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Domingos Geraldo Jorge
Identification of inhibitors of the human hv1 proton channel

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P40

Identification of inhibitors of the human hv1 proton channel

Geraldo Domingos1, Adam Feher1, Eva Korpos 1,2, Tibor G. Szanto1, Martina Piga3, Tihomir Tomasic3, Nace Zidar3, Adrienn Gyongyosi4, Judit Kallai4, Arpad Lanyi4, Ferenc Papp1, Katinka Gyuris1, Zoltan Varga1

1 Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Hungary,

2 MTA-DE Cell Biology and Signalling Research Group, Faculty of Medicine, University of Debrecen, Hungary

3 Department of Pharmaceutical Chemistry, University of Ljubljana, Slovenia, 4 Department of Immunology, Faculty of Medicine, University of Debrecen, Hungary

The human voltage-gated proton channel (hHV1) plays an important role in immune system and cancer cells being involved in functions such as proliferation, migration, and oxidative burst. HV1 does not have a conventional ion-conducting pore, the conduction occurs through the voltage-sensing domain. This difference may be the reason for the lack of selective hHV1 inhibitors. Currently, 5-chloro-2-guanidinobenzimidazole (ClGBI) is the most widely used inhibitor of HV1 but it has low selectivity for the channel. This could lead to misinterpretation of functional assays addressing the role of HV1 with the use of ClGBI. Thus, our aim was to find potent and more selective inhibitors for hHV1, which could be useful research tools and serve as lead molecules for the development of drug molecules targeting HV1.

We used patch-clamp to test the affinity and selectivity of potential inhibitors of HV1 on CHO and HEK cells expressing hHV1 and other channels. Seven “hit” molecules were identified among the NZ family of compounds of which NZ-13 has the best selectivity profile.

The widely-used HV1 inhibitor ClGBI blocks various ion channels and therefore is not a selective HV1 blocker. This must be considered in functional tests investigating the role of HV1 in healthy and pathological conditions.

We have identified a new family of hHV1 inhibitors, which have comparable affinities for the channel to ClGBI.

Most NZ molecules have low selectivity for hHV1, but NZ-13, the one with the highest selectivity, may be better suited for functional tests than ClGBI as it inhibits T cell proliferation less.

Papp Ferenc
A synthetic flavonoid derivate modulates the fluorescent signal of voltage-gated proton channels

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P49

A synthetic flavonoid derivate modulates the fluorescent signal of voltage-gated proton channels

Zoltán Pethő1,2, Gilman E. S. Toombes, Dávid Pajtás1, Martina Piga3, Zsuzsanna Magyar4, Nace Zidar3, György Panyi1, Zoltán Varga1 and Ferenc Papp1

1 Department of Biophysics and Cell Biology,Faculty of Medicine, University of Debrecen,

2 Institut für Physiologie II, Münster, Germany

3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana

4 Department of Physiology, Faculty of Medicine, University of Debrecen

The voltage-sensing domain (VSD) of voltage-gated proton channel (Hv1) serves as a pore for protons as well as a voltage sensor, which makes this channel unique among voltage-gated channels. Natural flavonoids, which are widely distributed and act as chemical messengers and physiological regulators in plants, modulate the function of some voltage-gated ion channels (EAG1, HCN2. etc.) in animal cells. We have designed synthetic flavonoid derivatives to inhibit the current of Hv1. We produced and tested tens of flavonoid derivatives on Ciona intestinalis Hv1 using the voltage-clamp fluorometry (VCF). The most potent compound, molecule #109, changed the originally negative VCF signal to positive and altered the biphasic VCF signal shape to monophasic. Also, this molecule caused a rightward shift in the conductance-voltage relationship in a concentration dependent manner. This flavonoid derivative quenched the TAMRA-MTS fluorescence in cuvette and on frog oocytes decreasing the baseline fluorescence, independently the oocyte expressed Hv1 or not. Furthermore, #109 could stain the membrane of HEK cells. These results indicate that #109 binds not directly to CiHv1 but to the cell membrane and in this way, it indirectly modifies the gating of Hv1 and the VCF signal, as a strong quenching molecule in close vicinity of the fluorophore. The latter was confirmed by our model calculations, in which we assumed that molecule #109, as a strong quencher, is embedded in the cell membrane close to TAMRA and during the conformational change due to depolarization, TAMRA is continuously moving away from this strong quencher molecule. Therefore, the VCF signal becomes a continuously increasing fluorescence change from the originally complex shape, which was overall a decreasing fluorescence change.

Acknowledgment

This work was supported by OTKA Bridging Fund 1G3DBKB0BFPF247 (FP); by János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00355/21/8) (FP); by the ÚNKP-21-5-DE-460 New National Excellence Program of the Ministry for Innovation and Technology (FP); OTKA 132906 (ZV). This study was also funded by the Slovenian Research Agency (Grant No. P1-0208) (NZ).