The Influence of Peptide Linkers on Functional Properties of Hybrid Structures with the Selective pH-Dependent Binding to Cancer Cells

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Abstract

Most of the modern cancer therapies are non-specific and have adverse side effects on the body. Nowadays, targeted cancer therapies are being developed, in particular using targeting peptides that selectively bind to cancer cells. The aim of the present work is to explore the prospects of using a peptide pHLIP that binds to cancer cells at decreased pH values, as a part of recombinant protein-peptide construct for cancer diagnosis and targeted therapy. Hybrid structures based on the fluorescent protein EGFP and a linker sequence connecting fluorescent protein to two different pHLIP variants were obtained. The effect of different linkers on the pH-dependent binding of the constructs to cells, as well as on the efficiency of EGFP chromophore synthesis within the hybrid construct was investigated.

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About the authors

A. Yu. Frolova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Author for correspondence.
Email: anastasiya_frolova_box@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

A. A. Pakhomov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: anastasiya_frolova_box@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

S. M. Deyev

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: anastasiya_frolova_box@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

V. I. Martynov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: anastasiya_frolova_box@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

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2. Fig. 1. Simplified scheme (a) and spatial model (b) of hybrid constructs. Hexahistidine tag is shown in blue, fluorescent protein – in green, linker – in purple, pHLIP – in orange.

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3. Fig. 2. Normalized absorption spectra of EGFP/pHLIP and EGFP constructs.

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4. Fig. 3. pH-dependent binding to HeLa cells of EGFP/pHLIPwt and EGFP/pHLIPvar3 constructs with GS and IEGRCGS linkers at different pH values. Median fluorescence intensity (MFI) values ​​are normalized to EGFP-IEGFCGS-pHLIPwt (the most efficient binding construct) and corrected for the degree of chromophore maturation.

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5. Fig. 4. pH-dependent binding of EGFP/pHLIPwt to HeLa cells determined by confocal microscopy. From left to right: green (EGFP, excitation 488 nm, emission 500–550 nm) and blue (Hoechst 33258, excitation 405 nm, emission 420–470 nm) fluorescence images, transmitted light, and their overlay. Scale bar 20 μm.

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