Kinase experiment:

Human sRAGE is immobilized (10 μg/mL) overnight at 4℃ in 96-well microtiter plates and blocked with 3% bovine serum albumin. 125I-labeled Aβ40, HMGB1, or S100B at 5 nM in the absence and presence of various concentrations of FPS2 or FPS-ZM1 (10 to 1,000 nM) is added to the wells containing immobilized sRAGE and incubated for 1 hour at room temperature in PBS. Wells are washed with cold PBS to remove unbound radiolabeled ligands, and the radioactivity is analyzed[1].

Cell experiment:

To determine whether FPS2 and FPS-ZM1 are toxic to CHO cells, the cells are treated for 72 hours with different concentrations of inhibitors ranging from 10 nM to 10 μM. The cellular toxicity is determined using the WST-8 Assay Kit[1].

Animal experiment:

Rats: Starting from 1 week before intrahippocampal injection, FZM1 and AGEs+FZM1 rats are intraperitoneally injected with FPS-ZM1 (1 mg/kg/d at a volume of 2 mL) for 4 weeks; rats in the AGEs and the control groups are intraperitoneally injected with normal saline with the same volume for 4 weeks. Three weeks after AGEs intrahippocampal injection, the escape latency time of rats is assayed with Morris water maze test, and then all rats are sacrificed[2]. Mice: FPS2 or FPS-ZM1 are administered i.v. (1 mg/kg) via the femoral vein and arterial blood samples (30 μL) collected at 1, 5, 10, 15, and 20 minutes via the cannulated femoral artery. Plasma is separated by centrifugation at 4℃ and immediately stored at -80℃ until analysis[1].

Ki: 25 nM

FPS-ZM1 is a RAGE Inhibitor.

The receptor for advanced glycation end products (RAGE) belongs to the immunoglobulin superfamily. RAGE has an extracellular V domain binding multiple ligands. The ligand-RAGE interactions result in sustained cellular perturbation in chronic diseases including diabetes, inflammation, as well as AD.

In vitro: FPS-ZM1 was identified as a high-affinity inhibitor of the receptor for advanced glycation end products. FPS-ZM1 could block the binding of amyloid β (Aβ) protein to RAGE and inhibit Aβ40- and Aβ42-induced cellular stress in RAGE-expressing cells [1].

In vivo: Animal study found that FPS-ZM1 was nontoxic to mice and readily crossed the blood-brain barrier (BBB). In aged APPsw/0 mice overexpressing human Aβ-precursor protein, FPS-ZM1 could inhibit RAGE-mediated influx of circulating Aβ40 and Aβ42 into the brain. FPS-ZM1 bound exclusively to RAGE In brain, which inhibited β-secretase activity and Aβ production and suppressed microglia activation and the neuroinflammatory response. Moreover, the blockade of RAGE actions at the BBB and in the brain could reduce Aβ40 and Aβ42 levels in brain in aged APPsw/0 mice [1].

Clinical trial: So far, no clinical study has been conducted.

Reference:
[1] Deane, R. ,Singh, I.,Sagare, A.P., et al. A multimodal RAGE-specific inhibitor reduces amyloid β-mediated brain disorder in a mouse model of Alzheimer disease. J. Clin. Invest. 122(4), 1377-1392 (2012).