ABSTRACT
Mutations to the gene encoding superoxide dismutase-1 (SOD1) were the first genetic elements discovered that cause motor neuron disease (MND). These mutations result in compromised SOD1 dimer stability, with one of the severest and most common mutations Ala4Val (A4V) displaying a propensity to monomerise and aggregate leading to neuronal death. We show that the clinically used ebselen and related analogues promote thermal stability of A4V SOD1 when binding to Cys111 only. We have developed a A4V SOD1 differential scanning fluorescence-based assay on a C6S mutation background that is effective in assessing suitability of compounds. Crystallographic data show that the selenium atom of these compounds binds covalently to A4V SOD1 at Cys111 at the dimer interface, resulting in stabilisation. This together with chemical amenability for hit expansion of ebselen and its on-target SOD1 pharmacological chaperone activity holds remarkable promise for structure-based therapeutics for MND using ebselen as a template.
Subject(s)
Azoles/chemistry , Azoles/pharmacology , Drug Design , Motor Neuron Disease/drug therapy , Organoselenium Compounds/chemistry , Organoselenium Compounds/pharmacology , Superoxide Dismutase-1 , Amino Acid Substitution/genetics , Azoles/chemical synthesis , Azoles/therapeutic use , Crystallography, X-Ray , Drug Discovery/methods , Drug Evaluation, Preclinical/methods , Humans , Isoindoles , Models, Molecular , Molecular Chaperones/chemical synthesis , Molecular Chaperones/chemistry , Molecular Chaperones/therapeutic use , Molecular Docking Simulation , Motor Neuron Disease/genetics , Motor Neuron Disease/metabolism , Motor Neuron Disease/pathology , Mutant Proteins/chemistry , Mutant Proteins/drug effects , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense , Organoselenium Compounds/chemical synthesis , Organoselenium Compounds/isolation & purification , Organoselenium Compounds/therapeutic use , Protein Folding/drug effects , Protein Multimerization/drug effects , Protein Stability/drug effects , Protein Structure, Tertiary , Sulfur Compounds/chemical synthesis , Sulfur Compounds/chemistry , Superoxide Dismutase-1/chemistry , Superoxide Dismutase-1/drug effects , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , ThermodynamicsABSTRACT
Current therapeutic options for cryptococcal meningitis are limited by toxicity, global supply, and emergence of resistance. There is an urgent need to develop additional antifungal agents that are fungicidal within the central nervous system and preferably orally bioavailable. The benzimidazoles have broad-spectrum antiparasitic activity but also have in vitro antifungal activity that includes Cryptococcus neoformans Flubendazole (a benzimidazole) has been reformulated by Janssen Pharmaceutica as an amorphous solid drug nanodispersion to develop an orally bioavailable medicine for the treatment of neglected tropical diseases such as onchocerciasis. We investigated the in vitro activity, the structure-activity-relationships, and both in vitro and in vivo pharmacodynamics of flubendazole for cryptococcal meningitis. Flubendazole has potent in vitro activity against Cryptococcus neoformans, with a modal MIC of 0.125 mg/liter using European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology. Computer models provided an insight into the residues responsible for the binding of flubendazole to cryptococcal ß-tubulin. Rapid fungicidal activity was evident in a hollow-fiber infection model of cryptococcal meningitis. The solid drug nanodispersion was orally bioavailable in mice with higher drug exposure in the cerebrum. The maximal dose of flubendazole (12 mg/kg of body weight/day) orally resulted in an â¼2 log10CFU/g reduction in fungal burden compared with that in vehicle-treated controls. Flubendazole was orally bioavailable in rabbits, but there were no quantifiable drug concentrations in the cerebrospinal fluid (CSF) or cerebrum and no antifungal activity was demonstrated in either CSF or cerebrum. These studies provide evidence for the further study and development of the benzimidazole scaffold for the treatment of cryptococcal meningitis.
Subject(s)
Antifungal Agents/therapeutic use , Drug Repositioning/methods , Mebendazole/analogs & derivatives , Meningitis, Cryptococcal/drug therapy , Meningitis, Cryptococcal/microbiology , Mycoses/drug therapy , Mycoses/microbiology , Animals , Antiparasitic Agents/therapeutic use , Benzimidazoles/therapeutic use , Cryptococcosis/drug therapy , Cryptococcosis/microbiology , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/pathogenicity , Female , Fluconazole/therapeutic use , Male , Mebendazole/therapeutic use , Mice , Microbial Sensitivity Tests , Rabbits , Rats , SwineABSTRACT
Screening of the GSK corporate collection, some 1.9 million compounds, against Plasmodium falciparum (Pf), revealed almost 14000 active hits that are now known as the Tres Cantos Antimalarial Set (TCAMS). Followup work by Calderon et al. clustered and computationally filtered the TCAMS through a variety of criteria and reported 47 series containing a total of 522 compounds. From this enhanced set, we identified the carbamoyl triazole TCMDC-134379 (1), a known serine protease inhibitor, as an excellent starting point for SAR profiling. Lead optimization of 1 led to several molecules with improved antimalarial potency, metabolic stabilities in mouse and human liver microsomes, along with acceptable cytotoxicity profiles. Analogue 44 displayed potent in vitro activity (IC50 = 10 nM) and oral activity in a SCID mouse model of Pf infection with an ED50 of 100 and ED90 of between 100 and 150 mg kg(-1), respectively. The results presented encourage further investigations to identify the target of these highly active compounds.
