In vitro and in vivo activities of HPi1, a selective antimicrobial against Helicobacter pylori

Abstract

A high-throughput screen (HTS) was performed to identify molecules specifically active against Helicobacter pylori, the causative agent of peptic ulcer and gastric carcinoma. Currently, treatment of H. pylori infection is suboptimal, with failure rates approaching 25%, despite triple therapy with two broad-spectrum antibiotics and a proton pump inhibitor or quadruple therapy with added bismuth. The HTS was performed in 384-well plates, and reduction of the metabolic indicator resazurin was used as a reporter for cell growth. Diverse molecules from commercial sources were identified as hits, and in vitro validations included measurements of MIC and time-dependent killing as well as anaerobic susceptibility testing against a panel of gut microbes. In vivo validation included testing in the mouse model of H. pylori infection. The small molecule HPi1 (3-hydrazinoquinoxaline-2-thiol) had excellent potency, with an MIC of 0.08 to 0.16 μg/ml and good selectivity for H. pylori compared to a panel of commensal bacteria. HPi1 was also effective in a mouse model of H. pylori infection, reducing colony counts to below the limit of detection after oral dosing of 25 mg/kg/day for 3 days. HPi1 is a promising lead in the search for more effective and specific H. pylori therapeutics.

FIG 1

Structure of HPi1 (3-hydrazinoquinoxaline-2-thiol).

FIG 2

Dose responses of clarithromycin, amoxicillin, and HPi1 against H. pylori ATCC 43504 in a resazurin growth inhibition assay.

FIG 3

Time-dependent killing of H. pylori by HPi1. Growing cultures of H. pylori ATCC 43504 were left untreated or exposed to HPi1 at 0.16 μg/ml, 0.8 μg/ml, and 1.6 μg/ml, which are equal to 1×, 5×, and 10× the MIC, respectively. Samples were removed, serially diluted, and plated to determine the viable counts for each starting culture and after 3, 6, 12, 18, 24, and 48 h of exposure to HPi1. The x axis is the limit of detection, and standard deviations are indicated by the error bars.

FIG 4

Effect of HPi1 in a mouse model of H. pylori infection. (a) Infection with H. pylori strain SS1 was confirmed using Giemsa staining histopathology (magnification, ×1,600). (b) Each mouse was dosed by oral gavage with HPi1, BMT triple therapy, or vehicle once per day for 3 days. After treatment, stomachs were harvested, weighed, and homogenized, and H. pylori burden was determined by plating dilutions on selective medium. The x axis is the limit of detection, and horizontal bars indicate the average for each group. If no colonies were present, calculations were made using the limit of detection. *, P = 0.01 to 0.05; ****, P < 0.0001.