The Mechanism Of Natural Product Derivative P-13 In Inhibiting STAT3 Signaling And In Treating Human Liver Injury

The JAK/STAT signaling pathway plays key roles in immune responses and cancer development by regulating cell growth and differentiation.Discovery of JAK/STAT pathway regulators have attracted much attention.The natural compounds in Chinese herbal medicines are numerous and varied in structure,many of which have been reported to regulate the JAK/STAT signaling pathway and have anti-inflammatory,anti-tumor activities.However,the mechanism of the Chinese herbal medicines and their natural compounds has not been fully revealed.Here,we aimed to find compounds from the Chinese herbal medicines that can regulate JAK/STAT signaling pathways,and to study their mode of action,and to explore the possible molecular mechanisms of Chinese herbal medicines in treating human diseases.First,we studied the mechanism of natural compound derivative P-13 in inhibiting STAT3 signaling.By using a STAT3-responsive luciferase gene reporter assay,we screened the natural components and their derivatives from the traditional Chinese Medicinal herb Carpesiu,m abrotanoides L.We identified a very potent compound 2-desoxy-4?-propylcarbamate-pulchellin(P-13),a derivative of the natural compound 2-desoxy-4-epi-pulchellin(PCL),belonging to sesquiterpene lactones.We found that P-13 inhibited the IL-6-induced,as well as the constitutive,STAT3 activation in a dose and time-dependent manner.In vitro kinase activity analyses demonstrated that P-13 directly inhibited JAK2 kinase activity.P-13 also inhibited IFN-a induced JAK1 and TYK2 activation,suggesting it was a pan-JAK inhibitors.The inhibitory function of P-13 on STAT3 signaling could be blocked by the reducing agent dithiothreitol or glutathione,indicating the reaction between the thiol and ?,?-unsaturated carbonyl group in P-13.Further mass spectrometry analysis and computer molecules docking revealed that P-13 covalently bound to C452 in the SH2 domain of JAK2.We also discussed the selectivity of P-13 on the JAK/STAT signaling pathway.In addition,P-13 inhibited the growth of many cancer cell lines and induced apoptosis,particularly those expressing constitutively activated STAT3.It also inhibited in vivo growth of human colon cancer cell xenografts.Taken together,our findings revealed that P-13 was a novel covalent inhibitor of JAK2 containing a Michael acceptor(a,(3-unsaturated carbonyl group)and proved to be a promising anti-cancer drug candidate.Second,we discussed the mechanism of the ubiquitous compounds containing Michael acceptors(?,?-unsaturated carbonyl group),that would accumulate in a long-lived protein to cure diseases.First,the structure of ingredients from the 8609 natural plants,49 traditional anti-inflammatory Chinese herbal medicines,and 21 classic traditional anti-inflammatory decoctions were classified according to whether there is a Michael receptor.We found that about 1/3 of the compounds from 8609 natural plants;1/5 of the compounds from anti-inflammatory Chinese herbal medicines;and 1/4 of the compounds from classical traditional anti-inflammatory decoction,that contained such a structure.Nearly all formulas contain this type of compounds.It was indicated that covalent compounds were common in nature and in traditional Chinese medicine.We used the P-alk,an alkynyl-labeled natural compound derivative containing an ?,?-unsaturated carbonyl group,as a probe,to investigate the interaction time between P-alk and different targets,as well as the accumulation effect,using a CuAAC reaction.In vitro,we found that the interaction time between P-alk and proteins was short and less different.P-alk had the volume effect and cumulative effect on repeated-dosing.In vivo,P-alk was mainly enriched in the liver after oral administration.The interaction time between P-alk and proteins was not the same in different tissues.In the liver,although most proteins interacted with P-alk for a shorter time,there are still a few proteins that interacted longer with P-alk,such as the JAK2 protein.In addition,we found that P-alk was capable of reversing ConA-induced liver damage in a dose-dependent manner.Finally,a lower dose of P-alk with no effect in a single administration could accumulate on the JAK2 upon repeated-dosing and block ConA-induced liver damage.Therefore,our findings revealed that covalent compounds could produce biological effects through long-term accumulation on long-lived proteins.Our study provided a possible new mechanism for the treatment of human diseases by traditional Chinese medicinal herb at the molecular level,that was covalent accumulation.The research also provides new theoretical guidance for us to better develop and utilize Chinese herbal medicines.