Rational drug design to identify a new urease inhibitor to treat urinary catheter blockage.

Heylen R1, Cusick N1, Jenkins T1

Research Type

Pure and Applied Science / Translational

Abstract Category

Continence Care Products / Devices / Technologies

Abstract 514
The Best of the Rest in Science
Scientific Podium Short Oral Session 33
Saturday 10th September 2022
12:30 - 12:37
Hall G1
Biochemistry Incontinence Infection, Urinary Tract
1. University of Bath
In-Person
Presenter
R

Rachel A. Heylen

Links

Abstract

Hypothesis / aims of study
Urinary catheter blockage is a frequent problem for long-term catheterized patients, who are often referred to as ‘blockers’. Encrustation of the catheter is generally caused by urease-positive microorganisms, such as Proteus mirabilis [1]. Urease metabolizes urea to ammonia, resulting in alkaline urine, causing precipitation of struvite and apatite crystals, and these encrustations then lead to catheter blockage [1]. Long-term catheterized patients often have asymptomatic infections present within the bladder, therefore the prescription of antibiotics is generally discouraged. Here, we examine the use of urease inhibitors as an anti-virulence mechanism to prevent catheter blockage. Acetohydroxamic acid (AHA) is a urease-inhibitor and is prescribed for the treatment of frequent catheter blockage, it is not licensed in the UK or EU and is rarely used owing to its toxic side effect. Urease inhibitors have been well-examined in the literature, often for use in agriculture. However, other than AHA, none have been licensed as a therapeutic for catheter blockage. Our study aims to identify new and improved urease inhibitors which can be prescribed to long-term urinary catheter users who are frequent blockers.
Study design, materials and methods
Computational modelling docking experiments were used to dock small molecules onto the target-site using the crystal structure of the enzyme, urease from Sporosarcina pasteruii (PDB: 4UBP) (Fig.1). Docking was carried out using Cresset Flare software. Compounds were designed around the structures of known inhibitors: 2-mercaptoacetamide (2-MA), thiourea, and the natural flavonoid, quercetin [2,3]. Analysis of the docking score and the contacts of the compound to the urease crystal structure were used to rank the compounds. Compounds were filtered by Lipinski’s rules and the ability to purchase the compounds, the potency of the compound was assessed in vitro using the Berthelot assay against purified Canavalia ensiformis urease and a whole-cell assay of P. mirabilis.
Results
From the virtual screening of high-ranking compounds, A5, A6, and A11, were identified and purchased. The ability of these compounds to inhibit urease was assessed using in vitro urease activity assays, with comparisons to known inhibitors (Fig. 2). Inhibitory concentration 50 (IC50) describes the ability of the compound to reduce the enzyme activity by 50%, IC50 can be used to determine the potency of the compounds.
Interpretation of results
Analysis of the IC50 showed that A11 appears to be 500-fold more potent than AHA against purified C. ensiformis urease (Fig. 2A). The whole-cell P. mirabilis assay informs on the ability of compounds to cross the bacterial membrane and access the intracellular urease. Results appear to show A11 to be 50-fold more potent than AHA (Fig. 2B).
Concluding message
We have demonstrated the small molecule, A11, as a potential therapeutic to treat recurrent catheter blockages. Future work is required to assess A11’s ability to prevent urinary catheter blockage using in vitro models of a catheterized urinary tract.
Figure 1 Figure 1: 2-mercaptoacetamide (2-MA) docked into the crystal structure of urease from Sporosarcina pasteruii. 2-MA’s chelate the nickel ions in the active site, as well as making additional contacts with amino acids, shown by dotted lines, and measured i
Figure 2 Figure 2A: Determination of IC50 against purified C. ensiformis urease. B: Determination of IC¬50 against whole-cell P. mirabilis, for the following compounds: AHA, 2-MA, Quercetin, A11, A5 and A6. Three independent biological repeats were carried out, er
References
  1. Stickler, D.J. Bacterial biofilms in patients with indwelling urinary catheters. Nat. Clin. Pract. Urol. 2008, 5, doi:10.1038/ncpuro1231.
  2. Milo, S.; Heylen, R.A.; Glancy, J.; Williams, G.T.; Patenall, B.L.; Hathaway, H.J.; Thet, N.T.; Allinson, S.L.; Laabei, M.; Jenkins, A.T.A. A small-molecular inhibitor against Proteus mirabilis urease to treat catheter-associated urinary tract infections. Sci. Rep. 2021, 11, 1–15, doi:10.1038/s41598-021-83257-2.
  3. Rego, Y.F.; Queiroz, M.P.; Brito, T.O.; Carvalho, P.G.; Queiroz, V.T. De; Fátima, Â. De; Macedo, F. A review on the development of urease inhibitors as antimicrobial agents against pathogenic bacteria q. J. Adv. Res. 2018, 13, 69–100, doi:10.1016/j.jare.2018.05.003.
Disclosures
Funding PhD funded by: Annette Trust and EPSRC Clinical Trial No Subjects None
Citation

Continence 2S2 (2022) 100465
DOI: 10.1016/j.cont.2022.100465

16/02/2024 19:28:04