Dual-action mode inhibitors of urease and their anti-virulent activity against Helicobacter pylori and Cryptococcus neoformans

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Principal Investigator: Artur Mucha

Project financed by National Science Centre, no 2018/31/B/NZ6/02017

Start date: September 2nd, 2019

Project duration: 36 months

Budget: 1 629 000 PLN

Abstract

Urease is an amidohydrolase involved in the degradation of urea to ammonia and carbamate, and then to carbon dioxide. The scientific developments on urease predestinate the enzyme to hold a special position among milestones of biochemistry. In 1926 it was the first ever enzyme crystallized and identified as a protein (Nobel Prize for James Sumner, 1946) and then demonstrated as nickel-dependent. Urease is also characteristic by an exceptional rate of hydrolysis enhancement (1014-fold). The functions of the hydrolase involve participation in global nitrogen circulation as it is present in plants, fungi and bacteria, such as Proteus mirabilis, Klebsiella aerogenes and Helicobacter pylori. This occurrence determines its significance to humans, in particular in public health context. The activity of bacterial ureases is related with colonization by microorganisms of the urinary and gastrointestinal tracts, and persistent infections. Accordingly, H. pylori is the main etiological agent of peptic ulcers in the stomach by producing the alkaline environment which facilitates the colonization of the mucosal lining (Nobel Prize for Barry Marshall, 2005).

Inhibition of ureolytic activity of microorganisms is an attractive approach to intelligent antimicrobial therapies based on specific interactions with the enzymatic target at a molecular level. Thus, tremendous effort are dedicated to develop the agents that affect the urease activity in order to control urease-dependent bacterial and fungal infections. Our research group is well-experienced in computer-aided design, chemical syntheses, kinetic characteristics and structure-activity analysis of such inhibitors. Organophosphorus compounds (phosphonic and phosphinic acid), mimicking the substrate in the transition state of the enzymatic process, have been the most intensively studied. They were found highly active towards ureases from Sporosarcina pasteurii, P. mirabilis and H. pylori, avoiding hydrolytical lability of phosphoramidates, the canonical phosphorus-based transition state analog inhibitors. Recently, a remarkable level of inhibition has been also achieved by cysteine-reactive compounds that target the thiol group of Cys322 residue forming a movable flap of the enzyme active site.

Following these developments we wish to envisage within the current project a concept of urease inhibition by dually acting compounds, constructed to combine the reactivity toward the SH of the cysteine with potent nickel-binding properties. An extensive set of new structures is planned to be obtained, their affinity to model and pathogenic ureases measured and the mode of action confirmed. Importantly, selected virulent bacterial and fungal strains will be targeted in whole-cell experiments. The results are expected to bring high-affinity compounds that will be validated as potent and selective agents targeted against Helicobacter infections and cryptococcosis.

Publications

  • Vassiliou, S.; Pagoni, A.; Węglarz-Tomczak, E.; Talma, M.; Tabor, W.; Grabowiecka, A.; Berlicki, Ł.; Mucha, A., Phosphinic acid-based enzyme inhibitors Phos. Sulph. Rel. El. 2022, 197, 451. View at Publisher
    Abstract

    The phosphinic acid functionality has emerged as an invaluable scaffold for the construction of biologically active compounds, in particular enzyme inhibitors. This article presents two examples of recent achievements in the preparation and application of phosphinic acids as ligands for hydrolases. Synthetic approaches to a dipeptide analog inhibitor of metalloaminopeptidases and a catechol-based inhibitor of urease are shown. The activity and mode of binding of phosphinic compounds to enzymes are further discussed..
  • Loharch, S.; Berlicki, Ł., Rational Development of Bacterial Ureases Inhibitors Chem. Rec. 2022, accepted. View at Publisher
    Abstract

    Urease, an enzyme that catalyzes the hydrolysis of urea, is a virulence factor of various pathogenic bacteria. In particular, Helicobacter pylori, that colonizes the digestive tract and Proteus spp., that can infect the urinary tract, are related to urease activity. Therefore, urease inhibitors are considered as potential therapeutics against these infections. This review describes current knowledge of the structures, activity, and biological importance of bacterial ureases. Moreover, the structure-based design of several classes of bacterial urease inhibitors is presented and discussed. Phosphinic and phosphonic acids were applied as transition-state analogues, while Michael acceptors and ebselen derivatives were applied as covalent binders of cysteine residue. This review incorporates bacterial urease inhibitors from literature published between 2008 and 2021..
  • Pagoni, A.; Grabowiecka, A.; Tabor, W.; Mucha, A.; Vassiliou, S.; Berlicki, Ł., Covalent Inhibition of Bacterial Urease by Bifunctional Catechol-Based Phosphonates and Phosphinates J. Med. Chem. 2021, 64, 404. View at Publisher
    Abstract

    In this study, a new class of bifunctional inhibitors of bacterial ureases, important molecular targets for antimicrobial therapies, was developed. The structures of the inhibitors consist of a combination of a phosphonate or (2-carboxyethyl)phosphinate functionality with a catechol-based fragment, which are designed for complexation of the catalytic nickel ions and covalent bonding with the thiol group of Cys322, respectively. Compounds with three types of frameworks, including β-3,4-dihydroxyphenyl-, α-3,4-dihydroxybenzyl-, and α-3,4-dihydroxybenzylidene-substituted derivatives, exhibited complex and varying structure-dependent kinetics of inhibition. Among irreversible binders, methyl β-(3,4-dihydroxyphenyl)-β-(2-carboxyethyl)phosphorylpropionate was observed to be a remarkably reactive inhibitor of Sporosarcina pasteurii urease (kinact/KI = 10 420 s–1 M–1). The high potential of this group of compounds was also confirmed in Proteus mirabilis whole-cell-based inhibition assays. Some compounds followed slow-binding and reversible kinetics, e.g., methyl β-(3,4-dihydroxyphenyl)-β-phosphonopropionate, with Ki* = 0.13 μM, and an atypical low dissociation rate (residence time τ = 205 min). .