Utilizing a Dual Human Transporter MDCKII-MDR1-BCRP Cell Line to Assess Efflux at the Blood Brain Barrier.

To facilitate the design of drugs readily able to cross the blood brain barrier (BBB), a Madin-Darby canine kidney (MDCK) cell line was established that over expresses both P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), the main human efflux transporters of the BBB. Proteomics analyses indicate BCRP is expressed at a higher level than Pgp in this cell line. This cell line shows good activity for both transporters [BCRP substrate dantrolene efflux ratio (ER) 16.3 ± 0.9, Pgp substrate quinidine ER 27.5 ± 1.2], and use of selective transporter inhibitors enables an assessment of the relative contributions to overall ERs. The MDCKII-MDR1-BCRP ER negatively correlates with rat unbound brain/unbound plasma ratio, Kpuu Highly brain penetrant compounds with rat Kpuu ≥ 0.3 show ERs ≤ 2 in the MDCKII-MDR1-BCRP assay while compounds predominantly excluded from the brain, Kpuu ≤ 0.05, demonstrate ERs ≥ 20. A subset of compounds with MDCKII-MDR1-BCRP ER < 2 and rat Kpuu < 0.3 were shown to be substrates of rat Pgp using a rat transfected cell line, MDCKII-rMdr1a. These compounds also showed ERs > 2 in the human National Institutes of Health (NIH) MDCKI-MDR1 (high Pgp expression) cell line, which suggests that they are weak human Pgp substrates. Characterization of 37 drugs targeting the central nervous system in the MDCKII-MDR1-BCRP efflux assay show 36 have ERs < 2. In drug discovery, use of the MDCKII-MDR1-BCRP in parallel with the NIH MDCKI-MDR1 cell line is useful for identification of compounds with high brain penetration. SIGNIFICANCE STATEMENT: A single cell line that includes both the major human efflux transporters of the blood brain barrier (MDCKII-MDR1-BCRP) has been established facilitating the rapid identification of efflux substrates and enabling the design of brain penetrant molecules. Efflux ratios using this cell line demonstrate a clear relationship with brain penetration as defined by rat brain Kpuu.

Quantitative investigation of the affinity of human respiratory syncytial virus phosphoprotein C-terminus binding to nucleocapsid protein.

BACKGROUND: There are no approved small molecule drug therapies for human respiratory syncytial virus (hRSV), a cause of morbidity and mortality in at-risk newborns, the immunocompromised, and the elderly. We have investigated as a potential novel hRSV drug target the protein-protein interaction between the C-terminus of the viral phosphoprotein (P) and the viral nucleocapsid protein (N), components of the ribonucleoprotein complex that contains, replicates, and transcribes the viral RNA genome. Earlier work by others established that the 9 C-terminal residues of P are necessary and sufficient for binding to N. METHODS: We used a fluorescence anisotropy assay, surface plasmon resonance and 2-D NMR to quantify the affinities of peptides based on the C terminus of P for RNA-free, monomeric N-terminal-truncated N(13-391). We calculated the contributions to the free energies of binding of P to N(13-391) attributable to the C-terminal 11 residues, phosphorylation of the C-terminal 2 serine residues, the C-terminal Asp-Phe, and the phenyl ring of the C-terminal Phe. RESULTS: Binding studies confirmed the crucial role of the phosphorylated C-terminal peptide D(pS)DNDL(pS)LEDF for binding of P to RNA-free, monomeric N(13-391), contributing over 90% of the binding free energy at low ionic strength. The phenyl ring of the C-terminal Phe residue contributed an estimated -2.7 kcal/mole of the free energy of binding, the C-terminal Asp-Phe residues contributed -3.8 kcal/mole, the sequence DSDNDLSLE contributed -3.1 kcal/mole, and phosphorylation of the 2 Ser residues contributed -1.8 kcal/mole. Due to the high negative charge of the C-terminal peptide, the affinity of the P C-terminus for N(13-391) decreased as the ionic strength increased. CONCLUSIONS: The results support the idea that the interaction of the C-terminal residues of P with N constitutes a protein-protein interaction hotspot that may be a suitable target for small-molecule drugs that inhibit viral genome replication and transcription.

Assessing the activity of cediranib, a VEGFR-2/3 tyrosine kinase inhibitor, against VEGFR-1 and members of the structurally related PDGFR family.

Cediranib is a potent inhibitor of the VEGF receptor (VEGFR)-2 and VEGFR-3 tyrosine kinases. This study assessed the activity of cediranib against the VEGFR-1 tyrosine kinase and the platelet-derived growth factor receptor (PDGFR)-associated kinases c-Kit, PDGFR-α, and PDGFR-ß. Cediranib inhibited VEGF-A-stimulated VEGFR-1 activation in AG1-G1-Flt1 cells (IC(50) = 1.2 nmol/L). VEGF-A induced greatest phosphorylation of VEGFR-1 at tyrosine residues Y1048 and Y1053; this was reversed by cediranib. Potency against VEGFR-1 was comparable with that previously observed versus VEGFR-2 and VEGFR-3. Cediranib also showed significant activity against wild-type c-Kit in cellular phosphorylation assays (IC(50) = 1-3 nmol/L) and in a stem cell factor-induced proliferation assay (IC(50) = 13 nmol/L). Furthermore, phosphorylation of wild-type c-Kit in NCI-H526 tumor xenografts was reduced markedly following oral administration of cediranib (≥1.5 mg/kg/d) to tumor-bearing nude mice. The activity of cediranib against PDGFR-ß and PDGFR-α was studied in tumor cell lines, vascular smooth muscle cells (VSMC), and a fibroblast line using PDGF-AA and PDGF-BB ligands. Both receptor phosphorylation (IC(50) = 12-32 nmol/L) and PDGF-BB-stimulated cellular proliferation (IC(50) = 32 nmol/L in human VSMCs; 64 nmol/L in osteosarcoma cells) were inhibited. In vivo, ligand-induced PDGFR-ß phosphorylation in murine lung tissue was inhibited by 55% following treatment with cediranib at 6 mg/kg but not at 3 mg/kg or less. In contrast, in C6 rat glial tumor xenografts in mice, ligand-induced phosphorylation of both PDGFR-α and PDGFR-ß was reduced by 46% to 61% with 0.75 mg/kg cediranib. Additional selectivity was showed versus Flt-3, CSF-1R, EGFR, FGFR1, and FGFR4. Collectively, these data indicate that cediranib is a potent pan-VEGFR kinase inhibitor with similar activity against c-Kit but is significantly less potent than PDGFR-α and PDGFR-ß.

Vascular endothelial growth factor receptors VEGFR-2 and VEGFR-3 are localized primarily to the vasculature in human primary solid cancers.

PURPOSE: Vascular endothelial growth factor (VEGF) signaling is key to tumor angiogenesis and is an important target in the development of anticancer drugs. However, VEGF receptor (VEGFR) expression in human cancers, particularly the relative expression of VEGFR-2 and VEGFR-3 in tumor vasculature versus tumor cells, is poorly defined. EXPERIMENTAL DESIGN: VEGFR-2- and VEGFR-3-specific antibodies were identified and used in the immunohistochemical analysis of human primary cancers and normal tissue. The relative vascular localization of both receptors in colorectal and breast cancers was determined by coimmunofluorescence with vascular markers. RESULTS: VEGFR-2 and VEGFR-3 were expressed on vascular endothelium but not on malignant cells in 13 common human solid tumor types (n > 400, bladder, breast, colorectal, head and neck, liver, lung, skin, ovarian, pancreatic, prostate, renal, stomach, and thyroid). The signal intensity of both receptors was significantly greater in vessels associated with malignant colorectal, lung, and breast than adjacent nontumor tissue. In colorectal cancers, VEGFR-2 was expressed on both intratumoral blood and lymphatic vessels, whereas VEGFR-3 was found predominantly on lymphatic vessels. In breast cancers, both receptors were localized to and upregulated on blood vessels. CONCLUSIONS: VEGFR-2 and VEGFR-3 are primarily localized to, and significantly upregulated on, tumor vasculature (blood and/or lymphatic) supporting the majority of solid cancers. The primary clinical mechanism of action of VEGF signaling inhibitors is likely to be through the targeting of tumor vessels rather than tumor cells. The upregulation of VEGFR-3 on tumor blood vessels indicates a potential additional antiangiogenic effect for dual VEGFR-2/VEGFR-3-targeted therapy.