Lowering of amyloid beta peptide production with a small molecule inhibitor of amyloid-ß precursor protein dimerization.

The amyloid ß precursor protein (APP) is a single-pass transmembrane glycoprotein that is ubiquitously expressed in many cell types, including neurons. Amyloidogenic processing of APP by ß- and γ-secretases leads to the production of amyloid-ß (Aß) peptides that can oligomerize and aggregate into amyloid plaques, a characteristic hallmark of Alzheimer's disease (AD) brains. Multiple reports suggest that dimerization of APP may play a role in Aß production; however, it is not yet clear whether APP dimers increase or decrease Aß and the mechanism is not fully understood. To better understand the relationship between APP dimerization and production of Aß, a high throughput screen for small molecule modulators of APP dimerization was conducted using APP-Firefly luciferase enzyme complementation to detect APP dimerization. Selected modulators identified from a compound library of 77,440 compounds were tested for their effects on Aß generation. Two molecules that inhibited APP dimerization produced a reduction in Aß levels as measured by ELISA. The inhibitors did not change sAPPα or γ-CTF levels, but lowered sAPPß levels, suggesting that blocking the dimerization is preventing the cleavage by ß-secretase in the amyloidogenic processing of APP. To our knowledge, this is the first High Throughput Screen (HTS) effort to identify small molecule modulators of APP dimerization. Inhibition of APP dimerization has previously been suggested as a therapeutic target in AD. The findings reported here further support that modulation of APP dimerization may be a viable means of reducing the production of Aß.

Identification of novel small molecules that elevate Klotho expression.

The absence of Klotho (KL) from mice causes the development of disorders associated with human aging and decreased longevity, whereas increased expression prolongs lifespan. With age, KL protein levels decrease, and keeping levels consistent may promote healthier aging and be disease-modifying. Using the KL promoter to drive expression of luciferase, we conducted a high-throughput screen to identify compounds that activate KL transcription. Hits were identified as compounds that elevated luciferase expression at least 30%. Following validation for dose-dependent activation and lack of cytotoxicity, hit compounds were evaluated further in vitro by incubation with opossum kidney and Z310 rat choroid plexus cells, which express KL endogenously. All compounds elevated KL protein compared with control. To determine whether increased protein resulted in an in vitro functional change, we assayed FGF23 (fibroblast growth factor 23) signalling. Compounds G-I augmented ERK (extracellular-signal-regulated kinase) phosphorylation in FGFR (fibroblast growth factor receptor)-transfected cells, whereas co-transfection with KL siRNA (small interfering RNA) blocked the effect. These compounds will be useful tools to allow insight into the mechanisms of KL regulation. Further optimization will provide pharmacological tools for in vivo studies of KL.