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.

Abnormal generation of neurotoxic amyloid-b peptide (Ab) 42/43 species due to mutations in the catalytic presenilin 1 (PS1) subunit of c-secretase is the major cause of familial Alzheimer's disease (FAD). Deeper mechanistic insight on the generation of Ab43 is still lacking, and it is unclear whether c-secretase modulators (GSMs) can reduce the levels of this Ab species. By comparing several types of Ab43-generating FAD mutants, we observe that very high levels of Ab43 are often produced when presenilin function is severely impaired. Altered interactions of C99, the precursor of Ab, are found for all mutants and are independent of their particular effect on Ab production. Furthermore, unlike previously described GSMs, the novel compound RO7019009 can effectively lower Ab43 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 Ab43-generating FAD mutations could in principle be treated by GSMs.

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.

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Genetic,p reclinical and clinical data link Parkinsonsd isease and Gauchersd isease and providearational entry point to disease modification therapyvia enhancement of b-Glucocerebrosidase (GCase) activity.W ed iscoveredanew 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 acellular assay. We obtained the first crystal structure for an activator and identified an ovel non-inhibitory binding mode at the interface of ad imer, rationalizing the observed structure-activity relationship (SAR). The compound binds GCase inducing formation of ad imeric state at both endoplasmic reticulum (ER) and lysosomal pHs,asconfirmed 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 provideadeeper mechanistic understanding of the requirements for enzymatic activation, pointing to the relevance of dimerization.