Team:Nanjing high school/Proof Of Concept

Nanjing_high_school

Proof of Concept

Inflammatory neurodegeneration means neuronal death/loss caused by inflammation. It found that this process (like energy depletion, protein aggregation, and excitotoxicity) contributes to neuronal loss or dysfunction in many different neurological diseases, including Alzheimer's Disease, Amyotrophic Laternal Sclerosis, and stroke.

It is found that PRIMA is functional in reducing the expression of specific markers of M1 inflammatory macrophage, and hence inhibiting the inflammatory factors, indicating the potential anti-inflammatory function of PRIMA. Thus, we are planning to explore the anti-inflammatory function of PRIMA in Microglia, a common immune cell in our central nervous system. We choose PRIMA as our compound screening target, based on which our goal is to find a compound that may inhibit the inflammatory response in the brain.

Supporting Experiment Results

Figure 1. SDS-PAGE assay

First of all, 6×His-PPM1A was purified with a Ni-NTA column followed by AKTA FPLC according to the protocol. Then we used SDS-PAGE to test the purity of 6×His-PPM1A. As shown in Figure 1, the 6×His-PPM1A was purified successfully.

Figure 2. PPM1A enzyme activity

Next, we screened the PPM1A activator in Lab in-house compound library by phosphatase enzyme activity assay. The effect of compounds 5 (0.01, 0.1, 1, 5, 10, 20, 40, 100 μM) on the PPM1A was detected by phosphatase activity assay with pNPP as the substrate. All data were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).

As indicated in Figure 2, among the compounds, Compound 5 was finally selected for its highest enzymatic activity against PPM1A. Besides, it could also tell that compound 5 dose-dependently enhanced PPM1A enzyme activity. These results thus implied that compound 5 was a PPM1A enzymatic activator.

Figure 3. Compound 5 suppressed inflammation in BV-2 cells

Finally, the qPCR assay was further carried out to verify the inhibitive effect of compound 5 against PPM1A. BV-2 cells were co-incubated with LPS and different concentrations (5, 10, 20 μm) of compound 5 for 24 h. Then the mRNA level of IL-1β and IL-6 were detected by the qPCR assay. All data were presented as mean±S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).

As shown in Figure 3, LPS effectively increased the mRNA level of IL-1β and IL-6, and compound 5 suppressed this increase effectively. Thus, these results confirmed that the suppressive effect of compound 5 against inflammation in BV-2 cells.

The experiment results above indicated that Compound 5 was demonstrated as a PPM1A activator and its anti-inflammatory effect was determined. We will investigate the feasibility of this compound to be further developed into drugs against inflammatory neurodegeneration related diseases, such as Alzheimer's Disease, Amyotrophic Laternal Sclerosis, and stroke.