Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jun;28(4):170-179.
doi: 10.1016/j.slasd.2023.03.003. Epub 2023 Mar 17.

A novel fluorogenic reporter substrate for 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2): Application to high-throughput screening for activators to treat Alzheimer's disease

Ramya Visvanathan  1 , Tadanobu Utsuki  2 , Daniel E Beck  2 , Emma Lendy  3 , Kuai-Lin Sun  4 , Yinghui Liu  4 , Kirk W Hering  4 , Andrew Mesecar  3 , Zhong-Yin Zhang  1 , Karson S Putt  5
Affiliations

A novel fluorogenic reporter substrate for 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2): Application to high-throughput screening for activators to treat Alzheimer's disease

Ramya Visvanathan et al. SLAS Discov. 2023 Jun.

Abstract

A rare coding variant in PLCγ2 (P522R) expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type. This mutation is reported to be protective against the cognitive decline associated with late-onset Alzheimer's disease (LOAD) and therefore, activation of wild-type PLCγ2 has been suggested as a potential therapeutic target for the prevention and treatment of LOAD. Additionally, PLCγ2 has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLCγ2 activity have been identified. Here, pharmacological inhibition may provide a therapeutic effect. In order to facilitate our investigation of the activity of PLCγ2, we developed an optimized fluorogenic substrate to monitor enzymatic activity in aqueous solution. This was accomplished by first exploring the spectral properties of various "turn-on" fluorophores. The most promising turn-on fluorophore was incorporated into a water-soluble PLCγ2 reporter substrate, which we named C8CF3-coumarin. The ability of PLCγ2 to enzymatically process C8CF3-coumarin was confirmed, and the kinetics of the reaction were determined. Reaction conditions were optimized to identify small molecule activators, and a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed with the goal of identifying small molecule activators of PLCγ2. The optimized screening conditions allowed identification of potential PLCγ2 activators and inhibitors, thus demonstrating the feasibility of this approach for high-throughput screening.

Keywords: Alzheimer's disease; C8CF3-coumarin; High throughput screening; PLCG2; Phospholipase C; Turn-on fluorophore.

PubMed Disclaimer

Conflict of interest statement

Conflicts of interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Fig. 1.
Fig. 1.
Enzymatic reactions of PLCγ2 and C8CF3-coumarin. (A) Scheme of enzymatic reaction of C8CF3-coumarin. (B) Enzymatic reaction profiles of C8CF3-coumarin (4 μM) with various concentrations of PLCγ2. (C) The slope (30 min) from the initial linear range of reaction profiles. The values are shown as the mean ± SD from three different experiments. (D) ATP titrations with PLCγ2 (2.5 nM) and C8CF3-coumarin (4 μM). Results were represented with relative slope of initial linear range (30 min) in reaction profiles (n == 3). (E) Average enzymatic profile from all runs of PLCγ2-WT (2.5 nM), PLCγ2-P522R (2.5 nM) and C8CF3-coumarin (4 μM). The values are shown as the mean ± SD from three different experiments.
Fig. 2.
Fig. 2.
Kinetics profiles in enzymatic reactions of PLCγ2 and PLCγ1 with C8CF3-coumarin. (A) PLCγ2 (1 nM) and C8CF3-coumarin (5–100 μM) (B) PLCγ1 (2 nM) and C8CF3-coumarin (5–100 μM). Results were represented with slope of initial linear range (30 min) in reaction profiles. The values are shown as the mean ± SD from four different experiments.
Fig. 3.
Fig. 3.
Profiles of Z’ score in enzymatic reactions of PLCγ2 with C8CF3-coumarin. PLCγ2 – 0 nM (green color), 2.5 nM (blue color) and 5.0 nM (red color) with C8CF3-coumarin (4.0 μM). Results represent the slope of the initial linear range (30 min) in the reaction profiles.
Fig. 4.
Fig. 4.
LOPAC1280 library screening. Doseresponse profiles of four compounds with and without sodium cholate (0.4% w/v) in enzymatic reactions of PLCγ2 and C8CF3-coumarin. Results represent the slope of the initial linear range (30 min) in the reaction profiles. The values are shown as the mean ± SD from three different experiments. Each experiment had four well replicates.**P < 0.01, * P < 0.05 represents significant differences versus the control group.
Scheme 1.
Scheme 1.
Novel synthesis of intermediate 2. Reagents and conditions: (a) NaH, 1-iodooctane, DMF, THF, 0 °C to rt to 50 °C, 29%.
Scheme 2.
Scheme 2.
Completion of novel substrate C8CF3-coumarin. Reagents and conditions: (a) triphosgene, NEt3, DCE, 90 °C; (b) NEt3, DMAP, 4 Å molecular sieves, DCM, rt, 86%; (c) i. TMSBr, DCM, 5 °C to rt; ii. MeOH, rt; iii. aq TEAB, rt, 71%.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Sims R, et al. Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial–mediated innate immunity in Alzheimer’s disease. Nat Genet 2017;49:1373–84. - PMC - PubMed
    1. Marioni RE, et al. GWAS on family history of Alzheimer’s disease. Transl Psychiatry 2018;8:99. - PMC - PubMed
    1. Bellenguez C, et al. New insights into the genetic etiology of Alzheimer’s disease and related dementias. Nat Genet 2022;54:412–36. - PMC - PubMed
    1. Efthymiou AG, Goate AM. Late onset Alzheimer’s disease genetics implicates microglial pathways in disease risk. Mol Neurodegener 2017;12:43. - PMC - PubMed
    1. Magno L, et al. Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alzheimer’s Res Ther 2019;11:16. - PMC - PubMed

Publication types