ABSTRACT
We describe the discovery and optimization of new, brain-penetrant T-type calcium channel blockers. We present optimized compounds with excellent efficacy in a rodent model of generalized absence-like epilepsy. Along the fine optimization of a chemical series with a pharmacological target located in the CNS (target potency, brain penetration, and solubility), we successfully identified an Ames negative aminopyrazole as putative metabolite of this compound series. Our efforts culminated in the selection of compound 20, which was elected as a preclinical candidate.
Subject(s)
Calcium Channel Blockers/therapeutic use , Calcium Channels, T-Type/drug effects , Drug Discovery , Epilepsy, Generalized/drug therapy , Animals , Calcium Channels, T-Type/physiology , Disease Models, Animal , Humans , Mice , RatsABSTRACT
The identification of new sleep drugs poses particular challenges in drug discovery owing to disease-specific requirements such as rapid onset of action, sleep maintenance throughout major parts of the night, and absence of residual next-day effects. Robust tools to estimate drug levels in human brain are therefore key for a successful discovery program. Animal models constitute an appropriate choice for drugs without species differences in receptor pharmacology or pharmacokinetics. Translation to man becomes more challenging when interspecies differences are prominent. This report describes the discovery of the dual orexin receptor 1 and 2 (OX1 and OX2) antagonist ACT-541468 out of a class of structurally related compounds, by use of physiology-based pharmacokinetic and pharmacodynamic (PBPK-PD) modeling applied early in drug discovery. Although all drug candidates exhibited similar target receptor potencies and efficacy in a rat sleep model, they exhibited large interspecies differences in key factors determining their pharmacokinetic profile. Human PK models were built on the basis of in vitro metabolism and physicochemical data and were then used to predict the time course of OX2 receptor occupancy in brain. An active ACT-541468 dose of 25 mg was estimated on the basis of OX2 receptor occupancy thresholds of about 65% derived from clinical data for two other orexin antagonists, almorexant and suvorexant. Modeling predictions for ACT-541468 in man were largely confirmed in a single-ascending dose trial in healthy subjects. PBPK-PD modeling applied early in drug discovery, therefore, has great potential to assist in the identification of drug molecules when specific pharmacokinetic and pharmacodynamic requirements need to be met.
Subject(s)
Brain/drug effects , Brain/physiology , Drug Discovery/methods , Imidazoles/pharmacokinetics , Orexin Receptor Antagonists/pharmacokinetics , Pyrrolidines/pharmacokinetics , Animals , CHO Cells , Cricetinae , Cricetulus , Dogs , Dose-Response Relationship, Drug , Double-Blind Method , Humans , Male , Rats , Rats, WistarABSTRACT
Understanding the mechanisms through which multicellular organisms regulate cell, organ and body growth is of relevance to developmental biology and to research on growth-related diseases such as cancer. Here we describe a new effector in growth control, the small GTPase Rheb (Ras homologue enriched in brain). Mutations in the Drosophila melanogaster Rheb gene were isolated as growth-inhibitors, whereas overexpression of Rheb promoted cell growth. Our genetic and biochemical analyses suggest that Rheb functions downstream of the tumour suppressors Tsc1 (tuberous sclerosis 1)-Tsc2 in the TOR (target of rapamycin) signalling pathway to control growth, and that a major effector of Rheb function is ribosomal S6 kinase (S6K).
Subject(s)
Cell Division/genetics , Drosophila Proteins/metabolism , Growth Substances/metabolism , Monomeric GTP-Binding Proteins/physiology , Neuropeptides/physiology , Ribosomal Protein S6 Kinases/metabolism , Animals , Cell Division/physiology , Cell Size/genetics , Cell Size/physiology , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Eye/ultrastructure , Female , Gene Deletion , Genes, Insect , Genes, Tumor Suppressor , Growth Substances/genetics , Monomeric GTP-Binding Proteins/genetics , Neuropeptides/genetics , Ras Homolog Enriched in Brain Protein , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Ribosomal Protein S6 Kinases/genetics , Signal Transduction , Transcriptional Activation , TransgenesABSTRACT
With the availability of complete genome sequences, new rapid and reliable strategies for positional cloning become possible. Single-nucleotide polymorphisms (SNPs) permit the mapping of mutations at a resolution not amenable to classical genetics. Here we describe a SNP mapping procedure that relies on resolving polymorphisms by denaturing HPLC without the necessity of determining the nature of the SNPs. With the example of mapping mutations to the Drosophila nicastrin locus, we discuss the benefits of this method, evaluate the frequency of closely linked and potentially misleading second site mutations, and demonstrate the use of denaturing high-performance liquid chromatography to identify mutations in the candidate genes and to fine-map chromosomal breakpoints. Furthermore, we show that recombination events are not uniformly dispersed over the investigated region but rather occur at hot spots.
Subject(s)
Chromatography, High Pressure Liquid/methods , Chromosome Mapping/methods , Polymorphism, Single Nucleotide , Animals , Drosophila/genetics , Female , Genes, Recessive , Male , Mutagenesis , Recombination, Genetic , ras Proteins/geneticsABSTRACT
Individual members of gene families often have partially redundant functions during nervous system development, making conventional genetic analysis problematic. Here we review experiments showing that several genes can be silenced together by injection of double-stranded RNAs into wild-type Drosophila embryos. By dye-labeling single neuroblasts in injected embryos, the effects of multigene silencing on individual CNS axon pathways can now be examined.
