CN104434909A - Application of desoxypodophyllotoxin as insulin-like growth factor acceptor antagonist - Google Patents

Abstract

The invention provides an application of desoxypodophyllotoxin as an insulin-like growth factor acceptor antagonist, which comprises the following steps: taking a 384 low volume white board as a reaction container, adding 2mumL enzyme solution, 2mumL substrate solution, 4mumL buffer (or 4mumL compound solution) and 2mumL ATP solution in each pore of a reaction system, wherein reaction system sum is 10mumL, reacting for assigned time under room temperature, adding 5mumL antibody and 5mumL allophycocyanin when the reaction stops, incubating for 1 hour at room temperature and then using Envision for detection, primary screening the compound after the model is established, secondary screening on the compound with good primary screening activity, and employing Prism5.0 (Graphpad Software, USA) statistics analysis software for calculating the active compound IC50. The application has the advantages that 1)fluorescence ratio obtained through determination is used for calculating inhibition rate of compound to kinases, thereby the strong and weak inhibition effect of compound to kinases can be determined; 2)sensitivity of the fluorescence detection value is higher, detection result accuracy is increased; and 3)analysis result is stable and reliable, and reappearance is good.

Description

A kind of deoxypodophyllotoxin is as IGF-1 antagonist purposes

Technical field

The present invention relates to the purposes of a kind of deoxypodophyllotoxin class for IGF-1 inhibitor.

Background technology

Deoxypodophyllotoxin is extraction purification and the compound obtained from Chinese medicine Chinese podophyllum root Sinopodophyllum emodi (Wall.), it is clear and definite to tumor cell In Vitro Anti proliferation function (Planta Medica that research shows that it has, 1993,59 (3): 246-249.), epoxidase, lipid peroxidase inhibition effect (Biol.Pharm.Bull, 2004,27 (6): 786-788.) and mice acetic acid twisting analgesic activity (PharmBiol.2013; 51 (5): 566-72.).

IGF-1 (insulin-like growth factor 1 receptor, IGF1R) receptor type tyrosine kinase (PTK) is belonged to, participate in regulating multiple process such as cell survival, propagation, apoptosis, migration, and play an important role in the drug resistance of tumor neogenetic and vicious transformation and oncotherapy.IGF1R is distributed widely on human tissue cell's film, is a member of Insulin Receptor Family.The tetramer structure (α 2, β 2) be made up of 2 α subunits and 2 β subunits, alpha subunit is the outer aglucon binding sites of born of the same parents, and β subunit cross-film distributes, and has after birth exterior domain, trans-membrane region and the intracellular region containing tyrosine kinase activity.The zones of different of β subunit can mediate the different biologic activity of IGFIR, promotes the formation of cell colony, makes cell obtain the biological functions such as transformation function, anti-apoptotic and mitogenesis.Preclinical study is had to show, IGF1R inhibitor and chemotherapeutics coupling can increase tumor cell to the sensitivity for the treatment of, as IGF1R micromolecular inhibitor NVP-AEW541 and chemotherapeutics vincristine, actinomycin D, when ifosfamide share, Canis familiaris L. can be increased in the sensitivity of body tumor cell to chemotherapeutics.Therefore, IGF1R becomes important antineoplaston target spot.Homogeneous phase time discrimination fluorescence technology is used in this experiment, the situation of IGF-1 activity is suppressed to carry out high flux screening to testing sample, select active lead compound preferably to enter molecular targeted antitumor drug deeply to develop, testing sample purchases ChemDiv company of U.S. high flux screening compound library.

Summary of the invention

New drug candidate is found in order to develop IGF-1 antagonist thus treat for antineoplastic, the present invention is at employing IGF-1 inhibitor high flux screening model, lead compound is found by functional authorization, find a para-insulin like growth factor acceptor inhibitor, for the exploitation of novel antibumor molecules targeted drug provides lead compound.The exploitation that the present invention can be this type of clinical antineoplastic medicine provides lead compound, for the exploitation of new antitumoral molecular targeted agents is given a clue and experimental basis.

Technical scheme of the present invention is: adopt homogeneous phase time discrimination fluorescence method establishment external IGF-1 antagonist high flux screening model, primary dcreening operation, and multiple sieve has found a kind of drug candidate with anti-tumor activity.Concrete steps are as follows:

Step one: set up IGF-1 inhibitor high flux screening model.

Step 2: positive drug test and model stability checking.

Step 3: set up high flux screening experimental program and carry out relevant testing sample screening active ingredients.

Accompanying drawing illustrates:

Fig. 1: IGF1R kinase concentration gradient optimizing experiment.

The time-optimized experiment of Fig. 2: 1GF1R kinase reaction.

Fig. 3: 1GF1R kinase substrate concentration optimization experiment.

Fig. 4: 1GF1R kinases ATP concentration optimization experiment.

Fig. 5: positive drug staurosporine amount effect curve.

Fig. 6: deoxypodophyllotoxin suppresses curve.

Detailed description of the invention

Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described:

1 detection method

This experiment adopts homogeneous phase time discrimination fluorescence method to detect reaction system, the method is divided into two steps: one is enzymatic reaction, namely after adding ATP, start reaction, kinases makes substrate generation phosphorylation reaction, has been connected to by phosphate radical on biotin labeled substrate, two is stop and testing process, EDTA terminates the carrying out of reaction in this process, there is europium rubidium marking anti phosphotyrosine antibody on the phosphate radical of substrate, the allophycocyanin of labelling XL665 is combined on the biotin labeling of substrate, resonance energy transfer is there is and produces fluorescence at 665nm place in two fluorophors in process adjacent to each other, fluorescence is there is in free TK antibody (labelling europium ion) at 620nm place, this signal can be used as background signal, and free SA-XL665 only produces of short duration fluorescence, can be ignored by delaying detection time (detecting again for after adding terminator 1 hour).In the optimization experiment of insulin like growth factor high flux screening model, enzyme concentration optimization experiment the results are shown in Figure 1, and the time-optimized experimental result of enzyme reaction is shown in Fig. 2, and the concentration of substrate optimization time the results are shown in Figure 3, ATP concentration optimization experimental result is shown in Fig. 4, and positive compound carries out Quality Control experimental result and sees Fig. 5.

2 models are set up and screening compound

With 384 low volume white board for reaction vessel, in reaction system, every hole adds 2 μ L enzymatic solution, 2 μ L substrate solutions, 4 μ L buffer (or 4 μ L compound solutions), 2 μ LATP solution, totally 10 μ L reaction systems, react the fixed time at ambient temperature.Add 5 μ L antibody-solutions during cessation reaction again, 5 μ L allophycocyanin solution, at room temperature hatch 60min.Detect with Envision afterwards.Reagent solution wherein in experiment reaction uses BiomekNXP automatization application of sample instrument and Multidrop automatic liquid separation device to carry out application of sample.Model is built up and has been carried out primary dcreening operation to micromolecular compound to be measured afterwards, carries out multiple sieve preferably to primary dcreening operation result activity, adopts Prism5.0 (Graphpad Software, USA) statistical analysis software calculated activity Compound I C ₅₀, lead compound Activity Results is shown in Fig. 6.

IGF-1 inhibitor activity screening experiment result

Screen its structure of IGF1R acceptor inhibitor and IC of obtaining ₅₀as follows:

Claims (1)

1. the compound of claimed formula I or its pharmaceutically acceptable salt for the preparation of the purposes of IGF-1 inhibitor.