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

Chemosensitivity profiling of osteosarcoma tumour cell lines identifies a model of BRCAness

Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS

Hi-JAK-ing the ubiquitin system : the design and physicochemical optimisation of JAK PROTACs

PROTACs have recently emerged as a novel paradigm in drug discovery.They can hijack existing biological machinery to selectively degrade proteins o finterest, in a catalytic fashion. Here we describe the design, optimisation and biological activity of a set of novel PROTACs targeting the Janus kinase family (JAK1, JAK2, JAK3 and TYK2) of proximal membrane-bound proteins.The JAK family proteins display membrane localisation by virtue of their association with cytoplasmic tails of cytokine receptors, and there are no reports of a successful PROTAC strategy being deployed against this class of proteins. JAK PROTACs from two distinct JAK chemotypes were designed, optimising the physicochemical properties for each template to enhance cell permeation. These PROTACs are capable of inducing JAK1 and JAK2 degradation, demonstrating an extension of the PROTAC methodology to an unprecedented class of protein targets. A number of known ligase binders were explored, and it was found that PROTACs bearing an inhibitor of apoptosis protein (IAP) ligand induced significantly more JAK degradation over VonHippel–Lindau (VHL) and Cereblon (CRBN. In addition, the mechanism of action of the JAK PROTACs was elucidated, and it was confirmed that JAKdegradation was both IAP-and proteasome-dependent

The Grafting of Universal T-Helper Epitopes Enhances Immunogenicity of HIV-1 Tat Concurrently Improving Its Safety Profile

<div><p>Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol₇₁₁ into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.</p></div

Mapping of the B-cell epitopes in Tat.

<p>The antisera collected from mice immunized with (A) WT-Tat or (B) PADRE-CRD proteins were diluted 500 and 1,000 times, respectively, and used in the pepscan analysis. Twenty-mer synthetic peptides with a 10-residue overlap and encompassing the full-length C-Tat consensus sequence were used in the assay. Peptides 1-9 represent full-length subtype C Tat protein. The gray bars (A and B) represent the amino acid sequences generated due to the grafting of the HTL in Tat. The data are presented as the mean absorbance + SD on the x-axis with corresponding peptides on the y-axis. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114155#pone.0114155.s002" target="_blank">Figure S2</a> for data pertaining to the other three HTL-Tat antisera.</p

Cross-clade reactivity of the anti-Tat antibodies.

<p>The antibody titers were determined by incubating serially diluted antisera in triplicate microtiter wells coated with recombinant Tat from subtypes A, B, C and D. The x-axis represents the serum dilution and the y-axis to mean absorbance + SD. Representative data of two or more independent experiments are shown.</p