Development and Optimization of a Redox Enzyme-Based Fluorescence Biosensor for the Identification of MsrB1 Inhibitors

Abstract

MsrB1 is a thiol-dependent enzyme that reduces protein methionine-R-sulfoxide and regulates inflammatory response in macrophages. Therefore, MsrB1 could be a promising therapeutic target for the control of inflammation. To identify MsrB1 inhibitors, we construct a redox protein-based fluorescence biosensor composed of MsrB1, a circularly permutated fluorescent protein, and the thioredoxin1 in a single polypeptide chain. This protein-based biosensor, named RIYsense, efficiently measures protein methionine sulfoxide reduction by ratiometric fluorescence increase. We used it for high-throughput screening of potential MsrB1 inhibitors among 6868 compounds. A total of 192 compounds were selected based on their ability to reduce relative fluorescence intensity by more than 50% compared to the control. Then, we used molecular docking simulations of the compound on MsrB1, affinity assays, and MsrB1 activity measurement to identify compounds with reliable and strong inhibitory effects. Two compounds were selected as MsrB1 inhibitors: 4-[5-(4-ethylphenyl)-3-(4-hydroxyphenyl)-3,4-dihydropyrazol-2-yl]benzenesulfonamide and 6-chloro-10-(4-ethylphenyl)pyrimido[4,5-b]quinoline-2,4-dione. They are heterocyclic, polyaromatic compounds with a substituted phenyl moiety interacting with the MsrB1 active site, as revealed by docking simulation. These compounds were found to decrease the expression of anti-inflammatory cytokines such as IL-10 and IL-1rn, leading to auricular skin swelling and increased thickness in an ear edema model, effectively mimicking the effects observed in MsrB1 knockout mice. In summary, using a novel redox protein-based fluorescence biosensor, we identified potential MsrB1 inhibitors that can regulate the inflammatory response, particularly by influencing the expression of anti-inflammatory cytokines. These compounds are promising tools for understanding MsrB1's role during inflammation and eventually controlling inflammation in therapeutic approaches.

Keywords: circularly permuted yellow fluorescence protein; high-throughput screening; inflammation; inhibitor; methionine sulfoxide reductase B1; redox protein-based fluorescence biosensor.

Figure 1

Characteristics of RIYsense biosensor. (A) Diagram of RIYsense protein and its potential mechanism of action. (B) Schematic view of the primary structure of the active and inactive form of RIYsense. Excitation and emission spectra of reduced and oxidized RIYsense in (C) the active form and (D) the inactive form.

Figure 2

Substrate specificity of RIYsense. (A) RFI of RIYsense incubated with N-AcMetO or N-AcMet. (B) The active and inactive forms of RIYsense were incubated with N-AcMetO (oxidized) or without N-AcMetO (reduced) at different pH levels, and (C) the relative RFI was presented as the ratio of oxidized to reduced forms of both active and inactive RIYsense across various pH levels. (D) Kinetic analysis of RIYsense incubated with various concentrations of N-AcMetO. (E) Kinetic analysis of the active form of RIYsense incubated with N-AcMetO, N-AcMet, or BSA, or the inactive form of RIYsense incubated with N-AcMetO. The R/R0 value represents the ratio of the RFI at t = 0 to the current time.

Figure 3

High-throughput screening using RIYsense biosensor protein. (A) Illustration of high-throughput screening using RIYsense to identify MsrB1 inhibitors. (B) Percentage of RFI decrease obtained by incubating the compounds with RIYsense.

Figure 4

Verification of MsrB1 inhibitors. (A) Docking simulation using AutoDock Vina and (B) NADPH consumption assay to verify MsrB1 inhibitors, A06, B03, B05, and D03. (C) MST binding assay to measure the binding affinity of MsrB1 to A06, B03, B05, D03, and G05.

Figure 5

MsrB1 activity assay with various concentrations of MsrB1 inhibitors. (A) Relative enzyme activity of MsrB1 was analyzed at 10 µM, 100 µM, and 500 µM MsrB1 inhibitor concentrations. (B) Compound structure of A06 and B03 were used in this assay as MsrB1 inhibitors. A06; 4-[5-(4-ethylphenyl)-3-(4-hydroxyphenyl)-3,4-dihydropyrazol-2-yl]benzenesulfonamide and B03; 6-chloro-10-(4-ethylphenyl)pyrimido[4,5-b]quinoline-2,4-dione. All experiments were performed three times.

Figure 6

Regulation of the inflammatory response by MsrB1 inhibitors. (A) mRNA expression levels of the anti-inflammatory cytokine genes IL-10 and IL-1rn in WT BMDMs treated with MsrB1 inhibitors and in MsrB1 KO BMDMs following LPS stimulation. (B) H&E staining was used to visualize the features of acetone-treated left auricles and TPA-treated right auricles of MsrB1 KO and WT mice administered 50 mL water containing 0.5 µM inhibitors or DMSO. The scale bar represents 100 μm. All experiments were repeated two times, and the average values are presented.