2-Guanidino-quinazoline promotes the readthrough of nonsense mutations underlying human genetic diseases.

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).

ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

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.

Discovery of compounds blocking the proliferation of Toxoplasma gondii and Plasmodium falciparum in a chemical space based on piperidinyl-benzimidazolone analogs.

A piperidinyl-benzimidazolone scaffold has been found in the structure of different inhibitors of membrane glycerolipid metabolism, acting on enzymes manipulating diacylglycerol and phosphatidic acid. Screening a focus library of piperidinyl-benzimidazolone analogs might therefore identify compounds acting against infectious parasites. We first evaluated the in vitro effects of (S)-2-(dibenzylamino)-3-phenylpropyl 4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)piperidine-1-carboxylate (compound 1) on Toxoplasma gondii and Plasmodium falciparum. In T. gondii, motility and apical complex integrity appeared to be unaffected, whereas cell division was inhibited at compound 1 concentrations in the micromolar range. In P. falciparum, the proliferation of erythrocytic stages was inhibited, without any delayed death phenotype. We then explored a library of 250 analogs in two steps. We selected 114 compounds with a 50% inhibitory concentration (IC50) cutoff of 2 µM for at least one species and determined in vitro selectivity indexes (SI) based on toxicity against K-562 human cells. We identified compounds with high gains in the IC50 (in the 100 nM range) and SI (up to 1,000 to 2,000) values. Isobole analyses of two of the most active compounds against P. falciparum indicated that their interactions with artemisinin were additive. Here, we propose the use of structure-activity relationship (SAR) models, which will be useful for designing probes to identify the target compound(s) and optimizations for monotherapy or combined-therapy strategies.

Inhibition of retrograde transport protects mice from lethal ricin challenge.

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.

Small-molecule inhibitors of sodium iodide symporter function.

The Na(+)/I(-) symporter (NIS) mediates iodide uptake into thyroid follicular cells. Although NIS has been cloned and thoroughly studied at the molecular level, the biochemical processes involved in post-translational regulation of NIS are still unknown. The purpose of this study was to identify and characterize inhibitors of NIS function. These small organic molecules represent a starting point in the identification of pharmacological tools for the characterization of NIS trafficking and activation mechanisms. The screening of a collection of 17,020 druglike compounds revealed new chemical inhibitors with potencies down to 40 nM. Fluorescence measurement of membrane potential indicates that these inhibitors do not act by disrupting the sodium gradient. They allow immediate and total iodide discharge from preloaded cells in accord with a specific modification of NIS activity, probably through distinct mechanisms.

A 96-well automated radioiodide uptake assay for sodium/iodide symporter inhibitors.

A high-throughput screening method based on radioiodide uptake in human embryonic kidney 293 cells expressing the human sodium/iodide symporter was developed. Central to assay development was a homogeneous cell culture in the 96-well microplate coupled with the use of scintillation proximity technology. The assay is fast and highly reproducible with a Z' greater than 0.8. The automated procedure allows the screening of 4,000 compounds per day. Using this methodology, several known substrates of the sodium/iodide symporter were evaluated in a single day. Inhibition of iodide uptake was shown to follow the series PF(6)(-) > ClO(4)(-) > BF(4)(-) > SCN(-) >> NO(3)(-) > IO(4)(-) > N(3)(-) >> Br(-), in accord with the literature. This method represents an initial approach to the search for inhibitors of iodide transport mediated by the sodium/iodide symporter.