The use of the cellular thermal shift assay for the detection of intracellular beta-site amyloid precursor protein cleaving enzyme-1 ligand binding.

Inhibition of the Alzheimer's disease associated protein ß-site amyloid precursor protein cleaving enzyme-1 (BACE1) remains a potential avenue for treatment of this disease. The cellular thermal shift assay (CETSA) is an attractive method of screening for protein binding molecules due to its ability to detect intracellular binding while avoiding the need to purify the protein in question. Here, the CETSA was carried out using the known BACE1 inhibitor verubecestat, where an increase in Tagg to 53.27 ± 0.89 °C from 49.53 ± 0.69 °C was observed. Three test compounds from the ChemBridge DiverSet compound library, identified to bind BACE1 using differential scanning fluorimetry, were then screened using the CETSA. Only compound C34 yielded a significant increase in Tagg (p value ≤ 0.05), indicative of intracellular binding. This is the first description of the cellular thermal shift assay being used to detect BACE1 binding molecules, with one novel BACE1 binding molecule being validated.

The amyloid precursor protein affects glyceraldehyde 3-phosphate dehydrogenase levels, organelle localisation and thermal stability.

Glyceraldehyde 3-phosphate dehydrogenase's (GAPDH) proapoptotic response to cellular oxidative stress has suspected implication for Alzheimer's disease (AD). Interestingly, the overexpression of the amyloid precursor protein (APP) can initiate oxidative stress responses within mammalian cell lines. Here, APP695 and APP770 overexpression significantly increased the level of GAPDH, while no effect was observed when the APP homologues APLP1 or APLP2 were used. Heterologous expression of APP695 was shown to increase the level of GAPDH within the cytoplasm by over 100% and within the mitochondria by approximately 50%. Moreover, a shift in organelle distribution from cytoplasm > nucleus > mitochondria in control cell lines to cytoplasm > mitochondria > nucleus in the APP695 overexpressing cell line was also observed. Further, the overexpression of APP695 increased GAPDH aggregation temperature by 3.09 ± 0.46 °C, indicative of greater thermal stability. These results demonstrate a clear correlation between APP overexpression and GAPDH levels, organelle distribution and thermal stability.

Inducing the Degradation of Disease-Related Proteins Using Heterobifunctional Molecules.

Current drug development strategies that target either enzymatic or receptor proteins for which specific small molecule ligands can be designed for modulation, result in a large portion of the proteome being overlooked as undruggable. The recruitment of natural degradation cascades for targeted protein removal using heterobifunctional molecules (or degraders) provides a likely avenue to expand the druggable proteome. In this review, we discuss the use of this drug development strategy in relation to degradation cascade-recruiting mechanisms and successfully targeted disease-related proteins. Essential characteristics to be considered in degrader design are deliberated upon and future development challenges mentioned.

The amyloid precursor protein: a converging point in Alzheimer's disease.

The decades of evidence that showcase the role of amyloid precursor protein (APP), and its fragment amyloidß (Aß), in Alzheimer's disease (AD) pathogenesis are irrefutable. However, the absolute focus on the single APP metabolite Aß as the cause for AD has resulted in APP and its other fragments that possess toxic propensity, to be overlooked as targets for treatment. The complexity of its processing and its association with systematic metabolism suggests that, if misregulated, APP has the potential to provoke an array of metabolic dysfunctions. This review discusses APP and several of its cleaved products with a particular focus on their toxicity and ability to disrupt healthy cellular function, in relation to AD development. We subsequently argue that the reduction of APP, which would result in a concurrent decrease in Aß as well as all other toxic APP metabolites, would alleviate the toxic environment associated with AD and slow disease progression. A discussion of those drug-like compounds already identified to possess this capacity is also included.

A superior loading control for the cellular thermal shift assay.

The cellular thermal shift assay (CETSA), as a method to determine protein-ligand interaction and cellular protein modification, has rapidly become routine laboratory practice. However, current options to determine that (1) sample was loaded in each lane of the analysed western blot and (2) the amount loaded was equal, are suboptimal. Here, we report that the αC-terminal fragment of the amyloid precursor protein (APP-αCTF), detected in several wild-type mammalian cell lines, is a highly stable, soluble protein equally present from 4 to 95 °C. We demonstrate that the level of traditional loading controls (vinculin, GAPDH, ß-actin, heat-shock chaperone 70 and superoxide dismutase-1) are all temperature sensitive. Additionally, both APP-CTFs (α and ß) behaved similarly upon temperature exposure while APP-ßCTF levels were not influenced by the presence of a binding ligand either. This emphasises that these proteins can be used as a loading control in the unlikely event of off-target binding during ligand screening. A working example is also presented for mitogen-activated protein kinase kinase in the presence of two inhibitors, PD184352 and U0126, where APP-αCTF was used to normalise the data across experimental replicates. A reduction in data variance and standard deviations was observed after normalisation. Conclusively, APP-αCTF is a superior CETSA loading control that can be used as a standard for this technique.

Determining the Protein Stability of Alzheimer's Disease Protein, Amyloid Precursor Protein.

Determining protein thermal stability is integral in biomedical research. Here, with the use of two thermal stability assays, we show the melting temperature of amyloid precursor protein, an Alzheimer's disease related protein. The average melting temperature for amyloid precursor protein of 55.9 °C was derived from differential scanning fluorometry (55.1 ± 0.3 °C) and cellular thermal melt (56.7 ± 0.7 °C). These experimental methods have significant application for Alzheimer's disease research including their use for amyloid precursor protein stability profiling and for the identification of additional binding partners to further elucidate novel protein functions.