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

Elżbieta Katarzyna Jagusztyn-Krynicka,Marija Luić,Agnieszka Bzowska,Verica Aleksić Sabo,Saša Kazazić,Petar Knežević,Marta Ilona Wojtyś,Radosław Jaźwiec,Ivana Leščić Ašler

doi:10.1007/s00253-021-11510-9

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.Key points• A method for assessing H. pylori cells penetration by drug candidates is described.• Three alternative detection assays that complement each other can be used.• The method may be adapted for other bacteria as well.

Highlights

The gram-negative microaerophilic bacterium Helicobacter pylori is a common pathogen affecting about 50% of the human population worldwide (Kamboj et al 2017)
We have previously shown that formycin A, the analogue of adenosine with C–C bond between the base and the pentose (Fig. 2), is an efficient inhibitor of purine nucleoside phosphorylase (PNP) from the H. pylori 26695 strain
Formycin uptake was already quantified by the liquid chromatography (LC)–mass spectrometry (MS) and UV absorption, we decided to check if inhibition of PNP could be used as a detection method as well

Summary

Introduction

The gram-negative microaerophilic bacterium Helicobacter pylori is a common pathogen affecting about 50% of the human population worldwide (Kamboj et al 2017). With the growing resistance of H. pylori to antimicrobial agents, especially to clarithromycin and metronidazole, in the triple therapy, these antibiotics are often replaced by other drugs, e.g., by levofloxacin, sitafloxacin, or rifabutin (Thung et al 2016; Suzuki and Mori 2018) Even with these modifications, standard triple therapy is no longer effective in most countries (Huang et al 2017). The 2016 Maastricht V/Florence Consensus Report (Malfertheiner et al 2017) and the 2016 Toronto Consensus (Fallone et al 2016) recommended a bismuth quadruple therapy as a first-line choice, especially when resistance to clarithromycin occurs This treatment includes a proton pump inhibitor, bismuth, metronidazole, and tetracycline; is well tolerated by patients; and shows effectiveness even against in vitro metronidazole-resistant strains. In the hybrid or reverse hybrid therapy, a proton pump inhibitor and amoxicillin are administrated for 10–14 days, while metronidazole and clarithromycin only for the first and the second half, respectively (Huang et al 2017)

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Conclusion