WO2023056715A1 - Stable polypeptide protein covalent inhibitor of papain-like protease (plpro) targeting 2019 novel coronavirus - Google Patents

Abstract

The present invention provides a stable polypeptide protein covalent inhibitor of papain-like protease (PLpro) targeting 2019 novel coronavirus. The present invention also provides use of the stable polypeptide protein covalent inhibitor in preparing a drug for inhibiting the enzyme activity of PLpro. According to the present invention, a method for forming a single sulfonium salt by reacting methionine-cysteine on polypeptide with a dialkylation reagent is adopted to stabilize the sulfonium salt cyclic peptide targeting PLpro. According to the present invention, a novel stable polypeptide protein covalent inhibitor targeting PLpro is invented by adopting a policy of coupling sulfonium salt-stabilized polypeptide to small molecules. The polypeptide protein covalent inhibitor of the present invention can effectively inhibit the activity of PLpro, thereby blocking the immune escape reaction generated by cutting ISG15 by PLpro.

Description

A stable polypeptide protein covalent inhibitor targeting the papain-like protease PLpro of 2019 novel coronavirus technical field

The invention belongs to the field of bioengineering and relates to a stable polypeptide protein covalent inhibitor, specifically a stable polypeptide protein covalent inhibitor targeting 2019 novel coronavirus papain-like protease PLpro and its application.

Background technique

The causative agent of COVID-19 is a new type of coronavirus called SARS-CoV-2. This coronavirus is highly contagious and pathogenic, and has spread widely around the world since it was first discovered in December 2019. So far, it has infected more than 200 million people and killed more than 4 million people. The incubation period of COVID-19 infection ranges from 2-14 days and can be as long as 24 days. These longer incubation periods, due to their transmissibility and asymptomatic nature, are responsible for the high number of infections. The growing number of COVID-19 cases reflects the seriousness of the current situation and the need for effective solutions, yet effective antiviral treatments are still lacking.

Current research suggests that this virus is similar to SARS-CoV. A potential antiviral drug target is the papain-like protease PLpro, a cysteine protease encoded by SARS-CoV-2. The replicase gene of SARS-CoV-2 encodes two proteins, pp1a and pp1ab, which are then processed into 16 nonstructural proteins (Nsps) for gene replication and RNA transcription. Proteolysis is carried out by two cysteine proteases - the papain-like protease (PLpro) and the major protease (Mpro/3CLpro). PLpro releases Nsp1-Nsp3 from the polyprotein N-terminus by recognizing the conserved sequence LXGG, and can also catalyze the removal of K48-linked ubiquitin on host cell proteins and remove interferon-stimulated gene 15 (ISG15) from host proteins, thereby interfering with the host's immune response.

Therapeutic drugs that people have paid attention to for a long time are mainly concentrated in two categories: small molecules and biologicals. The chemical space targeted by small molecule drugs has certain limitations. Protein drugs have poor stability and cannot penetrate cell membranes. Due to the limitations of their own biophysical properties, these two types of therapeutic drugs cannot effectively cover all of these. important molecular targets.

Peptide drugs are another class of targeting molecules that have attracted widespread attention and interest. Similar to biomacromolecules, polypeptide molecules also have higher binding force and selectivity for targets, and have smaller off-target effects than small molecule drugs. The metabolites of polypeptides in the body are amino acids, which minimizes toxicity. Due to the limited number of amino acid residues, traditional peptide drugs cannot effectively form complex secondary structures. They have a high degree of freedom in physiological solutions and are in an irregular linear state, which not only reduces their specificity but is also easily degraded by proteases. And the cell membrane penetration ability of peptide drugs is not very good. Modifying the polypeptide by chemical means to stabilize it into a conformation with a secondary structure can not only increase its stability to proteases, but also enhance the cell membrane penetration ability of the polypeptide, and can also reduce the entropy change when the polypeptide binds to the target. Thereby improving the ability of the polypeptide to bind to the target. Through various chemical modification methods, the secondary structural units involved in various protein-protein interactions are extracted and modified, not only by stabilizing their secondary conformation to simulate the interaction of the original protein, but more importantly, the modification can make These protein secondary structural units have the ability to penetrate cell membranes, thereby targeting intracellular protein-protein interactions. In 2019, our research group recently developed a peptide cyclization strategy through the dialkylation between Cys and Met, using the sulfur salt on the side chain of the polypeptide as a new warhead to interact with the protein Cys under the induction of the polypeptide ligand. Covalent reactions occur under conditions of spatial proximity. Based on this, this subject designed a series of sulfonium salt-stabilized peptides targeting PLpro, which can covalently bind to PLpro and inhibit the activity of PLpro, and remove interferon-stimulated gene 15 (ISG15) from host proteins in cells, Interfering with the host's immune response.

Contents of the invention

Aiming at the above-mentioned technical problems in the prior art, the present invention provides a stable polypeptide protein covalent inhibitor targeting 2019 novel coronavirus protein PLpro and its application. The described targeting 2019 novel coronavirus protein PLpro The stable polypeptide protein covalent inhibitor and its use should solve the technical problem that the drugs in the prior art are not effective in treating pneumonia caused by the new coronavirus.

The present invention provides a series of stable polypeptide protein covalent inhibitors targeting 2019 novel coronavirus protein PLpro, the structural formula of which is as follows,

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Further, its amino acid sequences are respectively:

polypeptide	sequence	linker small molecule	the
ECM	GRL0617-E-cyclic (CLRGM)	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.1
EMC	GRL0617-E-cyclic (MLRGC)	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.2
ELRGG	GRL0617-ELRGG	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.3
CM1	Ac-LRGG-cyclic (CAAAM),	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.4
CM2	Ac-LRGG-cyclic (MAAAC)	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.5
CM3	Ac-cyclic (MRGGC)	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.6
CM4	Ac-cyclic (CRGGM)	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.7
CM5	Ac-L-cyclic (MGGC)	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.8
CM6	Ac-L-cyclic (CGGM)	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.9
CM7	Ac-cyclic (MLRGC)	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.10
CM8	Ac-cyclic (CLRGM)	1,3-Dibromomethylbenzene	Shown in SEQ ID NO.11

The present invention also provides the use of the above-mentioned stable polypeptide protein covalent inhibitor in the preparation of drugs for inhibiting the enzyme activity of protein PLpro.

The present invention also provides the use of the above-mentioned stable polypeptide protein covalent inhibitor in the preparation of drugs for targeting the 2019 novel coronavirus protein PLpro.

The present invention also provides the use of the above-mentioned stable polypeptide protein covalent inhibitor in the preparation of a drug for treating pneumonia caused by novel coronavirus.

The present invention provides a stable polypeptide protein covalent inhibitor targeting 2019 novel coronavirus (SARS-CoV-2) papain-like protease (Papain-like protease, PLpro), whose amino acid sequence is derived from the PLpro recognition Conserved sequence LRGG. Enzyme activity experiments have fully proved that the covalent polypeptide inhibitor can effectively reduce the activity of papain-like protease PLpro.

The present invention adopts the method of forming a single sulfonium salt by reacting methionine-cysteine on the polypeptide with a dialkylating agent to stabilize the sulfonium salt cyclic peptide targeting PLpro. The method of selective covalent modification of protein cysteine based on sulfonium salt-stabilized polypeptide ligands reported in the previous literature is used. When the cyclic peptide ligand and the target protein PLpro recognize each other, the sulfonium salt on the cyclic peptide and the protein PLpro cysteine undergoes a nucleophilic reaction under spatial proximity to achieve covalent modification of proteins. SDS-PAGE analysis and mass spectrometry prove that the polypeptide reacts covalently with C111 of the target protein PLpro after mutual recognition with the target protein.

Through experiments such as fluorescence polarization detection and enzyme activity detection, the present invention proves that the polypeptide can be well combined with PLpro protein, and through the covalent reaction between the sulfonium salt on the polypeptide and the cysteine on the protein PLpro, the PLpro protein is blocked from interacting with the PLpro protein. Binding of the substrate molecule LRGG-AMC. The present invention also confirms that the polypeptide can well enter HCT116, A549 and other cells without causing massive cell death through experiments such as flow cytometry analysis and cell survival.

Description of drawings

Figure 1 is a schematic diagram of the synthesis method of polypeptide molecules of sulfonium salt covalent inhibitors (taking CM2 and EMC as examples).

Fig. 2 is a covalent binding map of polypeptide CM1-8 and PLpro protein.

Fig. 3 is a map of the covalent binding of polypeptide EMC to PLpro protein and PLpro ^C111S .

Fig. 4 is a mass spectrogram of the covalent binding of polypeptide ECM and PLpro protein.

Fig. 5 is a diagram showing the inhibition efficiency of polypeptide CM1-8 on PLpro enzyme activity.

Fig. 6 is a graph showing the inhibition efficiency of three PLpro enzyme activities by polypeptides EMC, EMC, ELRGG and GRL0617.

Fig. 7 is ISG15 immunoblotting verification that the polypeptide restores the level of ISG in cells.

Figure 8 shows the ability of different polypeptides to inhibit the proliferation of human non-small cell lung cancer cell A549 and normal cell HEK293T.

Fig. 9 is a schematic diagram of covalent inhibition of PLpro by sulfonium salt-stabilized polypeptides.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings.

Example 1

The present invention adopts the sulfonium salt stabilized polypeptide technology reported in previous literature (D.Wang, M.Yu, et al.Chem.Sci.10, 4966-4972), through the methionine, cysteine and dialkylene on the polypeptide The sulfonium salt cyclic peptide can be reacted with an alkylation reagent, which can not only stabilize the polypeptide, but also covalently modify the cysteine on the interaction site of the target protein, and block the papain-like protease PLpro and various non-structural proteins of the virus. Binding, inhibiting the replication of viral genes.

The present invention provides a series of stable polypeptide protein covalent inhibitors targeting 2019 novel coronavirus (SARS-CoV-2) papain-like protease (Papain-like protease, PLpro). The inventors synthesized a number of different polypeptides, as shown in Table 1.

Table 1: Molecular sequences of different stable polypeptide covalent inhibitors targeting the 2019-nCoV protein PLpro.

polypeptide	sequence	linker small molecule	molecular weight
ECM	GRL0617-E-cyclic (CLRGM)	1,3-Dibromomethylbenzene	1137.54
EMC	GRL0617-E-cyclic (MLRGC)	1,3-Dibromomethylbenzene	1137.54
ELRGG	GRL0617-ELRGG	1,3-Dibromomethylbenzene	858.50
CM1	Ac-LRGG-cyclic (CAAAM),	1,3-Dibromomethylbenzene	1421.60
CM2	Ac-LRGG-cyclic (MAAAC)	1,3-Dibromomethylbenzene	1421.60
CM3	Ac-cyclic (MRGGC)	1,3-Dibromomethylbenzene	1095.41
CM4	Ac-cyclic (CRGGM)	1,3-Dibromomethylbenzene	1095.41
CM5	Ac-L-cyclic (MGGC)	1,3-Dibromomethylbenzene	1052.60
CM6	Ac-L-cyclic (CGGM)	1,3-Dibromomethylbenzene	1052.60
CM7	Ac-cyclic (MLRGC)	1,3-Dibromomethylbenzene	1151.47
CM8	Ac-cyclic (CLRGM)	1,3-Dibromomethylbenzene	1151.47

Preparation and separation and purification steps of the polypeptide of embodiment 2:

The polypeptide of the present invention is synthesized in solid phase according to the amino acid sequence, which is a conventional technique and will not be repeated here. This example only describes the core steps for preparing the above-mentioned stable polypeptide as follows (taking CM1 as an example):

The specific operation steps (Figure 1) are:

(1) Polypeptide solid-phase synthesis: Weigh Rink amide MBHA resin into a peptide tube, add dichloromethane (DCM), and swell with nitrogen gas for 30 minutes. Add 50% (v/v) morpholine in N,N-dimethylformamide (DMF) solution, blow nitrogen gas for 30 minutes, and remove the Fmoc protecting group. After washing the resin alternately with DMF and DCM, the prepared Fmoc-AA-OH (5eq, 0.4M, DMF) solution, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea Fluorophosphate ester (HCTU) (5eq, 0.38M, DMF) solution and N,N-diisopropylethylamine (DIPEA) (10eq) were mixed well, then added to the resin and blown with nitrogen for 1h. Take out the reaction liquid, wash the resin according to the above method, and proceed to the next step. Subsequent amino acids are the same as above. The N-terminus of the polypeptide was acetylated with acetic anhydride and DIPEA (1:1.8 molar ratio), dissolved in DCM) for 30 minutes (twice).

(2) Intramolecular ring closure: deprotect the Trt group of the cysteine (Cys) side chain protecting group on the resin (deprotection solution: TFA/TIS/DCM=3/5/92, molar ratio), Take off in stages until the solution no longer turns yellow, and then wash with DMF and DCM for five times respectively. 1,3-bis(bromomethyl)benzene reagent (2 equivalents) and DIPEA (4 equivalents) are dissolved in DMF and added Reaction in resin for 2 hours.

(3) Polypeptide purification: use trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H ₂ O (v:v:v=9.5:0.25:0.25) to cut the polypeptide from the resin, Remove shear fluid. Purify and separate by high performance liquid chromatography, and finally confirm the molecular weight by mass spectrometry (MS). A pure polypeptide molecule is obtained, and the specific structural formula is as described above.

Example 3 Polypeptide Molecule and Protein PLpro Covalent in Vitro

The methionine and cysteine on different polypeptides with fluorescent groups react with dialkylating reagents to form sulfonium salt cyclic peptides and respectively co-incubate with the protein, the protein and the sulfonium salt polypeptide undergo a covalent reaction, and the protein can be seen Fluorescence is displayed on the band (Figure 2), indicating that CM1-8 polypeptide can covalently react with PLpro protein, while general linear peptides cannot produce covalent linkage.

We choose the peptide EM-C as an example to study reaction kinetics and stoichiometry studies. FAM-labeled peptide EM-C (10 μM) was reacted with SARS-CoV-2 PLpro (5 μM) in PBS buffer for different times (0.5, 1, 2, 3, 4 hours). The response was dose-dependent. Kinetic and stoichiometric studies clearly showed the high efficiency of binding (Fig. 3A,B).

The peptide has good specificity, and it mainly reacts covalently with the cysteine at position C111 of PLpro. If the cysteine at position 111 of PLpro is mutated to serine, the peptide basically does not react covalently with PLpro (Figure 3C) .

To assess the ability of peptides EM-C and EC-M to label PLpro in a complex proteomic environment, PLpro (5 μM) was added to 293T cell lysates (300 μg), followed by FAM-labeled peptides EM-C and EC-M (10 μM )deal with. The gel data showed clear single fluorescent bands with the correct molecular weight, indicating clean and selective conjugation of peptides EM-C and EC-M to PLpro (Fig. 3D).

In addition, we verified the binding sites of peptides and proteins by secondary mass spectrometry. MS/MS analysis of tryptic digested protein samples excised from SDS gels clearly showed peptide fragments from ECM and PLpro conjugation at Cys111. These results indicated that the peptide ECM was mainly covalently labeled with C111 of PLpro (Fig. 4).

Example 4 Polypeptide Molecule Inhibits PLpro Enzyme Activity Experiment

SARS-CoV-2PLpro has been tested to cleave the substrate LRGG-ACC to release the fluorophore ACC, increasing its fluorescence intensity. Use different concentrations of N-terminal acetylated polypeptide CM1-8 (0-800 μM), mix with PLpro protein (0.1 μM) in the experimental buffer (5mM NaCl, 20mM tris, 5mM DTT, pH=8.0), at 37°C After reacting in a water bath for 1 hour, the substrate LRGG-ACC (1 μM) was added, and the fluorescence emission intensity (λ _EX : 355nm, λ _EM : 460nm) was measured in a black bottom 96-well plate using a microplate reader. Sulfate-stabilized peptides were not effective in inhibiting SARS-CoV-2 PLpro (Fig. 5).

The sulfur-salt-stabilized peptide without GRL0617 could not effectively inhibit SARS-CoV-2PLpro, while the sulfur-salt-stable peptide and GRL0617 conjugates ECM and EMC had better inhibitory ability. However, the inhibitory effect was slightly worse than that of GRL0617 (Fig. 6A). The enzyme activity inhibition experiment of PLpro by different concentrations of polypeptide inhibitors proves that the inhibitory effect is dose-dependent. In addition, the inhibitory ability of the peptide-drug conjugates against SARS-CoV PLpro and MERS PLpro was tested. There is a high sequence identity (83%) between SARS-CoV-2PLpro and SARS-CoV PLpro, and EMC and ECM peptides can also inhibit SARS-CoV PLpro, and the inhibitory effect of the linear peptide ELRGG is weaker than that of the sulfur-salt-stabilized peptide (Figure 6B) . GRL0617 could not inhibit MERS PLpro, consistent with previous literature results, the same ECM, EMC and ELRGG had no inhibitory effect on MERS PLpro (Fig. 6C).

Example 5 Effect of Polypeptide Molecules on the De-ISG of Intracellular PLpro

An intracellular ISG15-based immunoblotting method was used to observe whether the cellular ISGylation level inhibited by PLpro could be restored, and the inhibitory effect of ECM and EMC on intracellular PLpro de-ISGylation activity was studied. Consistent with its activity in both drug screening assays, the peptide-drug conjugate could restore cellular ISGylation levels in a dose-dependent manner in a cell-based approach, suggesting that both peptide-drug conjugates can enter cells Inhibition of SARS-CoV-2 PLpro. Meanwhile, ECM showed higher potency in restoring cellular ISGylation levels than EMC ( FIG. 7 ).

The effect of embodiment 6 polypeptide on cell survival

In order to evaluate the killing ability of the polypeptide ECM, EMC and ELRGG on different cells, the cancer cell human non-small cell lung cancer cell A549 and the normal cell HEK293T were selected to exclude the non-specific toxicity of the polypeptide library.

Cell viability by MTT assay. Cells were inoculated at 4×10 ³ in a 96-well plate, treated with polypeptide dissolved in culture medium (5% serum) for 24 hours, and MTT was added to the culture medium and incubated for 4 hours. Then DMSO was added to dissolve the precipitate, and the absorbance was measured at 490 nm using a microplate reader. The survival rate of untreated cells was 100%.

The results showed that ECM, EMC and ELRGG had basically no toxic and side effects on human non-small cell lung cancer cell A549 and normal cell HEK293T ( FIG. 8 ).

Claims (5)

1. A stable polypeptide protein covalent inhibitor targeting papain-like protease PLpro of 2019 novel coronavirus, characterized in that its structural formula is as follows,

   

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2. A stable polypeptide protein covalent inhibitor targeting papain-like protease PLpro targeting 2019 novel coronavirus according to claim 1, characterized in that its amino acid sequence is as shown in any of SED ID NO.1-11 .
3. The use of the stable polypeptide protein covalent inhibitor according to claim 1 in the preparation of drugs for inhibiting protein PLpro enzyme activity.
4. The use of the stable polypeptide protein covalent inhibitor according to claim 1 in the preparation of a drug targeting the papain-like protease PLpro of the 2019 novel coronavirus.
5. The use of the stable polypeptide protein covalent inhibitor according to claim 1 in the preparation of medicines for the treatment of pneumonia caused by the 2019 novel coronavirus.

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