Overview

Availability of physiologically relevant in vitro models is one of the most important requirements determining the efficiency of drug screening process. Nowadays 2D monolayer cell culture represents one of the most employed in vitro methodologies for drug development.

In contrast to 2D cell culture, 3D spheroids are able to mimic some features of solid tumors, including their spatial organization, physiological responses and drug resistance mechanisms. In the case of normal cells, multicellular spheroids are especially useful for the screening of neuroprotective agents, 3D bioprinting and tissue regeneration.

An application of cyclo-RGDfK(TPP) peptide containing a triphenylphosphonium moiety, represents a novel and highly reproducible one-step approach for multicellular spheroids formation.² The peptide efficiently induces self-assembly in different tumour cell lines. Moreover, it also initiates the formation of 3D spheroids in several cell lines of both normal and stem cells. Those spheroids are valuable tools for the design of 3D in vitro models, as well as for tissue regeneration experiments.

Synthesis of rgd peptides inducing multicellular spheroids formation

Maria Leko^a , Anna Pokhvoshcheva^a , Marina Dorosh^a , Roman Akasov^b , Elena Markvicheva^b , Markus Weishaupt^c , Thomas Bruckdorfer^c , Sergey Burov^a.

Introduction

Availability of physiologically relevant in vitro models is one of the most important requirements determining the efficiency of drug screening process. Nowadays 2D monolayer cell culture represents one of the most employed in vitro methodologies for drug development. However, this simplified model with lack of cell-cell interactions is not able to adequately mimic the in vivo response.

Sutherland et al.¹ were first to propose multicellular tumor spheroids as a 3D model of small solid tumors. In contrast to 2D cell culture, 3D spheroids are able to mimic some features of solid tumors, including their spatial organization, physiological responses and drug resistance mechanisms. In the case of normal cells, multicellular spheroids (MS) are especially useful for the screening of neuroprotective agents, 3D bioprinting and tissue regeneration.

Recently we demonstrated the practical utility of a cyclic RGD peptide containing a triphenylphosphonium (TPP) moiety, namely cyclo-RGDfK(TPP), in a novel and highly reproducible one-step approach for MS formation.²

Here we describe an optimized procedure for the synthesis of cyclo-RGDfK(TPP) and related peptides using our novel hydrazone resin.³

Results and Discussion

There are several schemes for the synthesis of cyclo-RGDfK peptide described in the literature^4-6, including different variants of solution-phase and on-resin cyclization. The suggested protocols imply cyclization of protected peptide precursor followed by subsequent removal of side-chain protecting groups.

Recently we described synthesis of inexpensive hydrazone resin and its practical utility for the preparation of peptide hydrazides³. These results prompted us to test possible advantages of azide method for the synthesis of cyclo-RGDfK(TPP) and related peptides. In order to compare the efficiency of various reaction protocols, the synthesis was performed in parallel on different polymer supports, namely Wang resin, MBH-Br resin and Fmoc-hydrazono-pyruvoyl-aminomethylpolystyrene resin (Fig. 4). In preliminary experiments it was shown that attachment of the TPP moiety to the cyclic peptide in solution complicated the purification of the final product. Therefore, it was achieved by selective deprotection of Lys(Mtt)-containing peptidyl resin, followed by acylation with 4-carboxybutyltriphenyl phosphonium bromide. Unexpectedly, using MBH resin the Mtt-deprotection with 1% TFA in DCM resulted in peptide cleavage from the polymer support, while application of a mixture of AcOH/TFE/DCM (1:2:7) was inefficient (Fig. 6).

Synthesis on Wang resin provided linear peptide in reasonable purity. Its cyclization with HCTU proceeded smoothly without significant formation of dimeric side product. However, subsequent removal of the OBzl group from the aspartic acid residue by catalytic hydrogenolysis showed very low efficiency. The final peptide deprotection requires two
successive treatments with HBr/TFA for 30 min and 1 h.

The best results were obtained using hydrazone resin. Selective removal of the Mtt group had no influence on the stability of the hydrazone bond. Peptide cleavage from the solid support and removal of all protecting groups proceeded in one step. Cyclization of the resulting peptide hydrazide was achieved in good yield and purity using the azide
method (Fig. 3).

These data evidence the practical utility of hydrazone resin for the synthesis of cyclic RGD peptides or their analogs containing cargo molecules. The biological experiments have shown that both linear and cyclic RGD peptides containing TPP moiety efficiently suppress platelet aggregation (Table 1.). Withal, cyclo-RGDfK(TPP) is able to generate MS formation from tumor, normal or stem cell lines of various origin (Fig. 1,5). This novel one-step reproducible method of spheroid formation can be applied in the design of efficient in vitro models for the screening of novel drugs and drug delivery systems (Fig. 2, 7, 8).

References

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3. Chelushkin P.S., Polyanichko K.V., Leko M.V., Dorosh M.Yu., Bruckdorfer T., Burov S.V. Tetrahedron Lett. 2015, 56, 619–622.
4. Haubner R., Gratias R., Diefenbach B., Goodman S.L., Jonczyk A., Kessler H. J. Am. Chem. Soc. 1996, 118, 7461–7472.
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6. McCusker C.F., Kocienski P.J., Boyle F.T., Schätzlein A.G. Bioorg. Med. Chem. Lett. 2002, 12, 547–549.