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Overexpression of the receptor tyrosine kinase EphA2 is invariably associated with poor prognosis and development of aggressive metastatic cancers. Guided by our recently solved X-ray structure of the complex between an agonistic peptide and EphA2-LBD, we report on a novel agent, targefrin, that binds to EphA2-LBD with a 21 nM dissociation constant by isothermal titration calorimetry and presents an IC

Guided by a combination of nuclear magnetic resonance binding assays and computational docking studies, we synthesized a library of 5,5′ substituted Apogossypol derivatives as potent Bcl-XL antagonists. Each compound was subsequently tested for its ability to inhibit Bcl-XL in an in vitro fluorescence polarization competition assay and exert single-agent proapoptotic activity in human cancer cell lines. The most potent compound BI79D10 binds to Bcl-XL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively, and potently inhibits cell growth in the H460 human lung cancer cell line with an EC50 value of 680 nmol/L, expressing high levels of Bcl-2. BI79D10 also effectively induces apoptosis of the RS11846 human lymphoma cell line in a dose-dependent manner and shows little cytotoxicity against bax−/−bak−/− mouse embryonic fibroblast cells, in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that BI79D10 has little off-target effects. BI79D10 displays in vivo efficacy in transgenic mice, in which Bcl-2 is overexpressed in splenic B cells. Together with its improved plasma and microsomal stability relative to Apogossypol, BI79D10 represents a lead compound for the development of novel apoptosis-based therapies for cancer. [Mol Cancer Ther 2009;8(4):904–13]

Supplementary Data from Apogossypol derivatives as antagonists of antiapoptotic Bcl-2 family proteins

PDF file - 328K, Scheme 1. Synthesis of YNH motif and dYNH motif Scheme S2. Synthetic route for YSA-PTX Figure S1. In vivo prostate cancer xenograft studies. Figure S2. EphA2 expression in RENCA renal carcinoma cells and PC3M prostate cancer cells. Supplemental Table 1. Athymic nude mice bearing pre-established subcutaneous PC3M tumors (230 mm3 average volume) were treated three times weekly for a period of 18 days (8 total injections) with intravenous doses of vehicle (CT), PTX (10 mg/Kg) and DYP-PTX or YSA-PTX (at a dose equimolar to the taxol dose).

Purpose: YSA is an EphA2-targeting peptide that effectively delivers anticancer agents to prostate cancer tumors. Here, we report on how we increased the drug-like properties of this delivery system.Experimental Design: By introducing non-natural amino acids, we have designed two new EphA2 targeting peptides: YNH, where norleucine and homoserine replace the two methionine residues of YSA, and dYNH, where a D-tyrosine replaces the L-tyrosine at the first position of the YNH peptide. We describe the details of the synthesis of YNH and dYNH paclitaxel conjugates (YNH-PTX and dYNH-PTX) and their characterization in cells and in vivo.Results: dYNH-PTX showed improved stability in mouse serum and significantly reduced tumor size in a prostate cancer xenograft model and also reduced tumor vasculature in a syngeneic orthotopic allograft mouse model of renal cancer compared with vehicle or paclitaxel treatments.Conclusion: This study reveals that targeting EphA2 with dYNH drug conjugates could represent an effective way to deliver anticancer agents to a variety of tumor types. Clin Cancer Res; 19(1); 128–37. ©2012 AACR.

Supplementary Table S2. Outline of genetic changes in the tumors and cell lines used.

Supplementary Figure Legends 1-9

Supplementary Figures 1-9. Sabutoclax has greater efficacy than ABT-737. (S1) Sabutoclax causes a G1-S phase cell cycle arrest. (S2) Pancreatic cancer cells exhibit varying levels of sensitivity to Minocycline. (S3) Sabutoclax and Minocycline induce cytotoxicity in PANC-1 cells that is reversed with zVAD. (S4) Sabutoclax and Minocycline show synergy. Combination index (CI) values for the combination of Sabutoclax and Minocycline in MIA PaCa-2 cells. (S5) The cytotoxicity induced by Sabutoclax and Minocycline is caspase-dependent and dependent upon loss of Stat3 activation. (S6) Sabutoclax and Minocycline reduce tumor growth in a subcutaneous xenograft model of pancreatic cancer. (S7) Tumors from Pdx-1-Cre/K-rasLSL-G12D/p53flox/wt and Pdx-1-Cre/K-rasLSL-G12D/p53flox/flox mice overexpress Mcl-1 and show sensitivity to Sabutoclax and Minocycline. (S8) Tumors from KPC (Pdx-1-Cre/K-rasLSL-G12D/p53flox/flox )mice treated with Sabutoclax and Minocycline show decreased Stat3 activation. (S9)

Supplementary Figure 1, Table 1 from Mechanism by Which Mcl-1 Regulates Cancer-Specific Apoptosis Triggered by mda-7/IL-24, an IL-10–Related Cytokine

PDF file - 171K, Effect of ectopic expression of RKIP on cytoskeletal organization in melanoma cells

Melanoma development involves members of the AGC kinase family, including AKT, PKC, and, most recently, PDK1, as elucidated recently in studies of Braf::Pten mutant melanomas. Here, we report that PDK1 contributes functionally to skin pigmentation and to the development of melanomas harboring a wild-type PTEN genotype, which occurs in about 70% of human melanomas. The PDK1 substrate SGK3 was determined to be an important mediator of PDK1 activities in melanoma cells. Genetic or pharmacologic inhibition of PDK1 and SGK3 attenuated melanoma growth by inducing G1 phase cell-cycle arrest. In a synthetic lethal screen, pan-PI3K inhibition synergized with PDK1 inhibition to suppress melanoma growth, suggesting that focused blockade of PDK1/PI3K signaling might offer a new therapeutic modality for wild-type PTEN tumors. We also noted that responsiveness to PDK1 inhibition associated with decreased expression of pigmentation genes and increased expression of cytokines and inflammatory genes, suggesting a method to stratify patients with melanoma for PDK1-based therapies. Overall, our work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment. Cancer Res; 75(7); 1399–412. ©2015 AACR.

PDF file - 1.8MB, Expression of MDA-9 and RKIP in clinical samples

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Description of additional methods and procedures used in the study. Also includes Supplemental References.

PDF file - 155K, IL-8 neutralizing antibody blocks C8161.9-induced HuVEC proliferation, migration and tube formation.

Supplementary Table 1 from Identification of Small Molecules that Sensitize Resistant Tumor Cells to Tumor Necrosis Factor-Family Death Receptors

Supplementary Table and Figure Legends. Legend to Supplementary Tables S1-S2 and Supplementary Figures S1-S6.

SBI-756 inhibits AKT, mTOR signaling and alters cell cycle.

PDF file - 275K, Characterization of stable shmda-9/syntenin and mda-9/syntenin clones.

PDF file - 652K, Characterization of MDA-9 overexpressed FM-516 clones

Melanoma differentiation–associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacologic approaches were used to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma, and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, chorioallantoic membrane (CAM) assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several proangiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the extracellular matrix (ECM), activating Src and FAK resulting in activation by phosphorylation of Akt, which induces hypoxia inducible factor 1-α (HIF-1α). The HIF-1α activates transcription of insulin growth factor–binding protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell nonautonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (nonautonomous). Cancer Res; 73(2); 844–54. ©2012 AACR.

PDF file - 264K, Quantitation of protein expression.

Pharmacological properties of top four BI-69A11 analogs (S1); Blood chemistry following SBI-756 administration (S2); List of biotinylated-BI-69A11-bound proteins, identified by LC/MS/MS (S3); RPPA based analyses identify proteins that were significantly inhibited by SBI-756, compared to DMSO treatment at the 24 h time point (S4); Gene variants identified in SBI-756-resistant clones but not SBI-756-sensitive melanoma cell cultures (S5); The top 10 enriched canonical pathways identified using the Ingenuity Pathway Analysis of genes mutated in SBI-756 resistant melanoma (S6); Ingenuity Pathway Analysis of genes differentially expressed between SBI-756-sensitive and - resistant melanoma cells (S7).

PDF file - 89K, Graphical presentation of CD-31 expression in tumor sections.

PDF file - 413K, ERK activation negatively regulate RKIP expression

PDF file - 180K, Effect of RKIP expression on B-RAF mutant cells

PDF file - 746K, Quantitation of protein expression.

Supplementary Figures S1-S6. BrafV600E::Cdkn2a-/- delay development of tumors in PtenWT::Pdk-/-genotype (S1); Inhibition of SGK1 or SGK3 resembles changes in signaling seen upon PDK1 inhibition (S2); SGK and PDK1 inhibition affect cell cycle progression (S3); Synthetic lethal screen for PDK1i combination drugs (S4); RPPA analysis of signaling components that are affected upon inhibition of PDK1 (S5); PDK1 KD reduces the expression of genes involved in pigmentation and metastasis (S6).

Supplementary Figure 1 from Identification of Small Molecules that Sensitize Resistant Tumor Cells to Tumor Necrosis Factor-Family Death Receptors

PDF file - 622K, Effects of RKIP expression on cell growth

PDF file - 604K, MDA-9 activates ERK1/2

PDF file - 198K, Expression of different angiogenic factors in MDA-9/Syntenin expressing clones.

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PDF file - 205K, Expression of RKIP mRNA in different melanoma cells

PDF file - 212K, Human angiogenesis array coordinates.

PDF file - 173K, Graphical representations of the effects of IGFBP-2 on vasculogeneis.

Two major pathways for apoptosis have been identified, involving either mitochondria (intrinsic) or tumor necrosis factor (TNF)-family death receptors (extrinsic) as initiators of caspase protease activation and cell death. Because tumor resistance to TNF-family death receptor ligands is a common problem, helping malignant cells evade host immune defenses, we sought to identify compounds that selectively sensitize resistant tumor cells to death receptor ligands. We screened a 50,000-compound library for agents that enhanced anti-FAS antibody–mediated killing of FAS-resistant PPC-1 prostate cancer cell, then did additional analysis of the resulting hits to arrive at eight compounds that selectively sensitized PPC-1 cells to anti-FAS antibody (extrinsic pathway agonist) without altering sensitivity to staurosporine and etoposide (VP-16; intrinsic pathway agonists). These eight compounds did not increase Fas surface levels and also sensitized PPC-1 cells to apoptosis induced by TNF-family member TNF-related apoptosis-inducing ligand, consistent with a post-receptor mechanism. Of these, two reduced expression of c-FLIP, an intracellular antagonist of the extrinsic pathway. Characterization of the effects of the eight compounds on a panel of 10 solid tumor cell lines revealed two structurally distinct compounds that frequently sensitize to extrinsic pathway agonists. Structure-activity relation studies of one of these compounds revealed a pharmacophore from which it should be possible to generate analogues with improved potency. Altogether, these findings show the feasibility of identifying compounds that selectively enhance apoptosis via the extrinsic pathway, thus providing research tools for uncovering resistance mechanisms and a starting point for novel therapeutics aimed at restoring sensitivity of tumor cells to immune effector mechanisms. (Cancer Res 2006; 66(4): 2367-75)

We have recently reported on Lys-covalent agents that, based on aryl-sulfonyl fluorides, were designed to target binding site Lys 311 in the X-linked inhibitor of apoptosis protein (XIAP). Similar to XIAP, melanoma-IAP (ML-IAP), a less well-characterized IAP family protein, also presents a lysine residue (Lys 135), which is in a position equivalent to that of Lys 311 of XIAP. On the contrary, two other members of the IAP family, namely, cellular-IAPs (cIAP1 and cIAP2), present a glutamic acid residue in that position. Hence, in the present work, we describe the derivation and characterization of the very first potent ML-IAP Lys-covalent inhibitor with cellular activity. The agent can be used as a pharmacological tool to further validate ML-IAP as a drug target and eventually for the development of ML-IAP-targeted therapeutics.

Modulating disease-relevant protein-protein interactions (PPIs) using pharmacological tools is a critical step toward the design of novel therapeutic strategies. Over the years, however, targeting PPIs has proven a very challenging task owing to the large interfacial areas. Our recent efforts identified possible novel routes for the design of potent and selective inhibitors of PPIs using a structure-based design of covalent inhibitors targeting Lys residues. In this present study, we report on the design, synthesis, and characterizations of the first Lys-covalent BH3 peptide that has a remarkable affinity and selectivity for hMcl-1 over the closely related hBfl-1 protein. Our structural studies, aided by X-ray crystallography, provide atomic-level details of the inhibitor interactions that can be used to further translate these discoveries into novel generation, Lys-covalent pro-apoptotic agents.

Recently, it was reported that tetra-peptides cyclized via lactam bond between the amino terminus and a glutamic residue in position 4 (termed here N-lock) can nucleate helix formation in longer peptides. We applied such strategy to derive N-locked covalent BH3 peptides that were designed to selectively target the anti-apoptotic protein Bfl-1. The resulting agents were soluble in aqueous buffer and displayed a remarkable (low nanomolar) affinity for Bfl-1 and cellular activity. The crystal structure of the complex between such N-locked covalent peptide and Bfl-1 provided insights on the geometry of the N-locking strategy and of the covalent bond between the agent and Bfl-1.