Novel ß-Glucocerebrosidase Activators That Bind to a New Pocket at a Dimer Interface and Induce Dimerization.

Genetic, preclinical and clinical data link Parkinson's disease and Gaucher's disease and provide a rational entry point to disease modification therapy via enhancement of ß-Glucocerebrosidase (GCase) activity. We discovered a new class of pyrrolo[2,3-b]pyrazine activators effecting both Vmax and Km. They bind to human GCase and increase substrate metabolism in the lysosome in a cellular assay. We obtained the first crystal structure for an activator and identified a novel non-inhibitory binding mode at the interface of a dimer, rationalizing the observed structure-activity relationship (SAR). The compound binds GCase inducing formation of a dimeric state at both endoplasmic reticulum (ER) and lysosomal pHs, as confirmed by analytical ultracentrifugation. Importantly, the pyrrolo[2,3-b]pyrazines have central nervous system (CNS) drug-like properties. Our findings are important for future drug discovery efforts in the field of GCase activation and provide a deeper mechanistic understanding of the requirements for enzymatic activation, pointing to the relevance of dimerization.

Stepwise Design of γ-Secretase Modulators with an Advanced Profile by Judicious Coordinated Structural Replacements and an Unconventional Phenyl Ring Bioisostere.

Starting from RO6800020 (1), our former γ-secretase modulator (GSM) lead compound, we utilized sequential structural replacements to improve the potency (IC50), pharmacokinetic properties including the free fraction (fraction unbound (fu)) in plasma, and in vivo efficacy. Importantly, we used novel CF3-alkoxy groups as bioisosteric replacements of a fluorinated phenyl ring and properties such as lipophilicity, solubility, metabolic stability, and free fraction could be balanced, maintaining low Pgp efflux needed for CNS penetration. In addition, by reducing aromaticity, we prevented phototoxicity. Additional substitution in the triazolopyridine core disturbed the binding to phosphatidylinositol 4-kinase, catalytic ß (PIK4CB). We also introduced less lipophilic head heterocycles devoid of covalent binding (CVB) liability. After these changes, further modifications to the trifluoroethoxy bioisosteric replacement allowed rebalancing of properties, such as lipophilicity, and also potency. Our optimization strategy culminated with in vivo active RO7101556 (18B) having excellent properties and being selected as an advanced candidate.

Discovery of RO7185876, a Highly Potent γ-Secretase Modulator (GSM) as a Potential Treatment for Alzheimer's Disease.

γ-Secretase (GS) is a key target for the potential treatment of Alzheimer's disease. While inhibiting GS led to serious side effects, its modulation holds a lot of potential to deliver a safe treatment. Herein, we report the discovery of a potent and selective gamma secretase modulator (GSM) (S)-3 (RO7185876), belonging to a novel chemical class, the triazolo-azepines. This compound demonstrates an excellent in vitro and in vivo DMPK profile. Furthermore, based on its in vivo efficacy in a pharmacodynamic mouse model and the outcome of the dose range finding (DRF) toxicological studies in two species, this compound was selected to undergo entry in human enabling studies (e.g., GLP toxicology and scale up activities).

Aß43-producing PS1 FAD mutants cause altered substrate interactions and respond to γ-secretase modulation.

Abnormal generation of neurotoxic amyloid-ß peptide (Aß) 42/43 species due to mutations in the catalytic presenilin 1 (PS1) subunit of γ-secretase is the major cause of familial Alzheimer's disease (FAD). Deeper mechanistic insight on the generation of Aß43 is still lacking, and it is unclear whether γ-secretase modulators (GSMs) can reduce the levels of this Aß species. By comparing several types of Aß43-generating FAD mutants, we observe that very high levels of Aß43 are often produced when presenilin function is severely impaired. Altered interactions of C99, the precursor of Aß, are found for all mutants and are independent of their particular effect on Aß production. Furthermore, unlike previously described GSMs, the novel compound RO7019009 can effectively lower Aß43 production of all mutants. Finally, substrate-binding competition experiments suggest that RO7019009 acts mechanistically after initial C99 binding. We conclude that altered C99 interactions are a common feature of diverse types of PS1 FAD mutants and that also patients with Aß43-generating FAD mutations could in principle be treated by GSMs.

Diversity oriented biosynthesis via accelerated evolution of modular gene clusters.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

Design of Potent and Druglike Nonphenolic Inhibitors for Catechol O-Methyltransferase Derived from a Fragment Screening Approach Targeting the S-Adenosyl-l-methionine Pocket.

A fragment screening approach designed to target specifically the S-adenosyl-l-methionine pocket of catechol O-methyl transferase allowed the identification of structurally related fragments of high ligand efficiency and with activity on the described orthogonal assays. By use of a reliable enzymatic assay together with X-ray crystallography as guidance, a series of fragment modifications revealed an SAR and, after several expansions, potent lead compounds could be obtained. For the first time nonphenolic and small low nanomolar potent, SAM competitive COMT inhibitors are reported. These compounds represent a novel series of potent COMT inhibitors that might be further optimized to new drugs useful for the treatment of Parkinson's disease, as adjuncts in levodopa based therapy, or for the treatment of schizophrenia.

Selective naphthalene H(3) receptor inverse agonists with reduced potential to induce phospholipidosis and their quinoline analogs.

We reported earlier the refinement of our initial five-point pharmacophore model for the Histamine 3 receptor (H(3)R), with a new acceptor feature important for binding and selectivity against the other histamine receptor subtypes 1, 2 and 4. This approach was validated with a new series of H(3)R inverse agonists: the naphthalene series. In this Letter, we describe our efforts to overcome the phospholipidosis flag identified with our initial lead compound (1a). During the optimization process, we monitored the potency of our molecules toward the H(3) receptor, their selectivity against H(1)R, H(2)R and H(4)R, as well as some key molecular properties that may influence phospholipidosis. Encouraged by the promising profile of the naphthalene series, we used our deeper understanding of the H(3)R pharmacophore model to lead us towards the quinoline series. This series is perceived to have intrinsic advantages with respect to its amphiphilic vector.

5-hydroxyindole-2-carboxylic acid amides: novel histamine-3 receptor inverse agonists for the treatment of obesity.

Obesity is a major risk factor in the development of conditions such as hypertension, hyperglycemia, dyslipidemia, coronary artery disease, and cancer. Several pieces of evidence across different species, including primates, underscore the implication of the histamine 3 receptor (H(3)R) in the regulation of food intake and body weight and the potential therapeutic effect of H(3)R inverse agonists. A pharmacophore model, based on public information and validated by previous investigations, was used to design several potential scaffolds. Out of these scaffolds, the 5-hydroxyindole-2-carboxylic acid amide appeared to be of great potential as a novel series of H(3)R inverse agonist. Extensive structure-activity relationships revealed the interconnectivity of microsomal clearance and hERG (human ether-a-go-go-related gene) affinity with lipophilicity, artificial membrane permeation, and basicity. This effort led to the identification of compounds reversing the (R)-alpha-methylhistamine-induced water intake increase in Wistar rats and, further, reducing food intake in diet-induced obese Sprague-Dawley rats. Of these, the biochemical, pharmacokinetic, and pharmacodynamic characteristics of (4,4-difluoropiperidin-1-yl)[1-isopropyl-5-(1-isopropylpiperidin-4-yloxy)-1H-indol-2-yl]methanone 36 are detailed.

A new class of histamine H3 receptor antagonists derived from ligand based design.

Design and synthesis of highly potent and selective non-imidazole inverse agonists for the histamine H(3) receptor is described. The study validates a new pharmacophore model based on the merging of two previously described models. It also demonstrates that the removal of the basic center potentially interacting with ASP3.32 and common to both models leads to loss of activity, whereas the replacement of the second basic center by an acceptor retains the potency.