Activity of a novel, dual PI3-kinase/mTor inhibitor NVP-BEZ235 against primary human pancreatic cancers grown as orthotopic xenografts.

The phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway is frequently deregulated in pancreatic cancers, and is believed to be an important determinant of their biological aggression and drug resistance. NVP-BEZ235 is a novel, dual class I PI3K/mammalian target of rapamycin (mTor) inhibitor undergoing phase I human clinical trials. To simulate clinical testing, the effects of NVP-BEZ235 were studied in five early passage primary pancreatic cancer xenografts, grown orthotopically. These tumours showed activated PKB/Akt, and increased levels of at least one of the receptor tyrosine kinases that are commonly activated in pancreatic cancers. Pharmacodynamic effects were measured following acute single doses, and anticancer effects were determined in separate groups following chronic drug exposure. Acute oral dosing with NVP-BEZ235 strongly suppressed the phosphorylation of PKB/Akt, followed by recovery over 24 h. There was also inhibition of Ser235/236 S6 ribosomal protein and Thr37/46 4E-BP1, consistent with the effects of NVP-BEZ235 as a dual PI3K/mTor inhibitor. Chronic dosing with 45 mg kg(-1) of NVP-BEZ235 was well tolerated, and produced significant tumour growth inhibition in three models. These results predict that agents targeting the PI3K/Akt/mTor pathway might have anticancer activity in pancreatic cancer patients, and support the testing of combination studies involving chemotherapy or other molecular targeted agents.

Drug discovery approaches targeting the PI3K/Akt pathway in cancer.

The abnormal activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been validated by epidemiological and experimental studies as an essential step toward the initiation and maintenance of human tumors. Notable in this regard are the prevalent somatic genetic alterations leading to the inactivation of the tumor suppressor gene PTEN and gain-of-function mutations targeting PIK3CA--the gene encoding the catalytic phosphosinositide-3 kinase subunit p110 alpha. A number of the intracellular components of this pathway have been targeted as anticancer drug discovery activities leading to the current panoply of clinical trials of inhibitors of PI3K, Akt and HSP90 in man. This review summarizes current preclinical knowledge of modulators of the PI3K/Akt pathway in which drug discovery and development activities have been advanced focusing on both the relevant clinical stage inhibitors and other disclosed tool compounds targeting PI3K, PDK1, Akt and HSP90.

Signalling profile and antitumour activity of the novel Hsp90 inhibitor NVP-AUY922 in multiple myeloma.

We as well as others have recently shown that Hsp90 is overexpressed in multiple myeloma (MM) and critically contributes to tumour cell survival. Pharmacologic blockade of Hsp90 has consistently been found to induce MM cell death. However, most data have been obtained with MM cell lines whereas knowledge about the molecular effects of pharmacologic Hsp90 blockade in primary tumour cells is limited. Furthermore, these investigations have so far focused on geldanamycin derivatives. We analysed the biochemical effects of a novel diarylisoxazole-based Hsp90 inhibitor (NVP-AUY922) on signalling pathways and cell death in a large set of primary MM tumour samples and in MM cell lines. Treated cells displayed the molecular signature and pharmacodynamic properties for abrogation of Hsp90 function, such as downregulation of multiple survival pathways and strong upregulation of Hsp70. NVP-AUY922 treatment efficiently induced MM cell apoptosis and revealed both sensitive and resistant subgroups. Sensitivity was not correlated with TP53 mutation or Hsp70 induction levels and stromal cells from the bone marrow microenvironment were unable to abrogate NVP-AUY922-induced apoptosis of MM cells. Thus, NVP-AUY922 may be a promising drug for treatment of MM and clinical studies are warranted.

Identification of key residues involved in mediating the in vivo anti-tumor/anti-endothelial activity of Alphastatin.

BACKGROUND: We have recently shown that Alphastatin, a 24-amino-acid peptide (ADSGEGDFLAEGGGVRGPRVVERH) derived from human fibrinogen has anti-endothelial properties in vitro and in vivo. OBJECTIVES: The aim of this study was to determine the activity of a terminally modified (stabilized) form of Alphastatin in vitro and in vivo and to identify the key residues required for this activity. METHODS: The in vitro activity of modified Alphastatin, truncates and mutants was determined by endothelial cell (HuDMEC) tubule formation and migration. Active peptides were then assessed in vivo using syngeneic murine subcutaneous 4T1 mammary carcinomas. RESULTS: Modified Alphastatin-inhibited HuDMEC migration and tubule formation in response to multiple growth factors and caused a 45% inhibition in tumor growth when administered intravenously at 0.25 mg kg(-1) (three times per week). Intravenous (i.v.) administration proved non-toxic at all doses investigated, whereas oral and intraperitoneal (i.p.) administration demonstrated neither anti-tumor activity nor toxicity. Truncations of Alphastatin revealed an 11-amino-acid peptide (DFLAEGGGVRG), termed AHN419, which inhibited endothelial cell activity in vitro; however, intravenous AHN419 caused a non-significant growth inhibition in vivo. Single amino acid substitutions to alanine along the entire length of Alphastatin indicated that additional residues outside the AHN419 sequence were required for full activity. CONCLUSIONS: Terminal modification of Alphastatin altered the in vivo efficacy and these studies suggest that a hydrophobic cluster (Phe8, Leu9, Ala10 and Val15) is essential for the biological activity, but additional residues, including Ser3-Gly14, Pro18-Val20 and Arg23 are required for full inhibitory activity of Alphastatin.

Inhibition of IGF-I receptor in anchorage-independence attenuates GSK-3beta constitutive phosphorylation and compromises growth and survival of medulloblastoma cell lines.

We have previously reported that insulin-like growth factor-I (IGF-I) supports growth and survival of mouse and human medulloblastoma cell lines, and that IGF-I receptor (IGF-IR) is constitutively phosphorylated in human medulloblastoma clinical samples. Here, we demonstrate that a specific inhibitor of insulin-like growth factor-I receptor (IGF-IR), NVP-AEW541, attenuated growth and survival of mouse (BsB8) and human (D384, Daoy) medulloblastoma cell lines. Cell cycle analysis demonstrated that G1 arrest and apoptosis contributed to the action of NVP-AEW54. Interestingly, very aggressive BsB8 cells, which derive from cerebellar tumors of transgenic mice expressing viral oncoprotein (large T-antigen from human polyomavirus JC) became much more sensitive to NVP-AEW541 when exposed to anchorage-independent culture conditions. This high sensitivity to NVP-AEW54 in suspension was accompanied by the loss of GSK-3beta constitutive phosphorylation and was independent from T-antigen-mediated cellular events (Supplementary Materials). BsB8 cells were partially rescued from NVP-AEW541 by GSK3beta inhibitor, lithium chloride and were sensitized by GSK3beta activator, sodium nitroprusside (SNP). Importantly, human medulloblastoma cells, D384, which demonstrated partial resistance to NVP-AEW541 in suspension cultures, become much more sensitive following SNP-mediated GSK3beta dephosphorylation (activation). Our results indicate that hypersensitivity of medulloblastoma cells in anchorage-independence is linked to GSK-3beta activity and suggest that pharmacological intervention against IGF-IR with simultaneous activation of GSK3beta could be highly effective against medulloblastomas, which have intrinsic ability of disseminating the CNS via cerebrospinal fluid.

Inhibition of Aurora A in response to DNA damage.

Mitotic kinases are the ultimate target of pathways sensing genotoxic damage and impinging on the cell cycle machinery. Here, we provide evidence that Aurora A (AurA) was inhibited upon generation of double-strand breaks in DNA. We demonstrate that AurA was not downstream of CDK1 and that inhibition of AurA and CDK1 by DNA damage occurred independently. Using a cell line functionally deficient in Chk2, a selective Chk1 inhibitor and siRNA to Chk1, we show that DNA-damage signals were delivered to AurA through a Chk1-dependent pathway. With regard to the molecular mechanism of AurA inhibition, we found that the point mutation Ser(342)>Ala rendered AurA resistant to inhibition by DNA damage. By means of two distinct approaches we examined the impact of reconstitution of AurA activity in DNA-damaged cells: (i) transient expression of wild-type and Ser(342)>Ala mutant, but not kinase-dead, AurA led to bypass of the DNA damage block; (ii) direct transduction of highly active wt-AurA into G2 arrested cells precisely after induction of DNA damage resulted in mitotic entry. We show that the mechanism through which AurA allowed entry into mitosis was reactivation of CDK1, thus indicating that AurA plays a key role upstream of CDK1. A model depicting the possible role of AurA at the onset of mitosis and upon DNA damage is presented.

beta-Homolysine oligomers: a new class of Trojan carriers.

We describe the design, synthesis and cell-membrane translocation properties of a series of beta-peptides with the general sequence fluorescein-Adoa-(beta-homolysine)(n)-NH(2), n=5-8 and Adoa=8-amino-3,6-dioxaoctanoic acid. These beta-peptides are able to cross the cytoplasmic membrane and accumulate in the nucleus of mammalian cells.

Antagonists of the Src homology 2 (SH2) domains of Grb2, Src, Lck and ZAP-70.

Src homology 2 (SH2) domains are protein modules that mediate intracellular protein-protein interactions in signal transduction pathways. The specific association of an SH2 domain with a phosphotyrosine-containing sequence of another protein induces a cascade of molecular interactions that effect a wide range of cellular processes. Alterations in these signaling pathways have been associated with the development and progression of a broad range of pathologies. Because of the regulatory role of SH2 domains in these signal transduction pathways, specific SH2 domains can be ideal targets for intervention with therapeutic agents in many different disease indications (e.g. cancer, osteoporosis, disorders of the immune and cardiovascular systems). Among the SH2 domains pursued as drug discovery targets in the last few years are those of Grb2, Src, Lck and ZAP-70. This review focuses on contributions in the design and synthesis of antagonists of these particular SH2 domains. Specific examples have been selected to illustrate how structure-based design approaches have been used to progress in this area of research.

Coupling of the antennapedia third helix to a potent antagonist of the p53/hdm2 protein-protein interaction.

The use of cell-membrane translocating sequences for intracellular delivery of peptides can be a powerful approach to validate drug discovery targets in cellular settings. To accomplish this, a protocol has been implemented to couple the antennapedia third helix (residues 43-58) to a potent antagonist of the p53/hdm2 protein-protein interaction without affecting its in vitro inhibitory activity.

Phosphorylation disrupts the central helix in Op18/stathmin and suppresses binding to tubulin.

Protein phosphorylation represents a ubiquitous control mechanism in living cells. The structural prerequisites and consequences of this important post-translational modification, however, are poorly understood. Oncoprotein 18/stathmin (Op18) is a globally disordered phosphoprotein that is involved in the regulation of the microtubule (MT) filament system. Here we document that phosphorylation of Ser63, which is located within a helix initiation site in Op18, disrupts the transiently formed amphipathic helix. The phosphoryl group reduces tubulin binding 10-fold and suppresses the MT polymerization inhibition activity of Op18's C-terminal domain. Op18 represents an example where phosphorylation occurs within a regular secondary structural element. Together, our findings have implications for the prediction of phosphorylation sites and give insights into the molecular behavior of a globally disordered protein.

A new structural class of selective and non-covalent inhibitors of the chymotrypsin-like activity of the 20S proteasome.

We describe the identification and in vitro characterization of a series of 2-aminobenzylstatine derivatives that inhibit non-covalently the chymotrypsin-like activity of the 20S proteasome. Our initial SAR data demonstrate that the 2-aminobenzylstatine core structure can effectively serve as the basis for designing potent, selective and non-covalent inhibitors of the chymotrypsin-like activity of the 20S proteasome.

Modeling of the binding mode of a non-covalent inhibitor of the 20S proteasome. Application to structure-based analogue design.

The 2-aminobenzvlstatine derivative I is a 20S proteasome inhibitor of a novel chemical type identified by high throughput screening. The compound specifically inhibits the chymotrypsin-like catalytic activity of the human proteasome with an IC50 value in the micromolar range. Using the crystal structure of the yeast proteasome, we modeled the structure of the human proteasome in complex with 1. As one of the first applications of the model in our oncology programme targeting the proteasome, we designed an analogue of the inhibitor having enhanced stacking/hydrophobic interactions with the enzyme. One order of magnitude in inhibitory potency was gained.

A new Antennapedia-derived vector for intracellular delivery of exogenous compounds.

We describe the design, synthesis and cell translocation capacity of a peptide derived from the third alpha-helix of the homeodomain of Antennapedia. The new sequence appears to be an efficient and nontoxic means to deliver a covalently linked peptide cargo into cells.

An intrahelical salt bridge within the trigger site stabilizes the GCN4 leucine zipper.

We previously reported that a helical trigger segment within the GCN4 leucine zipper monomer is indispensable for the formation of its parallel two-stranded coiled coil. Here, we demonstrate that the intrinsic secondary structure of the trigger site is largely stabilized by an intrahelical salt bridge. Removal of this surface salt bridge by a single amino acid mutation induced only minor changes in the backbone structure of the GCN4 leucine zipper dimer as verified by nuclear magnetic resonance. The mutation, however, substantially destabilized the dimeric structure. These findings support the proposed hierarchic folding mechanism of the GCN4 coiled coil in which local helix formation within the trigger segment precedes dimerization.

Structure-based design of compounds inhibiting Grb2-SH2 mediated protein-protein interactions in signal transduction pathways.

Receptor protein tyrosine kinases are usually activated upon binding their growth factors, or other suitable ligands, to their extracellular domains. These activated receptors initiate cytoplasmic signalling cascades which, when aberrant, can result in different disease states, such as oncogenic transformation. Many receptor protein tyrosine kinases use Src homology 2 domains (SH2) to couple growth factor activation with intracellular signalling pathways to mediate cell control and other biological events. The characterization of the components involved in these signal transduction pathways has resulted in the identification of new attractive targets for therapeutic intervention. Such is the case for the protein-protein interactions involving the SH2 domain of growth factor receptor bound protein 2 (Grb2). Agents that specifically disrupt Grb2-SH2 binding interactions involved in aberrant signalling could potentially shut down these oncogenic pathways and thus block human malignancies. This paper reviews the structural characteristics of the Grb2-SH2 domain and the approaches which have been used to identify antagonists of the Grb2-SH2 domain. Examples have been selected from our own research to illustrate how the unique structural features of the ligand-bound Grb2-SH2 have been exploited to design potent and selective Grb2-SH2 antagonists.

Structure-based design and synthesis of phosphinate isosteres of phosphotyrosine for incorporation in Grb2-SH2 domain inhibitors. Part 1.

Based on X-ray crystal structure information, mono charged phosphinate isosteres of phosphotyrosine have been designed and incorporated in a short inhibitory peptide sequence of the Grb2-SH2 domain. The resulting compounds, by exploiting additional interactions, inhibit binding to the Grb2-SH2 domain as potently as the corresponding doubly charged (phosphonomethyl)phenylalanine analogue.

Structure-based design and synthesis of phosphinate isosteres of phosphotyrosine for incorporation in Grb2-SH2 domain inhibitors. Part 2.

A series of novel phosphinates, derived from 4-phosphonomethylphenylalanine, are described as isosteres of phosphotyrosine. Benzyl (or alkyl) phosphinomethylphenylalanine derivatives were prepared by alkylation of an amino acid P-H phosphinate.

Molecular approaches to receptors as targets for drug discovery.

Many receptors have been selected as viable drug discovery targets. One particular class of receptors that have received much interest and so far relatively good success are the receptor protein tyrosine kinases (RPTKs). Typically, RPTKs are activated following the binding of the peptide growth factor ligand to its receptor. The RPTKs play crucial roles in signal transduction pathways that regulate a number of cellular functions, such as cell differentiation and proliferation, both under normal physiological conditions as well as in a variety of pathological disorders. A variety of different tumour types have been shown to have dysfunctional RPTKs, either as a result of excess production of the growth factor, the receptor or both, or via mutations in the RPTKs structure. Irrespective of the cause, this leads to the over-activity of the particular RPTK system and in turn to the aberrant and inappropriate cellular signalling within the tumour cell. RPTKs are attractive targets in the search for therapeutic agents, not only against cancers but also against many other disease indications. Although an ever-increasing number of RPTKs have been selected as viable molecular targets for drug discovery programmes, four examples will be covered in this article. These are the epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGF-R), fibroblast growth factor receptor (FGR-R) and vascular endothelial growth factor receptor (VEGF-R), with the main emphasis of interest being on their role in oncology.

Synthesis and hybridization properties of polyamide based nucleic acid analogues incorporating pyrrolidine-derived nucleoamino acids.

Ndelta-Fmoc protected nucleoamino acids of type I (Base = T, C, A) have been synthesized and employed as building blocks for the construction of novel polyamide based nucleic acid analogues. Homopyrimidine oligomer A binds to complementary RNA with significant affinity and in a sequence-specific fashion, while no binding was observed to complementary DNA.