Potential repurposing of known drugs as potent bacterial β-glucuronidase inhibitors

Abstract

The active metabolite of the chemotherapeutic irinotecan, SN-38, is detoxified through glucuronidation and then excreted into the gastrointestinal tract. Intestinal bacteria convert the glucuronidated metabolite back to the toxic SN-38 using β-glucuronidase (GUS), resulting in debilitating diarrhea. Inhibiting GUS activity may relieve this side effect of irinotecan. In this study, we sought to determine whether any known drugs have GUS inhibitory activity. We screened a library of Food and Drug Administration-approved drugs with a cell-free biochemical enzyme assay using purified bacterial GUS. After triage, five drugs were confirmed to inhibit purified bacterial GUS. Three of these were the monoamine oxidase inhibitors nialamide, isocarboxazid, and phenelzine with average IC(50) values for inhibiting GUS of 71, 128, and 2300 nM, respectively. The tricyclic antidepressant amoxapine (IC(50) = 388 nM) and the antimalarial mefloquine (IC(50) = 1.2 µM) also had activity. Nialamide, isocarboxazid, and amoxapine had no significant activity against purified mammalian GUS but showed potent activity for inhibiting endogenous GUS activity in a cell-based assay using living intact Escherichia coli with average IC(50) values of 17, 336, and 119 nM, respectively. Thus, nialamide, isocarboxazid, and amoxapine have potential to be repurposed as therapeutics to reduce diarrhea associated with irinotecan chemotherapy and warrant further investigation for this use.

Fig. 1

Structures of studied drugs

Fig. 2. Potency determinations using an E. coli GUS enzyme assay.

Concentration response data for compounds was normalized to controls with and without enzyme and plotted as percent activity. Potency determinations of nialamide (●), isocarboxazid (▲), phenelzine (□), amoxapine (■), loxapine (○) and mefloquine (♦) resulted in average IC₅₀ values (nM) and SDs of 71 ± 32, 128 ± 56, 2,282 ± 1,041, 388 ± 98, >100,000 and 1,212 ± 234, respectively. The average Hill slope values derived from the IC₅₀ curves of all active drugs ranged from 0.93 – 1.26. Data points represent the average of three determinations per concentration and error bars represent standard deviation. Data are representative of three independent experiments.

Fig. 3. Activities in an E. coli cell-based GUS activity assay.

Concentration response data for compounds was normalized to controls with and without whole E. coli cells and plotted as percent activity. Potency determinations of nialamide (●), isocarboxazid (▲), phenelzine (□), amoxapine (■), loxapine (○) and mefloquine (♦) resulted in average IC₅₀ values (nM) and SDs of 17 ± 2, 336 ± 120, 7,123 ± 1,650, 119 ± 61, >100,000 and 5,961 ± 1,526, respectively. The average Hill slope values derived from the IC₅₀ curves of all active drugs ranged from 0.8 – 1.1. Data points represent the average of three determinations per concentration and error bars represent standard deviation. Data are representative of three independent experiments.

Fig. 4. Bacterial cytotoxicity assessment of studied drugs.

E. coli bacteria were treated with compounds at 10 and 100 μM, as indicated, for two hours. Viability was assessed with MTS viability reagent. Absorbance data was normalized to controls with and without whole E. coli cells and plotted as percent viability, with ‘no cells’ considered as 0% viability and DMSO only treated cells set at 100% viable. Kanamycin, a known cytotoxic antibiotic drug, was used as a control.