CN117045798A

CN117045798A - Anti-infective action of SHP2 allosteric inhibitor and application thereof

Info

Publication number: CN117045798A
Application number: CN202311235519.3A
Authority: CN
Inventors: 李天亮; 殷书磊; 韩超峰; 胡涵; 李楠
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2023-09-22
Filing date: 2023-09-22
Publication date: 2023-11-14

Abstract

The application relates to an anti-infective effect of an SHP2 allosteric inhibitor and application thereof, in particular to application of the SHP2 allosteric inhibitor (such as a compound SHP099 with a structure shown as a formula I or a derivative thereof) or pharmaceutically acceptable salt thereof in preparing a product for preventing and/or treating infectious diseases and related symptoms and/or symptoms of the subject. The application also relates to a composition comprising SHP2 allosteric inhibitionPharmaceutical compositions of agents (e.g., SHP099 or derivatives thereof) or pharmaceutically acceptable salts thereof, methods of preventing or treating infectious diseases and related conditions and/or symptoms using SHP2 allosteric inhibitors or pharmaceutically acceptable salts thereof.

Description

Anti-infective action of SHP2 allosteric inhibitor and application thereof

Technical Field

The application belongs to the field of medicine and biotechnology. In particular, the present application relates to anti-infective effects, mechanisms, methods of implementation and uses of SHP2 allosteric inhibitors (e.g., compound SHP099 and derivatives thereof) in infectious diseases (e.g., bacterial infections).

Background

Infection is a clinically common disease that can lead to local and systemic infectious manifestations in the body and even to severe consequences of infectious shock, endotoxic shock, and multiple organ dysfunction syndrome (multiple organ dysfunction syndrome, MODS). Shock or multiple organ failure due to severe infection has a mortality rate as high as 50-80%. Common clinical bacterial infections such as escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii, enterococcus faecalis infection and the like can cause local and systemic inflammatory reactions of organisms to cause infectious diseases of different degrees. For patients with bacterial infections, doctors typically use antibiotics, but abuse of antibiotics also increases the risk of multi-drug resistant bacterial infections.

The natural immune system is the first line of defense of the body against invading pathogens. When a pathogen invades a host, phagocytes such as neutrophils, monocytes, and macrophages can engulf and clear the pathogen, maintain cellular homeostasis, and promote specific pro-inflammatory and anti-inflammatory processes [ ostowski, p. Et al, dev cell.2016;38:135-146; doherty, g.j. Et al, annu Rev biochem.2009;78:857-902 ]. Endocytosis of pathogens has become a critical control step in the antibacterial signal transduction process. Mammalian endocytosis occurs through a variety of mechanisms including clathrin-dependent endocytosis, microcoytosis, phagocytosis and vesicle-dependent endocytosis.

After phagocytosis of the pathogen into cells, it is transported to lysosomes where it is degraded by hydrolytic enzymes in the acidic environment of lysosomal enzymes [ Moretti, J. Et al, curr Opin immunol.2014;26:100-110 ]. The partial degradation products of pathogens are presented to histocompatibility complex (MHC) molecules by antigen presenting cells such as macrophages, dendritic Cells (DCs), etc., then recognized by receptors on the T cell surface, thereby activating cd4+ and cd8+ T cells to initiate adaptive immunity [ Cao, X, etc., nat Rev immunol.2016;16:35-50 ]. Meanwhile, a Toll-like receptor, a RIG-I-like receptor, a NOD-like receptor and other Pattern Recognition Receptors (PRRs) on the surfaces of natural immune cells such as macrophages recognize pathogen-related molecular patterns (PAMP), activate a plurality of downstream signal paths, promote the generation and release of inflammatory cytokines [ Liu, J, and the like, and are immune.2016; 45:15-30 ]. Inflammatory cytokines can recruit and activate natural immune cells and lymphocytes, further promoting phagocytosis and clearance of pathogens [ Flannagan, r.s. etc., annu Rev pathl 2012;7:61-98 ].

If the body is unable to clear pathogens effectively in time, it may lead to excessive pathogen proliferation and thus to cytokine storms of the body, which are major potential factors leading to increased susceptibility to multiple organ failure and increased mortality [ Zhao, q. Et al, adv. Sci.2023;10: e2205097 ]. In the immunosuppressive phase of certain diseases (such as patients with advanced sepsis), patients have difficulty in clearing invading pathogens due to bacterial clearance disorder, making them susceptible to life threatening nosocomial infections [ Hoffman, d.immunity.2021;54:2712-2723 ]. This suggests that activation of phagocytes and efficient and timely clearance of bacteria may play a critical role in combating sepsis. Exploration of the regulatory mechanisms of macrophage endocytosis may provide new therapeutic targets for the treatment of bacterial infections and sepsis. Furthermore, untimely elimination of pathogens can also lead to chronic inflammation (such as tuberculosis, chronic hepatitis, chronic nephritis and chronic gastrointestinal diseases) in the body, causing dysfunction of the immune system of the body, and chronic inflammation can cause a number of secondary clinical diseases [ Cook, d.n. et al, nat immunol.2004,5:975-979 ]. Various autoimmune diseases, allergic diseases, atherosclerosis, and even various tumors are closely related to chronic inflammation, and pathogenesis thereof involves abnormal signaling of TLR [ Cook, D.N. et al, nat immunol.2004,5:975-979 ].

SHP2 is a member of a non-receptor tyrosine phosphatase. SHP2 is ubiquitously expressed, not only regulating cell surface kinase activity during normal cell development, but also playing an important role in signaling pathways associated with cell survival and proliferation. SHP2 has become a new target in the field of antitumor. SHP2 inhibitor SHP099 is a tumor inhibitor developed by nova that binds between the 2 SH2 domains and the PTP domain (known as the "tunnel" site) and inhibits SHP2 activity. On the basis of SHP099, various drug development teams have developed derivatives such as TNO155, SHP389, SHP394, RMC-4550, IACS-15414, RMC-4630, IACS-15509, etc. successively and demonstrated their SHP2 allosteric inhibitory activity and/or antitumor activity.

However, studies on the prevention and treatment of infectious diseases such as natural immunity, especially endocytosis, etc. of SHP2 inhibitors (e.g., SHP099 and its derivatives) have not been reported at present.

In view of the foregoing, there is a strong need in the art for the development of an immunologically active substance that is effective in promoting phagocytosis and clearance of infectious pathogens (e.g., bacteria) by cells.

Disclosure of Invention

Provided herein are novel uses of SHP2 allosteric inhibitors (e.g., SHP099 or derivatives thereof or pharmaceutically acceptable salts thereof) in anti-infective agents.

In some aspects herein, there is provided the use of an SHP2 allosteric inhibitor (e.g., SHP099, a derivative thereof, or a pharmaceutically acceptable salt thereof) in the manufacture of a product for preventing and/or treating an infectious disease (e.g., bacterial infection) and related conditions and/or symptoms thereof in a subject.

In some aspects herein, there is also provided an anti-infective pharmaceutical composition comprising:

(A) A therapeutically or prophylactically effective amount of an SHP2 allosteric inhibitor (e.g., SHP099, a derivative thereof, or a pharmaceutically acceptable salt thereof);

(B) Pharmaceutically or immunologically acceptable carriers or excipients;

(C) Optionally, one or more additional active agents for preventing or treating infectious diseases and related conditions and/or symptoms thereof.

In some embodiments, the additional active agent having activity in preventing or treating infection-related diseases and/or symptoms is administered prior to, concurrently with, or after the administration of the SHP2 allosteric inhibitor of the present application.

In some aspects herein, there is also provided a method of preventing or treating an infectious disease and its associated disorders and/or symptoms, the method comprising: administering to a subject in need of prevention or treatment an effective amount of an inhibitor of SHP2 allosteric.

Any combination of the technical solutions and features described above can be made by a person skilled in the art without departing from the inventive concept and the scope of protection of the present application. Other aspects of the application will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present application will be further described with reference to the accompanying drawings, wherein these drawings are provided only for illustrating embodiments of the present application and are not intended to limit the scope of the present application.

Fig. 1: SHP099 pretreated macrophages have enhanced phagocytic capacity:

a: e.coli clone number analysis in macrophages;

b: analysis of clone numbers of staphylococcus aureus and streptococcus pneumoniae in macrophages;

c: the left panel is representative cellular immunofluorescence maps of the experimental and control groups; the right panel shows the average number of bacteria phagocytosed per cell over 10 fields of view;

d: analyzing the average fluorescence intensity;

in the figure, "×" indicates P <0.05.

Fig. 2: SHP099 pretreated macrophages were enhanced in bactericidal capacity: the number of viable bacteria in macrophages is the proportion of the number of initial (0 hours) phagocytes (relative to CFU):

a: the ratio of the number of live E.coli in macrophages to the number of initial (0 hour) phagocytes (E.coli)

Relative CFU);

b: the ratio of the number of viable staphylococcus aureus in macrophages to the initial (0 hour) phagocytic count (gold

Staphylococcus yellow versus CFU);

in the figure, "×" indicates P <0.05, "×" indicates P <0.01.

Fig. 3: SHP099 pretreated mice have enhanced resistance to bacterial infection

A: survival rate analysis;

b: e.coli clone counts in spleen;

c: e.coli clone counts in liver;

d: ELISA analysis of TNF-alpha and IL-6 in serum 6h after E.coli infection;

in the figure, "" means P <0.05, "" means P <0.01, "" means P <0.001; "Vehicle" means physiological saline.

Fig. 4: pretreatment of the SHP2 allosteric agent TNO155 improves survival rate of mice infected with bacteria;

in the figure, "×" indicates P <0.01.

Fig. 5: survival of mice infected with bacteria was increased after treatment with SHP099 or TNO 155;

in the figure, "×" indicates P <0.05.

Description of the embodiments

Through a great number of researches and cell and animal model experiments, the inventor discovers that the SHP2 allosteric inhibitor (such as SHP099, TNO155 and the like) can effectively promote organism phagocytosis and bacteria removal, improve organ function states and increase survival rate of patients in infectious diseases. Thus, disclosed herein are novel functions of SHP2 allosteric inhibitors (including SHP099, derivatives thereof having SHP2 allosteric inhibition (e.g., TNO 155), or pharmaceutically acceptable salts thereof), represented by SHP099, namely anti-infective effects of promoting phagocytosis and clearance of pathogens by the body upon infection, protecting organ functions of the body, and improving survival rate of infected individuals. The application also provides a novel medicine which can effectively promote the capability of the body to phagocytose and clear pathogens during infection and improve the survival rate of infected individuals.

Specifically, application research on related drugs affecting phagocytic and clearance functions of macrophages is a hotspot of molecular biology and cell biology research, and application of compounds promoting phagocytic and clearance to prevention and treatment of infectious diseases is an effective technology, so that related drug research has wide application prospects.

The inventors found through research that: SHP099 and its derivatives (e.g., TNO 155) significantly enhance the ability of mouse macrophages to phagocytose and clear pathogens (e.g., bacteria). In an infection response induced by E.coli (E.coli), SHP099 was observed to significantly reduce pro-inflammatory cytokine production by macrophages. In addition, SHP099 can not only promote endocytosis and kill bacteria, but also maintain the body moderately producing inflammatory responses. In addition, in vivo experiments of animals further prove that SHP099 and derivatives thereof have anti-infection protection and treatment effects, and can remarkably improve the survival rate of animals under pathogen attack. These findings suggest that SHP2 allosteric inhibitors may have application prospects in the prevention and treatment of infectious diseases. Thus, the present application provides methods and strategies for using SHP099 class SHP2 allosteric inhibitors in the prevention and treatment of infectious diseases.

The application aims at novel SHP2 allosteric inhibitor (such as compound SHP099 and its derivative TNO 155) with anti-infective effect, researches on phagocytosis and clearance effect of macrophages on bacteria in immune cells, and verifies the treatment and protection effect of the compound on animals infected by pathogens. Experiments prove that: 1) SHP099 pretreated macrophages have enhanced ability to phagocytose and clear bacteria; 2) SHP099 pretreated mice were more resistant to infection; 3) SHP099 or TNO155 pretreatment and/or treatment can increase survival of bacteria infected mice.

The present application is directed to SHP2 allosteric inhibitors (e.g., SHP099, derivatives thereof, and pharmaceutically acceptable salts thereof) for promoting phagocytosis of macrophages and scavenging bacteria, and enhancing the ability of mice to resist bacterial infection. These applications demonstrate that SHP2 allosteric inhibitors are expected to be effective means for the treatment and prevention of infectious diseases.

All numerical ranges provided herein are intended to expressly include all values and ranges of values between the endpoints of the range. The features mentioned in the description or the features mentioned in the examples can be combined. All of the features disclosed in this specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

As used herein, "comprising," having, "or" including "includes" including, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …" and "consisting of … …" are under the notion of "containing", "having" or "including".

SHP2 allosteric inhibitors

As used herein, the term "SHP2" refers to Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP 2), a Protein Tyrosine Phosphatase (PTP) family member comprising two SH2 domains in tandem at the N-terminus (N-SH 2 and C-SH 2), one PTP domain with catalytic activity and a C-terminal tail comprising two tyrosine phosphorylation sites (Tyr 542 and Tyr 580).

SHP2 remains in a self-inhibited closed conformation in the resting state, where the D 'E loop and the flanking beta D' and beta E chains of the N-SH2 domain extend into the cleft of the PTP catalytic site, interacting with the P, pTyr and Q loops of the catalytic pocket, thereby preventing binding of the substrate to the catalytic site. When stimulated by growth factors or cytokines, the exposed phosphotyrosine (pTyr) recognition site on the surface of the two SH2 domains binds to a specific tyrosine motif on the surface of the RTK or cytokine receptor, resulting in separation of the N-SH2 domain from the PTP domain to induce a change in the closed conformation of SHP2 to an open conformation, allowing the substrate the opportunity to bind to the exposed catalytic site, ultimately effecting dephosphorylation.

As used herein, the term "SHP2 allosteric inhibitor" refers to a substance capable of inhibiting SHP2 conformational changes, such as SHP099, derivatives thereof, and pharmaceutically acceptable salts thereof.

In some embodiments, the SHP2 allosteric inhibitor is selected from the group consisting of:

(A) Formula (I)SHP099 (i.e., CAS:1801747-42-1;6- (4-amino-4-methyl-1-piperidinyl) -3- (2, 3-dichlorophenyl) -2-pyrazinamide; C) ₁₆ H ₁₉ Cl ₂ N ₅ )：

(B) SHP099 as a lead compound-derived SHP2 allosteric inhibitor;

(C) Pharmaceutically acceptable salts of the substances in (A) or (B).

In some embodiments, SHP 099-derived SHP2 allosteric inhibitors can be, for example, one or more SHP099 derivatives described in the following: research progress on anti-allergic inhibitors of SHP2, e.g., tung hong, pharmaceutical chemistry, 2022,10 (2), 211-223); chinese university of pharmacy patent CN113248449B (aromatic spiro derivative), patent application CN114539223a (aryl-aza seven membered ring derivative), patent application CN114478403a (aromatic guanidino derivative), patent CN114524772B (heterocyclic tandem derivative).

In some embodiments, SHP 099-lead compound-derived SHP2 allosteric inhibitors include, but are not limited to, SHP2 allosteric inhibitors selected from the group consisting of:

TNO 155SHP389/>SHP394RMC-4550/>IACS-15414RMC-4630、IACS-15509。

(a) SHP099 is a guanidine-terminated, formamidine-containing aryl spirocyclic derivative;

(b) Aromatic and aza seven-membered ring derivatives of SHP 099;

(c) An aromatic guanidine group-containing derivative of SHP 099;

(d) The terminal end of SHP099 has a derivative of heterocyclic tandem.

In some embodiments, the pharmaceutically acceptable salt is selected from an inorganic acid salt or an organic acid salt. In some embodiments, the pharmaceutically acceptable salt is selected from: a') inorganic acid salts, for example salts with inorganic acids selected from the group consisting of: hydrochloric acid, phosphoric acid, borohydric acid, nitric acid and sulfuric acid; or b') an organic acid salt, for example a salt with an organic acid selected from the group consisting of: lactic acid, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and acidic amino acids (e.g., glycine, aspartic acid and glutamic acid).

Macrophage phagocytosis promoting function and anti-infection application of SHP2 allosteric inhibitor

The application discloses the activity of the SHP2 allosteric inhibitor for promoting the phagocytic function of macrophages and preventing or treating infectious diseases for the first time and the role of the SHP2 allosteric inhibitor in resisting infection. Thus, in some aspects herein, there is provided the use of an SHP2 allosteric inhibitor in the preparation of a product for promoting phagocytosis of macrophages. In some aspects herein, there is provided the use of an SHP2 allosteric inhibitor in the manufacture of a product for the prevention and/or treatment of an infectious disease and related conditions and/or symptoms thereof in a subject. In some aspects herein, SHP2 allosteric inhibitors are provided for the prevention and/or treatment of infectious diseases and related conditions and/or symptoms thereof in a subject.

In some embodiments, the subject is a mammal, e.g., a human, a non-human primate, a pet (e.g., cat, dog, mouse), a livestock (e.g., horse, cow, sheep, pig, rabbit), and the like.

In some embodiments, the infection is caused by a pathogen, a chemical, a physical factor, or a combination thereof, e.g., the infection is caused by one or more pathogens selected from the group consisting of: bacteria, fungi, mycoplasma, chlamydia, and parasites.

In some embodiments, the pathogen is a pathogen that is capable of clearance by macrophages (e.g., clearance by phagocytosis of macrophages).

In some embodiments, the pathogen is a bacterium, such as a gram positive or gram negative bacterium.

In some embodiments, the pathogen is selected from one or more of the following group: coli, pseudomonas aeruginosa, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, enterococcus faecalis, bacillus, legionella pneumophila, haemophilus influenzae, helicobacter pylori, clostridium botulinum, bacillus anthracis, escherichia coli, neisseria, salmonella, shigella, candida, or combinations thereof.

In some embodiments, the related disorder and/or symptom is one or more selected from the group consisting of: the infection-related disorder and/or symptom is one or more selected from the group consisting of: overproduction of inflammatory factors following infection; endotoxic shock or death; inflammatory injury to organs caused by infection; multiple organ failure due to infection.

In some embodiments, the inflammatory factor is one or more selected from the group consisting of: TNFα, IL-1, IL-6, IFN- β, preferably TNFα, IFN- β or IL-6.

In some embodiments, the organ is selected from: liver, spleen, brain, kidney, heart, lung, stomach, intestine.

In some embodiments, the product is a pharmaceutical composition comprising an effective amount of an inhibitor of SHP2 allosteric (e.g., SHP099, a derivative thereof, or a pharmaceutically acceptable salt thereof), and a pharmaceutically acceptable carrier or excipient. Further description of the pharmaceutical combinations may be found below.

Pharmaceutical composition

The present application provides a pharmaceutical composition comprising an effective amount of an inhibitor of the present application of SHP2 allosteric (e.g., SHP099, a derivative thereof, or a pharmaceutically acceptable salt thereof), and a pharmaceutically acceptable carrier.

In preferred embodiments, the pharmaceutical compositions are useful in the treatment and prevention of infectious diseases known in the art, such as phagocytosis and clearance of bacteria in vivo after bacterial infection; endotoxic shock or death; inflammatory injury of organs; multiple organ failure.

As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.

As used herein, the term "effective amount" refers to an amount that is functional or active in and acceptable to a human and/or animal.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such agent carriers: they are not per se essential active ingredients and are not overly toxic after administration. Suitable vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington pharmaceutical sciences (Remington's Pharmaceutical Sciences, mack Pub.Co., N.J.1991).

The pharmaceutically acceptable carrier in the composition may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting or emulsifying agents, flavoring agents, pH buffering substances, etc. may also be present in these carriers. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8.

The SHP099 and pharmaceutically acceptable salt thereof in the composition of the application have the active ingredients accounting for 0.001 to 99.9 weight percent of the total weight of the composition; preferably 1 to 95wt%, more preferably 5 to 90wt%, and even more preferably 10 to 80wt% of the total weight of the composition. The rest is pharmaceutically acceptable carrier and other additives.

As used herein, the term "unit dosage form" refers to a dosage form that is required to prepare a composition of the present application for administration in a single administration, including but not limited to various solid (e.g., tablet), liquid, capsule, sustained release formulations.

In another preferred embodiment of the present application, the composition is in unit dosage form or multiple dosage form, and wherein the content of SHP099 and its pharmaceutically acceptable salts can be 0.01 to 2000 mg/dose, preferably 0.1 to 1500 mg/dose, more preferably 1 to 1000 mg/dose. In another preferred embodiment of the application, 1 to 6 doses of the composition of the application, preferably 1 to 3 doses, are administered daily; most preferably, the daily dosage is 1 dose.

It will be appreciated that the effective dose of SHP099 and pharmaceutically acceptable salts thereof used can vary with the severity of the subject to be administered or treated. The specific conditions are determined according to the individual condition of the subject (e.g., the subject's weight, age, physical condition, effect to be achieved), which is within the scope of judgment of a skilled physician.

The composition of the application can be solid (such as granules, tablets, freeze-dried powder, suppositories, capsules, sublingual tablets) or liquid (such as oral liquid) or other suitable shapes. The administration route can be as follows: (1) direct injection; (2) oral administration; (3) Complex formation with positively charged lipids to overcome the difficulty of crossing cell membranes due to negative charge of the phosphate backbone; (4) After the liposome is used for wrapping the SHP2 allosteric inhibitor, the liposome mediates to enter cells, thereby being beneficial to the smooth entry of macromolecules and avoiding the hydrolysis of various extracellular enzymes; (5) SHP2 allosteric inhibitor binding to cholesterol increases its cytoplasmic retention time by a factor of 10; (6) Specific transport to target tissues and cells is enabled by immunoliposome transport of SHP2 allosteric inhibitor; (7) In vitro transfection of SHP2 allosteric inhibitors into the transfected cells (e.g., fibroblasts) also provides for better loading into the target cells; (8) Electroporation (electro corporation), i.e., introduction of SHP2 allosteric inhibitors into target cells by means of electric current.

In addition, the compositions of the present application may contain other active substances for ameliorating and treating bacterial infectious diseases, said other active substances being selected from the group consisting of: one or more of antibiotics commonly used in clinic (including beta-lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, chloramphenicol, macrolides, antifungal antibiotics, antitubercular antibiotics).

The SHP2 allosteric inhibitors of the present application can be used in combination with each other, and also with other drugs and therapeutic means for the prevention and treatment of infectious diseases (e.g., bacterial infection).

Thus, the present application further provides the following embodiments:

in some embodiments, the product is a tablet, capsule, powder, granule, suspension, or injection. In some embodiments, the product is in a form suitable for administration in a manner selected from the group consisting of: oral, injection (e.g., direct injection, liposome-encapsulated compound injection), spray, inhalation.

(B) Pharmaceutically or immunologically acceptable carriers or excipients;

In some embodiments, other active agents that treat or prevent infectious diseases and their associated conditions and/or symptoms include, but are not limited to: one or more of antibiotics commonly used in clinic (including beta-lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, chloramphenicol, macrolides, antifungal antibiotics, antitubercular antibiotics).

In some embodiments, the SHP2 allosteric inhibitor in the pharmaceutical composition comprises from 0.001 to 99.9wt% of the total weight of the pharmaceutical composition.

In some embodiments, the SHP2 allosteric inhibitor in the pharmaceutical composition comprises 1 to 95wt%, preferably 5 to 90wt%, more preferably 10 to 80wt%, of the total weight of the pharmaceutical composition.

In some aspects herein, there is provided a method of preventing or treating an infectious disease and its associated disorders and/or symptoms, the method comprising: administering to a subject in need of prevention or treatment an effective amount of an inhibitor of SHP2 allosteric.

Examples

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Appropriate modifications and variations of the application may be made by those skilled in the art, and are within the scope of the application.

The experimental procedures described in the following examples, which are not explicitly described in the specification, may be carried out by methods conventional in the art, for example, by reference to the molecular cloning laboratory Manual (third edition, new York, cold spring harbor laboratory Press, new York: cold Spring Harbor Laboratory Press, 1989) or according to the conditions suggested by the suppliers.

Percentages and parts are by weight unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred methods and materials described herein are presented for illustrative purposes only.

Example 1: SHP099 pretreated macrophages have enhanced ability to phagocytize bacteria

1. Acquisition of macrophages

C57 mice (6-8 weeks, 18-22 g body weight, female) were intraperitoneally injected with 2ml of broth (available from Merc)k) Four days later, mice were sacrificed by cervical dislocation, the abdominal cavity was flushed with DMEM medium, and the medium was aspirated, placed in a centrifuge tube, and centrifuged at 1000rpm for 5min. The supernatant was discarded, resuspended in serum-bearing medium, and then air-beaten to mix to form a single cell suspension, after which the cells were counted and plated. Placing the cell plate at 37deg.C, 5% CO ₂ Culturing in incubator for 3 hr, and changing liquid to remove non-adherent cells, wherein the adherent cells are primary abdominal macrophages of the mice.

Pretreatment or post-treatment of SHP2 allosteric agent

SHP099 (cat No. S6388), TNO155 (cat No. S8987) were all purchased from Selleck. Coli (e.coli) (ATCC 25923), staphylococcus aureus (s.aureus) (ATCC 6538) or streptococcus pneumoniae (s.pneumoniae) (ATCC 6303) were all purchased from ACTT.

2.1. Cell experiment

Cell experiments for SHP099 pretreatment: cells were pretreated with SHP099 at the set concentration for 1 hour, the control group was treated with the same volume of solvent (DMSO) for 1 hour, and then escherichia coli, staphylococcus aureus, or streptococcus pneumoniae (MOI 10, respectively) were added, followed by subsequent experiments.

2.2. Animal experiment

Mice were randomly divided into control and experimental groups, all experiments using age, sex matched controls.

Mouse experiments with SHP099 or TNO155 pretreatment: SHP099 (50 mg/kg) or TNO155 (60 mg/kg) was injected intraperitoneally, the control group was injected intraperitoneally with the same volume of solvent (Vehicle, physiological saline), and E.coli (5X 10) was injected intraperitoneally after 24 hours ⁶ CFU/g) and then subsequent experiments were performed.

Mice experiments with SHP099 or TNO155 treatment after infection: coli (5×10) ⁶ CFU/g), SHP099 (50 mg/kg) or TNO155 (60 mg/kg) was injected intraperitoneally after 12 hours, and the control group was injected intraperitoneally with the same volume of solvent (physiological saline), and then the subsequent experiments were performed.

3. Test method

3.1. Bacterial clone number test for macrophage phagocytosis:

after pretreating macrophages with SHP099 at a set concentration for 1 hour, the mice were infected with peritoneal macrophages (bacteria: cell=10:1) of escherichia coli (e.coli), staphylococcus aureus (s.aureus) or streptococcus pneumoniae (s.pneumoniae), respectively, for 1 hour, and then the cells were placed on ice, cultured with gentamicin (100 μg/ml) for 30 minutes to remove extracellular viable bacteria, and then the colony formation Count (CFU) of the cell lysate (PBS solution containing 0.1% triton) was analyzed to examine the phagocytic capacity of the macrophages.

3.2. Immunofluorescence assay:

PI-labeled escherichia coli (e.coli) was incubated with mouse peritoneal macrophages (bacteria: cells=10:1) for 1h, washed twice with pre-chilled PBS on ice, then the cells were fixed with 4% paraformaldehyde, finally stained with DAPI, and phagocytosis of the macrophages by the escherichia coli was observed with a laser confocal microscope.

3.3. Zymosan cell phagocytosis assay:

after incubation with macrophages for 1h with fluorescent-labelled zymosan (zymosan, available from Merck under the designation IAK 0111), excess zymosan was washed off with PBS and the cells were digested and then detected by flow analysis.

4. Results and discussion

The number of bacterial clones phagocytosed by macrophages is shown in fig. 1 a and B: the number of macrophages phagocytosis by SHP099 pretreatment increased, which was dose dependent and formed a significant difference at 5 μm SHP099 treatment concentration from the untreated group.

Immunofluorescence results are shown in fig. 1C: the fluorescence generated by bacteria in SHP099 pretreated macrophages was significantly enhanced, indicating an increased number of phagocytic bacteria by macrophages.

The average fluorescence intensity measurement results of the zymosan cell phagocytic assay are shown in D of fig. 1: the number of SHP 099-pretreated macrophages phagocytosed the pathogen mimetic zymosan increased significantly, which was dose-dependent and formed a significant difference at 5 μm SHP 099-treated concentration from the untreated group. This result is consistent with the bacterial clone count test results of macrophage phagocytosis.

This example shows that: SHP099 enhances the ability of macrophages to phagocytose bacteria.

Example 2: SHP099 pretreated macrophages have enhanced ability to clear bacteria

1. Analysis of the sterilization efficiency in macrophages:

after the mice were infected with the E.coli or Staphylococcus aureus respectively for 1h with the abdominal macrophages (bacteria: cell=10:1), the number of phagocytic bacteria of the macrophages was calculated by CFU (recorded as 0 h), and after the extracellular bacteria were killed by culturing with gentamicin (100. Mu.g/ml) for 30min, the culturing was continued for 3h to 6h, and the percentage of the number of viable bacteria in the macrophages after 3h or 6h was calculated as the number of phagocytic bacteria of 0h, respectively.

2. Results and discussion:

the macrophage sterilization efficiency analysis is shown in fig. 2: macrophages pretreated with SHP099 can exhibit a very significantly reduced relative CFU at 3 hours, while still retaining significantly improved bactericidal effect at 6 hours. The result shows that SHP099 can significantly improve the sterilization efficiency of macrophages.

Example 3: SHP099 pretreatment enhances mice' ability to resist escherichia coli infection

1. Pretreatment of mice

C57 mice (10 animals each in female, experimental and control groups) of 6-8 weeks old were purchased from Pickie biotechnology development Co., ltd, and after one week of rearing in SPF-class animal houses at the university of navy medical science, the experimental group mice were intraperitoneally injected with SHP099 solution (50 mg/kg), and the control group mice were injected with an equivalent amount of solvent (Vehicle).

2. Test method

2.1. Mice survival rate observation:

after injection of SHP099 (50 mg/kg) or equivalent of solvent (Vehicle), the method was carried out using (5X 10) ⁶ CFU/g) mice were intraperitoneally injected with escherichia coli, and survival rate of the mice was dynamically observed;

2.2. serum inflammatory factor content assay:

blood was taken from the eye at various times and the serum was assayed for TNF- α and IL-6 levels using CBA (available from BD company);

2.3. spleen and liver bacteria number detection:

coli numbers (CFU) were measured in spleen and liver by bacterial culture.

3. Results and discussion

The survival rate of mice after infection with E.coli is shown in FIG. 3A. The results show that: SHP 099-pretreated mice began to die later, and the survival rate of SHP 099-pretreated mice was significantly higher than control mice.

The number of E.coli in spleen and liver is shown in FIGS. 4B and C. The results show that: the CFU of escherichia coli in SHP099 pretreated mice was significantly reduced, indicating that SHP099 limited bacterial proliferation and spread in mice.

The results of CBA analysis of the production of inflammatory factors TNFα, IL-6 in serum are shown in FIG. 3D. The results show that: SHP099 pretreated mice produced significantly reduced inflammatory factors tnfα, IL-6 after infection with escherichia coli.

This example shows that: SHP099 pretreated mice were enhanced in their ability to resist escherichia coli infection.

Example 4: TNO155 pretreatment enhances survival rate of bacteria infected mice

1. Animal treatment

C57 mice (9 mice in each of the experimental group and the control group) of 6-8 weeks old were purchased from Pickie biotechnology development Co., ltd, and after one week of rearing in SPF-class animal houses at the university of naval medical science, the experimental group mice were intraperitoneally injected with TNO155 solution (60 mg/kg) and the control group mice were injected with an equal volume of solvent (Vehicle).

2. Test method

After injection of TNO155 (60 mg/kg) or an equivalent amount of solvent (Vehicle), the method was carried out using (5X 10) ⁶ CFU/g) mice were intraperitoneally injected with escherichia coli, and survival rate of the mice was dynamically observed.

3. Results and discussion

The survival rate of mice after infection with E.coli is shown in FIG. 4A. The results show that: TNO155 pretreated mice began to die later and the survival rate of TNO155 pretreated mice was significantly higher than control mice.

This example shows that: TNO155 pretreatment greatly improves the survival rate of mice infected by bacteria.

Example 5: SHP099 and TNO155 treatment enhanced survival in mice infected with bacteria

1. Animal treatment

C57 mice (10 mice in each of the experimental group and the control group) of 6 to 8 weeks old were purchased from Pickie biotechnology development Co., ltd, and after one week of rearing in SPF-class animal houses at the university of naval medical science, the experimental group mice were intraperitoneally injected with SHP099 (50 mg/kg) or TNO155 solution (60 mg/kg), and the control group mice were injected with an equal volume of solvent (Vehicle).

2. Test method

By using (5X 10) ⁶ CFU/g) E.coli, TNO155 (60 mg/kg) or SHP099 (50 mg/kg) or equivalent solvent (Vehicle) was injected intraperitoneally into the mice after 12 hours, and the survival rate of the mice was dynamically observed;

3. results and discussion

The survival rate of mice after infection with E.coli is shown in FIG. 5A. The results show that: SHP099 or TNO155 treated mice began to die later and SHP099 or TNO155 treated mice survived significantly more than control mice.

This example shows that: treatment with SHP099 or TNO155 increased survival in mice infected with bacteria.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

Use of an shp2 allosteric inhibitor for the preparation of a product for the prevention and/or treatment of an infectious disease and related disorders and/or symptoms thereof in a subject.
2. Use according to claim 1, wherein the infection is caused by a pathogen, a chemical, a physical factor or a combination thereof, e.g. the infection is caused by one or more pathogens selected from the group consisting of: bacteria, fungi, mycoplasma, chlamydia and parasites; and/or

The subject is a mammal, e.g., a human, non-human primate, pet, livestock.
3. The use of claim 1, wherein the infection-related disorder and/or symptom is one or more selected from the group consisting of: overproduction of inflammatory factors following infection; endotoxic shock or death; inflammatory injury to organs caused by infection; multiple organ failure due to infection;

for example, the inflammatory factor overproduced after infection is one or more selected from the group consisting of: TNFα, IL-1, IL-6, IFN- β, preferably TNFα, IFN- β or IL-6;

for example, the organ is selected from: liver, spleen, brain, kidney, heart, lung, stomach, intestine.
4. The use of claim 2, wherein the pathogen is selected from one or more of the group consisting of: coli, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii, enterococcus faecalis, bacillus, legionella pneumophila, haemophilus influenzae, helicobacter pylori, clostridium botulinum, bacillus anthracis, escherichia coli, neisseria, salmonella, shigella, candida, or combinations thereof.
5. The use of claim 1, wherein the SHP2 allosteric inhibitor is selected from the group consisting of:

(A) SHP099 of formula (I):

(B) SHP099 is used as a precursor compound derived SHP2 allosteric inhibitor,

for example, SHP2 allosteric inhibitors selected from the group consisting of:

TNO 155SHP389/>

SHP394RMC-4550/>

IACS-15414RMC-4630, IACS-15509; or (b)

(C) Pharmaceutically acceptable salts of the substances in (A) or (B).
6. The use of claim 5, wherein the pharmaceutically acceptable salt is selected from the group consisting of: inorganic acid salts or organic acid salts, for example:

a') inorganic acid salts, for example salts with inorganic acids selected from the group consisting of: hydrochloric acid, phosphoric acid, borohydric acid, nitric acid and sulfuric acid; or (b)

b') an organic acid salt, for example a salt with an organic acid selected from the group consisting of: lactic acid, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and acidic amino acids (e.g., glycine, aspartic acid and glutamic acid).
7. Use according to claim 1, wherein the product is a pharmaceutical composition comprising an effective amount of an SHP2 allosteric inhibitor (e.g. SHP099, a derivative thereof or a pharmaceutically acceptable salt thereof), and a pharmaceutically acceptable carrier or excipient; and/or

The product is tablet, capsule, injection, powder, granule, suspension, suppository, or oral liquid; and/or

The product is in a form suitable for administration in a manner selected from the group consisting of: oral, injection (e.g., direct compound injection, liposome-encapsulated compound injection), spray, inhalation.
8. Use according to claim 1, wherein the product further comprises one or more other active substances for preventing or treating infectious diseases and their associated conditions and/or symptoms, such as clinically usual antibiotics (e.g. β -lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, chloramphenicol, macrolides, antifungal antibiotics, antitubercular antibiotics).
9. A pharmaceutical composition comprising:

(A) A therapeutically or prophylactically effective amount of an inhibitor of SHP2 allosteric, such as SHP099 or a pharmaceutically acceptable salt thereof;

(B) Pharmaceutically or immunologically acceptable carriers or excipients; and

(C) One or more other active agents for preventing or treating infectious diseases and related conditions and/or symptoms.
10. The pharmaceutical composition of claim 8, wherein the other active substance is selected from the group consisting of: antibiotics (e.g., beta-lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, chloramphenicol, macrolides, antifungal antibiotics, antitubercular antibiotics) are commonly used in clinic.