We describe the development of cell-permeable beta-secretase inhibitors that demonstratively inhibit the production of the secreted amino terminal fragment of an artificial amyloid precursor protein in cell culture. In addition to potent inhibition in a cell-based assay (IC50 < 100 nM), these inhibitors display impressive selectivity against other biologically relevant aspartyl proteases.

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain’s ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.

Radicicol (1) exhibits potent anticancer properties in vitro, which are likely to be mediated through its high affinity (20 nM) for the molecular chaperone Hsp90. Recently, we reported the results of a synthetic program targeting radicicol (1) and monocillin I (2), highlighted by the application of ring-closing metathesis to macrolide formation. These efforts resulted in a highly convergent synthesis of radicicol dimethyl ether but failed in the removal of the two aryl methyl ethers. Simple exchange of these methyl ethers with more labile functionalities disabled a key esterification in the initial route. Through extended experimentation, a successful route to both natural products was secured, along with some intriguing results that emphasize the implications of this design on a broad range of fused benzoaliphatic targets, including analogues of these natural products.

A small molecule nonpeptide inhibitor of beta-secretase has been developed, and its binding has been defined through crystallographic determination of the enzyme-inhibitor complex. The molecule is shown to bind to the catalytic aspartate residues in an unprecedented manner in the field of aspartyl protease inhibition. Additionally, the complex reveals a heretofore unknown S(3) subpocket that is created by the inhibitor. This structure has served an important role in the design of newer beta-secretase inhibitors.

␤-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid ␤ peptide A␤42, the putative neurotoxic species in Alzheimer's disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC 50 ϳ 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose-and timedependent lowering of brain A␤40 and A␤42. BACE1 inhibition also led to a robust brain secreted (s)APP␤ lowering that was accompanied by an increase in brain sAPP␣ levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (Ϫ/Ϫ) mice, in agreement with previous studies, this effect was also observed in 15-dayold heterozygous (ϩ/Ϫ) mice, but it was not evident in 30-dayold and 2-year-old BACE1 KO (Ϫ/Ϫ) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant A␤ lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major ␤-site cleavage enzyme for APP and that its inhibition can lower brain A␤ and redirect APP processing via the potentially nonamyloidogenic ␣-secretase pathway, without significantly altering NRG-1 processing.