Alba T. Macias

Computational Identification of Inhibitors of Protein-Protein Interactions

The ability to control protein-protein interactions (PPIs) for therapeutic purposes is attractive since many processes in cells involve such interactions. Recent successes in the discovery of small molecules that target protein-protein interactions for drug development have shown that targeting these interactions is indeed feasible. In the present review the use of computer-aided drug design (CADD) via database screening or docking algorithms for identifying inhibitors of protein-protein interactions is introduced. The principles of database screening and a practical protocol for targeting PPIs are described. The recent applications of these approaches to different systems involving protein-protein interactions, including BCL-2, S100B, ERK and p56lck, are presented and provide valuable examples of inhibitor discovery and design.

Fatty acid amide hydrolase inhibitors. Surprising selectivity of chiral azetidine ureas.

We report the discovery of a novel, chiral azetidine urea inhibitor of Fatty Acid Amide Hydrolase (FAAH,) and describe the surprising species selectivity of VER-156084 versus rat and human FAAH and also hCB1.

Mitogen activated protein (MAP) kinases: development of ATP and non-ATP dependent inhibitors.

Extracellular signals regulate most of the bodys physiological functions through the MAP kinase signaling pathways. These MAP kinase signaling pathways are normally under tight regulation such that activation and inactivation occurs only when needed. However, aberrant regulation observed with naturally occurring mutations in specific signaling proteins often results in constitutive activation of the MAP kinases and is involved in several pathophysiological conditions, such as cancer, neurodegeneration, and inflammation. As such, much effort has been expended to develop inhibitory molecules of the MAP kinase signaling pathways. Several compounds have been identified that inhibit MAP kinase signaling by targeting receptors or other proteins upstream of the MAP kinases. The development of specific inhibitors of the MAP kinases themselves has been less successful and only a few compounds, which interfere with ATP binding, have been identified. A common problem with kinase inhibitors that compete with ATP binding is their lack of specificity. Thus, alternative approaches to inhibit MAP kinase function are being sought. The MAP kinase proteins contain docking domains that direct the interactions with a variety of substrate proteins. Using the 3-dimensional structure of MAP kinases and computer modeling, molecules that target specific docking domains and selectively disrupt substrate interactions are being developed. This non-ATP interfering approach may allow the selective inhibition of MAP kinase substrates involved in disease processes while preserving MAP kinase functions associated with normal cells.

Fatty acid amide hydrolase inhibitors. 3: Tetra-substituted azetidine ureas with in vivo activity

We describe here our attempts to optimise the human fatty acid amide hydrolase (FAAH) inhibition and physicochemical properties of our previously reported tetrasubstituted azetidine urea FAAH inhibitor, VER-156084. We describe the SAR of a series of analogues and conclude with the demonstration of in vivo dose-dependant FAAH inhibition in an anandamide-loading study in rats.

Application of Off-Rate Screening in the Identification of Novel Pan-Isoform Inhibitors of Pyruvate Dehydrogenase Kinase.

Libraries of nonpurified resorcinol amide derivatives were screened by surface plasmon resonance (SPR) to determine the binding dissociation constant (off-rate, kd) for compounds binding to the pyruvate dehydrogenase kinase (PDHK) enzyme. Parallel off-rate measurements against HSP90 and application of structure-based drug design enabled rapid hit to lead progression in a program to identify pan-isoform ATP-competitive inhibitors of PDHK. Lead optimization identified selective sub-100-nM inhibitors of the enzyme which significantly reduced phosphorylation of the E1α subunit in the PC3 cancer cell line in vitro.

Abstract B155: VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and demonstrates context-dependent cytotoxicity to cancer cells.

Pyruvate dehydrogenase kinase (PDK) regulates the activity of the pyruvate dehydrogenase complex (PDC) through phosphorylation of three serine residues on the E1α subunit, resulting in decreased activity. The expression of all four mammalian isoforms of PDK have been shown to be up-regulated either under tumour relevant conditions (PDK-1 & PDK-3 by hypoxia) or by the loss of function of common tumour suppressor genes (PDK-2 by p53; PDK-4 by pRB). Furthermore, PDK-1 expression has been clinically correlated with poor prognosis in Gastric, HNSCC and Colon cancer. Previous studies employing RNAi have provided evidence for a survival role for PDK-1 as well as its importance in maintaining the glycolytic phenotype of cancer cells. DCA, a weak inhibitor of PDK, has also been used to study the role of PDK in cancer; however, interpretation of these studies has been complicated by conflicting data and the lack of specificity of this agent for PDK. Here we report the discovery of a novel, potent and selective pan-isoform inhibitor of PDK, VER-246608. Consistent with a PDK mediated MOA, treatment of PC-3 cells with VER-246608 resulted in increased PDC activity and oxygen consumption as well as reduced lactate production and glucose consumption. Interestingly, modulation of glycolytic activity required compound concentrations which achieved > 90 % reduction in E1α phosphorylation and this was only observed under glucose depleted conditions. Under normal culture conditions, VER-246608 showed little cytotoxicity to cancer cells. We hypothesised that the supraphysiological glucose concentrations present in cell culture media may limit the effect of PDK inhibition due to elevated intracellular pyruvate levels. We therefore performed cytotoxicity studies under conditions of limited nutrient availability. We found that combined depletion of glucose and glutamine or serum alone resulted in enhanced cytotoxicity vs normal media; however, hypoxic conditions had no effect. Furthermore, this differential cytotoxicity correlated with reduced pyruvate levels in the compound treated cells cultured in the above ‘austere’ conditions. VER-246608 also inhibited the growth of tumour spheroids with a potency that was comparable to cells grown in 2D culture. In addition, combination treatment studies revealed that VER-246608 potentiated anti-cancer agents which are known to influence mitochondrial function. In contrast, the lipoamide binding site inhibitor, Nov3r, showed no evidence of cytotoxicity under any of the above conditions and was ineffective in altering glycolytic activity. These studies suggest that PDK inhibition may be effective under the nutrient depleted conditions found in the tumour microenvironment and that combination treatments should be explored to reveal the full potential of this therapeutic strategy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B155. Citation Format: Jonathan Moore, Anna Staniszewska, Terence Shaw, Jalanie D9Alessandro, Ben Davis, Alan Surgenor, Lisa Baker, Natalia Massanova, James Murray, Alba Macias, Paul Brough, Mike Wood, Patrick C. Mahon. VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and demonstrates context-dependent cytotoxicity to cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B155.

Abstract A212: A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor‐induced apoptosis in HCT116 colon carcinoma cells

The role of the 70 kDa heat shock protein isoforms (Hsc70 and Hsp70) in cancer development and progression through their ability to inhibit apoptosis and via their role as Hsp90 co‐chaperones has been well documented. Dual targeting of Hsc70 and Hsp70 with siRNA has previously been demonstrated to induce proteasome‐dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition. The design and synthesis of novel adenosine‐derived inhibitors of Hsp70, guided by modelling and X‐ray crystallographic structures of these compounds in complex with Hsc70/BAG‐1, has been described.1 These were the first inhibitors described to target the ATPase binding domain of this family of chaperones. Many of these compounds exhibited submicromolar affinity for Hsp70, were highly selective over Hsp90, and displayed in vitro activity against a variety of human tumor cell lines. We further describe the in vitro mode of action of one of the most potent analogues, VER‐155008 in HCT116, HT29, BT474 and MDA‐MB‐468 carcinoma cell lines. Cell proliferation, cell cycle, cell apoptosis and caspase 3/7 activity was determined for VER‐155008 in the absence or presence of small molecule Hsp90 inhibitors. VER‐155008 inhibited the proliferation of human breast and colon cancer cell lines with GI50s in the range 5.3 to 14.4 M, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER‐155008 induced caspase‐3/7 dependent apoptosis in BT474 cells and non‐caspase dependent cell death in HCT116 and HT29 cells. VER‐155008 potentiated the apoptotic potential of the small molecule Hsp90 inhibitors VER‐821602 and 17‐AAG in HCT116 but not HT29 or MDA‐MB‐468 cells. In vivo, VER‐155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level. These data suggest that as a single agent, small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/Hsp70 inhibition remain to be determined. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A212.