Characterization and Optimization of Synthetic, VLDL Receptor Targeting, Antiangiogenic Peptides with TFPIc23-Like Activity.
Authors: Hembrough Todd , Yin Kevin , Hanson Art , Hahn-Dantona Elizabeth , Strickland Dudley , Plum Stacy , Zemede Genet , Agoston Greg , Holmes Christopher 
Published: April 2020

Abstract
We previously reported that a 23 amino acid fragment of tissue factor pathway inhibitor (TFPIc23) had significant antiangiogenic and antitumor activity in preclinical models. In in vitro studies, the very low density lipoprotein (VLDL) receptor was identified as the target for the activity of TFPIc23 (Blood103:3374). In order to confirm that the cellular target of TFPIc23 is the VLDL receptor, aortas from wildtype and VLDL receptor knockout (VLDLr −/−) mice were used for the ex vivo aorta ring model of angiogenesis. Vessel outgrowth from these rings was measured in the presence and absence of TFPIc23. In control studies, both wild type and VLDLr −/− mouse aorta rings grew vessels. When aorta rings from wild type mice were treated with TFPIc23, there was greater than 90% inhibition of vessel outgrowth. In contrast, VLDLr −/− aorta rings were insensitive to inhibition by the TFPIc23 peptide consistent with the hypothesis that the in vivo target of TFPIc23 activity is the VLDL receptor.
To further assess the role of the VLDL receptor in angiogenesis, chemical modification of the TFPIc23 sequence was performed to define the structure activity relationships between the peptide sequence, peptide binding affinity for the VLDL receptor, and inhibitory activity in HUVEC proliferation assays. The goals were to define a minimal peptide structure which retained antiproliferative activity, confirm the VLDL receptor as the target of action, and modify the peptide sequence and architecture to increase activity and differentiate the peptide from the native sequence.
151 peptides were synthesized and screened for VLDL receptor binding affinity, as well as inhibitory activity in the HUVEC assay. Several peptides of 11–15 amino acids that retained both VLDL receptor affinity, and antiproliferative activity were identified. In the HUVEC assay the IC50 value of some of the novel peptides was decreased from 15uM for TFPIc23 to &lt;1uM. The affinity of these peptides for the VLDL receptor was enhanced more than 10-fold. To confirm that the peptide’s antiproliferative activity was mediated by the VLDL receptor, blocking studies were performed with antibodies to the VLDL receptor. Blockade of the VLDL receptor abrogated the activity of several of the peptides with increase antiproliferative activity. Overall there was a good correlation between binding and activity, further supporting the VLDL receptor as mediating the antiangiogenic activity of these peptides.
Several novel peptide analogs of TFPIc23 that exhibited improved antiproliferative activity and VLDL receptor binding in vitro were then assessed in in vivo models. In the Matrigel angiogenesis model, one of these peptides significantly inhibited angiogenesis by ~75% at 200mg/ml and in the Lewis lung carcinoma metastatic model inhibited metastatic tumor growth by &gt;50% at 100mg/ml. In both cases these activities were significantly better than the activity of the parental TFPIc23 peptide. Taken together, these data show that SAR studies on novel synthetic peptides have yielded peptides with significantly better activity in preclinical tumor and angiogenesis models than TFPIc23. In addition, these results suggest that the VLDL receptor is a potential target for the development of antiangiogenic and antitumor agents.