WO2019180664A1 - Procédé de prévention ou de modulation de la fibrose et de la réponse fibreuse associée à la réponse intégrée au stress - Google Patents

Procédé de prévention ou de modulation de la fibrose et de la réponse fibreuse associée à la réponse intégrée au stress Download PDF

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WO2019180664A1
WO2019180664A1 PCT/IB2019/052321 IB2019052321W WO2019180664A1 WO 2019180664 A1 WO2019180664 A1 WO 2019180664A1 IB 2019052321 W IB2019052321 W IB 2019052321W WO 2019180664 A1 WO2019180664 A1 WO 2019180664A1
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sox9
substituted
unsubstituted
eif2oc
atf4
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Song Eng Kathryn CHEAH
Danny Chan
Cheng Wang
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The University Of Hong Kong
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • This invention relates to a method of preventing, ameliorating and/or treating fibrosis or fibrotic response associated with the integrated stress response arising from cellular stresses involving the phosphorylated eukaryotic initiation factor 2a (p-eIF2a) pathway.
  • the present invention provides a method which prevents or alleviates aberrant cell differentiation associated with the p-eIF2a pathway in various organs and tissues, where the aberrant cell differentiation causes a fibrosis or fibrotic response.
  • ISR Integrated Stress Response
  • ER intrinsic endoplasmic reticulum
  • ISR is primarily a pro-survival homeostatic program aiming to optimize the cellular adaptive response to stress, exposure to severe stress, either in intensity or in duration, will overwhelm the capacity of this adaptive response and drive signaling toward cell death.
  • the general translation initiation factor eIF2 is a major controller of protein synthesis at the level of translation.
  • eIF2 is a trimeric complex of a, b and g subunits that binds to both GTP and the initiator methionyl tRNA (Met-tRNA;) to form a ternary complex (cIF2*GTP*Mct- tRNAi).
  • eIF2B is a complex composed of five different subunits, eIF2B 1 , eIF2B2, eIF2B3, eIF2B4, eIF2B5 (also called the a, b, g, d, and e subunits).
  • eIF2B5 catalyzes the GDP/GTP exchange reaction and with eIF2B3, forms the‘catalytic core’.
  • eIF2B complex normally works to activate eIF2 via phosphorylation of eIF2 serine-51 (Ser-51).
  • Ser-51 phosphorylated on Ser-51
  • eIF2a-P dissociates from the eIF2B regulatory sub-complex and eIF2B is inactive.
  • Phosphorylated eIF2 blocks protein synthesis thus phosphorylation renders eIF2 an inhibitor of its own guanine nucleotide exchange factor (GEF).
  • GEF eIF2B catalyses release of eIF5 and GDP.
  • the core event in ISR is the phosphorylation of the alpha subunit of eIF2 (eIF2a) by one of four members of eIF2a kinase family: general control nonderepressible 2 (GCN2), protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK) and Fleme-regulated eIF2a kinase (F1RI) (2, 6, 9, 12, 84).
  • GCN22 general control nonderepressible 2
  • PLR protein kinase R
  • PERK PKR-like endoplasmic reticulum kinase
  • Fleme-regulated eIF2a kinase (F1RI) Fleme-regulated eIF2a kinase
  • ATF4 is the main effector of the ISR. It forms homodimers and heterodimers that bind to DNA targets to control the expression of genes involved in cellular adaptation, including cell death, cell survival or cell differentiation. It is likely that the duration and level of eIF2a phosphorylation, as well as ATF4 regulation, determine the balance between cell survival and cell death.
  • ECM extracellular matrix
  • integrins extracellular matrix
  • genes encoding collagen I cause osteogenesis imperfecta (OI) (13); and mutations-in fibrillins cause Marfan syndrome.
  • OI osteogenesis imperfecta
  • Many mutations in ECM genes result in misfolded proteins which can trigger the unfolded protein response as a result of the ER stress (22).
  • ER endoplasmic reticulum
  • URR adaptive unfolded protein response
  • Targeting the UPR is therefore a strategy for the treatment of disorders associated with ER stress.
  • type Schmid MCDS
  • CBZ carbamazepine
  • UPR Ultrastained activation of UPR has been implicated in the progression of a variety of diseases, including cancer, diabetes, inflammatory disease and neurodegenerative disorders (16). In the past few years, UPR has become an attractive target for drug discovery.
  • UPR Upon ER stress, UPR activates three independent ER stress sensors: inositol-requiring 1 (IRE1), PKR-like ER kinase (PERK), and membrane-tethered activating transcription factor 6 (ATF6) (17).
  • IRE1 inositol-requiring 1
  • PERK PKR-like ER kinase
  • ATF6 membrane-tethered activating transcription factor 6
  • ISR integrated stress response
  • Activation of the ISR and PERK signaling pathway is implicated in many diseases including cancer, diabetes, obesity, neurodegeneration and skeletal disorders (18, 71, 85).
  • the ISR has a central role in many forms of cellular stress, such as oxidative stress, hypoxia, ER stress, and its induction is associated with diverse common diseases, such as cancer, diabetes, lung disease, obesity, neurodegeneration and skeletal disorders and the associated induced fibrosis (26, 29). Recently, ISR has been implicated in intervertebral disc degeneration (19, 20), which is very common in humans and often causes low back pain (LBP).
  • LBP low back pain
  • SOX9 Sex-Determining Region Y-Box 9
  • Sox9 ATF4 directly transactivated Sex-Determining Region Y-Box 9
  • SOX9 is a potent transcription factor with key roles in cell fate determination in many cell types, not only in chondrocytes, but also in many other cell types, notably stem cells (dermal papilla, gonads, intestinal, neural etc.) (21) and is activated in acquired diseases such as cancer, obesity and fibrosis (22).
  • the present invention demonstrated for the first time a direct linkage between SOX9 and ISR, which may have broad implications for various diseases.
  • the eIF2a phosphorylation can be 1) either stimulated through chemical activators of eIF2a kinases by, for example, histidinol, asparaginase, halofuginone, arginine deiminase, BTdCPU, BEPP monohydrochloride and CCT020312, or prevented by indirubin-3’ -monoxime, SP600125, SyK, GSK260641, GSK2656157, C16, 2-aminopurine and aminopyrazolindane; 2) modulated via inhibiting eIF2a phosphatases, by, for example, guanabenz and Sephinl, to block GADD34, or nelfinavir to decrease CReP expression and disrupt binding of CReP-PPl complex to eIF2a; and most importantly, 3) reversing the consequences of eIF2a phosphorylation using Integrated Stress Response Inhibi
  • This invention relates to methods of preventing, ameliorating and/or treating fibrosis or fibrotic response associated with the integrated stress response involving eukaryotic Initiation Factor 2 (eIF2), phosphorylated eukaryotic initiation factor 2a (p-eIF2a) and PKR-like endoplasmic reticulum kinase (PERK) pathway arising from various cellular stresses, such as oxidative stress, hypoxia, ER stress, chronic or prolonged bio-mechanical stress and others.
  • eIF2 eukaryotic Initiation Factor 2
  • p-eIF2a phosphorylated eukaryotic initiation factor 2a
  • PERK PKR-like endoplasmic reticulum kinase pathway arising from various cellular stresses, such as oxidative stress, hypoxia, ER stress, chronic or prolonged bio-mechanical stress and others.
  • the present invention provides a method which prevents or alleviates aberrant cell differentiation caused by the activation of the integrated stress response and/or associated with the p-eIF2a and PERK pathway in various organs and tissues, and thereby prevents, ameliorates and/or treats fibrosis or fibrotic response.
  • the present invention provides a method of manipulating the inhibitory effects of p-eIF2a using a p-eIF2a modulator or a phosphorylated eukaryotic initiation factor 2b modulator (p-eIF2 -modulator) for the prevention, amelioration and/or treatment of fibrosis or fibrotic responses described herein.
  • FIG. 1 shows a schematic of the PERK signaling pathway in eukaryotes.
  • PERK is one of the kinases that phosphorylates eIF2a.
  • p-eIF2a acts to suppress global protein synthesis by suppressing Cap-dependent translation initiation while activating preferential translation of ISR-specific mRNAs, such as for transcription factor ATF4 and CHOP.
  • ATF4 in turn activates transcription of additional genes involved in cellular adaptation, including cell death, cell survival or cell differentiation.
  • Figures 2A-2Q show that small molecule IS RIB ameliorates skeletal deformities in l3del mice by preventing ATF4 induction under ER stress.
  • Figure 2A is a schematic timeline of the ISRIB (2.5 mg/kg) or vehicle (0.5% DMSO in 0.9% saline) administration in mice. ISRIB was administered by daily intraperitoneal injection starting on E13.5 and ending on p20. The animals were harvested at indicated time points.
  • Figures 2B and 2C demonstrate that treatment with ISRIB did not affect the body weight gain or body length growth in wild type mice.
  • Figures 2E and 2F show the radiographic analyses of WT and l3del mice demonstrating that skeletal deformities of l3del mice were alleviated at p20 by ISRIB treatment.
  • These alleviated skeletal deformities include the length of tibia, femur and spine (spine length here is measured by the length of 7 continuous vertebrae consisting of the last sacral vertebra and six tail vertebrae); pelvic bone deformation (Q1: the angle between ilium and pubis); Coxa Vara ( Q2 : the angle between the proximal head and the shaft of the femur) and Genu Varum ( Q3 : the angle between proximal head and distal head of tibia).
  • Figures 2G and 2H show the rescue of growth plate abnormalities in l3del mice by treatment with ISRIB at plO and p20, respectively, as demonstrated by histology (a-a”) and in vivo expression profiles of SOX9 (b-b” and c-c”), Col2al (d- d”) and Ppr (e-e”).
  • Figures 21 and 2J show the hypertrophic zone (HZ) length measurement and Sox9 + , Col2al + and Ppr + cells quantification in tested animals at indicated time points.
  • Figure 2K suggests that histology of plO growth plates was comparable between ISRIB-treated and vehicle-treated WT mice.
  • Figures 2L and 2M show the alleviation of the HZ expansion in caudal intervertebral disc (IVD) by ISRIB in l3del mice at plO and p20.
  • Figures 2N-2P show the results of in situ hybridization assays for different genes:
  • Figure 2N shows the alleviation of the growth plate deformities in caudal IVD by ISRIB in l3del mice as indicated by reduced number of the Sox9 (a-a”, c-c”) and the Col2al (b-b”, d-d”) expressing cells in the lower HZ at plO and p20.
  • Figure 20 shows that at plO, the transcripts of Atf4 ' (a-a”), Atf3 (b-b”), Chop (c-c”), Eroll (d-d”) and Fgf21 (e-e”) were down-regulated in HZ of ISRIB-treated l3del mice, while Rip (f-f”) was not affected.
  • Figure 2P shows that at plO, the protein level of ATF4 (a-b”), ATF3 (c-d”), CHOP (e-f”) and FGF21 (g-h”) were down-regulated in HZ of ISRIB- treated l3del mice.
  • Figure 2Q shows by TUNEL assays that ISRIB treatment did not induce apoptosis in l3del mice.
  • Figures 3A-3M show degenerative intervertebral disc (IVD) features in a human patient with metaphyseal chondrodysplasia, type Schmid (MCDS) and l3del mice.
  • Figure 3A shows the radiographic analysis that revealed early onset of Intervertebral Disc Degeneration (IDD) in a 20- years-old MCDS patient.
  • IDD Intervertebral Disc Degeneration
  • Figure 3B shows the FAST staining revealing swelling of the nucleus pulposus (middle arrow), endplate expansion (upper and lower dashed arrows) and accumulation of chondrocyte -like cells in the inner annulus fibrosus (iAF) in 13del mice (circled region) at 4 weeks (mouse age of 4 weeks is regarded as equivalent to human age of 14 years).
  • Figure 3C shows the radiographic analysis revealing severe intervertebral disc degeneration in tail region (T5/6, T6/7 and T7/8) in 7-, 9-, 12- and 16-month old 13del mice.
  • Figure 3D shows histological analysis demonstrating that the tail intervertebral discs (IVD) of 6-month old (upper panel) and 16-month old (lower panel) 13del mice exhibited significant characteristics of disc degeneration, including loss of nucleus pulposus/annulus fibrosus (NP/AF) boundary, disc bulging and widening of the AF interlamellar space.
  • the circled region clearly shows the inward bulging of inner AF (iAF) lamellae and significantly decreased volume of vascular canals in subchondral region between spinal growth plate and endplate in 6-month old 13del mice.
  • the 13del disc clearly exhibited (a) the altered NP structure and matrix, (b) the inward bulging of AF lamellae and the consequent fissure (boxed regions).
  • Figure 3E shows that excessive cell death (by TUNEL assay) in NP of the degenerated tail IVD of l6-month l3del mice compared to WT mice.
  • Figure 3F shows that the essential ER stress sensor BIP was ectopically upregulated in the core region of l3del NP at 6-month stage both transcriptionally (Rip, upper panel) and translationally (BIP, lower panel).
  • Figures 3G and 3H show that significant upregulation of p- eIF2oc, the most upstream event in ISR, was only observed in l3del degenerated tail discs (Figure 3G) but not in l3del lumbar discs (Figure 3H), indicating that ISR was only triggered in degenerated discs in spite of the transgene-bearing genetic background.
  • Figure 31 shows that concomitantly, although the transcriptional expression level of Atf4 was not changed, the protein level of ATF4 was significantly upregulated in NP of 6-month old l3del mice, indicating the contributory regulation of ISR.
  • Figure 3J shows that activation of ATF5, the vital transcription factor of mitochondria-dependent oxidative stress response, was observed in the core part of NP of 6-month old l3del mice (circled), indicating the induction of oxidative stress.
  • Figure 3K shows that in WT control mice, the peripheral nucleus pulposus cells (NPCs) highly expressed Sox9 and the level was much lower in cells within core region.
  • NPCs peripheral nucleus pulposus cells
  • the cell fate of NP cells was affected as indicated by the ectopic expression of Sox9 in cells within the NP core region.
  • FIG. 3L shows that OPN, a major component of NP extracellular matrix, was highly expressed in peripheral NPCs at young stage (plO, p20 and 4-month), the expression level is diminished at maturity (6-month) and absent at elderly stage (16-month) in WT mice.
  • OPN is a target of SOX9.
  • Figure 3M shows that similarly to Opn, oc-SMA marked the peripheral NPC at young stage (4-month) but became absent at 6-month in WT mice, while this marker was persistently expressed in l3del peripheral NPCs and was ectopically expressed in core NPCs.
  • Figures 4A-4C show that ISRIB ameliorated the IVD phenotypes of l3del mice.
  • Figure 4A shows that treatment with ISRIB (2.5mg/kg) eased the IVD abnormalities in l3del lumbar spine as demonstrated by the less expanded endplate and the more organized iAF structure.
  • Figure 4B shows that in l3del mice, treatment with ISRIB reduced the number of reprogrammed chondrocytes in the growth plates and endplates. Moreover, the ectopic expression of Opn in NP was greatly reduced.
  • FIG. 4C shows that in l3del lumbar IVD, ATF3 (a downstream target of ATF4) was significantly activated in the hypertrophic chondrocytes (HCs) of the growth plate and endplate (EP) as well as in the NP (arrows). No ATF3 expression was detected in lumbar IVD of l3del mice treated with ISRIB and there appeared to be fewer ATF3-expressing HCs.
  • Figures 5A-5B show morphological and fibrotic changes of punctured murine discs in transgenic mice in which NPCs are specifically labelled with EGFP.
  • annulus fibrosis (AF) puncture was induced at level 6 and level 8 of mouse tail discs.
  • Disc degeneration was observed by FAST staining of murine tail IVDs at different time points after AF puncture.
  • Figure 5B severe fibrotic changes in injured discs were demonstrated by the presence of oc-SMA, FAP-a and FSP-l stained cells, indicating they are myofibroblasts/fibroblast-like.
  • these accumulated myofibroblasts/fibroblast-like cells were EGFP-tagged indicating that these cells are derived from NP cells and fibrotic cell fate change of NP cells was induced by the puncture.
  • Figures 6A-6B show pathogenic changes in the kidney biopsy samples from patients with renal fibrosis (provided by Dr. Susan Yung, Prof. T. M. Chan, Department of Medicine, The University of Hong Kong).
  • Figure 6A lists the baseline clinical condition of the patients.
  • Figure 6B shows that severe fibrosis was observed in renal biopsy samples from patients with IgA nephropathy (Figure 6B-a’), diabetic nephropathy ( Figure 6B-a”) and Fupus Nephritis (Figure 6B-a”’), marked by oc-SMA.
  • oc-SMA was not only highly expressed in renal interstitium in all specimens, but also in the glomerular mesangium in the lupus nephritis specimen. Strikingly, ectopic expression of SOX9 was observed in all specimens ( Figure 6B-b, b’, b” and b’”), with highly tubular expression in IgA nephropathy and diabetic nephropathy patients, and tubulo interstitial as well as interstitial inflammatory cells expression in patients with lupus nephritis.
  • ISR was significantly activated, marked by the upregulation of p-eIF2oc (Figure 6B-d, d’, d” and d’”) and its downstream target ATF4 ( Figure 6B-c, c’, c” and c’”) in tubular interstitium in all specimen, indicating the etiological role of ISR in the genesis of renal fibrosis.
  • Figures 7A-7D demonstrate the putative ATF4 binding regions on Sox9 within the topologically associated domains (TAD), indicating the potential ISR-regulation of Sox9 by enhancers.
  • Figure 7A shows that human SOX9 ( hSOX9 ) and mouse Sox9 ( mSOX9 ) are located within the boundary region between 2 sub-TADs and share a highly conserved TAD pattern.
  • Figure 7B and 7C demonstrate the highly conserved CCCT C-binding factor (CTCF) insulator binding region presenting in human and mouse Sox9 gene locus.
  • CTCF CCCT C-binding factor
  • Figure 8 is a presentation of ATF4 ChIP peaks on regulatory regions (+/-2kb from transcriptional start site, TSS) of vital chondrogenic transcriptional factors (SOX, MEF2, RUNX, GLI and FOXA). The expression trends of these factors in WT and l3del chondrocytes were measured by normalized microarray expression profiles of the different populations of chondrocytes in the growth plate.
  • TSS transcriptional start site
  • chondrocytes isolated by fractionating the growth plate, include proliferating chondrocytes (PC), prehypertrophic chondrocytes (pHC), upper hypertrophic chondrocytes (UHC), middle hypertrophic chondrocytes (MHC) and lower hypertrophic chondrocytes (LHC) (82).
  • PC proliferating chondrocytes
  • pHC prehypertrophic chondrocytes
  • UHC upper hypertrophic chondrocytes
  • MHC middle hypertrophic chondrocytes
  • LHC lower hypertrophic chondrocytes
  • Figure 9A shows luciferase activities of Sox9 promoter reporters with different lengths of the 5’ flanking region of the gene (pSox9-2.7K, pSox9-l.8K and pSox9-0.8K) or ATF4 putative binding sites mutants (pSox9-l.8Ml, pSox9-l.8M2 and pSox9-l.8M3) responding to different dosages of ATF4 measured in ATDC5 cells.
  • Figure 9B shows ChIP-PCR demonstrating the direct binding of ATF4 to a putative motif on the Sox9 promoter in vivo, using the nuclear extracts from E15.5 WT and C10-ATF4 limbs. An ATF4 ChIP-seq peak (dark triangle) around this region has been reported in ER-stressed MEF cells.
  • Figures 10A-10C show the activation of the ISR in the SM/J mouse which is a natural model of early onset IDD.
  • Figure 10B shows that ISR was activated in degenerative SM/J tail IVDs compared to LG/J IVDs as revealed by ectopic expression of Atf4 and Chop.
  • Figure 10C shows ectopic expression of Sox9 and Col2al observed in degenerative SM/J tail IVDs compared to LG/J IVDs.
  • Figure 11A shows the histology of a chordoma sample (panel a is a low power image and panels b-e are high power images).
  • Figure 11B shows the histology of non-degenerated NP tissues..
  • Figure 12 shows different cell populations in human chordoma as revealed by single cell RNA sequencing. Two technologies were used: 10X Genomics and manual picking with Smartseq2. Panel a) shows two chordomas (cervical chordoma and sacrum chordoma) in 10X Genomics; panel b) shows a chordoma in smartseq2; panel c) shows sub-population 2 (S2) marked by Fibrosis markers; panel d) shows sub-population 2 (S2) of the third chordoma also marked by Fibrosis markers; panel e) shows two chordomas in 10X Genomics where the crosses indicate cells that were marked by co-expression of various markers; and panel f) shows a chordoma in smartseq2 where the crosses indicate cells that were marked by co-expression of various markers.
  • Figure 13 shows histological features of degenerated NP tissues. Note: clusters of round cells resembling chondrocytes (left panel) and spindle like cells resembling fibroblasts (right panel).
  • Figure 14 studies the co-expression of SOX9 with ISR effectors in human NP cells from degenerated discs.
  • Panel a) indicates SOX9 is expressed in non-degenerated NP tissue in chondrocyte like cells.
  • Panel b) indicates SOX9 expression is upregulated in the fibroblast-like cells in the degenerated NP tissue. Different from the notochordal-like and chondrocyte-like cells in non-degenerated NP, fibroblast-like cells were specifically identified the degenerated NP, characterized by their distinct cell morphology and higher expression level of SOX9.
  • Panels c) to f) represent tSNEplots of populations identified by single cell RNAsequencing.
  • DS refers to degenerated NP and DS01-05 and DS06 respectively refer to sample number.
  • Crosses Cells which were marked by co-expression of SOX9, CHOP, ATF4/ATF3/ATF6, and GADD34.
  • the present invention provides a method of preventing, ameliorating and/or treating fibrosis or fibrotic response associated with the integrated stress response (ISR) involving the phosphorylated eukaryotic initiation factor 2a (p-eIF2a) pathway arising from various cellular stresses, such as oxidative stress, hypoxia and ER stress, chronic or prolonged bio mechanical stress.
  • ISR integrated stress response
  • p-eIF2a phosphorylated eukaryotic initiation factor 2a pathway arising from various cellular stresses, such as oxidative stress, hypoxia and ER stress, chronic or prolonged bio mechanical stress.
  • the present invention provides a method of using a p-eIF2a-modulator for the prevention, amelioration and/or treatment of fibrosis or fibrotic response described herein.
  • phosphorylated eukaryotic initiation factor 2a pathways or p-eIF2a pathways include signaling pathways where de -phosphorylated eIF2a or phosphorylated eIF2a is involved, and include signaling pathways which are directly or indirectly affected by the de phosphorylation or phosphorylation of eIF2a.
  • ISR integrated stress response
  • the present invention represents the first mechanistic study in a model of human chondrodysplasia associated with ER stress that demonstrates causality and a direct link between the ISR and reprogrammed chondrocyte differentiation.
  • ISR signalling reverses hypertrophic chondrocyte differentiation via ATF4-directed trans activation of the transcription factor gene Sox9.
  • Sox9 a potent transcription factor gene
  • the present invention also discloses the dual action of CFlOP and ATF4 in promoting hypertrophic chondrocyte survival, establishing the critical role of CFlOP in partnership with ATF4 in enabling chondrocyte survival via the transactivation of Fgf21.
  • the present invention highlights the complex consequences of activating ISR, in part because of the distinct roles of ATF4 in controlling cell differentiation and proliferation depending on cell context.
  • the present invention further demonstrates that treatment of mutant l3del mice with a small molecule inhibitor of the ISR pathway, ISRIB (trans-N,N’ -(Cyclohexane- 1 ,4- diyl)bis(2-(4-chlorophenoxy)acetamide), which targets the interaction between eukaryotic initiation factor 2 (eIF2) and eukaryotic initiation factor 2B (eIF2B) and thereby suppresses ATF4 induction, prevents the differentiation defects and ameliorates chondrodysplasia in the l3del mice ( Figures 2A-2Q), and ameliorates the degenerative intervertebral disc (IVD) syndromes of the l3del mice ( Figures 4A-4C).
  • ISRIB trans-N,N’ -(Cyclohexane- 1 ,4- diyl)bis(2-(4-chlorophenoxy)acetamide
  • the present invention identifies a key causative role for the ISR in MCDS and demonstrates that targeting early in the pathway, i.e., at the level of PERK phosphorylation of eIF2a could be an effective therapeutic approach.
  • ISRIB antagonizes the preferential translation of ATF4 and other preferentially translated transcripts mediated by the ISR, thereby reversing or preventing reverted differentiation (i.e., reversing or preventing the transition of cells to a less mature stage).
  • ISR preferential translation of ATF4 and other preferentially translated transcripts mediated by the ISR
  • reversing or preventing reverted differentiation i.e., reversing or preventing the transition of cells to a less mature stage.
  • the net result is the improvement of murine skeletal development. This result may reflect the dominance of de-differentiation in the pathogenesis of the chondrodysplasia.
  • a dose of ISRIB is administered to a subject to achieve an optimal level of cell differentiation and cell survival.
  • the effective amount of ISRIB is 0.05-0.1, 0.1- 1, 1-5, 5-10, 10-20, 20-25, 25-50 or 50-100 mg/kg per day.
  • the subject is treated for 1 day or up to 365 days. In various embodiments, the subject is treated for 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 or 350 days.
  • the present invention provides a method of preventing, ameliorating and/or treating a disorder associated with integrated stress response involving the p-eIF2oc pathway in a subject using a molecule that targets the underlying p-eIF2oc-pathway.
  • molecules to be used in the present invention are modulators which directly or indirectly suppress the translational or transcriptional expression of ATF4 or SOX9.
  • SOX9 is a potent transcription factor with key roles in cell fate determination, not only in chondrocyte differentiation, but also in many other cell types, notably stem cells (e.g. dermal papilla, gonads, intestinal, and neural) and its overexpression or dysfunction results in many diseases including fibrosis and cancer (14).
  • stem cells e.g. dermal papilla, gonads, intestinal, and neural
  • its overexpression or dysfunction results in many diseases including fibrosis and cancer (14).
  • the present invention revealed that given the importance of ATF4 to normal development, simply preventing its expression globally may not work therapeutically. Rather, the present invention introduces for a novel and viable approach to alter cell differentiation or cell fate by targeting the translational control of ATF4 that leads to its over expression.
  • the present invention provides a method of preventing and/or ameliorating aberrant cell differentiation through the inhibition of ectopic expression of Sox9/SOX9 (referring to mouse transcription factor gene Sox9 and human transcription factor gene SOX9, respectively).
  • the present method prevents and/or alleviates conditions, disorders or diseases resulting from an aberrant cell differentiation.
  • aberrant cell differentiation includes any process whereby the cells undergo abnormal cell differentiation including without limitation de-differentiation, trans-differentiation and reverted differentiation.
  • the present method includes the use of a molecule which inhibits the ectopic expression of Sox9/SOX9.
  • the present method includes a use of a molecule which inhibits the ectopic expression of ATF4 which subsequently reduces the ectopic expression of Sox9/SOX9.
  • the molecule is a modulator that is capable of directly or indirectly inhibiting the transcriptional or translational expression of ATF4.
  • the modulator represented by Formula I is ISRIB having the formula
  • the modulator represented by
  • Formula I includes molecules that are described in WO 2014/144952, the entire contents of which are incorporated herein by reference into this application.
  • the modulator represented by Formula I is selected from the following molecules:
  • the modulator subject to the present invention is represented by Formula II: (II), wherein R 1 is bicycloheteroaryl, including but not limited to pyrrolopyrimidine, which may be unsubstituted or substituted with groups such as amino and alkyl; R 2 is heteroaryl, including but not limited to pyridyl, pyrrolyl and pyrazolyl, which may be unsubstituted or substituted with groups such as halogen, alkyl and trihaloalkyl, and R 3 is hydrogen or halogen.
  • the modulator is represented by Formula II which includes molecules described in WO2011/119663, the entire contents of which are incorporated herein by reference into this application.
  • the modulator represented by Formula II is GSK2656157 having the formula various embodiments, the modulator represented by Formula II is selected from the following molecules:
  • the modulator is Act2B
  • ER stress can increase cell death in injured tissues, induce epithelial-mesenchymal transition (EMT) and promote fibrotic remodeling instead of the restoration of normal tissue architecture (23).
  • EMT epithelial-mesenchymal transition
  • fibrotic remodeling instead of the restoration of normal tissue architecture (23).
  • Activation of ER stress and oxidative stress is a common pathological feature of fibrosis in a variety of organs, including lung (24), liver (25) and heart (26).
  • Fibrosis is an excessive accumulation of extracellular matrix (ECM) that can lead to distortion of tissue architecture and loss of organ function (27, 28). This pathology commonly results from a wound healing response to repeated or chronic injury or tissue damage, irrespective of the underlying etiology, and can occur in virtually any solid organ or tissue.
  • ECM extracellular matrix
  • a broad range of prevalent chronic diseases can give rise to fibrosis, including intervertebral disc degeneration (IDD) (62), osteoarthritis (63), diabetes, hypertension, viral and nonviral hepatitis, heart failure and cardiomyopathy, idiopathic pulmonary disease, scleroderma, and cancer.
  • Fibrosis resulting from these and other diseases can lead to failure of liver, lung, kidney, heart, or other vital organs as excessive ECM replaces and disrupts parenchymal tissue.
  • Ectopic expression of transcription factor Sex-Determining Region Y-Box 9 Sox9
  • Sox9 transcription factor Sex-Determining Region Y-Box 9
  • Fibrosis may be induced in other tissues such as liver, lung, cardiac, skin and so on (64).
  • the present invention provides a method of preventing, ameliorating and/or treating fibrosis or fibrotic response associated with the integrated stress response (ISR) involving the phosphorylated eukaryotic initiation factor 2a (p-eIF2a) pathway arising from various cellular stresses such as oxidative stress, hypoxia and ER stress, chronic or prolonged bio mechanical stress.
  • ISR integrated stress response
  • p-eIF2a phosphorylated eukaryotic initiation factor 2a pathway arising from various cellular stresses such as oxidative stress, hypoxia and ER stress, chronic or prolonged bio mechanical stress.
  • the present invention demonstrates for the first time a direct linkage between Sox9/SOX9 (mouse/human), ATF4 and ISR.
  • the present invention provides a novel approach for treating, preventing and/or ameliorating fibrosis or fibrotic response through the modulation of ectopic expression of ATF4 and its potential downstream mediators, such as Sox9/SOX9 and CFIOP.
  • the present invention provides a method for treating, preventing and/or ameliorating fibrosis or fibrotic response through the modulation of activity of ATF4 and its potential downstream mediators, such as Sox9/SOX9 and CFIOP.
  • the present method reduces the expression of Sox9 and/or CFlOP in an organ or tissue, thereby preventing or reducing the occurrence of fibrotic response in the organ or tissue.
  • presence or progress of fibrosis is indicated by an elevated level of one or more fibrotic factors as compared to normal subjects or cells (81).
  • the present invention provides a method of modulating the level of one or more fibrotic factors, which are indicative of the presence or progress of fibrosis, comprising a step of using a p-eIF2a- modulator described herein.
  • fibrotic factors include but are not limited to fibrillar proteins, glycoproteins, small leucine rich proteoglycans (SLRPs) and matricellular proteins.
  • fibrillar proteins include but are not limited to fibrillar collagen (e.g. type I-III, V, XI), fibronectin (e.g. ED-A, ED-B), and elastin.
  • glycoproteins include but are not limited to non-fibrillar collagen (e.g. type IV, VI- VIII, XIV), fibrillin (e.g. fibrillin 1-3), LTBP (e.g. LTBP 1-4), tenascin (e.g.
  • small leucine rich proteoglycans include but are not limited to biglycan, lumican, fibromodulin, dermatopontin and decorin.
  • matricellular proteins include but are not limited to CCN (CCN1-6), periostin, osteopontin and osteonectin (e.g. SPARC).
  • fibrotic factor is alpha smooth muscle actin (a-SMA), fibroblast activation protein alpha (FAP-a) and fibroblast specific protein 1 (FSP-l) or transforming growth factor b (TGF- b).
  • fibrotic factors include factors which involve the posttranslational modifications of the ECM, including but are not limited to lysyl oxidase (LOX), LOX-like 1-4 (LOXL-l-4), LH 1-3, transglutaminase 1-7 (e.g. TG 1-7), matrix metalloprotease, (e.g. MMP 1-3, 7- 17, 19-21, 23-28), tissue inhibitor of matrix metalloproteinase (e.g. TIMP 1-4) and plasmin- activation inhibitor (e.g. uPA, tPA, PAI-l, PAI-2). Rosenbloom et al. (2017) (64) and Dickens et al.
  • LOX lysyl oxidase
  • L-l-4 LH 1-3
  • transglutaminase 1-7 e.g. TG 1-7
  • matrix metalloprotease e.g. MMP 1-3, 7- 17, 19-21, 23-28
  • fibrotic responses include but are not limited to responses that initiate or advance fibrosis in organ or tissues described herein. In one embodiment, fibrotic responses are triggered by other complications, diseases or disorders such as obesity and cancer.
  • fibrosis to be treated, prevented and/or ameliorated by the present invention is a fibrosis occurs in an organ or a tissue.
  • fibrosis is a fibrosis that occurs in lung, liver, heart, brain, kidney, bone, skin, pancreas, intervertebral disc, cartilage and connective tissues.
  • fibrosis occurs as a result of trauma, e.g. in wound healing after burns.
  • fibrosis occurs as a result of other complications, diseases or disorders.
  • fibrosis is any fibrosis caused by, directly or indirectly, or associated with the activation of ISR involving the p-eIF2oc pathway.
  • the present method can be used to reverse fibrosis by, for example, reversing an established fibrosis, reducing the extent of excessive ECM or tissue deposited in a particular tissue, or reducing the extent of fibrotic process occurred and so on.
  • the present invention provides a method of preventing or ameliorating aberrant cell differentiation which results in an aberrant synthesis or accumulation of ECM, where the aberrant cell differentiation is caused by the activation of the ISR.
  • the present method prevents or ameliorates cell differentiation into a type of cell such as fibroblasts and myofibroblasts that synthesizes ECM.
  • the present method prevents or ameliorates an aberrant cell differentiation into a myofibroblastic lineage.
  • the present invention provides a method of using a p-eIF2oc-modulator described herein for the prevention, treatment and/or amelioration of fibrosis caused by an aberrant cell differentiation.
  • the present invention provides a method of using a p-eIF2oc-modulator described herein for the prevention, treatment and/or amelioration of aberrant accumulation of ECM caused by an aberrant cell differentiation. In one embodiment, the present invention provides a method of using a p-eIF2oc-modulator described herein for the prevention, treatment and/or amelioration of aberrant synthesis or accumulation of ECM that is caused by an aberrant cell differentiation and/or activation of transcription factor gene Sox9/SOX9.
  • the present invention provides a method of using a p-eIF2oc-modulator described herein for the prevention and/or amelioration aberrant synthesis or accumulation of ECM in a tissue or organ, where the aberrant accumulation of ECM is caused by the activation of the ISR.
  • the present invention provides a fibrotic mouse model and a method for screening candidate molecule for the ability to modulate fibrosis associated with integrated stress response (ISR) involving the p-eIF2oc pathway, the method comprising the steps of a) administering a candidate molecule to the mouse, and b) measuring one or more of fibrotic factors described herein, where changes in one or more fibrotic factors in the presence of the candidate molecule as compared to a control molecule indicates that the candidate molecule is capable of modulating fibrosis associated with ISR involving the p-eIF2oc pathway.
  • ISR integrated stress response
  • the fibrotic mouse model carries a Sox9 gene which can be conditionally knocked out (i.e., can be removed or inactivated in specific tissue(s) rather than universally removed or inactivated in the whole organism), thereby allowing to determine whether the effect of the candidate molecule on fibrosis is through its action on Sox9 in specific tissue(s).
  • the fibrotic mouse model can be obtained by crossing Sox9- flox mice with mice carrying Cre recombinase expressed from a tissue-specific promoter, which results in progeny with Sox9 knock out in a particular tissue.
  • the fibrotic mouse model is a Ddit3- null ( Ddit3 encodes protein CHOP).
  • the fibrotic mouse model carries a Ddit3 gene which can be conditionally knocked out in a similar manner as for 5ox9-conditional knockout.
  • the present invention provides a method of preventing, ameliorating and/or treating fibrosis or fibrotic response associated with integrated stress response involving the eukaryotic Initiation Factor 2 (eIF2), phosphorylated eukaryotic initiation factor 2a (p-eIF2a) and PKR-like endoplasmic reticulum kinase (PERK) pathway in a subject, comprising the step of administering to said subject an effective amount of a molecule which targets the p-eIF2a pathway (a“p-eIF2a modulator”).
  • eIF2 eukaryotic Initiation Factor 2
  • p-eIF2a phosphorylated eukaryotic initiation factor 2a
  • PERK PKR-like endoplasmic reticulum kinase
  • the present invention provides a use of a p- eIF2a modulator for the preparation of a medicament for preventing, ameliorating and/or treating fibrosis or fibrotic response associated with integrated stress response involving the p-eIF2a pathway.
  • the present invention provides molecules that are capable of modulating the p-eIF2a pathway for use in the treatment of diseases or modulation of conditions described herein.
  • the present invention provides a method of manipulating the inhibitory effects of p-eIF2a using a p-eIF2a modulator or a phosphorylated eukaryotic initiation factor 2 b- modulator (p-eIF2 -modulator) for the prevention, amelioration and/or treatment of fibrosis or fibrotic responses described herein.
  • the subject is a human including an adult and a child, or an animal.
  • “effective amount” means the amount of a molecule necessary to achieve a desired physiological effect.
  • the present invention provides a method of modulating the p-eIF2a pathway in a cell or a population of cells, the method comprising contacting the cell(s) with an effective amount of a p-eIF2a or a r-eIH2b modulator.
  • p-eIF2a modulators are small molecules, nucleic acids, proteins or other biomolecules.
  • p-eIF2a modulators are small molecules which are represented by Formula I or II described above.
  • p-eIF2a modulators are p- eIF2a inhibitors such as ISRIB, GSK2656157, Act2B and their analogs that inhibit one or more downstream molecules or signaling events in the p-eIF2a pathway.
  • p- eIF2a modulators are molecules such as Sulubrinal and Guanzbenz and their analogs that activate one or more downstream molecules or signaling events in the p-eIF2a pathway.
  • p-eIF2a modulators are molecules that alter one or more downstream molecules or signaling events in the p-eIF2a pathway (for example, those illustrated in Figure 1), and the p- eIF2a pathway can be part of the cellular stress responses such as oxidative stress, ER stress and hypoxia, or other chronic or prolonged biomechanical stress.
  • said p-eIF2oc modulators such as ISRIB and Act2B and their analogs are capable of targeting eIF2oc phosphorylation.
  • p-eIF2oc modulators are molecules which are capable of targeting GADD34-Pplc or promoting the assembly of GADD34- Pplc.
  • p-eIF2oc modulators are molecules which are capable of modulating the expression of ATF4 and its potential downstream factors, such as Sox9.
  • the effective amount of p-eIF2oc modulator such as ISRIB to be given to a subject is 2.5 mg/kg to 20 mg/kg per day.
  • the effective amount of p-eIF2oc modulator is 0.05-0.1, 0.1-1, 1-5, 5-10, 10-20, 20-25, 25-50 or 50-100 mg/kg per day.
  • the subject is treated for 1 day or up to 365 days. In various embodiments, the subject is treated for 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 or 350 days.
  • two or more p-eIF2oc pathway modulators are administered concurrently.
  • the second or subsequent p-eIF2oc pathway modulators are administered immediately or a certain period after the administration of the previous p-eIF2oc pathway modulator.
  • the present invention provides a method of inhibiting the ATF4/ISR mediated activation of transcription of murine .SY «9/human SOX9, thereby preventing, alleviating and/or treating fibrosis or fibrotic response in an organ or a tissue resulting from the overexpression of protein SOX9; the method comprises a step of contacting the cells with, or administering to a subject, a molecule that is capable of blocking the ATF4-binding site on the Sox9/SOX9 locus, or by interfering with molecules that modulate the ATF4-mediated transcription of Sox9//SOX9 (such as a molecule that enhances the binding between ATF4 and Sox9/SOX9 locus, i.e., an ATF4-binding enhancer).
  • a molecule that is capable of blocking the ATF4-binding site on the Sox9/SOX9 locus or by interfering with molecules that modulate the ATF4-mediated transcription of Sox9//SOX9 (such
  • Mouse Sox9 was found to be located at the boundary between two sub-TADs (topologically associated domains) on chromosome 11 (chrll :l l0760000-H4800000), represented by chr 11:111520000- 112200000 and chrll : ll3l60000-H4l60000 respectively (68, 63) (Fig 8A, lower panel). Binding sites for ATF4 in mouse embryonic fibroblasts have been reported (44).
  • the present invention has identified the putative binding site for ATF4 on the Sox9 locus in hypertrophic chondrocytes of mice - a region on chromosome 11 (loci: 112642927-112643074) which covers the promotor region of Sox9 ( Figure 8). It is thus possible to inhibit the transcription of Sox9 by using molecules that interfere with the entirety or a part of the putative ATF4-binding site and thereby modulate conditions resulting from the overexpression of SOX9.
  • the present invention provides a method of inhibiting the transcription of mouse Sox9 by using a molecule that blocks or interferes with one or more binding sites for ATF4 on the mouse Sox9 locus, thereby preventing, ameliorating and/or treating fibrosis or fibrotic response in an organ or a tissue.
  • said ATF4 binding site is located on mouse chromosome 11 (loci: 112642927-112643074) having the sequence TGTTGCAA (SEQ ID NO: 1).
  • said ATF4 binding site is located within the binding sites for ATF4 as reported in Flan:
  • the present invention further provides a method of inhibiting the transcription of human SOX9 by using a molecule that blocks or interferes with one or more binding sites for ATF4 on the human SOX9 locus, thereby preventing, ameliorating and/or treating fibrosis or fibrotic response in an organ or a tissue.
  • said ATF4 binding site is located on human chromosome 17 (chr 17:68609000-71514000).
  • said ATF4 binding site is TGTTGCAA (SEQ ID NO.: 3) (33) which is the consensus sequence of ATF4 binding site on human SOX9 locus.
  • ATF4-binding sites on Sox9/SOX9 or other related ATF4 potential downstream factors are mapped by the core Amino Acid Response Element (AARE) sequence TTgCaTCA (SEQ ID: 4), which is the complementary strand of SEQ ID NO.l.
  • AARE Amino Acid Response Element
  • open chromatin regions in cells expressing Sox9/SOX9 upon induction of the ISR and/or ATF4 over-expression are identified via ATAC-seq (34).
  • This method applies hyperactive Tn5 transposase, which inserts sequencing adapters into accessible regions of chromatin, to mark accessible regions of DNA, which are then sequenced.
  • GFP or other reporters
  • GFP are inserted into 3’ untranslated region of mouse or human loci and targeted so as to provide a readout of SOX9 activity, or, alternatively, the cells derived from Sox9 EGFP/+ mice are adopted (35). Cells are then subjected to ER stress, hypoxia or other stresses to induce ISR, or over-expression of ATF4 is induced.
  • regions in the mouse genome or human genome that are constitutively open and therefore not subject to position effects are used for assaying the enhancer activity (e.g. the TIGRE locus (36)).
  • Reporter loci are targeted in cell lines and transgenic mice with a vector comprising a minimal promoter (such as hsp68 or the minimal SOX9 promoter which has no activity in cells/transgenic mice) linked by a 2A peptide sequence (37) to a fluorescence reporter (e.g. GFP, RFP, YFP etc.) or other reporters (e.g. luciferase).
  • the enhancer interference assay is used for functional validation of enhancer elements by epigenome inhibition in vitro and in vivo, using a nuclease-deficient Cas9 (dCas9)-histone demethylase (38) fusion to inhibit the activity of candidate enhancer(s) by selectively altering the chromatin state of the target enhancer(s).
  • dCas9-histone demethylase (38) fusion to inhibit the activity of candidate enhancer(s) by selectively altering the chromatin state of the target enhancer(s).
  • Removal of H3K4mel/me2 modifications from specific active enhancer(s) using targeted catalytically inactive dead-Cas9 (dCas9) fused to the lysine-specific demethylase 1 (KDM1A/ESD1) results in‘inactivation’ of enhancer elements and down-regulation of gene expression from the associated loci.
  • the transgene containing dCas9-ESDl is targeted using CRISPR-Cas9 (39), in which a guide RNA (gRNA) is specifically designed to direct ESD1 to the putative Sox9/SOX9 enhancer(s).
  • gRNA guide RNA
  • the expression of ESD1 on targeted specific enhancer(s) silences the candidate enhancer(s) by demethylation of histone H3K4me2 and destruction of K27 acetylation (H3K27ac).
  • the targeted enhancer(s), resulting in loss of .VGA 9-dr ivcn EGFP expression when ISR is activated and/or when ATF4 is over expressed, are first identified in vitro.
  • the activities of identified ISR-inducible and or ATF4-inducible SOX9 enhancer(s) are assessed by: a) mutating the enhancer(s) in mice using CRISPR-Cas9; and b) targeting the enhancer(s) to the ISR reporter vector described above comprising a minimal hsp68 promoter, and testing for the enhancer’s ability to be activated upon ATF4 or ISR induction.
  • Enhancers that are active and specific to Sox9 can then be identified by determining whether the ISR is triggered and/or ATF4 is over-expressed.
  • the present invention provides a method of inhibiting the transcription of Sox9/SOX9 (such as murine Sox9/ Human SOX9 ) by using a molecule that blocks or interferes with one or more ATF4 binding enhancers which regulates the transcription of murine Sox9, said ATF4 binding enhancer comprises a sequence selected from the group consisting of SEQ ID NOs. : 5-29, thereby preventing, ameliorating and/or treating fibrosis or fibrotic response in an organ or a tissue.
  • Sox9/SOX9 such as murine Sox9/ Human SOX9
  • the present invention provides a method of inhibiting the transcription of human SOX9 by using a molecule that blocks or interferes with one or more ATF4 binding enhancers which regulate the transcription of human SOX9, thereby preventing, ameliorating and/or treating fibrosis or fibrotic response in an organ or tissue.
  • ATF4 ChIP-seq can be used in human fibroblasts, cancer cell lines, and any other cell lines where ISR induces SOX9 expression differentiated from human induced pluripotent stem cells such as chondrocytes.
  • the cells are treated with an ER stress-inducer such as tunicamycin (41) to activate the preferential translation of ATF4, and three biological replicates for each cell type are generated.
  • said ATF4 binding enhancers are located within human chromosome 17 (chr 17:68609000-71514000) .
  • the present invention provides a method of inhibiting the transcription of human SOX9 by using a molecule that blocks or interferes with one or more ATF4 binding enhancers which regulate the transcription of human SOX9, thereby preventing, ameliorating and/or treating fibrosis or fibrotic response in an organ or a tissue
  • said ATF4 binding enhancer comprises a sequence that could be similar/homologous to the murine sequence selected from the group consisting of SEQ ID Nos:. 5-29.
  • said ATF4 binding enhancer comprises a sequence corresponding to a sequence which is at least 70%, 75%, 80%, 85%, 90% or 95% homologous to the sequence selected from the group consisting of SEQ ID Nos. 5-29.
  • said ATF4 binding enhancer in human comprises a sequence corresponding to and showing high consensus to the murine sequence selected from the group consisting of SEQ ID Nos. 5-29. It is also possible that there will be human specific ATF4 binding enhancers not present in mouse. These will be detected by the ATAC-seq and ATF4 ChIP-seq approaches described above. Functional validation of the human enhancer activity will be tested by linking putative enhancer(s) to reporter (e.g.
  • Fuciferase/fluorescent proteins constructs and testing for their activation upon inducing the ISR in vitro (mouse or human cell-lines) and in vivo, using transgenic mice in which the ISR is induced, such as in 13del and/or SM/J.
  • ISRIB a selective modulator of phosphorylated-eukaryotic initiation factor (p- eIF2a) and eIF2B complex
  • p- eIF2a phosphorylated-eukaryotic initiation factor
  • eIF2B complex a selective modulator of phosphorylated-eukaryotic initiation factor (p- eIF2a) and eIF2B complex
  • ATF4 can transactivate Chop and ATF3, and form a heterodimer with ATF3 to modulate the expression of target genes, such as GADD34.
  • CHOP also acts upstream of GADD34, which encodes a regulatory subunit of the protein phosphatase complex that dephosphorylates p-eIF2a and restores protein translation.
  • ATF4, CHOP and GADD34 form a negative feedback loop to ensure transient attenuation of protein synthesis and later recovery of protein translation during ER stress response (42, 43).
  • the present invention discloses a novel approach in preventing or treating ISR-associated diseases, in particular to diseases where aberrant cell differentiation and over-synthesis and/or perturbed homeostasis of ECM proteins are the underlying cause.
  • the present invention provides a use of a modulator of a phosphorylated eukaryotic initiation factor 2a (p-eIF2a) for the manufacture of a medicament for the prevention, amelioration and/or treatment of fibrosis caused by the activation of the integrated stress response (ISR) involving the p-eIF2oc pathway in an organ or tissue, wherein the modulator is represented by Formula I:
  • each of Rl, R2, R3 and R4 is independently selected from a group consisting of hydrogen, halogen, -OCH3, -OCH 2 Ph, -C(0)Ph, -CH3, -CF3, -CCI3, -CN, -S(0)CH 3 , -OH, -NH 2 , -COOH, - CONH2, -NO2, -C(0)CH 3 , -CH(CH 3 ) 2 , -CCSi(CH 3 ) 3 , -CCH, -CH 2 CCH, -SH, SO3H, -S0 4 H, -
  • R 1 is pyrrolopyrimidine, which may be unsubstituted or substituted with amino or alkyl;
  • R 2 is pyridyl, pyrrolyl or pyrazolyl, which may be unsubstituted or substituted with halogen, alkyl or trihaloalkyl; and
  • R 3 is hydrogen or halogen.
  • an effective amount of the modulator is capable of one or more of the following: a) inhibiting the phosphorylation of eIF2oc;
  • the organ or tissue is selected from the group consisting of lung, liver, heart, brain, kidney, bone, skin, pancreas, intervertebral disc, cartilage and connective tissues.
  • the fibrosis is caused by an aberrant synthesis or accumulation of extracellular matrix (ECM) protein in the organ or tissue.
  • ECM extracellular matrix
  • the extracellular matrix (ECM) protein is synthesized by fibroblasts, myofibroblasts, or both.
  • the modulator is selected from the following:
  • the modulator is selected from the following:
  • the effective amount of the modulator is 0.1 mg/kg to 50 mg/kg per day.
  • the present invention provides a method of preventing and/or ameliorating aberrant synthesis or accumulation of extracellular matrix (ECM) protein in an organ or tissue of a subject, the method comprises a step of administering to the subject an effective amount of a molecule that is capable of inhibiting the p-eIF2oc pathway.
  • ECM extracellular matrix
  • the molecule is a small molecule represented by Formula I:
  • the molecule is capable of inhibiting the ectopic expression of Chop, Sox9/SOX9 and/or ATF4.
  • the molecule is capable of inhibiting the binding between ATF4 and a transcriptional regulatory element of mouse/human Sox9/SOX9.
  • the transcriptional regulatory element of Sox9/SOX9 comprises the nucleic acid sequence selected from the group consisting of SEQ ID NOs.: 1-3.
  • the transcriptional regulatory element of Sox9/SOX9 is a stress-induced or ATF-induced enhancer that regulates the transcription of Sox9/SOX9
  • the enhancer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs.: 5-29, or a nucleic acid sequence which is at least 80% homologous to the nucleic acid sequence selected from the group consisting of SEQ ID NOs.: 5-29.
  • the aberrant synthesis or accumulation of extracellular matrix (ECM) protein is caused by an aberrant cell differentiation in a population of cells caused by the activation of integrated stress response (ISR).
  • ISR integrated stress response
  • the population of cells comprises cells that can be differentiated to fibroblasts, myofibroblasts, or both.
  • the present invention provides a method of modulating the level of one or more fibrotic factors in an organ or tissue of a subject, wherein the fibrotic factors are indicative of the presence or progress of fibrosis, the method comprises a step of administering to the subject an effective amount of a p-eIF2oc modulator.
  • the fibrotic factor is selected from the group consisting of fibrillar collagen type I, II, III, V and XI, fibronectin ED-A and ED-B, elastin, non-fibrillar collagen type IV, VI, VII, VIII and XIV, fibrillin 1-3, LTBP 1-4, tenascin C (R, W and X), Hyaluronan form HA, versican V0-V3, syndecan 1-4, fibulin 1-7, biglycan, lumican, fibromodulin, dermatopontin, decorin, CCN1-6, periostin, osteopontin, osteonectin, SPARC, alpha smooth muscle actin, fibroblast activation protein alpha, fibroblast specific protein 1 and transforming growth factor b.
  • the fibrotic factor is selected from the group consisting of lysyl oxidase, LOX-like 1-4, LH 1-3, transglutaminase 1-7, matrix metalloprotease 1-3, 7- 17, 19-21 and 23-28, tissue inhibitor of matrix metalloproteinase 1-4 and plasmin-activation inhibitor uPA, tPA, PAI-l and PAI-2.
  • the present invention provides a method of preventing, ameliorating and/or treating fibrosis caused by the activation of the integrated stress response (ISR) involving the phosphorylated eukaryotic initiation factor 2a (p-eIF2a) signaling pathway in an organ or tissue of a subject, the method comprises a step of administering to the subject an effective amount of a p-eIF2a modulator.
  • ISR integrated stress response
  • p-eIF2a phosphorylated eukaryotic initiation factor 2a
  • an effective amount of the p-eIF2a modulator is capable of one or more of the following:
  • the organ or tissue is selected from the group consisting of lung, liver, heart, brain, kidney, bone, skin, pancreas, intervertebral disc, cartilage and connective tissues.
  • the fibrosis is caused by an aberrant synthesis or accumulation of extracellular matrix (ECM) protein in the organ or tissue.
  • ECM extracellular matrix
  • the extracellular matrix (ECM) protein is synthesized by fibroblasts, myofibroblasts, or both.
  • the p-eIF2oc modulator is represented by Formula I:
  • each of R1 , R2, R3 and R4 is independently selected from a group consisting of hydrogen, halogen, -OCH , -OCH 2 Ph, -C(0)Ph, -CH , -CF , -CC1 3 , -CN, -S(0)CH , -OH, -NH 2 , -COOH, -
  • the effective amount of the p-eIF2oc modulator is 0.1 mg/kg to 50 mg/kg per day.
  • the present invention provides a method of screening a candidate molecule for the ability to modulate fibrosis associated with integrated stress response (ISR) involving the p-eIF2oc pathway, the method comprises the steps of
  • the fibrotic mouse model is a transgenic mouse carrying a Sox9 gene which is conditionally knockout.
  • the fibrotic mouse model is a transgenic mouse carrying a Ddit3 gene which is conditionally knockout, or carrying no Ddit3 gene.
  • the fibrotic factors are selected from the group consisting of fibrillar collagen type I, II, III, V and XI, fibronectin ED- A and ED-B, elastin, non-fibrillar collagen type IV, VI, VII, VIII and XIV, fibrillin 1-3, LTBP 1-4, tenascin C (R, W and X), Hyaluronan form HA, versican V0-V3, syndecan 1-4, fibulin 1-7, biglycan, lumican, fibromodulin, dermatopontin, decorin, CCN1-6, periostin, osteopontin, osteonectin, SPARC, alpha smooth muscle actin, fibroblast activation protein alpha, fibroblast specific protein 1, transforming growth factor b, lysyl oxidase, LOX-like 1-4, LH 1-3, transglutaminase 1-7, matrix metalloprotease 1-3, 7- 17,
  • transitional term“comprising”, which is synonymous with“including”,“containing” or“characterized by”, is inclusive or open-ended, and does not exclude additional, un-recited elements or method steps.
  • the l3del transgenic mice were maintained in Fl (C57BL/6 x CBA) background.
  • the Chop-null mice and Fgf2l-null mice were reported previously (43, 44). Animal care and experiments performed were in accordance with the protocols approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong.
  • FAST staining refers to a multidye staining procedure using fast green, Alcian blue, Safranin-O, and tartrazine and was performed as described previously (45).
  • a AGACTAC A AA AGCTT CTTG (SEQ ID NO: 38) and 5’ -AAGAATTCTCATCGAAGTGCAA (SEQ ID NO: 39); and Fgf21, 5’- CAGGGGTCATTCAAATCCTG (SEQ ID NO: 40) and 5’- AGGAATCCTGCTTGGTCTTG (SEQ ID NO: 41).
  • Apoptotic cells in the growth plate of examined animals were detected by in situ terminal deoxynucleotidyltransferase deoxyuridine triphosphate nick end labeling (TUNEL) assay using the In situ Cell Death Detection Kit (Roche) following the manufacturer’s instructions.
  • TUNEL in situ terminal deoxynucleotidyltransferase deoxyuridine triphosphate nick end labeling
  • ISRIB (SML0843, Sigma) was dissolved in DMSO to make a 5 mg/ml stock and stored at 4°C. Animals were intraperitoneally injected with ISRIB (48, 49) (2.5 mg/kg, diluted in 0.9% saline) or vehicle (5% DMSO in saline) from E13.5 to p20. Animals were collected at plO and p20 for further analysis.
  • annulus puncture was performed in the tail discs of Foxa2mNE-Cre; 7/EG double transgenic mice. Briefly, 3-month- old mice, regardless of the gender, were anesthetized by intraperitoneal injection of Hypnorm and Dormicum at 1 ml/kg of body weight and the caudal disc levels were identified by X-ray (Model 43 855a; Faxitron Corp, IL, USA).
  • the tail skin was incised longitudinally and the C5/6 and C7/8 levels were punctured by inserting a 30G needle bevel into the dorsal annulus at 1 mm depth (BD biosciences) perpendicular to middle of the disc under the guidance of surgical microscope (Wild M691, Switzerland).
  • the C6/7 level was left untreated as control.
  • the mice were allowed to recover and have free activity in cage.
  • the operated animals were subjected to X-ray for disc height measurement.
  • the animals were euthanized and the spine was decalcified by EDTA and embedded in paraffin for histological analysis.
  • Luciferase assays were conducted using a dual luciferase reporter assay kit (Promega), according to the manufacturer’s protocol. Different promoter fragments of Sox9 were cloned into a pGL3 -basic vector (Promega) to drive the expression of firefly luciferase.
  • ATDC5 cells were plated at 2xl0 4 cells/well in 24-well plates. After l8-hours incubation, the cells were transfected with tested constructs with Renilla luciferase vector, which served as an internal control. Data presented are ratios of Luc/Renilla activity from at least three different experiments, and each experiment was performed in triplicate for each DNA sample.
  • fibrosis e.g. disc- and renal fibrosis
  • fibrosis e.g. disc- and renal fibrosis
  • the mouse models are prepared by crossing Sox9-flox mice (with tissue-specific Cre ) or Chop- null mice to l3del mice or mouse models where fibrosis is induced such as mouse models of kidney fibrosis (e.g. Lupus-prone/diabetic mice); mouse models of experimental induced liver fibrosis (e.g.
  • the present invention searched published ER stress-associated ATF4 ChIP-Seq data (40) for binding peaks in transcription factor genes, including members of SOX, RUNX, MEF2, GLI and FOXA families, and found ATF4 binding peaks in regulatory regions of Sox9, Sox5, Sox6, Runx2, GU2 and GU3, suggesting that the Sox family could be the regulatory targets of ATF4.
  • Transcription factor SOX9 is highly expressed in immature chondrocytes, transactivates critical cartilaginous matrix genes and regulates chondrocyte proliferation, differentiation and hypertrophy (50-54). It is required for the expression of SOX5 and SOX6, which cooperate with SOX9 to transactivate Col2al (54).
  • ISRIB Integrated Stress Response InhiBitor
  • ISRIB specifically reduced the amount of ATF4 and CHOP protein, and inhibited p- eIF2oc/ATF4/CHOP signaling transduction, marked by the down-regulation of the transcripts as well as the protein level of their downstream targets (ATF3, EROll and FGF21) ( Figures 20 and 2P).
  • inhibition of p-eIF2oc/ATF4/CHOP by ISRIB did not induce apoptosis ( Figure 2Q) in l3del HC.
  • ISRIB corrected the molecular, histological, and skeletal defects in l3del mice.
  • IDD Intervertebral disc degeneration
  • ISR Intervertebral disc degeneration
  • the tail intervertebral disc (IVD) of 13del mice exhibited significant characteristics of disc degeneration at adult stages ( Figure 3C), including altered NP structure and matrix, loss of NP/AF boundary, disc bulging, widening of the AF interlamellar space and the inward bulging of AF lamellae and consequently fissure ( Figure 3D).
  • Figure 3C tail intervertebral disc
  • Figure 3E excessive cell death was observed in l3del degenerated disc at l6-month stage, consistent with human IDD studies (61) ( Figure 3E).
  • ISRIB prevents the molecular chanees in the 13del IVD
  • the annulus puncturing protocol was used to induce disc degeneration in mouse tail discs.
  • the tail disc degeneration was observed after 2 weeks post puncturing and demonstrated that notochord descendants become fibroblasts and myofibroblasts by expressing alpha smooth muscle actin (oc-SMA), fibroblast activation protein alpha (FAP-a) and Fibroblast-specific protein 1 (FSP- 1) markers with their levels increasing from 4 weeks to 12 weeks post puncturing ( Figures 5A- 5B).
  • the present model could be used to study injury-induced fibrotic changes and subsequent disc degeneration in the IVD from activation of ISR in NP.
  • Lupus nephritis is a potentially reversible cause of severe acute kidney injury and is an important cause of end-stage renal failure in Asians and patients of African or Hispanic descent. It is characterized by aberrant exaggerated innate and adaptive immune responses, autoantibody production and their deposition in the kidney parenchyma, triggering complement activation, activation and proliferation of resident renal cells, and expression of pro-inflammatory and chemotactic molecules leading to the influx of inflammatory cells, all of which culminate in the destruction of normal nephrons and their replacement by fibrous tissue.
  • Anti-double-stranded DNA (anti-dsDNA) antibody level correlates with disease activity in most patients.
  • Fibrosis may also be induced in other tissues such as liver, lung, cardiac, skin and so on (64).
  • Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos genesis 45, 625-629 (2007).
  • Matrix metalloproteinase 12 is an indicator of intervertebral disc degeneration co-expressed with fibrotic markers. Osteoarthritis Cartilage. 24(10): 1826- 1836 (2016).
  • Osteopontin is a novel downstream target of SOX9 with diagnostic implications for progression of liver fibrosis in humans. Hepatology. 56(3): 1108-16 (2012). doi: 10.1002/hep.25758.

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Abstract

La présente invention concerne un procédé de prévention, d'amélioration et/ou de traitement d'une fibrose ou d'une réponse fibreuse associées à la réponse intégrée au stress (ISR) impliquant le facteur d'initiation eucaryote 2 (eIF2), le facteur d'initiation eucaryote phosphorylé 2α (p-eIF2α) et la voie PERK (PKR-like endoplasmic reticulum kinase) provenant de divers stress cellulaires, tels que le stress oxydatif, l'hypoxie et le stress ER, le stress biomécanique chronique ou prolongé et autres. Dans un mode de réalisation, l'invention concerne un procédé qui empêche ou atténue la différenciation cellulaire aberrante provoquée par l'activation de la réponse intégrée au stress, et ainsi empêche, traite ou atténue la fibrose ou la réponse fibreuse en résultant. Dans un autre mode de réalisation, la présente invention concerne un procédé d'utilisation d'un modulateur de p-eIF2α ou d'un modulateur du facteur d'initiation eucaryote phosphorylé 2β (modulateur p-eIF2β) pour la prévention, l'amélioration et/ou le traitement de la fibrose ou de la réponse fibreuse décrite ici.
PCT/IB2019/052321 2018-03-21 2019-03-21 Procédé de prévention ou de modulation de la fibrose et de la réponse fibreuse associée à la réponse intégrée au stress WO2019180664A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114672547A (zh) * 2022-01-24 2022-06-28 中山大学附属第一医院 Periostin基因在制备诊断和治疗椎间盘退变的产品中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014144952A2 (fr) * 2013-03-15 2014-09-18 Peter Walter Modulateurs de la voie eif2 alpha
WO2015120350A2 (fr) * 2014-02-07 2015-08-13 Effector Therapeutics, Inc. Compositions et méthodes pour traiter des maladies fibrosantes
WO2016025635A2 (fr) * 2014-08-13 2016-02-18 Epizyme, Inc. Polythérapie pour le traitement du cancer
WO2018055578A1 (fr) * 2016-09-22 2018-03-29 The University Of Hong Kong Approche préventive et thérapeutique ciblant la différenciation cellulaire aberrante et les maladies associées à l'isr

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014144952A2 (fr) * 2013-03-15 2014-09-18 Peter Walter Modulateurs de la voie eif2 alpha
WO2015120350A2 (fr) * 2014-02-07 2015-08-13 Effector Therapeutics, Inc. Compositions et méthodes pour traiter des maladies fibrosantes
WO2016025635A2 (fr) * 2014-08-13 2016-02-18 Epizyme, Inc. Polythérapie pour le traitement du cancer
WO2018055578A1 (fr) * 2016-09-22 2018-03-29 The University Of Hong Kong Approche préventive et thérapeutique ciblant la différenciation cellulaire aberrante et les maladies associées à l'isr

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114672547A (zh) * 2022-01-24 2022-06-28 中山大学附属第一医院 Periostin基因在制备诊断和治疗椎间盘退变的产品中的应用

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