A Small-Molecule Inhibitor of Gut Bacterial Urease Protects the Host from Liver Injury

Abstract

Hyperammonemia is characterized by the accumulation of ammonia within the bloodstream upon liver injury. Left untreated, hyperammonemia contributes to conditions such as hepatic encephalopathy that have high rates of patient morbidity and mortality. Previous studies have identified gut bacterial urease, an enzyme that converts urea into ammonia, as a major contributor to systemic ammonia levels. Here, we demonstrate use of benurestat, a clinical candidate used against ureolytic organisms in encrusted uropathy, to inhibit urease activity in gut bacteria. Benurestat inhibits ammonia production by urease-encoding gut bacteria and is effective against individual microbes and complex gut microbiota. When administered to conventional mice with liver injury induced by thioacetamide exposure, benurestat reduced gut and serum ammonia levels and rescued 100% of mice from lethal acute liver injury. Overall, this study provides an important proof-of-concept for modulating host ammonia levels and microbiota-driven risks for hyperammonemia with gut microbiota-targeted small-molecule inhibitors.

1

Inhibiting gut bacterial urease activity is a promising strategy for modulating host ammonia levels. (A) Urease enzymes convert urea into ammonia and carbon dioxide. (B) Structure of the Bacillus pasteurii urease (PDB: 4UBP) highlighting its bis-nickel metallocofactor bound to the clinically used urease inhibitor acetohydroxamic acid. Ni atoms represented as blue spheres. (C) Strategies for manipulation of urease activity in the gut microbiota including previously successful fecal microbiota transplant and proposed use of small-molecule urease inhibitors.

2

Acetohydroxamic acid (AHA) and benurestat inhibit ammonia production by urease-encoding gut bacteria. (A) Chemical structures of the FDA-approved urease inhibitor AHA and the clinical candidate benurestat. The key hydroxamate pharmacophore responsible for inhibitor activity is labeled in green. (B) Relative quantification of inhibition of ammonia production by cultured bacterial isolates grown with 8 mM urea and treated with AHA. Activity was normalized to a no inhibitor control and this experiment was performed in biological triplicate. (C) Relative quantification of ammonia production by cultured bacterial isolates grown with 8 mM urea and treated with benurestat. This experiment was performed in biological triplicate. (D) Benurestat inhibits urease activity in mouse fecal suspensions ex vivo. Error bars represent mean ± standard deviation of biological triplicate experiments for each concentration.

3

Benurestat treatment reduces fecal and serum ammonia levels in mice. (A) Study of conventional mice treated with vehicle or benurestat (administered 2 times a day for 3 days via oral gavage) and euthanized (labeled as X) on day 4. (B) Quantification of ammonia concentrations in fecal and serum samples collected from vehicle or benurestat treated conventional mice. (C) Study of a mouse model of liver injury (injection with 100 mg/kg TAA) treated with vehicle or benurestat (administered 2 times a day for 3 days via oral gavage) and euthanized on day 4. (D) Quantification of ammonia concentrations in fecal and serum samples collected from vehicle or benurestat treated conventional mice induced with hyperammonemia. P values were calculated using an unpaired t test to determine the significance between the individual groups. (**, p < 0.01; ****, p < 0.0001; ns, not statistically significant).

4

Benurestat treatment rescues mice from a lethal dose of TAA. (A) Schematic of benurestat treatment in a mouse model of acute liver injury. 7-week old Swiss Webster conventional mice were treated with 200 mg/kg of TAA on day 0 and given either vehicle or 100 mg/kg of benurestat 2 times a day over 3 days. (B) Survival analysis of vehicle or benurestat treatment in a model of acute liver injury.