WO2024019661A1 - Labdane based compounds and uses thereof - Google Patents
Labdane based compounds and uses thereof Download PDFInfo
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- WO2024019661A1 WO2024019661A1 PCT/SG2023/050487 SG2023050487W WO2024019661A1 WO 2024019661 A1 WO2024019661 A1 WO 2024019661A1 SG 2023050487 W SG2023050487 W SG 2023050487W WO 2024019661 A1 WO2024019661 A1 WO 2024019661A1
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 185
- LEWJAHURGICVRE-AISVETHESA-N labdane Chemical compound CC1(C)CCC[C@]2(C)[C@@H](CC[C@H](C)CC)[C@@H](C)CC[C@H]21 LEWJAHURGICVRE-AISVETHESA-N 0.000 title description 6
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Classifications
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
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- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4192—1,2,3-Triazoles
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- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/443—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
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- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
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- C07D307/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
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- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/58—One oxygen atom, e.g. butenolide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
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- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/64—Sulfur atoms
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
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- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/66—Nitrogen atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
- C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
Definitions
- the present invention relates, in general terms, labdane based compounds and their uses thereof.
- MAPK-activated protein kinase 2 is a direct downstream substrate of p3gMAPK a nc j as been recognized as a key driver of inflammation. MK2 is often found up-regulated and phosphorylated in chronic inflammation involved in asthma, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, Alzheimer's disease, ischemic heart, and brain diseases, as well as cancer.
- the kinase primarily functions as a master regulator of RNA-binding proteins (RBPs), indirectly modulating cytokine production by increasing the stability and translation of the corresponding mRNAs.
- RBPs RNA-binding proteins
- MK2 phosphorylates the anti-inflammatory RBP tristetraprolin (TTP), inhibits TTP-destabilizing activity towards adenylate/uridylate-rich elements (AREs)-containing mRNAs e.g., TNF-a), thus permitting their translation.
- TTP anti-inflammatory RBP tristetraprolin
- AREs adenylate/uridylate-rich elements
- MK2 is currently considered as a promising target for disease-modifying anti-rheumatic drugs (DMARDs) and a possible alternative to p38 MAPK for anti-inflammatory therapy development.
- DMARDs disease-modifying anti-rheumatic drugs
- MK2 knock-out mice are viable and fertile, and do not display any abnormalities apart from the intended impairment in cytokine biosynthesis.
- Targeting MK2 leaves intact the important p38 MAPK feedback loops, such as TAB1-TAK1, and the activation of downstream p38 MAPK anti-inflammatory substrates such as MSK1 and MSK2. Therefore, targeting MK2 could reproduce the beneficial effects of p38 MAPK inhibition while potentially sparing the accompanying side effects.
- PF-3644022 the first orally available small molecule MK2 inhibitor developed by Pfizer, was demonstrated to be effective in numerous acute and chronic models of inflammation including rheumatoid arthritis, inflammatory and fibrotic pulmonary diseases.
- biochemical efficiency of PF-3644022 and other MK2 inhibitors is rather poor as compared to most drugs on the market. Hence, the search for further small molecule drugs against MK2 is necessary.
- the present invention is predicated on the understanding that degradation, rather than inhibition, of MK2 protein may produce a more durable and sustained anti-inflammatory effect.
- the present invention provides a method of promoting the ubiquitination of MK2 protein, comprising contacting the MK2 protein with a compound of Formula (I) or a salt, solvate, stereoisomer or prodrug thereof: wherein
- Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino; 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
- R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
- the compound of Formula (I) can act as a MK2 protein regulator through ubiquitination and degradation of MK2 protein.
- Compounds of Formula (I) may bind to the activation loop of MK2 located at the interface of the p38a-MK2 complex, causing complex dissociation and the loss of MK2.
- Ri is selected from optionally substituted acyloxy, optionally substituted arylthio, optionally substituted heteroarylthio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryl oxy, and optionally substituted heteroarylamino.
- Ri is a moiety having moderate-to-good leaving group ability and/or possesses aromatic moieties, the compounds were found to be more active.
- the compound of Formula (I) is characterised by a pKaH of about 3 to about 10.
- the MK2 protein is characterised by a half-life of less than 4 h.
- the method is an in vivo method or an in vitro method.
- the compound of Formula (I) is a compound of Formula (Ic): wherein
- Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino;
- R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
- R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
- the compound of Formula (I) is a compound of Formula (Id): wherein
- X is a heteroatom
- R4 is selected from optionally substituted acyl, optionally substituted aryl, and optionally substituted heteroaryl;
- R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
- R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
- X is selected from 0, S, N.
- FU is selected from optionally substituted phenyl and optionally substituted 6 membered heteroaryl.
- FU is selected from phenyl optionally substituted at a para position and 6 membered heteroaryl optionally substituted at a para position.
- the compound of Formula (I) is a compound of Formula (le):
- Het is an optionally substituted heteroaryl
- F is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
- Fb is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
- the heteroaryl is bonded via its heteroatom to C-12 position of Formula (I).
- the heteroaryl is selected from oxazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolinyl, quinolinyl, phthalazinyl, naphthylpyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, acridinyl, phenanthrolinyl, isothiazolyl, phenazinyl, isoxazolyl, isothiazolyl, phenoxazinyl, phenothiazinyl, thiazolyl, thiadiazolyl
- the compound of Formula (I) is a compound of Formula (Ic 1 ): wherein
- Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino; 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and 3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optional
- the compound of Formula (I) is a compound of Formula (Ic"): wherein
- Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino; 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy,
- the compound is selected from one of the following:
- the present invention also provides a compound of Formula (Ic) or a salt, solvate, stereoisomer or prodrug thereof: wherein
- Ri is selected from
- X is a heteroatom selected from N, 0, S; 4 is selected from optionally substituted aryl, optionally substituted heteroaryl; or X- 4 together is optionally substituted acyloxy;
- Het is a optionally substituted heteroaryl, wherein the heteroatom on the heteroaryl is bonded to the C-12 position of Formula (I); 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and 3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
- the compound of Formula (I) is selected from:
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
- the present invention also provides a method of treating of a disease or condition associated with MK2 protein, comprising administrating to a subject in need thereof a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable a salt, solvate, stereoisomer or prodrug thereof.
- the present invention also provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for use in treating a disease or condition associated with MK2 protein.
- the present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament for the treatment of a disease or condition associated with MK2 protein.
- the treatment of a disease or condition associated with MK2 protein comprises ubiquitination of MK2 protein.
- the disease or condition associated with MK2 protein is an inflammatory disease or condition.
- the disease or condition associated with MK2 protein is a chronic respiratory disease or condition.
- the disease or condition associated with MK2 protein is selected from acute lung injury, chronic inflammation in asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease, atherosclerosis, Alzheimer's disease, ischemic heart, brain disease, cancer, chronic obstructive pulmonary disease (COPD), lung fibrosis, pneumonia, long COVID.
- acute lung injury chronic inflammation in asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease, atherosclerosis, Alzheimer's disease, ischemic heart, brain disease, cancer, chronic obstructive pulmonary disease (COPD), lung fibrosis, pneumonia, long COVID.
- COVID chronic obstructive pulmonary disease
- the present invention also provides a method of inducing an anti-inflammatory response in a subject in need thereof, comprising administrating to a subject in need thereof a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable a salt, solvate, stereoisomer or prodrug thereof.
- the present invention also provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for inducing an antiinflammatory response in a subject in need thereof.
- the present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament for inducing an anti-inflammatory response in a subject in need thereof.
- FIG. 1 MK2 is highly expressed in macrophages and is essential for production of inflammatory mediators.
- A Protein expression levels of MK2 in various cell lines. Total cell lysates were analyzed by immunoblotting. The images shown are representative of two independent experiments.
- B and C Validation of MK2 expression levels in MK2-knockdown (KD) and MK2-overexpression (OE) RAW264.7 cells compared with negative control cells (NC).
- KD MK2-knockdown
- OE MK2-overexpression
- NC negative control cells
- B MK2-KD cells were generated with siRNA to MK2 (si-MK2) and
- C MK2-OE cells were generated with CDNA-MK2. Cell lysates were assayed by immunoblotting. The images shown are representative of two independent experiments.
- AD down-regulates MK2 protein levels and blocks MK2-mediated inflammatory responses in macrophages.
- a and B Dose-dependent effect of AD on MK2 protein levels in RAW264.7 cells.
- A Cells were incubated with increasing concentrations of AD (i.e., 5, 10, 20, 50 pM) for 4 h.
- B Cells were pre-incubated with increasing concentrations of AD (i.e., 5, 10, 20, 50 pM) for 1 h and then treated with LPS (100 ng/ml) for 4 h. Cell lysates were analysed by immunoblotting. The images shown are representative of three independent experiments.
- C Time-dependent effect of CHX and AD on MK2 protein levels.
- D and E Effects of AD on LPS-induced (D) mRNA expression level and (E) secretion levels of TNF-a and MCP-1.
- D RAW264.7 cells were pre-incubated with increasing concentrations of AD for 1 h and then treated with LPS (100 ng/ml) for 4 h. Quantitative PCR was performed. Data are shown as mean ⁇ SEM of three independent experiments.
- FIG. 3 The anti-inflammatory effects of AD are MK2-dependent and involve TTP.
- a and B Effects of AD on LPS-induced secretion levels of (A) TNF-a and (B) MCP- 1 by NC and MK2-OE RAW264.7 cells. The cell supernatant way assayed by ELISA. Data are shown as mean ⁇ SEM of three independent experiments.
- C and D The transfection efficiency of siRNA to TTP (si-TTP).
- C Cell lysates were assayed by immunoblotting. GAPDH was used as the loading control. The images shown are representative of two independent experiments.
- D Cells were challenged with or without LPS (100 ng/ml) for 4 h and quantitative PCR was performed.
- AD promotes MK2 down-regulation in vivo and attenuates inflammatory responses in LPS-induced acute lung injury (ALI).
- a and B Schematic diagram of in vivo AD treatment and LPS-induced ALI.
- C Effects of AD on MK2 total protein expression in primary AMs and lung tissues. AMs were isolated from lung lobes. Total cells lysates and lung homogenates were analysed by immunoblotting. P-actin was used as the loading control. The images shown are representative of three independent experiments.
- D Effects of LPS stimulation on AMs isolated from mice pretreated with AD. Mice were treated with AD (1 mg/kg, i.t.) for the indicated times and AMs were isolated from lung lobes.
- AD promotes the ubiquitination and proteasomal degradation of MK2.
- A Effects of AD on mRNA expression of MK2 in RAW264.7 cells. Cells were incubated with increasing concentrations of AD ( .e., 5, 10, 20, 50 pM) for 4 h and quantitative PCR was performed. The amount of mRNA was normalized to p-actin expression and was presented as fold of untreated control. Data are shown as mean ⁇ SEM of four independent experiments.
- B Effects of AD on MK2 protein expression levels in the presence of MG132. Cells were pre-incubated with MG132 (1 pM) for 1 h and then treated with AD (50 pM) for another 2 h.
- AD binds to the activation loop of MK2 located at the interface between p38a and MK2.
- A X-ray crystallography of p38a-MK2 complex obtained from PDB (ID: 2OZA).
- B Docking of AD on MK2 (chain A). Blind docking (rigid residues) was performed using iGEMDOCK v.2.1. with 100 solutions. When the 100 conformers were sorted according to their energies, 90% of the conformers with the lowest energies docked around a site adjacent to the activation loop.
- C The binding site residues are colored. The figures were generated using the program Chimera (reference).
- D CETSA for MK2.
- AD was pre-incubated with recombinant MK2 10 min prior to the addition of p38a (4: 1 molar ratio) and ATP (150 pM). The amount of phosphorylated MK2 was chased with phosphosite-specific antibodies that recognize Thr222 or Thr334.
- the images shown are representative of three independent in vitro kinase assays.
- Recombinant MK2 was pre-incubated with DMSO, AD, and PF3644022 in increasing concentrations 10 min prior to the addition of HSP27 peptide substrate and ATP. The kinase reaction was allowed for 1 h before an ADP-Glo kinase assay. Data are shown as mean ⁇ SEM of three independent experiments.
- FIG. 7 Pharmacological difference between protein degrader (AD) and inhibitor (PF3644022) against MK2.
- A Recovery of MK2 level after washout of AD. After treatment for 4 h with AD (50 pM), RAW264.7 cells were washed with fresh culture medium and further cultured for the indicated times. The images shown are representative of three independent experiments.
- B Effects of pre-treatment time on the cytokine inhibitory effects of AD and PF3644022. RAW264.7 cells were preincubated with test compounds for 1, 6 or 12 h and then treated with LPS (100 ng/ml) for another 4 h. The cell supernatant way assayed by ELISA. Data are shown as mean ⁇ SEM of three independent experiments.
- FIG. 10 (A-B) Inhibitory effects of various test compounds on LPS-stimulated TNF- a and IL-6 release. (C) Cytotoxic effects of test compounds on non-stimulated RAW264.7 cells. (*) Derivatives carry aromatic substituents at C-12 position.
- FIG. 13 Total and differential cell counts in bronchoalveolar lavage fluids (BALF) of mice stimulated with HDM and treated with 4, or vehicle control. *p ⁇ 0.05, **p ⁇ 0.01 indicate statistically significant difference as compared to HDM-treated group. #p ⁇ 0.05 indicates statistically significant difference as compared to saline-treated group.
- BALF bronchoalveolar lavage fluids
- FIG. 14 Secretion levels of (A) MIP-2 and (B) KC in NC and siMK2-transfected RAW264.7 cells with or without LPS challenge. The cell supernatants were assayed by ELISA (4 h for MIP-2 and 24 h for KC after LPS challenge). Data are shown as mean ⁇ SEM of three independent experiments. **, P ⁇ 0.01; NC, negative control.
- FIG. 15 Effects of AD on the cell viability of RAW264.7 cells and primary AMs. Cells were treated with increasing concentrations of AD (0.1 to 50 pM) for the indicated times. Cell viability was assayed by MTS. Data are shown as mean ⁇ SEM of three independent experiments.
- FIG. 16 Effects of AD on the protein levels of p38a, TTP, MKK6 in primary AMs (A) and lung tissues (B). AMs were isolated from lung lobes. Total cells lysates and lung homogenates were analysed by immunoblotting, p-actin was used as the loading control. The images shown are representative of three independent experiments.
- FIG. 1 Time-dependent effect of CHX on MK2 protein levels.
- RAW264.7 cells were incubated with CHX (1 pg/mL) for the indicated times up to 8 h. Cell lysates were analysed by immunoblotting. Band intensities were analyzed by Image!.
- FIG. 18 Dose-dependent effects of (A) AD and (B) PF-3644022 on LPS-induced production of TNF-a.
- RAW264.7 cells were pre-treated with increasing concentrations of test compounds for 1 h before stimulated with LPS (100 ng/mL) for 4 h. The cell supernatants were assayed by ELISA. LPS-only treated cells were counted as 100%. IC50 value is determined as the concentration at which test compounds inhibit 50% of TNF-a production. Data are shown as mean ⁇ SEM of three independent experiments.
- FIG. 19 The immunofluorescent staining for DAPI (blue) and MK2 (red) in primary AMs showed that MK2 is ubiquitously expressed in both cytoplasmic and nuclear compartments.
- B Effects of AD on the phosphorylation of p38a in RAW264.7 cells. Cells were incubated with increasing concentrations of AD (i.e., 5, 10, 20, 50 pM) for 4 h. Cell lysates were analysed by immunoblotting. The images shown are representative of three independent experiments
- Alkyl refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 10 carbon atoms or more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, /so-propyl, n-butyl, /so- butyl, n-hexyl, and the like.
- Alkynyl refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1, and preferably from 1-2, carbon to carbon, triple bonds.
- Alkoxy refers to the group alkyl-O- where the alkyl group is as described above. Examples include, methoxy, ethoxy, n-propoxy, /so-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
- Alkenyloxy refers to the group alkenyl-O- wherein the alkenyl group is as described above.
- Alkynyloxy refers to the group alkynyl-O- wherein the alkynyl groups is as described above.
- Halo or "halogen” refers to fluoro, chloro, bromo and iodo.
- Haloalkyl refers to an alkyl group wherein the alkyl group is substituted by one or more halo group as described above.
- haloalkenyl refers to an alkyl group wherein the alkyl group is substituted by one or more halo group as described above.
- haloalkenyl refers to an alkyl group wherein the alkyl group is substituted by one or more halo group as described above.
- haloalkenyl haloalkynyl
- haloalkoxy are likewise defined.
- Aryl refers to an unsaturated aromatic carbocyclic group having a single ring (eg. phenyl) or multiple condensed rings (eg. naphthyl or anthryl), preferably having from 6 to 14 carbon atoms.
- aryl groups include phenyl, naphthyl and the like.
- Heteroaryl refers to a monovalent aromatic heterocyclic group which fulfils the Huckel criteria for aromaticity (ie. contains 4n + 2 n electrons) and preferably has from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur within the ring (and includes oxides of sulfur, selenium and nitrogen).
- Such heteroaryl groups can have a single ring (eg. pyridyl, pyrrolyl or N- oxides thereof or furyl) or multiple condensed rings (eg. indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl or benzothienyl).
- heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiophene, benzo[b]thiophene, triazole, imidazopyridine,
- Aryloxy refers to the group aryl-O- wherein the aryl group is as described above.
- Arylalkyl refers to -alkylene-aryl groups preferably having from 1 to 10 carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl and the like.
- Acyl refers to groups H-C(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl- C(O)- and heterocyclyl-C(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
- Oxyacyl refers to groups HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)-, and heterocyclyl-OC(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
- Amino refers to the group -NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- Aminoacyl refers to the group -C(O)NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- Acylamino refers to the group -NR"C(O)R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
- Acyloxy refers to the groups -OC(O)-alkyl, -OC(O)-aryl, -C(O)O-heteroaryl, and - C(O)O-heterocyclyl where alkyl, aryl, heteroaryl and heterocyclyl are as described herein.
- Aminoacyloxy refers to the groups -OC(O)NR"-alkyl, -OC(O)NR"-aryl, -OC(O)NR"- heteroaryl, and -OC(O)NR"-heterocyclyl where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- Oxyacylamino refers to the groups -NR"C(O)O-alkyl, -NR"C(O)O-aryl, -NR"C(O)O- heteroaryl, and NR"C(O)O-heterocyclyl where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- Oxyacyloxy refers to the groups -OC(O)O-alkyl, -O-C(O)O-aryl, -OC(O)O- heteroaryl, and -OC(O)O-heterocyclyl where alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
- Acylimino refers to the groups -C(NR")-R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
- Acyliminoxy refers to the groups -O-C(NR")-R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
- Oxyacylimino refers to the groups -C(NR")-OR” where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
- Cycloalkyl refers to cyclic alkyl groups having a single cyclic ring or multiple condensed rings, preferably incorporating 3 to 11 carbon atoms.
- Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, indanyl, 1,2,3,4-tetrahydronapthalenyl and the like.
- Cycloalkenyl refers to cyclic alkenyl groups having a single cyclic ring or multiple condensed rings, and at least one point of internal unsaturation, preferably incorporating 4 to 11 carbon atoms.
- suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclohex-4-enyl, cyclooct- 3-enyl, indenyl and the like.
- Heterocyclyl refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur, oxygen, selenium or phosphorous within the ring. The most preferred heteroatom is nitrogen. It will be understood that where, for instance, 2 or R' is an optionally substituted heterocyclyl which has one or more ring heteroatoms, the heterocyclyl group can be connected to the core molecule of the compounds of the present invention, through a C-C or C-heteroatom bond, in particular a C-N bond.
- heterocyclyl and heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1, 2, 3, 4-tetra hydroisoquinoline, 4,5,6,7-t
- Thio refers to groups H-S-, alkyl-S-, cycloalkyl-S-, aryl-S-, heteroaryl-S-, and heterocyclyl-S-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
- Thioacyl refers to groups H-C(S)-, alkyl-C(S)-, cycloalkyl-C(S)-, aryl-C(S)-, heteroaryl-C(S)-, and heterocyclyl-C(S)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
- Oxythioacyl refers to groups HO-C(S)-, alkylO-C(S)-, cycloalkylO-C(S)-, arylO- C(S)-, heteroarylO-C(S)-, and heterocyclylO-C(S)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
- Oxythioacyloxy refers to groups HO-C(S)-O-, alkylO-C(S)-O-, cycloalkylO-C(S)-O- , arylO-C(S)-O-, heteroarylO-C(S)-O-, and heterocyclylO-C(S)-O-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
- Thioacyloxy refers to groups H-C(S)-O-, alkyl-C(S)-O-, cycloalkyl-C(S)-O-, aryl- C(S)-O-, heteroaryl-C(S)-O-, and heterocyclyl-C(S)-O-, where alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
- Sulfinyl refers to groups H-S(O)-, alkyl-S(O)-, cycloalkyl-S(O)-, aryl-S(O)-, heteroaryl-S(O)-, and heterocyclyl-S(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
- Sulfonyl refers to groups H-S(O)2-, alkyl-S(O)2-, cycloalkyl-S(O)2-, aryl-S(O)2-, heteroaryl-S(O)2-, and heterocyclyl-S(O)2-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
- “Sulfinylamino” refers to groups H-S(O)-NR"-, alkyl-S(O)-NR"-, cycloalkyl-S(O)-NR"- , aryl-S(O)-NR"-, heteroaryl-S(O)-NR”-, and heterocyclyl-S(O)-NR"-, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- “Sulfonylamino” refers to groups H-S(O)2-NR"-, alkyl-S(O)2-NR”-, cycloalkyl-S(O)2- NR"-, aryl-S(O)2-NR"-, heteroaryl-S(O)2-NR”-, and heterocyclyl-S(O)2-NR”-, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- Oxysulfinylamino refers to groups HO-S(O)-NR"-, alkylO-S(O)-NR"-, cycloalkylO- S(O)-NR"-, arylO-S(O)-NR"-, heteroarylO-S(O)-NR"-, and heterocyclylO-S(O)-NR"-, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- Oxysulfonylamino refers to groups HO-S(O)2-NR"-, alkylO-S(O)2-NR"-, cycloalkylO-S(O)2-NR"-, arylO-S(O)2-NR"-, heteroarylO-S(O)2-NR”-, and heterocyclylO-S(O)2-NR”-, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- Aminothioacyl refers to groups R"R"N-C(S)-, where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- Thioacylamino refers to groups H-C(S)-NR"-, alkyl-C(S)-NR"-, cycloalkyl-C(S)- NR"-, aryl-C(S)-NR"-, heteroaryl-C(S)-NR”-, and heterocyclyl-C(S)-NR"-, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- Aminosulfinyl refers to groups R"R"N-S(O)-, where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- Aminosulfonyl refers to groups R"R"N-S(O)2-, where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
- a group may or may not be further substituted or fused (so as to form a condensed polycyclic group) with one or more groups selected from hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, phosphono, sulfo, phosphorylamino, phosphinyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino, aminoacyloxy, trihalomethyl, trialkyl, alkoxy, alkenyl, alken
- isomers includes especially optical isomers (for example essentially pure enantiomers, essentially pure diastereomers, and mixtures thereof) as well as conformation isomers
- Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers.
- the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
- Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses.
- HPLC high pressure liquid chromatography
- the invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
- "Optically-enriched,” as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer.
- the compound of the present invention is made up of at least about 90% by weight of a preferred enantiomer.
- the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer.
- Preferred enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses.
- HPLC high pressure liquid chromatography
- Jacques et al. Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972).
- AD andrographolide
- NF-KB inhibition prevails as the main hypothetical mechanism responsible for the anti-inflammatory action of AD.
- AD AD-induced cytokine release
- N F-KB suppression suggesting that there may be other mechanisms underlying the anti-inflammatory actions of AD, which in turn means that AD and its derivatives can be use in medical treatment in a different manner.
- AD and its derivatives can be use in medical treatment in a different manner.
- AD labdane based compounds
- the p38 MAPK -MK2 signaling axis serves as a point of convergence both downstream of receptors for inflammatory stimuli and upstream of the synthesis of pro- inflammatory signaling molecules, allowing itself to function as an amplifier of inflammation.
- Targeting the p38 MAPK -MK2 signaling axis represents a viable approach for therapeutic intervention of inflammatory diseases.
- AD andrographolide
- AD binds the MK2 activation loop located at the interface of the p38 MAPK -MK2 biomolecular complex, disrupting the complex formation and causing irreversible loss of MK2.
- the induced MK2 degradation accelerated pro-inflammatory mediator mRNAs decay, blocked downstream MK2-mediated inflammatory responses in in-vitro and was efficacious in attenuating inflammation in mice with acute lung injury.
- the results show that not only MK2 is a viable substrate for post-translational degradation, but also that the signaling inactivation achieved by MK2 degrader is more durable and sustained than that achieved via MK2 inhibition.
- the present invention provides a method of promoting the ubiquitination of MK2 protein, comprising contacting the MK2 protein with a compound of Formula (I) or a salt, solvate, stereoisomer or prodrug thereof:
- Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino;
- R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
- Rs is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
- the compound of Formula (I) can act as a MK2 protein regulator through ubiquitination and degradation of MK2 protein.
- Compounds of Formula (I) may bind to the activation loop of MK2 located at the interface of the p38a-MK2 complex, causing complex dissociation and the loss of MK2.
- the method is a method of promoting the degradation of MK2 protein. In some embodiments, the method is a method of promoting the ubiquitination and degradation of MK2 protein.
- proteins are marked for degradation by the attachment of ubiquitin to the amino group of the side chain of a lysine residue. Additional ubiquitins can be added to form a multiubiquitin chain, which are then recognized and degraded by a large, multisubunit protease complex, called the proteasome. In this sense, the primary structure is destroyed as covalent peptide bonds are broken. In contrast, denaturation only involves the unfolding of a protein, where quaternary, tertiary and secondary structures are disrupted but primary structure remains intact. Protein inhibition refers to a process of stopping or slowing an activity of a protein by the binding of a molecule to the protein's active site or alternative site. Such inhibition may be reversible or irreversible.
- Ri is selected from optionally substituted acyloxy, optionally substituted arylthio, optionally substituted heteroarylthio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryl oxy, and optionally substituted heteroarylamino.
- the optional substituent on Ri is selected from alkyl, aryl, alkyloxyacyl, alkylacylamino, alkylacyloxy, and alkylaminoacyl. In some embodiments, the optional substituent on Ri is selected from methyl, ethyl, propyl, phenyl, methyloxyacyl, ethyloxyacyl, propyloxyacyl, methylacylamino, ethylacylamino, propylacylamino, methylacyloxy, ethylacyloxy, propylacyloxy, methylaminoacyl, ethylaminoacyl, and propylaminoacyl.
- 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, and optionally substituted thio. In some embodiments, 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, and optionally substituted acyloxy. In some embodiments, 2 is selected from H, halogen, and hydroxyl. In some embodiments, 2 is hydroxyl.
- R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, and optionally substituted thio. In some embodiments, R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, and optionally substituted acyloxy. In some embodiments, 3 is selected from H, halogen, and hydroxyl. In some embodiments, 3 is hydroxyl.
- the compound of Formula (I) is characterised by a pKaH of about
- the pKaH is less than 10.
- the MK2 protein is characterised by a half-life of less than 4 h.
- the method of promoting the degradation of MK2 protein occurs in a cell.
- the method is an in vivo method, an in vitro method or an ex vivo method.
- the method is an in vitro method.
- Ex vivo refers to experimentation or measurements done in or on tissue from an organism in an external environment with minimal alteration of natural conditions. Testing the effect of compounds on skin biopsies is an example of ex vivo research, while isolating the primary cells from that biopsy and creating a 3D cell culture model is an example of in vitro research.
- the compound of Formula (I) is a compound of Formula (la):
- F is selected from optionally substituted acyl, optionally substituted aryl, and optionally substituted heteroaryl;
- R2 and R3 are as disclosed herein.
- X is selected from 0, S, N.
- the heteroaryl is selected from pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, dioxinyl, oxazinyl, thiazinyl, indolyl, isoindolyl, indazolyl, benzimidazolyl, azaindolyl, purinyl, isobenzofuranyl, benzothiophenyl, benzoisoxazolyl, benzoisothiazolyl, benzothiadiazolyl, quinolinyl, isoquinolinyl, and naphthalenyl.
- R4 is selected from optionally substituted phenyl and optionally substituted 6 membered heteroaryl.
- the 6 membered heteroaryl is selected from pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, dioxinyl, oxazinyl, and thiazinyl.
- FU is selected from phenyl optionally substituted at a para position and 6 membered heteroaryl optionally substituted at a para position.
- the optional substituent on FU is selected from alkyl, alkyloxyacyl, alkylacylamino, alkylacyloxy, and alkylaminoacyl. In some embodiments, the optional substituent on FU is selected from methyl, ethyl, propyl, phenyl, methyloxyacyl, ethyloxyacyl, propyloxyacyl, methylacylamino, ethylacylamino, propylacylamino, methylacyloxy, ethylacyloxy, propylacyloxy, methylaminoacyl, ethylaminoacyl, and propylaminoacyl.
- the compound of Formula (I) is a compound of Formula (lb): wherein
- Het is an optionally substituted heteroaryl; and FU and F are as disclosed herein.
- the heteroatom on the heteroaryl is bonded to the C-12 position of Formula (I).
- the heteroatom is linked via its heteroatom to the Markush structure.
- the C-12 position of the Formula (I) is shown below.
- the heteroaryl is selected from oxazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolinyl, quinolinyl, phthalazinyl, naphthylpyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, acridinyl, phenanthrolinyl, isothiazolyl, phenazinyl, isoxazolyl, isothiazolyl, phenoxazinyl, phenothiazinyl, thiazolyl, thiadiazolyl
- the optional substituent on the heteroaryl is selected from alkyl, alkyloxyacyl, alkylacylamino, alkylacyloxy, and alkylaminoacyl. In some embodiments, the optional substituent on the heteroaryl is selected from methyl, ethyl, propyl, phenyl, methyloxyacyl, ethyloxyacyl, propyloxyacyl, methylacylamino, ethylacylamino, propylacylamino, methylacyloxy, ethylacyloxy, propylacyloxy, methylaminoacyl, ethylaminoacyl, and propylaminoacyl.
- the compound of Formula (I) is a compound of Formula (I 1 ): wherein Ri, 2 and 3 are as disclosed herein.
- the compound of Formula (I) is a compound of Formula (I"): wherein Ri, R2 and R3 are as disclosed herein.
- the compound of Formula (I) is a compound of Formula (Ic): wherein Ri, R2 and R3 are as disclosed herein.
- the compound of Formula (I) is a compound of Formula (Id) : wherein
- X, R2, R3 and R4 are as disclosed herein.
- the compound of Formula (I) is a compound of Formula (le): wherein Het, R2, and R3 are as disclosed herein.
- the compound of Formula (I) is a compound of Formula (Ic 1 ):
- the compound of Formula (I) is a compound of Formula (Ic"): wherein Ri, R2 and R3 are as disclosed herein.
- the compound is selected from one of the following:
- the compound is selected from one of the following:
- the present invention also provides a compound of Formula (Ic) or a salt, solvate, 5 stereoisomer or prodrug thereof: wherein
- Ri is selected from
- X is a heteroatom selected from N, 0, S; 4 is selected from optionally substituted aryl, optionally substituted heteroaryl; or X- 4 together is optionally substituted acyloxy;
- Het is an optionally substituted heteroaryl, wherein the heteroatom on the heteroaryl is bonded to the C-12 position of Formula (I); 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and 3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
- 2 is hydroxyl. In some embodiments, R3 is hydroxyl.
- the compound of Formula (I) is selected from:
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
- the present invention also provides a method of treating of a disease or condition associated with MK2 protein, comprising administrating to a subject in need thereof a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable a salt, solvate, stereoisomer or prodrug thereof.
- the disease or condition associated with MK2 protein is treated by degrading the MK2 protein, and not inhibit the MK2 protein.
- the present invention also provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for use in treating a disease or condition associated with MK2 protein.
- the present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament.
- the present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament for the treatment of a disease or condition associated with MK2 protein.
- the treatment of a disease or condition associated with MK2 protein comprises degradation of MK2 protein.
- the disease or condition associated with MK2 protein is an inflammatory disease or condition.
- the disease or condition associated with MK2 protein is a chronic respiratory disease or condition.
- the disease or condition associated with MK2 protein is selected from acute lung injury, chronic inflammation in asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease, atherosclerosis, Alzheimer's disease, ischemic heart, brain disease, cancer, COPD, lung fibrosis, pneumonia, and long COVID.
- the disease or condition associated with MK2 protein is selected from acute lung injury, chronic inflammation in asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease, atherosclerosis, Alzheimer's disease, ischemic heart, brain disease, chronic obstructive pulmonary disease (COPD), lung fibrosis, pneumonia, and long COVID.
- the present invention also provides a method of inducing an anti-inflammatory response in a subject in need thereof, comprising administrating to a subject in need thereof a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable a salt, solvate, stereoisomer or prodrug thereof.
- the present invention also provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for inducing an antiinflammatory response in a subject in need thereof.
- the present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament for inducing an anti-inflammatory response in a subject in need thereof.
- the anti-inflammatory response is induced by degradation of MK2 protein.
- Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
- pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric
- Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.
- the present invention includes within its scope cationic salts eg sodium or potassium salts, or alkyl esters (eg methyl, ethyl) of the phosphate group.
- Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
- lower alkyl halide such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
- dialkyl sulfates like dimethyl and diethyl sulfate; and others.
- prodrug any compound that is a prodrug of the compound of formula (I) is also within the scope and spirit of the invention.
- the compound of the invention can be administered to a subject in the form of a pharmaceutically acceptable pro-drug.
- pro-drug is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compound of the invention. Such derivatives would readily occur to those skilled in the art.
- Other texts which generally describe prodrugs (and the preparation thereof) include: Design of Prodrugs, 1985, H. Bundgaard (Elsevier); The Practice of Medicinal Chemistry, 1996, Camille G.
- the compound can be substituted with a carboxylic acid moiety or a hydroxyl moiety at the C-12 position. This can be protected as a prod-drug through the formation of an ester group.
- the compound of the invention may be in crystalline form either as the free compound or as a solvate (e.g. hydrate) and it is intended that both forms are within the scope of the present invention.
- Methods of solvation are generally known within the art.
- a therapeutically effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of macular degeneration.
- the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.
- Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.
- the compound of the invention may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition.
- the formulation of such compositions is well known to those skilled in the art.
- the composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.
- compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
- Injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion.
- Carriers can include, for example, water, saline (e.g., normal saline (NS), phosphate-buffered saline (PBS), balanced saline solution (BSS)), sodium lactate Ringer's solution, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances, such as wetting or emulsifying agents, buffers, and the like can be added.
- saline e.g., normal saline (NS), phosphate-buffered saline (PBS), balanced saline solution (BSS)
- NS normal saline
- PBS phosphate-buffered saline
- BSS balanced saline solution
- Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants.
- the compound, composition or combination can be dissolved in a pharmaceutically effective carrier and be injected into the vitreous of the eye with a fine gauge hollow bore needle (e.g., 30 gauge, 1/2 or 3/8 inch needle) using a temporal approach (e.g., about 3 to about 4 mm posterior to the limbus for human eye to avoid damaging the lens).
- a person skilled in the art will appreciate that other means for injecting and/or administering the compound, composition or combinations to the vitreous of the eye can also be used.
- These other means can include, for example, intravitreal medical delivery devices.
- These devices and methods can include, for example, intravitreal medicine delivery devices, and biodegradable polymer delivery members that are inserted in the eye for long term delivery of medicaments.
- These devices and methods can further include transscleral delivery devices.
- solutions or suspensions of the compound or composition of the invention may be formulated as eye drops, or as a membranous ocular patch, which is applied directly to the surface of the eye.
- Topical application typically involves administering the compound of the invention in an amount between 0.1 ng and 10 mg.
- the compound or composition of the invention may also be suitable for intravenous administration.
- a compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof may be administered intravenously at a dose of up to 16 mg/m 2 .
- the compound or composition of the invention may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
- the active ingredient may also be presented as a bolus, electuary or paste.
- the compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug is orally administerable.
- a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
- Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent.
- a binder e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent.
- Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
- the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
- the compound or composition of the invention may be suitable for topical administration in the mouth including lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
- lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum
- pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum
- mouthwashes comprising the active ingredient in a suitable liquid carrier.
- the compound or composition of the invention may be suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like.
- suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
- Transdermal patches may also be used to administer the compounds of the invention.
- the compound or composition of the invention may be suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the compound, composition or combination isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
- the compound or composition may be presented in unitdose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- Preferred unit dosage composition or combinations are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.
- composition of this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents.
- suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine.
- Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.
- Suitable flavouring agents include peppermint oil, oil of Wintergreen, cherry, orange or raspberry flavouring.
- Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten.
- Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
- Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc.
- Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
- MK2 is highly expressed in macrophages and is essential for inflammatory cytokine/chemokine production.
- MK2 Due to the high level of MK2 expression and the low level of MK3, RAW264.7 cells and primary alveolar macrophages (AMs) were selected as the model systems for our studies. Macrophages are the main source of inflammatory cytokines and chemokines. Although the role of MK2 in regulating cytokine production has been studied in detail, its impact on chemokine secretion in macrophages has not yet well-established. We thus set out to re-investigate the functional effects of MK2 on macrophage activation using both transient knock-down and over-expression approaches.
- RAW 264.7 cells were transfected with small interfering RNA (siRNA) to MK2 (siMK2) or scrambled control (scRNA) and exposed to lipopolysaccharide (LPS) (25 ng/ml; 4 h).
- siRNA small interfering RNA
- siMK2 MK2
- scRNA scrambled control
- LPS lipopolysaccharide
- siMK2-treated or MK2-knockdown cells showed a significant decrease in LPS- mediated up-regulation of Ml macrophage marker e.g., /NOS), inflammatory cytokine (e.g., TNF-a, GM-CSF) and chemokine (e.g., MCP-1, MIP-ip, MIP-2, KC) gene expression ( Figure ID).
- Ml macrophage marker e.g., /NOS
- inflammatory cytokine e.g., TNF-a, GM-CSF
- chemokine e.g., MCP-1, MIP-ip, MIP-2, KC
- AD Andrographolide
- AD induced more than 50% reduction in MK2 levels as compared to the basal level after a 4-hour treatment. Similar effects were also observed in LPS-stimulated RAW264.7 cells (Figure 2B). In a time-course experiment, AD was found to induce MK2 down-regulation as early as after 30 min of compound incubation ( Figure 2C). Both isoforms of MK2 were affected. Cycloheximide (CHX), an inhibitor of de novo protein synthesis, was used to determine the half-life of the endogenous MK2. The level of MK2 protein in the total protein extract was gradually decreased with increased chase time points (from 0.5 to 24 h), yielding a characteristic exponential decay curve, in cells treated with CHX (1 pg/ml).
- CHX Cycloheximide
- AD anti-inflammatory effects of AD are MK2-dependent and involve MK2 downstream effector tristetraprolin (TTP)
- MK2 is a key repressor of anti-inflammatory RBPs such as TTP. Phosphorylation by MK2 deactivates TTP, reduces its ability to promote deadenylation and mRNA decay, leading to excessive cytokine/chemokine production and aberrant activation of immune responses. Therefore, MK2 deficiency induced by AD would in principle stabilize TTP, enhance its mRNA-destabilizing activity, thus attenuating the production of downstream inflammatory mediators ( Figure 31). In other words, the anti-inflammatory property of AD can be expected to be mediated in part via the anti-inflammatory action of TTP.
- TTP-knockdown cell lines were created by transfection with siRNA to TTP (siTTP) and the inhibitory effects of AD on LPS-induced cytokine production were evaluated in both scRNA- and siTTP-treated cells. It is expected that, under TTP-knockdown conditions, the cytokine inhibitory effects of AD will be weakened.
- siRNA to TTP induced approximately 30% reduction in TTP mRNA levels, which was translated to a moderate reduction in its protein levels (Figure 3C).
- the effects of siTTP on TTP mRNA levels were more prominent in cells treated with LPS ( Figure 3D). Consistent with published data, LPS-induced TNF-a production was significantly higher in cells with diminished TTP expression.
- TNF-a release was observed in TTP-KD cells as compared to scRNA-treated controls.
- Treatment with AD strongly suppressed the induced TNF-a release in scRNA-treated cells with an ICso value of approximately 5 pM.
- the inhibition was markedly weakened in cells with low TTP levels (/.e., siTTP-treated cells).
- the former two compounds control cytokine gene expression at the upstream transcriptional levels but not post-transcriptional events, and thus their inhibitory effects on cytokine gene expression should be minimally affected by the changes in the TTP protein expression levels.
- the cytokine inhibition by PF3644022 should be diminished in the absence of TTP, consistent with the behaviour of AD.
- the anti-inflammatory effects of dexamethasone and p50-i were comparable in both scRNA- and siTTP-treated cell lines, while that of PF3644022 was significantly weakened in siTTP-treated cells (Figure 3F). This further validate the involvement of TTP, a downstream effector of MK2, in the anti-inflammatory mechanism of AD.
- AD Andrographolide
- ALI LPS-induced acute lung injury
- BALF bronchoalveolar lavage fluid
- ALI is characterized by pulmonary neutrophil infiltration, which is mediated by important chemokines such as MCP-1 and MIP-2 that are released from AMs.
- the ALI model was established by intratracheal injection of LPS (10 pg/mice) (Figure 4B), which led to significant immune cell accumulation and neutrophil infiltration in BALF ( Figure 4E and F). This was prevented by AD (1 mg/kg, intraperitoneal (i.p.)) when administered every 12 h for 3 consecutive days. AD mainly blocked the recruitment of neutrophils, but not other immune cells (e.g., macrophages, lymphocytes) ( Figure 4G and H).
- AD Andrographolide
- AD increases MK2 proteasomal degradation by promoting its ubiquitination.
- p38a activation in response to stress, has been proposed to induce irreversible MK2 loss by causing p38a-MK2 complex dissociation.
- the ability of AD to activate p38 MAPK pathway has been documented in a number of studies. In our study, we observed the activation of p38 MAPK by AD at the high concentration of 50 pM . This led us to hypothesize that perhaps AD induces p38a activation, which, in turn, disrupts the complex, and results in reduced protein stability and the loss of MK2.
- AD Andrographolide
- the activation loop of MK2 contains Thr222 residue which serves as one of its main phosphorylation sites and is located at the interface between p38a and docked MK2 ( Figure 6A).
- CETSA and molecular docking suggest the direct binding of AD to the activation loop of MK2.
- Such interaction may affect the phosphorylation of MK2 at the Thr222 residue.
- MK2 was phosphorylated by active p38a in vitro and the kinetics of phosphorylation at these sites were monitored by using MK2 phosphositespecific antibodies (Figure 6E). It was observed that Thr334 phosphorylation happened ahead of Thr222 phosphorylation, which is consistent with previous reports. When MK2 was pre-incubated with AD 10 mins in advance of the addition of active p38a, phosphorylation of MK2 was inhibited specifically at the Thr222 residue, but not Thr334 ( Figure 6E). In a separate experiment, we analysed the kinase activity of MK2 using an ADP-Glo kinase assay.
- RAW264.7 cells were treated with the high concentration of AD (50 pM) for 4 h to induce significant reduction in MK2 level, and then were washed and incubated in compound-free medium for up to 48 h ( Figure 7A). Cells were collected at indicated time points (i.e., 0, 6, 12, 24, 48 h) after compound removal and the levels of MK2 were determined. We found that, in the absence of AD, MK2 took up to 24 h to recover to the basal level of the untreated cells. This observation suggests that MK2 is a viable target for post-translational degradation approach as the protein has a slow re-synthesis rate.
- PF-3644022 inhibited the production of TNF-a in LPS-stimulated RAW264.7 cells with an IC50 value of 0.3 pM, which was higher in activity than AD with an IC50 value of 15 pM. It appears that, under continuous exposure to the drugs, the inhibition of MK2 kinase activity is more involved in the shut-down of downstream inflammatory responses.
- a 24-hour pre-treatment resulted in a nearly 8-fold increase in IC50 value (i.e., 2.5 pM) as compared to the 1-hour pre-treatment regimen (i.e., 15 pM), demonstrating a timedependent characteristic.
- IC50 value i.e., 2.5 pM
- the 1-hour pre-treatment regimen i.e., 15 pM
- PF-3644022 showed comparable potency irrespective of the pre-treatment duration, characteristic of a non-covalent inhibitor. This finding suggests that although the potency of AD is seemingly lower than PF-3644022, its timedependent attribute allows lower concentrations to be used to achieve similar magnitude of effects as a competitive inhibitor when time permits, minimizing the potential off- target side effects.
- MK2 is a serine-threonine kinase downstream to p38a and is activated directly through phosphorylation of p38a under stress and inflammatory stimuli.
- MK2 has been identified as a promising alternative molecular target to p38a in order to block the pathway activation and downstream inflammatory responses. The assumption is that this approach would show similar efficacy as that of p38a inhibitors, with lesser toxicity concerns that are associated with the inhibition of other beneficial p38 substrates.
- MK2 is highly expressed in macrophages and is responsible for the production of various inflammatory cytokines and chemokines.
- An important downstream target of MK2 is TTP, an RBP that functions essentially as an antiinflammatory protein by limiting cytokine production from macrophages upon exposure to endotoxin and other inflammatory stimuli.
- TTP targets inflammation-associated mRNAs for degradation via a process that involves shortening of the poly(A) tail, deadenylation, and is indispensable for the resolution of inflammation.
- Activation of MK2 disables the mRNA destabilizing activity of TTP, leading to excessive cytokine production and aberrant immune responses.
- AD natural product - andrographolide
- AD blocked the induced production of key cytokine TNF-a, and chemokines MCP-1, MIP-2 from activated AMs, and attenuated the development of LPS-induced ALI in mice.
- MK2 induced by AD allows TTP to take actions in destabilizing its target mRNAs and dampen inflammation. This constitutes the downstream part of the anti-inflammatory mechanism of AD by harnessing the mRNA destabilizing activity of TTP.
- we showed that the anti-inflammatory property of AD is partially dependent on TTP as excessive TNF-a release is not modulated by AD-induced MK2 deficiency in the absence of TTP (Figure 3E and F).
- AD engendered MK2 downregulation in macrophages.
- CETSA computational docking
- sitespecific phosphorylation experiments we found that AD is likely to bind to the activation loop of MK2 which contains a phosphorylation site Thr222. This region is located at the interface between MK2 and p38a and is essential for the complex assembly.
- Such binding of AD appears to disrupt the p38a-MK2 complex, freeing both p38a and MK2.
- free MK2 is rapidly degraded by the proteasome, thus explaining the shortened half-life of MK2 in the presence of AD.
- the MK2 pathway is often targeted therapeutically by kinase inhibitors (e.g., PF3644022) which operate via an occupancy-driven pharmacology model.
- kinase inhibitors e.g., PF3644022
- occupancy-driven pharmacology model Much recently, it is reported an additional mode p38a-MK2 pathway regulation by specifically blocking p38a activation of MK2, which was shown to be efficacious in inhibiting inflammatory diseases in pre-clinical and clinical studies. Occupancy-driven pharmacology is predicated upon blocking protein functions via inhibition, i.e., by applying high concentrations of inhibitor (Figure 8).
- Event-driven pharmacology offers an alternative approach that, upon drug binding, the target protein is tagged for elimination, allowing for similar cellular outcomes as inhibitors.
- AD pharmacological difference of AD as a MK2 degrader
- PF-3644022 as a kinase inhibitor
- results illustrate the advantages of MK2 degradation, which results in more sustained effects than the kinase inhibition. In the latter, high concentrations of the inhibitors should be maintained to ensure active-site occupancy and to sustain the inhibitory effect.
- degraders reduce the target protein level and as such, cellular outcome is halted until sufficient amounts of MK2 accumulate in the cells by de novo synthesis, leading to prolonged duration of action.
- the current MK2 inhibitors suffer major limitations due to their poor drug-like properties such as scarce solubility, low cell permeation, kinase selectivity, as well as biochemical efficiency; hence, their clinical development is still limited.
- AD and A paniculata extracts have been validated to be effective in treating various inflammatory human diseases in the clinics.
- the compound displays good drug-like properties.
- Our delineation of the anti-inflammatory action of AD via MK2 degradation not only provides an additional mode for modulating the p38 MAPK -MK2 axis, but also presents the first example of small molecule MK2 degrader. This discovery paves the way for the development of novel anti-inflammatory agents targeting MK2 for degradation by harnessing the privileged scaffold of AD.
- the target compounds were prepared as 3,19-acetonide intermediates before an acid-catalysed deprotection to yield the final products.
- Treatment of 18 with PDC in CH2CI2 led to rearrangement of the C-14 hydroxyl group and formation of 12-hydroxy-14- dehydroandrographolide intermediate 19 in nearly quantitative yield.
- Esterification of 19 with acid anhydride furnished acetyl intermediate 20.
- Reaction of andrographolide with aqueous bisulfite solution under reflux facilitated a rare sulfurous nucleophilic attack and yielded derivative 14 in almost quantitative yield.
- Scheme 2 depicts the synthesis of C-12 derivatives with substituents of low leaving ability.
- Reaction of 18 with DPPA in the presence of DBU resulted in azide 33, which was converted to triazole 34 following a copper(I)-catalysed click chemistry with phenylacetylene.
- CeCh unexpectedly led to the formation of intermediate 35.
- Prolonged exposure of 18 to acidified methanol yielded the methoxy intermediate 36.
- Benzyl 13 was synthesized following 1,4-addition of benzyl magnesium bromide to the a,p-unsaturated lactone system in 18 with a catalytic amount of copper halide.
- the Grignard reagent was freshly prepared, and all hydroxyl groups in AD were protected prior to the reaction.
- Silyl protected intermediate 38 yielded a highly regioselective Grignard reaction, but not the 3,19-acetonide 37.
- Removal of TBS with fluoride anions (TBAF) led to isomerization of the terminal A 8 ' 17 double bond and was substituted by a milder acidic deprotection using p-TsOH in aqueous AcOH to afford the final product 13.
- Hydrogenation of 10 yielded compound 16 in almost quantitative yield. All acetonide intermediates were finally deprotected in acidic environment to yield the corresponding test compounds 1 - 15.
- the synthesized C-12 derivatives were first evaluated for thiol reactivity to identify and filter out highly reactive molecules prior to biological evaluation.
- Representative C-12 derivatives carrying substituents of various leaving potential were selected for the screening.
- Figure 9 summarizes the rates of GSH addition to various C-12 compounds at equimolar concentrations. It was observed that the thiol reactivity of C-12 derivatives followed the trend in leaving group ability, in which functional groups with lower pKaH showed much faster rate of reaction. Acetyl 1 appears to be the most reactive compound in C-12 series, with slightly higher reactivity as compared to AD. When pKaH values of the C-12 substituents were greater than 10, minimal reaction was observed as seen with 10 and 12. Derivatives with no leaving functional groups e.g.
- the synthesized C-12 derivatives were next evaluated for inhibitory effects against LPS- induced TNF-a and IL-6 production in an in vitro model of inflammation.
- Bacterial LPS was used to stimulate the production of pro-inflammatory cytokines in RAW 264.7 macrophages and the anti-inflammatory effects was measured as percentage of cytokine inhibition compared to vehicle-treated groups.
- Figure 10A-B summarizes the inhibitory effects of various test compounds on TNF-a and IL-6 release after 4h and 24h LPS stimulation, respectively.
- Compounds 13, 14 and 15 carrying non-leaving functional groups were inactive against both cytokines.
- Derivative 12 carries a phenylamino substituent that has very low leaving potential (pKaH ⁇ 25) yet displays relatively high potency, comparable to that AD. This observation suggests that the anti-inflammatory effects of C-12 derivatives are not governed solely by their chemical reactivity, but also by the physiochemical properties of substituents at C-12 position.
- C-12 derivatives bearing aromatic substituents such as phenyl-mercapto (-S-Ph) in 2, phenyl-hydroxy (-O-Ph) in 4, 5 and 7 and phenyl-amino (-NH-Ph) in 12 exhibited much stronger inhibitory activity as compared to the non-aromatic compounds, with the exception of the phenyl-methyl (-CH2-Ph) 13.
- phenyl-methyl (-CH2-Ph) 13 Changing the connecting heteroatoms (S, 0, N) to a carbon in 13 completely diminished the activity, despite minimal change in the structural conformation of these compounds.
- C-12 derivatives depend on multiple factors including: (1) a privileged ent-labdane structure with appropriate substituents at C-12 positions, in which aromatic substituents seem to confer best target recognition, (2) a Michael acceptor system and (3) a C-12 substituent capable of leaving under nucleophilic attack, if any, to ensure prolonged target engagement via a covalent attachment.
- TCIs targeted covalent inhibitors
- the synthesized C-12 derivatives were also evaluated for cytotoxicity in non-LPS- stimulated RAW 264.7 cells (Figure IOC). Most test compounds showed lower cytotoxicity as compared to AD, except for 2. Overall, the C-12 derivatives display more favorable therapeutic profiles and thus would be more attractive lead compounds for anti-inflammatory drug development based on the ent-labdane andrographolide (AD).
- C-12 andrograoholide derivatives were next evaluated for their inhibitory effects on LPS-induced cytokine mRNA expression levels. Specifically, RAW264.7 macrophages were pre-treated with test compounds (at 10 mM) for 1 h followed by LPS stimulation for another 4 h. The cytokine mRNA expression levels were quantified using qRT-PCR against the respective primer pairs. As shown in Figure 11, treatment with active C-12 derivatives effectively reduced the mRNA expression levels of TNF-a and IL-6.
- C-12 andrographolide derivatives blocked pro-inflammatory cytokine production by reducing their mRNA expression levels, possibly via transcriptional or post-trancriptional control.
- C-12 derivatives with aromatic substituents e.g. 2, 4, 5, 7 and 12
- aromatic substituents displayed much higher potency as compared to the non-aromatic compounds.
- an aromatic sulfur substituent is preferable for high potency followed by amino > oxygen >>> carbon.
- Derivative 13, 14 and 15 carrying non-leaving substituents showed no observable effects.
- derivative 4 exerted highest potency with more than 90% reduction in both TNF-a and IL-6 mRNA levels as compared to LPS-control groups, consistent with its strong inhibitory effects on cytokine protein release.
- the para-substitution on the phenol ring in 4 and 5 appeared to contribute significantly to the anti-inflammatory activity, as 7 lacking a substituent at this position was far less potent.
- Derivative 4 were selected for further in vivo evaluation. As excessive cytokine production has been shown to amplify inflammation in individuals with asthma, we evaluated the anti-inflammatory effects of 4 in a HDM-induced asthmatic models. Female mice of 6-8 weeks old were stimulated with HDM for 14 days to induce asthmatic response prior to treatment with 4 at 0.1, 1 and 3 mg/kg. Figure 13 displayed the total and differential cell counts from Bronchoalveolar lavage fluid (BALF). The number of white blood cells from BALF is a reliable indicator of airway inflammatory responses. It was observed that treatment with 4 significantly alleviated HDM-induced airway inflammation at dose as low as 1 mg/kg. HDM induced significant production of eosinophils, a hallmark of severe asthma, which was effectively blocked by 4. These data suggest the potential of 4 as a safe and effective anti-inflammatory therapeutic for the treatment of severe asthma.
- BALF Bronchoalveolar lavage fluid
- substituents at the C-12 position of AD can affect their anti-inflammatory activity. Specifically, we observed that substituents with better leaving group ability and aromatic character give rise to compounds with better anti-inflammatory profiles. To the best of our knowledge, this is the first report on the C-12 derivatives of AD as MK2 degraders and as potential anti-inflammatory agents. Their ability to inhibit pro- inflammatory cytokine release at high potency offer the possibility to replace current p38 inhibitor approach, without incurring side-effects that are commonly associated with direct blocking of p38 protein.
- Our study identified compound 4 as potent anti- inflamamtory agent that showed excellent in vivo efficacy at low dose of lmg/kg in a severe asthmatic mouse model. The therapeutic potential of compound 4 will also be evaluated in other chronic inflammatory conditions e.g., COPD).
- Mouse macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (Manassas, VA). Dexamethasone 21-phosphate disodium salt (>98%), andrographolide (>98%), LPS (Escherichia coli 0111 :B4), PF-3644022 hydrate (>98%), and DMSO (>99% cell culture grade) were obtained from Sigma-Aldrich (St. Louis, MO). p50 inhibitor peptide was obtained from Novusbio (Centennial, CO).
- DMEM Opti-MEMTM I Reduced Serum Medium
- FBS FBS
- M-PER mammalian protein extraction reagent M-PER mammalian protein extraction reagent
- Pierce protease and phosphatase inhibitor mini tablets Pierce IP lysis buffer came from ThermoScientific (Rockford, IL).
- Dynabeads Protein G, DynaMag-Spin Magnet, Bradford protein assay kit, SYBR Green qPCR MasterMix, Maxima first strand cDNA synthesis kit were purchased from ThermoFisher Scientific (Waltham, MA).
- Mini-PROTEAN® TGX precast protein gels were from Bio-Rad Laboratories (Singapore).
- Anti-MK2 (Cat# 12155, RRID: AB_2797831), anti-phospho-MK2 (Thr334, Cat# 3007, RRID: AB_490936), anti-phospho-MK2 (Thr222, Cat# 3316, RRID: AB_2141311), anti-TTP (Cat# 71632, RRID: AB_2799806), anti-KHSRP (Cat# 13398, RRID: AB_2798208), anti-p38a (Cat# 9218, RRID: AB_10694846), anti-p38 (Cat# 8690, RRID: AB_10999090), anti-phospho-p38 (Thrl80/Tyrl82, Cat# 4511, RRID: AB_2139682), anti-MKK6 (Cat# 8550, RRID: AB_11220227), anti-HSP27 (Cat# 95357, RRID: AB_2800246), anti-phospho-HSP
- Anti-p-actin (HRP-conjugated, Cat# HRP-60008, RRID: AB_2819183), anti-GAPDH (HRP-conjugated, Cat# HRP-60004, RRID: AB_2737588) antibodies were from Proteintech (Rosemont, IL).
- Mouse anti-Siglec-F microbeads, LS-column, and MACS Separator were obtained from Miltenyi biotech (Singapore).
- RNAIater solution, SYBR Green qPCR MasterMix, collagenase type I, and DNase I were purchased from Life Technologies (Carlsbad, CA).
- Mouse recombinant proteins MK2 and p38a, and kinase assay buffer III were obtained from Signalchem Biotech (Richmond, BC).
- Mouse KC and MIP-2 DuoSet ELISA kits were generated by R8 ⁇ D Systems (Minneapolis, MN).
- Mouse TNF-a, MCP-1 and IL-6 Mono/Mono OptEIATM ELISA kits were generated by BD Biosciences (Franklin Lakes, NJ).
- MK2 ADP-Glo kinase assay kit was from Promega (Madison, WI).
- mice 6- to 8-week-old Female BALB/c mice 6- to 8-week-old (InVivos, Singapore) were maintained in a 12-h light/dark cycle with food and water available ad libitum. Animal experiments were performed according to institutional guidelines of the Animal Care and Use Committee (IACUC) of the National University of Singapore.
- IACUC Animal Care and Use Committee
- Andrographolide (1 mg/kg in 2% DMSO), or vehicle (2% DMSO) in 40 pL saline was administered intratracheally for the time-dependent study. 10 pg LPS in 40 pl saline was instilled intratracheally to induce ALL One hour later, andrographolide (1 mg/kg in 2% DMSO), or vehicle (2% DMSO) in 0.1 ml PBS was administered by intraperitoneal injection twice daily for 3 consecutive days. Twelve hours after the last injection, mice were sacrificed to collect lung samples for various biochemical analyses.
- BAL fluid total cell counts were performed blinded. Differential cell count was determined by using the BD Fortessa flow cytometer and analyzed with FlowJo software (BD, Franklin Lakes, NJ, US). Leukocytes were identified as CD45+ cells, neutrophils as CDllb+/Gr-l+ cells, macrophage as CDllc+/Siglec-F+ cells. Mouse lungs were excised. Lung lobes were snap frozen in liquid nitrogen and then stored at -80°C for protein isolation.
- Mouse whole lung was perfused with 5 ml PBS and minced into 2- to 3-mm pieces. Lungs were digested with DMEM medium containing collagenase type I (2 mg/ml) and DNase I (100 U/ml) and further homogenised with the gentleMACS (Miltenyi) prior to passage through a 70 pm strainer to obtain single cell suspension. Cells were counted, resuspended in MACS buffer, and incubated with mouse anti-Siglec-F microbeads for 10 minutes at 4°C. Cells were run through an LS-column on the magnetic field of a MACS Separator. The positively selected fraction was collected and plated. Alveolar macrophages were allowed to adhere for a minimum of 2 hours, washed with DMEM medium supplemented with 10% FBS, and collected for further studies or analysis.
- the mouse macrophage cell line RAW264.7 or isolated primary mouse lung alveolar macrophages were cultured in DMEM medium supplemented with 10% FBS. All cell cultures were maintained in a humidified 37°C, 5% CO2 incubator. The cells were treated with various concentrations of andrographolide, PF-3644022, dexamethasone, p50-i, as well as LPS for indicated time points.
- mice /3-actirr forward 5'-GTG ACG TTG ACA TCC GTA AAG A-3', reverse 5'-GCC GGA CTC ATC GTA CTC C-3' ; mouse TNF-a: forward 5'-TCT GTC TAC TGA ACT TCG GGG TGA-3', reverse 5'-TTG TCT TTG AGA TCC ATG CCG TT-3' ; mouse MCP-1 : forward 5'- TAA AAA CCT GGA TCG GAA CCA AA-3', reverse 5' -GCA TTA GCT TCA GAT TTA CGG GT- 3'; mouse MK2 ⁇ forward 5'-GTT CCC CCA GTT CCA CGT CAA G-3', reverse 5'-CTA AAG AGC TCT CCA CCA TCG-3'; mouse TTP ⁇ forward 5'-CCA GGC TGG CTT TGA ACT CA-3', reverse 5' -ACC TGT AAC CCC AGA ACT TGT AAC CCC AGA ACT
- mouse MZP- 10 forward 5'-CAG CCC TGA TGC TTC TCA CT-3', reverse 5'-GGG AGA CAC GCG TCC TAT AAC-3';
- Anti-p-actin HRP- conjugated
- Anti-GAPDH HRP-conjugated
- anti-MK2, anti-phospho-MK2 Thr334)
- anti-phospho-MK2 Thr222
- anti-TTP anti-KHSRP
- anti-p38a anti-p38
- anti-phospho- p38 anti-phospho- p38 (Thrl80/Tyrl82)
- anti-MKK6, anti-HSP27, anti-phospho-HSP27 (Ser82) antibodies were used for immunoblotting.
- Total RAW264.7 cell lysates were prepared by using 200 pl of Pierce IP lysis buffer containing protease and phosphatase inhibitor. Cell lysates were pre-cleared with 20 pl Dynabeads Protein G for 30 min. After removal of the beads with DynaMag-Spin Magnet, the cell lysates were incubated with 2-pg anti-MK2, or anti-rabbit IgG antibodies overnight at 4°C, and with 20 pl Dynabeads Protein G for another 2 hours with gentle rotation. The immune complexes were pelleted with DynaMag-Spin Magnet, washed twice with 200 pl of immunoprecipitation buffer before immunoblotting.
- Recombinant MK2 protein (1.6 ng, 2 pL) were pre-incubated with test compounds (1 pl) for 1 hour at room temperature. A 2 pl mixture of HSP27 peptide (0.2 pg/pl) and ATP (50 pM) was then added, and the resulting mixtures (5 pL in total) were incubated for another 1 hour. Total MK2 activities were assayed using an ADP-Glo Kinase Assay according to the manufacturer's protocol. The raw data were normalized to the untreated control. Percent MK2 activity was calculated using the normalized values.
- CETSA Cellular thermal shift assay
- RAW264.7 cells were lysed in M-PER mammalian protein extraction reagent containing protease and phosphatase inhibitors at 3,000,000 cells/100 pl lysis buffer.
- Cell lysates were spiked with test compounds to the final concentration of 50 pM or with DMSO as vehicle control. After incubating at room temperature for 1 hour, the spiked lysates were aliquoted equally for gradient heat treatment in a 96-well thermocycler (Applied Biosystems) for 3 min. The denatured proteins were removed by centrifugation at
- RAW264.7 cells were plated at a density of 5 x 10 3 cells per well in 96-well plates or 1 x 10 5 cells per well in 6-well plates.
- Oligomer-lipofectamine complexes were prepared by mixing Lipofectamine 2000 (4 pl in 200 pl of Opti-MEM I Reduced Serum Medium, pre-incubated for 15 min) with siRNA (100 pmol in 200 pl of Opti-MEM I Reduced Serum Medium) or cDNA (1 pg in 200 pl of Opti-MEM I Reduced Serum Medium) and incubating for 20 min in the dark.
- Phosphorylation of MK2 by p38a in the absence or presence of AD was performed by first pre-incubating recombinant MK2 protein (0.5 pg) with AD for 10 mins at room temperature prior to the addition of recombinant p38a protein (0.2 pg). The reaction was started by addition of ATP to the final concentration of 150 pM. The phosphorylation was carried out in kinase assay buffer III (K03-09) at the final volume of 125 pl. Aliquots were taken at 0.5-, 5-, 15-, 30-, and 45-min time intervals and quenched with SDS sample buffer. Immunoblotting was performed using anti-phospho-MK2 antibodies.
- the crystal structure of MK2 (PDB ID: 2OZA, chain A) was used for docking with AD using iGEMDOCK v.2.1.
- the 3D structure of AD was generated using Spartan'20 v.1.1. "Accurate docking" was used, with a population size of 800 and generations of 80. The number of solutions was increased to 100 for more reliable clustering. This was a blind docking without specifying the locations of the binding site or the interacting residues.
Abstract
The present disclosure concerns a method of promoting the ubiquitination of MK2 protein, comprising contacting the MK2 protein with a compound of Formula (I) or a salt, solvate, stereoisomer or prodrug thereof. The present disclosure also concerns compounds of Formula (I) and their use in treating a disease or condition associated with MK2 protein.
Description
LABDANE BASED COMPOUNDS AND USES THEREOF
Technical Field
The present invention relates, in general terms, labdane based compounds and their uses thereof.
Background
The p38 mitogen-activated protein kinase (p38MAPK) signaling axis has been an attractive target for therapeutic intervention of inflammatory conditions due to its involvement in the regulation and expression of multiple cytokines and inflammatory signaling molecules. MAPK-activated protein kinase 2 (MK2) is a direct downstream substrate of p3gMAPK a ncj as been recognized as a key driver of inflammation. MK2 is often found up-regulated and phosphorylated in chronic inflammation involved in asthma, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, Alzheimer's disease, ischemic heart, and brain diseases, as well as cancer. The kinase primarily functions as a master regulator of RNA-binding proteins (RBPs), indirectly modulating cytokine production by increasing the stability and translation of the corresponding mRNAs. Upon activation, MK2 phosphorylates the anti-inflammatory RBP tristetraprolin (TTP), inhibits TTP-destabilizing activity towards adenylate/uridylate-rich elements (AREs)-containing mRNAs e.g., TNF-a), thus permitting their translation.
MK2 is currently considered as a promising target for disease-modifying anti-rheumatic drugs (DMARDs) and a possible alternative to p38MAPK for anti-inflammatory therapy development. In fact, as opposed to targeted p38 deletion that leads to embryonic lethality and compromised fertility, MK2 knock-out mice are viable and fertile, and do not display any abnormalities apart from the intended impairment in cytokine biosynthesis. Targeting MK2 leaves intact the important p38MAPK feedback loops, such as TAB1-TAK1, and the activation of downstream p38MAPK anti-inflammatory substrates such as MSK1 and MSK2. Therefore, targeting MK2 could reproduce the beneficial effects of p38MAPK inhibition while potentially sparing the accompanying side effects. To date, only a few potent and selective MK2 inhibitors are described. PF-3644022, the first orally available small molecule MK2 inhibitor developed by Pfizer, was demonstrated to be effective in numerous acute and chronic models of inflammation including rheumatoid
arthritis, inflammatory and fibrotic pulmonary diseases. However, the biochemical efficiency of PF-3644022 and other MK2 inhibitors is rather poor as compared to most drugs on the market. Hence, the search for further small molecule drugs against MK2 is necessary.
It would be desirable to overcome or ameliorate at least one of the above-described problems.
Summary
The present invention is predicated on the understanding that degradation, rather than inhibition, of MK2 protein may produce a more durable and sustained anti-inflammatory effect.
The present invention provides a method of promoting the ubiquitination of MK2 protein, comprising contacting the MK2 protein with a compound of Formula (I) or a salt, solvate, stereoisomer or prodrug thereof:
wherein
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino; 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally
substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
It was found that the compound of Formula (I) can act as a MK2 protein regulator through ubiquitination and degradation of MK2 protein. Compounds of Formula (I) may bind to the activation loop of MK2 located at the interface of the p38a-MK2 complex, causing complex dissociation and the loss of MK2.
In some embodiments, Ri is selected from optionally substituted acyloxy, optionally substituted arylthio, optionally substituted heteroarylthio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryl oxy, and optionally substituted heteroarylamino.
In particular, when Ri is a moiety having moderate-to-good leaving group ability and/or possesses aromatic moieties, the compounds were found to be more active.
In some embodiments, the compound of Formula (I) is characterised by a pKaH of about 3 to about 10.
In some embodiments, the MK2 protein is characterised by a half-life of less than 4 h.
In some embodiments, the method is an in vivo method or an in vitro method.
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally
substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
X is a heteroatom; and
R4 is selected from optionally substituted acyl, optionally substituted aryl, and optionally substituted heteroaryl;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
In some embodiments, FU is selected from optionally substituted phenyl and optionally substituted 6 membered heteroaryl.
In some embodiments, FU is selected from phenyl optionally substituted at a para position and 6 membered heteroaryl optionally substituted at a para position.
Het is an optionally substituted heteroaryl;
F is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
Fb is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
In some embodiments, the heteroaryl is bonded via its heteroatom to C-12 position of Formula (I).
In some embodiments, the heteroaryl is selected from oxazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolinyl, quinolinyl, phthalazinyl, naphthylpyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, acridinyl, phenanthrolinyl, isothiazolyl, phenazinyl, isoxazolyl, isothiazolyl, phenoxazinyl, phenothiazinyl, thiazolyl, thiadiazolyl, oxadiazolyl, oxatriazolyl, tetrazolyl, thiophenyl,
benzothiophenyl, triazolyl, and imidazopyridinyl.
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino; 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and 3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino; 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio. In some embodiments, the compound is selected from one of the following:
In some embodiments, the compound is selected from one of the following:
The present invention also provides a compound of Formula (Ic) or a salt, solvate, stereoisomer or prodrug thereof:
wherein
X is a heteroatom selected from N, 0, S; 4 is selected from optionally substituted aryl, optionally substituted heteroaryl; or X- 4 together is optionally substituted acyloxy;
Het is a optionally substituted heteroaryl, wherein the heteroatom on the heteroaryl is bonded to the C-12 position of Formula (I); 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and 3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
The present invention also provides a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer
or prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
The present invention also provides a method of treating of a disease or condition associated with MK2 protein, comprising administrating to a subject in need thereof a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable a salt, solvate, stereoisomer or prodrug thereof.
The present invention also provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for use in treating a disease or condition associated with MK2 protein.
The present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament for the treatment of a disease or condition associated with MK2 protein.
In some embodiments, the treatment of a disease or condition associated with MK2 protein comprises ubiquitination of MK2 protein.
In some embodiments, the disease or condition associated with MK2 protein is an inflammatory disease or condition.
In some embodiments, the disease or condition associated with MK2 protein is a chronic respiratory disease or condition.
In some embodiments, the disease or condition associated with MK2 protein is selected from acute lung injury, chronic inflammation in asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease, atherosclerosis, Alzheimer's disease, ischemic heart, brain disease, cancer, chronic obstructive pulmonary disease (COPD), lung fibrosis, pneumonia, long COVID.
The present invention also provides a method of inducing an anti-inflammatory response in a subject in need thereof, comprising administrating to a subject in need thereof a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable a salt, solvate, stereoisomer or prodrug thereof.
The present invention also provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for inducing an antiinflammatory response in a subject in need thereof.
The present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament for inducing an anti-inflammatory response in a subject in need thereof.
Brief description of the drawings
Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:
Figure 1. MK2 is highly expressed in macrophages and is essential for production of inflammatory mediators. (A) Protein expression levels of MK2 in various cell lines. Total cell lysates were analyzed by immunoblotting. The images shown are representative of two independent experiments. (B and C) Validation of MK2 expression levels in MK2-knockdown (KD) and MK2-overexpression (OE) RAW264.7 cells compared with negative control cells (NC). (B) MK2-KD cells were generated with siRNA to MK2 (si-MK2) and (C) MK2-OE cells were generated with CDNA-MK2. Cell lysates were assayed by immunoblotting. The images shown are representative of two independent experiments. (D and E) mRNA expression of inflammatory mediator genes in NC, (D) si-MK2- or (E) cDNA-MK2-transfected RAW264.7 cells. Cells were challenged with or without LPS (100 ng/ml) for 4 h and quantitative PCR was performed. Data are shown as mean ± SEM of four independent experiments. (F and G) Secretion levels of TNF-a and MCP-1 in NC, (F) si-MK2- or (G) cDNA-MK2-transfected RAW264.7 cells with or without LPS challenge. The cell supernatant way assayed by ELISA (4 h for TNF-a and 24 h for MCP-1 after LPS challenge). Data are shown as mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; NC, negative control.
Figure 2. AD down-regulates MK2 protein levels and blocks MK2-mediated inflammatory responses in macrophages. (A and B) Dose-dependent effect of AD on MK2 protein levels in RAW264.7 cells. (A) Cells were incubated with increasing
concentrations of AD (i.e., 5, 10, 20, 50 pM) for 4 h. (B) Cells were pre-incubated with increasing concentrations of AD (i.e., 5, 10, 20, 50 pM) for 1 h and then treated with LPS (100 ng/ml) for 4 h. Cell lysates were analysed by immunoblotting. The images shown are representative of three independent experiments. (C) Time-dependent effect of CHX and AD on MK2 protein levels. RAW264.7 cells were incubated with CHX (1 pg/ml) or AD (50 pM) for the indicated times. Cell lysates were analysed by immunoblotting. Band intensities were analyzed by Image!, and the MK2/GAPDH ratios relative to t = 0 min are indicated. Data are shown as mean of three independent experiments. (D and E) Effects of AD on LPS-induced (D) mRNA expression level and (E) secretion levels of TNF-a and MCP-1. (D) RAW264.7 cells were pre-incubated with increasing concentrations of AD for 1 h and then treated with LPS (100 ng/ml) for 4 h. Quantitative PCR was performed. Data are shown as mean ± SEM of three independent experiments. (E) RAW264.7 cells were pre-incubated with increasing concentrations of AD for 1 h and then treated with LPS (100 ng/ml). The cell supernatant was assayed by ELISA (4 h for TNF-a and 24 h for MCP-1 after LPS challenge). Data are shown as mean ± SEM of four independent experiments. (F) Effect of AD (10 pM) on MK2 protein levels in primary alveolar macrophages (AMs) with or without LPS (100 ng/ml) challenge. Cell lysates were analysed by immunoblotting. The images shown are representative of three independent experiments. (G) Effects of AD on LPS-induced TNF-a, MCP-1, MIP- 2, IL-6 production by primary alveolar macrophages. The cell supernatant way assayed by ELISA. Data are shown as mean ± SEM of four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; Veh: vehicle (DMSO). (H) Effect of AD on members of the MAPK axis and PAM pathway. RAW264.7 cells were incubated with increasing concentrations of AD (i.e., 5, 10, 20, 50 pM) for 4 h. Cell lysates were analysed by immunoblotting (n = 3).
Figure 3. The anti-inflammatory effects of AD are MK2-dependent and involve TTP. (A and B) Effects of AD on LPS-induced secretion levels of (A) TNF-a and (B) MCP- 1 by NC and MK2-OE RAW264.7 cells. The cell supernatant way assayed by ELISA. Data are shown as mean ± SEM of three independent experiments. (C and D) The transfection efficiency of siRNA to TTP (si-TTP). (C) Cell lysates were assayed by immunoblotting. GAPDH was used as the loading control. The images shown are representative of two independent experiments. (D) Cells were challenged with or without LPS (100 ng/ml) for 4 h and quantitative PCR was performed. Data are shown as mean ± SEM of three independent experiments. (E) Effects of AD on LPS-induced mRNA expression level of TNF-a in NC and si-TTP-transfected RAW 264.7 cells. Cells were pre-incubated with
increasing concentrations of AD for 1 h and then treated with LPS (100 ng/ml) for 4 h. Quantitative PCR was performed. The amount of mRNA was normalized to p-actin expression and was presented as fold of control. Data are shown as mean ± SEM of three independent experiments. (F) Effects of AD, PF-3644022, Dex and p50-i on LPS- induced TNF-a production by NC and si -TTP-transfected RAW264.7 cells. Cells were preincubated with test compounds for 1 h and then treated with LPS (100 ng/ml) for 24 h. The cell supernatant way assayed by ELISA. Data are shown as mean ± SEM of three independent experiments. (G and H) Effects of AD on mRNA expression and protein levels of TTP in RAW264.7 cells. (G) Cells were incubated with increasing concentrations of AD (/.e., 5, 10, 20, 50 pM) for 4 h and quantitative PCR was performed. The amount of mRNA was normalized to p-actin expression and was presented as fold of untreated control. Data are shown as mean ± SEM of four independent experiments. (H) Cells were incubated with AD for the indicated times. Cell lysates were analysed by immunoblotting. GAPDH was used as the loading control. The images shown are representative of three independent experiments. (I) Immunoprecipitation with MK2 antibody (IP:MK2). RAW264.7 cells were treated with or without AD (50 pM) for 30 min, immunoprecipitated with MK2 antibody, and then analyzed by immunoblotting. The images shown are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NC, negative control. (J) Schematic diagram illustrating the proposed mechanism of action of AD.
Figure 4. AD promotes MK2 down-regulation in vivo and attenuates inflammatory responses in LPS-induced acute lung injury (ALI). (A and B) Schematic diagram of in vivo AD treatment and LPS-induced ALI. (C) Effects of AD on MK2 total protein expression in primary AMs and lung tissues. AMs were isolated from lung lobes. Total cells lysates and lung homogenates were analysed by immunoblotting. P-actin was used as the loading control. The images shown are representative of three independent experiments. (D) Effects of LPS stimulation on AMs isolated from mice pretreated with AD. Mice were treated with AD (1 mg/kg, i.t.) for the indicated times and AMs were isolated from lung lobes. Cells were then challenged with LPS (100 ng/ml) and the cell supernatant way assayed by ELISA (4 h for TNF-a and 24 h for MCP-1 after LPS challenge). Data are shown as mean ± SEM of four independent experiments. (E) Effects of AD on LPS-induced ALI. Mice were intratrachea lly injected with LPS (0.5 mg/kg) or equal volume of saline, followed by AD (1 mg/kg, i.p.) treatment twice a day for 3 consecutive days. Differential cell staining in BALF was performed. Data are shown as mean ± SEM (n = 6 animals/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 5. AD promotes the ubiquitination and proteasomal degradation of MK2. (A) Effects of AD on mRNA expression of MK2 in RAW264.7 cells. Cells were incubated with increasing concentrations of AD ( .e., 5, 10, 20, 50 pM) for 4 h and quantitative PCR was performed. The amount of mRNA was normalized to p-actin expression and was presented as fold of untreated control. Data are shown as mean ± SEM of four independent experiments. (B) Effects of AD on MK2 protein expression levels in the presence of MG132. Cells were pre-incubated with MG132 (1 pM) for 1 h and then treated with AD (50 pM) for another 2 h. Cell lysates were analysed by immunoblotting. GAPDH was used as the loading control. Band intensities were analyzed by Image!, and the MK2/GAPDH ratios relative to DMSO control are indicated. Data are shown as mean ± SEM of three independent experiments. (C) Immunoprecipitation with MK2 antibody (IP:MK2). RAW264.7 cells were treated with or without AD (50 pM) for 2 h, immunoprecipitated with MK2 antibody, and then analyzed by immunoblotting. The images shown are representative of two independent experiments. (D) Effects of AD on MK2 protein expression levels in the presence of SB203580. Cells were pre-incubated with SB203580 (10 pM) for 1 h and then treated with AD (50 pM) for another 2 h. Cell lysates were analysed by immunoblotting. GAPDH was used as the loading control. The images shown are representative of three independent experiments. (E) Effects of AD on p38-MK2 complex assembly. Recombinant MK2 protein was incubated with increasing concentrations of AD (/.e., 50, 100, 200 pM) for 10 mins before an equimolar amount of recombinant p38a protein was added (MK2:p38a = 1 : 1). The mixtures were incubated overnight and then immunoprecipitated with MK2 antibody. Samples were analysed by immunoblotting (n = 3). The images shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; Veh, vehicle (DMSO). (F) Time-dependent effect of CHX and AD on MK2 protein levels. RAW264.7 cells were incubated with CHX (1 pg/mL) or AD (50 pM) for the indicated times. Cell lysates were analysed by immunoblotting. Band intensities were analyzed by Image!, and the MK2/GAPDH ratios relative to t = 0 min are indicated. Data are mean, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001; Veh, vehicle (DMSO).
Figure 6. AD binds to the activation loop of MK2 located at the interface between p38a and MK2. (A) X-ray crystallography of p38a-MK2 complex obtained from PDB (ID: 2OZA). (B) Docking of AD on MK2 (chain A). Blind docking (rigid residues) was performed using iGEMDOCK v.2.1. with 100 solutions. When the 100 conformers were sorted according to their energies, 90% of the conformers with the lowest energies docked around a site adjacent to the activation loop. (C). The binding site residues are
colored. The figures were generated using the program Chimera (reference). (D) CETSA for MK2. Total RAW264.7 cell lysates were treated with DMSO (0.05%), AD (50 pM) and PF-3644022 (10 pM) for 1 h. Aliquots were subjected to gradient heat treatment (37 to 53°C) and the soluble protein fraction was analyzed by immunoblotting. KHSRP was used as the loading control. Band intensities were analyzed by Image! and normalized to that at 37°C to obtain the CETSA melt curves of MK2. Data are shown as mean ± SEM of three independent experiments. (E) Effects of AD on the phosphorylation of MK2 by constitutively active p38a. AD was pre-incubated with recombinant MK2 10 min prior to the addition of p38a (4: 1 molar ratio) and ATP (150 pM). The amount of phosphorylated MK2 was chased with phosphosite-specific antibodies that recognize Thr222 or Thr334. The images shown are representative of three independent in vitro kinase assays. (F) Kinase activity of MK2 measured by ADP-Glo kinase assay. Recombinant MK2 was pre-incubated with DMSO, AD, and PF3644022 in increasing concentrations 10 min prior to the addition of HSP27 peptide substrate and ATP. The kinase reaction was allowed for 1 h before an ADP-Glo kinase assay. Data are shown as mean ± SEM of three independent experiments.
Figure 7. Pharmacological difference between protein degrader (AD) and inhibitor (PF3644022) against MK2. (A) Recovery of MK2 level after washout of AD. After treatment for 4 h with AD (50 pM), RAW264.7 cells were washed with fresh culture medium and further cultured for the indicated times. The images shown are representative of three independent experiments. (B) Effects of pre-treatment time on the cytokine inhibitory effects of AD and PF3644022. RAW264.7 cells were preincubated with test compounds for 1, 6 or 12 h and then treated with LPS (100 ng/ml) for another 4 h. The cell supernatant way assayed by ELISA. Data are shown as mean ± SEM of three independent experiments. (C and D) Effects of wash-out on the cytokine inhibitory effects of AD and PF3644022. RAW264.7 cells were pre-incubated with test compounds for 24 h, washed with fresh culture medium and further cultured for the indicated times. Cells were then treated with LPS (100 ng/ml) for another 4 h. The cell supernatant way assayed by ELISA. Data are shown as mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NC, negative control.
Figure 8. Occupancy-and event-driven pharmacology models.
Figure 9. Intrinsic thiol reactivity of AD and representative C-12 derivatives measured against GSH as a surrogate
Figure 10. (A-B) Inhibitory effects of various test compounds on LPS-stimulated TNF- a and IL-6 release. (C) Cytotoxic effects of test compounds on non-stimulated RAW264.7 cells. (*) Derivatives carry aromatic substituents at C-12 position.
Figure 11. (A-B) Inhibitory effects of various test compounds on LPS-stimulated TNF- a and IL-6 mRNA expression levels. (*) Derivatives carry aromatic substituents at C-12 position.
Figure 12. Modulatory effects of C-12 andrographolide analogues on MK2 protein expression levels.
Figure 13. Total and differential cell counts in bronchoalveolar lavage fluids (BALF) of mice stimulated with HDM and treated with 4, or vehicle control. *p < 0.05, **p < 0.01 indicate statistically significant difference as compared to HDM-treated group. #p < 0.05 indicates statistically significant difference as compared to saline-treated group.
Figure 14. Secretion levels of (A) MIP-2 and (B) KC in NC and siMK2-transfected RAW264.7 cells with or without LPS challenge. The cell supernatants were assayed by ELISA (4 h for MIP-2 and 24 h for KC after LPS challenge). Data are shown as mean ± SEM of three independent experiments. **, P < 0.01; NC, negative control.
Figure 15. Effects of AD on the cell viability of RAW264.7 cells and primary AMs. Cells were treated with increasing concentrations of AD (0.1 to 50 pM) for the indicated times. Cell viability was assayed by MTS. Data are shown as mean ± SEM of three independent experiments.
Figure 16. Effects of AD on the protein levels of p38a, TTP, MKK6 in primary AMs (A) and lung tissues (B). AMs were isolated from lung lobes. Total cells lysates and lung homogenates were analysed by immunoblotting, p-actin was used as the loading control. The images shown are representative of three independent experiments.
Figure 17. (A) Time-dependent effect of CHX on MK2 protein levels. RAW264.7 cells were incubated with CHX (1 pg/mL) for the indicated times up to 8 h. Cell lysates were analysed by immunoblotting. Band intensities were analyzed by Image!. (B) The MK2/GAPDH ratios relative to t = 0 min are indicated. Data are shown as mean of three independent experiments.
Figure 18. Dose-dependent effects of (A) AD and (B) PF-3644022 on LPS-induced production of TNF-a. RAW264.7 cells were pre-treated with increasing concentrations of test compounds for 1 h before stimulated with LPS (100 ng/mL) for 4 h. The cell supernatants were assayed by ELISA. LPS-only treated cells were counted as 100%. IC50 value is determined as the concentration at which test compounds inhibit 50% of TNF-a production. Data are shown as mean ± SEM of three independent experiments.
Figure 19. (A) The immunofluorescent staining for DAPI (blue) and MK2 (red) in primary AMs showed that MK2 is ubiquitously expressed in both cytoplasmic and nuclear compartments. (B) Effects of AD on the phosphorylation of p38a in RAW264.7 cells. Cells were incubated with increasing concentrations of AD (i.e., 5, 10, 20, 50 pM) for 4 h. Cell lysates were analysed by immunoblotting. The images shown are representative of three independent experiments
Detailed description
"Alkyl" refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 10 carbon atoms or more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, /so-propyl, n-butyl, /so- butyl, n-hexyl, and the like.
"Alkenyl" refers to a monovalent alkenyl group which may be straight chained or branched and preferably have from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and have at least 1 and preferably from 1-2, carbon to carbon, double bonds. Examples include ethenyl (-CH=CH2), n-propenyl (-CH2CH = CH2), /so-propenyl (-C(CH3)=CH2), but-2-enyl (-CH2CH = CHCH3), and the like.
"Alkynyl" refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1, and preferably from 1-2, carbon to carbon, triple bonds. Examples of alkynyl groups include ethynyl (-C= CH), propargyl (-CH3C= CH), pent-2-ynyl (-CH2C=CCH2-CH3), and the like.
"Alkoxy" refers to the group alkyl-O- where the alkyl group is as described above. Examples include, methoxy, ethoxy, n-propoxy, /so-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
"Alkenyloxy" refers to the group alkenyl-O- wherein the alkenyl group is as described above.
Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.
"Oxo/hydroxy" refers to groups =0, HO-.
"Haloalkyl" refers to an alkyl group wherein the alkyl group is substituted by one or more halo group as described above. The terms "haloalkenyl", "haloalkynyl" and "haloalkoxy" are likewise defined.
"Aryl" refers to an unsaturated aromatic carbocyclic group having a single ring (eg. phenyl) or multiple condensed rings (eg. naphthyl or anthryl), preferably having from 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl and the like.
"Heteroaryl" refers to a monovalent aromatic heterocyclic group which fulfils the Huckel criteria for aromaticity (ie. contains 4n + 2 n electrons) and preferably has from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur within the ring (and includes oxides of sulfur, selenium and nitrogen). Such heteroaryl groups can have a single ring (eg. pyridyl, pyrrolyl or N- oxides thereof or furyl) or multiple condensed rings (eg. indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl or benzothienyl).
Examples of heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiophene, benzo[b]thiophene, triazole, imidazopyridine and the like.
"Aryloxy" refers to the group aryl-O- wherein the aryl group is as described above.
"Arylalkyl" refers to -alkylene-aryl groups preferably having from 1 to 10 carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl and the like.
"Acyl" refers to groups H-C(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl- C(O)- and heterocyclyl-C(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Oxyacyl" refers to groups HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)-, and heterocyclyl-OC(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Amino" refers to the group -NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Aminoacyl" refers to the group -C(O)NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Acylamino" refers to the group -NR"C(O)R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
"Acyloxy" refers to the groups -OC(O)-alkyl, -OC(O)-aryl, -C(O)O-heteroaryl, and - C(O)O-heterocyclyl where alkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Aminoacyloxy" refers to the groups -OC(O)NR"-alkyl, -OC(O)NR"-aryl, -OC(O)NR"- heteroaryl, and -OC(O)NR"-heterocyclyl where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Oxyacylamino" refers to the groups -NR"C(O)O-alkyl, -NR"C(O)O-aryl, -NR"C(O)O- heteroaryl, and NR"C(O)O-heterocyclyl where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Oxyacyloxy" refers to the groups -OC(O)O-alkyl, -O-C(O)O-aryl, -OC(O)O- heteroaryl, and -OC(O)O-heterocyclyl where alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
"Acylimino" refers to the groups -C(NR")-R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
"Acyliminoxy" refers to the groups -O-C(NR")-R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
"Oxyacylimino" refers to the groups -C(NR")-OR" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
"Cycloalkyl" refers to cyclic alkyl groups having a single cyclic ring or multiple condensed rings, preferably incorporating 3 to 11 carbon atoms. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, indanyl, 1,2,3,4-tetrahydronapthalenyl and the like.
"Cycloalkenyl" refers to cyclic alkenyl groups having a single cyclic ring or multiple condensed rings, and at least one point of internal unsaturation, preferably incorporating 4 to 11 carbon atoms. Examples of suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclohex-4-enyl, cyclooct- 3-enyl, indenyl and the like.
"Heterocyclyl" refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur, oxygen, selenium or phosphorous within the ring. The most preferred heteroatom is nitrogen. It will be understood that where, for instance, 2 or R' is an optionally substituted heterocyclyl which has one or more ring heteroatoms, the heterocyclyl group can be connected to the core
molecule of the compounds of the present invention, through a C-C or C-heteroatom bond, in particular a C-N bond.
Examples of heterocyclyl and heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1, 2, 3, 4-tetra hydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiazolidine, thiophene, benzo[b]thiophene, morpholino, piperidinyl, pyrrolidine, tetra hydrofuranyl, triazole, and the like.
"Thio" refers to groups H-S-, alkyl-S-, cycloalkyl-S-, aryl-S-, heteroaryl-S-, and heterocyclyl-S-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Thioacyl" refers to groups H-C(S)-, alkyl-C(S)-, cycloalkyl-C(S)-, aryl-C(S)-, heteroaryl-C(S)-, and heterocyclyl-C(S)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Oxythioacyl" refers to groups HO-C(S)-, alkylO-C(S)-, cycloalkylO-C(S)-, arylO- C(S)-, heteroarylO-C(S)-, and heterocyclylO-C(S)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Oxythioacyloxy" refers to groups HO-C(S)-O-, alkylO-C(S)-O-, cycloalkylO-C(S)-O- , arylO-C(S)-O-, heteroarylO-C(S)-O-, and heterocyclylO-C(S)-O-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Thioacyloxy" refers to groups H-C(S)-O-, alkyl-C(S)-O-, cycloalkyl-C(S)-O-, aryl- C(S)-O-, heteroaryl-C(S)-O-, and heterocyclyl-C(S)-O-, where alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
"Sulfinyl" refers to groups H-S(O)-, alkyl-S(O)-, cycloalkyl-S(O)-, aryl-S(O)-, heteroaryl-S(O)-, and heterocyclyl-S(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Sulfonyl" refers to groups H-S(O)2-, alkyl-S(O)2-, cycloalkyl-S(O)2-, aryl-S(O)2-, heteroaryl-S(O)2-, and heterocyclyl-S(O)2-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
"Sulfinylamino" refers to groups H-S(O)-NR"-, alkyl-S(O)-NR"-, cycloalkyl-S(O)-NR"- , aryl-S(O)-NR"-, heteroaryl-S(O)-NR"-, and heterocyclyl-S(O)-NR"-, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Sulfonylamino" refers to groups H-S(O)2-NR"-, alkyl-S(O)2-NR"-, cycloalkyl-S(O)2- NR"-, aryl-S(O)2-NR"-, heteroaryl-S(O)2-NR"-, and heterocyclyl-S(O)2-NR"-, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Oxysulfinylamino" refers to groups HO-S(O)-NR"-, alkylO-S(O)-NR"-, cycloalkylO- S(O)-NR"-, arylO-S(O)-NR"-, heteroarylO-S(O)-NR"-, and heterocyclylO-S(O)-NR"-, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Oxysulfonylamino" refers to groups HO-S(O)2-NR"-, alkylO-S(O)2-NR"-, cycloalkylO-S(O)2-NR"-, arylO-S(O)2-NR"-, heteroarylO-S(O)2-NR"-, and heterocyclylO-S(O)2-NR"-, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Aminothioacyl" refers to groups R"R"N-C(S)-, where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Thioacylamino" refers to groups H-C(S)-NR"-, alkyl-C(S)-NR"-, cycloalkyl-C(S)- NR"-, aryl-C(S)-NR"-, heteroaryl-C(S)-NR"-, and heterocyclyl-C(S)-NR"-, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Aminosulfinyl" refers to groups R"R"N-S(O)-, where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
"Aminosulfonyl" refers to groups R"R"N-S(O)2-, where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
In this specification "optionally substituted" is taken to mean that a group may or may not be further substituted or fused (so as to form a condensed polycyclic group) with one or more groups selected from hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, phosphono, sulfo, phosphorylamino, phosphinyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino, aminoacyloxy, trihalomethyl, trialkylsilyl, pentafluoroethyl, trifluoromethoxy, difluoromethoxy, trifluoromethanethio, trifluoroethenyl, mono- and di-alkylamino, mono-and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclyl amino, and unsymmetric di-substituted amines having different substituents selected from alkyl, aryl, heteroaryl and heterocyclyl, and the like, and may also include a bond to a solid support material, (for example, substituted onto a polymer resin). For instance, an "optionally substituted amino" group may include amino acid and peptide residues.
"Isomer" includes especially optical isomers (for example essentially pure enantiomers, essentially pure diastereomers, and mixtures thereof) as well as conformation isomers
(i.e. isomers that differ only in their angles of at least one chemical bond), position isomers (particularly tautomers), and geometric isomers (e.g. cis-trans isomers).
Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers. "Optically-enriched," as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments the compound of the present invention is made up of at least about 90% by weight of a preferred enantiomer. In other embodiments the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer. Preferred enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972).
The present invention is predicated on the understanding that andrographolide (AD), a labdane diterpene isolated from the plant Andrographis paniculata, has been demonstrated to exhibit potent anti-inflammatory effects in various inflammatory disease models. Reports have confirmed the therapeutic potential of AD in treatment of various chronic inflammatory conditions such as asthma, COPD, psoriasis, Alzheimer's diseases, and cancer. Despite continuous efforts to elucidate the anti-inflammatory mechanism of AD, its specific mechanism is not entirely clear. Among the diverse signaling pathways being investigated, NF-KB inhibition prevails as the main
hypothetical mechanism responsible for the anti-inflammatory action of AD. However, and in contrast to current views, the inventors have found that the ability of AD to inhibit LPS-induced cytokine release is minimally linked to its N F-KB suppression, suggesting that there may be other mechanisms underlying the anti-inflammatory actions of AD, which in turn means that AD and its derivatives can be use in medical treatment in a different manner. Recently, there have been accumulating reports on the modulatory effect of AD on p38MAPK pathway; yet its link to the anti-inflammatory property of AD remains unclear.
Without wanting to be bound by theory, the inventors have found that labdane based compounds (such as AD) promotes the ubiquitination and degradation of MK2, thereby blocking downstream MK2-mediated inflammatory responses. Investigations was focused on activated macrophages as a model system due to their high level of expression of MK2. It is shown that AD binds to the activation loop of MK2 located at the interface of the p38a-MK2 complex, causing complex dissociation and the loss of MK2. AD inhibits the production of inflammatory cytokines such as TNF-a by promoting the mRNA destabilizing effects of TTP, a downstream substrate of MK2. Selective downregulation of MK2 by labdane based compounds (such as AD), which may be desirable from a safety standpoint, is not only efficacious in inhibiting inflammatory responses but also produces more sustained effects as compared to a conventional MK2 inhibitor approach (e.g., PF-3644022). The results illustrate an additional mode of modulating the p38MAPK-MK2 signaling axis, which can be harnessed for effective anti-inflammatory drug development.
In particular, the p38MAPK-MK2 signaling axis serves as a point of convergence both downstream of receptors for inflammatory stimuli and upstream of the synthesis of pro- inflammatory signaling molecules, allowing itself to function as an amplifier of inflammation. Targeting the p38MAPK-MK2 signaling axis represents a viable approach for therapeutic intervention of inflammatory diseases. The mechanism of action of andrographolide (AD), a small-molecule natural product belonging to the ent-labdane diterpene family, was explored as an inhibitor of the p38MAPK-MK2 signaling axis. AD binds the MK2 activation loop located at the interface of the p38MAPK-MK2 biomolecular complex, disrupting the complex formation and causing irreversible loss of MK2. The induced MK2 degradation accelerated pro-inflammatory mediator mRNAs decay, blocked downstream MK2-mediated inflammatory responses in in-vitro and was
efficacious in attenuating inflammation in mice with acute lung injury. The results show that not only MK2 is a viable substrate for post-translational degradation, but also that the signaling inactivation achieved by MK2 degrader is more durable and sustained than that achieved via MK2 inhibition. These findings have translational clinical implications as AD also offers the potential to mitigate the side effects associated with global p38a inhibitors that result from their inhibition of non-MK2 substrates involved in housekeeping and anti-inflammatory responses.
Additionally, a series of labdane based compounds was developed using an integrated strategy that focuses on both reactivity and pharmacophore optimization. Their warhead reactivity were fine-tuned to minimize potential off-target binding and side effects, while enhancing the intended anti-inflammatory activities. These compounds were been tested for in vivo efficacy in an animal model of severe asthma. The compounds exhibit significantly improved anti-inflammatory activity relative to AD. Moreover, these compounds display low reactivity towards glutathione, a frequent off target of AD, and thus are associated with lower cytotoxicities. For example, at doses of lmg/kg, compound 4 effectively alleviates allergic airway inflammation in mice, with no observable side-effects. The compounds may also be synthesized using highly reproducible synthetic route with good yields.
Accordingly, the present invention provides a method of promoting the ubiquitination of MK2 protein, comprising contacting the MK2 protein with a compound of Formula (I) or a salt, solvate, stereoisomer or prodrug thereof:
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally
substituted heteroarylamino;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
Rs is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
It was found that the compound of Formula (I) can act as a MK2 protein regulator through ubiquitination and degradation of MK2 protein. Compounds of Formula (I) may bind to the activation loop of MK2 located at the interface of the p38a-MK2 complex, causing complex dissociation and the loss of MK2.
In some embodiments, the method is a method of promoting the degradation of MK2 protein. In some embodiments, the method is a method of promoting the ubiquitination and degradation of MK2 protein.
In general, proteins are marked for degradation by the attachment of ubiquitin to the amino group of the side chain of a lysine residue. Additional ubiquitins can be added to form a multiubiquitin chain, which are then recognized and degraded by a large, multisubunit protease complex, called the proteasome. In this sense, the primary structure is destroyed as covalent peptide bonds are broken. In contrast, denaturation only involves the unfolding of a protein, where quaternary, tertiary and secondary structures are disrupted but primary structure remains intact. Protein inhibition refers to a process of stopping or slowing an activity of a protein by the binding of a molecule to the protein's active site or alternative site. Such inhibition may be reversible or irreversible.
In some embodiments, Ri is selected from optionally substituted acyloxy, optionally substituted arylthio, optionally substituted heteroarylthio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryl oxy, and optionally substituted heteroarylamino.
In some embodiments, the optional substituent on Ri is selected from alkyl, aryl, alkyloxyacyl, alkylacylamino, alkylacyloxy, and alkylaminoacyl. In some embodiments, the optional substituent on Ri is selected from methyl, ethyl, propyl, phenyl, methyloxyacyl, ethyloxyacyl, propyloxyacyl, methylacylamino, ethylacylamino, propylacylamino, methylacyloxy, ethylacyloxy, propylacyloxy, methylaminoacyl, ethylaminoacyl, and propylaminoacyl.
In particular, when Ri is a moiety having moderate-to-good leaving group ability and/or possesses aromatic moieties, the compounds were found to be more active; i.e. stronger binding of compound of Formula (I) to MK2.
In some embodiments, 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, and optionally substituted thio. In some embodiments, 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, and optionally substituted acyloxy. In some embodiments, 2 is selected from H, halogen, and hydroxyl. In some embodiments, 2 is hydroxyl.
In some embodiments, R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, and optionally substituted thio. In some embodiments, R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, and optionally substituted acyloxy. In some embodiments, 3 is selected from H, halogen, and hydroxyl. In some embodiments, 3 is hydroxyl.
Without wanting to be bound by theory, the inventors postulated that anti-inflammatory activities may be retained when there are modifications at the 2 and 3 positions. This is based on parallel studies conducted with the C-14 analogues, which shows that modifications at these positions does not adversely affect the intended function of the C-14 analogues.
In some embodiments, the compound of Formula (I) is characterised by a pKaH of about
In some embodiments, the MK2 protein is characterised by a half-life of less than 4 h.
In some embodiments, the method of promoting the degradation of MK2 protein occurs in a cell. In some embodiments, the method is an in vivo method, an in vitro method or an ex vivo method. In other embodiments, the method is an in vitro method. Ex vivo refers to experimentation or measurements done in or on tissue from an organism in an external environment with minimal alteration of natural conditions. Testing the effect of compounds on skin biopsies is an example of ex vivo research, while isolating the primary cells from that biopsy and creating a 3D cell culture model is an example of in vitro research.
F is selected from optionally substituted acyl, optionally substituted aryl, and optionally substituted heteroaryl; and
R2 and R3 are as disclosed herein.
In some embodiments, X is selected from 0, S, N.
In some embodiments, the heteroaryl is selected from pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, dioxinyl, oxazinyl, thiazinyl, indolyl, isoindolyl, indazolyl, benzimidazolyl, azaindolyl, purinyl, isobenzofuranyl, benzothiophenyl, benzoisoxazolyl, benzoisothiazolyl, benzothiadiazolyl, quinolinyl, isoquinolinyl, and naphthalenyl.
In some embodiments, R4 is selected from optionally substituted phenyl and optionally substituted 6 membered heteroaryl. In some embodiments, the 6 membered heteroaryl
is selected from pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, dioxinyl, oxazinyl, and thiazinyl.
In some embodiments, FU is selected from phenyl optionally substituted at a para position and 6 membered heteroaryl optionally substituted at a para position.
In some embodiments, the optional substituent on FU is selected from alkyl, alkyloxyacyl, alkylacylamino, alkylacyloxy, and alkylaminoacyl. In some embodiments, the optional substituent on FU is selected from methyl, ethyl, propyl, phenyl, methyloxyacyl, ethyloxyacyl, propyloxyacyl, methylacylamino, ethylacylamino, propylacylamino, methylacyloxy, ethylacyloxy, propylacyloxy, methylaminoacyl, ethylaminoacyl, and propylaminoacyl.
Het is an optionally substituted heteroaryl; and FU and F are as disclosed herein.
In some embodiments, the heteroatom on the heteroaryl is bonded to the C-12 position of Formula (I). In other words, the heteroatom is linked via its heteroatom to the Markush structure. For clarity, the C-12 position of the Formula (I) is shown below.
In some embodiments, the heteroaryl is selected from oxazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolinyl, quinolinyl, phthalazinyl, naphthylpyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, acridinyl, phenanthrolinyl, isothiazolyl, phenazinyl, isoxazolyl, isothiazolyl, phenoxazinyl, phenothiazinyl, thiazolyl, thiadiazolyl, oxadiazolyl, oxatriazolyl, tetrazolyl, thiophenyl, benzothiophenyl, triazolyl, and imidazopyridinyl.
In some embodiments, the optional substituent on the heteroaryl is selected from alkyl, alkyloxyacyl, alkylacylamino, alkylacyloxy, and alkylaminoacyl. In some embodiments, the optional substituent on the heteroaryl is selected from methyl, ethyl, propyl, phenyl, methyloxyacyl, ethyloxyacyl, propyloxyacyl, methylacylamino, ethylacylamino, propylacylamino, methylacyloxy, ethylacyloxy, propylacyloxy, methylaminoacyl, ethylaminoacyl, and propylaminoacyl.
In some embodiments, the compound of Formula (I) is a compound of Formula (I1):
wherein Ri, 2 and 3 are as disclosed herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (I"):
wherein Ri, R2 and R3 are as disclosed herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (Ic):
wherein Ri, R2 and R3 are as disclosed herein.
X, R2, R3 and R4 are as disclosed herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (le):
wherein Het, R2, and R3 are as disclosed herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (Ic1):
In some embodiments, the compound of Formula (I) is a compound of Formula (Ic"):
wherein Ri, R2 and R3 are as disclosed herein.
In some embodiments, the compound is selected from one of the following:
The present invention also provides a compound of Formula (Ic) or a salt, solvate, 5 stereoisomer or prodrug thereof:
wherein
X is a heteroatom selected from N, 0, S; 4 is selected from optionally substituted aryl, optionally substituted heteroaryl; or X- 4 together is optionally substituted acyloxy;
Het is an optionally substituted heteroaryl, wherein the heteroatom on the heteroaryl is bonded to the C-12 position of Formula (I); 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and 3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
In some embodiments, 2 is hydroxyl. In some embodiments, R3 is hydroxyl.
The present invention also provides a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
The present invention also provides a method of treating of a disease or condition associated with MK2 protein, comprising administrating to a subject in need thereof a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable a salt, solvate, stereoisomer or prodrug thereof.
The disease or condition associated with MK2 protein is treated by degrading the MK2 protein, and not inhibit the MK2 protein.
The present invention also provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for use in treating a disease or condition associated with MK2 protein.
The present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament.
The present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament for the treatment of a disease or condition associated with MK2 protein.
In some embodiments, the treatment of a disease or condition associated with MK2 protein comprises degradation of MK2 protein.
In some embodiments, the disease or condition associated with MK2 protein is an inflammatory disease or condition. In some embodiments, the disease or condition associated with MK2 protein is a chronic respiratory disease or condition. In some embodiments, the disease or condition associated with MK2 protein is selected from acute lung injury, chronic inflammation in asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease, atherosclerosis, Alzheimer's disease, ischemic heart, brain disease, cancer, COPD, lung fibrosis, pneumonia, and long COVID. In some
embodiments, the disease or condition associated with MK2 protein is selected from acute lung injury, chronic inflammation in asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease, atherosclerosis, Alzheimer's disease, ischemic heart, brain disease, chronic obstructive pulmonary disease (COPD), lung fibrosis, pneumonia, and long COVID.
The present invention also provides a method of inducing an anti-inflammatory response in a subject in need thereof, comprising administrating to a subject in need thereof a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable a salt, solvate, stereoisomer or prodrug thereof.
The present invention also provides a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for inducing an antiinflammatory response in a subject in need thereof.
The present invention also provides a use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament for inducing an anti-inflammatory response in a subject in need thereof.
In some embodiments, the anti-inflammatory response is induced by degradation of MK2 protein.
The compound of the invention can be administered to a subject as a pharmaceutically acceptable salt thereof. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and
alkylammonium. In particular, the present invention includes within its scope cationic salts eg sodium or potassium salts, or alkyl esters (eg methyl, ethyl) of the phosphate group.
Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
It will be appreciated that any compound that is a prodrug of the compound of formula (I) is also within the scope and spirit of the invention. Thus the compound of the invention can be administered to a subject in the form of a pharmaceutically acceptable pro-drug. The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compound of the invention. Such derivatives would readily occur to those skilled in the art. Other texts which generally describe prodrugs (and the preparation thereof) include: Design of Prodrugs, 1985, H. Bundgaard (Elsevier); The Practice of Medicinal Chemistry, 1996, Camille G. Wermuth et al., Chapter 31 (Academic Press); and A Textbook of Drug Design and Development, 1991, Bundgaard et al., Chapter 5, (Harwood Academic Publishers). For example, the compound can be substituted with a carboxylic acid moiety or a hydroxyl moiety at the C-12 position. This can be protected as a prod-drug through the formation of an ester group.
The compound of the invention may be in crystalline form either as the free compound or as a solvate (e.g. hydrate) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.
The compound of the invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof is administered to the patient in a therapeutically effective amount. As used herein, a therapeutically effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of macular degeneration.
As used herein, the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks,
months or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.
Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.
The compound of the invention may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.
The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
Injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion. Carriers can include, for
example, water, saline (e.g., normal saline (NS), phosphate-buffered saline (PBS), balanced saline solution (BSS)), sodium lactate Ringer's solution, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances, such as wetting or emulsifying agents, buffers, and the like can be added. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. By way of example, the compound, composition or combination can be dissolved in a pharmaceutically effective carrier and be injected into the vitreous of the eye with a fine gauge hollow bore needle (e.g., 30 gauge, 1/2 or 3/8 inch needle) using a temporal approach (e.g., about 3 to about 4 mm posterior to the limbus for human eye to avoid damaging the lens).
A person skilled in the art will appreciate that other means for injecting and/or administering the compound, composition or combinations to the vitreous of the eye can also be used. These other means can include, for example, intravitreal medical delivery devices. These devices and methods can include, for example, intravitreal medicine delivery devices, and biodegradable polymer delivery members that are inserted in the eye for long term delivery of medicaments. These devices and methods can further include transscleral delivery devices.
Other modes of administration including topical or intravenous administration may also be possible. For example, solutions or suspensions of the compound or composition of the invention may be formulated as eye drops, or as a membranous ocular patch, which is applied directly to the surface of the eye. Topical application typically involves administering the compound of the invention in an amount between 0.1 ng and 10 mg.
The compound or composition of the invention may also be suitable for intravenous administration. For example, a compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof may be administered intravenously at a dose of up to 16 mg/m2.
The compound or composition of the invention may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as
an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug is orally administerable.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
The compound or composition of the invention may be suitable for topical administration in the mouth including lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
The compound or composition of the invention may be suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal patches may also be used to administer the compounds of the invention.
The compound or composition of the invention may be suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the
compound, composition or combination isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compound or composition may be presented in unitdose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage composition or combinations are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.
It should be understood that in addition to the active ingredients particularly mentioned above, the composition of this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of Wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
A detailed description of the workings of the invention is laid out below. In the embodiments that follow, the invention is described in relation to some conditions for consistency to showcase the present invention. However, the skilled person would understand that the invention is not limited to such.
MK2 is highly expressed in macrophages and is essential for inflammatory cytokine/chemokine production.
To identify a suitable model to study the biological function of MK2, we examined the expression of native MK2 in various cell types including non-tumorigenic and tumorigenic epithelial cell lines, monocytes and macrophage-like cells, and primary macrophages. The results in Figure 1A showed that MK2 is highly expressed in macrophages as compared to other types of cells, while the expression of the long and the short isoforms of MK2 was more variable among the tested cell lines. The protein expression of MK3, another closely-related MAPKAP kinase to MK2 that shares similar function, was found to be trivial in macrophages. Due to the high level of MK2 expression and the low level of MK3, RAW264.7 cells and primary alveolar macrophages (AMs) were selected as the model systems for our studies. Macrophages are the main source of inflammatory cytokines and chemokines. Although the role of MK2 in regulating cytokine production has been studied in detail, its impact on chemokine secretion in macrophages has not yet well-established. We thus set out to re-investigate the functional effects of MK2 on macrophage activation using both transient knock-down and over-expression approaches.
RAW 264.7 cells were transfected with small interfering RNA (siRNA) to MK2 (siMK2) or scrambled control (scRNA) and exposed to lipopolysaccharide (LPS) (25 ng/ml; 4 h). MK2 protein was in large part eliminated in the presence of siRNA to MK2 (Figure IB). siMK2-treated or MK2-knockdown cells (MK2-KD) showed a significant decrease in LPS- mediated up-regulation of Ml macrophage marker e.g., /NOS), inflammatory cytokine (e.g., TNF-a, GM-CSF) and chemokine (e.g., MCP-1, MIP-ip, MIP-2, KC) gene expression (Figure ID). The reduced mRNA levels in MK2-KD cells were accompanied by the reduced levels of the corresponding proteins as exemplified by TNF-a, MCP-1 (Figure IF), MIP-2, and KC (Figure 14). To complement the observations from the knock-down studies, over-expression studies of MK2 were carried out. A plasmid encoding mouse MK2 cDNA was transiently transfected into RAW 264.7 cells, resulting in the over-expression of both MK2 long and short isoforms (Figure 1C). Under unstimulated conditions, over-expressing MK2 did not affect the function, nor cell proliferation, of naive macrophages, similar to the knock-down of MK2. However, in the presence of LPS, MK2 over-expressed cells showed increased expression of inflammatory mediators at both mRNA (Figure IE) and protein (Figure 1G) levels.
Collectively, these results confirm the major role MK2 as a key regulator of cytokine and chemokine production in macrophages.
Andrographolide (AD) down-regulates MK2 protein levels and blocks MK2- mediated inflammatory responses
Numerous studies have tracked down the p38MAPK signaling axis as the target pathway for the pharmacological actions of AD, mainly as an anti-aging, antioxidant, and antimicrobial agent. In an attempt to elucidate the potential involvement of p38MAPK pathway in the anti-inflammatory action of AD, the inventors were intrigued by the observations that AD induced marked down-regulation of MK2 protein levels in macrophages, while sparing other members of the p38 MAPK axis (Figure 2H). As shown in Figure 2A and B, treatment of naive RAW264.7 macrophages with AD resulted in a dose-dependent decrease in MK2 protein level. At 50 pM, AD induced more than 50% reduction in MK2 levels as compared to the basal level after a 4-hour treatment. Similar effects were also observed in LPS-stimulated RAW264.7 cells (Figure 2B). In a time-course experiment, AD was found to induce MK2 down-regulation as early as after 30 min of compound incubation (Figure 2C). Both isoforms of MK2 were affected. Cycloheximide (CHX), an inhibitor of de novo protein synthesis, was used to determine the half-life of the endogenous MK2. The level of MK2 protein in the total protein extract was gradually decreased with increased chase time points (from 0.5 to 24 h), yielding a characteristic exponential decay curve, in cells treated with CHX (1 pg/ml). The half-life of MK2 in RAW264.7 cells was determined to be 7 h, which was significantly shortened upon treatment with AD (c.a. 4 h) (Figure 2C). These data indicate that AD reduces MK2 protein stability, thus protein levels in macrophages, which may constitute a regulatory mechanism to shut down its downstream kinase activity and dependent inflammatory responses.
In fact, LPS markedly induced TNF-a and MCP-1 mRNA and protein expression in RAW264.7 cells; these responses correlated with p38a and MK2 activation and were inhibited by AD in a dose-dependent manner (Figure 2D and E). Consistent with the observations in RAW264.7 cells, treatment with AD profoundly down-regulated MK2 protein in primary alveolar macrophages (Figure 2F), which was accompanied by a decrease in the pro-inflammatory cytokine and chemokine productions by the activated AMs (Figure 2G). AD was not cytotoxic at efficacious concentrations tested in these
studies (Figure 15). Altogether, these results establish a correlation between the MK2 protein levels and the anti-inflammatory outcome of AD.
The anti-inflammatory effects of AD are MK2-dependent and involve MK2 downstream effector tristetraprolin (TTP)
We hypothesize that the AD-induced down-regulation of induced by AD could lead to the destabilization of inflammatory cytokine and chemokine mRNAs, impair their biosynthesis, thus resulting in the observed anti-inflammatory effects of AD. To establish the causation between MK2 down-regulation and the anti-inflammatory effects of AD, we tested the ability of AD to inhibit TNF-a and MCP-1 release in MK2 overexpressed RAW274.7 cells. As shown in Figure 3A and B, the inhibitory effects of AD on both TNF-a and MCP-1 release were abrogated in cells that over-expressed MK2, strongly linking its mechanism of anti-inflammatory action to the ability to down- regulate MK2.
MK2 is a key repressor of anti-inflammatory RBPs such as TTP. Phosphorylation by MK2 deactivates TTP, reduces its ability to promote deadenylation and mRNA decay, leading to excessive cytokine/chemokine production and aberrant activation of immune responses. Therefore, MK2 deficiency induced by AD would in principle stabilize TTP, enhance its mRNA-destabilizing activity, thus attenuating the production of downstream inflammatory mediators (Figure 31). In other words, the anti-inflammatory property of AD can be expected to be mediated in part via the anti-inflammatory action of TTP. To investigate this hypothesis, TTP-knockdown cell lines (TTP-KD) were created by transfection with siRNA to TTP (siTTP) and the inhibitory effects of AD on LPS-induced cytokine production were evaluated in both scRNA- and siTTP-treated cells. It is expected that, under TTP-knockdown conditions, the cytokine inhibitory effects of AD will be weakened. siRNA to TTP induced approximately 30% reduction in TTP mRNA levels, which was translated to a moderate reduction in its protein levels (Figure 3C). The effects of siTTP on TTP mRNA levels were more prominent in cells treated with LPS (Figure 3D). Consistent with published data, LPS-induced TNF-a production was significantly higher in cells with diminished TTP expression. As shown in Figure 3F, more than 50% increase in TNF-a release was observed in TTP-KD cells as compared to scRNA-treated controls. Treatment with AD strongly suppressed the induced TNF-a release in scRNA-treated cells with an ICso value of approximately 5 pM. However, the inhibition was markedly weakened in cells with low TTP levels (/.e., siTTP-treated cells).
This was supported by the observations that AD effectively blocked the increase in TNF- a mRNA levels in LPS-induced scRNA-treated cells but failed to do so in siTTP-treated cells (Figure 3E). These results corroborate the importance of TTP in mediating the antiinflammatory effects of AD and suggest that AD is likely to control the production of pro-inflammatory cytokines by destabilizing their mRNAs. As a control, we tested two anti-inflammatory agents that presumably work via different mechanisms from AD - a glucocorticoid receptor agonist, dexamethasone, and a direct NF-KB peptide inhibitor, p50-i - and a MK2 inhibitor that in principle behaves similarly to AD at the downstream. - PF3644022. Mechanistically, the former two compounds control cytokine gene expression at the upstream transcriptional levels but not post-transcriptional events, and thus their inhibitory effects on cytokine gene expression should be minimally affected by the changes in the TTP protein expression levels. In contrast, the cytokine inhibition by PF3644022 should be diminished in the absence of TTP, consistent with the behaviour of AD. As expected, the anti-inflammatory effects of dexamethasone and p50-i were comparable in both scRNA- and siTTP-treated cell lines, while that of PF3644022 was significantly weakened in siTTP-treated cells (Figure 3F). This further validate the involvement of TTP, a downstream effector of MK2, in the anti-inflammatory mechanism of AD.
Additionally, we observed that treatment with AD strongly promoted the accumulation of TTP in cells in a time-course manner (Figure 3G). Although MK2 inhibits the activity of TTP, it has been reported to be essential for the stabilization of TTP protein in cells. The accumulation of TTP in cells treated with AD, despite the lack of MK2, was reconciled by the observation that treatment of AD strongly induced TTP gene expression (Figure 3H). This preceded MK2 down-regulation as evidenced by the accumulated TTP protein bound to MK2 upon co-immunoprecipitation (Figure 31), at the time point when MK2 level was minimally affected by AD treatment (/.e., 30 min). Taken together, these results suggest that AD exerts its anti-inflammatory properties, in part, by promoting the gene expression of the anti-inflammatory protein TTP, in addition to boosting its mRNA-destabilizing activity via MK2 down-regulation.
Andrographolide (AD) promotes MK2 down-regulation in alveolar macrophages in vivo and attenuates inflammatory responses in LPS-induced acute lung injury (ALI)
To examine if the in vitro effects of AD on MK2 down-regulation could be translated into in vivo efficacy, naive BALB/c mice were treated with AD (1 mg/kg, intratracheal (i.t.)) and alveolar macrophages (AMs) were obtained (Figure 4A). A time-course treatment was employed to study the kinetics of the effects of AD on MK2 expression in AMs and lung tissue, in comparison with other members of the p38MAPK axis. AD was found to specifically reduce MK2 protein levels in AMs, but not lung tissue, with the peak effect observed at 6 h after i.t. administration (Figure 4C). By 12 h, the level of MK2 was recovered to the basal level in the control group. Other members of the p38MAPK axis including p38a, MKK6, TTP, HSP27 were minimally affected by AD in both AMs and lung tissue (Figure 16). The isolated AMs at indicated time points were exposed to LPS stimulation and the extent of macrophage activation was determined by measuring their cytokine/chemokine production. In line with the reduced MK2 levels, the production of inflammatory cytokine TNF-a, as well as chemokines MCP-1 and MIP-2 (Figure 4D) was also decreased in AMs isolated at 2 and 6 h. This was reversed at the later time points (/.e., 12 and 24 h) in accordance with the recovery in MK2 protein level, suggesting that down-regulation of MK2 is a conserved mechanism for inhibiting macrophage activation by AD.
AMs play an important role in the pathogenesis of LPS-induced acute lung injury (ALI). Recent evidence reveals that conditional MK2 deletion in AMs, but not lung tissue, attenuates the development of ALI and inhibits cytokine and chemokine accumulation in bronchoalveolar lavage fluid (BALF). ALI is characterized by pulmonary neutrophil infiltration, which is mediated by important chemokines such as MCP-1 and MIP-2 that are released from AMs. Given the deleting effects of AD on MK2 protein level in AMs and corresponding macrophage activation, the protective effect of AD against ALI was investigated. The ALI model was established by intratracheal injection of LPS (10 pg/mice) (Figure 4B), which led to significant immune cell accumulation and neutrophil infiltration in BALF (Figure 4E and F). This was prevented by AD (1 mg/kg, intraperitoneal (i.p.)) when administered every 12 h for 3 consecutive days. AD mainly blocked the recruitment of neutrophils, but not other immune cells (e.g., macrophages, lymphocytes) (Figure 4G and H).
To investigate the mechanisms by which AD down-regulates MK2 in macrophages, the mRNA expression of MK2 was determined. At the same exposure time as in Figure 2A, treatment with AD did not greatly affect the mRNA levels of MK2 (Figure 5A) at the same treatment duration as used in Figure 2H experiment. At the higher concentration of 50 pM, a slight increase in MK2 mRNA levels (less than 1.5-fold) could be observed, which can be attributed to a feedback mechanism triggered by the significant loss of MK2 protein levels at this concentration. These results suggest that AD does not down- regulate MK2 by modulating its mRNA expression; rather potentially affecting the protein at the level of translation. This was further supported by the previous observation that MK2 down-regulation by AD occurred rather rapidly and the turnover rate of MK2 was shortened in the presence of AD (Figure 2C).
In a time-course experiment (Figure 5F), it was observed that MK2 downregulation by AD occurred rather rapidly, as early as 30 min after compound treatment. Both isoforms of MK2 were affected with more than 50% reduction after 4 h. To determine the biological half-life of endogenous MK2, we treated macrophages with cycloheximide (CHX), an inhibitor of de novo protein synthesis. Cells treated with CHX (1 pg/mL) showed diminished MK2 levels upon increased chase time points (from 0.5 to 4 h). A characteristic protein decay curve was obtained and the half-life of MK2 was determined to be approximately 7.5 h (Figure 17), which was significantly shortened upon treatment with AD (c.a. 4 h, Figure 5F). These data, coupled with the unchanged MK2 mRNA levels, suggest that AD may affect MK2 protein stability via a post-translational process.
In eukaryotic cells, endogenous MK2 degradation is mediated by the ubiquitin- proteosome system. To address how MK2 is reduced in AD-treated cells, we utilized MG132, a potent proteasome inhibitor, to block the proteolytic activity of the 26S proteasome complex and examined the effects of AD on MK2. We found that inhibition of the proteasome prevented MK2 down-regulation in macrophages following treatment with AD (Figure 5B). These results suggest that AD promotes MK2 degradation via a proteasome-mediated mechanism. To confirm whether AD increases MK2 ubiquitination, resulting in its degradation, we performed IP experiments to pull down MK2 from the total protein lysates and identified the ubiquitinated MK2. As shown in Figure 5C, the levels of ubiquitinated Nrf2 increased when the RAW264.7 cells were treated with AD for 30 min; the time point at which MK2 level was minimally affected.
This suggests that AD increases MK2 proteasomal degradation by promoting its ubiquitination. p38a activation, in response to stress, has been proposed to induce irreversible MK2 loss by causing p38a-MK2 complex dissociation. The ability of AD to activate p38MAPK pathway has been documented in a number of studies. In our study, we observed the activation of p38MAPK by AD at the high concentration of 50 pM . This led us to hypothesize that perhaps AD induces p38a activation, which, in turn, disrupts the complex, and results in reduced protein stability and the loss of MK2. To examine this, we pre-treated cells with SB203580 to block the enzymatic activity of p38a before exposing to AD and immunoblotting MK2 (Figure 5D). Surprisingly, inhibiting p38a activity failed to reverse the effects of AD on MK2 degradation, suggesting that the rapid degradation of MK2 by AD occurred independently of p38a activation.
Studies have shown that, while p38a is stable independently of the p38a-MK2 complex status, the half-life of free MK2 is reduced compared to that of MK2 in complex with p38a. To investigate the possibility if AD can directly interfere with the complex formation, recombinant proteins of p38a and MK2 were used to study the binding affinities of the two proteins in the presence and absence of AD. p38a was effectively co-immunoprecipitated with MK2 from the protein mixtures (Figure 5E-left). Strikingly, this process was inhibited when AD was added in increasing concentrations (Figure 5E- right). This finding suggests that, upon exposure of cells to AD, the compound may interfere with the endogenous p38a-MK2 complex formation, releasing the free MK2 which is then subjected to ubiquitination and rapid proteosome-mediated degradation.
Andrographolide (AD) binds to the activation loop of MK2 located at the interface between p38a and MK2.
In resting cells, p38a forms a physiological complex with MK2, which prevents MK2 degradation. When the complex dissociates, both p38a and MK2 become free and MK2 undergoes rapid proteasomal degradation. To search for promising binding sites of AD on p38a-MK2 complex (Figure 6A), molecular docking simulation experiments were performed between AD and individual proteins of the complex (PDB ID: 2OZA) using iGEMDOCK v.2.1 software. One hundred runs were performed using the "accurate docking" parameters, and 90% of the docked conformers were found to be located in close vicinity to each other on the MK2 protein (Figure 6B). The lowest energy
conformers formed a major cluster in the activation loop of MK2, which comprised the phosphorylation site - Thr222 (Figure 6C).
To confirm whether MK2 is indeed a direct target of AD, we employed a cellular thermal shift assay (CETSA) that allows the investigation of ligand-target engagement on a proteome-wide scale. As shown in Figure 6D, treatment with AD significantly shifted the melting curve of MK2 to the right-hand side as compared to the vehicle control. The average Tm value was increased from 49°C in DMSO group to greater than 53°C in AD- treated group, suggesting a direct binding of AD to MK2. To reinforce this observation, PF-3644022 was used as a positive control because it directly targets MK2. Consistently, PF-3644022 protected MK2 from thermal destabilization. KHSPR, a thermostable protein, was used as an internal control to ensure equal sample loading.
The activation loop of MK2 contains Thr222 residue which serves as one of its main phosphorylation sites and is located at the interface between p38a and docked MK2 (Figure 6A). CETSA and molecular docking suggest the direct binding of AD to the activation loop of MK2. Such interaction, in principle, may affect the phosphorylation of MK2 at the Thr222 residue. To investigate this possibility, we examined if there is a discrepancy in MK2 phosphorylation at different sites (/.e., Thr 222 and Thr334) in the presence and absence of AD. MK2 was phosphorylated by active p38a in vitro and the kinetics of phosphorylation at these sites were monitored by using MK2 phosphositespecific antibodies (Figure 6E). It was observed that Thr334 phosphorylation happened ahead of Thr222 phosphorylation, which is consistent with previous reports. When MK2 was pre-incubated with AD 10 mins in advance of the addition of active p38a, phosphorylation of MK2 was inhibited specifically at the Thr222 residue, but not Thr334 (Figure 6E). In a separate experiment, we analysed the kinase activity of MK2 using an ADP-Glo kinase assay. We observed that pre-incubation of recombinant MK2 protein with AD did not affect its activity as measured by the extent of phosphorylation of HSP27 peptide substrate (Figure 6F). In contrast, PF-3644022 strongly inhibited MK2 kinase activity by targeting the ATP-binding site. This finding further supports the idea that AD binds to the pocket adjacent to Thr222 on MK2, which is far apart from its ATP-binding site. Using recombinant proteins, we also showed that the p38a-MK2 complex formation was inhibited in the presence of AD (Figure 5E). Collectively, these results confirm the binding of AD to the activation loop of MK2, which does not directly affect its activity, but rather results in p38a-MK2 complex separation and rapid degradation of MK2. The
loss of MK2 constitutes a regulatory mechanism to shut down its downstream kinase activity in cells.
Pharmacological difference between degrader and inhibitor against MK2
Traditional drug discovery approach relies on the ability of a compound to block the activity of a protein - termed protein inhibitor. An alternative to the inhibition of protein activity is the down-regulation of protein levels, which results in similar biological outcomes. This constitutes the concept of occupancy- and event-driven pharmacology (Figure 8). To understand the pharmacological difference between AD as a degrader of MK2 and PF-3644022 as an inhibitor of MK2, we performed a head-to-head comparison of the anti-inflammatory effects of AD and PF-3644022 following extended-treatment period and wash-out experiments. First, we set out to determine the recovery rates of the endogenous MK2 following treatment with AD. RAW264.7 cells were treated with the high concentration of AD (50 pM) for 4 h to induce significant reduction in MK2 level, and then were washed and incubated in compound-free medium for up to 48 h (Figure 7A). Cells were collected at indicated time points (i.e., 0, 6, 12, 24, 48 h) after compound removal and the levels of MK2 were determined. We found that, in the absence of AD, MK2 took up to 24 h to recover to the basal level of the untreated cells. This observation suggests that MK2 is a viable target for post-translational degradation approach as the protein has a slow re-synthesis rate. PF-3644022 inhibited the production of TNF-a in LPS-stimulated RAW264.7 cells with an IC50 value of 0.3 pM, which was higher in activity than AD with an IC50 value of 15 pM. It appears that, under continuous exposure to the drugs, the inhibition of MK2 kinase activity is more involved in the shut-down of downstream inflammatory responses.
Next, we established the potency profile for AD and PF-3644022 in LPS-induced RAW264.7 cells. PF-3644022 inhibited LPS-induced TNF-a production with an IC50 value of 0.2 pM, which was much higher in activity than AD (e.g., IC50 = 17 pM) (Figure 18) when cells were pre-treated for the same period of time (i.e., 1 h). Strikingly, we found that the potency of AD increased as the pre-treatment time increased (Figure 7B). A 24-hour pre-treatment resulted in a nearly 8-fold increase in IC50 value (i.e., 2.5 pM) as compared to the 1-hour pre-treatment regimen (i.e., 15 pM), demonstrating a timedependent characteristic. This could be explained by the increased loss of MK2 when cells are exposed to AD for longer time, which translates to higher degrees of TNF-a release inhibition. In contrast, PF-3644022 showed comparable potency irrespective of
the pre-treatment duration, characteristic of a non-covalent inhibitor. This finding suggests that although the potency of AD is seemingly lower than PF-3644022, its timedependent attribute allows lower concentrations to be used to achieve similar magnitude of effects as a competitive inhibitor when time permits, minimizing the potential off- target side effects.
A compound's effect is often linked to its concentration at the site of action. When a compound is cleared, its effects will be waned off. To determine the duration of action of AD and PF-3644022, we examined the effect of wash-out on their anti-inflammatory properties. RAW264.7 cells were pre-treated with 2 times higher concentrations than the IC50 of each compound for 24 h. The cells were then washed and incubated in compound-free medium for various duration (i.e., 0, 6, 12, 24 h). After 24-hour treatment, PF-3644022 strongly blocked the induced TNF-a release; however, the cells resumed rapid cytokine production immediately following compound removal (Figure 7C). In contrast, AD continued to inhibit TNF-a release even after the compound was removed from the medium for up to 24 h. These results clearly demonstrate the advantages gained from MK2 degradation compared with kinase inhibition with regard to the prolonged duration of action.
Discussion
The p38MAPK pathway has been at the center of interest for anti-inflammatory drug discovery for many years. MK2 is a serine-threonine kinase downstream to p38a and is activated directly through phosphorylation of p38a under stress and inflammatory stimuli. Studies have delineated the role of MK2 as an important post-transcriptional regulator of genes encoding pro-inflammatory cytokines, chemokines and protooncogenes. Disruption of MK2 signalling leads to a significant reduction in the level of pro-inflammatory mediators as evident from various knock-out studies. For these reasons, MK2 has been identified as a promising alternative molecular target to p38a in order to block the pathway activation and downstream inflammatory responses. The assumption is that this approach would show similar efficacy as that of p38a inhibitors, with lesser toxicity concerns that are associated with the inhibition of other beneficial p38 substrates.
We showed that MK2 is highly expressed in macrophages and is responsible for the production of various inflammatory cytokines and chemokines. An important
downstream target of MK2 is TTP, an RBP that functions essentially as an antiinflammatory protein by limiting cytokine production from macrophages upon exposure to endotoxin and other inflammatory stimuli. TTP targets inflammation-associated mRNAs for degradation via a process that involves shortening of the poly(A) tail, deadenylation, and is indispensable for the resolution of inflammation. Activation of MK2 disables the mRNA destabilizing activity of TTP, leading to excessive cytokine production and aberrant immune responses.
Given the importance of MK2 in many human diseases, efforts have been spent looking for effective kinase inhibitors against MK2. Herein, we discover a small molecule natural product - andrographolide (AD) - that, instead of inhibiting the kinase activity of MK2, targets MK2 for degradation, thereby blocking its downstream inflammatory outcomes. The reduced MK2 levels upon treatment with AD were observed in various macrophage models both in vitro and in vivo and linked to the anti-inflammatory action of AD. NF- KB inhibition has been proposed as the prevailing mechanism responsible for antiinflammatory action of AD; however, we show here that MK2 down-regulation is more involved in this behavior. When tested in vivo, AD specifically reduced MK2 levels in AMs, but not in total lung tissue. AD blocked the induced production of key cytokine TNF-a, and chemokines MCP-1, MIP-2 from activated AMs, and attenuated the development of LPS-induced ALI in mice. Mechanistically, the loss of MK2 induced by AD allows TTP to take actions in destabilizing its target mRNAs and dampen inflammation. This constitutes the downstream part of the anti-inflammatory mechanism of AD by harnessing the mRNA destabilizing activity of TTP. In fact, we showed that the anti-inflammatory property of AD is partially dependent on TTP as excessive TNF-a release is not modulated by AD-induced MK2 deficiency in the absence of TTP (Figure 3E and F).
We attempted to delineate the mechanism by which AD engendered MK2 downregulation in macrophages. On the basis of computational docking, CETSA, and sitespecific phosphorylation experiments, we found that AD is likely to bind to the activation loop of MK2 which contains a phosphorylation site Thr222. This region is located at the interface between MK2 and p38a and is essential for the complex assembly. Such binding of AD appears to disrupt the p38a-MK2 complex, freeing both p38a and MK2. Unlike p38a that is stable independently of the complex status, free MK2 is rapidly degraded by the proteasome, thus explaining the shortened half-life of MK2 in the
presence of AD. It has been reported that, in resting non-immune cells, p38a forms a physiological complex with MK2, which is located primarily in the nucleus. However, we observed that, in macrophages, MK2 is ubiquitously present in both the cytoplasmic and nuclear compartment of the cells (Figure 19A). This explains the effects of AD on MK2 degradation even in resting macrophages (Figure 2A). It should also be noted that, although AD degrades MK2, it is observed to activate p38a at higher concentration (Figure 19B). p38MAPK pathway has both pro- and anti-inflammatory functions, depending on the downstream substrates that are activated. By targeting the pro- inflammatory side of p38MAPK pathway via MK2 degradation, AD potentially spares the anti-inflammatory side of p38MAPK activation, further contributing to its ability to attenuate inflammation.
The MK2 pathway is often targeted therapeutically by kinase inhibitors (e.g., PF3644022) which operate via an occupancy-driven pharmacology model. Much recently, it is reported an additional mode p38a-MK2 pathway regulation by specifically blocking p38a activation of MK2, which was shown to be efficacious in inhibiting inflammatory diseases in pre-clinical and clinical studies. Occupancy-driven pharmacology is predicated upon blocking protein functions via inhibition, i.e., by applying high concentrations of inhibitor (Figure 8). Although this strategy has been proven to be very successful, high systemic exposures are typically required to maintain sufficient target engagement in vivo, thus increasing the risk of undesired off-target side effects. Event-driven pharmacology offers an alternative approach that, upon drug binding, the target protein is tagged for elimination, allowing for similar cellular outcomes as inhibitors. To examine pharmacological difference of AD as a MK2 degrader, and PF-3644022 as a kinase inhibitor, we conducted a head-to-head comparison on their anti-inflammatory effects in LPS-stimulated macrophages. Shortterm treatment with AD showed long-lasting suppression of TNF-a release, whereas PF- 3644022 lost its efficacy after compound removal. These results illustrate the advantages of MK2 degradation, which results in more sustained effects than the kinase inhibition. In the latter, high concentrations of the inhibitors should be maintained to ensure active-site occupancy and to sustain the inhibitory effect. In contract, degraders reduce the target protein level and as such, cellular outcome is halted until sufficient amounts of MK2 accumulate in the cells by de novo synthesis, leading to prolonged duration of action.
The current MK2 inhibitors suffer major limitations due to their poor drug-like properties such as scarce solubility, low cell permeation, kinase selectivity, as well as biochemical efficiency; hence, their clinical development is still limited. In contrast, AD and A paniculata extracts have been validated to be effective in treating various inflammatory human diseases in the clinics. The compound displays good drug-like properties. Our delineation of the anti-inflammatory action of AD via MK2 degradation not only provides an additional mode for modulating the p38MAPK-MK2 axis, but also presents the first example of small molecule MK2 degrader. This discovery paves the way for the development of novel anti-inflammatory agents targeting MK2 for degradation by harnessing the privileged scaffold of AD.
Development of Labdane based compounds as anti-inflammatory drugs
Design of target compound library based on the ent-labdane andrograoholide (AD)
enf-iabdane scaffold Andrographolide (AD)
eoxy andrographolide derivatives Our study shows that the anti-inflammatory activity of C-12 andrographolide derivatives depends on the leaving group ability of substituents at the C-12 position. Hence, a selection of andrographolide derivatives carrying various leaving groups at C-12 position (1 - 16) were designed and synthesized (Table 1). pKaH of the conjugate acid is the most predictive tool of leaving group ability. Hence, the choice of C-12 substituents
were made to cover as broad the pKaH range as possible within synthetic feasibility. Compound 16, representing a non-electrophilic system, was also included to examine the importance of the A13'14 double bond for chemical reactivity and cellular activity.
pKa values of R-H cannot be predicted
Synthesis of the design compounds
Where possible, the target compounds were prepared as 3,19-acetonide intermediates before an acid-catalysed deprotection to yield the final products. The key intermediate, 3,19-isopropylidene andrographolide (18), was synthesized following a literature reported procedure by refluxing andrographolide with 2, 2, -dimethoxy-propane in catalytic amount of PPTS (Scheme 1). Treatment of 18 with PDC in CH2CI2 led to rearrangement of the C-14 hydroxyl group and formation of 12-hydroxy-14- dehydroandrographolide intermediate 19 in nearly quantitative yield. Esterification of 19 with acid anhydride furnished acetyl intermediate 20. Reaction of andrographolide with aqueous bisulfite solution under reflux facilitated a rare sulfurous nucleophilic attack and yielded derivative 14 in almost quantitative yield.
Mitsunobu reaction have been previously utilised to convert AD to various C-14 derivatives, mainly the carboxylates. During the course of reaction condition optimization, we noted that both C-14 and C-12 derivatives could be obtained from one
single Mitsunobu reaction. The C-12/C-14 product ratios depend on various factors including the acid pKaH, the bulkiness of the conjugate base, solvent polarity and reaction temperature. For example, carboxylic acids (pKaH from 3 to 5) yielded exclusively C-14 derivatives with inversion of stereochemistry as seen with 21. In contrast, a mixture of both C-14 and C-12 isomers was obtained when phenolic nucleophiles, such as phenol, methyl-4-hydroxyl-benzoate, and acetaminophen (pKaH from 8.5 to 10), were used. The C-12/C-14 ratios for phenolic nucleophiles increased when the acidity reduced (higher pKaH). Reaction with methyl-4-hydroxyl-benzoate (pKa ~ 8.6) gave less than 5% of the C-12 derivative (26), while phenol (pKa ~ 10) yielded almost 1 : 1 product mixture. When DCM was used instead of THF, the yield of C-12 derivatives increased. The reaction with methyl-4-hydroxyl-benzoate yielded approximately 30% of the C-12 product 26 in DCM, compared to less than 5% in THF. It appears that solvents play a significant role in regulating the regioselectivity of Mitsunobu reaction. The C-12/C-14 product ratios also depends on the reaction temperature. A lower temperature (e.g. 0°C) resulted in larger C-12/C-14 product ratios. Overall, it appears that nucleophiles of higher pKaH favours the formation of C-12 derivatives. Exceptions remained as Mitsunobu reaction with N-hydroxyphthalimide proceeded slowly and only C-12 isomer was obtained, despite a relatively low pKa ~ 6.3. It is possible that the bulkiness of the nucleophile may also play a significant role in determining the product ratios. Interestingly, attempts to couple with other heteroatoms, such as sulfur and nitrogen in thiophenol, thioacetic acid and 5-fluoro- uracil, resulted in no reaction. This indicates the chemo-specificity of Mitsunobu reaction for oxygen nucleophiles.
To further diversify the substituents at C-12 position, various chemical reactions were employed. It was found that sulfur and nitrogen nucleophiles readily conjugated with the Michael acceptor in 18 in a Michael addition-elimination manner when protic solvents such as MeOH, EtOH, and water, were used. The reaction was greatly accelerated and proceeded to completion when the C-14 hydroxyl in 18 was converted to an acetyl in 29/30. As opposed to Mitsunobu reaction, this condition yielded exclusively C-12 products (31 - 32, 8 - 9) and was impervious to oxygen nucleophiles.
Scheme 2 depicts the synthesis of C-12 derivatives with substituents of low leaving ability. Reaction of 18 with DPPA in the presence of DBU resulted in azide 33, which was converted to triazole 34 following a copper(I)-catalysed click chemistry with
phenylacetylene. Attempts to saturate the A12'13 double bond in 18 with NaBFU/cat. CeCh unexpectedly led to the formation of intermediate 35. Prolonged exposure of 18 to acidified methanol yielded the methoxy intermediate 36. Benzyl 13 was synthesized following 1,4-addition of benzyl magnesium bromide to the a,p-unsaturated lactone system in 18 with a catalytic amount of copper halide. The Grignard reagent was freshly prepared, and all hydroxyl groups in AD were protected prior to the reaction. Silyl protected intermediate 38 yielded a highly regioselective Grignard reaction, but not the 3,19-acetonide 37. Removal of TBS with fluoride anions (TBAF) led to isomerization of the terminal A8'17 double bond and was substituted by a milder acidic deprotection using p-TsOH in aqueous AcOH to afford the final product 13. Hydrogenation of 10 yielded compound 16 in almost quantitative yield. All acetonide intermediates were finally deprotected in acidic environment to yield the corresponding test compounds 1 - 15.
Scheme 1. Synthetic scheme of the designed C-12 andrographolide derivatives. Reagents and conditions: (a) 2,2-dimethoxy-propane, cat. PPTS, DCM, reflux, 4 h; (b) PDC, DCM, RT, overnight; (c) Ac?O, cat. DMAP, DCM, 0°C - RT; (d) corresponding pronucleophiles, PPh3, DIAD, THF/DCM, 0°C - RT; (e) AcOH/THF/H2O (v/v 7/1/1), RT, overnight; (f) corresponding nucleophiles, EtOH, RT, overnight; (g) Na2S2O3, H2SCU, RT, overnight.
Scheme 2. Synthetic scheme of the designed C-12 andrographolide derivatives. Reagents and conditions: (a) DPPA, DMF, reflux; (b) sodium ascorbate, phenylacetylene, EtOH, H2O, RT; (c) NaBH4, MeOH, 0°C - RT; (d) cat. p-Ts RT, overnight; (e) TBSOTf, 2,6-lutidine, DCM, 0°C - RT; (f) PhMgBr, THF, 0
Reactivity and biological evaluation of the synthesized C-12 derivatives Evaluation of the intrinsic thiol reactivity
The synthesized C-12 derivatives were first evaluated for thiol reactivity to identify and filter out highly reactive molecules prior to biological evaluation. Representative C-12 derivatives carrying substituents of various leaving potential were selected for the screening. Figure 9 summarizes the rates of GSH addition to various C-12 compounds at equimolar concentrations. It was observed that the thiol reactivity of C-12 derivatives followed the trend in leaving group ability, in which functional groups with lower pKaH showed much faster rate of reaction. Acetyl 1 appears to be the most reactive compound in C-12 series, with slightly higher reactivity as compared to AD. When pKaH values of the C-12 substituents were greater than 10, minimal reaction was observed as seen with 10 and 12. Derivatives with no leaving functional groups e.g. 13, 14 and 15) showed no apparent interaction with GSH. The generally low thiol reactivity of C-12 derivatives suggests that these compounds are less likely to bind indiscrimately to off- target bio-nucleophiles, and thus exhibit lesser side effects, as opposed to AD.
Anti-inflammatory activity and cytotoxicity screening
The synthesized C-12 derivatives were next evaluated for inhibitory effects against LPS- induced TNF-a and IL-6 production in an in vitro model of inflammation. Bacterial LPS was used to stimulate the production of pro-inflammatory cytokines in RAW 264.7 macrophages and the anti-inflammatory effects was measured as percentage of cytokine inhibition compared to vehicle-treated groups. Figure 10A-B summarizes the inhibitory effects of various test compounds on TNF-a and IL-6 release after 4h and 24h LPS stimulation, respectively. Overall, it can be seen that only thiol-reactive C-12 derivatives as determined from the above chemical assay show significant antiinflammatory effects. Compounds 13, 14 and 15 carrying non-leaving functional groups were inactive against both cytokines. A loss of activity was also observed when the endocyclic Michael acceptor double bond was saturated in 16. These findings suggest that both the Michael acceptor system and the leaving functional group at C-12 are necessary for activity of C-12 andrographolide derivatives. From these observations, it would appear that the mechanism of action of these compounds may involve covalent chemistry, possibly in a Michael addition-elimination manner similar to the mechanism Of AD.
The cytokine inhibitory effects appear to follow the trend in leaving group potential, as C-12 substituents of lower pKaH values tend to exert higher potency. It was also observed that C-12 derivatives with substituents in the pKaH range from 3 - 10 were
more active than AD at inhibiting both TNF-a and IL-6, despite their lower intrinsic thiol reactivity. These observations suggest that the C-12 structure provides a better pharmacophore for anti-inflammatory action as compared to the C-14 structure in AD. For substituents of low leaving potential (pKaH > 10) such as hydroxyl in 10 and methoxy in 11, their cytokine inhibitory activities were minimal except for 12. Derivative 12 carries a phenylamino substituent that has very low leaving potential (pKaH ~ 25) yet displays relatively high potency, comparable to that AD. This observation suggests that the anti-inflammatory effects of C-12 derivatives are not governed solely by their chemical reactivity, but also by the physiochemical properties of substituents at C-12 position. Looking at the SAR trend, it was noted that C-12 derivatives bearing aromatic substituents such as phenyl-mercapto (-S-Ph) in 2, phenyl-hydroxy (-O-Ph) in 4, 5 and 7 and phenyl-amino (-NH-Ph) in 12 exhibited much stronger inhibitory activity as compared to the non-aromatic compounds, with the exception of the phenyl-methyl (-CH2-Ph) 13. Changing the connecting heteroatoms (S, 0, N) to a carbon in 13 completely diminished the activity, despite minimal change in the structural conformation of these compounds. It appears that the antiinflammatory actions of C-12 derivatives depend on multiple factors including: (1) a privileged ent-labdane structure with appropriate substituents at C-12 positions, in which aromatic substituents seem to confer best target recognition, (2) a Michael acceptor system and (3) a C-12 substituent capable of leaving under nucleophilic attack, if any, to ensure prolonged target engagement via a covalent attachment. The necessity for these structural features highlights the potential of C-12 derivatives to act as targeted covalent inhibitors (TCIs), in which both non-covalent (target recognition) and covalent (target engagement) binding steps are essential for the activity of the inhibitors.
The synthesized C-12 derivatives were also evaluated for cytotoxicity in non-LPS- stimulated RAW 264.7 cells (Figure IOC). Most test compounds showed lower cytotoxicity as compared to AD, except for 2. Overall, the C-12 derivatives display more favorable therapeutic profiles and thus would be more attractive lead compounds for anti-inflammatory drug development based on the ent-labdane andrographolide (AD).
Effects of C-12 andrograoholide derivatives on LP5-induced cytokine mRNA expression To understand the mechanism by which C-12 derivatives suppress pro-inflammatory cytokine production, selected compounds were next evaluated for their inhibitory effects on LPS-induced cytokine mRNA expression levels. Specifically, RAW264.7 macrophages
were pre-treated with test compounds (at 10 mM) for 1 h followed by LPS stimulation for another 4 h. The cytokine mRNA expression levels were quantified using qRT-PCR against the respective primer pairs. As shown in Figure 11, treatment with active C-12 derivatives effectively reduced the mRNA expression levels of TNF-a and IL-6. This observation suggests that C-12 andrographolide derivatives blocked pro-inflammatory cytokine production by reducing their mRNA expression levels, possibly via transcriptional or post-trancriptional control. C-12 derivatives with aromatic substituents (e.g. 2, 4, 5, 7 and 12) displayed much higher potency as compared to the non-aromatic compounds. When comparing the activity of 2, 7, 12 and 13, it appears that an aromatic sulfur substituent is preferable for high potency followed by amino > oxygen >>> carbon. Derivative 13, 14 and 15 carrying non-leaving substituents showed no observable effects. Amongst the test compounds, derivative 4 exerted highest potency with more than 90% reduction in both TNF-a and IL-6 mRNA levels as compared to LPS-control groups, consistent with its strong inhibitory effects on cytokine protein release. The para-substitution on the phenol ring in 4 and 5 appeared to contribute significantly to the anti-inflammatory activity, as 7 lacking a substituent at this position was far less potent.
Effects of the C-12 andrographolide derivatives on MK2 protein expression
The effects of various C-12 analogues on the protein expression of MK2 were next investigated. Briefly, RAW264.7 cells were treated with the test compounds at 10 pM and the MK2 protein levels were quantified using Western blot analysis. As shown in Figure 12, cells that were treated with the active C-12 analogues (/.e., 1 - 12) showed significantly lower levels of MK2 proteins as compared to the untreated control. Several key observations were made: (1) a substituent with moderate-to-good leaving group ability at the C-12 position is necessary for the activity, (2) compounds that possess aromatic substituents at the C- 12 position (/.e., 2 - 7, 12) were more active as compared to the non-aromatic compounds (/.e., 1, 8, 10) and (3) compounds bearing substituents with poor leaving group ability (/.e., 13 and 14) showed no effects on MK2 downregulation. Of the compounds tested, compound 4 is the most potent. At 10 pM, 4 induced more than 50% reduction in the protein levels of MK2. Taken together, our results showed a positive correlation between MK2 downregulation and the antiinflammatory activity of the C-12 analogues (/.e., the ability to inhibit cytokine release and their mRNA expression). This suggests that the mechanisms of the anti-
inflammatory actions of these compounds are likely to involve the downregulation of the MK2 protein.
Evaluation of in vivo efficacy of derivative 4
Derivative 4 were selected for further in vivo evaluation. As excessive cytokine production has been shown to amplify inflammation in individuals with asthma, we evaluated the anti-inflammatory effects of 4 in a HDM-induced asthmatic models. Female mice of 6-8 weeks old were stimulated with HDM for 14 days to induce asthmatic response prior to treatment with 4 at 0.1, 1 and 3 mg/kg. Figure 13 displayed the total and differential cell counts from Bronchoalveolar lavage fluid (BALF). The number of white blood cells from BALF is a reliable indicator of airway inflammatory responses. It was observed that treatment with 4 significantly alleviated HDM-induced airway inflammation at dose as low as 1 mg/kg. HDM induced significant production of eosinophils, a hallmark of severe asthma, which was effectively blocked by 4. These data suggest the potential of 4 as a safe and effective anti-inflammatory therapeutic for the treatment of severe asthma.
Conclusions
In general, substituents at the C-12 position of AD can affect their anti-inflammatory activity. Specifically, we observed that substituents with better leaving group ability and aromatic character give rise to compounds with better anti-inflammatory profiles. To the best of our knowledge, this is the first report on the C-12 derivatives of AD as MK2 degraders and as potential anti-inflammatory agents. Their ability to inhibit pro- inflammatory cytokine release at high potency offer the possibility to replace current p38 inhibitor approach, without incurring side-effects that are commonly associated with direct blocking of p38 protein. Our study identified compound 4 as potent anti- inflamamtory agent that showed excellent in vivo efficacy at low dose of lmg/kg in a severe asthmatic mouse model. The therapeutic potential of compound 4 will also be evaluated in other chronic inflammatory conditions e.g., COPD).
Experimental methods Materials
Mouse macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (Manassas, VA). Dexamethasone 21-phosphate disodium salt (>98%), andrographolide (>98%), LPS (Escherichia coli 0111 :B4), PF-3644022 hydrate (>98%),
and DMSO (>99% cell culture grade) were obtained from Sigma-Aldrich (St. Louis, MO). p50 inhibitor peptide was obtained from Novusbio (Centennial, CO). DMEM, Opti-MEM™ I Reduced Serum Medium, FBS, M-PER mammalian protein extraction reagent, and Pierce protease and phosphatase inhibitor mini tablets, Pierce IP lysis buffer came from ThermoScientific (Rockford, IL). Dynabeads Protein G, DynaMag-Spin Magnet, Bradford protein assay kit, SYBR Green qPCR MasterMix, Maxima first strand cDNA synthesis kit were purchased from ThermoFisher Scientific (Waltham, MA). Mini-PROTEAN® TGX precast protein gels were from Bio-Rad Laboratories (Singapore). Anti-MK2 (Cat# 12155, RRID: AB_2797831), anti-phospho-MK2 (Thr334, Cat# 3007, RRID: AB_490936), anti-phospho-MK2 (Thr222, Cat# 3316, RRID: AB_2141311), anti-TTP (Cat# 71632, RRID: AB_2799806), anti-KHSRP (Cat# 13398, RRID: AB_2798208), anti-p38a (Cat# 9218, RRID: AB_10694846), anti-p38 (Cat# 8690, RRID: AB_10999090), anti-phospho-p38 (Thrl80/Tyrl82, Cat# 4511, RRID: AB_2139682), anti-MKK6 (Cat# 8550, RRID: AB_11220227), anti-HSP27 (Cat# 95357, RRID: AB_2800246), anti-phospho-HSP27 (Ser82, Cat# 9709, RRID: AB_11217429), antirabbit IgG (Cat# 2729, RRID:AB_1031062) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-p-actin (HRP-conjugated, Cat# HRP-60008, RRID: AB_2819183), anti-GAPDH (HRP-conjugated, Cat# HRP-60004, RRID: AB_2737588) antibodies were from Proteintech (Rosemont, IL). Mouse anti-Siglec-F microbeads, LS-column, and MACS Separator were obtained from Miltenyi biotech (Singapore). RNAIater solution, SYBR Green qPCR MasterMix, collagenase type I, and DNase I were purchased from Life Technologies (Carlsbad, CA). Mouse recombinant proteins MK2 and p38a, and kinase assay buffer III (K03-09) were obtained from Signalchem Biotech (Richmond, BC). Mouse KC and MIP-2 DuoSet ELISA kits were generated by R8<D Systems (Minneapolis, MN). Mouse TNF-a, MCP-1 and IL-6 Mono/Mono OptEIA™ ELISA kits were generated by BD Biosciences (Franklin Lakes, NJ). MK2 ADP-Glo kinase assay kit was from Promega (Madison, WI). RNA was extracted using RNAzol RT reagent from Molecular Research Center (Cincinnati, OH), and cDNA was synthesized using Maxima first strand cDNA synthesis kit from ThermoFisher Scientific (Waltham, MA). All primer pairs were synthesized by Integrated DNA Technologies (Coralville, IA). siRNAs for MK2 (m) (CAT# sc-35856), TTP (m) (CAT# sc- 36761), and scramble siRNA (CAT# sc-374305) were purchased from Santa Cruz Biotechnology (Dallas, TX). ORF cDNA clone (CAT# MC203417) came from Origene (Rockville, MD).
Animals
Female BALB/c mice 6- to 8-week-old (InVivos, Singapore) were maintained in a 12-h light/dark cycle with food and water available ad libitum. Animal experiments were performed according to institutional guidelines of the Animal Care and Use Committee (IACUC) of the National University of Singapore.
Administration of andrographolide and LPS into mice
Andrographolide (1 mg/kg in 2% DMSO), or vehicle (2% DMSO) in 40 pL saline was administered intratracheally for the time-dependent study. 10 pg LPS in 40 pl saline was instilled intratracheally to induce ALL One hour later, andrographolide (1 mg/kg in 2% DMSO), or vehicle (2% DMSO) in 0.1 ml PBS was administered by intraperitoneal injection twice daily for 3 consecutive days. Twelve hours after the last injection, mice were sacrificed to collect lung samples for various biochemical analyses.
Bronchoalveolar lavage fluid and lung tissue harvest
Tracheotomy was performed and a cannula was inserted into the mouse trachea. Ice- cold PBS (0.5 ml 3x) was instilled into the lungs, and bronchoalveolar lavage (BAL) fluid was collected and kept at -80°C for analysis. BAL fluid total cell counts were performed blinded. Differential cell count was determined by using the BD Fortessa flow cytometer and analyzed with FlowJo software (BD, Franklin Lakes, NJ, US). Leukocytes were identified as CD45+ cells, neutrophils as CDllb+/Gr-l+ cells, macrophage as CDllc+/Siglec-F+ cells. Mouse lungs were excised. Lung lobes were snap frozen in liquid nitrogen and then stored at -80°C for protein isolation.
Lung alveolar macrophage preparations
Mouse whole lung was perfused with 5 ml PBS and minced into 2- to 3-mm pieces. Lungs were digested with DMEM medium containing collagenase type I (2 mg/ml) and DNase I (100 U/ml) and further homogenised with the gentleMACS (Miltenyi) prior to passage through a 70 pm strainer to obtain single cell suspension. Cells were counted, resuspended in MACS buffer, and incubated with mouse anti-Siglec-F microbeads for 10 minutes at 4°C. Cells were run through an LS-column on the magnetic field of a MACS Separator. The positively selected fraction was collected and plated. Alveolar macrophages were allowed to adhere for a minimum of 2 hours, washed with DMEM medium supplemented with 10% FBS, and collected for further studies or analysis.
Cell culture and treatment in vitro
The mouse macrophage cell line RAW264.7 or isolated primary mouse lung alveolar macrophages were cultured in DMEM medium supplemented with 10% FBS. All cell cultures were maintained in a humidified 37°C, 5% CO2 incubator. The cells were treated with various concentrations of andrographolide, PF-3644022, dexamethasone, p50-i, as well as LPS for indicated time points.
RNA extraction and qPCR
Total RNA was extracted from cells using RNAzol according to the manufacturer's instructions. cDNAs were synthesized using Maxima first strand cDNA synthesis kit. cDNA synthesis was performed by the Biometra gradient thermal cycler (Goettingen, Germany). qPCR was performed with SYBR Green qPCR MasterMix as a detection dye in the ABI 7900 Real-Time PCR machine and presented as fold differences over the controls by the 2-aact method. The efficiency of all used primer pairs was pre-tested. The fold change of mRNA level was adjusted to the efficiency of primer pairs used. Mouse p-actin gene was used as an endogenous control. All primers were synthesized by Integrated DNA Technologies (Coralville, IA). The primer pairs are listed as following : mouse /3-actirr. forward 5'-GTG ACG TTG ACA TCC GTA AAG A-3', reverse 5'-GCC GGA CTC ATC GTA CTC C-3' ; mouse TNF-a: forward 5'-TCT GTC TAC TGA ACT TCG GGG TGA-3', reverse 5'-TTG TCT TTG AGA TCC ATG CCG TT-3' ; mouse MCP-1 : forward 5'- TAA AAA CCT GGA TCG GAA CCA AA-3', reverse 5' -GCA TTA GCT TCA GAT TTA CGG GT- 3'; mouse MK2\ forward 5'-GTT CCC CCA GTT CCA CGT CAA G-3', reverse 5'-CTA AAG AGC TCT CCA CCA TCG-3'; mouse TTP\ forward 5'-CCA GGC TGG CTT TGA ACT CA-3', reverse 5' -ACC TGT AAC CCC AGA ACT TGG A-3' ; mouse //VOS: forward 5'-CGG GCA AAC ATC ACA TTC AGA TCC CG-3', reverse 5'-TAT ATT GCT GTG GCT CCC ATG TT-3'; mouse GM-CSF: forward 5'-GGC CTT GGA AGC ATG TAG AGG-3', reverse 5'-GGA GAA CTC GTT AGA GAC GAC TT-3'; mouse KC\ forward 5'-CTT GAA GGT GTT GCC CTC AGS', reverse 5'- GTC AGA AGC CAG CGT TCA C-3'; mouse MIP-2-. forward 5'-AAG TTT GCC TTG ACC CTG AA-3', reverse 5'-AGG CAC ATC AGG TAC GAT CC-3'; mouse MZP- 10: forward 5'-CAG CCC TGA TGC TTC TCA CT-3', reverse 5'-GGG AGA CAC GCG TCC TAT AAC-3';
Total lysate, nuclear fractionation, and immunoblotting
Cells were lysed in M-PER mammalian protein extraction reagent containing protease and phosphatase inhibitor mini tablets. The protein concentrations were determined
using Bradford protein assay. Protein extracts were separated by 10% SDS-PAGE or 4 - 15% Mini-PROTEAN® TGX precast protein gels, transferred to PVDF membranes, and probed with antibodies. Immunoblots were visualized and documented with ChemiDoc Touch Gel Imaging System (Bio-Rad Laboratories). Band intensity was quantitated using Image Lab software (Bio-Rad Laboratories J, RRID: SCR_014210). Anti-p-actin (HRP- conjugated), Anti-GAPDH (HRP-conjugated), anti-MK2, anti-phospho-MK2 (Thr334), anti-phospho-MK2 (Thr222), anti-TTP, anti-KHSRP, anti-p38a, anti-p38, anti-phospho- p38 (Thrl80/Tyrl82), anti-MKK6, anti-HSP27, anti-phospho-HSP27 (Ser82) antibodies were used for immunoblotting.
Immunoprecipitation
Total RAW264.7 cell lysates were prepared by using 200 pl of Pierce IP lysis buffer containing protease and phosphatase inhibitor. Cell lysates were pre-cleared with 20 pl Dynabeads Protein G for 30 min. After removal of the beads with DynaMag-Spin Magnet, the cell lysates were incubated with 2-pg anti-MK2, or anti-rabbit IgG antibodies overnight at 4°C, and with 20 pl Dynabeads Protein G for another 2 hours with gentle rotation. The immune complexes were pelleted with DynaMag-Spin Magnet, washed twice with 200 pl of immunoprecipitation buffer before immunoblotting.
MK2 kinase activity assay
Recombinant MK2 protein (1.6 ng, 2 pL) were pre-incubated with test compounds (1 pl) for 1 hour at room temperature. A 2 pl mixture of HSP27 peptide (0.2 pg/pl) and ATP (50 pM) was then added, and the resulting mixtures (5 pL in total) were incubated for another 1 hour. Total MK2 activities were assayed using an ADP-Glo Kinase Assay according to the manufacturer's protocol. The raw data were normalized to the untreated control. Percent MK2 activity was calculated using the normalized values.
Cellular thermal shift assay (CETSA)
RAW264.7 cells were lysed in M-PER mammalian protein extraction reagent containing protease and phosphatase inhibitors at 3,000,000 cells/100 pl lysis buffer. Cell lysates were spiked with test compounds to the final concentration of 50 pM or with DMSO as vehicle control. After incubating at room temperature for 1 hour, the spiked lysates were aliquoted equally for gradient heat treatment in a 96-well thermocycler (Applied Biosystems) for 3 min. The denatured proteins were removed by centrifugation at
20,000 g for 20 min at 4°C, and the supernatant containing soluble fractions of proteins was collected for immunoblotting. siRNA and cDNA transfection
RAW264.7 cells were plated at a density of 5 x 103 cells per well in 96-well plates or 1 x 105 cells per well in 6-well plates. Oligomer-lipofectamine complexes were prepared by mixing Lipofectamine 2000 (4 pl in 200 pl of Opti-MEM I Reduced Serum Medium, pre-incubated for 15 min) with siRNA (100 pmol in 200 pl of Opti-MEM I Reduced Serum Medium) or cDNA (1 pg in 200 pl of Opti-MEM I Reduced Serum Medium) and incubating for 20 min in the dark. Cells were washed once with Opti-MEM and the oligomer- lipofectamine complexes were added dropwise to cells in Opti-MEM to the final concentration of 100 nM siRNA in a total volume of 50 pl in 96-well plates and 1 ml in 6-well plates. The transfection was allowed for 6 hours before the medium was replaced with DMEM supplemented with 10% FBS. Cells were harvested 48 hours after transfection for studies.
Phosphorylation of MK2 by p38a
Phosphorylation of MK2 by p38a in the absence or presence of AD was performed by first pre-incubating recombinant MK2 protein (0.5 pg) with AD for 10 mins at room temperature prior to the addition of recombinant p38a protein (0.2 pg). The reaction was started by addition of ATP to the final concentration of 150 pM. The phosphorylation was carried out in kinase assay buffer III (K03-09) at the final volume of 125 pl. Aliquots were taken at 0.5-, 5-, 15-, 30-, and 45-min time intervals and quenched with SDS sample buffer. Immunoblotting was performed using anti-phospho-MK2 antibodies.
Computer Docking
The crystal structure of MK2 (PDB ID: 2OZA, chain A) was used for docking with AD using iGEMDOCK v.2.1. The 3D structure of AD was generated using Spartan'20 v.1.1. "Accurate docking" was used, with a population size of 800 and generations of 80. The number of solutions was increased to 100 for more reliable clustering. This was a blind docking without specifying the locations of the binding site or the interacting residues.
Data are presented as mean ± SEM. Significant difference between different groups was determined by one-way ANOVA followed by Dunnett's test. All statistical analyses were conducted with GraphPad Prism software.
It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is/are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Claims
1. A method of promoting the ubiquitination of MK2 protein, comprising contacting the MK2 protein with a compound of Formula (I) or a salt, solvate, stereoisomer or prodrug thereof:
wherein
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and s is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
2. The method according to claim 1, wherein Ri is selected from optionally substituted acyloxy, optionally substituted arylthio, optionally substituted heteroarylthio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino.
4. The method according to any one of claims 1 to 3, wherein the MK2 protein is characterised by a half-life of less than 4 h.
5. The method according to any one of claims 1 to 4, wherein the method is an in vivo method or an in vitro method.
6. The method according to any one of claims 1 to 5, wherein, the compound of Formula (I) is a compound of Formula (Ic):
wherein
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and 3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
7. The method according to any one of claims 1 to 6, wherein the compound of Formula (I) is a compound of Formula (Id):
wherein
X is a heteroatom; and
F is selected from optionally substituted acyl, optionally substituted aryl, and optionally substituted heteroaryl;
F is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
Fb is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
8. The method according to claim 7, wherein X is selected from 0, S, N.
9. The method according to claim 7 or 8, wherein FU is selected from optionally substituted phenyl and optionally substituted 6 membered heteroaryl.
10. The method according to any one of claims 7 to 9, wherein FU is selected from phenyl optionally substituted at a para position and 6 membered heteroaryl optionally substituted at a para position.
11. The method according to any one of claims 1 to 6, wherein the compound of Formula (I) is a compound of Formula (le):
R3 de) wherein
Het is an optionally substituted heteroaryl;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
12. The method according to claim 11, wherein the heteroatom on the heteroaryl is bonded to C-12 position of Formula (I).
13. The method according to claim 12 or 13, wherein the heteroaryl is selected from oxazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolinyl, quinolinyl, phthalazinyl, naphthylpyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, acridinyl, phenanthrolinyl, isothiazolyl, phenazinyl, isoxazolyl, isothiazolyl, phenoxazinyl, phenothiazinyl, thiazolyl, thiadiazolyl, oxadiazolyl, oxatriazolyl, tetrazolyl, thiophenyl, benzothiophenyl, triazolyl, and imidazopyridinyl.
14. The method according to any one of claims 1 to 6, wherein the compound of Formula (I) is a compound of Formula (Ic1):
wherein
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino; 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
15. The method according to any one of claims 1 to 6, wherein the compound of Formula (I) is a compound of Formula (Ic"):
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy,
optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
17. The method according to any one of claims 1 to 16, wherein the compound is selected from one of the following:
X is a heteroatom selected from N, 0, S; 4 is selected from optionally substituted aryl, optionally substituted heteroaryl; or X- 4 together is optionally substituted acyloxy;
Het is a optionally substituted heteroaryl, wherein the heteroatom on the heteroaryl is bonded to the C-12 position of Formula (I); 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and 3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
20. A pharmaceutical composition comprising a compound of Formula (I) or a
pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient:
wherein
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino; 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
21. A method of treating of a disease or condition associated with MK2 protein, comprising administrating to a subject in need thereof a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable a salt, solvate, stereoisomer or prodrug thereof:
wherein
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino; 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
22. A compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for use in treating a disease or condition associated with MK2 protein:
wherein
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally
substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
23. A use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament for the treatment of a disease or condition associated with MK2 protein:
wherein
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino,
optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
24. The method, compound or use according to any one of claims 21 to 23, wherein the treatment of a disease or condition associated with MK2 protein comprises ubiquitination of MK2 protein.
25. The method, compound or use according to any one of claims 21 to 24, wherein the disease or condition associated with MK2 protein is an inflammatory disease or condition.
26. The method, compound or use according to any one of claims 21 to 24, wherein the disease or condition associated with MK2 protein is a chronic respiratory disease or condition.
27. The method, compound or use according to any one of claims 21 to 26, wherein the disease or condition associated with MK2 protein is selected from acute lung injury, chronic inflammation in asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease, atherosclerosis, Alzheimer's disease, ischemic heart, brain disease, cancer, COPD, lung fibrosis, pneumonia, long COVID.
28. A method of inducing an anti-inflammatory response in a subject in need thereof, comprising administrating to a subject in need thereof a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable a salt, solvate, stereoisomer or prodrug thereof:
wherein
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy,
optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
29. A compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof for inducing an anti-inflammatory response in a subject
wherein
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino;
R2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally
substituted aminoacyloxy, and optionally substituted thio.
30. A use of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof in a manufacture of a medicament for inducing an anti-inflammatory response in a subject in need thereof:
Ri is selected from optionally substituted acyloxy, optionally substituted thio, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroaryloxy, and optionally substituted heteroarylamino; 2 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio; and
R3 is selected from H, halogen, hydroxyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted acyliminoxy, optionally substituted ocyacyloxy, optionally substituted aminoacyloxy, and optionally substituted thio.
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US20080139622A1 (en) * | 2006-12-11 | 2008-06-12 | Hutchison Medipharma Limited | Decahydronaphthalene Compounds |
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DATABASE REGISTRY ANONYMOUS : "- 2(5H)-Furanone, 3-[2-[(1R,4aS,5R,6R,8aS)-decahydro-6-hydroxy-5- (hydroxymethyl)-5,8a-dimethyl-2-methylene-1-naphthalenyl]-1-(2- thienylthio)ethyl]-, rel- (CA INDEX NAME)", XP093135928, retrieved from STN * |
GUTIERREZ-PRAT NURIA, CUBILLOS-ROJAS MONICA, CÁNOVAS BEGOÑA, KUZMANIC ANTONIJA, GUPTA JALAJ, IGEA ANA, LLONCH ELISABET, GAESTEL MA: "MK2 degradation as a sensor of signal intensity that controls stress-induced cell fate", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 118, no. 29, 20 July 2021 (2021-07-20), XP093135929, ISSN: 0027-8424, DOI: 10.1073/pnas.2024562118 * |
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