GALA3-containing modular nanotransporters are capable of delivering Keap1 monobody to target cells and inhibiting the formation of reactive oxygen species in the cells

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Abstract

In the previously created modular nanotransporter (MNT) capable of delivering a monobody to Keap1 into the cytosol, the translocation domain of diphtheria toxin (DTox) was replaced by the endosomolytic peptide GALA3. It was found that this substitution more than doubles the lifetime of MNT in the blood. Using confocal microscopy, it was shown that MNT with GALA3 was internalized into AML12 cells mainly due to binding to the epidermal growth factor receptor, and is also able to exit from endosomes into the cytosol. Using cellular thermal shift assay, it was shown that MNT with GALA3 and MNT with DTox are equally effective in disrupting the formation of the Nrf2 complex with Keap1, which led to similar protection of AML12 cells from the action of hydrogen peroxide. The obtained results allow not only to optimize the systemic use of MNT, but can also serve as a basis for creating agents aimed at treating diseases associated with oxidative stress.

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About the authors

Y. V. Khramtsov

Institute of Gene Biology, RAS

Email: alsobolev@yandex.ru
Russian Federation, Moscow

E. S. Bunin

Institute of Gene Biology, RAS

Email: alsobolev@yandex.ru
Russian Federation, Moscow

A. V. Ulasov

Institute of Gene Biology, RAS

Email: alsobolev@yandex.ru
Russian Federation, Moscow

T. N. Lupanova

Institute of Gene Biology, RAS

Email: alsobolev@yandex.ru
Russian Federation, Moscow

G. P. Georgiev

Institute of Gene Biology, RAS

Email: alsobolev@yandex.ru

Corresponding Member of the RAS

Russian Federation, Moscow

A. S. Sobolev

Institute of Gene Biology, RAS; Lomonosov Moscow State University

Author for correspondence.
Email: alsobolev@yandex.ru

Academician of the RAS

Russian Federation, Moscow; Moscow

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2. Fig. 1. Schematic structure of the MNT used in this work. The description of the modules is given in the text.

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3. Fig. 2. Evaluation of the time dependence of the concentration of MNTDk-AF488 and MNTGk-AF488 in the blood of BALB/c mice based on the AF488 fluorescence intensity. The fluorescence of MNTDk-AF488 and MNTGk-AF488 immediately after intravenous administration was taken as 100%. Mean values ​​± standard error are shown (n = 4–5).

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4. Fig. 3. Dependence of the proportion of free Nrf2 at physiological temperatures (37°C) on the incubation time of 500 nM MNTG or MNTD with AML12 cells. Data are presented as mean values ​​± standard deviation.

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5. Fig. 4. Effect of preincubation of AML12 cells with 500 nM MNTG or MNTD for 5 min on cDCF fluorescence induced by adding 10 μM hydrogen peroxide to the cells for 15 min. The time between MNT addition and fluorescence measurement is indicated. Data are presented as mean ± SEM.

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