Neuropilin-1 drives tumor-specific uptake of chlorotoxin

Abstract

Background Chlorotoxin (Cltx) isolated from scorpion venom is an established tumor targeting and antiangiogenic peptide. Radiolabeled Cltx therapeutic ( 131 I-TM601) yielded promising results in human glioma clinical studies, and the imaging agent tozuleristide, is under investigation in CNS cancer studies. Several binding targets have previously been proposed for Cltx but none effectively explain its pleiotropic effects; its true target remains ambiguous and is the focus of this study. Methods A peptide-drug conjugate (ER-472) composed of Cltx linked to cryptophycin as warhead was developed as a tool to probe the molecular target and mechanism of action of Cltx, using multiple xenograft models. Results Neuropilin-1 (NRP1), an endocytic receptor on tumor and endothelial cells, was identified as a novel Cltx target, and NRP1 binding by Cltx increased drug uptake into tumor. Metabolism of Cltx to peptide bearing free C-terminal arginine, a prerequisite for NRP1 binding, took place in the tumor microenvironment, while native scorpion Cltx with amidated C-terminal arginine did not bind NRP1, and instead acts as a cryptic peptide. Antitumor activity of ER-472 in xenografts correlated to tumor NRP1 expression. Potency was significantly reduced by treatment with NRP1 blocking antibodies or knockout in tumor cells, confirming a role for NRP1-binding in ER-472 activity. Higher cryptophycin metabolite levels were measured in NRP1-expressing tumors, evidence of NRP1-mediated enhanced drug uptake and presumably responsible for the superior antitumor efficacy. Conclusions NRP1 was identified as a novel Cltx target which enhances tumor drug uptake. This finding should facilitate tumor selection for chlorotoxin-based therapeutics and diagnostics. Electronic supplementary material The online version of this article (10.1186/s12964-019-0368-9) contains supplementary material, which is available to authorized users.

Structural basis for the binding of the cancer targeting scorpion toxin, ClTx, to the vascular endothelia growth factor receptor neuropilin-1

“…The NMR chemical shift mapping experiments reveal a distinct binding interface which includes the basic patch spanning R25, K27 and R36. These results are in qualitative agreement with the report by McGonigle et al., where the binding of ClTx to NRP1-b1 was first reported ( McGonigle et al., 2019 ). However, we find that the ITC binding data using our label free NRP1-b1 to deamidated ClTx shows a notably weaker K d (143 ​μM) than measured by biolayer-interferometry of a biotinylated ClTx to NRP1 (1–644) (using an Octet system – K d 240 ​nM).…”

“…While binding to the peptide N-terminus or K15 and K23 are, based on our data, predicted not to affect NRP1 binding, we expect that the modification of K27 may adversely affect this binding. In the study by McGonigle et al., for example, they used a mixture of N-terminal and K27 biotinylated ClTx for NRP1 binding studies ( McGonigle et al., 2019 ). Our model would predict that the two species would show different binding constants.…”

“…Most recently, the vascular endothelial growth factor (VEGF) receptor, Neuropilin-1 (NRP1) was identified as the likely receptor, responsible for tumour uptake ( McGonigle et al., 2019 ). The extracellular b1 domain of NRP1 (NRP1-b1) is known to bind to VEGF via a C-terminal basic region including a terminal arginine residue.…”

“…Although ClTx does not contain the [R/K]XX[R/K] motif it was shown to bind the NRP1-b1 domain, but only when the peptide was deamidated to expose the carboxy group of its C-terminal arginine residue ( McGonigle et al., 2019 ). This raises the question of how ClTx can bind to NRP1, despite not adhering to the C-end rule.…”

Receptor-Targeted Fluorescence-Guided Surgery With Low Molecular Weight Agents

“…By contrast, tumorspecific FGS was 75% sensitive and 100% specific, but when the analysis was limited to FRa+ adenomas, sensitivity and specificity were both 100% (Table2). The CTX targeting motif, a 36 amino acid peptide with four disulfide bridges derived from scorpion venom, has been postulated to bind to a number of targets overexpressed in tumors including matrix metalloproteinases, Annexin A2, chloride ion channels, and others(56). The rationale for selecting CTX for FGS drug development came from several studies showing selective targeting of glioma cells compared with non-neoplastic cells or normal brain(57).…”