Nonsteroidal Anti-Inflammatory Drug-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells

The intestinal epithelium provides a critical barrier that separates the gut microbiota from host tissues. Nonsteroidal anti-inflammatory drugs (NSAIDs) are efficacious analgesics and antipyretics and are among the most frequently used drugs worldwide. In addition to gastric damage, NSAIDs are toxic to the intestinal epithelium, causing erosions, perforations, and longitudinal ulcers in the gut. Here, we use a unique <i>in vitro</i> human primary small intestinal cell monolayer system to pinpoint the intestinal consequences of NSAID treatment. We found that physiologically relevant doses of the NSAID diclofenac (DCF) are cytotoxic because they uncouple mitochondrial oxidative phosphorylation and generate reactive oxygen species. We also find that DCF induces intestinal barrier permeability, facilitating the translocation of compounds from the luminal to the basolateral side of the intestinal epithelium. The results we outline here establish the utility of this novel platform, representative of the human small intestinal epithelium, to understand NSAID toxicity, which can be applied to study multiple aspects of gut barrier function including defense against infectious pathogens and host–microbiota interactions

Gut Microbial β-Glucuronidase Inhibition via Catalytic Cycle Interception

Microbial β-glucuronidases (GUSs) cause severe gut toxicities that limit the efficacy of cancer drugs and other therapeutics. Selective inhibitors of bacterial GUS have been shown to alleviate these side effects. Using structural and chemical biology, mass spectrometry, and cell-based assays, we establish that piperazine-containing GUS inhibitors intercept the glycosyl-enzyme catalytic intermediate of these retaining glycosyl hydrolases. We demonstrate that piperazine-based compounds are substrate-dependent GUS inhibitors that bind to the GUS–GlcA catalytic intermediate as a piperazine-linked glucuronide (GlcA, glucuronic acid). We confirm the GUS-dependent formation of inhibitor–glucuronide conjugates by LC–MS and show that methylated piperazine analogs display significantly reduced potencies. We further demonstrate that a range of approved piperazine- and piperidine-containing drugs from many classes, including those for the treatment of depression, infection, and cancer, function by the same mechanism, and we confirm through gene editing that these compounds selectively inhibit GUS in living bacterial cells. Together, these data reveal a unique mechanism of GUS inhibition and show that a range of therapeutics may impact GUS activities in the human gut

Human carboxylesterase 1 active site structure.

<p>Active site of human carboxylesterase 1 covalently inhibited via S221 with cyclosarin (magenta) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017441#pone.0017441-Hemmert1" target="_blank">[8]</a>. The other catalytic residues, in addition to S221, are H468 and E354 (yellow), and are surrounded by hydrophobic residues (grey surface) including V146 and L363 (light blue), as well as the oxyanion hole (white).</p

Catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m) of engineered enzymes towards hemisubstrates.

a<p>(16).</p>b<p>(32).</p>c<p>(32).</p

Bimolecular rates of inhibition, Michaelis-Menten constants, and rates of reactivation for wild-type and V146H/L363E hCE1 against racemic cyclosarin and stereoisomers of cyclosarin model compounds.

<p>N = 3, s.d., N.D. is not determined, N.R. is no reactivation, pH 7.4, 25°C.</p>a<p>Racemic <i>bona fide</i> cyclosarin,</p>b<p>stereoisomers of cyclosarin model compounds,</p>c<p>(8).</p

Mechanism of reactivation by V146H/L363E hCE1 after cyclosarin binding.

<p><b>A</b>. Model of V146H/L363E (cyan) hCE1 with P<i>_R</i> cyclosarin (magenta) including a water molecule (red) between E363 and the central phosphorus. <b>B</b>. Proposed mechanism for enhanced reactivation following cyclosarin inhibition. <b>C</b>. pH dependence of V146H/L363E (black) and L363E (grey) hCE1 dephosphonylation following cyclosarin inhibition.</p

Organophosphate (OP) inhibition of human carboxylesterase 1 (hCE1).

<p><b>A</b>. Three G-type OP nerve agents and OP model compound (R represents respective <i>O</i>-alkoxy groups). Wild-type hCE1 preferentially binds the stereoisomers shown (7). <b>B</b>. Schematic mechanism of OP hydrolysis by hCE1. X represents the leaving group and * denotes a non-reactive state.</p

Reactivation of hCE1 following nerve agent exposure.

<p><b>A</b>. Spontaneous reactivation of V146H/L363E hCE1 following inhibition by racemic sarin (blue), soman (green), or cyclosarin (red). Wild type hCE1 (grey) only reactivates following sarin inhibition (7). n = 6, s.d. <b>B</b>. Rates of dephosphonylation for hCE1 variants in the presence of sarin (blue), soman (green) and cyclosarin (red). n = 3, s.d.</p

Inhibition and Michaelis-Menten constants for wild-type and V146H/L363E hCE1 against stereoisomers of sarin and soman model compounds.

<p>(N = 3, s.d.)</p>a<p>(7).</p