N-terminal modification of VEGF-A C terminus-derived peptides delineates structural features involved in neuropilin-1 binding and functional activity.

The interaction between VEGF-A and its neuropilin (NRP) receptors mediates a number of important biological effects. NRP1 and the related molecule NRP2 are widely expressed on multiple tumour types and throughout the tumour vasculature, and are emerging as critical molecules required for the progression of angiogenic diseases. Given the increasing evidence supporting a role for NRP1 in tumour development, there is growing interest in developing inhibitors of NRP1 interactions with VEGF and its other ligands. In order to probe the interaction we synthesised a number of exon 7- and 8-derived bicyclic peptides with N-terminal lipophilic groups and found a simple N-octanoyl derivative (EG00086) to be the most potent and functionally active. Detailed modelling studies indicated that new intramolecular hydrogen bonds were formed, stabilising the structure and possibly contributing to the potency. Removal of a salt bridge between D142 and R164 implicated in VEGF-A binding to neuropilin-1 had a minor effect on potency. Isothermal calorimetry was used to assess binding of EG00086 to NRP1 and NRP2, and the stability of the peptide in serum and in vivo was investigated. EG00086 is a potent blocker of VEGF-promoted cellular adhesion to extracellular matrices, and phosphorylation of p130Cas contributes to this effect.

Ligand- and structure-based virtual screening for clathrodin-derived human voltage-gated sodium channel modulators.

Voltage-gated sodium channels (VGSC) are attractive targets for drug discovery because of the broad therapeutic potential of their modulators. On the basis of the structure of marine alkaloid clathrodin, we have recently discovered novel subtype-selective VGSC modulators I and II that were used as starting points for two different ligand-based virtual screening approaches for discovery of novel VGSC modulators. Similarity searching in the ZINC database of drug-like compounds based on compound I resulted in five state-dependent Na(v)1.3 and Na(v)1.7 modulators with improved activity compared to I (IC50 < 20 µM). Compounds 2 and 16 that blocked sodium permeation in Na(v)1.7 with IC50 values of 7 and 9 µM, respectively, are among the most potent clathrodin analogs discovered so far. In the case of compound II, 3D similarity searching in the same database was followed by docking of an enriched compound library into our human Na(v)1.4 open-pore homology model. Although some of the selected compounds, e.g., 31 and 32 displayed 21% and 22% inactivated state I(peak) block of Na(v)1.4 at 10 µM, respectively, none showed better Na(v)1.4 modulatory activity than compound II. Taken together, virtual screening yielded compounds 2 and 16, which represent novel scaffolds for the discovery of human Na(v)1.7 modulators.

VEGF binding to NRP1 is essential for VEGF stimulation of endothelial cell migration, complex formation between NRP1 and VEGFR2, and signaling via FAK Tyr407 phosphorylation.

In endothelial cells, neuropilin-1 (NRP1) binds vascular endothelial growth factor (VEGF)-A and is thought to act as a coreceptor for kinase insert domain-containing receptor (KDR) by associating with KDR and enhancing VEGF signaling. Here we report mutations in the NRP1 b1 domain (Y297A and D320A), which result in complete loss of VEGF binding. Overexpression of Y297A and D320A NRP1 in human umbilical vein endothelial cells reduced high-affinity VEGF binding and migration toward a VEGF gradient, and markedly inhibited VEGF-induced angiogenesis in a coculture cell model. The Y297A NRP1 mutant also disrupted complexation between NRP1 and KDR and decreased VEGF-dependent phosphorylation of focal adhesion kinase at Tyr407, but had little effect on other signaling pathways. Y297A NRP1, however, heterodimerized with wild-type NRP1 and NRP2 indicating that nonbinding NRP1 mutants can act in a dominant-negative manner through formation of NRP1 dimers with reduced binding affinity for VEGF. These findings indicate that VEGF binding to NRP1 has specific effects on endothelial cell signaling and is important for endothelial cell migration and angiogenesis mediated via complex formation between NRP1 and KDR and increased signaling to focal adhesions. Identification of key residues essential for VEGF binding and biological functions provides the basis for a rational design of antagonists of VEGF binding to NRP1.

Small molecule inhibitors of the neuropilin-1 vascular endothelial growth factor A (VEGF-A) interaction.

We report the molecular design and synthesis of EG00229, 2, the first small molecule ligand for the VEGF-A receptor neuropilin 1 (NRP1) and the structural characterization of NRP1-ligand complexes by NMR spectroscopy and X-ray crystallography. Mutagenesis studies localized VEGF-A binding in the NRP1 b1 domain and a peptide fragment of VEGF-A was shown to bind at the same site by NMR, providing the basis for small molecule design. Compound 2 demonstrated inhibition of VEGF-A binding to NRP1 and attenuated VEGFR2 phosphorylation in endothelial cells. Inhibition of migration of endothelial cells was also observed. The viability of A549 lung carcinoma cells was reduced by 2, and it increased the potency of the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination. These studies provide the basis for design of specific small molecule inhibitors of ligand binding to NRP1.

Prediction of ion channel activity using binary kernel discrimination.

Voltage-gated ion channels are a diverse family of pharmaceutically important membrane proteins for which limited 3D information is available. A number of virtual screening tools have been used to assist with the discovery of new leads and with the analysis of screening results. One such tool, and the subject of this paper, is binary kernel discrimination (BKD), a machine-learning approach that has recently been applied to applications in chemoinformatics. It uses a training set of compounds, for which both structural and qualitative activity data are known, to produce a model that can then be used to rank another set of compounds in order of likely activity. Here, we report the use of BKD to build models for the prediction of five different ion channel targets using two types of activity data. The results obtained suggest that the approach provides an effective way of prioritizing compounds for acquisition and testing.

Discovery of inhibitors of the pentein superfamily protein dimethylarginine dimethylaminohydrolase (DDAH), by virtual screening and hit analysis.

An efficient process for the discovery of inhibitors of DDAH enzymes, without the requirement for high throughput screening, is described. Physicochemical filtering of a 308,000-compound library according to drug likeness followed by reciprocal nearest neighbour selection produced a representative subset of 35,000 compounds. Virtual screening on a dual processor PC using FlexX, followed by biological screening, identified two hit series. Similarity searches of commercial databases and chemical re-synthesis of pure compounds resulted in SR445 as an inhibitor of Pseudomonas aeruginosa DDAH at 2 microM.

Characterization of a bicyclic peptide neuropilin-1 (NP-1) antagonist (EG3287) reveals importance of vascular endothelial growth factor exon 8 for NP-1 binding and role of NP-1 in KDR signaling.

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (K(i) 1.2 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-gamma tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1.

Aurora A and B kinases as targets for cancer: will they be selective for tumors?

Aurora A and B kinases are closely related kinases involved in regulating separate points in the cell cycle. This review highlights the rationale for Aurora kinases as cancer targets and examines the currently known Aurora kinase inhibitors in the patent and scientific literature. The known crystal structures of the Aurora kinases are described with relevance to bound ligand interactions and the prospect of the generation of drug-resistant mutant forms. The potential for selectivity versus primary cells will also be discussed. The status of the inhibitors in clinical development is described.

Imatinib binding and cKIT inhibition is abrogated by the cKIT kinase domain I missense mutation Val654Ala.

Several activating mutations in the cKIT receptor tyrosine kinase are associated with the development and progression of gastrointestinal stromal tumors (GIST). Treatment of GIST with the tyrosine kinase inhibitor imatinib (Gleevec, STI571; Novartis, Basel, Switzerland) increases patient survival. However, many patients develop resistance to imatinib following initial responses. We sequenced cKIT exons from two patients with GIST after the development of imatinib resistance, revealing a point mutation in kinase domain I (exon 13), Val654Ala, which has been associated previously with relapse and resistance. Molecular modeling of cKIT-imatinib complexes shows that this residue is located in the drug-binding site and that the Val654Ala mutation disrupts drug binding by removing hydrophobic contacts with the central diaminophenyl ring of imatinib. Loss of these contacts results in a destabilizing effect on two key hydrogen bonds between imatinib and Asp310 and Thr670 of cKIT. Calculations based on published crystallography data show an estimated destabilization energy of 2.25 kcal/mol in the Val654Ala cKIT compared with wild type. When present on the same cKIT allele as an oncogenic mutation, the Val654Ala mutation abolishes imatinib-mediated inhibition of cKIT phosphoactivation in vitro. These results highlight some of the structural and functional consequences of the Val654Ala mutation in relapsing imatinib-resistant GIST and emphasize the importance of tumor genetics in drug development and patient-specific cancer treatment regimens.

Copper (II)-mediated arylation with aryl boronic acids for the N-derivatization of pyrazole libraries.

A N-derivatized 3-dimethylaminopropyloxypyrazole library was prepared using solution-phase parallel synthesis. The library was designed using physicochemical constraints designed to remove non-membrane-permeable molecules. Cupric acetate-mediated N-arylation with aryl boronic acids proceeded regioselectively to form the N-2-substituted derivatives. The presence of the 3-dimethylaminopropyloxy group was found to completely control the regioselectivity of the arylation. Presence of a dimethylaminoethyloxy or dimethylaminobutyloxy group gave a lesser degree of regioselectivity. The scope of the method as applied to library synthesis is discussed.