CN106699897B - Fusion protein for screening MdmX inhibitor or testing MdmX inhibitor inhibitory activity - Google Patents

Abstract

The invention discloses a fusion protein for screening an MdmX inhibitor or testing the inhibitory activity of the MdmX inhibitor, which comprises a test sequence, a binding sequence and a connecting arm sequence; wherein the test sequence is a MdmX sequence or a MdmX fragment comprising a p53 binding domain in the MdmX sequence; the binding sequence is the p53 sequence or a p53 fragment comprising the MdmX binding domain in the p53 sequence; the linker arm sequence is a peptide fragment which is linked upstream and downstream to one of said test sequence and said binding sequence, respectively, and which has an amino acid sequence which does not participate in the formation of the secondary structure of the fusion protein, and which has a length and flexibility sufficient to allow any surface portion of the steric structure of said test sequence to come into steric contact with any surface portion of the steric structure of said binding sequence. When the MdmX inhibitor competitively binds to the p53 binding domain in the binding sequence, the tryptophan in the released binding sequence is fully exposed to the aqueous phase and the emission spectra of the tryptophan differ between the two states, thereby effectively determining the degree of MdmX inhibitor inhibition.

Description

Fusion protein for screening MdmX inhibitor or testing MdmX inhibitor inhibitory activity

Technical Field

The invention belongs to the technical field of biology, and relates to preparation and application of a recombinant protein for screening an MdmX inhibitor or testing the inhibitory activity of the MdmX inhibitor, in particular to preparation of a p53 polypeptide, an MdmX amino-terminal fusion protein and an analogue thereof, and application of the p53 polypeptide, the MdmX amino-terminal fusion protein and the analogue thereof in screening the MdmX inhibitor or testing the inhibitory activity of the MdmX inhibitor.

Background

p53 is a cancer suppressor protein, plays a role in biological processes such as cell cycle arrest, DNA damage repair and apoptosis, and has become an important tumor treatment target. Inhibition of the canceration activity of p53 was inhibited by intracellular overexpression of MdmX and Mdm2, and was associated with a majority of cancers. Relieving the inhibition of p53 by MdmX and Mdm2 is a target for developing novel cancer therapeutic drugs.

MdmX and Mdm2 are important regulatory gene proteins downstream of p53, and MdmX and Mdm2 are homologous proteins, which are highly similar in structure, but have different mechanisms for inhibiting p 53. Currently, the screening and design of Mdm2 inhibitors are mature and many have entered the clinical research stage, for example, nutlin-3a modified molecule RG7112 will enter phase II clinical research (Ray-Coquardi, Blay J Y, Italiano A, et al. Effect of the MDM2antagonist RG7112on the P53pathway in Patients with MDM2-amplified, well-differentiated ordered clinical laboratory of an expression of-mechanism assay [ J ]. the clinical assay 2012,13(11): 1133-1140). However, effective screening and design of MdmX inhibitors has progressed slowly. Due to the resistance problem of Mdm2 inhibitors (Lane D P, Cheok C F, Lann S.p53-based cancer therapy [ J ]. Cold Spring Harbor therapeutics in biology,2010,2(9): a001222.), MdmX in cancer cells is further overexpressed, exacerbating the cancer cells. Therefore, the MdmX inhibitor and the MdmX/Mdm2 dual inhibitor designed based on MdmX are effective medicines for treating nearly half of cancers.

The N-terminal of MdmX has a binding domain of p53, and the p53 binding domain of MdmX can be combined with the transcription activation domain of p53 protein to form a p53/MdmX complex, which can inhibit the transcription activity of p53 but does not mediate the degradation of p53, so that the p53 protein is inactivated to induce tumor.

If a compound competes with the p53-MdmX complex to release the transcriptional activity of p53, or binds free MdmX competitively or irreversibly, the compound can release or partially release the inhibition of p53 by MdmX, so that p53 can exert a cancer-inhibiting effect, and thus the compound can be considered as a drug for treating or alleviating cancer.

Several protocols for testing MdmX inhibitors by competitively binding to MdmX with p53 are disclosed in the prior art.

Chinese patent document CN105936646 (published: 2016.09.14) discloses a kind of mini-protein and its application. The mini-protein takes a 3 alpha helical bundle of an albumin binding domain as a framework protein, introduces cationic amino acids at least one of the three-dimensional structure surface, N end and C end of the framework protein, and is connected with at least a key amino acid site for binding with MDM2/MDMX through the cationic amino acids. The mini-protein has the capacity of entering cells and combining with albumin, MDM2/MDMX, can well inhibit the interaction between p53 and Mdm2/MdmX, can be used for preparing anti-cancer drugs and the like, and can be used for large-scale production and application. Fluorescence polarization detection for binding of small proteins to Mdm 2/MdmX.

Chinese patent document CN103923067 (published: 2014.07.16) discloses a small molecule inhibitor of MdmX/Mdm2, and also relates to a preparation method of the small molecule inhibitor of MdmX/Mdm2, the small molecule inhibitor compound can inhibit the interaction of MdmX protein and p53 protein, and also can inhibit the interaction of Mdm2 protein and p53 protein, and the MdmX inhibitor screening in the method is determined by a fluorescence polarization method.

There are some deficiencies in the above model for detecting inhibitors of MdmX:

when the p53 fragment and the MdmX fragment are added to the test system separately, the molar ratio of the two fragments is difficult to determine accurately, and large systematic errors or random errors are caused.

2. The p53 fragment is labeled by fluorescein in the scheme, so that the molar ratio of fluorescein molecules to p53 fragment molecules in each labeling operation is not completely consistent, larger random errors are brought, and accurate quantitative research is more difficult to carry out.

3. In the scheme, a p53 fragment is marked by adopting fluorescein, and the fluorescein is easy to decompose and denature under visible light and loses the fluorescent function, so that the corresponding operation is required to be protected from light, the accuracy of the detection method is reduced by photodecomposition, and the operation difficulty and the experiment cost are increased by the requirement of protecting from light.

4. Due to the hydrophobic nature of fluorescein, the accuracy of the detection method and data is reduced.

The p53 fragment and MdmX need to be prepared separately, which increases the complexity of the procedure.

6. Although the Mdm2 and the Mdm2 are homologous proteins and have high similarity in three-dimensional structures, the existing Mdm2 small-molecule inhibitor has weak inhibition effect on the MdmX.

7. The same model was used for testing MdmX inhibitors of significantly different forces, failing to distinguish strong from weak MdmX inhibitors, and more importantly, the weaker MdmX inhibitors may not be detected due to their inability to effectively competitively bind the p53 domain.

Disclosure of Invention

The protein has a light absorption phenomenon in an absorption wavelength range of 270-300 nm, and the light absorption property mainly comes from tryptophan, tyrosine and phenylalanine in the protein, so that the protein containing the amino acid has a natural fluorescence property. When tryptophan is not present in the protein, the maximum emission wavelength is approximately 304nm, and the position of the fluorescence spectrum is not affected by the conformation of the macromolecule; when tryptophan exists in the protein, the maximum fluorescence emission wavelength of the protein has a clear relationship with the degree of hydrophobic environment in which the tryptophan is located, and the maximum fluorescence emission wavelength is in the range of 308-355 nm (Vivian, J.T.Callis, P.R., Mechanisms of tryptophan fluorescence shifts in proteins. HYPERLINK:// www.ncbi.nlm.nih.gov/PMC/articules/PMC 1301402/"Biophys J.2001, 80(5): 2093-.

The resolved crystal structure of the complex formed by MdmX (23-111 sequences) and p53 (15-29 sequences) (PDB ID:3DAB, see Popowicz G, Czarna A, Holak T.Structure of the human Mdmxprotein bound to the p53 promoter transcriptional domain [ J ]. CellCycle,2008,7(15):2441-3.) shows that the transcriptional activation region (15-29 sequences) of p53 interacts with the N-terminal hydrophobic region of MdmX in an alpha-helical fashion through 3 key amino acids (Phe19, Trp23 and Leu 26). The 3 key sites of action on MdmX, together with their adjacent amino acids, make up three binding pockets, namely the F19 pocket, the W23 pocket and the L26 pocket.

The domain corresponding to the N-terminal domain (23-111 sequences) of human MdmX is referred to herein as the p53 binding domain (defined as N-MdmX), and the 15-29 sequences of human p53 are referred to as the MdmX binding domain (defined as p53 p).

To solve at least the above technical problems, the present invention provides a fusion protein for screening for or testing for an inhibitory activity of a MdmX inhibitor. The core concept is as follows.

The p53p was linked to N-MdmX by a linker arm amino acid sequence (defined as XX) to form a fusion protein in which the tryptophan Trp23 of the p53p fragment (23 here representing the tryptophan originating from position 23 of the wild-type p53 protein and not the position in the fusion protein, the same applies hereinafter) is both the key amino acid for binding to N-MdmX and an endogenous fluorescent probe. Since this tryptophan is deeply buried in the hydrophobic cavity of the N-MdmX binding pocket (Popowicz G, Czarna A, Holak T. Structure of the humanMdmx protein bound to the p53 promoter activity domain [ J ]. CellCycle,2008,7(15):2441-3.) its maximum fluorescence emission wavelength is at 321nm, after the MdmX inhibitor competitively binds to the p53 binding domain of the MdmX moiety in the fusion protein, part or all of the p53p will separate from the N-MdmX, when tryptophan Trp23 is fully exposed to the aqueous phase, the fluorescence signal intensity at 321nm will vary significantly with increasing inhibitor concentration and in certain mathematical correlations, while the fluorescence emission wavelength is shifted towards 350 nm.

Therefore, the magnitude of the inhibitory activity/degree of inhibition of the MdmX inhibitor on MdmX can be calculated by measuring the change of the 321nm emitted light intensity in the fusion protein system before and after the addition of the MdmX inhibitor to be tested, and further, the fusion protein plays a role in screening the MdmX inhibitor or testing the inhibitory activity of the MdmX inhibitor.

In contrast to the prior art, the fusion protein has at least some of the following advantages:

the p53 binding domain of MdmX and the MdmX binding domain in p53 are on the same peptide chain in equal molar amounts, corresponding to a fixed relative content of the p53 moiety to MdmX that competes with the MdmX inhibitor, which reduces the random error between batch runs. Such a 1: 1 can also more accurately reflect the ability of the screened ligand to compete with the p53p moiety for binding to the hydrophobic cavity on the surface of the MdmX protein.

The fusion protein does not need to label a fluorescent probe, and errors caused by different degrees of fluorescent labeling can be avoided. Tryptophan in the fusion protein is not decomposed by visible light, the molar ratio of tryptophan to fusion protein is fixed, and the emission spectra of the corresponding tryptophan are different when p53p is in bound or free state with MdmX bound tryptophan. Therefore, no light-shielding device is required, and in the above fusion protein, the free tryptophan Trp23 is equimolar with the free p53 moiety and the MdmX inhibitor bound to the MdmX moiety (it is first assumed that the molar ratio of the MdmX inhibitor to the MdmX is 1: 1), the bound tryptophan Trp23 is equimolar with the complex of the p53 moiety and the MdmX moiety, the corresponding emitted light is about 321nm, that is, the change in the intensity of the emitted light at 321nm before and after the addition of the MdmX inhibitor corresponds to the extent of competitive binding with the MdmX inhibitor, and there is less systematic error or random error compared with the prior art.

To achieve the above object, the core technical solution for screening MdmX inhibitors or fusion proteins tested for MdmX inhibitor inhibitory activity is:

the fusion protein comprises a test sequence, a binding sequence and a connecting arm sequence;

the test sequence is a MdmX sequence or a MdmX fragment comprising a p53 binding domain in the MdmX sequence;

a mutant of a p53p sequence, a p53 fragment comprising the MdmX binding domain in the p53 sequence, or a p53 fragment comprising the MdmX binding domain in the p53 sequence which retains binding activity to MdmX;

the connecting arm sequence is a peptide segment, and the upstream and downstream of the connecting arm are respectively connected with one of the test sequence and the binding sequence; the amino acid sequence of the linker arm is not involved in the formation or maintenance of the secondary structure of the fusion protein; the length and flexibility of the linker arm are sufficient to provide any surface portion of the spatial structure of the test sequence in spatial contact with any surface portion of the spatial structure of the binding sequence.

Optionally, the fusion protein has a tag sequence upstream or downstream of the fusion protein for protein isolation and/or purification.

Further, since there are four tryptophanes in human p53(GenBank: AB082923.1), only one of them falls within its MdmX binding domain; seven tryptophans were present in the human MdmX gene (GeneBank: AF007111.1), none of which fell into the p53 binding domain. In a further technical scheme, the MdmX domain of p53 and the p53 domain of MdmX are connected through a connecting arm, and the connecting arm does not contain tryptophan. Thus, there is only a single tryptophan residue in the entire fusion protein, and the fluorescence spectrum of this tryptophan varies with the environment of the tryptophan. Specifically, when the p53 binding domain is combined with the MdmX binding domain, the only tryptophan residue in the fusion protein is hidden in the hydrophobic cavity, and the fluorescence emission wavelength is around 321 nm. When the MdmX inhibitor binds to the p53 binding domain in MdmX, the only tryptophan residue in part or all of the fusion protein is exposed to the aqueous phase, with a fluorescence emission wavelength around 350 nm. Under the condition, the 321nm emitted light intensity in the fusion protein system is measured before and after the MdmX inhibitor to be detected is added, so that the influence of tryptophan which does not change the light-emitting characteristic along with the combination of the MdmX inhibitor on the tryptophan light-emitting spectrum is avoided, and the experimental test result can be more sensitive.

Specifically, for the purpose, the technical scheme is as follows:

the fusion protein is represented by the formula p53p-XX-N-MdmX,

p53p represents the binding sequence SEQ ID NO.1, specifically: SQETFSDLWKLLPEN, respectively;

XX represents the connecting arm sequence SEQ ID NO.2, specifically: GSGSSENLYFQ, respectively;

N-MdmX represents the test sequence SEQ ID NO.3, and specifically: GSQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT, respectively;

the primary structure of amino acids in the p53p-XX-N-MdmX fusion protein indicates that only one tryptophan is present in the sequence, and that it is also the key amino acid corresponding to the interaction between the p53 polypeptide and N-MdmX, and small molecule compounds that compete with the p53 polypeptide for binding MdmX in the p53p-XX-N-MdmX fusion protein can be screened for based on the endogenous fluorescence characteristics of tryptophan.

For convenience of purification after expression of the fusion protein, a histidine tag sequence may be fused upstream of the fusion protein, and the fusion protein sequence fused with the histidine tag may be:

GSSHHHHHHGSSQETFSDLWKLLPENGSGSSENLYFQGSQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT

different MdmX inhibitors will bind to MdmX differently, and will compete well with the p53 moiety for binding to the MdmX moiety for more potent MdmX inhibitors. Whereas for a less binding-competent MdmX inhibitor it may not be sufficient to allow a detectable degree of competitive binding. Therefore, the above-mentioned fusion protein cannot effectively detect the MdmX inhibitor.

To further enable the above-described fusion proteins to detect weakly binding MdmX inhibitors, one approach is to weaken the binding force between the MdmX binding domain and the p53 binding domain by mutating the MdmX binding domain, for example by mutating one of the p53p key amino acids Phe19 or Leu26 (these 19 or 26 represent the position of the corresponding amino acid in the wild type of p53p, the same applies) that binds the MdmX binding domain to the p53 binding domain, so that the less binding MdmX inhibitor is allowed to competitively bind to the p53 binding domain with the MdmX binding domain, enabling the fusion protein to be used for screening for less binding MdmX inhibitors or testing for inhibitory activity of less binding mmx inhibitors. Using these two models of weakness, one can further screen for small molecule compounds with affinities in the micromolar concentration range.

Specifically, the technical scheme is as follows:

the binding sequence represented by p53p was mutated to: SQETASDLWKLLPEN are provided.

The binding sequence represented by p53p was mutated to: SQETFSDLWKLAPEN are provided.

The present invention also provides a nucleotide sequence capable of expressing a fusion protein for screening or testing for MdmX inhibitors for inhibitory activity as described in the various schemes above.

Alternatively, the nucleotide sequence is SEQ ID NO.04 (for expressing the base sequence of p53 p-XX-N-MdmX), SEQ ID NO.05 (for expressing p53p)^F19ABase sequence of-XX-N-MdmX) or SEQ ID NO.06 (for expression of p53p^L26ABase sequence of-XX-N-MdmX).

The present invention also provides an expression vector for screening for or testing for an inhibitory activity of a MdmX inhibitor, said expression vector being capable of expressing, by means of a host, a fusion protein for screening for or testing for an inhibitory activity of a MdmX inhibitor as described in the various schemes above.

Alternatively, the vector is a pET28a vector and the host is e.coli BL21(DE 3).

The present invention also provides the use of a fusion protein as described in each of the above protocols for screening for or testing for inhibitory activity of a MdmX inhibitor, wherein the MdmX inhibitor is an inhibitor capable of competitively binding to MdmX with P53, in screening for or testing for inhibitory activity of a MdmX inhibitor.

More specifically, nutlin-3a is used as a well-known strong Mdm2 inhibitor and has a certain inhibiting effect on MdmX, and nutlin-3a, MdmX and K of Mdm2_dValues of 28. mu.M, 0.7. mu.M (Laurie N A, Donovan S L, Shih C S, et al. Inactivity of the p53path in retinoblasma. [ J. ]]Nature,2006,444(7115): 61-6). Nutlin-3a is reasonable as a representative small molecule to identify the effectiveness of the p53p-XX-N-MdmX fusion protein model.

In addition, this model was further characterized by 32 Mdm2 small molecule inhibitors that were commercially available.

In order to achieve the purpose, the invention adopts the following technical measures:

the invention constructs p53p-XX-N-MdmX and p53p by molecular cloning^F19A-XX-N-MdmX and p53p^L26AAn expression plasmid of-XX-N-MdmX fusion protein, and is prepared by utilizing escherichia coli engineering bacteria BL21(DE3) to express and purify. Analysis of nutlin-3a and 32 Using F-7000 FluorospectrophotometerThe competitive binding capacity of each Mdm2 inhibitor and p53p-XX-N-MdmX fusion protein is used for verifying the feasibility and effectiveness of screening the inhibitor by the model. Then, the key amino acids Phe19 and Leu26 are respectively mutated into Ala to construct p53p^F19A-XX-N-MdmX and p53p^L26AThe XX-N-MdmX two fusion protein screening models verify the stability of the mutant fusion protein by using protein denaturant guanidine hydrochloride, the result also proves that the binding capacity of p53p and the MdmX amino terminal is weakened after the key amino acid mutation, and then nutlin-3a and 32 Mdm2 inhibitors are simultaneously screened by an LS45/55 type fluorescence/phosphorescence/luminescence spectrophotometer of PerkinElmer by using three models, and the screening result shows that the improved model is more effective for screening the inhibitors with low affinity.

Drawings

FIG. 1 is a tertiary structure diagram of p53p-XX-N-MdmX fusion protein.

FIG. 2 shows three-step PCR reaction conditions for constructing p53p-XX-N-MdmX fusion protein expression plasmid.

FIG. 3 shows SDS-PAGE gel (a) after purification of samples by Ni-NTA chromatography column, AKTA (b) and SDS-PAGE gel (c) after further purification by gel chromatography column (Suprenex 16/60075pg) during purification of p53p-XX-N-MdmX fusion protein.

FIG. 4 shows p53p-XX-N-MdmX fusion protein at lambda_EXAt 278nm, λ_EMFluorescence spectrum curve (a) in the range of 290-500 nm and at lambda_EMAt 321nm, λ_EXExcitation light spectrum curve (b) at 245nm to 300 nm.

FIG. 5 shows the titer of nutlin-3a as OD_280nm0.1 p53p-XX-N-MdmX fusion protein, lambda_EXAt 278nm, λ increased with nutlin-3a concentration_EMThe change curve (a) and the lambda of the fluorescence intensity in the range of 290nm to 500nm_EM(ii) a curve fitted to the trend of the fluorescence intensity peak at 321nm (b).

FIG. 6 is a graph of the competitive binding pattern of nutlin-3a to p53p-XX-N-MdmX fusion protein.

FIG. 7 is λ_EXAt 278nm, nutlin-3a (a) and p53p (b) are at λ_EMFluorescence emission spectrum curve in the range of 290 nm-500 nm.

FIG. 8 shows the OD concentration of DMSO (without nutlin-3a) as the titration concentration_280nm0.1 p53p-XX-N-MdmX fusion protein, lambda_EXAt 278nm, λ increased with DMSO concentration_EMThe change curve (a) and the lambda of the fluorescence intensity in the range of 290nm to 500nm_EM(ii) a curve fitted to the fluorescence intensity peak at 321nm (b).

FIG. 9 is a comparison of the competitive inhibition constant Ki values for nutlin-3a and 32 Mdm2 small molecule inhibitors against p53p-XX-N-MdmX fusion protein, using nutlin-3a as a control.

FIG. 10 shows p53p^F19A-XX-N-MdmX (a) and p53p^L26A-XX-N-MdmX (b) fusion protein tertiary structure simulation diagram.

FIG. 11 shows three-step PCR reaction conditions for construction of p53p-XX-N-MdmX fusion protein mutant expression plasmid.

FIG. 12 shows p53p^F19A-DNA gel during molecular cloning of XX-N-MdmX fusion protein.

FIG. 13 is a_EXAt 278nm, with increasing guanidine hydrochloride concentration, p53p-XX-N-MdmX (a), p53p^F19A-XX-N-MdmX (b) and p53p^L26AFluorescence emission spectrum of-XX-N-MdmX (c) fusion protein at lambda_EMThe variation trend is 290-500 nm.

FIG. 14 shows that the three fusion proteins are at lambda with increasing guanidine hydrochloride concentration_maxShows the trend of the peak change of fluorescence intensity at 321nm, 331nm and 330 nm.

FIG. 15 shows a p53p-XX-N-MdmX fusion protein as a control, using p53p^F19A-XX-N-MdmX and p53p^L26AThe XX-N-MdmX fusion protein model screens 32 Mdm2 small molecule inhibitors and nutlin-3a to show the change of the binding capacity of the fusion protein to the small molecule after mutation.

Detailed Description

In order to better explain the technical scheme of the invention, the following detailed description of various embodiments of the invention is combined with the accompanying drawings. The following examples are intended to further illustrate the invention but should not be construed as being limitations or restrictive thereon. Unless otherwise specified, technical features used in the embodiments may be replaced by other technical features known in the art having equivalent or similar functions or effects without departing from the inventive concept, and any combination of different modules of the present invention falls within the scope of the present invention.

Example 1: construction of p53p-XX-N-MdmX fusion protein expression plasmid

Based on the published crystal Structure of the MdmX binding domain p53p of p53 and the p53 binding domain at the amino terminus of MdmX (PDB ID: 3DAB) (see Structure of the human Mdmx protein bound to the p53 promoter transactivation domain, Popowicz, G.M., Czarna, A., Holak, T.A. (2008) CellCycle7:2441-2443), we established the following p53p-XX-N-MdmX fusion protein model.

The amino acid sequence of the p53p-XX-N-MdmX fusion protein is shown below (the mimic diagram of the tertiary structure of the protein using PymolWin after removal of the tag GSSHHHHHHGS is shown in FIG. 1):

GSSHHHHHHGSSQETFSDLWKLLPENGSGSSENLYFQGSQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT

wherein SQETFSDLWKLLPEN is derived from the MdmX binding domain sequence of p53, GSQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT is derived from the p53 binding domain sequence of MdmX, and GSGSSENLYFQ is the linker arm amino acid sequence. GSSHHHHHHGS is a tag sequence that facilitates the isolation and purification of fusion proteins, where the GSS or GS at both ends is a flexible amino acid fragment.

In order to be able to construct the above amino acid sequences by molecular cloning, the DNA sequence of N-MdmX was synthesized according to the codon bias of E.coli BL21(DE3), the nucleotide coding sequence of which is shown in SEQ ID NO. 4.

The nucleotide was ligated to pET28a plasmid (double restriction with Nco I and EcoR I) by double restriction with Nco I and EcoR I, and a recombinant plasmid containing the coding sequence of N-MdmX was obtained by molecular cloning procedures and named pET28 a-N-MdmX.

To construct and express the above fusion protein, the following primer sequences were designed based on the constructed pET28a-N-MdmX plasmid:

Primer1:5'-CAT GCC ATG GGC AGC AGC CAT CAC CAT CAT CAC CAC GGC AGC-3'

Primer2:5'-GTT TCC ACA GAT CGC TAA AGG TTT CCT GGC TGC TGC CGT GGTGAT GAT G-3'

Primer3:5'-TTA GCG ATC TGT GGA AAC TGC TGC CGG AAA ATG GCA GCG GCAGCA GCG A-3'

Primer4:5'-CGG GAT CCC TGA AAA TAC AGG TTT TCG CTG CTG CCG CTG CCA-3'

the designed primers were used to amplify the fragments according to the following system.

PCR system 1:

the PCR1 reaction conditions are shown in FIG. 2-a;

PCR System 2:

the PCR2 reaction conditions are shown in FIG. 2-b;

PCR System 3:

the PCR3 reaction conditions are shown in FIG. 2-c;

the PCR3 product was recovered in a kit, and the recovered product was digested simultaneously with Nco I and BamH I as inserts.

And (3) PCR recovery product enzyme digestion system:

and (4) recovering an enzyme digestion product Kit (OMEGA, Plasmid Mini Kit I) at 37 ℃ for 3 h.

In the three PCR reactions, no template is needed in the reaction systems of the PCR1 and the PCR2, and two primers are subjected to complementary pairing extension to form two nucleotide sequences without recovering products. The PCR3 experiment was performed by performing fusion PCR using PCR1 and PCR2 as templates. Further, a fusion nucleotide sequence containing a histidine tag, p53p and XX was obtained, and ligated to pET28a-N-MdmX plasmid after digestion.

pET28a-N-MdmX plasmid was double digested with Nco I and BamH I as a vector.

Plasmid vector restriction system:

and (4) recovering the digestion product by using a Gel cutting Kit (kang is a century, Gel Extraction Kit) at 37 ℃ for 3 h.

The Ligation reaction was carried out by mixing the vector DNA and the insert DNA at a molar ratio of 1:3, adding high-efficiency DNA ligase (TOYOBO, Ligation high Ver.2) as same as the system, and ligating at 16 ℃ for 3 h.

Transferring all the ligation products into 50 mu l of DH5 alpha (top10) escherichia coli, carrying out ice bath for 30min, immediately returning to the ice bath after carrying out heat shock for 90s at 42 ℃, carrying out activation for 2min, adding 200 mu l of LB liquid culture medium, carrying out shake culture at 37 ℃, carrying out 1h recovery at 200rpm, then coating an LB solid culture medium plate containing kanamycin, carrying out inverted culture at 37 ℃ for 12-16 h, and observing the growth condition of colonies.

And (3) carrying out PCR identification on a single colony, carrying out PCR identification on a bacterial solution, carrying out a correct band, carrying out quality-improved particle pET28a-p53p-XX-N-MdmX, carrying out enzyme digestion identification on a plasmid, carrying out plasmid sequencing, carrying out a correct sequencing result, and preserving a strain for later use.

The nucleotide sequence of sequencing result of pET28a-p53p-XX-N-MdmX is shown in SEQ ID NO. 4.

Example 2: expression and purification of p53p-XX-N-MdmX fusion protein

Plasmid-transformation of pET28a-p53p-XX-N-MdmX into E.coli BL21(DE 3); selecting single colony to 2ml LB K⁺(Kanamycin) culture medium, 37 degrees C, 200rpm overnight culture; collecting 500 μ l of the strainThe solution was transferred to 50ml LB K⁺Culturing in culture medium at 37 deg.C and 200rpm for 3 hr; transferring 50ml of bacterial liquid into 1L of LB K⁺Culturing in culture medium at 37 deg.C and 200rpm until the thallus concentration reaches OD_280nmWhen the concentration was about 0.8, IPTG (final concentration: 0.4mM) was added for induction, and the cells were collected for about 20 hours (centrifuge parameters for collecting cells were set to 3500rpm, 30min, 4 ℃).

The ultrasonic cell crusher crushes cells. According to the volume of the thallus: buffer volume 1:5, buffeA buffer (50mM Na) was added₂HPO₄200mM NaCl, 10mM imidazole, 1mM BME (β -mercaptoethanol), ph8.0), with the parameters set to: the total time is 2 min; ultrasonic time is 2 s; the gap time is 4 s; the temperature is alarmed for 24 ℃; the ultrasonic power is 40% (JY 92-IIN ultrasonic cell crusher of Ningbo Xinzhi Biotechnology GmbH). Repeating for 2-3 times until the thallus is completely broken. The cell disruption solution was aliquoted and balanced in a 50ml centrifuge tube, centrifuged at 18000rpm at 4 ℃ for 30min, and the supernatant and the precipitate were collected in a reagent bottle (tube), respectively.

Passing the crushed supernatant through AKTA pure Ni-NTA chromatographic column (FIG. 3a) and gel chromatographic column (Superdex 16/60075pg or Superdex 26/60075pg, FIGS. 3b, c), collecting purified protein, concentrating, packaging, and freezing with liquid nitrogen at 200 μ l per cell, and freezing at-80 deg.C in refrigerator.

Example 3: p53p-XX-N-MdmX fusion protein model fluorescence spectroscopy analysis

A frozen stock of 200. mu.l p53p-XX-N-MdmX protein was taken, thawed rapidly, and the protein concentration was diluted to OD with phosphate buffer_280nm0.1. 400 μ l were taken into a quartz cuvette and fluorescence scanned on an F-7000 fluorescence spectrophotometer. Taking lambda according to the endogenous fluorescence characteristics of tryptophan_EX278nm, scan λ_EMFluorescence intensity in the range of 290nm to 500nm, the maximum fluorescence intensity was found to be 321nm (FIG. 4 a). So as to determine lambda_EMAt 321nm, scan λ_EXThe fluorescence excitation spectrum curve was 245nm to 300nm, and the result is also shown in FIG. 4b_EXAt 278nm, the fluorescence intensity is maximum, so the excitation wavelength is 278 nm.

Example 4: fluorescence determination of nutlin-3a affinity for MdmX using p53p-XX-N-MdmX fusion protein

Freezing p53p-XX-N-MdmX protein at-80 deg.C, thawing rapidly, and diluting with phosphate buffer to OD_280nm0.1, 400. mu.l of the sample are placed in a quartz cuvette and lambda is measured on an F-7000 spectrofluorometer_EXIs 278nm, lambda_EMFluorescence scanning is carried out in the range of 290 nm-500 nm. Titration was performed according to the nutlin-3a (stock 2.5mM in DMSO) concentration gradient as in Table 1 below (mixing was done for each addition of nutlin-3 a).

TABLE 1 nutlin-3a titration p53p-XX-N-MdmX fusion protein concentration gradient

A graph of the competitive binding pattern of Nutlin-3a to p53p-XX-N-MdmX fusion protein, as shown in FIG. 6.

As shown in FIG. 5a, the effect of different nutlin-3a concentrations on the fluorescence spectrum of the fusion protein, the fluorescence intensity at the maximum emission wavelength of 321nm changes significantly as the nutlin-3a titration concentration increases. In FIG. 5b, a trend graph of the change of the fluorescence intensity value at 321nm with the change of the concentration of nutlin-3a is fitted to the trend curve based on the standard of the fluorescence intensity peak at 0. mu.M of nutlin-3a, and the fitting result can obtain the inhibition constant (K) of nutlin-3a to N-MdmX (K-MdmX)_iValues, competition binding dissociation constant), the fitting results are shown in fig. 5 b.

The fitting formula is: f. of_i＝I/[I+K_i(1+L_T/K_d)](refer to Chen Y, Prusoff W. relative displacement between the inhibition restriction and the restriction of an inhibition of a 50% inhibition of an enzyme reaction [ J]Biochem Pharmacol,1973,22: 3099-3108); wherein f is_iIs the partial degree of inhibition of N-MdmX competition by the inhibitor (as the relative intensity of fluorescence change, in percent, as specified below). I is increased in competitive inhibitionInhibitor concentration (μ M); ki is the competitive dissociation constant, i.e., the affinity dissociation constant of the inhibitor for competitive binding to N-MdmX with p53 p; l is_TIs the concentration of p53p in the fusion protein (. mu.M); k_dIs the dissociation constant, i.e., the affinity dissociation constant of p53p for N-MdmX.

And the degree of competitive inhibition f of the inhibitor on N-MdmX_iCan be reflected by the attenuation value of the fluorescence, i.e. f_i(a1-y)/(a1-a2) where a1 is the maximum fluorescence intensity on the fitted curve, a2 is the minimum fluorescence intensity on the fitted curve, y is any point on the fitted curve, and the formula after integration is: y ═ a1-X (a1-a2)/[ X + Ki (1+ L)_T/K_d)]。

From FIG. 5a, it can be clearly judged that as the concentration of nutlin-3a increases, there are two distinct peak-shaped fluctuations in the graph, λ_EMIs 321nm and lambda_EMIs 350nm to 450nm, wherein lambda_EMAt 321nm is a peak with a gradually decreasing fluorescence intensity, which has been clearly analyzed above, and the change in this peak is the change in the intensity of the emitted fluorescence of tryptophan. And in the range of 350 nm-450 nm

Is a side peak with gradually increased fluorescence intensity, and the side peak in the range of 350nm to 450nm is judged to be caused by the fluorescence of nutlin-3a or p53p after competitive combination with p53p through independently and respectively verifying the fluorescence intensity peak of small molecules of nutlin-3a and p53p (synthesized polypeptide containing fifteen amino acids: SQETFSDLWKLLPEN) in a phosphate system (no p53p-XX-N-MdmX, other conditions are consistent).

The same test conditions as for nutlin-3a titration of p53p-XX-N-MdmX, take lambda_EXIs 278nm, lambda_EMThe particle size is 290-500 nm, and the results are shown in FIGS. 7a (nutlin-3a) and 7b (p53 p). Shown in FIG. 7a, λ_max375nm, shown in FIG. 7b, λ_maxAt 350nm, it was determined that the side peak at the fluorescence emission wavelength of 350nm to 450nm is the intensity of the fluorescence introduced by nutlin-3a as nutlin-3a titrates, and thus is negligible in the experimental test and calculation results.

Taking 400 μ l and buffering with phosphateDiluting the solution to OD_280nmA blank control experiment without nutlin-3a was performed with p53p-XX-N-MdmX protein 0.1, except that the conditions were identical to those described above. The results of the experiment are shown in FIG. 8a (change in fluorescence intensity) and FIG. 8b (change in fluorescence intensity peak with concentration).

Nutlin-3a titration results show that the competitive binding relationship between Nutlin-3a and p53p to the binding site of MdmX obviously reduces the peak emission fluorescence intensity of tryptophan at 321 nm. The invention will be further validated with 32 small molecule compounds of known binding capacity.

Example 5: screening of small molecule compound libraries by p53p-XX-N-MdmX fusion protein fluorescence and polarized Fluorescence (FP)

(1) The inhibitors used were tested:

the library of small molecule compounds contained 32 commercially available Mdm2 small molecule inhibitors. The structure can be seen in table 2.

TABLE 2 Structure of Compounds in Small molecule Compound library and two Ks thereof_iMeasurement results

(2) Fluorescence screening of p53p-XX-N-MdmX fusion protein:

referring to the nutlin-3a titration protocol (see example 4 for details), p53p-XX-N-MdmX protein was diluted to OD with phosphate buffer _280nm400. mu.l of the resulting solution was placed in a quartz cuvette. The 32 small molecules were each titrated as a concentration gradient as in table 3 below (the 32 small molecule stock solutions were each 2.5mM in DMSO).

TABLE 332 Small molecule titration p53p-XX-N-MdmX fusion protein concentration gradient

Obtained after titration as the concentration of small molecules increases, at λ_EMThe Ki value (small molecule dissociation constant) of the fluorescence intensity peak at 321nm as a function of concentration was fitted linearly according to the formula. The Ki values for the individual molecules are specified in the third column of table 2.

The fitted Ki values for 32 small molecules were compared to nutlin-3a for summary and then compared to nutlin-3a for competitive binding. The comparative results are shown in FIG. 9.

(2) p53p and N-MdmX polarized Fluorescence (FP) screening:

the p53p polypeptide (amino acids 15-29) was labeled with fluorescein (fluorescein-GSGSSQETFSDLWKLLPEN, Flu-p53 p).

pET28a-N-MdmX was transformed into BL21(DE3) by plasmid granulation, induced expression and purification with 0.4mM IPTG (see example 2 for expression and purification method), frozen in liquid nitrogen, and frozen at-80 ℃ in a refrigerator.

The buffer for the FP experiment was PBS (137mM NaCl; 2.7mM KCl; 10mM Na)₂HPO₄；2mM KH₂PO₄；pH7.4)、0.025％Tween-20；0.5％BSA；pH7.5。

32 small molecule compounds and nutlin-3a were diluted in DMSO to a final concentration of 2 mM.

Mu.l of the small molecule compound diluted in DMSO and nutlin-3a were taken and 47. mu.l of buffer was added to dilute the small molecule to a final concentration of 0.12 mM. And (3) taking 20 mu l of the small molecule compound by using a pipette, placing the small molecule compound in a 96-well plate according to the marked sequence, and repeating each small molecule for 66 wells in total. 4 wells were prepared with DMSO under the same conditions without addition of small molecule compound as a control.

Taking N-MdmX protein to rapidly thaw at 14000rpm for 10min, and centrifuging at 4 ℃.

Preparation solution 1 contained 15nM Flu-p53p polypeptide and OD_280nmSolution 2 contained 15nM of Flu-p53p polypeptide solution, 0.1 in N-MdmX protein.

Adding solution 1 to two of 66 wells with small molecule compound and a control well without small molecule compound (as a negative control), 40 μ l per well; solution 2 was added to 2 of the control wells (as a positive control), 40 μ l per well.

The 96-well plate was centrifuged at 200g for 2min and mixed in the dark at room temperature for 30 min.

The detection is carried out by using an EnVision multi-label micropore plate detector of Perkinelmer, and the detection is carried out by taking excitation light at 555nm and fluorescence at 632 nm. The analysis formula of the detection result is as follows: percent inhibition (negative control-sample value)/(negative control-positive control) (Zhang Q, Lu h. identification of small molecules infection p53-MDM2/MDMXinteraction by fluorescence polarization [ J ]. p53Protocols,2013:95-111.), results are shown in the fourth column of table 2.

The following is an analysis of the results reflected in fig. 9 and table 2. Taking Cpd20, Cpd23, Cpd28 and Cpd31 as examples, the four compounds are designed to be one by taking 1,4-benzodiazepine-2,5- (1H, 4H) -diketone as a frameworkThe compounds (a class of Mdm2 inhibitors, Novel 1,4-benzodiazepine-2,5-diones as Hdm2 antagnones with improved cellular activity, are designed by the framework), and the Ki values of the compounds and the MdmX measured by FP are respectively 6.32 mu M, 10.33 mu M, 3.58 mu M and 10.63 mu M, and the results are matched with K for screening the four compounds by using p53p-XX-N-MdmX fusion protein_iThe values were substantially the same for 5.90. mu.M, 9.79. mu.M, 2.07. mu.M and 8.29. mu.M, respectively.

Using Cpd4 and CPd6 as examples, it can be seen from the figure that K of Cpd4 and Cpd6_iThe values are very different, with Ki values of 1.85. mu.M and 67.3. mu.M for them and MdmX, respectively, measured by FP, and 1.49. mu.M and 51.73. mu.M for the fluorimetric assay Ki of the p53p-XX-N-MdmX fusion protein, respectively, indicating that the fluorimetric method of the invention and the FP method of the prior art measure K_iThe values are also consistent.

Comparison of the results shown in Table 2 shows that the results of the titration experiments with nutlin-3a and 32 small molecules have certain errors, but the established p53p-XX-N-MdmX fusion protein model for screening suitable small molecule inhibitors is basically consistent with the results of the FP method in the prior art, which indicates that the fusion protein screening system is effective.

Example 6: plasmid construction of p53p-XX-N-MdmX fusion protein mutants

p53p^F19A-XX-N-MdmX and p53p^L26A-XX-N-MdmX is a mutant model of the p53p-XX-N-MdmX fusion protein, which is based on the p53p-XX-N-MdmX fusion protein, in which Phe19 in the p53p peptide is mutated to Ala19 and Leu26 to Ala26, respectively (19 and 26 refer to the positions of the corresponding amino acids in the wild-type p53 polypeptide). Two amino acids in three key amino acid residues combined in the three binding pockets are respectively mutated to respectively weaken the binding capacities of the p53p polypeptide segment and the MdmXN-terminal domain, and a foundation is provided for screening out a novel small molecular drug framework with weaker binding capacity.

Two mutant models were established based on the p53p-XX-N-MdmX fusion protein model as follows:

p53p^F19Athe amino acid sequence of-XX-N-MdmX is shown below (the tertiary structure is simulated by PymolWin, and the simulated diagram is shown in the figure10a, not comprising GSSHHHHHHGS): GSSHHHHHHGSSQETASDLWKLLPENGSGSSENLYFQGSQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT

p53p^L26AThe amino acid sequence of-XX-N-MdmX is as follows (the mimic of the tertiary structure using PymolWin is shown in FIG. 10b, which does not include GSSHHHHHHGS): GSSHHHHHHGSSQETFSDLWKLAPENGSGSSENLYFQGSQINQVRPKLPLLKILHAAGA QGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT

To construct the above fusion protein model, the following primer sequences were designed based on the existing plasmid pET28a-p53 p-XX-N-MdmX:

F19A-forward：5’-GGC AGC AGC CAG GAA ACC GCT AGC GAT CTG TGG AAA-3’

F19A-reverse：5’-TTT CCA CAG ATC GCT AGC GGT TTC CTG GCT GCT GCC-3’

L26A-forward:5’-AGC GAT CTG TGG AAA CTG GCG CCG GAA AAT GGC AGC-3’

L26A-reverse:5’-GCT GCC ATT TTC CGG CGC CAG TTT CCA CAG ATC GCT-3’

p53p^F19A-XX-N-MdmX fusion protein construction:

PCR system 1:

the PCR1 reaction conditions are shown in FIG. 11 a;

the DNA gel map is shown in FIG. 12 a;

PCR System 2:

the PCR2 reaction conditions are shown in FIG. 11 b;

the DNA gel map is shown in FIG. 12 b;

PCR System 3:

the PCR3 reaction conditions are shown in FIG. 11 c;

the DNA gel map is shown in FIG. 12 c;

the PCR product was recovered, and the recovered product was digested simultaneously with Nco I and EcoR I to prepare an insert.

This is fusion PCR. Two nucleotide sequences were obtained by PCR1 and PCR2, and PCR3 was a fusion PCR and was mutated for only a single amino acid.

And (3) PCR recovery product enzyme digestion system:

and (4) recovering the enzyme digestion product kit at 37 ℃ for 3 h.

pET28a-N-MdmX plasmid was digested simultaneously with Nco I and EcoRI, and the resulting product was used as a vector after cutting the gel.

And (3) performing ligation reaction according to the molar ratio of the vector DNA to the insert DNA of 1:3, mixing, adding high-efficiency ligase (TOYOBO, Ligation high Ver.2) which is the same as the system, and connecting for 3 hours at 16 ℃.

Transferring all the ligation products into 50 mu l of DH5 alpha (top10) escherichia coli, carrying out ice bath for 30min, immediately returning to the ice bath after carrying out heat shock for 90s at 42 ℃, carrying out activation for 2min, adding 200 mu l of LB liquid culture medium, carrying out shake culture at 37 ℃, carrying out 1h recovery at 200rpm, then coating an LB solid culture medium plate containing kanamycin, carrying out inverted culture at 37 ℃ for 12-16 h, and observing the growth condition of colonies. Extraction of pET28a-p53p^F19AAnd (4) carrying out sequencing on the-XX-N-MdmX plasmid, wherein the sequencing result is correct, and the strain is preserved. p53p^F19A-XX- -N-MdmX with p53p^L26AMolecular cloning of-XX-N-MdmX was performed under identical conditions except for the primer sequence during PCR.

p53p^F19AThe nucleic acid coding sequence of-XX-N-MdmX is shown in SEQ ID NO.5, p53p^L26AThe nucleic acid coding sequence of-XX-N-MdmX is shown in SEQ ID NO. 6.

Example 7: p53p^F19A-XX-N-MdmX fusion protein and p53p^L26AExpression and purification of (E) -XX-N-MdmX fusion protein

pET28a-p53p^F19A-XX-N-MdmX and pET28a-p53p^L26A-XX-The N-MdmX plasmid was chemically transformed into E.coli BL21(DE3), respectively; selecting single colony to 2ml LB K⁺Culturing in culture medium at 37 deg.C and 200rpm overnight; 500. mu.l of the bacterial solution was transferred to 50ml of LB K⁺Culturing in culture medium at 37 deg.C and 200rpm for 3 hr; transferring 50ml of bacterial liquid into 1L of LB K⁺Culturing in culture medium at 37 deg.C and 200rpm until the thallus concentration reaches OD₂₈₀When the concentration was about 0.8, IPTG (final concentration: 0.4mM) was added for induction, and the cells were collected for about 20 hours (centrifuge parameters for cell collection were set to 3500rpm, 30min, 4 ℃).

The ultrasonic cell crusher crushes cells. According to the volume of the thallus: buffer volume 1:5, buffeA buffer (50mM Na) was added₂HPO₄200mM NaCl, 10mM imidazole, 1mM BME, PH8.0), parameters set to: the total time is 2 min; ultrasonic time is 2 s; the gap time is 4 s; the temperature is alarmed for 24 ℃; the ultrasonic power is 40%. Repeating for 2-3 times (observing the viscosity degree of the crushed bacteria to determine the crushing times) until the bacteria are completely crushed. The cell disruption solution was aliquoted and balanced in a 50ml centrifuge tube, centrifuged at 18000rpm at 4 ℃ for 30min, and the supernatant and the precipitate were collected in a reagent bottle (tube), respectively.

Passing the supernatant through AKTA pure Ni-NTA chromatographic column and Gel-filtration (sSuperdex 16/60075pg or Superdex 26/60075pg), collecting purified protein, concentrating, packaging, quick freezing with liquid nitrogen, and freezing at-80 deg.C.

Example 8: verification of p53p-XX-N-MdmX, p53p^F19A-XX-N-MdmX and p53p^L26AStability variability of the three-XX-N-MdmX

At room temperature, 3-4 mol/L guanidine hydrochloride can make the protein change from a natural state to a denatured state, usually increasing the concentration of a denaturant can increase the degree of denaturation, and usually 6mol/L guanidine hydrochloride can make the protein completely change to the denatured state.

P53p compared to p53p-XX-N-MdmX fusion protein^F19A-XX-N-MdmX fusion protein and p53p^L26Athe-XX-N-MdmX fusion protein is formed by changing Phe19 to Ala19 and Leu26 to Ala26 of a p53p polypeptide fragment on the basis of p53 p-XX-N-MdmX. Two amino acids among the three key amino acid residues bound in the three binding pockets are mutated separatelyIn addition, the binding capacity of p53p and the MdmX N-terminal domain is weakened respectively, so that the stability of the fusion protein is weakened, and the small molecule inhibitor with a novel framework structure and weak competitive binding capacity is screened.

100ml of guanidine hydrochloride solution having a concentration of 8M was prepared with ultrapure water.

Take 4 200. mu.l/branch of p53p-XX-N-MdmX (OD)₂₈₀1.0), 3p 53p of 200. mu.l/branch^F19A-XX-N-MdmX(OD₂₈₀1.38), 2 p53p at 200. mu.l/branch^L26A-XX-N-MdmX(OD₂₈₀2.0) fusion protein was thawed fast, mixed, 14000rpm, 10min, and centrifuged.

P53p-XX-N-MdmX and p53p were prepared at different guanidine hydrochloride concentrations according to the following ratios in Table 4, Table 5 and Table 6^F19A-XX-N-MdmX and p53p^L26A-XX-N-MdmX fusion protein solution.

TABLE 4 solution of p53p-XX-N-MdmX fusion protein at 0-4M guanidine hydrochloride concentration

TABLE 5p 53p^F19A-N-MdmX fusion protein solution at 0-4M guanidine hydrochloride concentration

TABLE 6 p53p^L26A-N-MdmX fusion protein solution at 0-4M guanidine hydrochloride concentration

According to the guanidine hydrochloride concentration gradient, 400 μ l of sample is put into a quartz cuvette, the cuvette is put into an F-7000 fluorescence spectrophotometer, and the lambda is taken_EXIs 278nm, lambda_EMTaking 290-500 nm, p53p-XX-N-MdmX and p53p according to the concentration change of guanidine hydrochloride^F19A-XX-N-MdmX and p53p^L26AThe fluorescence intensity profiles obtained for-XX-N-MdmX are shown in FIGS. 13a, b and c.

The results of the fluorescence intensity peak values of the three fusion proteins as a function of the guanidine hydrochloride concentration were collated as shown in FIG. 14, based on the change in the fluorescence intensity peak values:

according to FIGS. 13a,13b and 13c, the peak of the fluorescence emission wavelength is p53 p-XX-N-MdmX: lambda [ alpha ]_EM＝321nm、p53p^F19A-XX-N-MdmX：λ_EM331nm and p53p^L26A-XX-N-MdmX：λ_EMThe tryptophan on p53p can be judged to be combined in the hydrophobic cavity of N-MdmX according to the depth sequence of the tryptophan combined in the hydrophobic cavity of the N-MdmX and the sequence of p53p-XX-N-MdmX and p53p in the order of 330nm^L26A-XX-N-MdmX、p53p^F19A-XX-N-MdmX, i.e. the strength of the stability of the fusion protein can be preliminarily judged as follows: p53p-XX-N-MdmX, p53p^L26A-XX-N-MdmX and p53p^F19A-XX-N-MdmX。

FIG. 14 shows that the peak fluorescence intensity of the three fusion proteins varies with the concentration of guanidine hydrochloride, which is a protein denaturing agent, and the peak fluorescence intensity IC is analyzed₅₀It was found that when the peak fluorescence intensity was reduced by half, the required guanidine hydrochloride concentrations were in the order of: p53p-XX-N-MdmX, p53p^L26A-XX-N-MdmX、p53p^F19A-XX-N-MdmX, i.e.the strength of the stability of the fusion protein can be further judged as follows: p53p-XX-N-MdmX, p53p^L26A-XX-N-MdmX and p53p^F19A-XX-N-MdmX。

The results show that p53p^L26A-XX-N-MdmX and p53p^F19Athe-XX-N-MdmX structure is less stable than the p53p-XX-N-MdmX structure, indicating that the former has a weaker affinity between the two domains and is therefore more readily competitively bound by the less avidity MdmX inhibitor.

This indicates the use of p53p^L26A-XX-N-MdmX and p53p^F19AIt is theoretically possible to screen for small molecules with weak affinity by using-XX-N-MdmX as a screening model.

Example 9: p53p-XX-N-MdmX, p53p using the 32 small molecules and nutlin-3a^F19A-XX-N-MdmX and p53p^L26ATitration was performed by-XX-N-MdmX to verify the change in binding capacity.

The fusion protein was thawed rapidly at 14000rpm for 10min, centrifuged and the precipitated protein due to denaturation was discarded.

The concentration of the small molecule mother solution is 10mM, and 1. mu.l of the small molecule mother solution is diluted to 1mM by adding 9. mu.l of DMSO.

According to the final concentration of small molecules of 10 μ M and the protein concentration of OD_280nmThe solution was added to a 96-well black plate at this concentration (with the same volume of DMSO added as a blank) and mixed in a refrigerator at 4 ℃ for 2 h. Reading the fluorescence data of a 96-well plate by using a Perkinelmer LS-45/55 fluorescence/phosphorescence/luminescence spectrophotometer, and firstly taking lambda_EXAt 278nm, p53p-XX-N-MdmX lambda_EMAt 321nm, p53p^F19A-XX-N-MdmXλ_EMAt 331nm, p53p^L26A-XX-N-MdmXλ_EMThe fluorescence intensity value of the three fusion proteins can be read when the excitation light is 278nm and is 330nm, the excitation grating is 15nm, the fluorescence grating is 20nm, and the threshold value (cut off) is 290 nm. Then by λ_EXAt 278nm, p53p-XX-N-MdmX, p53p^F19A-XXN-MdmX、p53p^L26A-N-MdmXλ_EMAt 375nm, the threshold (cut off) of 350nm can be read to the fluorescence intensity value of 278nm of the excitation light for the three fusion proteins. The measurement is carried out every 4h, and the measurement is repeated twice each time.

The 96-well plate can simultaneously test the inhibition effect of 32 inhibitors and nutlin-3a on three fusion proteins at most once (a group of parallel experiments can be performed).

Taking p53p-XX-N-MdmX fusion protein as an example, at lambda_EM321nm, and the fluorescence intensity read at a cutoff wavelength of 290nm is the fluorescence intensity after addition of the small molecule at lambda_EMThe fluorescence intensity read when the cutoff wavelength is 350nm is 375nm, the fluorescence intensity read when the cutoff wavelength is 350nm is the fluorescence intensity value introduced by nutlin-3a (or other compounds), the difference value of the fluorescence intensity values read twice is the fluorescence intensity change value of tryptophan after the micromolecules are added, the fluorescence intensity value after the DMSO with the same volume is added is used as a blank control, the difference value between the fluorescence intensity change value of tryptophan after the micromolecules are added and the blank control is the free degree of tryptophan caused by competitive combination with p53p after the micromolecules are added, the larger the negative value is, the larger the free degree of tryptophan is, the stronger the binding capacity of the micromolecules is, the difference value is taken as the ordinate, and if the difference value is the positive value, the tryptophan binding enhancement is realizedI.e., there may be interactions that make the N-MdmX domain more stable, the affinity of p53p and N-MdmX is enhanced. The results of this experiment are shown in FIG. 15, with the small molecule numbers as the abscissa, and 1# -32# and nutlin-3a in this order.

The results in FIG. 15 show that the negative results for p53p-XX-N-MdmX are less than for p53p for single small molecules^F19A-XX-N-MdmX and p53p^L26ANegative values of-XX-N-MdmX, indicating that the ability of the same small molecule to compete for the p53 moiety in p53p-XX-N-MdmX is weaker than that of p53p^F19A-XX-N-MdmX and p53p^L26AXX-N-MdmX, which indicates that the binding strength of the p53 binding domain to the MdmX binding domain is greater in p53p-XX-N-MdmX, and the result is consistent with the design object of the present invention.

At the same time, the same inhibitor molecule pair p53p^F19A-XX-N-MdmX and p53p^L26AThe stronger competitive binding of the N-MdmX moiety in-XX-N-MdmX, meaning p53p^F19A-XX-N-MdmX and p53p^L26A-XX-N-MdmX may be suitable for screening or testing of small molecule inhibitors that bind less strongly to the N-MdmX moiety. This further illustrates the results consistent with the design objectives of the present invention. Furthermore, the affinity of the p53p-XX-N-MdmX fusion protein screening model in FIG. 15 for 32 small molecule inhibitors is substantially identical to that reflected in FIG. 8, further illustrating the effectiveness of the model.

The above embodiments are only used for further illustration of the present invention, and are not intended to limit the scope of the present invention, and all equivalent modifications and obvious modifications made based on the concept of the present invention fall within the scope of the present invention.

SEQUENCE LISTING

<110> Hubei university of industry

<120> A fusion protein for screening or testing the inhibitory activity of MdmX inhibitor

<130>2016-12-07

<160>6

<170>PatentIn version 3.5

<210>1

<211>15

<212>PRT

<213> Artificial sequence

<400>1

Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn

1 5 10 15

<210>2

<211>11

<212>PRT

<213> Artificial sequence

<400>2

Gly Ser Gly Ser Ser Glu Asn Leu Tyr Phe Gln

1 5 10

<210>3

<211>91

<212>PRT

<213> Artificial sequence

<400>3

Gly Ser Gln Ile Asn Gln Val Arg Pro Lys Leu Pro Leu Leu Lys Ile

1 5 10 15

Leu His Ala Ala Gly Ala Gln Gly Glu Met Phe Thr Val Lys Glu Val

20 25 30

Met His Tyr Leu Gly Gln Tyr Ile Met Val Lys Gln Leu Tyr Asp Gln

35 40 45

Gln Glu Gln His Met Val Tyr Cys Gly Gly Asp Leu Leu Gly Glu Leu

50 55 60

Leu Gly Arg Gln Ser Phe Ser Val Lys Asp Pro Ser Pro Leu Tyr Asp

65 70 75 80

Met Leu Arg Lys Asn Leu Val Thr Leu Ala Thr

85 90

<210>4

<211>384

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<213> Artificial sequence

<400>4

ggcagcagcc atcaccatca tcaccacggc agcagccagg aaacctttag cgatctgtgg60

aaactgctgc cggaaaatgg cagcggcagc agcgaaaacc tgtattttca gggatcccag 120

attaaccagg tgcgtccgaa actgccgctg ctgaaaattc tgcatgcggc gggcgcgcag 180

ggcgaaatgt ttaccgtgaa agaagtgatg cattatctgg gccagtatat tatggtgaaa 240

cagctgtatg atcagcagga acagcacatg gtgtattgcg gcggcgatct gctgggcgaa 300

ctgctgggcc gtcagagctt tagcgtgaaa gatccgagcc cgctgtatga tatgctgcgt 360

aaaaacctgg tgaccctggc gacc 384

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ggcagcagcc atcaccatca tcaccacggc agcagccagg aaaccgctag cgatctgtgg 60

aaactgctgc cggaaaatgg cagcggcagc agcgaaaacc tgtattttca gggatcccag 120

attaaccagg tgcgtccgaa actgccgctg ctgaaaattc tgcatgcggc gggcgcgcag 180

ggcgaaatgt ttaccgtgaa agaagtgatg cattatctgg gccagtatat tatggtgaaa 240

cagctgtatg atcagcagga acagcacatg gtgtattgcg gcggcgatct gctgggcgaa 300

ctgctgggcc gtcagagctt tagcgtgaaa gatccgagcc cgctgtatga tatgctgcgt 360

aaaaacctgg tgaccctggc gacc 384

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<212>DNA

<213> Artificial sequence

<400>6

ggcagcagcc atcaccatca tcaccacggc agcagccagg aaacctttag cgatctgtgg 60

aaactggcgc cggaaaatgg cagcggcagc agcgaaaacc tgtattttca gggatcccag 120

attaaccagg tgcgtccgaa actgccgctg ctgaaaattc tgcatgcggc gggcgcgcag 180

ggcgaaatgt ttaccgtgaa agaagtgatg cattatctgg gccagtatat tatggtgaaa 240

cagctgtatg atcagcagga acagcacatg gtgtattgcg gcggcgatct gctgggcgaa 300

ctgctgggcc gtcagagctt tagcgtgaaa gatccgagcc cgctgtatga tatgctgcgt 360

aaaaacctgg tgaccctggc gacc 384

Claims (1)

1. A nucleic acid molecule having a nucleotide sequence capable of expressing a fusion protein for screening for or testing for an inhibitory activity of MdmX inhibitors,

wherein the fusion protein comprises a test sequence, a binding sequence and a linker arm sequence, and is represented by the formula p53 p-XX-N-MdmX;

p53p represents the binding sequence;

the test sequence is a MdmX sequence or a MdmX fragment comprising a p53 binding domain in the MdmX sequence;

the binding sequence is SQETASDLWKLLPEN or SQETFSDLWKLAPEN;

the connecting arm sequence is a peptide segment, and the upstream and downstream of the connecting arm are respectively connected with one of the test sequence and the binding sequence; the amino acid sequence of the linker arm is not involved in the formation or maintenance of the secondary structure of the fusion protein; the length and flexibility of the linker arm are sufficient to make any surface portion of the spatial structure of the test sequence in spatial contact with any surface portion of the spatial structure of the binding sequence; XX represents the connecting arm sequence SEQ ID NO.2, specifically: GSGSSENLYFQ, respectively;

N-MdmX represents the test sequence SEQID number 3, and specifically comprises the following steps: GSQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT are provided.

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