ABSTRACT
Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.
Subject(s)
Benzamides/pharmacology , Benzodiazepinones/pharmacology , Cytoprotection , Protein Transport , Ricin/antagonists & inhibitors , Thiophenes/pharmacology , Administration, Intranasal , Animals , Benzamides/chemistry , Benzodiazepinones/chemistry , Cell Line, Tumor , Cell Membrane/metabolism , Endocytosis , Endosomes/metabolism , Golgi Apparatus/metabolism , HeLa Cells , High-Throughput Screening Assays , Humans , Mice , Qa-SNARE Proteins/metabolism , Ricin/administration & dosage , Ricin/toxicity , Shiga Toxins/antagonists & inhibitors , Shiga Toxins/toxicity , Thiophenes/chemistry , trans-Golgi Network/metabolismABSTRACT
Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.
Subject(s)
Adamantane/analogs & derivatives , Benzyl Compounds/pharmacology , Endosomes/drug effects , Ricin/antagonists & inhibitors , Toxins, Biological/antagonists & inhibitors , Adamantane/chemistry , Adamantane/pharmacology , Animals , Benzyl Compounds/chemistry , Benzylamines , Cell Compartmentation/drug effects , Endoplasmic Reticulum/drug effects , Golgi Apparatus/drug effects , HeLa Cells , Humans , Lysosomes/drug effects , Mice , Ricin/drug effects , Ricin/toxicity , Toxins, Biological/chemistry , Toxins, Biological/toxicityABSTRACT
A long-sought milestone in the defense against bioterrorism is the development of rapid, simple, and near-patient assays for diagnostic and theranostic purposes. Here, we present a powerful test based on a host response to a biological weapon agent, namely the ricin toxin. A signature for exposure to ricin was extracted and characterized in mice and then integrated into a plastic microfluidic cartridge. This enabled early diagnosis of exposure to ricin in mice using a drop of whole blood in less than 1 h and 30 min. The cartridge stores the reagents and implements all of the steps of the analysis, including mRNA extraction from a drop of blood, followed by tens of parallel RT-qPCR reactions. The simple and low-cost microfluidic cartridge developed here may find other applications in point-of-care diagnostics.
Subject(s)
Biological Warfare Agents , Lab-On-A-Chip Devices , Point-of-Care Systems , Ricin/poisoning , Theranostic Nanomedicine , Animals , Humans , Mice , Mice, Inbred BALB C , Theranostic Nanomedicine/instrumentation , Theranostic Nanomedicine/methodsABSTRACT
The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC(50) of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD(50)). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention.