Radhakrishna Pillai

Tuftsin Binds Neuropilin-1 through a Sequence Similar to That Encoded by Exon 8 of Vascular Endothelial Growth Factor

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF165-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.

A phospholipid-PEG2000 conjugate of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeting heterodimer peptide for contrast-enhanced ultrasound imaging of angiogenesis.

The transition of a targeted ultrasound contrast agent from animal imaging to testing in clinical studies requires considerable chemical development. The nature of the construct changes from an agent that is chemically attached to microbubbles to one where the targeting group is coupled to a phospholipid, for direct incorporation to the bubble surface. We provide an efficient method to attach a heterodimeric peptide to a pegylated phospholipid and show that the resulting construct retains nanomolar affinity for its target, vascular endothelial growth factor receptor 2 (VEGFR2), for both the human (kinase insert domain-containing receptor - KDR) and the mouse (fetal liver kinase 1 - Flk-1) receptors. The purified phospholipid-PEG-peptide isolated from TFA-based eluents is not stable with respect to hydrolysis of the fatty ester moieties. This leads to the time-dependent formation of the lysophospholipid and the phosphoglycerylamide derived from the degradation of the product. Purification of the product using neutral eluent systems provides a stable product. Methods to prepare the lysophospholipid (hydrolysis product) are also included. Biacore binding data demonstrated the retention of binding of the lipopeptide to the KDR receptor. The phospholipid-PEG2000-peptide is smoothly incorporated into gas-filled microbubbles and provides imaging of angiogenesis in a rat tumor model.