CN110638846B

CN110638846B - Application of RNase L inhibitor

Info

Publication number: CN110638846B
Application number: CN201910905538.XA
Authority: CN
Inventors: 黄昊; 汤金乐; 刘志宏; 付子阳
Original assignee: Peking University Shenzhen Graduate School
Current assignee: Peking University Shenzhen Graduate School
Priority date: 2019-09-24
Filing date: 2019-09-24
Publication date: 2021-08-03
Anticipated expiration: 2039-09-24
Also published as: CN110638846A

Abstract

The invention discloses application of an RNase L inhibitor, which comprises application of specific compounds in preparation of RNase L inhibitors, oncolytic virus anti-tumor synergists and anti-tumor products and pharmaceutical compositions, wherein the specific compounds have targeted specificity on RNase L. The prepared RNase L inhibitor can effectively inhibit the activity of RNase L, prevent oncolytic virus from being degraded, ensure that the oncolytic virus can be successfully replicated in host tumor cells and kill the tumor cells, and effectively improve the anti-tumor effect of the oncolytic virus. On the basis, the RNase L inhibitor and the specific oncolytic virus can be compounded into an anti-tumor product, so that the normal exertion of the effect of killing tumor cells by the oncolytic virus is ensured.

Description

Application of RNase L inhibitor

Technical Field

The invention relates to the technical field of anticancer, in particular to application of an RNase L inhibitor.

Background

RNase L is a protein molecule that plays an important role in the protection of higher animals against virus invasion and the innate immunity of the human body. Higher animals secrete interferon when invaded by virus, the interferon induces the expression of 2-5A Synthetase (OAS), the 2-5A Synthetase is activated after being combined with virus double-stranded RNA, and then ATP is synthesized into 2 '-5' linked oligoadenylate (2 '-5' linked oligoadenylate, 2-5A). The chemical structure of 2-5A comprises three linear linked adenylates (a): px5 ' A (2 ' p5 ' A) n, where x is 1-3 and n is 2 or more. The generated 2-5A is combined with RNase L to activate RNase L molecules, so that the RNase L has the function of degrading virus and host single-stranded RNA, and the virus cannot be replicated, thereby achieving the effect of resisting viruses of human bodies. RNase L has a poor substrate selectivity and degrades many single-stranded RNA sequences, but the rate of degradation varies. For example, it still tends towards cleavage sites for UN ^ N (N stands for arbitrary nucleotide) sequences, i.e.cleavage between the two N's after the uracil ribonucleotide U. In different UN combinations, the enzyme cutting rate is UU > UA > > UG > UC. Overall, RNase L tends to cleave behind the UU and UA sites. RNase L has a broad spectrum of antiviral activity against viruses including encephalomyocarditis virus, West Nile virus, coronavirus, etc.

RNase L is not only an important factor in the antiviral immune response of human body, but also a target of antitumor auxiliary drugs. There is currently a new cancer Therapy, called anti-cancer Viral Therapy (Anticancer Viral Therapy), that utilizes a low activity virus to selectively attack cancer cells. In 2015, Amgen's Oncolytic Virus Therapy (oncocytic Virus Therapy) was officially approved by the FDA in the united states for surgical removal of poorly effective melanoma. The drug Talimogene laherparevec (also known as Talimogene lahervec) used in this oncolytic virus therapy

Or T-VEC), is a genetically engineered Herpes Simplex Virus (Herpes Simplex Virus). However, when cancer cells in the human body are attacked by viruses in vitro, the human autoimmune system resists the invasion of the viruses, thereby weakening the attack of the viruses on the cancer cells. For example, when using Vesicular Stomatitis Virus (VSV) to develop anti-cancer oncolytic Virus therapy, researchers have found that RNase L attacks the VSV Virus used, resulting in poor cancer cell suppression by the VSV Virus. Meanwhile, scientists at the medical center of Cleveland USA find that small molecule anticancer drug Sunitinib (Sunitinib) can inhibit two important proteins RNase L and PKR in human innate immune system, wherein the inhibition effect of Sunitinib on RNase L is 1.4 μ M (IC 50), and the inhibition effect of Sunitinib on PKR is 0.3 μ M (IC 50). Further experimental results show that the combination of VSV and the inhibitor Sunitinib of RNase L can effectively inhibit the antiviral immune response of human body, so that the virus therapy has very good effect on various tumors, such as bladder cancer, breast cancer and renal cancer. However, Sunitinib is a small molecule inhibitor of tyrosine kinases that does not possess RNase L by targeting multiple receptor tyrosine kinases to inhibit cellular signalingHas specific inhibiting effect. Therefore, there is a need to develop specific inhibitors targeting RNase L.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides the application of the specific compound in preparing RNase L inhibitor, oncolytic virus anti-tumor synergist and anti-tumor products, wherein the specific compound has targeted specificity on RNase L.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention there is provided the use of a compound, or a pharmaceutically acceptable salt thereof, in the preparation of an RNase L inhibitor, the compound being selected from:

at least one of (1).

In a second aspect of the present invention there is provided the use of a compound, or a pharmaceutically acceptable salt thereof, for the manufacture of an oncolytic anti-tumour potentiating agent, the compound being selected from:

wherein the oncolytic virus anti-tumor synergist comprises an RNase L inhibitor, and the RNase L inhibitor is the compound or a pharmaceutically acceptable salt thereof. An oncolytic virus anti-tumor synergist is a product which is used in combination with an oncolytic virus in an oncolytic virus therapy to reduce the anti-viral immune response of an immune system, thereby enhancing the inhibitory effect of the oncolytic virus on cancer cells.

In a third aspect of the present invention, there is provided the use of a compound or a pharmaceutically acceptable salt thereof, for the manufacture of an anti-tumour product, the compound being selected from:

the anti-tumor product comprises oncolytic virus and an RNase L inhibitor used as an anti-tumor synergist of the oncolytic virus, wherein the RNase L inhibitor is the compound or pharmaceutically acceptable salt thereof.

According to an embodiment of the present invention, the oncolytic virus is at least one of herpes simplex virus, oncolytic adenovirus, newcastle disease virus, vaccinia virus, vesicular stomatitis virus, encephalomyocarditis virus, myocarditis virus, west nile virus, coronavirus.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) an oncolytic virus;

(b) a compound I or a pharmaceutically acceptable salt thereof, the compound I being selected from:

at least one of (1).

In a fifth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) an oncolytic virus;

(c) a compound II, which is obtained by modifying or transforming (b) as a precursor;

wherein (b) is compound i or a pharmaceutically acceptable salt thereof, compound i is selected from:

at least one of (1).

Wherein the modification or modification includes, but is not limited to, side chain addition, deletion, fragment supplement, etc., using the above compound as a parent substance, so as to

For example, reference is made to the following formula:

R₁-R₃、R₅-R₇each independently selected from any one of halo (including F, Cl, Br, I), unsubstituted C1 to C3 saturated alkyl, C1 to C3 saturated alkyl substituted with 1 or more halo;

R₄any one selected from hydroxyl, halogen (including F, Cl, Br, I), unsubstituted saturated alkyl of C1-C3, and phenyl. As a result of the modification, a modification method known in the art is used, and the activity of the drug-like small molecule is not significantly affected, and a person skilled in the art can presume that the modified compound has similar chemical properties based on the published 10G5, 10G-1 and 10G5-2, and can be used as an RNase L inhibitor.

In a sixth aspect of the present invention, there is provided a method for screening an RNase L inhibitor, which comprises the step of evaluating the interaction between a drug binding pocket of the RNase L, which targets a pseudokinase domain of the RNase L, and amino acid residues constituting the drug binding pocket A373, R398, D502 and K503, and a compound to be screened.

According to an embodiment of the invention, the original side chain position of the amino acid residue R398 of the RNase L pseudokinase domain is occupied by 10G5, and the R398 side chain is located above the molecular plane of the fragment in the co-crystal, forming the Pi-ligation interaction.

According to an embodiment of the present invention, the amino acid residues constituting the drug binding pocket are charged residues except A373.

The drug binding pocket is adjacent to the ATP binding site of the pseudokinase domain, and fragment ligation can be used to design the leptin compounds.

The embodiment of the invention has the beneficial effects that:

the compound provided by the invention can specifically inhibit the activity of RNase L, and can be applied to preparation of RNase L inhibitors. The prepared RNase L inhibitor can effectively inhibit the activity of RNase L, prevent oncolytic virus from being degraded, ensure that the oncolytic virus can be successfully replicated in host tumor cells and kill the tumor cells, and effectively improve the anti-tumor effect of the oncolytic virus. On the basis, the RNase L inhibitor and the specific oncolytic virus can be compounded into an anti-tumor product, so that the normal exertion of the effect of killing tumor cells by the oncolytic virus is ensured.

Drawings

FIG. 1 shows the RNase L small molecule inhibitor enzyme activity screening and the corresponding half maximal effective Inhibitory Concentration (IC) according to one embodiment of the present invention₅₀) And (6) measuring results. A is the result of Coomassie blue staining experiment in the process of full-length RNase L expression and purification, B is the result of relative fluorescence intensity detection, C is the result of drug-like molecule screening, and D is IC₅₀As a result of the measurement, E is the structural formula of the two types of drug molecules screened out.

FIG. 2 shows the results of analysis of 10G5 complex crystals with RNase L and the binding pocket of the drug according to one embodiment of the present invention. A is the crystal and diffraction data of the complex of 10G5 and RNase L, B is the eutectic structure of 10G5 and RNase L, and C is the result of MST technique to measure the binding constants of RNaseL and 10G 5.

FIG. 3 shows the results of binding constant measurements for 10G5-1 and 10G5-2 in another embodiment of the present invention. A is a molecular docking scheme of the 10G5 derivative based on a eutectic structure, and B and C are results of MST method measurement of binding constants of the derivative 10G5-1/10G5-2 and RNaseL, respectively.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

The following example uses a library of fragments of Maybrid Ro3 (containing 1000 drug-like molecules) from Maybrid corporation under the Seimer Feilai. These 1000 drug-like molecules contain strong pharmacophores and have superior ADME (absorption, distribution, metabolism, and excretion) properties; the structure is wide in diversity, contains groups easy to combine, and accords with the performance of 'Rule of Three' (the fat-water distribution coefficient logP is less than 3, the molecular weight is less than 300Da, no more than Three hydrogen bond donors, no more than Three hydrogen bond acceptors, no more than Three rotatable bonds and the like); therefore, the fragment molecules with biological activity screened by the Maybridge Ro3 fragment library have excellent lead compound performance and drug development potential.

In the examples, each raw material reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Example 1

1 method of experiment

1.1 preparation of RNase L protein sample

Prokaryotic expression vectors originally stored in the laboratory for the expression of RNase L were pGEX-FL-RNase L (constructed by inserting the nucleotide sequence fragment NM-001097512 of RNase L into the BamHI and XhoI multiple cloning sites of vector pGEX-4-T1) and pGEX-N21732, which contained the wild-type RNase L gene sequence (used to resolve the full-length crystal structure of RNase L: a fragment containing all domains, N-and C-termini with the exception of a small portion of no secondary structure). The relevant plasmid is transformed into prokaryotic expression escherichia coli strain BL-21. Single clones were picked for expansion culture and finally induced overnight at low temperature by addition of IPTG. Crushing the obtained thalli at high pressure and centrifuging at high speed to obtain protein supernatant, mixing the obtained supernatant with GST affinity chromatography medium for low-temperature incubation, cleaning the chromatography medium by using a binding buffer solution, directly adding TEV protease for low-temperature enzyme digestion overnight, collecting RNase L protein suspension obtained by enzyme digestion the next day, and finally purifying by SEC molecular chromatography to obtain high-purity RNase L and mutant protein thereof.

1.2 RNase L Activity detection method

Enzyme activity detection protocols based on Fluorescence Resonance Energy Transfer (FRET) techniques are employed. The two ends of the RNA substrate are respectively marked with a fluorescent group and a quenching group to form the RNA probe, when the complete RNA probe is excited by exciting light, energy is transferred to the quenching group at the near end, and after the RNA probe is cut, the quenching group is far away from the fluorescent group, and after the fluorescent group is excited by the exciting light, emitted light is detected by a fluorescence photometer, so that the RNA substrate can be shown to be sheared by ribonuclease L.

1.2.1 substrate Material

RNA fluorescent probe substrate: 6-FAM-UUAUCAAAUUCUUAUUUGCCCCAUUUUUUUGGUUUA-BHQ-1; FAM (carboxyfluorescein) is a fluorescent reporter group and BHQ1 is a fluorescence quencher group, which is derived from the RNA sequence of the respiratory syncytial virus genome, contains multiple cleavage sites (UU and UA), and is highly sensitive to cleavage by RNase L enzyme.

1.2.2 enzyme Activity assays

RNase L protein (25 pg/. mu.L), 2-5A (1nM), RNA fluorescent probe substrate (100nM) were mixed and incubated at 22 ℃ for 1 h. Fluorescence signals were detected using a multiwell microplate reader (PerkinElmer Envision 2104) at 480nm excitation light and 535nm emission light wavelengths, with the read intensity reduction set to 0%, and the relative fluorescence intensity (RFU) values were determined.

1.3 RNase L Small molecule fragment inhibitor screening and identification

The library of Maybrid Ro3 fragments was screened for RNase L inhibitors using the above described enzyme activity assay.

1.4 obtaining and verifying protein cocrystal

1.4.1 obtaining of protein Co-crystals

Cocultivation or crystal soaking to obtain eutectic of the inhibiting fragment and RNase L, and Micro Scale Thermophoresis (MST) to determine the binding constant of the inhibiting fragment and RNase L.

(1) Co-incubation method

Incubating the positive inhibition fragment obtained by screening with protein RNase L crystal, wherein the ratio of the compound to the molecule is 20-40, incubating at room temperature for 2 hr, dropping the crystal by hanging drop method, and dropping the crystal by hanging drop method (crystal condition: 0.1M Tris (7.0-8.5), 0.15M (NH)₄)₂SO₄PEG4000 (15% -20%), 5mM DTT). Standing at 20 deg.C for 2-3 days to allow crystal growth. Selecting 6-8 crystals with 20% ethylene glycol as protective solution, freezing the crystals in liquid nitrogen, transferring to Shanghai synchrotron radiation center, and collecting data by diffraction.

(2) Crystal soaking method

RNase L crystals were grown directly, and the crystals were soaked in a 50mM compound and left to stand at 20 ℃ for 2 to 4 hours or even overnight. Selecting 3-4 soaked crystals, freezing, and sending to Shanghai synchrotron radiation center to collect data.

2. Results of the experiment

2.1 screening results of Small molecule fragment inhibitors

26 positive small molecule fragments are obtained by primary screening, and then three times of repeated tests are carried out on the 26 small molecule fragments, so that 2 small molecule fragments with inhibiting effect are obtained. And half of the effective inhibitory concentration IC50 of the 2 molecular fragments was obtained as determined by the concentration gradient assay, the results of which are shown in FIG. 1. FIG. 1 shows the RNase L small molecule inhibitor enzyme activity screening and the corresponding half maximal effective Inhibitory Concentration (IC) according to one embodiment of the present invention₅₀) And (6) measuring results. A is the result of Coomassie blue staining experiment in the process of full-length RNase L expression and purification, B is the result of relative fluorescence intensity detection, C is the result of drug-like molecule screening, and D is IC₅₀As a result of the measurement, E is the structural formula of the two types of drug molecules screened out. As can be seen, the relative molecular mass of the RNase L protein is 83.9kDa, and the wild type RNase L protein with high concentration is obtained and has high purity. Mixing the RNase L and the RNA probe, reacting at 25 ℃, and detecting the relative fluorescence intensity once every 3 minutes to obtain a curve in the B. As can be seen, the RNase L protein of the experimental group has good enzyme activity curve characteristics, which indicates that the RNase L protein with biological activity is successfully obtained. The screening result showed that the IC of 10G5₅₀IC at 85. mu.M, 11F5₅₀681 μm, all reach micromolar grade.

2.2 results of eutectic analysis

FIG. 2 shows the results of analysis of 10G5 complex crystals with RNase L and the binding pocket of the drug according to one embodiment of the present invention. A is the crystal and diffraction data of the complex of 10G5 and RNase L, B is the eutectic structure of 10G5 and RNase L, and C is the result of MST technique to measure the binding constants of RNaseL and 10G 5. It can be seen that the 10G5 binding site has two key positively charged amino acid residues R398 and K503, which is very close to the ADP binding pocket of the pseudokinase domain. The binding constant of RNaseL and 10G5 measured by the MST technique was 93. mu.M, which was consistent with the constant measured by the enzyme activity method (83. mu.M). The drug binding pocket of the RNase L takes a pseudokinase domain of the RNase L as a target, the amino acid residues forming the drug binding pocket are A373, R398, D502 and K503, the original side chain position of the amino acid residue R398 of the RNase L pseudokinase domain is occupied by 10G5, and the side chain of R398 in the eutectic is positioned above the molecular plane of the fragment to form Pi-ligation interaction. The amino acid residues constituting the drug binding pocket are charged residues except A373. The drug binding pocket is adjacent to the ATP binding site of the pseudokinase domain, and fragment ligation can be used to design the leptin compounds.

In the embodiment, an international advanced fragment-based drug development method (FBDD) is adopted to screen out drug-like molecules capable of effectively inhibiting the activity of a target protein RNase L enzyme, so that a clear direction is provided for further developing a specific inhibitor of the RNase L enzyme for enhancing the anticancer effect of an oncolytic virus therapy, and a corresponding antitumor product can be prepared.

Example 2

Two 10G5 derivatives, 10G-1 and 10G-2, were used as two inhibitory fragments, and the binding constants of the inhibitory fragments to RNase L were determined by micro-scale thermophoresis (MST). The results are shown in FIG. 3, which is a graph showing the results of measurement of binding constants of 10G5-1 and 10G5-2 in another example of the present invention. A is a molecular docking scheme of the 10G5 derivative based on a eutectic structure, and B and C are results of MST method measurement of binding constants of the derivative 10G5-1/10G5-2 and RNaseL, respectively. As can be seen from the graph, the binding constants of 10G5-1 and 10G5-2 were 14. mu.M and 10. mu.M, respectively.

Example 3

An antitumor pharmaceutical composition comprising encephalomyocarditis virus and

example 4

An antitumor pharmaceutical composition comprises West Nile virus and

example 5

An antitumor pharmaceutical composition comprising vaccinia virus and

the embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention.

Claims

1. The application of a compound or pharmaceutically acceptable salt thereof in preparing an oncolytic virus anti-tumor synergist is as follows:

。

2. the use according to claim 1, wherein the oncolytic virus is at least one of herpes simplex virus, oncolytic adenovirus, newcastle disease virus, vaccinia virus, vesicular stomatitis virus, encephalomyocarditis virus, myocarditis virus, west nile virus, coronavirus.

3. A pharmaceutical composition, comprising:

(a) an oncolytic virus;

(b) a compound I or a pharmaceutically acceptable salt thereof, said compound I being:

。

4. the pharmaceutical composition of claim 3, wherein the oncolytic virus is at least one of herpes simplex virus, oncolytic adenovirus, newcastle disease virus, vaccinia virus, vesicular stomatitis virus, encephalomyocarditis virus, myocarditis virus, west nile virus, and coronavirus.