positive MDR colon cancer cells and indirectly regulates P-gp, which provides a clue for CS compound design and a novel strategy to obviate P-gp-mediated colon cancer MDR by re-sensitizing heterogeneous tumors with CS agents. Citation Format: Zi-Ning Lei, Zhongzhi Wu, Qiu-Xu Teng, Min Xiao, Wei Li, John N. Wurpel, Zhe-Sheng Chen. Selective toxicity of MX-106-4C, a survivin inhibitor, in P-glycoprotein-mediated multidrug resistant colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5264.

Purpose: Castration-resistant prostate cancer (CRPC) may occur by several mechanisms including the upregulation of androgen receptor (AR), coactivators, and steroidogenic enzymes, including aldo keto reductase 1C3 (AKR1C3). AKR1C3 converts weaker 17-keto androgenic precursors to more potent 17-hydroxy androgens and is consistently the major upregulated gene in CRPC. The studies in the manuscript were undertaken to examine the role of AKR1C3 in AR function and CRPC. Experimental Design: LNCaP cells stably transfected with AKR1C3 and VCaP cells endogenously expressing AKR1C3 were used to understand the effect of AKR1C3 on prostate cancer cell and tumor growth in nude mice. Chromatin immunoprecipitation, confocal microscopy, and co-immunoprecipitation studies were used to understand the recruitment of AKR1C3, intracellular localization of AKR1C3 and its interaction with AR in cells, tumor xenograft, and in Gleason sum 7 CRPC tissues. Cells were transiently transfected for AR transactivation. Novel small-molecule AKR1C3-selective inhibitors were synthesized and characterized in androgen-dependent prostate cancer and CRPC models. Results: We identified unique AR-selective coactivator- and prostate cancer growth-promoting roles for AKR1C3. AKR1C3 overexpression promotes the growth of both androgen-dependent prostate cancer and CRPC xenografts, with concomitant reactivation of androgen signaling. AKR1C3 interacted with AR in prostate cancer cells, xenografts, and in human CRPC samples and was recruited to the promoter of an androgen-responsive gene. The coactivator and growth-promoting functions of AKR1C3 were inhibited by an AKR1C3-selective competitive inhibitor. Conclusions: AKR1C3 is a novel AR-selective enzymatic coactivator and may represent the first of more than 200 known nuclear hormone receptor coactivators that can be pharmacologically targeted. Clin Cancer Res; 19(20); 5613–25. ©2013 AACR.

1α,20S-Dihydroxyvitamin D3 [1,20S(OH)2D3], a natural and bioactive vitamin D3 metabolite, was chemically synthesized for the first time. X-ray crystallography analysis of intermediate 15 confirmed its 1α-OH configuration. 1,20S(OH)2D3 interacts with the vitamin D receptor (VDR), with similar potency to its native ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] as illustrated by its ability to stimulate translocation of the VDR to the nucleus, stimulate VDRE-reporter activity, regulate VDR downstream genes (VDR, CYP24A1, TRPV6 and CYP27B1), and inhibit the production of inflammatory markers (IFNγ and IL1β). However, their co-crystal structures revealed differential molecular interactions of the 20S-OH moiety and the 25-OH moiety to the VDR, which may explain some differences in their biological activities. Furthermore, this study provides a synthetic route for the synthesis of 1,20S(OH)2D3 using the intermediate 1α,3β-diacetoxypregn-5-en-20-one (3), and provides a molecular and biological basis for the development of 1,20S(OH)2D3 and its analogs as potential therapeutic agents.

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.

Nonsteroidal estrogens have been known since the 1930s. However, the relatively recent (1996) discovery of estrogen receptor subtype beta (ERbeta) suggested a possible paradigm shift away from SERM-like selectivity. Selective ERbeta agonism would potentially allow expansion of estrogenic targeting into new indications (discussed herein) currently precluded by the thrombogenic and hyperproliferative effects of nonselective estrogens.ERbeta agonist design has been very successful. Pharmacophores for ERbeta selective nonsteroidal estrogens are generally diphenolic compounds that achieve an inter-phenolic distance and geometry similar to 17beta-estradiol with few restraints on the nature of the element linking the phenols (or phenol mimetics). The tremendously chemodiverse ERbeta agonist patent literature is reviewed, segregating the agonists into structurally similar compounds based on their interphenolic linking elements.A comprehensive understanding of the chemotype landscape of this field and an assessment of its maturation.Subtype selective ERbeta agonist therapy seems very promising. However, more clinical testing is needed to firmly establish its therapeutic potential. At this point, ERbeta is a promising target in search of an indication.

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.

New boronated unnatural cyclic amino acids, 1–6, were synthesized for potential use in neutron capture therapy. In order to understand the effect of molecular lipophilicity on the biological activity, different linkers were introduced between the boronic acid and 1-aminocycloalkanecarboxylic acid moieties. The key step in the syntheses was the preparation of a series of alkenyl-substituted cycloalkanones, which were subsequently converted to amino acids via the Bucherer–Strecker reaction. The introduction of the boronic acid function into hydrantoins 19–24 was realized by hydroboration reactions using diisopinocampheylborane (Ipc2BH). The target boronated amino acids were modeled after 1-aminocyclobutanecarboxylic acid and 1-amino-3-boronocyclopentanecarboxylic acid, which have previously demonstrated high uptake in tumors. Copyright © 2008 John Wiley & Sons, Ltd.

A boron trihalide mediated alkyne-aldehyde coupling reaction leading to stereodefined 1,3,5-triaryl-1,5-dihalo-1,4-pentadienes is described. The study led to the discovery of a direct substitution of hydroxyl groups by stereodefined alkenyl moieties using alkenylboron dihalides. During the investigation, it was also discovered that, at low temperatures, the reaction of BCl3 with alkynes produces monovinylboron dichloride rather than the reported divinylboron chloride. A modified reaction mechanism for the boron trichloride mediated alkyne-aldehyde coupling reaction is now proposed. The reaction temperature and mode of addition have been found to have dramatic affects on the stereochemistry of the diene products.

Cyclopentadienyl-free disilyl amide complexes [(Me2N)3M(SiButPh2)2]- (M = Zr, 1; Hf, 2 as [Li(THF)4]+ salts), K(18-crown-6)3/2{(Me2N)3M[(Me3Si)2Si-(CH2)2-Si(SiMe3)2]} (M = Zr, 3; Hf, 4), (Me2N)3Ta[Si(SiMe3)3]2 (5), (Me2N)3Ta(SiButPh2)2 (6), and (Me2N)3Ta(SiButPh2)[Si(SiMe3)3] (7) have been prepared. The structures of 1−4 have been determined by X-ray single-crystal diffraction. The two −Si(SiMe3)3- ligands in (Me2N)3Ta[Si(SiMe3)3]2 (5) were replaced sequentially by the −SiButPh2- anions to give (Me2N)3Ta(SiButPh2)[Si(SiMe3)3] (7) and (Me2N)3Ta(SiButPh2)2 (6). The silyl ligand in (Me2N)3Zr-Si(SiMe3)3 was found to undergo a reversible exchange with SiButPh2-, probably through a disilyl intermediate, to reach the following equilibrium: (Me2N)3ZrSi(SiMe3)3 + SiButPh2- ⇌ (Me2N)3ZrSiButPh2 + Si(SiMe3)3- with ΔH° = 4.6(0.5) kcal/mol and ΔS° = −7(2) eu. A similar exchange involving [(Me2N)3M(SiButPh2)2]- (M = Zr, 1; Hf, 2) was observed: (Me2N)3ZrSiButPh2 + SiButPh2- ⇌ 1 with the estimated free energy of activat...

Bis(alkylidene) complex W(CH-t-Bu)2(CH2-t-Bu)(Si-t-BuPh2) (1a) has been found to be in equilibrium with its alkyl alkylidyne tautomer W(⋮C-t-Bu)(CH2-t-Bu)2(Si-t-BuPh2) (1b). Bis(alkylidene) complex...

Reaction of (ArN)2MoCl2(DME) (Ar=2,6-Pri 2C6H3, DME=dimethoxyethane) with LiNMe2 and Li(THF)3Si(SiMe3)3 was found to give a Mo imide amide silyl complex (ArN)2Mo(NMe2)[Si(SiMe3)3] (1). Imide amide complexes (ArN)2Mo(NMe2)2 (2) and (ArN)2Mo(NMe2)[N(SiMe3)2] (4) were prepared from the reactions of (ArN)2MoCl2(DME) with lithium amide reagents and characterized. X-ray structural studies of 1, 2 and 4 are presented. An intermediate to 4, (ArN)2MoCl[N(SiMe3)2] (3), was obtained from the reaction of (ArN)2MoCl2(DME) with LiN(SiMe3)2 and structurally characterized. In addition, a new X-ray structure of previously reported complex (ArN)2Mo(NHAr)2 (5) is reported.

Several new organic transformations have been achieved utilizing boron halide reagents. Aryl aldehydes are conveniently converted to gem-dichloromethylbenzenes using boron trichloride. Aryl aldehydes are alkylated by alkylboron chlorides in a Grignard-like fashion to generate the corresponding arylalkanols or alkylboron chlorides. Aryl aldehydes react with divinylboron halides (generated via the haloboration of alkynes) to produce 1,5-di-halo-1,4-dienes in excellent yields.

Dialkynylboron chlorides couple smoothly with aryl aldehydes at room temperature to afford 1,4-dialkynes in good to excellent yields. Dialkynylboron halides act simultaneously as Lewis acid and reactant in this coupling reaction.

Alkylidene complexes (Me 3 SiCH 2 ) 3 Ta(PMe 3 )=CHSiMe 3 (1) and Me 3 SiCH 2 Ta(PMe 3 ) 2 (=CHSiMe 3 ) 2 (3a) were found to react with phenylsilanes H 2 SiR'Ph (R' = Me, Ph) and (PhSiH 2 ) 2 CH 2 to give disilyl-substituted alkylidenes (Me 3 SiCH 2 ) 3 Ta=C(SiMe 3 )(SiHR'Ph) (2) and novel metallasilacyclobutadiene and metalladisilacyclohexadiene complexes. Silyl-substituted alkylidene complex (Bu t CH 2 ) 2 W(=O)[=C(But)(SiPh 2 Bu t )] (5a) was prepared from the reaction of O 2 with an equilibrium mixture (Bu t CH 2 )W(=CHBu t ) 2 (SiPh 2 Bu t ) (4b) ⇔ (Bu t CH 2 ) 2 W(≡CBu t )(SiPh 2 Bu t ) (4a). Our recent studies of the preparation of these complexes and mechanistic pathways in the formation of these silyl-substituted alkylidene complexes are summarized.

The synthesis and characterization of amido silyl complexes of tantalum(V) free of π-anionic ligands are reported. The amido silyl chloride complexes (Me2N)3Ta(SiR3)Cl [SiR3 = Si(SiMe3)3 (1a), SiPh2But (2)] were prepared from (Me2N)3TaCl2 and the corresponding silyllithium reagents Li(THF)3Si(SiMe3)3 and Li(THF)3SiPh2But. The amido silyl complexes (Me2N)4Ta(SiR3) [SiR3 = Si(SiMe3)3 (3), SiPh2But (4)] were synthesized by the reactions of (Me2N)4TaCl with Li(THF)3SiR3. Complex 3 was found to react with 1 equiv of O2 to give an oxidation product (Me2N)3Ta(η2-ONMe2)[OSi(SiMe3)3] (5), and the structure of 5 was confirmed by X-ray crystallography. The spectroscopic data and crystal structure determination reveal that the coordination geometry around Ta metal in 1a and 2−4 is trigonal bipyramid with silyl ligands in an equatorial position.

Reactions of aromatic aldehydes with alkylboron chloride derivatives in the presence of oxygen have been investigated. Dialkylboron chlorides react with aryl aldehydes to produce arylalkylmethanols in good to excellent yields. Under the same reaction conditions, alkylboron dichlorides lead to the formation of arylalkyl chlorides.

Reactions of aryl aldehydes with styrenes in the presence of boron trichloride in CH2Cl2 at 0°C produce 1,3-dichloro-1,3-diarylpropanes in excellent yields. Reactions carried out using boron tribromide generate the corresponding 1,3-dibromo-1,3-diarylpropane in good yields.

The reaction of aryl aldehydes with alkylboron chlorides has been investigated. Monoalkylboron dichlorides react with aryl aldehydes in hexane under reflux conditions to give a mixture of dichloroarylmethane and benzyl chloride. Under the same reaction conditions, dialkylboron chlorides lead to formation of a mixture of benzyl chloride and the chloroalkylation product. In the presence of a base such as 2,6-lutidine, the reactions of monoalkylboron dichlorides with aryl aldehydes yield small amounts of the desired alkylation products at room temperature. Dialkylboron chlorides react with aryl aldehydes in hexane in the presence of base to generate the corresponding arylalkylmethanols in good yields.

Reactions of silanes with d0 Ta alkylidene and Group 4 amide complexes were found to involve the initial attack on silicon atoms in the silanes by nucleophilic alkylidene carbon or amide nitrogen atoms. The reaction of (Me3SiCH2) 3Ta (PMe 3) [=CHSiMe3] (1) with H2SiR'Ph (R' = Me, Ph) gave (Me3SiCH2) 3Ta[=C (SiMe 3) SiHR'Ph] (2a-b). Similar reactions of these two silanes and (H2PhSi) 2CH2 with (Me3SiCH2) Ta (PMe3) 2[=CHR]2 (R = SiMe3, 3; CMe3, 4) yielded novel metallasilacyclobutadienes and a metalladisilacyclohexadiene, respectively. In comparison, the reactions between M (NMe2) 4 (M = Ti, Zr, Hf) and silanes were found to give aminosilanes and metal amide hydride species which were perhaps involved in the formation of titanium-silicon-nitride (Ti-Si-N) ternary materials.

The reductive bromination of aromatic aldehydes with isopinocampheyl boron dibromide in hexane at room temperature produces the corresponding benzyl bromides in excellent yields.

Alkoxide C-O bond cleavage occurs readily at room temperature in the presence of titanium(IV) halide. Capture of the resultant carbocation by alkynes provides an efficient route to trisubstituted (E)-alkenyl halides with high stereoselectivity.

The reactions of alkyl silyl complexes (Me3ECH2)3ZrSi(SiMe3)3 [E = C (1), Si (2)] with 2,6-dimethylphenyl isocyanide (ArNC) have been investigated. The first ArNC was found to insert exclusively into the Zr−Si bond, and the second and third ArNC into the Zr−C bonds in 1 and 2. 1 and 2 react with up to 3 equiv of ArNC to give (Me3ECH2)3Zr{η2-C[Si(SiMe3)3]NAr} [E = C (3), Si (4)], (Me3ECH2)2Zr[η2-C(CH2EMe3)NAr]{η2-C[Si(SiMe3)3]NAr} [E = C (5), Si (6)], and (Me3ECH2)Zr[η2-C(CH2EMe3)NAr]2{η2-C[Si(SiMe3)3]NAr} [E = C (7), Si (8)]. The tri-insertion complexes 7 and 8 are inert to excess ArNC. The structures of 3 and 7 have been determined by X-ray crystallography. In the structure of 3, the α-hydrogen atoms on one neopentyl ligand lie in close contact (av 2.41 A) with the metal center giving rise to Zr−C−Hα bond angles of 90 and 92°. The crystal structure of the precursor 1 has also been determined.

The chlorobis(trimethylsiloxy)zirconium silyl derivative (Me3SiO)2Zr(SiPh2But)Cl·2THF (2) and its precursor (Me3SiO)2ZrCl2·2THF (1) have been synthesized and characterized by X-ray crystallography. The Zr−Si bond distance of 2.848(3) A in 2 is, to our knowledge, the longest reported Zr−Si bond to date.

Reactions of 3 and 2 equiv of lithium dimethylamide with ZrCl4 in THF afford dinuclear complexes (Me2N)3Zr(μ-Cl)2(μ-NMe2)Zr(NMe2)2(THF) (1) and Cl(Me2N)2Zr(μ-Cl)2(μ-NMe2)Zr(NMe2)2(THF) (2), respectively. These complexes were found to have unusual structures containing dichloro(amido) bridges between Zr atoms. Both complexes 1 and 2 crystallize in the monoclinic space group P21/c (a = 8.735(3) A, b = 21.498(7) A, c = 13.997(5) A, β = 92.56(3)°, Z = 4 for 1; a = 8.550(3) A, b = 20.673(7) A, c = 14.229(6) A, β = 95.11(3)°, Z = 4 for 2). Each Zr atom in 1 and 2 has a trigonal antiprismatic coordination geometry. Reaction of 1 equiv of lithium dimethylamide with ZrCl4 in THF gave the monosubstituted complex as the LiCl adduct (Me2N)(THF)ZrCl2(μ-Cl)2Li(THF)2 (4), which crystallizes in the triclinic space group P1 (a = 10.078(6) A, b = 11.003(7) A, c = 11.039(6) A, α = 84.75(5)°, β = 78.38(5)°, γ = 66.65(5)°, Z = 2). In 4, the Zr atom has a distorted octahedral coordination geometry. These new amido complexes a...

Dialkynylboron chlorides couple smoothly with aryl aldehydes at room temperature to afford 1,4-dialkynes in good to excellent yields. Dialkynylboron halides act simultaneously as Lewis acid and reactant in this coupling reaction.

Reactions of aryl aldehydes with styrenes in the presence of boron trihalides produce 1,3-diaryl-1,3-dihalopropanes in excellent yields.

Receptor tyrosine kinases (RTKs), in response to their growth factor ligands, phosphorylate and activate downstream signals important for physiological development and pathological transformation. Increased expression, activating mutations and rearrangement fusions of RTKs lead to cancer, inflammation, pain, neurodegenerative diseases, and other disorders. Activation or over-expression of ALK, ROS1, TRK (A, B, and C), and RET are associated with oncogenic phenotypes of their respective tissues, making them attractive therapeutic targets. Cancer cDNA array studies demonstrated over-expression of TRK-A and ROS1 in a variety of cancers, compared to their respective normal tissue controls. We synthesized a library of small molecules that inhibit the above indicated RTKs with picomolar to nanomolar potency. The lead molecule GTx-186 inhibited RTK-dependent cancer cell and tumor growth. In vitro and in vivo growth of TRK-A-dependent IMR-32 neuroblastoma cells and ROS1-overexpressing NIH3T3 cells were inhibited by GTx-186. GTx-186 also inhibited inflammatory signals mediated by NFκB, AP-1, and TRK-A and potently reduced atopic dermatitis and air-pouch inflammation in mice and rats. Moreover, GTx-186 effectively inhibited ALK phosphorylation and ALK-dependent cancer cell growth. Collectively, the RTK inhibitor GTx-186 has a unique kinase profile with potential to treat cancer, inflammation, and neuropathic pain.

A new straightforward procedure has been developed for the synthesis of polysubstituted naphthalene derivatives. The reaction of alpha-aryl-substituted carbonyl compounds with terminal or internal alkynes in the presence of TiCl4 regioselectively generates substituted naphthalene derivatives in good to excellent yields.

Reactions of aryl aldehydes with styrene and (E)-β-methylstyrene in the presence of phenylboron dichloride regioselectively generate 1,3-diaryl-3-chloro-1-propanols and 1,3-diaryl-3-chloro-2-methyl-1-propanols in good to excellent yields with high stereoselectivity.