Molecular docking virtual screening of JAK3 specific inhibitors based on TCMSP database
Janus kinases (JAKs) belong to the intracellular tyrosine kinase family, consisting of four members: JAK1, JAK2, JAK3, and TYK2. They play a central role in the signaling process of many cytokine receptors. By activating downstream target genes through signal transducers and activators of transcription (STATs), they exert their regulatory effects on cellular biological functions. The JAK-STAT signaling pathway is involved in important biological processes such as cell proliferation, differentiation, apoptosis, and immune regulation. Abnormal activation of the JAK-STAT signaling pathway can accelerate the proliferation process of cancer cells, such as acute promyelocytic leukemia, whose cell line HT93A induces differentiation and activates JAK-STAT, leading to accelerated proliferation. JAK-STAT channel is also activated by NADPH oxidase, which reduces the expression level of liver X receptor, greatly affects the expression of ATP adenosine triphosphate binding cassette (AB-CA1), inhibits the cholesterol excretion of THP-1 foam cells, and leads to the pathogenesis of atherosclerosis. At present, research in the field of medicine is mainly focused on cancer, inflammation, and cardiovascular diseases, among which inflammatory diseases mainly include ulcerative colitis, rheumatoid arthritis, and Crohn’s disease.
As an important protein in the human body, JAK protein has many small molecule inhibitors designed to target it. However, most of them are in different stages of clinical research due to low oral bioavailability and poor selectivity. So far, only two JAK inhibitors have been approved by regulatory authorities for marketing. Tofacitinib is a novel JAK1/3 inhibitor developed by Pfizer Inc. in the United States. It was approved for market by the US FDA on November 6, 2021, and later by the Japanese PMDA on March 25, 2013. Tofacitinib, as a first generation JAK inhibitor, blocks cytokine signal transduction by inhibiting downstream signals of cytoplasmic receptors, thereby interfering with the JAK-STAT signaling pathway and affecting DNA transcription. It has played a significant role in anti proliferation and anti-inflammatory effects. Although the drug has been widely used in clinical practice and has good therapeutic effects, it has also produced adverse reactions, including but not limited to gastrointestinal discomfort, nausea, vomiting, anemia, fever, upper respiratory tract infections, elevated liver enzymes and blood lipids, and in severe cases, respiratory distress. Not only that, the combination of tofacitinib with potent immunosuppressants such as tacrolimus and cyclosporine also increases the risk of immunosuppression, leading to exacerbation of drug side effects.

JAK3 is the only family member in the JAK family associated with the cytokine receptor gamma c-chain. Due to the extremely important role of JAK3 in cytokine signaling and its expression only in specific tissues, inhibiting JAK3 activity leads to immune suppression without causing further abnormal physiological changes, making JAK3 an important target for studying immunosuppressants. Therefore, there is still an urgent need to develop JAK3 inhibitor drugs with definite therapeutic effects and mild toxic side effects. Through molecular docking virtual screening, small molecules of traditional Chinese medicine components with similar binding sites to tofacitinib are expected to obtain JAK3 specific inhibitors.

The above docking results show that the binding ability of small molecule glutaric acid, folic acid, secologanin, crotonoside, 2-O – (β – D-glucopyranosyl) – ascorbic acid (2-O – (β – D-glucopyranosyl) – ascorbic acid), tryptamine, and vulganthin-I, which are Chinese herbal ingredients, to JAK3 kinase is stronger than that of Tofacitinib, and they can be used as candidate JAK3 kinase inhibitors. A summary analysis of the binding sites revealed that the amino acid residues Arg935, Asp967, Lys830, Ala966, and Asn954 in the active cavity of JAK3 kinase are prone to form hydrogen bonds with the H and O atoms of small molecule inhibitors, which is crucial for the development and design of JAK3 inhibitors.
Among the seven small molecules, secologanin has poor binding affinity with other JAK family proteins and exhibits high selectivity for binding to JAK3 proteins. It has the value of further research and design as a JAK3 kinase specific inhibitor. Malvaceae glycoside is found in plants such as honeysuckle and Malvaceae. Honeysuckle (Lonicera japonica Flos) is a traditional Chinese medicine in China, which has the effects of enhancing immunity, anti-tumor, lowering blood sugar and lipid levels, anti-inflammatory, and antipyretic. It has a long history of medicinal use and has a wide range of clinical applications; Strychnosnux vomica L. has been found to affect the JAK-STAT pathway associated with the onset of rheumatoid arthritis. According to the results of this study, catarrhizin may have a pharmacological mechanism similar to tofacitinib, which inhibits JAK phosphorylation by binding to JAK3 protein, thereby inhibiting STAT phosphorylation, blocking the JAK-STAT signaling pathway, inhibiting the synthesis and secretion of various inflammatory cytokines, and achieving anti-inflammatory and immune regulatory effects. Tofacitinib can exert inhibitory effects on both JAK1 and JAK3 kinases, while the binding of kaempferol to JAK3-STAT is highly selective, making it more specific as a JAK3 kinase inhibitor. In addition, paeoniflorin is also a commonly used component of traditional Chinese medicine such as honeysuckle, which has the characteristics of greenness, easy accessibility, and minimal toxic side effects. It is worth further research as a JAK3 specific inhibitor.