Sylvestor A. Moses

Discovery of a novel class of AKT pleckstrin homology domain inhibitors

AKT, a phospholipid-binding serine/threonine kinase, is a key component of the phosphoinositide 3-kinase cell survival signaling pathway that is aberrantly activated in many human cancers. Many attempts have been made to inhibit AKT; however, selectivity remains to be achieved. We have developed a novel strategy to inhibit AKT by targeting the pleckstrin homology (PH) domain. Using in silico library screening and interactive molecular docking, we have identified a novel class of non–lipid-based compounds that bind selectively to the PH domain of AKT, with “in silico” calculated KD values ranging from 0.8 to 3.0 μmol/L. In order to determine the selectivity of these compounds for AKT, we used surface plasmon resonance to measure the binding characteristics of the compounds to the PH domains of AKT1, insulin receptor substrate-1, and 3-phosphoinositide–dependent protein kinase 1. There was excellent correlation between predicted in silico and measured in vitro KDs for binding to the PH domain of AKT, which were in the range 0.4 to 3.6 μmol/L. Some of the compounds exhibited PH domain–binding selectivity for AKT compared with insulin receptor substrate-1 and 3-phosphoinositide–dependent protein kinase 1. The compounds also inhibited AKT in cells, induced apoptosis, and inhibited cancer cell proliferation. In vivo, the lead compound failed to achieve the blood concentrations required to inhibit AKT in cells, most likely due to rapid metabolism and elimination, and did not show antitumor activity. These results show that these compounds are the first small molecules selectively targeting the PH domain of AKT. [Mol Cancer Ther 2008;7(9):2621–32]

Molecular Pharmacology and Antitumor Activity of PHT-427, a Novel Akt/Phosphatidylinositide-Dependent Protein Kinase 1 Pleckstrin Homology Domain Inhibitor

Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non–small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity. Mol Cancer Ther; 9(3); 706–17

In vitro and In vivo Activity of Novel Small-Molecule Inhibitors Targeting the Pleckstrin Homology Domain of Protein Kinase B/AKT

The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties.

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.

Development of sulfonamide AKT PH domain inhibitors.

Disruption of the phosphatidylinositol 3-kinase/AKT signaling pathway can lead to apoptosis in cancer cells. Previously we identified a lead sulfonamide that selectively bound to the pleckstrin homology (PH) domain of AKT and induced apoptosis when present at low micromolar concentrations. To examine the effects of structural modification, a set of sulfonamides related to the lead compound was designed, synthesized, and tested for binding to the expressed PH domain of AKT using a surface plasmon resonance-based competitive binding assay. Cellular activity was determined by means of an assay for pAKT production and a cell killing assay using BxPC-3 cells. The most active compounds in the set are lipophilic and possess an aliphatic chain of the proper length. Results were interpreted with the aid of computational modeling. This paper represents the first structure-activity relationship (SAR) study of a large family of AKT PH domain inhibitors. Information obtained will be used in the design of the next generation of inhibitors of AKT PH domain function.

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.

Neural Foraminal Lesions: An Imaging Overview

The neural foramina allow passage of spinal nerves, arteries, veins, and lymphatics from the spinal canal to the periphery [1,2] and are formed by various surrounding osseous and nonosseous structures. The anterior border is made up of the posterior aspect of adjacent vertebral bodies, the intervertebral disk, and the posterior longitudinal ligament. The superior and inferior borders of the foramen are formed by the vertebral notches of the superior and inferior vertebral pedicles. The facet, or zygapophyseal, joints form the posterior border [1,2]. Laterally, the foramen is covered by overlying psoas muscle and fascia [2].

Abstract 5563: Targeting the PH domain of TIAM1 to inhibit prostate cancer metastasis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC TIAM1 (T-lymphoma invasion and metastasis-inducing protein-1) is a highly conserved guanine nucleotide exchange factor and contains an Dbl Homology (DH)/C-terminal Pleckstrin Homology (PH) domain. The crystal structure of DH-PH of TIAM1 in complex with Rac-1 has been reported. TIAM1 has been found to be over-expressed cancers such as breast, colon and prostate cancers. An increase in TIAM1 expression has been shown to be associated with increased metastatic potential of breast cancer cell lines and is also correlated with poor prognosis of patients with prostate cancer. TIAM1 is thus a novel PH domain-containing drug target directly related to cancer progression, metastasis and patient survival. We have identified and characterized novel small molecule inhibitors targeting the phosphoinositide lipid binding PH domain of TIAM1 in order to selectively inhibit cell migration, invasion and survival. An In Silico screen of our internal and Maybridge library using the crystal structure of TIAM1 has identified 11 compounds that bind to the PH domain. In vitro assays revealed that compounds TPH-3 and TPH-15 significantly reduced the amount of active Rac1 in PC-3 prostate cancer cells (EC50 of 2.73±0.13 and 2.38±0.98 μM) by binding to PH-TIAM1 with high affinity (KD in the micromolar range) using surface plasmon resonance (SPR) spectrometry. Both compounds displaced PtdIns-3,4,5-P3 in a SPR competitive binding assay. TPH-3 and TPH-15 inhibited cell proliferation with EC50 of 19.8±3.2 and 28.7±1.7 μM in prostate cancer cells LnCaP and EC50 of 18.6±1.8 and 33.3±6.7 μM in PC-3. Wound healing assays and lamellipodia formation were both inhibited by the compounds. TPH-15 inhibited ≈70% of PC-3 invasion using a matrigel invasion assay. Finally, TPH-15 exhibits anti-tumor properties in a PC-3 mouse xenograft study (%T/C≈38.8) with good pharmacokinetic properties (T1/2≈5 hours). Cardio-injection of prostate PC-3 cells in SCID mice was used to test the effects of TPH-15 in the prevention of bone metastasis. Mice were treated with 50mg/kg ip of TPH-15 once a day for 5 days. TPH-15-treated mice developed significantly less pelvic bone lesions than the untreated group and TPH-15 treated mice lived longer, on average, than the untreated group. Overall, we have identified novel compounds which exhibit the ability to reduce prostate cancer bone metastasis by binding to the PH domain of TIAM1, an important GEF in the process of metastasis. Citation Format: Emmanuelle J. Meuillet, Sylvestor A. Moses, Jana Jandova, Eugene A. Mash, Shuxing Zhang. Targeting the PH domain of TIAM1 to inhibit prostate cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5563. doi:10.1158/1538-7445.AM2013-5563

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