Zuohe Song

Direct inhibition of hypoxia-inducible transcription factor complex with designed dimeric epidithiodiketopiperazine.

Selective blockade of hypoxia-inducible gene expression by designed small molecules would prove valuable in suppressing tumor angiogenesis, metastasis and altered energy metabolism. We report the design, synthesis, and biological evaluation of a dimeric epidithiodiketopiperazine (ETP) small molecule transcriptional antagonist targeting the interaction of the p300/CBP coactivator with the transcription factor HIF-1alpha. Our results indicate that disrupting this interaction results in rapid downregulation of hypoxia-inducible genes critical for cancer progression. The observed effects are compound-specific and dose-dependent. Controlling gene expression with designed small molecules targeting the transcription factor-coactivator interface may represent a new approach for arresting tumor growth.

Computational modeling of novel inhibitors targeting the Akt pleckstrin homology domain

Computational modeling continues to play an important role in novel therapeutics discovery and development. In this study, we have investigated the use of in silico approaches to develop inhibitors of the pleckstrin homology (PH) domain of AKT (protein kinase B). Various docking/scoring schemes have been evaluated, and the best combination was selected to study the system. Using this strategy, two hits were identified and their binding behaviors were investigated. Robust and predictive QSAR models were built using the k nearest neighbor (kNN) method to study their cellular permeability. Based on our in silico results, long flexible aliphatic tails were proposed to improve the Caco-2 penetration without affecting the binding mode. The modifications enhanced the AKT inhibitory activity of the compounds in cell-based assays, and increased their activity as in vivo antitumor testing.

Sodium Selenite Increases the Activity of the Tumor Suppressor Protein, PTEN, in DU-145 Prostate Cancer Cells

Epidemiological and clinical data suggest that selenium may prevent prostate cancer; however, the cellular effects of selenium in malignant prostate cells are not well understood. We previously reported that the activity of the tumor suppressor PTEN is modulated by thioredoxin (Trx) in a RedOx-dependent manner. In this study, we demonstrated that the activity of Trx reductase (TR) is increased by sevenfold in the human prostate cancer cell line, DU-145, after 5 days of sodium selenite (Se) treatment. The treatment of DU-145 cells with increasing concentrations of Se induced an increase in PTEN lipid phosphatase activity by twofold, which correlated with a decrease in phospho-ser 473 -Akt, and an increase in phospho-Ser 370 -PTEN levels. Se also increased casein kinase-2 (CK2) activity; and the use of apigenin, an inhibitor of CK2, revealed that the regulation of the tumor suppressor PTEN by Se may be achieved via both the Trx-TR system and the RedOx control of the kinase involved in the regulation of PTEN activity.

Novel Inhibitors Induce Large Conformational Changes of GAB1 Pleckstrin Homology Domain and Kill Breast Cancer Cells

The Grb2-associated binding protein 1 (GAB1) integrates signals from different signaling pathways and is over-expressed in many cancers, therefore representing a new therapeutic target. In the present study, we aim to target the pleckstrin homology (PH) domain of GAB1 for cancer treatment. Using homology models we derived, high-throughput virtual screening of five million compounds resulted in five hits which exhibited strong binding affinities to GAB1 PH domain. Our prediction of ligand binding affinities is also in agreement with the experimental K D values. Furthermore, molecular dynamics studies showed that GAB1 PH domain underwent large conformational changes upon ligand binding. Moreover, these hits inhibited the phosphorylation of GAB1 and demonstrated potent, tumor-specific cytotoxicity against MDA-MB-231 and T47D breast cancer cell lines. This effort represents the discovery of first-in-class GAB1 PH domain inhibitors with potential for targeted breast cancer therapy and provides novel insights into structure-based approaches to targeting this protein.

Abstract 2835: Discovery of novel inhibitors for ECT2 as a novel therapeutic strategy for lung cancer

The Epithelial Cell Transforming sequence 2 (ECT2) proto-oncogene is a Guanine Exchange Factor (GEF) for RhoA, Rac1 and Cdc42 and is essential to the regulation of cytokinesis. ECT2 contains the Dbl homology and pleckstrin homology (PH) domains, which are the hallmarks of GEFs. ECT2 is over-expressed in primary non-small cell lung cancer (NSCLC) tumors, and injection of ECT2 transfectants into nude mice efficiently induces tumor formation. High level of ECT2 expression is associated with poor prognosis for patients with NSCLC. Knock down of ECT2 expression by small interfering RNAs effectively suppresses lung cancer cell growth, suggesting a specific role of ECT2 in lung cancer development. Taken together, ECT2 may represent an attractive molecular target for inhibiting lung tumor growth. Our studies are based on the hypothesis that ECT2 plays an important role in lung cancer progression and is a novel target for the development of drugs to treat lung cancer. We have built a model for ECT2 PH domain using protein homology modeling. Docking of new compounds selected from commercial drug-like libraries has led to the identification of novel inhibitors of ECT2 PH domain. We have identified several compounds that bind ECT2 PH domain in the low micromolar range (KD Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2835. doi:1538-7445.AM2012-2835

Abstract 2166: In silico identification and biological evaluation of potent small molecule inhibitors targeting GAB1 pleckstrin homology domain

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The Grb2-associated binder-1 (GAB1) over-expressed in many cancers represents a novel therapeutic target that integrates signals from different systems such as MET and PI3K/Akt pathways. It mediates processes involved in tumor progression and metastasis. In this study we have identified novel inhibitors targeting the pleckstrin homology (PH) domain of GAB1 for cancer treatment. To this end, a homology model of GAB1 PH domain was constructed, followed by structural refinement using molecular dynamics. The resulting structure was applied to high-throughput virtual screening of 5 million compounds and 19 hits were selected for experimental evaluation. We found that 6 compounds bound to the PH domain of GAB1 as measured by surface plasmon resonance spectroscopy, with a KD. Out of these 6 hits, all of them inhibited the HGF-I induced phosphorylation of GAB1 Tyr627 in T47D and inhibited T47D and MDA-MB-231 breast cancer cell lines proliferation. Three of them exhibited selective binding to GAB1 PH domain as compared to the PH domain of IRS-1 and AKT. This demonstrates for the first time the identification of novel GAB1 PH domain inhibitors which may be used for targeted breast cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2166. doi:1538-7445.AM2012-2166

Abstract 1559: Targeting the PH domain of Tiam-1 to inhibit breast cancer metastasis

TIAM-1 (T-lymphoma invasion and metastasis-inducing protein-1) is a highly conserved guanine nucleotide exchange factor and contains an N-terminal pleckstrin homology domain (PHn) and a Dbl homology (DH)/C-terminal PH domain (PHc). While no crystal structure exists for the PHn of TIAM-1, the crystal structure of DH-PHc of TIAM-1 in complex with Rac-1 has been reported. TIAM-1 has been found to be over-expressed in breast, colon and prostate cancers. An increase in TIAM-1 expression has been shown to be associated with increased metastatic potential of breast cancer cell lines. TIAM-1 is thus an ideal PH domain drug target directly related to cancer progression, metastasis and patient survival. In this study, we have identified novel small molecule inhibitors targeting the phosphoinositide (PtdIns) lipid binding PHc domain of TIAM-1 in order to selectively inhibit cell migration and survival. Utilizing an In Silico screen of our internal and Maybridge library and the crystal structure of TIAM-1, we have found 29 compounds that bind to PHc TIAM-1. In vitro assays have revealed that compound PH-210 and #10 significantly reduced the amount of active Rac1 and binds to PHc-TIAM-1 with high affinity (KD in the micromolar range) using surface plasmon resonance (SPR) spectrometry. Both compounds displace PtdIns-3,4,5-P3 in a SPR competitive binding assay. Ongoing in vivo studies using MDA-231 breast cancer cell lines will determine the anti-tumor activity of these compounds. Overall, these novel compounds could exhibit the ability to significantly reduce breast cancer metastasis by binding to PHc-TIAM-1 and could have the potential to be used with other chemotherapeutic compounds as part of an effective breast cancer treatment regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1559.

Abstract 3565: Novel inhibitors of mPGES-1 exhibit anti-tumor activity in colorectal and lung cancers

Chronic inflammation is associated with 25% of all cancers. In the inflammation-cancer axis, prostaglandin E2 (PGE2) is one of the major players involved. Prostaglandin E synthase (PGES) is an enzyme downstream of the cyclooxygenases (COXs) in the PGE2 biosynthesis pathway. Among the three PGES isoforms, microsomal prostaglandin E synthase-1 (mPGES-1) is inducible by proinflammatory stimuli and constitutively expressed in colon, lung, and gastric cancers. The potential role for this enzyme in tumorigenesis has also been reported. Since inhibition of COXs may lead to some side effects due to the global reduction of other key prostaglandins, targeting downstream mPGES-1 might be a better therapeutic or chemopreventive strategy for cancers. In order to identify novel small molecule inhibitors of mPGES-1, we have screened in silico a small library of compounds. As a result, 14 lead compounds were identified by molecular docking simulations of the chemicals based on the crystal structure of mPGES-1. These compounds were tested for their ability to inhibit cell growth, induce apoptosis and decrease cytokine-stimulated PGE2 production in various colorectal and lung cancer cell lines. Using surface plasmon resonance (SPR) spectroscopy, we show the binding of the compounds PGE001 and PGE011 to human mPGES-1 recombinant protein with good affinity (KD at low micromolar range). These two compounds also showed the ability to reduce the IL-1beta-induced PGE2 production in HT29 and SW837 colon cancer cells and A549 lung cancer cell line with EC50 at low micromolar range. Furthermore, the compounds exhibit significant anti-tumor activity in the SW837 xenograft mouse model with T/C (relative size of treated and control tumors)=40.3% for compound PGE001 and T/C=68.7% for compound PGE011 (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3565.

Abstract B139: Molecular pharmacology and antitumor activity of PHT‐427 a novel AKT/PDPK1 pleckstrin homology domain inhibitor

The pleckstrin homology (PH) domain is a highly conserved three dimensional superfold found in many high valued pharmaceutical target proteins. While for the majority of PH domain proteins PtdIns binding is weak and non‐specific, a subclass of approximately 40 PH domain containing proteins show high affinity for phosphatidylinositol‐3‐phosphates (PtdIns‐3‐P) causing their translocation to the plasma membrane and/or activation. Akt and PDKP1 are two such PH domain containing serine threonine kinase members of the PtdIns‐3‐kinase signaling pathway that plays a critical role in activating survival and anti‐apoptotic signaling in cancer cells. This pathway is constitutively activated through mutation, overexpression and loss of the tumor suppressor PTEN in many different cancer types. There is emerging evidence that Akt and PDKP1 play complimentary yet independent roles in signaling by the pathway. Attempts to develop ATP catalytic site inhibitors of Akt and PDKP1 have generally resulted in toxic agents because of inhibition of off‐target kinase family members. Through a process of reiterative molecular docking and structure refinement using a proprietary computational platform, and measurement of binding affinities by surface plasmon resonance (SPR) spectroscopy, we have developed inhibitors of these proteins that bind to the PH domain. We report here the development of PHT‐427 a compound that binds to the PH domain of AKT and PDKP1. To develop PHT‐427 an initial pharmacophore that bound to the PH domain PtdIns binding pocket was first identified. During the docking/modeling we identified a channel whithin the PH domains of both AKT and PDPK1 that could accommodate the binding of an alkyl chain. A series of analogs with C‐4 to C‐16 alkyl chain length were therefore synthesized and PHT‐427 (C‐12 chain) was found to bind with the highest affinity to the PH domains of both PDPK1 and AKT. PHT‐427 gives a transient inhibition of AKT signaling in cells and tumor xenografts and a longer lasting inhibition of PDPK1 signaling. This inhibition correlated with decreased activation of downstream signaling targets for both proteins. PHT‐427 given ias an oral formulation on a twice daily schedule inhibited the growth rate of human tumor xenografts in immunodeficient mice with up to an 80% inhibition in the most sensitive tumors and showed greater activity than analogs with C4, C6 or C8 alkyl chains. Tumors with a K‐Ras mutation were less sensitive to PHT‐427 as it has been seen for other inhibitors of the PtdIns‐3‐K signaling pathway. Combination antitumor studies showed that PHT‐427 has greater than additive activity with paclitaxel in breast cancer and with erlotinib in non small cell lung cancer. There was minimal toxicity of PH‐427 at doses that gave antitumor activity. PHT‐427 given twice daily for 5 days caused no weight loss or change in blood chemistry. In summary we have identified a novel PH domain binding inhibitor of PDPK1/AKT signaling that has minimal toxicity and antitumor activity against human tumor xenografts.. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B139.