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

Founding: 2018/31/B/NZ6/02017

Budget: 1 629 000 PLN

PI: Artur Mucha

Team: Katarzyna Macegoniuk, Agnieszka Grabowiecka, Michał Talma, Saurabh Loharch, Marta Maślanka, Wojciech Tabor, Łukasz Berlicki

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.  evaluated.

Results

Urease is an enzyme that hydrolyzes urea to ammonia and carbon dioxide. The enzyme occurs commonly in plants, bacteria, and fungi and plays a key role in the global nitrogen cycle. At the same time, urease is the etiological factor for infections associated with pathogens such as Proteus mirabilis, Klebsiella aerogenes, Helicobacter pylori, and Cryptococcus neoformans, as it facilitates the survival of microorganisms in the environment and colonization of the host organism. Therefore, specific inhibition of ureolytic activity is a perspective approach to alter virulence and introduce smart therapies, e.g., in combination with the use of classic antibiotics. 

The aim of the project was to develop innovative tools to control urease activity. These were low-molecular weight heteroorganic compounds that combined chemical reactivity toward the SH group of the characteristic cysteine residue of the protein with complexing properties toward nickel ions present in the active site. 

In order to achieve the assumed goals, synthetic procedures leading to structurally complex, multifunctional compounds, mainly phosphonic and phosphinic acids and esters, containing a catechol or organoselenium fragment reactive to thiols, were developed. Multiple target structures, not described in the literature, were synthesized, among them, very effective inhibitors of the model Sporosarcina pasteurii urease. These organoselenium compounds showed unique picomolar values of inhibition constants (K i < 20 pM). A series of inhibitors of urease that was isolated from C. neoformans, the fungus responsible for cryptococcosis, was also obtained and systematically characterized for the first time. Numerous compounds excellently controlled ureolysis in whole cells of pathogenic microorganisms: Gram-negative bacteria P. mirabilis (IC 50 < 100 nM) and H. pylori (IC 50 < 60 nM), as well as yeast C. neoformans (IC 50 < 200 nM). The supporting studies determined the influence of selected products on the homeostasis of microbial cells, their redox status, protein biosynthesis, and the impact on the growth and viability of the bacterial biofilm. It was also evidenced that the developed inhibitors showed satisfactory chemical stability and low cytotoxicity to mammalian cells. On the basis of X-ray structural studies and molecular modeling, mechanisms of their action at the molecular level were proposed. In the case of organoselenium compounds and S. pasteurii urease, a unique product of covalent diselenation of the cysteine residue, crucial for the conformational changes of the enzyme during substrate hydrolysis, was found. The characteristics achieved allow us to propose the inhibitors developed as active and specific components in prospective combination antimicrobial therapies. A synergistic effect of co-administration with commonly used antibiotics may reduce the growing problem of drug resistance in microorganisms.

Publications

1. Aikaterini Pagoni, Agnieszka Grabowiecka, Wojciech Tabor, Artur Mucha, Stamatia Vassiliou, Łukasz Berlicki, Covalent inhibition of bacterial urease by bifunctional catechol-based phosphonates and phosphinates. Journal of Medicinal Chemistry. 2021, vol. 64, nr 1, pp. 404-416.
2. Stamatia Vassiliou, Aikaterini Pagoni, Ewelina Węglarz-Tomczak, Michał Talma, Wojciech Tabor, Agnieszka Grabowiecka, Łukasz Berlicki, Artur Mucha, Phosphinic acid-based enzyme inhibitors. Phosphorus, Sulfur, and Silicon and the Related Elements. 2022, vol. 197, nr 5/6, pp. 451-456.
3. Saurabh Loharch, Łukasz Berlicki, Rational development of bacterial ureases inhibitors. Chemical Record. 2022, vol. 22, nr 8, art. e202200026, pp. 1-15.
4. Marta Maślanka, Artur Mucha, Antibacterial activity of ebselen. International Journal of Molecular Sciences. 2023, vol. 24, nr 2, art. 1610, pp. 1-15.
5. Katarzyna M. Macegoniuk, Wojciech Tabor, Luca Mazzei, Michele Cianci, Mirosław Giurg, Kamila Olech, Małgorzata Burda-Grabowska, Rafał Kaleta, Agnieszka Grabowiecka, Artur Mucha, Stefano Ciurli, Łukasz Berlicki, Optimized ebselen-based inhibitors of bacterial ureases with nontypical mode of action. Journal of Medicinal Chemistry. 2023, vol. 66, nr 3, pp. 2054-2063.
6. Marta Maślanka, Wojciech Tabor, Paweł Krzyżek, Agnieszka Grabowiecka, Łukasz Berlicki, Artur Mucha, Inhibitory activity of catecholic phosphonic and phosphinic acids against Helicobacter pylori ureolysis. European Journal of Medicinal Chemistry. 2023, vol. 257, art. 115528, pp. 1-11.

Conference Communications

1. Wojciech Tabor, Aikaterini Pagoni, Agnieszka Grabowiecka, Artur Mucha, Stamatia Vassiliou, Łukasz Berlicki "Covalent inhibition of bacterial urease by bifunctional catechol-based phosphonates and phosphinates", International Symposium on Medicinal Chemistry (EFMC-ISMC) and the EFMC Young Medicinal Chemists' Symposium of European Federation for Medicinal Chemistry and Chemical Biology, 09.09.2020
2. Marta Maślanka, Artur Mucha "Synthesis of multifunctional organophosphorus compounds as a route to new urease inhibitors with dual mechanism of interaction with the enzyme", ChemBiotIC (Chemistry & Biotechnology International Conference), Wrocław, 24-25.06.2021
3. Artur Mucha "Phosphinic acid-based enzyme inhibitors", 3rd International Conference on Phosphorus Chemistry, Częstochowa, 5-9.07.2021
4. Marta Maślanka, Artur Mucha "Phosphorus derivatives of catechol and ebselen as an innovative approach to urease inhibitors", 23rd International Conference on Phosphorus Chemistry, Częstochowa, 5-9.07.2021
5. Saurabh Loharch, Wojciech Tabor, Łukasz Berlicki "Organoselenium based compounds as potent inhibitors of bacterial urease", XXVI EFMC International Symposium on Medicinal Chemistry, 29.08-02.09.2021
6. Marta Maślanka, Artur Mucha "Inhibitory ureazy o hybrydowym mechanizmie działania", XIV Kopernikańskie Seminarium Doktoranckie, Toruń, 20-22.09.2021
7. Marta Maślanka, Artur Mucha "Synteza oraz charakterystyka inhibitorów ureazy o dualnym mechanizmie oddziaływania z enzymem", XIV Kopernikańskie Seminarium Doktoranckie, Toruń, 20-22.09.2021
8. Wojciech Tabor, Paweł Krzyżek, Urszula Nawrot, Artur Mucha, Łukasz Berlicki, Agnieszka Grabowiecka "Ebselen based inhibitors of urease affect the viability of Cryptococcus neoformans and Helicobacter pylori", ACCORD Interdisciplinary Conference on Drug Sciences, Warszawa, 26-28.05.2022
9. Marta Maślanka, Wojciech Tabor, Agnieszka Grabowiecka, Artur Mucha „Synteza i aktywność biologiczna nowych pochodnych benzizoselenazol-3(2H)-onu wobec ureazy bakteryjnej”, „Na pograniczu chemii i biologii” - XVIII Ogólnopolskie Seminarium dla Doktorantów i Studentów, Smardzewice, 12-15.06.2022
10. Marta Maślanka, Wojciech Tabor, Agnieszka Grabowiecka, Artur Mucha „Nowe fosforoorganiczne inhibitory ureazy – synteza, stabilność i aktywność biologiczna”, XV Kopernikańskie Seminarium Doktoranckie, Toruń, 20-22.06.2022
11. Marta Maślanka, Wojciech Tabor, Agnieszka Grabowiecka, Artur Mucha, „Fosforoorganiczne pochodne benzizoselenazolu-3(2H)-onu i katecholu jako inhibitory ureazy o hybrydowym mechanizmie działania”, 64. Zjazd Naukowy Polskiego Towarzystwa Chemicznego, Lublin, 11-16.09.2022
12. Wojciech Tabor, Katarzyna Macegoniuk, Agnieszka Grabowiecka, Stefano Ciurli, Luca Mazzei, Artur Mucha, „Ebselen derivatives as picomolar inhibitors of bacterial ureases”, ESCMID/ASM Conference on Drug Development to Meet the Challenge of Antimicrobial Resistance, Dublin, Irlandia, 4-7.10.2022
13. Agnieszka Grabowiecka, Wojciech Tabor, Paweł Krzyżek, Urszula Nawrot, Artur Mucha, Łukasz Berlicki „Organoselenium inhibitors reduce ureolysis and viability of C. neoformans and H. pylori”, ESCMID/ASM Conference on Drug Development to Meet the Challenge of Antimicrobial Resistance, Dublin, Irlandia, 4-7.10.2022
14. Marta Maślanka, Wojciech Tabor, Agnieszka Grabowiecka, Artur Mucha „Katecholowe fosfoniany i fosfiniany – synteza i właściwości antyureolityczne wobec Helicobacter pylori”, „Na pograniczu chemii i biologii” - XIX Ogólnopolskie Seminarium dla Doktorantów i Studentów, Trzebieszowice, 04-07.06.2023.
15. Artur Mucha, Agnieszka Grabowiecka, Katarzyna Macegoniuk, Marta Maślanka, Wojciech Tabor, Mirosław Giurg, Luca Mazzei, Stefano Ciurli, Łukasz Berlicki „Urease inhibition by cysteine modification” 28th American Peptide Symposium, Scottsdale, USA, 24-29.06.2013
16. Wojciech Tabor, Marta Maślanka, Agnieszka Grabowiecka, Urszula Nawrot, Paweł Krzyżek, Artur Mucha, Łukasz Berlicki "Ebselen-based compounds act as covalent inhibitors of microbial ureases in nanomolar concentrations", 23rd Tetrahedron Symposium, Goteborg, Szwecja, 27-30.06.2023
17. Agnieszka Grabowiecka, Marta Maślanka, Wojciech Tabor, Paweł Krzyżek, Łukasz Berlicki, Artur Mucha "Catecholic phosphonic and phosphinic acid inhibitors for the control of ureolytic microbial pathogens", 23rd Tetrahedron Symposium, Goteborg, Szwecja, 27-30.06.2023
18. Marta Maślanka, Wojciech Tabor, Łukasz Berlicki, Agnieszka Grabowiecka, Artur Mucha „Synthesis of phosphonic acid-functionalized benzisoselenazolones and catechols, and their antiureolityc activity”, 22nd European Symposium on Organic Chemistry, Ghent, Belgia, July 9-13.07.2023