Quantifying the phosphorylation timescales of receptor-ligand complexes: a Markovian matrix-analytic approach.

Cells interact with the extracellular environment by means of receptor molecules on their surface. Receptors can bind different ligands, leading to the formation of receptor-ligand complexes. For a subset of receptors, called receptor tyrosine kinases, binding to ligand enables sequential phosphorylation of intra-cellular residues, which initiates a signalling cascade that regulates cellular function and fate. Most mathematical modelling approaches employed to analyse receptor signalling are deterministic, especially when studying scenarios of high ligand concentration or large receptor numbers. There exist, however, biological scenarios where low copy numbers of ligands and/or receptors need to be considered, or where signalling by a few bound receptor-ligand complexes is enough to initiate a cellular response. Under these conditions stochastic approaches are appropriate, and in fact, different attempts have been made in the literature to measure the timescales of receptor signalling initiation in receptor-ligand systems. However, these approaches have made use of numerical simulations or approximations, such as moment-closure techniques. In this paper, we study, from an analytical perspective, the stochastic times to reach a given signalling threshold for two receptor-ligand models. We identify this time as an extinction time for a conveniently defined auxiliary absorbing continuous time Markov process, since receptor-ligand association/dissociation events can be analysed in terms of quasi-birth-and-death processes. We implement algorithmic techniques to compute the different order moments of this time, as well as the steady-state probability distribution of the system. A novel feature of the approach introduced here is that it allows one to quantify the role played by each kinetic rate in the timescales of signal initiation, and in the steady-state probability distribution of the system. Finally, we illustrate our approach by carrying out numerical studies for the vascular endothelial growth factor and one of its receptors, the vascular endothelial growth factor receptor of human endothelial cells.

Beyond conventional dose-response curves: Sensorgram comparison in SPR allows single concentration activity and similarity assessment.

Previously we have introduced two SPR-based assay principles (dual-binding assay and bridging assay), which allow the determination of two out of three possible interaction parameters for bispecific molecules within one assay setup: two individual interactions to both targets, and/or one simultaneous/overall interaction, which potentially reflects the inter-dependency of both individual binding events. However, activity and similarity are determined by comparing report points over a concentration range, which also mirrors the way data is generated by conventional ELISA-based methods So far, binding kinetics have not been specifically considered in generic approaches for activity assessment. Here, we introduce an improved slope-ratio model which, together with a sensorgram comparison based similarity assessment, allows the development of a detailed, USP-conformal ligand binding assay using only a single sample concentration. We compare this novel analysis method to the usual concentration-range approach for both SPR-based assay principles and discuss its impact on data quality and increased sample throughput.

Preliminary in vitro and in vivo assessment of a new targeted inhibitor for choroidal neovascularization in age-related macular degeneration.

Choroidal neovascularization (CNV) in age-related macular degeneration usually causes blindness. We established a novel targeted inhibitor for CNV in age-related macular degeneration. The inhibitor CR2-sFlt 1 comprises a CR2-targeting fragment and an anti-vascular endothelial growth factor (VEGF) domain (sFlt 1). The targeting of CR2-sFlt 1 was studied using the transwell assay in vitro and frozen sections in vivo using green fluorescent labeling. Transwell assay results showed that CR2-sFlt 1 migrated to the interface of complement activation products and was present in the retinal tissue of the CR2-sFlt 1-treated CNV mice. Treatment effects were assessed by investigating the VEGF concentration in retinal pigmented epithelial cell medium and the thickness of the CNV complex in the mice treated with CR2-sFlt 1. CR2-sFlt 1 significantly reduced the VEGF secretion from retinal pigmented epithelial cells in vitro and retarded CNV progress in a mouse model. Expression analysis of VEGF and VEGFRs after CR2-sFlt 1 intervention indicated the existence of feedback mechanisms in exogenous CR2-sFlt 1, endogenous VEGF, and VEGFR interaction. In summary, we demonstrated for the first time that using CR2-sFlt 1 could inhibit CNV with clear targeting and high selectivity.

Vascular Endothelial Growth Factor Peptide Ligands Explored by Competition Assay and Isothermal Titration Calorimetry.

The v114* cyclic peptide has been identified as a tight vascular endothelial growth factor (VEGF) ligand. Here we report on the use of isothermal titration calorimetry (ITC), 96-well plate competition assay, and circular dichroism (CD) to explore the binding determinants of a new set of related peptides. Anti-VEGF antibodies are currently used in the clinic for regulating angiogenesis in cancer and age-related macular degeneration treatment. In this context, our aim is to develop smaller molecular entities with high affinity for the growth factor by a structure activity relationship approach. The cyclic disulfide peptide v114* was modified in several ways, including truncation, substitution, and variation of the size and nature of the cycle. The results indicated that truncation or substitution of the four N-terminal amino acids did not cause severe loss in affinity, allowing potential peptide labeling. Increase of the cycle size or substitution of the disulfide bridge with a thioether linkage drastically decreased the affinity, due to an enthalpy penalty. The leucine C-terminal residue positively contributed to affinity. Cysteine N-terminal acetylation induced favorable ΔΔG° and ΔΔH° of binding, which correlated with free peptide CD spectra changes. We also propose a biochemical model to extrapolate Ki from IC50 values measured in the displacement assay. These calculated Ki correlate well with the Kd values determined by extensive direct and reverse ITC measurements.

Modulation, bioinformatic screening, and assessment of small molecular peptides targeting the vascular endothelial growth factor receptor.

Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) are important factors in tumor growth and metastasis. Molecular probes or drugs designed to target VEGF/VEGFR interactions are crucial in tumor molecular imaging and targeted therapy. Bioinformatic methods enable molecular design based on the structure of bio-macromolecules and their interactions. This study was aimed to identify tumor-targeting small-molecule peptides with high affinity for VEGFR using bioinformatics screening. The VEGFR extracellular immunoglobulin-like modules Ig1-Ig3 were used as the target to systematically alter the primary peptide sequence of VEGF125-136. Molecular docking and surface functional group interaction methods were combined in an in silico screen for polypeptides, which in theory, would have higher affinities for VEGFR. In vitro receptor competition binding assays were used to assess the affinity of the putative VEGFR-binding polypeptides. Rhodamine-conjugated peptides were used to label and visualize peptide-binding sites on A549 cells. Using bioinformatic screening, we identified 20 polypeptides with potentially higher affinity for VEGFR. The polypeptides were capable of inhibiting the binding of (125)I-VEGF to VEGFR in a dose-dependent manner. The IC50 values of QKRKRKKSRKKH and RKRKRKKSRYIVLS (80 and 185 nmol/L, respectively) were significantly lower than that of VEGF125-136 (464 nmol/L); thus, the affinity of these peptides for VEGFR was 6- and 2.5-fold higher, respectively, than that of VEGF125-136. Rhodamine labeling of A549 cells revealed peptide binding mainly on the plasma membrane and in the cytoplasm. Bioinformatic approaches hold promise for the development of molecular imaging probes. Using this approach, we designed two peptides that showed higher affinity toward VEGFR. These polypeptides may be used as molecular probes or drugs targeting VEGFR, which can be utilized in molecular imaging and targeted therapy of certain tumors.

High-pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: refolding optimization, and feasibility assessment.

High-pressure has been established as an effective technique for refolding proteins at high concentrations. In this study, high hydrostatic pressure (1-3 kbar) was utilized to refold a homodimeric protein from inclusion bodies and the process was evaluated for large-scale manufacturing feasibility. This research focused on increasing protein concentration while maximizing yield and product quality. Refolding yields of 29-42% were achieved in the absence of urea at 2 kbar and at a protein concentration of 6 g/L. Optimization of the refolding buffer composition via multivariate design of experiments and other process parameters such as refolding pressure, gas sparging, and time under pressure are discussed. Although high-pressure refolding can be considered a viable technology for manufacturing if the gains are clearly identified, in this particular case, the benefits that the high-pressure technology offers do not compensate for the drawbacks of implementing new equipment in an existing facility, and unknown impact of scale-up for this molecule.

111In- or 99mTc-labeled recombinant VEGF bioconjugates: in vitro evaluation of their cytotoxicity on porcine aortic endothelial cells overexpressing Flt-1 receptors.

INTRODUCTION: The aims of this study were to (a) synthesize and characterize a novel vascular endothelial growth factor (VEGF-2K) recombinant protein expressed in Pichia pastoris and (b) compare its cytotoxicity when labeled with the Auger electron emitter (111)In or (99m)Tc, both of which are in the nanometer-micrometer range, toward porcine aortic endothelial (PAE) cells transfected with the flt-1 gene to overexpress Flt-1 receptors (PAE-Flt-1). METHODS: The gene for the VEGF(165) isoform was fused to a sequence encoding an extended flexible peptide (KGGGGSK) with two accessible lysines for preferential derivatization with diethylenetriaminepentaacetic acid (DTPA) for complexing (111)In and a sequence for a His(6) affinity tag that bound the [(99m)Tc(CO)(3)(H(2)O)(3)](+) tricarbonyl complex. P. pastoris strain KM71H was transfected with the recombinant gene, the VEGF-2K protein expressed with methanol induction, and then purified by metal-affinity chromatography. VEGF-2K was modified with 13-mer peptides [CGYGPKKKRKVGG] containing the nuclear localization sequence (NLS) of SV-40 large T-antigen (underlined) to promote nuclear uptake following its receptor-mediated internalization. RESULTS: (99m)Tc-DTPA-VEGF-2K bound strongly and preferentially to PAE-Flt-1 cells compared with non-transfected PAE cells, but NLS modification diminished the ratio of PAE-Flt-1 to PAE binding to 2.3-fold. Nuclear accumulation of (99m)Tc-labeled DTPA-VEGF-2K was not enhanced by NLS modification but was enhanced by 1.5-fold for (111)In-DTPA-VEGF-2K-NLS. However, confocal microscopy revealed intranuclear distribution of DTPA-VEGF-2K-NLS, whereas DTPA-VEGF-2K distribution was mainly perinuclear. (111)In-DTPA-VEGF-2K-NLS was the most cytotoxic to PAE-Flt-1 cells, reducing their clonogenic survival by 4-fold. (111)In-DTPA-VEGF-2K, (99m)Tc-DTPA-VEGF-2K or (99m)Tc-DTPA-VEGF-2K-NLS had less effect on the clonogenic survival of PAE-Flt-1 or PAE cells. The strong cytotoxicity of (111)In-DTPA-VEGF-2K-NLS toward PAE-Flt-1 cells was associated with a 27-fold increase in nuclear foci of immunofluorescence for phosphorylated histone-2AX corresponding to sites of unrepaired DNA double-strand breaks. Monte Carlo modeling revealed that radionuclide decay in the nucleus would provide a 5-fold higher radiation absorbed dose for (111)In than for (99m)Tc, explaining their differential cytotoxicity, and intranuclear localization would amplify the radiation dose delivered by (111)In by 3-fold, explaining the greater potency of (111)In-DTPA-VEGF-2K-NLS compared with (111)In-DTPA-VEGF-2K. CONCLUSIONS: We conclude that targeted Auger electron radiotherapy aimed at Flt-1 receptors is a promising strategy that should be explored further for treatment of tumors in which this angiogenic pathway is up-regulated. (111)In is a more cytotoxic radionuclide than (99m)Tc, unless DNA delivery can be achieved, due to the short range of the electrons emitted.