Shim Hyun Bo, Lee Hyunjeong, Cho Hwa Yeon, Jo Young Ho, Tarrago Lionel, Kim Hyunggee, Gladyshev Vadim N, Lee Byung Cheon
College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea.
College of Engineering, Institute of Green Manufacturing Research Center, Korea University, Seoul 02841, Republic of Korea.
Antioxidants (Basel). 2024 Nov 2;13(11):1348. doi: 10.3390/antiox13111348.
MsrB1 is a thiol-dependent enzyme that reduces protein methionine--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 and , 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.
MsrB1是一种硫醇依赖性酶，可还原蛋白质甲硫氨酸亚砜并调节巨噬细胞中的炎症反应。因此，MsrB1可能是控制炎症的一个有前景的治疗靶点。为了鉴定MsrB1抑制剂，我们构建了一种基于氧化还原蛋白的荧光生物传感器，该传感器由MsrB1、环状排列的荧光蛋白和硫氧还蛋白1组成，位于一条单一的多肽链中。这种基于蛋白质的生物传感器名为RIYsense，通过比率荧光增加有效地测量蛋白质甲硫氨酸亚砜的还原。我们用它在6868种化合物中高通量筛选潜在的MsrB1抑制剂。基于与对照相比能够将相对荧光强度降低50%以上的能力，共选择了192种化合物。然后，我们对化合物与MsrB1进行分子对接模拟、亲和力测定和MsrB1活性测量，以鉴定具有可靠且强效抑制作用的化合物。选择了两种化合物作为MsrB1抑制剂：4-[5-(4-乙基苯基)-3-(4-羟基苯基)-3,4-二氢吡唑-2-基]苯磺酰胺和6-氯-10-(4-乙基苯基)嘧啶并[4,5-b]喹啉-2,4-二酮。对接模拟显示，它们是具有取代苯基部分的杂环多芳族化合物，与MsrB1活性位点相互作用。在耳部水肿模型中，发现这些化合物会降低抗炎细胞因子如 和 的表达，导致耳廓皮肤肿胀和厚度增加，有效地模拟了在MsrB1基因敲除小鼠中观察到的效果。总之，通过使用一种新型的基于氧化还原蛋白的荧光生物传感器，我们鉴定出了潜在的MsrB1抑制剂，它们可以调节炎症反应，特别是通过影响抗炎细胞因子的表达。这些化合物是理解MsrB1在炎症过程中的作用并最终在治疗方法中控制炎症的有前景的工具。
