David L. Selwood

Disruption of methylarginine metabolism impairs vascular homeostasis.

Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both L-NMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. However, despite considerable interest in this pathway and in the role of ADMA as a cardiovascular risk factor, there is little evidence to support a causal role of ADMA in pathophysiology. Here we reveal the structure of human DDAH-1 and probe the function of DDAH-1 both by deleting the DDAH1 gene in mice and by using DDAH-specific inhibitors which, as we demonstrate by crystallography, bind to the active site of human DDAH-1. We show that loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. Our results also suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.

HIV-1 evades innate immune recognition through specific cofactor recruitment

Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

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 (Ki 1.2μm) and effectively (>95% inhibition at 100 μm) 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-γ 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.

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.

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.

Small-molecule mimics of an alpha-helix for efficient transport of proteins into cells.

We designed and synthesized small-molecule mimics of an alpha-helical peptide protein transduction domain (PTD). These small-molecule carriers, which we termed SMoCs, are easily coupled to biomolecules, and efficiently deliver dye molecules and recombinant proteins into a variety of cell types. We designed the SMoCs using molecular modeling techniques. As an example of a protein cargo, we applied this new technology to the internalization of the DNA replication licensing repressor geminin, in vitro, providing evidence that extracellularly delivered SMoC-geminin can have an antiproliferative effect on human cancer cells. Uptake of SMoC-geminin was inhibited at 4 °C and by chlorpromazine, a compound that induces misassembly of clathrin-coated pits at the cell surface. Thus the mechanism of uptake is likely to be clathrin-mediated endocytosis.

Neuropilin-1 antagonism in human carcinoma cells inhibits migration and enhances chemosensitivity

Background:Neuropilin-1 (NRP1) is a non-tyrosine kinase receptor for vascular endothelial growth factor (VEGF) recently implicated in tumour functions.Methods:In this study we used a specific antagonist of VEGF binding to the NRP1 b1 domain, EG3287, to investigate the functional roles of NRP1 in human carcinoma cell lines, non-small-cell lung A549, kidney ACHN, and prostate DU145 cells expressing NRP1, and the underlying mechanisms involved.Results:EG3287 potently displaced the specific binding of VEGF to NRP1 in carcinoma cell lines and significantly inhibited the migration of A549 and ACHN cells. Neuropilin-1 downregulation by siRNA also decreased cell migration. EG3287 reduced the adhesion of A549 and ACHN cells to extracellular matrix (ECM), and enhanced the anti-adhesive effects of a β1-integrin function-blocking antibody. EG3287 increased the cytotoxic effects of the chemotherapeutic agents 5-FU, paclitaxel, or cisplatin on A549 and DU145 cells, through inhibition of integrin-dependent cell interaction with the ECM.Conclusions:These findings indicate that NRP1 is important for tumour cell migration and adhesion, and that NRP1 antagonism enhances chemosensitivity, at least in part, by interfering with integrin-dependent survival pathways. A major implication of this study is that therapeutic strategies targeting NRP1 in tumour cells may be particularly useful in combination with other drugs for combating tumour survival, growth, and metastatic spread independently of an antiangiogenic effect of blocking NRP1.

Mitochondrial targeting of cyclosporin A enables selective inhibition of cyclophilin-D and enhanced cytoprotection after glucose and oxygen deprivation.

CsA (cyclosporin A) is a hydrophobic undecapeptide that inhibits CyPs (cyclophilins), a family of PPIases (peptidylprolyl cis–trans isomerases). In some experimental models, CsA offers partial protection against lethal cell injury brought about by transient ischaemia; this is believed to reflect inhibition of CyP-D, a mitochondrial isoform that facilitates formation of the permeability transition pore in the mitochondrial inner membrane. To evaluate this further, we have targeted CsA to mitochondria so that it becomes selective for CyP-D in cells. This was achieved by conjugating the inhibitor to the lipophilic triphenylphosphonium cation, enabling its accumulation in mitochondria due to the inner membrane potential. In a cell-free system and in B50 neuroblastoma cells the novel reagent (but not CsA itself) preferentially inhibited CyP-D over extramitochondrial CyP-A. In hippocampal neurons, mitochondrial targeting markedly enhanced the capacity of CsA to prevent cell necrosis brought about by oxygen and glucose deprivation, but largely abolished its capacity to inhibit glutamate-induced cell death. It is concluded that CyP-D has a major pathogenic role in ‘energy failure’, but not in glutamate excitotoxicity, where cytoprotection primarily reflects CsA interaction with extramitochondrial CyPs and calcineurin. Moreover, the therapeutic potential of CsA against ischaemia/reperfusion injuries not involving glutamate may be improved by mitochondrial targeting.

siRNA Delivery: From Lipids to Cell‐penetrating Peptides and Their Mimics

To deliver siRNA for therapeutic use, several hurdles must be addressed. Metabolic degradation must be blocked, and the RNAi cellular machinery is located in the cytoplasm, while double‐stranded siRNA is large, highly charged and impermeable to cell membranes. To date, the solutions to the delivery issues have mostly involved different forms of lipid particle encapsulation. Cell‐penetrating peptides and their mimics or analogues offer a different approach and this is an emerging field with the first in vivo examples now reported. Recent reports point to lipid receptors being involved in the cellular uptake of both types of transporter. This review examines the delivery of siRNA with a focus on cell‐penetrating peptides and their small molecule and oligomeric mimics. The current status of siRNA delivery methods in clinical trials is examined. It now seems that the goal of delivering siRNA therapeutically is achievable but will they form part of a sustainable healthcare portfolio for the future.

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. [Mol Cancer Ther 2005;4(12):2005–15]