Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential.

Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite that enhances both platelet responsiveness and in vivo thrombosis potential in animal models, and TMAO plasma levels predict incident atherothrombotic event risks in human clinical studies. TMAO is formed by gut microbe-dependent metabolism of trimethylamine (TMA) moiety-containing nutrients, which are abundant in a Western diet. Here, using a mechanism-based inhibitor approach targeting a major microbial TMA-generating enzyme pair, CutC and CutD (CutC/D), we developed inhibitors that are potent, time-dependent, and irreversible and that do not affect commensal viability. In animal models, a single oral dose of a CutC/D inhibitor significantly reduced plasma TMAO levels for up to 3 d and rescued diet-induced enhanced platelet responsiveness and thrombus formation, without observable toxicity or increased bleeding risk. The inhibitor selectively accumulated within intestinal microbes to millimolar levels, a concentration over 1-million-fold higher than needed for a therapeutic effect. These studies reveal that mechanism-based inhibition of gut microbial TMA and TMAO production reduces thrombosis potential, a critical adverse complication in heart disease. They also offer a generalizable approach for the selective nonlethal targeting of gut microbial enzymes linked to host disease limiting systemic exposure of the inhibitor in the host.

Slc39a14 gene encodes ZIP14, a metal/bicarbonate symporter: similarities to the ZIP8 transporter.

The mouse and human genomes contain 14 highly conserved SLC39 genes. Viewed from an evolutionary perspective, SLC39A14 and SLC39A8 are the most closely related, each having three noncoding exons 1. However, SLC39A14 has two exons 4, giving rise to Zrt- and Irt-related protein (ZIP)ZIP14A and ZIP14B alternatively spliced products. C57BL/6J mouse ZIP14A expression is highest in liver, duodenum, kidney, and testis; ZIP14B expression is highest in liver, duodenum, brain, and testis; and ZIP8 is highest in lung, testis, and kidney. We studied ZIP14 stably retroviral-infected mouse fetal fibroblast cultures and transiently transfected Madin-Darby canine kidney (MDCK) polarized epithelial cells. Our findings include: 1) ZIP14-mediated cadmium uptake is proportional to cell toxicity, but manganese is not; 2) ZIP14B has a higher affinity than ZIP14A toward Cd(2+) (K(m) = 0.14 versus 1.1 microM) and Mn(2+) uptake (K(m) = 4.4 versus 18.2 microM); 3) ZIP14A- and ZIP14B-mediated Cd(2+) uptake is most inhibited by Zn(2+), and next by Mn(2+) and Cu(2+); 4) like ZIP8, ZIP14A- and ZIP14B-mediated Cd(2+) uptake is dependent on extracellular HCO(3)(-); 5) like ZIP8, ZIP14 transporters are localized on the apical surface of MDCK-ZIP cells; and 6) like ZIP8, ZIP14 proteins are glycosylated. Tissues such as intestine and liver, located between the environment and the animal, show high levels of ZIP14; given the high affinity for ZIP14, Cd(2+) is likely to act as a rogue hitchhiker-displacing Zn(2+) or Mn(2+) and entering the body to cause unwanted cell damage and disease.

Cd2+ versus Zn2+ uptake by the ZIP8 HCO3--dependent symporter: kinetics, electrogenicity and trafficking.

The mouse Slc39a8 gene encodes the ZIP8 transporter, which has been shown to be a divalent cation/HCO3- symporter. Using ZIP8 cRNA-injected Xenopus oocyte cultures, we show herein that: [a] ZIP8-mediated cadmium (Cd(2+)) and zinc (Zn(2+)) uptake have V(max) values of 1.8+/-0.08 and 1.0+/-0.08 pmol/oocyte/h, and K(m) values of 0.48+/-0.08 and 0.26+/-0.09 microM, respectively; [b] ZIP8-mediated Cd(2+) uptake is most inhibited by Zn(2+), second-best inhibited by Cu(2+), Pb(2+) and Hg(2+), and not inhibited by Mn(2+) or Fe(2+); and [c] electrogenicity studies demonstrate an influx of two HCO3- anions per one Cd(2+) (or one Zn(2+)) cation, i.e. electroneutral complexes. Using Madin-Darby canine kidney (MDCK) polarized epithelial cells retrovirally infected with ZIP8 cDNA and tagged with hemagglutinin at the C-terminus, we show that-similar to ZIP4-the ZIP8 eight-transmembrane protein is largely internalized during Zn(2+) homeostasis, but moves predominantly to the cell surface membrane (trafficking) under conditions of Zn(2+) depletion.