WO2024030332A1 - Methods of treating neurological disorders with activators of unc51-like autophagy activating kinase 1 (ulk1) - Google Patents
Methods of treating neurological disorders with activators of unc51-like autophagy activating kinase 1 (ulk1) Download PDFInfo
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
Definitions
- compositions useful in the methods described herein include agents that increase the expression of and/or activate Unc51-like autophagy activating kinase 1 (ULK1).
- ULK1 Unc51-like autophagy activating kinase 1
- ULK1 Unc51-like autophagy activating kinase 1 is the serine/threonine protein kinase of the ULK1 complex, and along with Unc-51 like autophagy activating kinase 2 (ULK2), is a critical regulatory node that induces autophagy through phosphorylation of downstream targets.
- ULK1 is known to be essential for autophagy initiation under nutrient deprivation conditions (Lazarus et al. 2015. “Structure of the Human Autophagy Initiating Kinase ULK1 in Complex with Potent Inhibitors.” ACS Chemical Biology 10 (1): 257-61).
- ULK1 phosphorylates Beclin-1 which activates the PIK3 class 3 (PIK3C3/VPS34) lipid kinase complex necessary for full autophagic induction and autophagosome maturation (Russell et al. 2013. “ULK1 Induces Autophagy by Phosphorylating Beclin-1 and Activating VPS34 Lipid Kinase.” Nature Cell Biology 15 (7): 741-50).
- ULK1 interacts with ATG13, ATG101, and FIP200 to form a complex critical for the initiation of autophagy.
- ULK1 may have an additional role in promoting autophagosome-lysosome fusion via STX17-SNAP29 (See Figure 1, Hansen et al. 2018.
- ULK1 promotes mitophagy via phosphorylation and stabilization of BNIP3 - LC3 complexes (Poole et al. 2021. “ULK1 promotes mitophagy via phosphorylation and stabilization of BNIP3.” Scientific Reports 11 :20526) and phosphorylation of Parkin (Hung et al. 2021. “AMPK/ULK1 -Mediated Phosphorylation of Parkin ACT Domain Mediates an Early Step in Mitophagy.” Science Advances 7 (15): 1-15.).
- ULK1 The activity of ULK1 is regulated by phosphorylation by mTORCl (mammalian target of rapamycin complex 1) and AMPK (AMP-activated protein kinase).
- mTORCl mimmalian target of rapamycin complex 1
- AMPK AMP-activated protein kinase
- mTORCl phosphorylates ULK1 at S757 to inhibit autophagy.
- Cellular stress or nutrient deprivation inhibits mTORC, relieving inhibition from ULK1, and activates AMPK to induce ULK1 phosphorylation at other sites to induce autophagy (Zhu et al. 2019. “Balancing MTOR Signaling and Autophagy in the Treatment of Parkinson’s Disease.” International Journal of Molecular Sciences 20 (3): 1-15; Kim et al. 2011. “AMPK and mTOR regulate autophagy through direct phosphorylation of Ulkl.” Nature Cell Biology 13(2): 132-141).
- ULK1 is in complex with C9ORF72 and coordinates autophagy with TBK1, two genes associated with amyotrophic lateral sclerosis (ALS).
- C9ORF72 regulates expression and activity of ULK1 (Yang et al. 2016. “A C9ORF72/SMCR8- containing complex regulates ULK1 and plays a dual role in autophagy.” Science Advances 2(9): el601167)
- ULK1 also plays a role in mitophagy (Egan et al. 2011. “Phosphorylation of ULK1 (HATG1) by AMP -Activated Protein Kinase Connects Energy Sensing to Mitophagy.” Science 331 (6016): 456-61.). After mitochondrial damage, ULK1 rapidly phosphorylates and activates Parkin, a core ubiquitin ligase in mitophagy. Mutation of Parkin phospho-sites, ULK1 depletion, or ULK1 inhibition cause deficits in mitophagy (Hung et al. 2021.
- ULK1 is recruited to damaged mitochondria by autophagy receptors NDP52 (also known as calcium binding and coiled-coil domain 2 (CALCOCO2) and OPTN (optineurin), which are recruited by ubiquitin kinase PINK1 (Lazarou et al. 2015. “The Ubiquitin Kinase PINK1 recruits Autophagy Receptors to Induce Mitophagy.” Nature 524 (7565): 309-14).
- ULK1 phosphorylated mitochondrial membrane proteins FUNDCI (FUN14 domain containing 1), an integral mitochondrial outer-membrane protein (and a receptor for hypoxia-induced mitophagy) and BNIP3 (Poole et al.
- ULK1 activation is protective in several Parkinson disease (PD) models by activation of autophagy.
- PD Parkinson disease
- ULK1 activates starvation-induced autophagy in SH-SY5Y cells, a process which is regulated by two microRNAs (Chen et al. 2015. “Identification of ULK1 as a Novel Biomarker Involved in MIR-4487 and MIR-595 Regulation in Neuroblastoma SH-SY5Y Cell Autophagy.” Scientific Reports 5 (January): 1-10).
- Small molecule activators ofULKl, 33i (BL-918) and LYN1604 induce autophagy, which is neuroprotective in MPP treated SH-SY5Y cells. (Zhang et al. 2017. “Discovery of a small molecule targeting ULK1 -modulated cell death of triple negative breast cancer in vitro and in vivo ” Chemical Science 8(4):2687-2701.
- ULK1 is negatively regulated by ubiquitin specific peptidase-24 (USP24), a Parkinson disease 10 (PARK10) locus gene, and inhibition of USP24 improves neurite arborization in aged induced pluripotent stem cell-derived dopaminergic (iPSC-DA) neurons.
- ULK1 also protects mouse embryonic fibroblasts (MEFs) against paraquat toxicity (Anandhan et al. 2017.
- GM1 (monosialotetrahexosylganglioside), a ganglioside, protects MPTP treated mice from motor dysfunction and increased dopamine; and rescues a-syn accumulation, mitochondrial dysfunction, and oxidative stress in MPP+ treated SH-SY5Y cells and a-syn A53T overexpressing PC12 cells via induction of autophagy through ULK1 (Guo et al. 2021. “Autophagy-Dependent Removal of a-Synuclein: A Novel Mechanism of GM1 Ganglioside Neuroprotection against Parkinson’ s Disease.” Acta Pharmacologica Sinica 42 (4): 518-28).
- Urate induces autophagy by mTOR/ULKl to degrade a-syn (Sheng et al. 2017. “Urate Promotes SNCA/a-Synuclein Clearance via Regulating MTOR-Dependent Macroautophagy.” Experimental Neurology 297 (July): 138-47).
- ULK1 plays a role in mitophagy in a shared pathway with other PD and ALS targets, including Parkin, PINK1, and OPTN (optineurin), further supporting the hypothesis that increasing ULK1 activity may be beneficial.
- ULK1 is a regulator of neurological pathways, such as involved in PD (See, e.g., Figs 1A-1C), progressive supranuclear palsy (PSP) (Figs 2A-2C) and ALS (Figs 6A-6B and 7A-7C). Accordingly, in some embodiments, methods of treating a neurological disorder, such as a neurodegenerative disease, are provided, wherein the methods comprise administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1.
- a neurological disorder such as a neurodegenerative disease
- Embodiment l is a method of treating a neurological disorder, the method comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1.
- Embodiment 2 is the method of embodiment 1, wherein the neurological disorder is a neurological disorder in which the expression and/or activity levels of ULK1 detected in the subject is different from a normal control.
- Embodiment 3 is the method of embodiment 1 or 2, wherein the agent increases the expression of ULK1.
- Embodiment 4 is the method of embodiment 1 or 2, wherein the agent activates ULK1.
- Embodiment 5 is the method of any one of embodiments 1 to 4, wherein the agent is a small molecule, an antibody or active fragment thereof, a peptide, or PROTAC.
- Embodiment 6 is the method of any one of embodiments 1 to 5, wherein the agent is a small molecule.
- Embodiment 7 is the method of embodiment 6, wherein the increased expression level of ULK1 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control.
- Embodiment 8 is the method of embodiment 7, wherein the increased activity of ULK1 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control.
- Embodiment 9 is the method of any one of embodiments 1 to 8, wherein the neurological disorder is Parkinson’s Disease (PD), Parkinson’s Disease with Lewy bodies, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), primary lateral sclerosis (PLS), Charcot-Marie-Tooth (CMT; including type 4J (CMT4J)), and Yunis-Varon syndrome, autophagy, polymicrogyria (including polymicrogyria with seizures), temporo-occipital polymicrogyria, Pick’s disease, dementia with Lewy bodies, Lewy body disease, diseases of neuronal nuclear inclusions of polyglutamine and intranuclear inclusion bodies, disease of Marinesco and Hirano bodies, tauopathy, Alzheimer’s disease, neurodegeneration, spongiform neurodegeneration, peripheral neuropathy, leukoencephalopathy, motor neuropathy, sensory neuropathy, abnormal lyso
- Embodiment 10 is the method of any one of embodiments 1 to 9, wherein the neurological disorder is a neurodegenerative disease.
- Embodiment 11 is the method of embodiment 10, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), Alzheimer’s disease, Parkinson’s disease (PD), Huntington’s disease, prion disease, Lewy body disease, Friedreich’s ataxia, or spinal muscular atrophy.
- ALS amyotrophic lateral sclerosis
- FTD frontotemporal dementia
- PSP progressive supranuclear palsy
- Alzheimer’s disease Parkinson’s disease
- PD Parkinson’s disease
- Huntington’s disease prion disease
- Lewy body disease Friedreich’s ataxia
- spinal muscular atrophy or spinal muscular atrophy.
- Embodiment 12 is the method of embodiments 10 or 11, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
- ALS amyotrophic lateral sclerosis
- Embodiment 13 is the method of embodiments 10 or 11, wherein the neurodegenerative disease is progressive supranuclear palsy (PSP).
- PSP progressive supranuclear palsy
- Embodiment 14 is the method of embodiments 10 or 11, wherein the neurodegenerative disease is Alzheimer’s disease.
- Embodiment 15 is the method of embodiments 10 or 11, wherein the neurodegenerative disease is Parkinson’s Disease (PD).
- PD Parkinson’s Disease
- Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the subject is a human.
- Embodiment 17 is the method of embodiment 16, wherein the neurological disorder is a neurodegenerative disease.
- Embodiment 18 is a composition comprising an agent that increases the expression of and/or activates ULK1 for use in treating a neurological disorder in a subject in need thereof.
- Embodiment 19 is the composition of embodiment 18, further comprising a pharmaceutically acceptable carrier.
- Embodiment 20 is the use of an agent that increases the expression of and/or activates ULK1 in the manufacture of a medicament for treating a neurological disorder in a subject in need thereof.
- Figure 1A shows a representation of ULK1 involvement in regulating the gene expression network identified in brains from Parkinson’s disease (PD) patients
- Figure IB shows a representation of the regulatory effects of the knockdown of ULK1 in mouse fibroblasts.
- Figure 1C shows ULK1 ranking in a genetic scoring method.
- Figure ID shows a representation of the convergence of genetic risk factors of Parkinson’s Disease (PD) on the ULKl-mediated autophagy pathway, listing roles of ULK1 in autophagy initiation and downstream lysosome fusion.
- PD Parkinson’s Disease
- Figure 2A is a representation of a gene coexpression network computationally discovered in putamen tissue of PSP patients obtained postmortem.
- Figure 2B shows bidirectional dysregulation of this network in putamen tissue of PSP patients relative to neurotypical controls.
- FIG. 2C shows that the ULK1 gene is a member of this coexpression network. Protein interactions with network members suggest ULKl’s regulatory effect in PSP.
- Figures 3A and 3B show a normalized cell viability count versus the concentration of ULK1 activator, demonstrating that ULK1 activator rescue PD relevant toxicity.
- SH-SY5Y cells pre-treated with LYN- 1604 for 3 days, then LYN- 1604 with 200pM MPP+ (Fig. 3A) or lOOnM rotenone for 2 days (Fig. 3B).
- Figures 4A to 4C show cell viability (as a percent of control) versus ULK1 activator concentration, demonstrating that ULK1 activators rescue against MPP+ toxicity in SH-SY5Y cells. 24h preincubation with BL-918 (Fig.
- Figures 5A and 5B show the results of treating a fibroblast cell model of Parkinson’s disease with ULK1 activators.
- a 20h exposure with LYN-1604 increases autolysosome (Fig. 5A) and autophagosome (Fig. 5B) number in human dermal fibroblasts from control subjects (left bars) and in Parkinson’s disease patients carrying a G2019S allele in LRRK2, one of the most prevailing genetic mutations.
- FIGS. 6A to 6C demonstrate ULK1 activators enhance autophagy in iPSC derived motoneurons (iMNs) derived from healthy subjects.
- Fig. 6A shows iMNs untreated, treated with the positive control compound Apilimod, and treated with LYN- 1604.
- p62 and LAMP1 puncta colocalization marks the autolysosomal structures.
- Fig. 6B shows quantitative results with LYN- 1604 and Apilimod treatment increased the number of p62 positive autophagosomes.
- Fig. 6C shows LYN- 1604 and Apilimod treatment results in an increased number of autolysosomes.
- Chloroquine is a lysosomotropic agent used to visualize autophagic flux. Chloroquine treatment increases the number of autoplysosomes through increase of lysosomal pH of cells. Autolysosome number is further elevated with exposure to Apilimod and LYN- 1604 (Fig 6B,C). Thus, ULK1 activators increase autophagic flux.
- Figures 7A to 7C show a comparative analysis of iPSC derived motorneurons from healthy subject and amyotrophic lateral sclerosis (ALS) patient carrying a C9ORF72 expansion mutation.
- p62 and LAMP1 puncta colocalization marks the autolysosomal structures, and p62 the autophagosomes.
- Fig. 7A shows control and ALS mutant (c9orf72 expansion) motoneurons treated with LYN-1604.
- ALS mutant c9orf72 iMNs have a reduced number of p62 positive autophagosomes (Fig. 7B) and autolysosomes indicative of reduced autophagy (Fig. 7C).
- ULK1 activator LYN- 1604 enhances autophagy in healthy ALS motoneurons and show rescue effects in ALS diseased iPSC.
- AGC-kinases refers to the subgroup of Ser/Thr protein kinases named after three representative families, the cAMP-dependent protein kinase (PKA) family, the cGMP-dependent protein kinase (PKG) family, and the protein kinase C (PKC) family.
- PKA cAMP-dependent protein kinase
- PKG cGMP-dependent protein kinase
- PKC protein kinase C
- the AGC-kinases subgroup contains more than 60 human protein kinases which have been highly conserved throughout eukaryotic evolution and can be classified into 14 subfamilies.
- a unique feature of AGC kinases is the presence of a C-terminal segment containing a hydrophobic motif within the catalytic domain whereas the selectivity and specificity in the regulation of AGC kinases is predominantly derived from the regions located N- and C-terminal to the catalytic core.
- ULK1 or “Unc-51 Like Autophagy Activating Kinase 1”, refers to a protein coding gene. Among its related pathways are Autophagy and Mitophagy pathways and mTOR signaling. Gene Ontology (GO) annotations related to this gene include transferase activity, transferring phosphorus-containing groups and protein tyrosine kinase activity. A paralog of this gene is ULK2. Aliases for ULK1 include ATG1, ATG1 A, Serine/Threonine- Protein Kinase ULK1, Autophagy -Related Protein 1 Homolog, EC 2.7.11.1, HATG1, ATG1 Autophagy Related 1 Homolog (S.
- activator refers to an agent that increases or enhances the expression of ULK1 and/or activates ULK1.
- an activator of ULK1 enhances or increases the activity and/or expression level of ULK1.
- an activator may interact directly with ULK1, thereby activating ULK1.
- an activator of ULK1 may also activate a signaling pathway that is downstream of ULK1.
- an activator may instead interact with a protein that interacts with or affects ULK1, thereby activating ULK1 indirectly.
- treatment refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying, or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. Treat can be used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition and can contemplate a range of results directed to that end, including but not restricted to prevention of the condition entirely.
- an “effective amount” of an agent refers to the amount of the agent, at dosages and for periods of time necessary, sufficient to provide a beneficial effect or to otherwise reduce a detrimental non-beneficial event to the individual to whom the composition is administered.
- an agent described herein to a subject in need thereof depends on several factors including, for example but not limited to, the health of the subject, the specific disease or condition of the subject, the grade or level of a specific disease or condition of the subject, the additional therapeutics the subject is being or has been administered, and the like.
- a “subject” refers to any member of the animal kingdom.
- subject refers to humans.
- subject refers to non-human animals.
- subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms.
- the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a horse, a primate, and/or a pig).
- a subject may be a transgenic animal, genetically engineered animal, and/or a clone.
- the subject is an adult, an adolescent, or an infant.
- terms “individual” or “patient” are used and are intended to be interchangeable with “subject.”
- the phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
- ULK1 is a regulator of neurological pathways (Figs. 1A-1C and Figs. 2A-2C). While the autophagy pathways in the traditional sense are not PD or PSP specific, the gene expression network that has been computationally identified is specific to, although not limited to, PD and PSP. ULK1 is a hub of the Parkinson’s and PSP gene network and ULK1 activation is predicted to restore the Parkinson’s and PSP networks. (Fig. 1A). PD genetic scoring highlights ULK1 in the topranked 1% of PD targets. (Fig. 1C), and the top target in PSP (Fig. 2C) (See Nalls et al. 2019.
- Figs. 3-7) Provided herein are methods of treating a neurological disorder, the method comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1 (or “the ULK1 activator” hereinafter).
- the methods of treatment and compositions described herein are for use with a subject having or suspected of having a neurological disorder.
- the methods of treatment and compositions are for use with a subject having or suspected of having a neurodegenerative disease.
- the subject is human.
- the ULK1 activator is administered to a subject in need of treatment for a neurological disorder, and the ULK1 activator is able to bind to ULK1 or a homolog thereof and increase the expression of and/or activate ULK1 or a homolog thereof. In some embodiments, the activator activates ULK1 or a homolog thereof.
- the neurological disorder is a neurological disorder in which the expression and/or activity levels of ULK1 detected in the subject is lower than a healthy subject (i.e., a normal control).
- ULK1 is a 112-kDa protein. It contains a N-terminal kinase domain, a serine-proline rich region, and a C-terminal interacting domain.
- the serine-proline rich region has been shown experimentally to be the site of phosphorylation by mTORCl and AMPK — a negative and positive regulator of ULK1 activity, respectively.
- the C-terminal domain contains two microtubule-interacting and transport (MIT) domains and acts as a scaffold which links ULK1, ATG13, and FIFP200 together to form a complex that is essential to initiate autophagy.
- MIT microtubule-interacting and transport
- MIT domains Early autophagy targeting/tethering (EAT) domains in the C-terminus are arranged as MIT domains consisting of two three-helix bundles. MIT domains also mediate interactions with membranes.
- the N-terminus contains a serine-threonine kinase domain.
- ULK1 also contains a large activation loop between the N and C terminus that is positively charged. This region may regulate kinase activity and play a role in recognizing different substrates.
- ULK1 and ULK2 share significant homology in both the C-terminal and N-terminal domains.
- ULK1 is phosphorylated by AMPK on Ser317 and Ser777 to activate autophagy; mTOR participates in inhibitory phosphorylation of ULK1 on Ser757. Additionally, ULK1 can autophosphorylate itself at Thrl80 to facilitate self-activation. Phosphorylation of ULK1-S555 by AMPK allows ULK1 to translocate to mitochondria.
- the neurological disorder is Parkinson’s Disease (PD), Parkinson’s Disease with Lewy bodies, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), primary lateral sclerosis (PLS), Charcot-Marie-Tooth (CMT; including type 4J (CMT4J)), and Yunis-Varon syndrome, autophagy, polymicrogyria (including polymicrogyria with seizures), temporo-occipital polymicrogyria, Pick’s disease, dementia with Lewy bodies, Lewy body disease, diseases of neuronal nuclear inclusions of polyglutamine and intranuclear inclusion bodies, disease of Marinesco and Hirano bodies, tauopathy, Alzheimer’s disease, neurodegeneration, spongiform neurodegeneration, peripheral neuropathy, leukoencephalopathy, motor neuropathy, sensory neuropathy, abnormal lysosomal storage syndrome, myotubular myopathy, muscle weakness
- the neurological disorder is a neurodegenerative disease.
- the neurodegenerative disease is a central nervous system (CNS) neurodegenerative disease.
- CNS central nervous system
- the neurodegenerative disease include, but are not limited to, PD, PSP, ALS, FID, Alzheimer’s disease, Huntington’s disease, prion disease, Lewy body disease, Friedreich’s ataxia, or spinal muscular atrophy.
- the neurodegenerative disease is PD.
- the neurological disorder or neurodegenerative disease is PSP.
- the neurodegenerative disease is ALS.
- the neurodegenerative disease is Alzheimer’s Disease.
- the neurological disorder or neurodegenerative disease is PD.
- a method of treating PD comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1. In some embodiments, the effective amount of said agent activates ULK1.
- the neurological disorder or neurodegenerative disease is ALS.
- a method of treating ALS comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1. In some embodiments, the effective amount of said agent activates ULK1.
- the neurological disorder or neurodegenerative disease is PSP.
- a method of treating PSP comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1.
- the effective amount of said agent activates ULK1.
- An ULK1 activator disclosed herein may have an IC50, Ki, or Kd of less than 500 nM when determined at a physiologically relevant concentration of adenosine triphosphate (ATP).
- ATP adenosine triphosphate
- the ULK1 activator is a small molecule, an antibody or active fragment thereof, a peptide, a proteolysis targeting chimera (PROTAC), a complementary DNA (cDNA).
- the antibody is a humanized antibody or active fragment thereof.
- the PROTAC comprises a protein of interest (POI) ligand and an E3 ubiquitin ligase (E3) recruiting ligand, wherein the POI ligand and the E3 are linked.
- the ULK1 activator is a small molecule. Also encompassed are approaches that genetically modify a cell to increase ULK1 levels, such as, for example, AAV gene therapy, cDNA therapy, and gene editing such as CRISPR.
- the ULK1 activator binds to ULK1.
- the ULK1 activator is an irreversible activator of ULK1.
- the irreversibility leads to a permanent activation of the action of an enzyme. In some embodiments this can be achieved by tight binding of the activator to the protein, conferring activation.
- activation of ULK1 results in increased expression of ULK1.
- activation of ULK1 results in an increased expression level of ULK1, which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control.
- activation of ULK1 results in increased activity of ULK1.
- activation of ULK1 results in increased activity of ULK1 that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control.
- the control can be determined by those of skill in the art as applicable to the particular situation.
- the control is an industry standard agreed upon by those of skill as being a level or range of levels that is typical of a subject having a neurological disorder before the treatment.
- the control is a reference level of ULK1 from the same individual taken at a time point before the treatment and whether the subject has increased level of ULK1 after the treatment is determined based on a sample from that same individual taken before the treatment.
- levels of ULK1 are measured in the in vitro or in vivo systems.
- levels of ULK1 are measured in cells, e.g., motor neuronal cells, in plasma, or in cell culture media.
- levels of ULK1 are measured from a plasma sample.
- levels of ULK1 are measured from a serum sample.
- the ULK1 activator described herein may be for use in treating a neurological disorder in a subject in need thereof. In some embodiments, the ULK1 activator described herein may be for use in treating a neurodegenerative disease in a subject in need thereof. Examples of the neurological disorders and neurodegenerative diseases are disclosed herein.
- the neurological disorder is a neurological disorder in which the expression and/or activity levels of ULK1 detected in a subject is higher than a normal control.
- the activity level of ULK1 is measured or determined by the change in binding to a partner protein, such as STX17, SNAP29 or VAMP8, BNIP3 and Parkin.
- the activity level of ULK1 is measured or determined by the extent of protein phosphorylation by ULK1.
- the activity level of ULK1 is measured or determined by the extent of protein phosphorylation of a ULK1 substrate, such as ATG13, In some embodiments, the extent of protein phosphorylation by ULK1 in a subject having a neurological disorder is different than the extent of protein phosphorylation by ULK1 in a normal control. In some embodiments, the extent of protein phosphorylation by ULK1 in a subject having a neurological disorder is higher than in a normal control. In some embodiments, the activity level of ULK1 is measured or determined by a change in a cellular phenotype, such as autophagy or mitophagy. In some embodiments, the cellular phenotype, such as autophagy or mitophagy, is increased.
- the normal control can be determined by those of skill in the art as applicable to the particular situation.
- the normal control is an industry standard agreed upon by those of skill as being a level or range of levels that is typical of an individual without an ULK1- associated condition.
- the normal control is a reference level of ULK1 from the same individual taken at a time point and whether the subject has elevated ULK1 is determined based on a sample from that same individual taken at a different, typically later, time point.
- compositions that are useful in the methods described herein include any agent that activates ULK1 or increases ULK1 expression (i.e., an ULK1 activator).
- compositions may further comprise a pharmaceutically acceptable carrier.
- Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such
- agents that increase the expression of and/or activate ULK1 for use in the manufacture of a medicament for treating a neurological disorder in a subject in need thereof are provided, optionally wherein the neurological disorder is PD, PSP or ALS.
- the ULK1 activator (or a pharmaceutical composition comprising the same described herein) of the present disclosure is administered in an effective amount by any of the accepted modes of administration for agents that serve similar utilities. Effective amounts of will depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound being utilized, the route and form of administration, and other factors.
- the ULK1 activator (or a pharmaceutical composition comprising the same) described herein that can be used in treatment can be formulated and dosages established in a fashion consistent with good medical practice taking into account the disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners.
- administration of the compounds or pharmaceutical compositions can include routes of administration, non-limiting examples of administration routes include intravenous, intraarterial, subcutaneous, subdural, intramuscular, intracranial, intrastemal, intratumoral, or intraperitoneally.
- routes of administration include intravenous, intraarterial, subcutaneous, subdural, intramuscular, intracranial, intrastemal, intratumoral, or intraperitoneally.
- a pharmaceutical composition or compound can be administered to a subject by additional routes of administration, for example, by inhalation, oral, dermal, intranasal, or intrathecal administration. The appropriate formulation and route of administration may be selected according to the intended application.
- compositions or agents of the present disclosure can be administered to a subject in need thereof in a first administration, and in one or more additional administrations.
- the one or more additional administrations can be administered to the subject in need thereof minutes, hours, days, weeks, or months following the first administration. Any one of the additional administrations can be administered to the subject in need thereof less than 21 days, or less than 14 days, less than 10 days, less than 7 days, less than 4 days or less than 1 day after the first administration.
- the one or more administrations can occur more than once per day, more than once per week, or more than once per month.
- the agents or pharmaceutical compositions can be administered to the subject in need thereof in cycles of 21 days, 14 days, 10 days, 7 days, 4 days, or daily over a period of one to seven days (e.g., daily over a period of 1, 2, 3, 4, 5, 6, or 7 days).
- a co-expression module enriched for genes associated with PD was identified. Specifically, gene expression was measured from Substantia nigra and dorsolateral prefrontal cortex tissue from a patient cohort collected by that included 207 Parkinson’s patient and 118 non-neurological controls. This data was combined with gene expression from five publicly-available bulk brain datasets from PD patients (NIH GEO accession numbers GSE20163, GSE20292, GSE7621, GSE20164, and GSE20168). Taken together, these gene expression data were used to identify a gene co-expression network, using the methods disclosed in Langfelder and Horvath, BMC Bioinformatics, 9:559 (2008).
- Module #6 (a.k.a. 8946-6) was identified as being enriched for genes associated with PD using genome-wide association studies (GWAS), transcriptome-wide association studies (TWAS), or related studies.
- GWAS genome-wide association studies
- TWAS transcriptome-wide association studies
- ULK1 was identified using an approach to predict genes that regulate the expression levels of genes in network 8946-6, described in Example 1. Overall, 16,051 human genes were scored according to their co-expression with network 8946-6 and their known genetic association with neurodegenerative indications.
- ULK1 ranks #434-th out of 16,051 for its correlation to the eigengene defining network 8946-6, where correlation is measured according to the methods described by Langefelder and Horvath (Example 1).
- ULK1 After summing the genetic and network ranks into a meta-rank , ULK1 ranks #42 overall out of 16,051 genes. After filtering this list for genes that can be drugged, ULK1 was prioritized as a top candidate target for Parkinson’s therapeutic development.
- Example 3 Identification of Co-expression Modules progressive supranuclear palsy (PSP [0098] Gene expression was measured by RNA-seq of cryopreserved post-mortem putamen tissue from a patient cohort that included 28 PSP patients and 22 neurotypical controls. A gene coexpression network of 113 genes, referred to as module 14239.42.5.11, was identified that exhibited bidirectional dysregulation in PSP patient tissue compared to controls. The module is enriched for familial PSP risk genes (Fig. 2A).
- ULK1 Ranking all detected human genes by strength of evidence to associate with network 14239.42.5.11 and filtering for genes encoding proteins that have existing small molecule ligands, ULK1 ranks #1 of 14,789 genes, and was therefore prioritized as a candidate drug target for PSP (Fig. 2C).
- Figs. 3A and 3B show results of experiments with SH-SY5Y cells pre-treated with LYN-1604 for 3 days, then LYN-1604 with 200pM MPP+ (Fig. 3A) or lOOnM rotenone (Fig. 3B) for 2 additional days.
- SH-SY5Y cells were differentiated for 5 days in DMEM-F12 media with 5% FBS (Fetal bovine serum ) with retinoic acid, then split into a Poly-D lysine (PDL) coated 96 well plate at 30,000 cells per well.
- FBS Fetal bovine serum
- Fig. 4A shows results of experiments with undifferentiated SH-SY5Y cells seeded at 40,000 cells/well in 96w plate for 24hrs.
- SH-SY5Y cells were preincubated with ULK1 activator BL-918 for 6hrs, followed with 48hrs incubation of 250pM mitochondrial toxins MPP+ in the continuous presence of BL918.
- Cell viabilities were measured in an absorbance based platereader assay using the CellTiter-Glo reagent. Data are mean of 3 technical replicates ⁇ SEM. These data suggest that ULK1 activation can protect from death induced by PD associated toxins.
- Figs. 4B and 4C show results of experiments with differentiated SH-SY5Y neurons
- SH-SY5Y cells were seeded at 30,000 cells/well in 96w plate and differentiated for 12 days. They were then pre-incubated for 2hrs with ULK1 activators BL-918 (Fig. 4B) or LYN-1604 (Fig. 4C) followed with 48hrs incubation of 200 pM mitochondrial toxins MPP+. Cell viabilities were measured in an absorbance based plate-reader assay using the CellTiter-Glo reagent. Data are mean of 3 technical replicates ⁇ SEM.
- Example 5 ULK1 activator LYN-1604 enhances autophagy in fibroblast from healthy and PD patients expressing the LRRK2 G2019S (LRRK2 GS) mutation
- Fig. 5A shows quantification of autolysosomes measured as Lampl and p62 colocalization area per fibroblast treated for 20 hours with different concentrations of the ULK1 activator LYN- 1604.
- Fig. 5B shows quantification of autophagosomes measured as p62 area per fibroblast treated for 20 hours with different concentrations of the ULK1 activator LYN-1604.
- This experiment shows that autophagy can be induced in cells derived from healthy and PD patients.
- Example 6 ULK1 activator LYN-1604 enhances autophagy in iPSC derived motoneurons from healthy subjects
- Fig. 6A shows iPSC derived motoneurons (iMNs) derived from healthy subjects were seeded at 20,000 cells/well in 96w plate and grown for 7 days.
- ULK1 activator LYN-1604 (4 pM) was added on day 8 for 48 hr followed by 20 hr Chloroquine (25 pM) treatment.
- IpM PIKFyve inhibitor Apilimod
- Cells were fixed with 4% paraformaldehyde (PF A) and 4% Sucrose on day 10 and stained for lysosomal marker LAMP1, autophagosomal marker p62 and motoneuron marker Hb9.
- p62 and LAMP1 puncta colocalization marks the autolysosomal structures.
- Chloroquine treatment inhibits autophagic flux by blocking lysosomal degradation and thereby revealing the amount of basal autophagy.
- Fig. 6B shows LYN- 1604 and Apilimod treatment increased the number of p62 positive autophagosomes.
- Chloroquine treatment shows both LYN1604 and Apilimod increases basal autophagy.
- LYN-1604 and Apilimod treatment also increased number of autolysosomes indicative of increased autophagy. Data are shown as mean ⁇ SD.
- Example 7 ULK1 activator LYN-1604 enhances autophagy in ALS patient derived motoneurons
- Fig. 7A shows control and ALS mutant (carrying a c9orf72 expansion) motoneurons seeded at 20,000 cells/well in 96w plate and grown for 8 days.
- ULK1 activator LYN-1604 (4 pM) was then added for 48 hr.
- Cells were fixed with 4% PFA & 4% Sucrose on day 10 and stained for lysosomal marker LAMP1, autophagosomal marker p62 and nuclear marker DAPI.
- p62 and LAMP1 puncta colocalization marks the autolysosomal structures.
- ALS c9orf72 mutant iMNs have reduced number of p62 positive autophagosomes (Fig.
- LYN- 1604 treatment increased the number of autophagosomes and autolysosomes in control iMNs and rescued autophagy in c9orf72 mutant iMNs. Data are shown as mean ⁇ SD.
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Abstract
The present disclosure relates to methods for treating a neurological disorder, such as Parkinson's Disease (PD), progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), and Alzheimer's Disease. The methods include administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1.
Description
METHODS OF TREATING NEUROLOGICAL DISORDERS WITH ACTIVATORS OF UNC51-LIKE AUTOPHAGY ACTIVATING KINASE 1 (ULK1)
Cross-Reference to Related Applications
[001] This application claims priority to U.S. Provisional Application No. 63/394,092, filed on August 1, 2022, the disclosure of which is hereby incorporated by reference in its entirety.
Technical Field
[002] The present disclosure provides methods for treating neurological disorders, in particular, neurodegenerative diseases. Compositions useful in the methods described herein include agents that increase the expression of and/or activate Unc51-like autophagy activating kinase 1 (ULK1).
Introduction and Summary
[003] ULK1 (Unc51-like autophagy activating kinase 1) is the serine/threonine protein kinase of the ULK1 complex, and along with Unc-51 like autophagy activating kinase 2 (ULK2), is a critical regulatory node that induces autophagy through phosphorylation of downstream targets. ULK1 is known to be essential for autophagy initiation under nutrient deprivation conditions (Lazarus et al. 2015. “Structure of the Human Autophagy Initiating Kinase ULK1 in Complex with Potent Inhibitors.” ACS Chemical Biology 10 (1): 257-61). Notably, ULK1 phosphorylates Beclin-1 which activates the PIK3 class 3 (PIK3C3/VPS34) lipid kinase complex necessary for full autophagic induction and autophagosome maturation (Russell et al. 2013. “ULK1 Induces Autophagy by Phosphorylating Beclin-1 and Activating VPS34 Lipid Kinase.” Nature Cell Biology 15 (7): 741-50). ULK1 interacts with ATG13, ATG101, and FIP200 to form a complex critical for the initiation of autophagy. ULK1 may have an additional role in promoting autophagosome-lysosome fusion via STX17-SNAP29 (See Figure 1, Hansen et al. 2018. “Autophagy as a promoter of longevity: insights from model organisms.” Nat Rev. Mol. Biol. 19: 579-593). ULK1 promotes mitophagy via phosphorylation and stabilization of BNIP3 - LC3 complexes (Poole et al. 2021. “ULK1 promotes mitophagy via phosphorylation and stabilization of BNIP3.” Scientific Reports 11 :20526) and phosphorylation of Parkin (Hung et al. 2021. “AMPK/ULK1 -Mediated Phosphorylation of Parkin ACT Domain Mediates an Early Step in Mitophagy.” Science Advances 7 (15): 1-15.).
[004] The activity of ULK1 is regulated by phosphorylation by mTORCl (mammalian target of rapamycin complex 1) and AMPK (AMP-activated protein kinase). For instance, mTORCl phosphorylates ULK1 at S757 to inhibit autophagy. Cellular stress or nutrient deprivation inhibits mTORC, relieving inhibition from ULK1, and activates AMPK to induce ULK1 phosphorylation at other sites to induce autophagy (Zhu et al. 2019. “Balancing MTOR Signaling and Autophagy in the Treatment of Parkinson’s Disease.” International Journal of Molecular Sciences 20 (3): 1-15; Kim et al. 2011. “AMPK and mTOR regulate autophagy through direct phosphorylation of Ulkl.” Nature Cell Biology 13(2): 132-141).
[005] ULK1 is in complex with C9ORF72 and coordinates autophagy with TBK1, two genes associated with amyotrophic lateral sclerosis (ALS). C9ORF72 regulates expression and activity of ULK1 (Yang et al. 2016. “A C9ORF72/SMCR8- containing complex regulates ULK1 and plays a dual role in autophagy.” Science Advances 2(9): el601167)
[006] ULK1 also plays a role in mitophagy (Egan et al. 2011. “Phosphorylation of ULK1 (HATG1) by AMP -Activated Protein Kinase Connects Energy Sensing to Mitophagy.” Science 331 (6016): 456-61.). After mitochondrial damage, ULK1 rapidly phosphorylates and activates Parkin, a core ubiquitin ligase in mitophagy. Mutation of Parkin phospho-sites, ULK1 depletion, or ULK1 inhibition cause deficits in mitophagy (Hung et al. 2021. “AMPK/ULK1 -Mediated Phosphorylation of Parkin ACT Domain Mediates an Early Step in Mitophagy.” Science Advances 7 (15): 1-15.). Translocation of ULK1 to mitochondria is dependent upon phosphorylation at Ser555 by AMPK, and is necessary for mitophagy in response to hypoxia (Tian et al. 2015. “Phosphorylation of ULK1 by AMPK Regulates Translocation of ULK1 to Mitochondria and Mitophagy.” FEBS Letters 589 (15): 1847-54.). ULK1 is recruited to damaged mitochondria by autophagy receptors NDP52 (also known as calcium binding and coiled-coil domain 2 (CALCOCO2) and OPTN (optineurin), which are recruited by ubiquitin kinase PINK1 (Lazarou et al. 2015. “The Ubiquitin Kinase PINK1 Recruits Autophagy Receptors to Induce Mitophagy.” Nature 524 (7565): 309-14). After translocation to damaged mitochondria, ULK1 phosphorylated mitochondrial membrane proteins FUNDCI (FUN14 domain containing 1), an integral mitochondrial outer-membrane protein (and a receptor for hypoxia-induced mitophagy) and BNIP3 (Poole et al. 2021. “ULK1 promotes mitophagy via phosphorylation and stabilization of BNIP3.” Scientific Reports 11 :20526), to facilitate its interaction with LC3 (light chain 3) and promote mitophagy (Wu et al. 2014. “ULK1 Translocates to Mitochondria and Phosphorylates FUNDCI to Regulate Mitophagy.” EMBO Reports 15 (5): 566-75). FUNDCI is an integral mitochondrial outer-membrane protein and a
receptor for hypoxia-induced mitophagy. BNIP3 locates to the mitochondrial outer membrane, modulating membrane potential, where it may also interact with pro-apoptotic factors such as BAX, BAK and BCL2 (Zhang & Ney 2009. “Role of BNIP3 and NIX in cell death, autophagy, and mitophagy.” Cell Death & Differntiation 16: 939-946.)
[007] ULK1 activation is protective in several Parkinson disease (PD) models by activation of autophagy. For example, ULK1 activates starvation-induced autophagy in SH-SY5Y cells, a process which is regulated by two microRNAs (Chen et al. 2015. “Identification of ULK1 as a Novel Biomarker Involved in MIR-4487 and MIR-595 Regulation in Neuroblastoma SH-SY5Y Cell Autophagy.” Scientific Reports 5 (January): 1-10). Small molecule activators ofULKl, 33i (BL-918) and LYN1604, induce autophagy, which is neuroprotective in MPP treated SH-SY5Y cells. (Zhang et al. 2017. “Discovery of a small molecule targeting ULK1 -modulated cell death of triple negative breast cancer in vitro and in vivo ” Chemical Science 8(4):2687-2701.
[008] They also rescue dopaminergic neuron loss and motor dysfunction through autophagy activation in an MPTP (l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine) mouse model (Ouyang et al. 2018. “Small-Molecule Activator of UNC-51-Like Kinase 1 (ULK1) That Induces Cytoprotective Autophagy for Parkinson’s Disease Treatment.” Journal of Medicinal Chemistry 61 (7): 2776-92). ULK1 expression protects PC-12 cells from hypoxia-induced apoptosis by promoting autophagy (Wang et al. 2018. “Ulkl/FUNDCl Prevents Nerve Cells from Hypoxia- Induced Apoptosis by Promoting Cell Autophagy.” Neurochemical Research 43 (8): 1539-48). [009] ULK1 is negatively regulated by ubiquitin specific peptidase-24 (USP24), a Parkinson disease 10 (PARK10) locus gene, and inhibition of USP24 improves neurite arborization in aged induced pluripotent stem cell-derived dopaminergic (iPSC-DA) neurons. ULK1 also protects mouse embryonic fibroblasts (MEFs) against paraquat toxicity (Anandhan et al. 2017. “Glucose Metabolism and AMPK Signaling Regulate Dopaminergic Cell Death Induced by Gene (a- Synuclein)-Environment (Paraquat) Interactions.” Molecular Neurobiology 54 (5): 3825-42). [0010] Clearance of alpha-synuclein (a-syn), a neuronal protein that may contribute to PD pathogenesis, can be induced by several molecules that act through ULK1. GM1 (monosialotetrahexosylganglioside), a ganglioside, protects MPTP treated mice from motor dysfunction and increased dopamine; and rescues a-syn accumulation, mitochondrial dysfunction, and oxidative stress in MPP+ treated SH-SY5Y cells and a-syn A53T overexpressing PC12 cells via induction of autophagy through ULK1 (Guo et al. 2021. “Autophagy-Dependent Removal of a-Synuclein: A Novel Mechanism of GM1 Ganglioside Neuroprotection against Parkinson’ s Disease.” Acta Pharmacologica Sinica 42 (4): 518-28).
Urate induces autophagy by mTOR/ULKl to degrade a-syn (Sheng et al. 2017. “Urate Promotes SNCA/a-Synuclein Clearance via Regulating MTOR-Dependent Macroautophagy.” Experimental Neurology 297 (July): 138-47).
[0011] Although the role of mTOR mediated autophagy is complex (Norwitz et al. 2020. “MTOR Mysteries: Nuances and Questions About the Mechanistic Target of Rapamycin in Neurodegeneration.” Frontiers in Neuroscience 14 (July): 1-10), it is believed that promoting autophagy and mitophagy through activating or overexpression of ULK1 could be therapeutic for PD patients. Furthermore, ULK1 plays a role in mitophagy in a shared pathway with other PD and ALS targets, including Parkin, PINK1, and OPTN (optineurin), further supporting the hypothesis that increasing ULK1 activity may be beneficial.
[0012] The present disclosure is based in part on the findings that ULK1 is a regulator of neurological pathways, such as involved in PD (See, e.g., Figs 1A-1C), progressive supranuclear palsy (PSP) (Figs 2A-2C) and ALS (Figs 6A-6B and 7A-7C). Accordingly, in some embodiments, methods of treating a neurological disorder, such as a neurodegenerative disease, are provided, wherein the methods comprise administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1.
[0013] In some embodiments, the following embodiments are provided.
[0014] Embodiment l is a method of treating a neurological disorder, the method comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1.
[0015] Embodiment 2 is the method of embodiment 1, wherein the neurological disorder is a neurological disorder in which the expression and/or activity levels of ULK1 detected in the subject is different from a normal control.
[0016] Embodiment 3 is the method of embodiment 1 or 2, wherein the agent increases the expression of ULK1.
[0017] Embodiment 4 is the method of embodiment 1 or 2, wherein the agent activates ULK1. [0018] Embodiment 5 is the method of any one of embodiments 1 to 4, wherein the agent is a small molecule, an antibody or active fragment thereof, a peptide, or PROTAC.
[0019] Embodiment 6 is the method of any one of embodiments 1 to 5, wherein the agent is a small molecule.
[0020] Embodiment 7 is the method of embodiment 6, wherein the increased expression level of ULK1 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control.
[0021] Embodiment 8 is the method of embodiment 7, wherein the increased activity of ULK1 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control.
[0022] Embodiment 9 is the method of any one of embodiments 1 to 8, wherein the neurological disorder is Parkinson’s Disease (PD), Parkinson’s Disease with Lewy bodies, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), primary lateral sclerosis (PLS), Charcot-Marie-Tooth (CMT; including type 4J (CMT4J)), and Yunis-Varon syndrome, autophagy, polymicrogyria (including polymicrogyria with seizures), temporo-occipital polymicrogyria, Pick’s disease, dementia with Lewy bodies, Lewy body disease, diseases of neuronal nuclear inclusions of polyglutamine and intranuclear inclusion bodies, disease of Marinesco and Hirano bodies, tauopathy, Alzheimer’s disease, neurodegeneration, spongiform neurodegeneration, peripheral neuropathy, leukoencephalopathy, motor neuropathy, sensory neuropathy, abnormal lysosomal storage syndrome, myotubular myopathy, muscle weakness, cleidocranial dysplasia, Lewy body disease, inclusion body disease, corticobasal syndrome, chronic traumatic encephalopathy, traumatic brain injury (TBI), cerebral ischemia, Guillain-Barre Syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, a lysosomal storage disease, Fabry’s disorder, Gaucher’s disorder, Niemann Pick C disease, Tay-Sachs disease, and Mucolipidosis type IV, neuropathy, Coffin-Lowry Syndrome (CLS), X-linked mental retardation disorder (XLMR), intellectual disability, Huntington’s disease, a psychiatric disorder, ADHD, schizophrenia, a mood disorder, major depressive disorder, depression, bipolar disorder I, or bipolar disorder II.
[0023] Embodiment 10 is the method of any one of embodiments 1 to 9, wherein the neurological disorder is a neurodegenerative disease.
[0024] Embodiment 11 is the method of embodiment 10, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), Alzheimer’s disease, Parkinson’s disease (PD), Huntington’s disease, prion disease, Lewy body disease, Friedreich’s ataxia, or spinal muscular atrophy.
[0025] Embodiment 12 is the method of embodiments 10 or 11, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
[0026] Embodiment 13 is the method of embodiments 10 or 11, wherein the neurodegenerative disease is progressive supranuclear palsy (PSP).
[0027] Embodiment 14 is the method of embodiments 10 or 11, wherein the neurodegenerative disease is Alzheimer’s disease.
[0028] Embodiment 15 is the method of embodiments 10 or 11, wherein the neurodegenerative disease is Parkinson’s Disease (PD).
[0029] Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the subject is a human.
[0030] Embodiment 17 is the method of embodiment 16, wherein the neurological disorder is a neurodegenerative disease.
[0031] Embodiment 18 is a composition comprising an agent that increases the expression of and/or activates ULK1 for use in treating a neurological disorder in a subject in need thereof. [0032] Embodiment 19 is the composition of embodiment 18, further comprising a pharmaceutically acceptable carrier.
[0033] Embodiment 20 is the use of an agent that increases the expression of and/or activates ULK1 in the manufacture of a medicament for treating a neurological disorder in a subject in need thereof.
Brief Description of the Figures
[0034] Figure 1A shows a representation of ULK1 involvement in regulating the gene expression network identified in brains from Parkinson’s disease (PD) patients
[0035] Figure IB shows a representation of the regulatory effects of the knockdown of ULK1 in mouse fibroblasts.
[0036] Figure 1C shows ULK1 ranking in a genetic scoring method.
[0037] Figure ID shows a representation of the convergence of genetic risk factors of Parkinson’s Disease (PD) on the ULKl-mediated autophagy pathway, listing roles of ULK1 in autophagy initiation and downstream lysosome fusion.
[0038] Figure 2A is a representation of a gene coexpression network computationally discovered in putamen tissue of PSP patients obtained postmortem.
[0039] Figure 2B shows bidirectional dysregulation of this network in putamen tissue of PSP patients relative to neurotypical controls.
[0040] Figure 2C shows that the ULK1 gene is a member of this coexpression network. Protein interactions with network members suggest ULKl’s regulatory effect in PSP.
[0041] Figures 3A and 3B show a normalized cell viability count versus the concentration of ULK1 activator, demonstrating that ULK1 activator rescue PD relevant toxicity. SH-SY5Y cells pre-treated with LYN- 1604 for 3 days, then LYN- 1604 with 200pM MPP+ (Fig. 3A) or lOOnM rotenone for 2 days (Fig. 3B).
[0042] Figures 4A to 4C show cell viability (as a percent of control) versus ULK1 activator concentration, demonstrating that ULK1 activators rescue against MPP+ toxicity in SH-SY5Y cells. 24h preincubation with BL-918 (Fig. 4A), and 2h preincubation with BL-918 (Fig. 4B) or LYN-1604 (Fig. 4C), followed with incubation of 250pM mitochondrial toxins MPP+ shows increase in cell number relative to control cells not exposed to ULK1 activators.
[0043] Figures 5A and 5B show the results of treating a fibroblast cell model of Parkinson’s disease with ULK1 activators. A 20h exposure with LYN-1604 increases autolysosome (Fig. 5A) and autophagosome (Fig. 5B) number in human dermal fibroblasts from control subjects (left bars) and in Parkinson’s disease patients carrying a G2019S allele in LRRK2, one of the most prevailing genetic mutations.
[0044] Figures 6A to 6C demonstrate ULK1 activators enhance autophagy in iPSC derived motoneurons (iMNs) derived from healthy subjects. Fig. 6A shows iMNs untreated, treated with the positive control compound Apilimod, and treated with LYN- 1604. p62 and LAMP1 puncta colocalization marks the autolysosomal structures. Fig. 6B shows quantitative results with LYN- 1604 and Apilimod treatment increased the number of p62 positive autophagosomes. Fig. 6C shows LYN- 1604 and Apilimod treatment results in an increased number of autolysosomes.
[0045] Chloroquine is a lysosomotropic agent used to visualize autophagic flux. Chloroquine treatment increases the number of autoplysosomes through increase of lysosomal pH of cells. Autolysosome number is further elevated with exposure to Apilimod and LYN- 1604 (Fig 6B,C). Thus, ULK1 activators increase autophagic flux.
[0046] Figures 7A to 7C show a comparative analysis of iPSC derived motorneurons from healthy subject and amyotrophic lateral sclerosis (ALS) patient carrying a C9ORF72 expansion mutation. p62 and LAMP1 puncta colocalization marks the autolysosomal structures, and p62 the autophagosomes. Fig. 7A shows control and ALS mutant (c9orf72 expansion) motoneurons treated with LYN-1604. ALS mutant c9orf72 iMNs have a reduced number of p62 positive autophagosomes (Fig. 7B) and autolysosomes indicative of reduced autophagy (Fig. 7C). ULK1 activator LYN- 1604 enhances autophagy in healthy ALS motoneurons and show rescue effects in ALS diseased iPSC.
Detailed Description
[0047] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be
described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.
[0048] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any literature incorporated by reference contradicts any term defined in this specification, this specification controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
A. Definitions
[0049] Unless otherwise stated, the following terms used in the specification and claims are defined for the purposes of this disclosure and have the following meanings.
[0050] As used herein, “ AGC-kinases” refers to the subgroup of Ser/Thr protein kinases named after three representative families, the cAMP-dependent protein kinase (PKA) family, the cGMP-dependent protein kinase (PKG) family, and the protein kinase C (PKC) family. The AGC-kinases subgroup contains more than 60 human protein kinases which have been highly conserved throughout eukaryotic evolution and can be classified into 14 subfamilies. A unique feature of AGC kinases is the presence of a C-terminal segment containing a hydrophobic motif within the catalytic domain whereas the selectivity and specificity in the regulation of AGC kinases is predominantly derived from the regions located N- and C-terminal to the catalytic core.
[0051] As used herein, “ULK1” or “Unc-51 Like Autophagy Activating Kinase 1”, refers to a protein coding gene. Among its related pathways are Autophagy and Mitophagy pathways and mTOR signaling. Gene Ontology (GO) annotations related to this gene include transferase activity, transferring phosphorus-containing groups and protein tyrosine kinase activity. A paralog of this gene is ULK2. Aliases for ULK1 include ATG1, ATG1 A, Serine/Threonine- Protein Kinase ULK1, Autophagy -Related Protein 1 Homolog, EC 2.7.11.1, HATG1, ATG1 Autophagy Related 1 Homolog (S. Cerevisiae), Unc-51 (C. Elegans)-Like Kinase 1, Unc-51- Like Kinase 1 (C. Elegans), ATG1 Autophagy Related 1 Homolog, Unc-51 -Like Kinase 1, EC 2.7.11, KIAA0722, Unc51.1, and UNC51.
[0052] As used herein, “activator” refers to an agent that increases or enhances the expression of ULK1 and/or activates ULK1. For example, an activator of ULK1 enhances or increases the activity and/or expression level of ULK1. In one example, an activator may interact directly with ULK1, thereby activating ULK1. In this way, an activator of ULK1 may also activate a signaling pathway that is downstream of ULK1. In another example, an activator may instead interact with a protein that interacts with or affects ULK1, thereby activating ULK1 indirectly. [0053] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying, or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. Treat can be used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition and can contemplate a range of results directed to that end, including but not restricted to prevention of the condition entirely.
[0054] As used herein, an “effective amount” of an agent refers to the amount of the agent, at dosages and for periods of time necessary, sufficient to provide a beneficial effect or to otherwise reduce a detrimental non-beneficial event to the individual to whom the composition is administered. One of ordinary skill in the art would understand that the amount, duration, and frequency of administration of an agent described herein to a subject in need thereof depends on several factors including, for example but not limited to, the health of the subject, the specific disease or condition of the subject, the grade or level of a specific disease or condition of the subject, the additional therapeutics the subject is being or has been administered, and the like. [0055] As used herein, a “subject” refers to any member of the animal kingdom. In some embodiments, subject refers to humans. In some embodiments, subject refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In certain embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a horse, a primate, and/or a pig). In some embodiments, a subject may be a transgenic animal, genetically engineered animal, and/or a clone. In certain embodiments, the subject is an adult, an adolescent, or an infant. In some embodiments, terms “individual” or “patient” are used and are intended to be interchangeable with “subject.”
[0056] As used herein, the phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of
sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0057] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. [0058] It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a conjugate” includes a plurality of conjugates and reference to “a cell” includes a plurality of cells and the like.
[0059] “Optional” or “optionally” means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.
[0060] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings. [0061] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).
[0062] The terms “or a combination thereof’ and “or combinations thereof’ as used herein refers to any and all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, and so forth.
The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
[0063] “ Or” is used in the inclusive sense, i.e., equivalent to “and/or,” unless the context requires otherwise.
B. Methods of Treatment
[0064] The present disclosure is based in part on the findings that ULK1 is a regulator of neurological pathways (Figs. 1A-1C and Figs. 2A-2C). While the autophagy pathways in the traditional sense are not PD or PSP specific, the gene expression network that has been computationally identified is specific to, although not limited to, PD and PSP. ULK1 is a hub of the Parkinson’s and PSP gene network and ULK1 activation is predicted to restore the Parkinson’s and PSP networks. (Fig. 1A). PD genetic scoring highlights ULK1 in the topranked 1% of PD targets. (Fig. 1C), and the top target in PSP (Fig. 2C) (See Nalls et al. 2019. “Identification of novel risk loci, causal insights, and heritable risk for Parkinson’s disease: a meta-genome wide association study” Lancet Neurol. 18(12): 1091-1102 ; Alfradique-Dunham et al., 2021. “Genome-Wide Association Study Meta-Analysis for Parkinson Disease Motor Subtypes” Neurol Genet 7:e557; and Iwaki et al., 2019 "Genome-Wide Association Study of Parkinson’s Disease Clinical Biomarkers in 12 Longitudinal Patients’ Cohorts” Mov Disord. 34(12): 1839-1850 regarding PD cognitive impairment progression).
[0065] Computational analysis of transcriptomic data from putamen tissue of PSP patients, and comparing the results to that from neurotypical controls, identified ULK1 as a hub of the PSP gene network, and ULK1 activation is predicted to restore the PSP network (Figs. 2A-C). [0066] Knockdown of ULK1 has widespread regulatory effects across the PD module in a gene expression analysis of mouse fibroblasts, suggesting that UKL1 activation would do the reverse. (Saleiro et al. 2015. “Central Role of ULK1 in Type I Interferon Signaling” Cell Reports 11(4): 605-617). (Fig. IB). In vitro experiments in models of PD and ALS support the beneficial effects of ULK1 activation across neurodegenerative disease indications (Figs. 3-7) [0067] Provided herein are methods of treating a neurological disorder, the method comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1 (or “the ULK1 activator” hereinafter). The methods of treatment and compositions described herein are for use with a subject having or suspected of having a neurological disorder. In some embodiments, the methods of treatment and
compositions are for use with a subject having or suspected of having a neurodegenerative disease. In some embodiments, the subject is human.
[0068] In some embodiments, the ULK1 activator is administered to a subject in need of treatment for a neurological disorder, and the ULK1 activator is able to bind to ULK1 or a homolog thereof and increase the expression of and/or activate ULK1 or a homolog thereof. In some embodiments, the activator activates ULK1 or a homolog thereof. In some embodiments, the neurological disorder is a neurological disorder in which the expression and/or activity levels of ULK1 detected in the subject is lower than a healthy subject (i.e., a normal control).
C. ULK1
[0069] ULK1 is a 112-kDa protein. It contains a N-terminal kinase domain, a serine-proline rich region, and a C-terminal interacting domain. The serine-proline rich region has been shown experimentally to be the site of phosphorylation by mTORCl and AMPK — a negative and positive regulator of ULK1 activity, respectively. The C-terminal domain contains two microtubule-interacting and transport (MIT) domains and acts as a scaffold which links ULK1, ATG13, and FIFP200 together to form a complex that is essential to initiate autophagy. Early autophagy targeting/tethering (EAT) domains in the C-terminus are arranged as MIT domains consisting of two three-helix bundles. MIT domains also mediate interactions with membranes. The N-terminus contains a serine-threonine kinase domain. ULK1 also contains a large activation loop between the N and C terminus that is positively charged. This region may regulate kinase activity and play a role in recognizing different substrates. ULK1 and ULK2 share significant homology in both the C-terminal and N-terminal domains.
[0070] ULK1 is phosphorylated by AMPK on Ser317 and Ser777 to activate autophagy; mTOR participates in inhibitory phosphorylation of ULK1 on Ser757. Additionally, ULK1 can autophosphorylate itself at Thrl80 to facilitate self-activation. Phosphorylation of ULK1-S555 by AMPK allows ULK1 to translocate to mitochondria.
D. Neurological Disorders
[0071] In some embodiments, the neurological disorder is Parkinson’s Disease (PD), Parkinson’s Disease with Lewy bodies, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), primary lateral sclerosis (PLS), Charcot-Marie-Tooth (CMT; including type 4J (CMT4J)), and Yunis-Varon syndrome, autophagy, polymicrogyria (including polymicrogyria with seizures), temporo-occipital
polymicrogyria, Pick’s disease, dementia with Lewy bodies, Lewy body disease, diseases of neuronal nuclear inclusions of polyglutamine and intranuclear inclusion bodies, disease of Marinesco and Hirano bodies, tauopathy, Alzheimer’s disease, neurodegeneration, spongiform neurodegeneration, peripheral neuropathy, leukoencephalopathy, motor neuropathy, sensory neuropathy, abnormal lysosomal storage syndrome, myotubular myopathy, muscle weakness, cleidocranial dysplasia, Lewy body disease, inclusion body disease , corticobasal syndrome, chronic traumatic encephalopathy, traumatic brain injury (TBI), cerebral ischemia, Guillain- Barre Syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, a lysosomal storage disease, Fabry’s disorder, Gaucher’s disorder, Niemann Pick C disease, Tay- Sachs disease, and Mucolipidosis type IV, neuropathy, Coffin-Lowry Syndrome (CLS), X- linked mental retardation disorder (XLMR), intellectual disability, Huntington’s disease, a psychiatric disorder, ADHD, schizophrenia, a mood disorder, major depressive disorder, depression, bipolar disorder I, or bipolar disorder II.
[0072] In some embodiments, the neurological disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is a central nervous system (CNS) neurodegenerative disease. Examples of the neurodegenerative disease include, but are not limited to, PD, PSP, ALS, FID, Alzheimer’s disease, Huntington’s disease, prion disease, Lewy body disease, Friedreich’s ataxia, or spinal muscular atrophy. In some embodiments, the neurodegenerative disease is PD. In some embodiments, the neurological disorder or neurodegenerative disease is PSP. In some embodiments, the neurodegenerative disease is ALS. In some embodiments, the neurodegenerative disease is Alzheimer’s Disease.
[0073] In some embodiments, the neurological disorder or neurodegenerative disease is PD. Thus, in some embodiments, a method of treating PD is provided comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1. In some embodiments, the effective amount of said agent activates ULK1. [0074] In some embodiments, the neurological disorder or neurodegenerative disease is ALS. Thus, in some embodiments, a method of treating ALS is provided comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1. In some embodiments, the effective amount of said agent activates ULK1. [0075] In some embodiments, the neurological disorder or neurodegenerative disease is PSP. Thus, in some embodiments, a method of treating PSP is provided comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1. In some embodiments, the effective amount of said agent activates ULK1.
E. ULK1 Activators
[0076] An ULK1 activator disclosed herein may have an IC50, Ki, or Kd of less than 500 nM when determined at a physiologically relevant concentration of adenosine triphosphate (ATP).
[0077] Some examples of molecules known to activate ULK1 are provided in Table 1 :
[0078] In some embodiments, the ULK1 activator is a small molecule, an antibody or active fragment thereof, a peptide, a proteolysis targeting chimera (PROTAC), a complementary DNA (cDNA). In some embodiments, the antibody is a humanized antibody or active fragment thereof. In some embodiments, the PROTAC comprises a protein of interest (POI) ligand and an E3 ubiquitin ligase (E3) recruiting ligand, wherein the POI ligand and the E3 are linked. In some embodiments, the ULK1 activator is a small molecule. Also encompassed are approaches that genetically modify a cell to increase ULK1 levels, such as, for example, AAV gene therapy, cDNA therapy, and gene editing such as CRISPR.
[0079] In some embodiments, the ULK1 activator binds to ULK1. In some embodiments, the ULK1 activator is an irreversible activator of ULK1. In some instances, the irreversibility leads to a permanent activation of the action of an enzyme. In some embodiments this can be achieved by tight binding of the activator to the protein, conferring activation.
[0080] In some embodiments, activation of ULK1 results in increased expression of ULK1. In some embodiments, activation of ULK1 results in an increased expression level of ULK1, which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control. In some embodiments, activation of ULK1 results in increased activity of ULK1. In some embodiments, activation of ULK1 results in increased activity of ULK1 that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control.
[0081] The control can be determined by those of skill in the art as applicable to the particular situation. In some instances, the control is an industry standard agreed upon by those of skill as being a level or range of levels that is typical of a subject having a neurological disorder before the treatment. In some instances, the control is a reference level of ULK1 from the same individual taken at a time point before the treatment and whether the subject has increased level of ULK1 after the treatment is determined based on a sample from that same individual taken before the treatment. In some embodiments, levels of ULK1 are measured in the in vitro or in vivo systems. In some embodiments, levels of ULK1 are measured in cells, e.g., motor neuronal cells, in plasma, or in cell culture media. In some embodiments, levels of ULK1 are measured from a plasma sample. In some embodiments, levels of ULK1 are measured from a serum sample.
[0082] In some embodiments, the ULK1 activator described herein may be for use in treating a neurological disorder in a subject in need thereof. In some embodiments, the ULK1 activator
described herein may be for use in treating a neurodegenerative disease in a subject in need thereof. Examples of the neurological disorders and neurodegenerative diseases are disclosed herein.
[0083] In some embodiments, the neurological disorder is a neurological disorder in which the expression and/or activity levels of ULK1 detected in a subject is higher than a normal control. In some embodiments, the activity level of ULK1 is measured or determined by the change in binding to a partner protein, such as STX17, SNAP29 or VAMP8, BNIP3 and Parkin. In some embodiments, the activity level of ULK1 is measured or determined by the extent of protein phosphorylation by ULK1. In some embodiments, the activity level of ULK1 is measured or determined by the extent of protein phosphorylation of a ULK1 substrate, such as ATG13, In some embodiments, the extent of protein phosphorylation by ULK1 in a subject having a neurological disorder is different than the extent of protein phosphorylation by ULK1 in a normal control. In some embodiments, the extent of protein phosphorylation by ULK1 in a subject having a neurological disorder is higher than in a normal control. In some embodiments, the activity level of ULK1 is measured or determined by a change in a cellular phenotype, such as autophagy or mitophagy. In some embodiments, the cellular phenotype, such as autophagy or mitophagy, is increased.
[0084] The normal control can be determined by those of skill in the art as applicable to the particular situation. In some instances, the normal control is an industry standard agreed upon by those of skill as being a level or range of levels that is typical of an individual without an ULK1- associated condition. In some instances, the normal control is a reference level of ULK1 from the same individual taken at a time point and whether the subject has elevated ULK1 is determined based on a sample from that same individual taken at a different, typically later, time point.
F. Compositions
[0085] Compositions that are useful in the methods described herein include any agent that activates ULK1 or increases ULK1 expression (i.e., an ULK1 activator).
[0086] The compositions may further comprise a pharmaceutically acceptable carrier.
[0087] Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)
malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
[0088] In some embodiments, agents that increase the expression of and/or activate ULK1 for use in the manufacture of a medicament for treating a neurological disorder in a subject in need thereof are provided, optionally wherein the neurological disorder is PD, PSP or ALS.
[0089] In general, the ULK1 activator (or a pharmaceutical composition comprising the same described herein) of the present disclosure is administered in an effective amount by any of the accepted modes of administration for agents that serve similar utilities. Effective amounts of will depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound being utilized, the route and form of administration, and other factors. The ULK1 activator (or a pharmaceutical composition comprising the same) described herein that can be used in treatment can be formulated and dosages established in a fashion consistent with good medical practice taking into account the disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners.
[0090] Often, administration of the compounds or pharmaceutical compositions can include routes of administration, non-limiting examples of administration routes include intravenous, intraarterial, subcutaneous, subdural, intramuscular, intracranial, intrastemal, intratumoral, or intraperitoneally. Additionally, a pharmaceutical composition or compound can be administered to a subject by additional routes of administration, for example, by inhalation, oral, dermal, intranasal, or intrathecal administration. The appropriate formulation and route of administration may be selected according to the intended application.
[0091] Pharmaceutical compositions or agents of the present disclosure can be administered to a subject in need thereof in a first administration, and in one or more additional administrations. The one or more additional administrations can be administered to the subject in need thereof minutes, hours, days, weeks, or months following the first administration. Any one of the additional administrations can be administered to the subject in need thereof less than 21 days,
or less than 14 days, less than 10 days, less than 7 days, less than 4 days or less than 1 day after the first administration. The one or more administrations can occur more than once per day, more than once per week, or more than once per month. The agents or pharmaceutical compositions can be administered to the subject in need thereof in cycles of 21 days, 14 days, 10 days, 7 days, 4 days, or daily over a period of one to seven days (e.g., daily over a period of 1, 2, 3, 4, 5, 6, or 7 days).
Examples
[0092] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.
Example 1: Identification of Co-expression Modules for Parkinson’s Disease (PD)
[0093] Using a consensus eigengene network, a co-expression module enriched for genes associated with PD was identified. Specifically, gene expression was measured from Substantia nigra and dorsolateral prefrontal cortex tissue from a patient cohort collected by that included 207 Parkinson’s patient and 118 non-neurological controls. This data was combined with gene expression from five publicly-available bulk brain datasets from PD patients (NIH GEO accession numbers GSE20163, GSE20292, GSE7621, GSE20164, and GSE20168). Taken together, these gene expression data were used to identify a gene co-expression network, using the methods disclosed in Langfelder and Horvath, BMC Bioinformatics, 9:559 (2008). This network, referred to as network #8946, has 22 gene modules. Module #6 (a.k.a. 8946-6) was identified as being enriched for genes associated with PD using genome-wide association studies (GWAS), transcriptome-wide association studies (TWAS), or related studies.
Example 2: Identification of ULK1 as Candidate Targets in PD
[0094] ULK1 was identified using an approach to predict genes that regulate the expression levels of genes in network 8946-6, described in Example 1. Overall, 16,051 human genes were scored according to their co-expression with network 8946-6 and their known genetic association with neurodegenerative indications.
[0095] Using this method, ULK1 ranks #434-th out of 16,051 for its correlation to the eigengene defining network 8946-6, where correlation is measured according to the methods described by Langefelder and Horvath (Example 1).
[0096] ULK1 ranks #391 out of 16,051 for its genetic associations to Parkinson’s disease and other neurodegenerative indications. Specifically, ULK1 is a significant hit (P = 5e-5) in a previous genome-wide association study for Parkinson’s progression of cognitive impairment phenotypes (Iwaki et al., Movement Disorders 2019). ULK1 is also a sub-significant hit (p = 2e- 3) for a genome wide association study for Parkinson’s subtypes (Alfradique-Dunham et al., Neurology Genetics 2021; data was available to Verge pre-publication).
[0097] After summing the genetic and network ranks into a meta-rank , ULK1 ranks #42 overall out of 16,051 genes. After filtering this list for genes that can be drugged, ULK1 was prioritized as a top candidate target for Parkinson’s therapeutic development.
Example 3: Identification of Co-expression Modules progressive supranuclear palsy (PSP [0098] Gene expression was measured by RNA-seq of cryopreserved post-mortem putamen tissue from a patient cohort that included 28 PSP patients and 22 neurotypical controls. A gene coexpression network of 113 genes, referred to as module 14239.42.5.11, was identified that exhibited bidirectional dysregulation in PSP patient tissue compared to controls. The module is enriched for familial PSP risk genes (Fig. 2A).
[00110] ULK1 was identified as one of the 113 members of coexpression network 14239.42.5.11 with a correlation coefficient of 0.916 to the eigengene of the network. Using a combination of publicly available protein interaction databases, ULK1 was also found to be enriched in known protein to protein interactions with other network member gene encoded proteins (Fisher’s exact test, odds ratio = 4.09, p-value = 0.00476). (Figs. 2B and 2C).
[00111] Ranking all detected human genes by strength of evidence to associate with network 14239.42.5.11 and filtering for genes encoding proteins that have existing small molecule ligands, ULK1 ranks #1 of 14,789 genes, and was therefore prioritized as a candidate drug target for PSP (Fig. 2C).
Example 4 ULK1 activators BL-918 and LYN-1604 rescue against MPP+ toxicity in SH- SY5Y cells
[0099] Figs. 3A and 3B show results of experiments with SH-SY5Y cells pre-treated with LYN-1604 for 3 days, then LYN-1604 with 200pM MPP+ (Fig. 3A) or lOOnM rotenone (Fig. 3B) for 2 additional days. SH-SY5Y cells were differentiated for 5 days in DMEM-F12 media with 5% FBS (Fetal bovine serum ) with retinoic acid, then split into a Poly-D lysine (PDL) coated 96 well plate at 30,000 cells per well. The day after plating (day 6 after starting
differentiation), the media was changed to neuronal media (Neurobasal media with B27 supplement, IX Glutamax, BDNF, cAMP) and cells were matured. Cells were treated with drugs on day 9 and 12, with the addition of toxins (lOOpM MPP+ (l-methyl-4- phenylpyridinium) or lOOnM rotenone) on day 12. 48 hours after toxin treatment, on day 14, cell viability was assessed with a cell viability reagent (AlamarBlue™) in an absorbance-based plate-reader assay. Cell viability was assessed using a cell viability reagent in an absorbancebased plate-reader assay. (AlamarBlue™). Data are expressed as mean SEM (standard error of mean). n=3 replicates. Data are representative graphs from 1 of 3 independent experiments showing similar results.
[00100] Fig. 4A shows results of experiments with undifferentiated SH-SY5Y cells seeded at 40,000 cells/well in 96w plate for 24hrs. SH-SY5Y cells were preincubated with ULK1 activator BL-918 for 6hrs, followed with 48hrs incubation of 250pM mitochondrial toxins MPP+ in the continuous presence of BL918. Cell viabilities were measured in an absorbance based platereader assay using the CellTiter-Glo reagent. Data are mean of 3 technical replicates ± SEM. These data suggest that ULK1 activation can protect from death induced by PD associated toxins.
[00101] Figs. 4B and 4C show results of experiments with differentiated SH-SY5Y neurons SH-SY5Y cells were seeded at 30,000 cells/well in 96w plate and differentiated for 12 days. They were then pre-incubated for 2hrs with ULK1 activators BL-918 (Fig. 4B) or LYN-1604 (Fig. 4C) followed with 48hrs incubation of 200 pM mitochondrial toxins MPP+. Cell viabilities were measured in an absorbance based plate-reader assay using the CellTiter-Glo reagent. Data are mean of 3 technical replicates ± SEM.
[00102] These experiments show that ULK1 activators rescue PD relevant toxicity in acute (2h; Figs. 4 B and 4C) and chronic (3 days; Figs. 3A and 4A) exposure settings.
Example 5: ULK1 activator LYN-1604 enhances autophagy in fibroblast from healthy and PD patients expressing the LRRK2 G2019S (LRRK2 GS) mutation
[00103] Fig. 5A shows quantification of autolysosomes measured as Lampl and p62 colocalization area per fibroblast treated for 20 hours with different concentrations of the ULK1 activator LYN- 1604. Fig. 5B shows quantification of autophagosomes measured as p62 area per fibroblast treated for 20 hours with different concentrations of the ULK1 activator LYN-1604.
[00104] This experiment shows that autophagy can be induced in cells derived from healthy and PD patients.
Example 6: ULK1 activator LYN-1604 enhances autophagy in iPSC derived motoneurons from healthy subjects
[00105] Fig. 6A shows iPSC derived motoneurons (iMNs) derived from healthy subjects were seeded at 20,000 cells/well in 96w plate and grown for 7 days. ULK1 activator LYN-1604 (4 pM) was added on day 8 for 48 hr followed by 20 hr Chloroquine (25 pM) treatment. As a positive control, cells were treated with PIKFyve inhibitor Apilimod (IpM) for 24 hr. Cells were fixed with 4% paraformaldehyde (PF A) and 4% Sucrose on day 10 and stained for lysosomal marker LAMP1, autophagosomal marker p62 and motoneuron marker Hb9. p62 and LAMP1 puncta colocalization marks the autolysosomal structures. Chloroquine treatment inhibits autophagic flux by blocking lysosomal degradation and thereby revealing the amount of basal autophagy. Fig. 6B shows LYN- 1604 and Apilimod treatment increased the number of p62 positive autophagosomes. Chloroquine treatment shows both LYN1604 and Apilimod increases basal autophagy. In Fig. 6C, LYN-1604 and Apilimod treatment also increased number of autolysosomes indicative of increased autophagy. Data are shown as mean ± SD.
[00106] This experiment shows that ULK1 activators enhance autophagy, suggesting the potential use of ULK1 activators in treating ALS patients
Example 7: ULK1 activator LYN-1604 enhances autophagy in ALS patient derived motoneurons
[00107] Fig. 7A shows control and ALS mutant (carrying a c9orf72 expansion) motoneurons seeded at 20,000 cells/well in 96w plate and grown for 8 days. ULK1 activator LYN-1604 (4 pM) was then added for 48 hr. Cells were fixed with 4% PFA & 4% Sucrose on day 10 and stained for lysosomal marker LAMP1, autophagosomal marker p62 and nuclear marker DAPI. p62 and LAMP1 puncta colocalization marks the autolysosomal structures. ALS c9orf72 mutant iMNs have reduced number of p62 positive autophagosomes (Fig. 7B) and autolysosomes indicative of reduced autophagy (Fig. 7C). LYN- 1604 treatment increased the number of autophagosomes and autolysosomes in control iMNs and rescued autophagy in c9orf72 mutant iMNs. Data are shown as mean ± SD.
[00108] This experiment shows that autophagy is inhibited in motorneurons derived from C9ORF72 patient relative to the healthy subject cells, and that this inhibition can be normalized
to the control levels after ULK1 activation, supporting the notion that ULK1 activation could be a potential avenue for patient treatment
[00109] The foregoing disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding. Therefore, it is to be understood that the above description is intended to be illustrative and not restrictive. The scope of the disclosure should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled.
Claims
1. A method of treating a neurological disorder, the method comprising administering to a subject in need thereof an effective amount of an agent that increases the expression of and/or activates ULK1.
2. The method of claim 1, wherein the neurological disorder is a neurological disorder in which the expression and/or activity levels of ULK1 detected in the subject is different from a normal control.
3. The method of claim 1 or 2, wherein the agent increases the expression of ULK1.
4. The method of claim 1 or 2, wherein the agent activates ULK1.
5. The method of any one of claims 1 to 4, wherein the agent is a small molecule, an antibody or active fragment thereof, a peptide, or PROTAC.
6. The method of any one of claims 1 to 5, wherein the agent is a small molecule.
7. The method of claim 6, wherein the increased expression level of ULK1 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control.
8. The method of claim 7, wherein the increased activity of ULK1 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control.
9. The method of any one of claims 1 to 8, wherein the neurological disorder is Parkinson’s Disease (PD), Parkinson’s Disease with Lewy bodies, progressive supranuclear palsy (PSP) amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), primary lateral sclerosis (PLS), Charcot-Marie-Tooth (CMT; including type 4J (CMT4J)), and Yunis-Varon syndrome, autophagy, polymicrogyria (including polymicrogyria with seizures), temporo-occipital polymicrogyria, Pick’s disease, dementia with Lewy bodies, Lewy body disease, diseases of neuronal nuclear inclusions of polyglutamine and intranuclear inclusion bodies, disease of Marinesco and Hirano bodies, tauopathy, Alzheimer’s disease, neurodegeneration, spongiform neurodegeneration, peripheral neuropathy, leukoencephalopathy, motor neuropathy, sensory neuropathy, abnormal lysosomal storage syndrome, myotubular myopathy, muscle weakness, cleidocranial dysplasia, Lewy body disease, inclusion body disease , corticobasal syndrome, chronic traumatic encephalopathy, traumatic brain injury (TBI), cerebral ischemia, Guillain- Barre Syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, a lysosomal storage disease, Fabry’s disorder, Gaucher’s disorder, Niemann Pick C disease, Tay-
Sachs disease, and Mucolipidosis type IV, neuropathy, Coffin-Lowry Syndrome (CLS), X- linked mental retardation disorder (XLMR), intellectual disability, Huntington’s disease, a psychiatric disorder, ADHD, schizophrenia, a mood disorder, major depressive disorder, depression, bipolar disorder I, or bipolar disorder II.
10. The method of any one of claims 1 to 9, wherein the neurological disorder is a neurodegenerative disease.
11. The method of claim 10, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), Alzheimer’s disease, Parkinson’s disease (PD), Huntington’s disease, prion disease, Lewy body disease, Friedreich’s ataxia, or spinal muscular atrophy.
12. The method of claims 10 or 11, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
13. The method of claims 10 or 11, wherein the neurodegenerative disease progressive supranuclear palsy (PSP).
14. The method of claims 10 or 11, wherein the neurodegenerative disease is Alzheimer’s disease.
15. The method of claims 10 or 11, wherein the neurodegenerative disease is Parkinson’s Disease (PD).
16. The method of any one of claims 1 to 15, wherein the subject is a human.
17. The method of claim 16, wherein the neurological disorder is a neurodegenerative disease.
18. A composition comprising an agent that increases the expression of and/or activates ULK1 for use in treating a neurological disorder in a subject in need thereof.
19. The composition of claim 18, further comprising a pharmaceutically acceptable carrier.
20. Use of an agent that increases the expression of and/or activates ULK1 in the manufacture of a medicament for treating a neurological disorder in a subject in need thereof.
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