A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by H. pylori

Wojtyś, Marta Ilona; Jaźwiec, Radosław; Kazazić, Saša; Leščić Ašler, Ivana; Knežević, Petar; Aleksić Sabo, Verica; Luić, Marija; Jagusztyn- Krynicka, Elżbieta Katarzyna; Bzowska, Agnieszka (2021) A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by H. pylori. Applied microbiology and biotechnology, 105 . pp. 7949-7967. ISSN 0175-7598

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Abstract

Due to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium’s recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori — two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori.

Item Type:	Article
Uncontrolled Keywords:	Helicobacter pylori ; Cells penetration by drug candidates ; Salvage pathway enzymes ; Formycin ; Hadacidin
Subjects:	NATURAL SCIENCES > Biology
Divisions:	Division of Physical Chemistry
Projects:	Project title Project leader Project code Project type Enzimi purinskog reciklirajućeg ciklusa iz Helicobacter pylori i Escherichie coli Luić, Marija IP-2013-11-7423 HRZZ
Depositing User:	Saša Kazazić
Date Deposited:	15 Nov 2021 09:49
URI:	http://fulir.irb.hr/id/eprint/6706
DOI:	10.1007/s00253-021-11510-9

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