Multiple bactericidal mechanisms of the zinc ionophore PBT2

Published 18 March 2020Globally, more antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance (AMR). The development of novel ionophores, a class of antimicrobials used exclusively in animals, holds promise as a strategy to replace or reduce essential human antimicrobials in veterinary practice. PBT2 is a zinc ionophore with recently demonstrated antibacterial activity against several Gram-positive pathogens, although the underlying mechanism of action is unknown. Here, we investigated the bactericidal mechanism of PBT2 in the bovine mastitis-causing pathogen, Streptococcus uberis In this work, we show that PBT2 functions as a Zn2+/H+ ionophore, exchanging extracellular zinc for intracellular protons in an electroneutral process that leads to cellular zinc accumulation. Zinc accumulation occurs concomitantly with manganese depletion and the production of reactive oxygen species (ROS). PBT2 inhibits the activity of the manganese-dependent superoxide dismutase, SodA, thereby impairing oxidative stress protection. We propose that PBT2-mediated intracellular zinc toxicity in S. uberis leads to lethality through multiple bactericidal mechanisms: the production of toxic ROS and the impairment of manganese-dependent antioxidant functions. Collectively, these data show that PBT2 represents a new class of antibacterial ionophores capable of targeting bacterial metal ion homeostasis and cellular redox balance. We propose that this novel and multitarget mechanism of PBT2 makes the development of cross-resistance to medically important antimicrobials unlikely.IMPORTANCE More antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance. Therefore, the elimination of antimicrobial crossover between human and veterinary medicine is of great interest. Unfortunately, the development of new antimicrobials is an expensive high-risk process fraught with difficulties. The repurposing of chemical agents provides a solution to this problem, and while many have not been originally developed as antimicrobials, they have been proven safe in clinical trials. PBT2, a zinc ionophore, is an experimental therapeutic that met safety criteria but failed efficacy checkpoints against both Alzheimer's and Huntington's diseases. It was recently found that PBT2 possessed potent antimicrobial activity, although the mechanism of bacterial cell death is unresolved. In this body of work, we show that PBT2 has multiple mechanisms of antimicrobial action, making the development of PBT2 resistance unlikely.Nichaela Harbison-Price, Scott A. Ferguson, Adam Heikal, George Taiaroa, Kiel Hards ... Christopher A. McDevitt ... et al

Two Stereoisomers of the Rat Toxicant Norbormide

The structures of two diastereoisomers of norbormide (C₃₃H₂₅N₃O₃, one as an ethyl acetate hemi-solvate C₃₃H₂₅N₃O₃·0.5(C₄H₈O₃) have been unambiguously determined. They differ in the relative stereochemistry of the exocyclic double bond and the relative conformations of the aryl rings. Each is involved in both intra- and inter-molecular hydrogen bonding

Replacement of the CysA7-CysB7 disulfide bond with a 1,2,3-triazole linker causes unfolding in insulin glargine

Two analogues of insulin glargine containing a 1,4-disubstituted 1,2,3-triazole group in place of the CysA7–CysB7 disulfide bond were prepared using CuAAC click chemistry to efficiently join the peptide chains.</p

Glicentin-related pancreatic polypeptide inhibits glucose-stimulated insulin secretion from the isolated pancreas of adult male rats.

Several peptides derived from the glucagon gene Gcg, for example glucagon and glucagon-like peptide 1 (GLP-1), act as major regulators of fuel metabolism. GLP-1 is an incretin which has given rise to two new classes of anti-diabetic agents, the GLP-1 agonists and the DPP4 inhibitors. Glucagon is another potent Gcg-derived glucoregulatory hormone whose main regulatory role is to increase glucose output from the liver. Peptides derived from Gcg are thus of major interest in the regulation of intermediary metabolism, the pathogenesis of diseases such as type-2 diabetes and obesity, and their therapeutic management. Glicentin-related pancreatic polypeptide (GRPP) is a further, 30 amino-acid Gcg-derived peptide identified in human, mouse, rat and pig. A closely-related peptide, GRPP-like peptide (GRPP-LP) has also been identified in rat islets. The potential glucoregulatory functions of these peptides are largely unknown. We synthesized rat GRPP (rGRPP) and rat GRPP-LP (rGRPP-LP) and determined their actions in the liver and pancreas of adult male rats by employing isolated-perfused organ preparations. Rat GRPP and rGRPP-LP did not affect glucose output from the liver but both elicited potent inhibition of glucose-stimulated insulin secretion from the rat pancreas. This action is unlikely to be mediated by glucagon or GLP-1 receptors as rGRPP and rGRPP-LP did not stimulate cyclic adenosine monophosphate (cAMP) production from the glucagon or GLP-1 receptors, nor did they antagonize glucagon- or GLP-1-stimulated cAMP-production at either receptor. GRPP and GRPP-LP may be novel regulators of insulin secretion, acting through an as-yet undefined receptor

PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery