Original ArticleNRPa-308, a new neuropilin-1 antagonist, exerts in vitro anti-angiogenic and anti-proliferative effects and in vivo anti-cancer effects in a mouse xenograft model
Introduction
The main Vascular Endothelial Growth Factor (VEGF) over-expressed in tumour tissues is VEGF-A165, which exerts pro-angiogenic and tumour growth effects through its binding to structurally related type III tyrosine kinase receptors: VEGF-R1, VEGF-R2, and VEGF-R3 [1]. However, current anti-angiogenic therapies targeting this signalling pathway fail to eradicate cancers, since tumours always relapse. This underlies the need for alternative anti-angiogenic strategies [2].
In the last decade, another VEGF-A165 receptor has been identified: Neuropilin-1 (NRP-1), a 120–130 KDa transmembrane glycoprotein without catalytic activity. NRP-1 has been extensively studied in the control of neuronal guidance [3], but its involvement in tumour growth, cell migration, and survival emerged recently [4]. Nonetheless, its over-expression is related in vivo to a high metastatic potential and is associated to poor prognosis [5]. NRP-1 is also involved in the control of immunological synapses and in immunosuppression through its binding to semaphorin-3A [6], [7].
These results point out NRP-1 as an indisputable therapeutic target for cancer treatment [8]. The humanized monoclonal antibody MNRP1685A, directed towards the NRP-1 extracellular domain, is the most advanced NRP-1 antagonist (NRPa). It down-regulates angiogenesis and exhibits synergic effects when used in association with anti-angiogenic agents and/or conventional cytotoxic drugs in a non-small cell lung cancer xenograft mice model [9]. MNRP1685A reached the phase Ib clinical trials on patients suffering incurable solid tumours, and no marked adverse effects have been reported [10].
Another NRPa of interest is compound EG00229, developed by Selwood and co-workers [11]. This pseudo-peptide inhibits 125I VEGF A165 binding to porcine aortic endothelial cells (KI = 1.2 μM), reduces viability of lung carcinoma cells (A549), and enhances paclitaxel and 5-fluorouracil cytotoxicity in vitro. NRP-1 ablation or inhibition by EG00229 result in slower disease progression in a glioma xenograft mice model, highlighting the relevance of small-sized NRPas for cancer treatment [12].
However, as the use of pseudo-peptides as drugs remains partly questionable due to their lack of stability [13], we have developed the pioneering multi-step screening procedure leading to the first non-peptidic NRPas, 1a (NRPa-47) and 1b (NRPa-48) (Chart 1) [14]. These molecules exert in vitro anti-angiogenic effects, and 1a reduces angiogenesis, tumour growth, and metastases formation in mice xenografted with human breast cancer cells. Recently, an alternative screening has been reported by Perret, revealing four new NRPas [15]. Nevertheless, their evaluation in vitro on tumours cells, and in vivo in xenografted animals should be reported.
In line with our previous discoveries, we report herein a new family of 18 NRPas, sharing a common scaffold. The most promising molecule, compound 2a (renamed NRPa-308) exerts in vitro anti-angiogenic effect and reduces cell viability of human solid tumours and haematological malignancies. Moreover, 2a has been assayed in vivo on mice xenografted with human aggressive and metastatic breast tumour cells (MDA-MB-231). At a dose of 50 mg/kg, 2a significantly reduces tumour growth and improves animal survival (median survival increases by 20%). Altogether these results highlight 2a as hit for the further development of a new and versatile anti-cancer agent.
Section snippets
Structure based virtual ligand screening (SB-VLS)
We reported the SB-VLS [14a], which is summarized in Supporting Information (Fig. S1).
Chemicals
The compounds used for the cellular and molecular screening were purchased from ChemBridge (San Diego, CA) and belong to the “Building Block Collection”. Their purity is guaranteed to be over 95% (www.hit2lead.com). Sunitinib malate has been purchased from Tocris.
Molecular assay: VEGF-A165 binding inhibition to NRP-1
96-well plates were coated overnight at 4 °C, with PBS buffer containing 2 μg/mL of NRP-1 (Bio-Techne®, France). After 3 washes with PBS 0.5% Tween
Selection of 2a as new “hit” compound
Our NRPas selection procedure, which provided 1a [14a], allowed the unreported emergence of a short list of 18 structurally-related NRPas, distinct from 1a, and from already the known NRPas [11], [14], [15], [17]. These 18 molecules share a scaffold including two aromatic rings (“A”, “C”) linked to a third one (“B”) through amide and sulfonamide bonds (Fig. 1A). Among these molecules (2a-r), 16 share a methyl group in the R′ position of “B” (except 2n R’ = Cl, and 2r R’ = H). Finally, only one (
Discussion
While NRP-1 has been validated as a target in oncology, identification of small-sized non-peptidic NRPa remains challenging. We have developed a pioneering NRPas selection procedure, leading to compounds 1a and 1b, the first non-peptidic NRPas [14]. We report herein a new series of 18 structurally-related NRPas, sharing in their scaffold three aromatic motives linked through carboxamide and sulfonamide bond. Among them, 2a emerged as the most promising molecule.
Our docking predictions suggest
Conclusion
We report herein the identification of a new series including 18 small-sized NRPas. Among them, 2a emerged as a really promising agent. Its biological evaluation confirms the major importance of small-sized NRPas to specifically target tumour cells. This study highlights 2a as a new member of the short-list of the small-sized NRPas active in vivo, which includes the monoclonal antibody MNRP1685A, the pseudopepdide EG00229, and our previous “hit” 1a. Furthermore, hit-to-lead optimization of 2a
Conflict of interest
None.
Acknowledgements
This study was supported by grants from: (i) the “Institut National Du Cancer” (INCa “Angiomed”, 2007–2010 grant), and (ii) the “Canceropôle Provence Alpes Cote d’Azur” (“TANUKI”, emergence 2016 grant). We would like to thank Drs. Yves Collette and R. Castellano (TrGET, preclinical assay platform, CRCM, INSERM U1068-Institut Paoli-Calmette, Marseille, France) for in vivo experiments. We thank Prof. Jain for generously providing the Surflex package, and Molsoft LLC for providing ICM suite
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These authors contributed equally to this work.