Study On The Interactions Of Natural Drug Molecules With Heme Proteins By Mass Spectrometry-based Experimental And Calculation Methods

In various diseases,drugs bind to intracellular proteins to achieve therapeutic goals or regulate therapeutic effects.These interactions are via covalent or non-covalent bindings.Among them,the non-covalent interaction between drugs and proteins has important effects on the free concentration,distribution,metabolism,and toxicity of drugs.It is the basis of many biological processes and pharmacological mechanisms.It can assess the mediation of drugs at the protein level,and is of great significance in designing new therapies for the treatment of protein-related diseases.It also provides references for research on biosafety,drug response and efficacy.The non-covalent interaction involves the changes in energy and structure of proteins.The determination of the binding constant,number,mechanism,and mode are key factors in analyzing the association.Myoglobin(Mb)is a biomarker for early tumor diagnosis,kidney injury and acute myocardial infarction.Cytochrome C(Cyt C)is an intracellular regulator that acts on oxidative stress,metabolism and apoptosis in cells.The two proteins have unique ligand binding properties and are of importance in pathology,pharmacology and clinical diagnosis,so that they are ideal model proteins for the study of ligand-protein complexes.Natural products are an important source of lead compounds and clinical drugs,and about 40% of clinical drugs are natural products or natural product derivatives.They have rich pharmacological effects,pharmacological mechanisms and chemical diversity.Therefore,the study on natural products with good pharmaceutical properties combining with Mb and Cyt C would be helpful for the discovery and development of new therapeutic drugs,and various protein biomarkers acting on natural products involved in diseases and natural product-derived drugs.In this paper,a new multiple analysis method,combining mass spectrometry,spectroscopy,and theoretical calculations,namely Native ESI-MS,HDX-MS,CD,FS and MD,has been developed to systematically study the interactions of Mb,Cyt C and of the natural products with similar skeleton structures.The structure-activity relationship between the molecular structures of different natural product ligands and protein binding ability was studied.The experimental and calculation results had good consistency,revealing the possible binding location and binding manner of ligand and protein.1)The interactions of Mb with four indole alkaloids(IAs,extracted from Ophiorrhiza japonica)were studied by Native MS.The experiment conditions,electrospray ionization source parameters,and the addition of suitable stabilizer on the ion intensity of protein complexes were investigated.By ESI-MS analysis,under the same experimental conditions and instrument parameters,the addition of 0.1% DMSO led to the charge distribution of Mb shifting to lower charged ions.As for the binding of Mb with IAs,Ophiorrhine A,Ophiorrhine B,Compound C,Compound D bound with Mb to form Mb-IA complexes.Comparing the calculated binding constants by ESI-MS data,the order of the binding ability of four IAs and Mb was as Ophiorrhine B > Compound C > Ophiorrhine A > Compound D.MD,complemented MS measurement,probed that the binding sites and binding modes of four IAs to Mb.CD and HDX-MS proved that Mb binding with IAs could change the conformation of Mb by reducing the α-helix content of Mb and more deuteration numbers.2)The interactions of Mb and four Torreya fargesii Franch extracts were studied.It was observed that LMT-2,LMT-11,LMT-14,and LMT-31 could bind with Mb to form Mb-LMT complexes.The competition experiments and concentration gradient experiments of Mb-LMT compexes were verified to exclude the non-specfic interactions.By comparing the binding constants calculated by the ESI-MS data,the order of the binding ability of four LMT and Mb was LMT-14 > LMT-31 > LMT-2 > LMT-11.MD,as the supplement to the MS measurement,explored the binding sites and binding modes of the four LMT and Mb.CD results proved that the addition of LMT reduced the α-helix content of Mb,and the wavelength of the negative peak of the spectrum had red shift,which indicated that polarity around the Trp residue increased and the hydrophobicity decreased.FS results showed that after adding LMT ligands,the distance between Trp and heme in Mb increased,which suppressed the quenching reaction between them and fluorescence intensity increased significantly.3)The interactions of Cyt C with three IAs(5-oxodolichantoside,Ophiorrhine B,strictosamide)were studied.ESI-MS results observed that 5-oxodolichantoside,Ophiorrhine B,strictosamide bound with Mb,forming up to 1:2,1:2,1:3 complexes,respectively.HDX-MS of Cyt C and Cyt C-IA were measured at different HDX time,and the overall deuteration number was observed to increase slightly with the addition of the IAs ligand,indicating that Cyt C structure became less compact.The interactions of the target natural drug ligand(including IAs and Torreya fargesii Franch extracts)with Mb and Cyt C have been studied for the first time,and the top-down HDX technique has been used to explore protein conformation at the intact protein level.The analytical methods developed in this thesis can be also applied to the study of other ligand-protein complexes and the understanding of their structure-activity relationships,facilitating the discovery of new drugs,drug localization and drug design.It would provide useful information for drug pharmacology,active drug screening,and drug target protein research.