US20200087648A1 - Parkin ligase activation methods and compositions - Google Patents

Parkin ligase activation methods and compositions Download PDF

Info

Publication number
US20200087648A1
US20200087648A1 US16/193,184 US201816193184A US2020087648A1 US 20200087648 A1 US20200087648 A1 US 20200087648A1 US 201816193184 A US201816193184 A US 201816193184A US 2020087648 A1 US2020087648 A1 US 2020087648A1
Authority
US
United States
Prior art keywords
cancer
cell
tumors
parkin
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US16/193,184
Inventor
Jennifer Johnston
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nysnobio Ireland Dac
Original Assignee
An2h Discovery Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by An2h Discovery Ltd filed Critical An2h Discovery Ltd
Priority to US16/193,184 priority Critical patent/US20200087648A1/en
Assigned to An2H Discovery Limited reassignment An2H Discovery Limited ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JOHNSTON, JENNIFER
Publication of US20200087648A1 publication Critical patent/US20200087648A1/en
Assigned to NYSNOBIO IRELAND DAC reassignment NYSNOBIO IRELAND DAC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: An2H Discovery Limited
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • 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
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Definitions

  • the present invention relates to methods of activating Parkin ligase by disrupting zinc finger domains for therapeutic benefit.
  • Ubiquitin-Proteasome Pathway System is a critical pathway that regulates key regulator proteins and degrades misfolded or abnormal proteins. UPS is central to multiple cellular processes, and if defective or imbalanced, it leads to pathogenesis of a variety of diseases. Posttranslational modification of proteins by ubiquitin is a fundamental cellular mechanism that regulates protein stability and activity and underlies a multitude of functions, from almost every aspect of biology. The covalent attachment of ubiquitin to specific protein substrates is achieved through the action of E3 ubiquitin ligases. These ligases comprise over 500 different proteins and are categorized into multiple classes defined by the structural element of their E3 functional activity.
  • both HECT and RING ligases transfer an activated ubiquitin from a thioester to the e-amino acid group of a lysine residue on a substrate; however, HECT ligases have an active site cysteine that forms an intermediate thioester bond with ubiquitin, while RING ligases function as a scaffold to allow direct ubiquitin transfer from the E2 to substrate.
  • a subfamily of RING ligases the RING-between-RING (RBR) family, may contain a catalytic cysteine residue 1,2 in addition to a canonical RING domain. (Riley et al. 2013 . Nat Commun. 4:1982, “Riley et al.”), which is herein incorporated by reference in its entirety.
  • Deubiquitinating proteins and ubiquitin-specific proteases (DUBs and USPs) and E3 Ligases play a vital role in the UPS. These proteins are supported by flexible Zinc Finger (ZnF) domains which stabilize the binding of ubiquitin (Ub) for specialized functions.
  • ZnF Zinc Finger
  • Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection.
  • the individual RING domains for Parkin have been the subject of much debate, in regards to the specific residues that coordinate Zn ions, as well as their relationship to canonical RING crossbrace structures defining classical E2-binding domains.
  • R0 is a novel domain structure, but is more similar to Zn-finger domains than to E3 RING domains (Riley et al. 2013 . Nat Commun. 4:1982)
  • the present invention is directed towards a novel approach of disrupting Zn-finger domains that provide a therapeutic benefit for various diseases and disorders, including oncology and neurology disorders.
  • the present invention relates to modulating the structures and/or functions of ligases in the UPS by binding to zinc ions and/or cysteine residues in their ZnF domains, for therapeutic benefit. This mechanism is distinct from binding to the active sites of ligases, which receive the tail of Ub.
  • the present invention is directed to a method for activating Parkin ligase by coordination of small molecules to zinc ions in Parkin ZnF domains, or by chemical reactions of small molecules with cysteine residues in Parkin ZnF domains.
  • the coordination of small molecules to zinc ions may or may not remove the zinc ions from the ZnF domains.
  • the chemical reactions of small molecules with cysteine residues may be reversible or irreversible.
  • Specific embodiments of the present invention include methods of activating a Parkin ligase.
  • the Parkin ligase may be activated by administering to a subject a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger.
  • the compound can coordinate with a Zn ion, and/or bind or react with a cysteine.
  • the compound may react with the thiol group in the cysteine.
  • the activated Parkin ligase suppresses one or more tumors. In another specific embodiment, the activated Parkin ligase provides dopamine neuron protection.
  • Compounds that can coordinate to a Zn ion include, but are not limited to, a monodentate, bidentate, or tridentate ligand.
  • Compounds that can react with the thiol group in the cysteine residue include, but are not limited to an alkylator, oxidant, Michael acceptor, another unsaturated structure, or a disulfide.
  • the compound eliminates damaged mitochondria, increases cell viability during cellular stress, decreases tumor transformation and/or mitigates alpha-synuclein in cells.
  • the subject has been diagnosed with cancer.
  • cancer is glioblastoma, small cell lung carcinoma, breast cancer or prostate cancer.
  • the patient has been diagnosed with a neuro-degenerative disease.
  • the neurodegenerative disease is Parkinson's disease, dementia, Amyotrophic lateral sclerosis (ALS) or Huntington's disease.
  • the dementia is dementia with Lewy bodies (DLB), multiple system atrophy (MSA) or Progressive supranuclear palsy (PSP).
  • the compound substantially disrupts the structure of at least one zinc finger in the Parkin ligase.
  • at least one zinc finger is selected from one or more of the group consisting of the domains defined by R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465.
  • the amino acid residues of at least one zinc finger corresponds to or aligns within one or more domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase.
  • the zinc finger comprises four cysteine residues.
  • the compound may be a zinc chelator.
  • the compound can bind or react with one or more cysteine residues. In another specific embodiment, the compound can bind or react with one or more cysteine residues selected from the group consisting of C59 and C377 of human Parkin Ligase.
  • the compound may substantially disrupt a structure of at least one zinc finger in the Parkin ligase.
  • the zinc finger in the Parkin ligase may be located within one or more domains selected from the group consisting of R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase.
  • the zinc finger that is substantially disrupted is located in IBR amino acids 328-377 of the Parkin ligase.
  • the compound may act synergistically with Phospho Ubiquitin (pUB) in activating the Parkin ligase.
  • pUB Phospho Ubiquitin
  • Parkin ligase activation alters ubiquitination.
  • activating the Parkin ligase treats or reduces the incidence of one or more diseases or ailments selected from the group consisting of Alzheimer's Dementia, Parkinson's disease, Huntington Disease, Amyotrophic Lateral Sclerosis (ALS), Freidreich's ataxia, Spinocerebellar Ataxia, Multiple Systems Atrophy, PSP, Tauopathy, Diffuse Lewy Body Disease, Lewy Body dementia, any disorder characterized by abnormal accumulation of ⁇ -synuclein, disorders of the aging process, stroke, bacterial infection, viral infection, Mitochondrial related disease, mental retardation, deafness, blindness, diabetes, obesity, cardiovascular disease, multiple sclerosis, Sjogrens syndrome, lupus, glaucoma, including pseudoexfoliation glaucoma, Leber's Hereditary Optic Neuropathy, and rheumatoid arthritis.
  • diseases or ailments selected from the group consisting of Alzheimer's Dementia, Parkinson's disease, Huntington Disease, Amyotrophic Lateral
  • the bacterial infection is Mycobacterium infection.
  • the viral infection is Hepatitis C infection.
  • the Mitochondrial related disease is selected from one or more of the group consisting of Alpers Disease, Barth Syndrome/LIC (Lethal Infantile Cardiomyopathy), Beta-oxidation Defects, Carnitine-Acyl-Carnitine Deficiency, Carnitine Deficiency, Creatine Deficiency Syndromes, Co-Enzyme Q10 Deficiency, Complex I Deficiency, Complex II Deficiency, Complex III Deficiency, Complex IV Deficiency/COX Deficiency, Complex V Deficiency, CPEO, CPT I Deficiency, CPT II Deficiency, KSS, Lactic Acidosis, LBSL—Leukodystrophy, LCAD, LCHAD, Leigh Disease or Syndrome, Gut Disease, MAD/Glutaric Aciduria Type II, MCAD, MELAS, MERRF, MIRAS
  • Another embodiment of the invention includes methods of treating and/or reducing the incidence of cancer.
  • a specific embodiment includes administering to a subject in need thereof a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger and induces Parkin ligase activity, wherein the compound can coordinate with a Zn ion and/or react with a thiol group in a cysteine.
  • activating the Parkin ligase suppresses one or more tumors.
  • the compound eliminates damaged mitochondria, increases cell viability during cellular stress, decreases tumor transformation and/or mitigates ⁇ -synuclein in cells.
  • the cancer is glioblastoma, small cell lung carcinoma, breast cancer or prostate cancer.
  • Another embodiment of the present invention includes methods for treating and/or reducing the incidence of Parkinson's disease.
  • a specific embodiment for treating and/or reducing the incidence of Parkinson's disease includes administering to a subject in need thereof a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger and induces Parkin ligase activity, wherein the compound can coordinate with a Zn ion and/or react with a thiol group in a cysteine.
  • Parkin ligase activation alters ubiquitination, as defined by the ability of Parkin to modify a substrate protein by covalent attachment of ubiquitin, a substrate protein being Parkin itself, or another protein such as Mitofusion 1 or 2, FBW7, or other publicly reported substrates of Parkin ligase.
  • the pharmaceutical formulations activate Parkin ligase.
  • the pharmaceutical formulations may comprise an effective amount of a compound or its salt thereof that disrupts at least one Parkin ligase zinc finger, and a pharmaceutically acceptable carrier, wherein the compound or its salt thereof can coordinate a Zn ion, and/or react with the thiol group in a cysteine.
  • the compound can bind or react with one or more cysteine residues selected from the group consisting of C59 and C377 of human Parkin Ligase.
  • the pharmaceutical composition is in a formulation selected from the group consisting of a solid, powder, liquid and a gel.
  • FIG. 1 indicates that N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide, a chelator compound (identified as AH001 or compound 76 in Table 2) increases the Parkin Ligase reaction with the Activity-based Ubiquitin vinyl sulfone probe.
  • a chelator compound identified as AH001 or compound 76 in Table 2
  • FIG. 2 indicates that 6-benzyl-2,5-dimethyl-3-phenylpyrazolo[1,5-a]pyrimidine-7-thiol, an electrophile and chelator compound (identified as AH007 or compound 77 in Table 2) increases the Parkin Ligase reaction with the Activity-based Ubituitin vinyl sulfone probe.
  • FIG. 3 indicates that compound N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide, a chelator compound (AH001) increases Parkin activity in an auto-ubiquitination assay.
  • AH001 a chelator compound
  • FIGS. 4A and 4B indicate that N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide (AH001) with pUB synergistically increases parkin activation in an auto-ubiquitination assay and allows for a lower concentration of pUB to activate parkin.
  • AH001 N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide
  • FIG. 5 indicates that 6-benzyl-2,5-dimethyl-3-phenylpyrazolo[1,5-a]pyrimidine-7-thiol, an electrophile compound (AH007) increases Parkin activity in an auto-ubiquitination assay.
  • Pharmaceutically acceptable salts include those obtained by reacting the active compound functioning as a base, with an inorganic or organic acid to form a salt, for example, salts of hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, camphorsulfonic acid, oxalic acid, maleic acid, succinic acid, citric acid, formic acid, hydrobromic acid, benzoic acid, tartaric acid, fumaric acid, salicylic acid, mandelic acid, carbonic acid, etc.
  • acid addition salts may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods.
  • treating means one or more of relieving, alleviating, delaying, reducing, reversing, improving, or managing at least one symptom of a condition in a subject.
  • the term “treating” may also mean one or more of arresting, delaying the onset (i.e., the period prior to clinical manifestation of the condition) or reducing the risk of developing or worsening a condition.
  • an “effective amount” means the amount of a formulation according to the invention that, when administered to a patient for treating a state, disorder or condition is sufficient to effect such treatment.
  • the “effective amount” will vary depending on the active ingredient, the state, disorder, or condition to be treated and its severity, and the age, weight, physical condition and responsiveness of the mammal to be treated.
  • terapéuticaally effective applied to dose or amount refers to that quantity of a compound or pharmaceutical formulation that is sufficient to result in a desired clinical benefit after administration to a patient in need thereof.
  • substantially refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result.
  • an object that is “substantially” enclosed would mean that the object is either completely enclosed or nearly completely enclosed.
  • the exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking, the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained.
  • the use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of action, characteristic, property, state, structure, item, or result.
  • compositions that is “substantially free of” other active agents would either completely lack other active agents, or so nearly completely lack other active agents that the effect would be the same as if it completely lacked other active agents.
  • a composition that is “substantially free of” an ingredient or element or another active agent may still contain such an item as long as there is no measurable effect thereof
  • the “alignment” of two or more protein/amino acid sequences may be performed using the alignment program ClustalW2, available at the ebi website.
  • Ubiquitin Proteasome Pathway System relates to the ubiquitin proteasome pathway, conserved from yeast to mammals, and is required for the targeted degradation of most short-lived proteins in the eukaryotic cell. Targets include cell cycle regulatory proteins, whose timely destruction is vital for controlled cell division, as well as proteins unable to fold properly within the endoplasmic reticulum. Ubiquitin modification is an ATP-dependent process carried out by three classes of enzymes.
  • a “ubiquitin activating enzyme” (E1) forms a thio-ester bond with ubiquitin, a highly conserved 76-amino acid protein.
  • E3 ligases can be single- or multi-subunit enzymes. In some cases, the ubiquitin-binding and substrate binding domains reside on separate polypeptides brought together by adaptor proteins or culling. Numerous E3 ligases provide specificity in that each can modify only a subset of substrate proteins. Further specificity is achieved by post-translational modification of substrate proteins, including, but not limited to, phosphorylation.
  • Effects of monoubiquitination include changes in subcellular localization. However, multiple ubiquitination cycles resulting in a polyubiquitin chain are required for targeting a protein to the proteasome for degradation.
  • the multisubunit 26S proteasome recognizes, unfolds, and degrades polyubiquitinated substrates into small peptides. The reaction occurs within the cylindrical core of the proteasome complex, and peptide bond hydrolysis employs a core threonine residue as the catalytic nucleophile. It has been shown that an additional layer of complexity, in the form of multiubiquitin chain receptors, may lie between the polyubiquitination and degradation steps.
  • Protein degradation through the ubiquitin-proteasome system is the major pathway of non-lysosomal proteolysis of intracellular proteins. It plays important roles in a variety of fundamental cellular processes such as regulation of cell cycle progression, division, development and differentiation, apoptosis, cell trafficking, and modulation of the immune and inflammatory responses.
  • the central element of this system is the covalent linkage of ubiquitin to targeted proteins, which are then recognized by the 26S proteasome, an adenosine triphosphate-dependent, multi-catalytic protease. Damaged, oxidized, or misfolded proteins as well as regulatory proteins that control many critical cellular functions are among the targets of this degradation process. Aberration of this system leads to the dysregulation of cellular homeostasis and the development of multiple diseases (Wang et al. Cell Mol Immunol. 2006 August; 3(4):255-61).
  • Parkin Ligase or “Parkin” as used herein relates to a protein which in humans is encoded by the PARK2 gene.
  • Parkin or “Parkin” as used herein relates to a protein which in humans is encoded by the PARK2 gene.
  • “Ligase” as used herein, is an enzyme that can catalyze the joining of two or more compounds or biomolecules by bonding them together with a new chemical bond.
  • the “ligation” of the two usually with accompanying hydrolysis of a small chemical group dependent to one of the larger compounds or biomolecules, or the enzyme catalyzing the linking together of two compounds, e.g., enzymes that catalyze joining of groups C—O, C—S, C—N, etc.
  • Ubiquitin-protein (E3) ligases are a large family of highly diverse enzymes selecting proteins for ubiquitination.
  • “Ub Ligases” are involved in disease pathogenesis for oncology, inflammation & infectious disease. E3 ligase belonging to the RING-between-RING (RBR) family of E3 ligases containing both canonical RING domains and a catalytic cysteine residue usually restricted to HECT E3 ligases; termed ‘RING/HECT hybrid’ enzymes. Mutations in Parkin linked to Parkinson's disease, cancer and mycobacterial infection. Parkin is recognized as a neuroprotective protein with a role in mitochondrial integrity. Human genetic data implicate loss of Parkin activity as a mechanism for pathogenesis of Parkinson's Disease (PD).
  • PD Parkinson's Disease
  • ZnF Zinc Finger (ZnF) Domain
  • DUBs Deubiquitinating Enzymes
  • E3 Ligases
  • Ligands as used herein bind to metal via one or more atoms in the ligand, and are often termed as chelating ligands.
  • a ligand that binds through two sites is classified as bidentate, and three sites as tridentate.
  • the “bite angle” refers to the angle between the two bonds of a bidentate chelate.
  • Chelating ligands are commonly formed by linking donor groups via organic linkers.
  • a classic bidentate ligand is ethylenediamine, which is derived by the linking of two ammonia groups with an ethylene (—CH2CH2-) linker.
  • a classic example of a polydentate ligand is the hexadentate chelating agent EDTA, which is able to bond through six sites, completely surrounding some metals.
  • the binding affinity of a chelating system depends on the chelating angle or bite angle.
  • Many ligands are capable of binding metal ions through multiple sites, usually because the ligands have lone pairs on more than one atom. Some ligands can bond to a metal center through the same atom but with a different number of lone pairs.
  • the bond order of the metal ligand bond can be in part distinguished through the metal ligand bond angle (M-X-R). This bond angle is often referred to as being linear or bent with further discussion concerning the degree to which the angle is bent.
  • an imido ligand in the ionic form has three lone pairs.
  • One lone pair is used as a sigma X donor, the other two lone pairs are available as L type pi donors. If both lone pairs are used in pi bonds then the M-N-R geometry is linear. However, if one or both these lone pairs is non-bonding then the M-N-R bond is bent and the extent of the bend speaks to how much pi bonding there may be. It was found that few heteroatoms, such as nitrogen, oxygen, and sulfur atoms, interacted with zinc, ideal distances between the zinc and these heteroatoms were identified.
  • Simple organic species are also very common, be they anionic (RO ⁇ and RCO 2 ⁇ ) or neutral (R 2 O, R 2 S, R 3-x NH x , and R 3 P).
  • Complexes of polydentate ligands are called chelate complexes. They tend to be more stable than complexes derived from monodentate ligands. This enhanced stability, the chelate effect, is usually attributed to effects of entropy, which favors the displacement of many ligands by one polydentate ligand.
  • the chelating ligand forms a large ring that at least partially surrounds the central atom and bonds to it, leaving the central atom at the center of a large ring. The more rigid and the higher its denticity, the more inert will be the macrocyclic complex.
  • “Chelator” as used herein relates to a binding agent that suppresses chemical activity by forming a chelate (a coordination compound in which a metal atom or ion is bound to a ligand at two or more points on the ligand, so as to form, for example, a heterocyclic ring containing a metal atom).
  • a chelate a coordination compound in which a metal atom or ion is bound to a ligand at two or more points on the ligand, so as to form, for example, a heterocyclic ring containing a metal atom.
  • “Chelation” as used herein relates to a particular way that ions and molecules bind metal ions. According to the International Union of Pure and Applied Chemistry (IUPAC), chelation involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central atom. Usually these ligands are organic compounds, and are called chelants, chelators, chelating agents, or sequestering agents.
  • Electrophile as used herein relates to species that is attracted to an electron rich center.
  • an electrophile is a reagent attracted to electrons. It participates in a chemical reaction by accepting an electron pair in order to bond to a nucleophile. Because electrophiles accept electrons, they are Lewis acids. Most electrophiles are positively charged, have an atom that carries a partial positive charge, or have an atom that does not have an octet of electrons.
  • Ubiquitin-protein (E3) ligases are a large family of enzymes that select various proteins for ubiquitination. These ubiquitin ligases, called “Ub ligases” are known to have a role in various diseases and conditions, including but not limited to, cancer, inflammation and infectious diseases.
  • Parkin ligase is a component of a multiprotein “E3” ubiquitin ligase complex, which in turn is part of the ubiquitin-proteasome system that mediates the targeting of proteins for degradation. Although the specific function of Parkin ligase is not known, mutations in Parkin ligase are linked to various diseases, such as Parkinson's disease, cancer and mycobacterial infection. Parkin ligase is thus an attractive target for therapeutic intervention.
  • ligases particularly ligases involved in the Ubiquitin-Proteasome Pathway System (UPS), are known to have Zinc Finger (ZnF) domains that stabilize critical protein binding regions in that ligase.
  • UPS Ubiquitin-Proteasome Pathway System
  • ZnF domains coordinate zinc ions and this coordination stabilizes functional activity of the protein.
  • the functional activity provided by proteins with ZnF domains can include the regulation of important cellular signaling pathways, such as recognizing ubiquitins, regulation of DNA, such as transcription and repair, and acting as cellular redox sensors.
  • the binding of zinc to ZnF domains, or simply just regulating how zinc interacts with the ZnF domains, are essential to ligases involved in the UPS.
  • Parkin ligase is known to have one or more ZnF domains.
  • the present invention focuses on two different strategies for modulating ZnF domains in Parkin ligase.
  • One strategy of the present invention includes using chelating compounds that bind to the ZnF domains and thus disallow the binding of zinc, or cause the dissociation of zinc, such as Zn, or Zn 2+ , from the ZnF domain.
  • Another strategy of the present invention includes using compounds that bind or react with a cysteine amino acid residue in the ZnF domain.
  • One or more cysteine residues are essential in ZnF domains for binding to and/or coordinating to the zinc ion.
  • the zinc ion usually Zn 2+ ) can coordinate with multiple cysteine or histidine residues.
  • the more cysteine residues there are in the domain the more flexible is the ZnF domain.
  • Ligases, such as Parkin ligase are thought to have multiple cysteine residues coordinated with zinc in their ZnF domains. This flexibility in the ZnF domains of Parkin ligase is thought allow the domain to be reversible, and is thus is one possible mechanism for regulating Parkin ligase.
  • the present invention thus relates to the use of one or more agents or one or more compounds with electrophilic, chelation or both electrophilic and chelation properties that can interact with the zinc ion and/or the cysteine residue(s) in a Parkin ligase and thus modulate Parkin ligase's activity. Specifically, it is believed that not allowing a zinc ion to coordinate in at least one of Parkin ligase's ZnF domains induces its activity.
  • the present invention is thus directed to a method for activating Parkin ligase by the chelation of Zn followed by its removal from the ZnF domain, or through electrophilic attack at the cysteine amino acid(s) that holds the Zn in place.
  • the methods of activating a Parkin ligase include administering to a subject in need thereof a therapeutically effective amount of one or more compounds that disrupt at least one Parkin ligase zinc finger.
  • the methods of activating a Parkin ligase include administering to a subject two or more compounds that disrupt at least one Parkin ligase zinc finger.
  • the compounds of the present invention may be an electrophile or a chelator.
  • the compounds of the present invention may be able to function as both an electrophile and as a chelator.
  • the compounds of the present invention can include multiple functional groups wherein a functional group has chelating properties and a functional group that has electrophilic properties.
  • the compound is selected from one or more of the group consisting of the compounds in Table 1 and Table 2 or a salt or ester thereof.
  • the compounds in Table 1 or Table 2 may be chelators, electrophiles or both.
  • compounds 76 and 97 from Table 2 act as a chelator, but compound 113 of Table 2 acts as a thiol-reactive electrophile.
  • compounds from Table 1 or Table 2 can act as both an electrophile and as a nucleophile.
  • compounds 91 and 107 of Table 2 are both chelators, but can possibly also act as thiol-reactive electrophiles.
  • the compounds of the present invention are an electrophile, chelator or both an electrophile and a chelator selected from one or more of the group consisting of the compounds in Table 1 and Table 2 or a salt or ester thereof.
  • the compounds in Table 1 and Table 2 or a salt or ester thereof bind and active Parkin ligase.
  • the compound can be 2-(4-benzylpiperazin-1-yl)-N-[(2-hydroxy-3-prop-2-enyl-phenyl)methylideneamino]acetamide (also referred to as Pac-1) or a salt or ester thereof.
  • Pac-1 2-(4-benzylpiperazin-1-yl)-N-[(2-hydroxy-3-prop-2-enyl-phenyl)methylideneamino]acetamide (also referred to as Pac-1) or a salt or ester thereof.
  • Pac-1 provided as compound 114 in Table 2 below, is believed to be a chelator and may also have the ability to increase the Parkin Ligase reaction.
  • Pac-1, or a salt or thereof may also be an electrophile and/or be both a chelator and an electrophile.
  • the compound may be a derivative or analogue of Pac-1.
  • the compound may be a compound as described in PCT Application Publication Nos. WO2010/091382, WO2013/131089, WO2013/124407, WO2014/022858, U.S. Application Publication Nos. US2015/0210659, US2015/0231132, US2014/0073609, US20150017264, US2015/0099759, U.S. Pat. Nos. 8,916,705, 9,102,661, 8,592,584, and 8,778,945, the disclosures of which are incorporated by reference herein in their entirety.
  • Activity-based probe assays and mass spectrometry analysis indicate that some candidate compounds in Table 1 and/or Table 2 can bind or react with multiple cysteine residues in human Parkin ligase.
  • mass spectrometry analysis shows that AH007 binds to at least cysteine residue 59 (C59) and cysteine residue 377 (C377) of Parkin ligase.
  • methods of activating a Parkin ligase include administering to a subject in need thereof a therapeutically effective amount of one or more compounds that disrupt at least one Parkin ligase zinc finger.
  • the one or more compounds are selected from Table 1 and/or Table 2, or a salt or ester thereof.
  • the compound may be a chelator, an electrophile or both a chelator and an electrophile.
  • the one or more compounds can coordinate with a Zn ion, and/or bind or react with one or more cysteine residues.
  • the Zn ion may be either a Zn + or a Zn 2+ ion.
  • the compound can coordinate to a Zn ion is a monodentate, bidentate, or tridentate ligand.
  • the compound can bind and/or react with a thiol group in more than one cysteine residues. In another embodiment, the compound can bind and/or react with a thiol group in two cysteine residues. In another embodiment, the compound can bind and/or react with a thiol group in three cysteine residues. In another embodiment, the compound can bind and/or react with a thiol group in four cysteine residues. In another specific embodiment, the compound can bind or react with one or more cysteine residues in one or more domains selected from the group consisting amino acids 141-225, amino acids 238-293, amino acids 313-377, and amino acids 418-449 of human Parkin Ligase. See 060260 at the uniport website.
  • the compound can bind or react with one or more cysteine residues selected from the group consisting of C182, C258 and C377 of human Parkin Ligase. In another specific embodiment, the compound can bind or react with one or more cysteine residues selected from the group consisting of C59 and C377 of human Parkin Ligase. In a specific embodiment, the compound can react with C377 of human Parkin Ligase. In another specific embodiment, the compound is AH007.
  • the compound can bind or react with one or more cysteine residues selected from one or more residues of a parkin ligase, parkin ligase derivative, or parkin ligase homologue that correspond to or align with C182, C258 and/or C377 of human Parkin Ligase.
  • the compound can bind or react with one or more cysteine residues selected from one or more residues of a parkin ligase, parkin ligase derivative, or parkin ligase homologue that correspond to or align with C59 and/or C377 of human Parkin Ligase.
  • the compound is AH007.
  • the IBR domain may play a key role in regulating Parkin activity. It is believed that the RO domain includes at least one ZnF domain that as discussed above, could be involved in one possible mechanism for regulating Parkin ligase. Accordingly, in a specific embodiment, the structure of at least one ZnF domain located in the IBR domain (amino acids 328-377) may be substantially disrupted by the administration of a compound to a subject in need thereof. In another embodiment, one or more compounds selected from Table 1 and/or Table 2, or a salt or ester thereof, may substantially disrupt the structure of at least one ZnF domain located in the IBR domain (amino acids 328-377).
  • the compound can bind and/or react with a cysteine residue, including any histidine residue(s) in or near the ZnF domain.
  • the compounds may substantially disrupt the structure of at least one zinc finger (or ZnF domain) in the Parkin ligase. In another embodiment, the compounds of the present invention may disrupt one or more ZnF domains in Parkin ligase.
  • Riley et al. describes a human Parkin ligase of 465 amino acids that includes multiple functional areas with Zn coordination residues (amino acid sequence provided in Table 3 (SEQ ID NO:1) and identified in the ncbi website (See sequence NM_004562.2)).
  • Riley et al. discusses 4 domains designated as R0, R1, IBR and R2.
  • R0, R1 and R2 which were previously designated as RING domains.
  • Riley et al. questions whether the R0, IBR and R2 domains are actual, or traditional RING domains: “R0 is a novel domain structure, but is more similar to Zn-finger domains than to E3 RING domains” Riley et al.
  • the R0, IBR and R2 domains look like ideal domain candidates for regulating the activity of Parkin Ligase.
  • the R0, IBR and R2 domains refer to amino acids 141-216, amino acids 328-377, and amino acids 415-465 of human Parkin Ligase, respectively.
  • the at least one zinc finger that may be substantially disrupted correspond to or align with one or more domains selected from the group consisting amino acids 141-216, amino acids 328-377, and amino acids 415-465 of human Parkin Ligase.
  • the amino acids from the at least one zinc finger may overlap in an alignment with one or more of the R0, IBR and R2 domains from human Parkin Ligase.
  • the at least one zinc finger that may be substantially disrupted correspond to or align with one or more domains selected from the group consisting amino acids 141-225, amino acids 238-293, amino acids 313-377, and amino acids 418-449 of human Parkin Ligase. See O60260 at the uniport website.
  • At least one of the zinc fingers in the Parkin ligase comprises, one, two, three or four cysteine residues.
  • the disruption of at least one zinc finger induces the activity of the Parkin ligase.
  • at least one of the zinc fingers in the Parkin ligase comprises, one, two, three or four cysteine residues from amino acids 141-225, amino acids 238-293, amino acids 313-377, and amino acids 418-449 of human Parkin Ligase.
  • a compound can react and thus disrupt one or more zinc fingers by binding or reacting to one or more cysteine residues selected from the group consisting of C182, C258 and C377 of human Parkin Ligase.
  • the methods of the present invention also include activating auto-ubiquitinization of a Parkin ligase by administering to a subject in need thereof a therapeutically effective amount of one or more compounds.
  • the one or more compounds disrupt at least one Parkin ligase zinc finger.
  • the compounds in Table 1 and/or Table 2 may be used to activate auto-ubiquitinization of Parkin ligase.
  • the compounds in Table 1 and/or Table 2 may be used in addition with other compounds to activate auto-ubiquitinization of Parkin ligase.
  • Phospho Ubiquitin an endogenous cellular regulator of Parkin
  • Parkin ligase an endogenous cellular regulator of Parkin
  • one or more compounds in Table 1 and/or Table 2, or salts and esters thereof may be administered to a subject in need thereof that acts synergistically with Phospho Ubiquitin (pUB) in activating the Parkin ligase. See, e.g., Example 5.
  • one or more compounds may be administered with pUB to synergistically increase the activation of Parkin ligase and/or its auto-ubiquitinization.
  • the compound may be a chelator and/or an electrophile.
  • the one or more compounds are selected from Table 1 and/or Table 2, or a salt or ester thereof.
  • the activation of the Parkin ligase treats or reduces the incidence of one or more diseases or ailments selected from the group consisting of Alzheimer's Dementia, Parkinson's disease, Huntington Disease, Amyotrophic Lateral Sclerosis (ALS), Freidreich's ataxia, Spinocerebellar Ataxia, Multiple Systems Atrophy, PSP, Tauopathy, Diffuse Lewy Body Disease, Lewy Body dementia, any disorder characterized by abnormal accumulation of ⁇ -synuclein, disorders of the aging process, stroke, bacterial infection, viral infection, Mitochondrial related disease, mental retardation, deafness, blindness, diabetes, obesity, cardiovascular disease, multiple sclerosis, Sjogrens syndrome, lupus, glaucoma, including pseudoexfoliation glaucoma, Leber's Hereditary Optic Neuropathy, and rheumatoid arthritis.
  • Alzheimer's Dementia Parkinson's disease, Huntington Disease, Amyotrophic Lateral Sclerosis (ALS), Freidreich'
  • the bacterial infection is Mycobacterium infection.
  • the viral infection is HIV, Hepatitis B infection or Hepatitis C infection.
  • Another embodiment of the present invention includes methods of treating and/or reducing the incidence of cancer, specifically comprising administering to a subject in need thereof a therapeutically effective amount of one or more compounds that disrupt at least one Parkin ligase zinc finger and induces Parkin ligase activity.
  • the activated Parkin ligase suppresses the growth of one or more tumors and/or prevents metastasis of one or more tumors.
  • the cancer may be selected from one or more of the group consisting of Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma, Lymphoma, Anal Cancer, Appendix Cancer, Astrocytomas, Childhood Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Skin Cancer (Nonmelanoma), Childhood Bile Duct Cancer, Extrahepatic Bladder Cancer, Bone Cancer, Ewing Sarcoma Family of Tumors, Osteosarcoma and Malignant Fibrous Histiocytoma, Brain Stem Glioma, Brain Tumors, Embryonal Tumors, Germ Cell Tumors, Craniopharyngioma, Ependymoma, Bronchial Tumors, Burkitt Lymphoma (Non-Hodgkin Lymphoma), Carcinoid Tumor, Gastrointestinal
  • Islet Cell Tumors Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma, Kidney Cancer, Renal Cell Cancer, Wilms Tumor and Other Childhood Kidney Tumors, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Chronic Lymphocytic Cancer, Chronic Myelogenous Cancer, Hairy Cell Cancer, Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In situ (LCIS), Lung Cancer, Non-Small Cell Cancer, Small Cell Cancer, Lymphoma, Cutaneous T-Cell (Mycosis Fungoides and Sézary Syndrome), Hodgkin Cancer, Non-Hodgkin Cancer, Macroglobulinemia, Waldenström, Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Intraocular (Eye) Cancer, Merkel Cell Carcinoma, Mesothelioma, Malignant, Metastatic Squa
  • the cancer is glioblastoma, small cell lung carcinoma, breast cancer and/or prostate cancer.
  • the administration of the Parkin ligase suppresses one or more tumors in the subject.
  • the compound eliminates damaged mitochondria, increases cell viability during cellular stress, decreases tumor transformation and/or mitigates alpha-synuclein in cells.
  • the methods of the present invention include treating and/or reducing the incidence of Parkinson's disease, specifically by administering to a subject in need thereof a therapeutically effective amount of one or more compounds that disrupt at least one Parkin ligase zinc finger and induces Parkin ligase activity, wherein the compound can coordinate with a Zn ion and/or react with a thiol group in a cysteine(s).
  • the one or more compounds eliminate damaged mitochondria, increases cell viability during cellular stress and/or mitigates alpha-synuclein in cells. “Somatic Mutations of the Parkinson's disease-associated gene PARK2 in glioblastoma and other human malignancies” ( Nature Genetics January 2010 42(1)77-82).
  • the Parkin ligase activation alters ubiquitination.
  • the alteration of ubiquitination is caused by the ability of Parkin to modify a substrate protein by covalent attachment of Ubiquitin, a substrate protein being Parkin itself, or another protein such as Mitofusion 1 or 2, FBW7, or other publicly reported substrates of Parkin ligase.
  • the methods of the present invention include treating and/or reducing the incidence of cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger and induces Parkin ligase activity, wherein the compound can coordinate with a zinc ion and/or bind or react with a cysteine.
  • the compound is from Table 1 or Table 2 or a salt or ester thereof.
  • the Parkin ligase suppresses the growth of one or more tumors and/or prevents metastasis of one or more tumors.
  • the compound eliminates damaged mitochondria, increases cell viability during cellular stress, decreases tumor transformation and/or mitigates alpha-synuclein in cells.
  • the cancer is glioblastoma, small cell lung carcinoma, breast cancer or prostate cancer.
  • the compound for treating and/or reducing the incidence of cancer can coordinate to a Zn ion as a monodentate, bidentate, or tridentate ligand.
  • the compound for treating and/or reducing the incidence of cancer can coordinate to a Zn ion substantially which disrupts the structure of at least one zinc finger in the Parkin ligase.
  • the amino acid residues of at least one zinc finger corresponds to or aligns with one or more amino acid domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase.
  • the compound substantially disrupts the structure of at least one zinc finger located in the IBR domain (amino acids 328-377).
  • the zinc finger comprises four cysteine residues.
  • the Parkin ligase activation alters ubiquitination.
  • the compound binds or reacts with the thiol group in a cysteine.
  • the cysteine is selected from one or more of the group consisting of C59 and C377 of human Parkin ligase.
  • the cysteine is C377 of human Parkin ligase.
  • the compound the compound is AH001 and/or AH007.
  • the compound for treating and/or reducing the incidence of cancer is an alkylator, oxidant, Michael acceptor, another unsaturated structure, and/or has a disulfide.
  • this compound also substantially disrupts the structure of at least one zinc finger in the Parkin ligase.
  • the amino acid residues of the at least one zinc finger corresponds to or aligns with one or more amino acid domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase.
  • the zinc finger comprises four cysteine residues.
  • the methods of the present invention include treating and/or reducing the incidence of Parkinson's disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger and induces Parkin ligase activity, wherein the compound can coordinate with a zinc ion and/or bind or react with a cysteine.
  • the compound is from Table 1 or Table 2 or a salt or ester thereof.
  • the compound eliminates damaged mitochondria, increases cell viability during cellular stress and/or mitigates alpha-synuclein in cells.
  • the compound that can coordinate to a zinc ion is a monodentate, bidentate, or tridentate ligand.
  • the amino acid residues of at least one zinc finger corresponds to or aligns with one or more amino acid domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase.
  • the compound substantially disrupts the structure of at least one zinc finger located in the IBR domain (amino acids 328-377).
  • the zinc finger comprises four cysteine residues.
  • the Parkin ligase activation alters ubiquitination.
  • the compound binds or reacts with the thiol group in a cysteine.
  • the cysteine is selected from one or more of the group consisting of C59 and C377 of human Parkin ligase.
  • the cysteine is C377 of human Parkin ligase.
  • the compound the compound is AH001 and/or AH007.
  • the Parkin ligase activation alters ubiquitination wherein the alteration of ubiquitination is caused by the ability of Parkin to modify a substrate protein by covalent attachment of Ubiquitin, a substrate protein being Parkin itself, or another protein such as Mitofusion 1 or 2, FBW7, or other publicly reported substrates of Parkin ligase.
  • a compound induces Parkin ligase activation.
  • the compound is from Table 1 or Table 2 or a salt or ester thereof.
  • the compound is an alkylator, oxidant, Michael acceptor, another unsaturated structure, or has a disulfide.
  • the compound substantially disrupts the structure of at least one zinc finger in the Parkin ligase.
  • the amino acid residues of at least one zinc finger corresponds to or aligns with one or more amino acid domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase.
  • the compound substantially disrupts the structure of at least one zinc finger located in the IBR domain (amino acids 328-377).
  • the zinc finger comprises four cysteine residues.
  • the Parkin ligase activation alters ubiquitination.
  • the compound binds or reacts with the thiol group in a cysteine.
  • the cysteine is selected from one or more of the group consisting of C59 and C377 of human Parkin ligase. In another embodiment, the cysteine is C377 of human Parkin ligase. In another embodiment, the compound the compound is AH001 and/or AH007.
  • proteins While proteins are built-up to cater for the structural and biochemical requirements of the cell, they are also broken-down in a highly-regulated process serving more purposes than just destruction and space management. Proteins have different half-lives, determined by the nature of the amino acids present at their N-termini. Some will be long-lived, while other will rapidly be degraded. Proteolysis not only enables the cell to dispose of misfolded or damaged proteins, but also to fine-tune the concentration of essential proteins within the cell, such as the proteins involved in the cell cycle. This rapid, highly specific degradation can be achieved through the addition of one to several ubiquitin molecules to a target protein. The process is called ubiquitination.
  • Ubiquitination is crucial for a plethora of physiological processes, including cell survival and differentiation and innate and adaptive immunity.
  • considerable progress has been made in the understanding of the molecular action of ubiquitin in signaling pathways and how alterations in the ubiquitin system lead to the development of distinct human diseases. It has been shown that ubiquitination plays a role in the onset and progression of cancer, metabolic syndromes, neurodegenerative diseases, autoimmunity, inflammatory disorders, infection and muscle dystrophies (Popovic et al. Nature Medicine 20, 1242-1253 (2014)).
  • Some embodiments of the present invention relate to methods of treating, preventing, or ameliorating one or more symptoms of diseases or disorders associated with but not limited to solid tumors, such as glioma (oligodendrogliomas, mixed gliomas and glioblastomas), lung cancer, breast cancer, prostate cancer, ovarian cancer, and Warburg effect in tumors (restoration of Parkin activity to prevent Warburg effect).
  • Solid tumors such as glioma (oligodendrogliomas, mixed gliomas and glioblastomas), lung cancer, breast cancer, prostate cancer, ovarian cancer, and Warburg effect in tumors (restoration of Parkin activity to prevent Warburg effect).
  • Human genetic and pathology data support Parkin protein as a high value target. If there is not enough activated Parkin, then cell death and loss of dopamine neurons occurs. (“Familial Parkinson Disease Gene Product, Parkin, Is a Ubiquitin-Protein Ligase” Nature Genetics 25, 302-305, 1 Jul. 2000).
  • FIG. 1 For embodiments of the present invention, relate to methods of treating, preventing, or ameliorating one or more symptoms associated with neurological diseases or disorders including but not limited to Alzheimer's Dementia, Parkinson's disease, Huntington Disease, Amyotrophic Lateral Sclerosis (ALS), Freidreich's ataxia, Spinocerebellar Ataxia, Multiple Systems Atrophy, PSP, Tauopathy, Diffuse Lewy Body Disease, Lewy Body dementia, any disorder characterized by abnormal accumulation of ⁇ -synuclein, disorders of the aging process, and stroke.
  • ALS Amyotrophic Lateral Sclerosis
  • ALS Freidreich's ataxia
  • Spinocerebellar Ataxia Multiple Systems Atrophy
  • PSP Tauopathy
  • Diffuse Lewy Body Disease Lewy Body dementia
  • Lewy Body dementia any disorder characterized by abnormal accumulation of ⁇ -synuclein, disorders of the aging process, and stroke.
  • inventions relate to methods of treating, preventing, or ameliorating one or more symptoms associated with but not limited to mental retardation, deafness, blindness, diabetes, obesity, cardiovascular disease, and autoimmune diseases such as multiple sclerosis, Sjogrens syndrome, lupus, glaucoma, including pseudoexfoliation glaucoma, Leber's Hereditary Optic Neuropathy, and rheumatoid arthritis.
  • autoimmune diseases such as multiple sclerosis, Sjogrens syndrome, lupus, glaucoma, including pseudoexfoliation glaucoma, Leber's Hereditary Optic Neuropathy, and rheumatoid arthritis.
  • Increased Parkin activity eliminates damaged mitochondria (red) and increases cell viability during cellular stress, decreasing tumor transformation and mitigating ⁇ -synuclein in cells.
  • Multiple diverse Parkin activating compounds were identified that remove zinc from Parkin. Activation after ZnF unfolding is similar to other known ZnF proteins.
  • the present invention also includes pharmaceutical compositions for activating a Parkin ligase in a subject.
  • the pharmaceutical compositions comprise one or more compounds or the salts thereof that disrupt at least one Parkin ligase zinc finger.
  • the one or more compounds or the salts thereof can coordinate with a Zn ion, and/or react with at least one thiol group in a cysteine.
  • the pharmaceutical compositions may comprise one or more compounds selected from the group consisting of the compounds in Table 1 and Table 2.
  • the compounds, methods and pharmaceutical compositions in the present invention may be from one or more of following drug classes: 8-hydroxyquinolines; alpha-hydroxyketone; aminomethyl benzimidazoles; aminomethyl indoles; barbiturates; benzisothiazolones, carboxylate, dithiobisbenzamides, dithiocarbamates, formamides, hydrazides, hydroxamates, hydroxypyridinones/hydroxypyranones, hydroxysulfonamides, imidazoles, ketone hydrates, N-acyl ortho-phenylenediamines, N-hydroxyureas, O-substituted phosphamates, phosphamates, phosphones, sulfamates, sulfamides, sulfodiimines, sulfonamides, thiadiazines, thiadiazolothiones, and thiols.
  • 8-hydroxyquinolines alpha-hydroxyketone
  • the present invention also relates to the pharmaceutical formulations for activating Parkin ligase in a subject comprising an effective does of an agent that disrupts at least one Parkin ligase zinc finger, wherein the agent or the compound that can coordinate a zinc ion, or the agent or the compound that can react with the thiol group in a cysteine.
  • compositions further comprising a pharmaceutically acceptable excipient or adjuvant.
  • the methods of the present invention include any clinically-acceptable route of administration of the composition to the subject.
  • the route of administration is systemic, e.g., oral or by injection.
  • the agents or compounds, or pharmaceutically acceptable salts or derivatives thereof are administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperintoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally, intraportally, and parenterally.
  • the route of administration is local, e.g., topical, intra-tumor and peri-tumor.
  • the compound is administered orally.
  • the agents disclosed herein are administered by the intravenous route.
  • the parenteral administration may be provided in a bolus or by infusion.
  • composition is administered dependent, in part, upon the cause and/or location.
  • One skilled in the art will recognize the advantages of certain routes of administration.
  • the method includes administering an effective amount of the agent or compound (or composition comprising the agent or compound) to achieve a desired biological response, e.g., an amount effective to alleviate, ameliorate, or prevent, in whole or in part, a symptom of a condition to be treated, e.g., oncology and neurology disorders.
  • a desired biological response e.g., an amount effective to alleviate, ameliorate, or prevent, in whole or in part, a symptom of a condition to be treated, e.g., oncology and neurology disorders.
  • the amount of the compound of any one of structural formulas shown in Table 1 and or Table 2, or salt or ester thereof administered is about 0.001 mg/kg to about 100 mg/kg body weight (e.g., about 0.01 mg/kg to about 10 mg/kg or about 0.1 mg/kg to about 5 mg/kg).
  • the concentration of a disclosed compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic characteristics of the compound(s) employed, and the route of administration.
  • the agent may be administered in a single dose or in repeat doses.
  • the dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. Treatments may be administered daily or more frequently depending upon a number of factors, including the overall health of a patient, and the formulation and route of administration of the selected compound(s). An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • the compound or composition of the invention may be manufactured and/or administered in single or multiple unit dose forms.
  • compositions comprising the compound of any one of structural formulas shown in Table 1, and or Table 2 and a pharmaceutically-acceptable carrier, e.g., a pharmaceutically-acceptable excipient, carrier, binder, and/or diluent.
  • a pharmaceutically-acceptable carrier e.g., a pharmaceutically-acceptable excipient, carrier, binder, and/or diluent.
  • the compounds described herein, and the pharmaceutically acceptable salts, solvates, hydrates, and prodrugs thereof are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent.
  • Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions.
  • the composition comprises one or more additional therapeutic agents. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.
  • compositions of the present invention comprise one or more excipients.
  • excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
  • a pharmaceutical composition of the present invention is prepared using known techniques, including, but not limited to mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.
  • Additional embodiments relate to the pharmaceutical formulations wherein the formulation is selected from the group consisting of a solid, powder, liquid and a gel.
  • a pharmaceutical composition of the present invention is a liquid (e.g., a suspension, elixir and/or solution).
  • a liquid pharmaceutical composition is prepared using ingredients known in the art, including, but not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.
  • a pharmaceutical composition of the present invention is a solid (e.g., a powder, tablet, and/or capsule).
  • a solid pharmaceutical composition comprising one or more ingredients known in the art, including, but not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents.
  • a pharmaceutical composition of the present invention is formulated as a depot preparation. Certain such depot preparations are typically longer acting than non-depot preparations. In certain embodiments, such preparations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. In certain embodiments, depot preparations are prepared using suitable polymeric or hydrophobic materials (for example an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical composition of the present invention comprises a delivery system.
  • delivery systems include, but are not limited to, liposomes and emulsions.
  • Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds.
  • certain organic solvents such as dimethylsulfoxide are used.
  • a pharmaceutical composition of the present invention comprises a co-solvent system.
  • co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • co-solvent systems are used for hydrophobic compounds.
  • a non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80 and 65% w/v polyethylene glycol 300.
  • the proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.
  • co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • a pharmaceutical composition of the present invention comprises a sustained-release system.
  • a sustained-release system is a semi-permeable matrix of solid hydrophobic polymers.
  • sustained-release systems may, depending on their chemical nature, release pharmaceutical agents over a period of hours, days, weeks or months.
  • a pharmaceutical composition of the present invention is prepared for oral administration.
  • a pharmaceutical composition is formulated by combining one or more agents and pharmaceutically acceptable carriers. Certain of such carriers enable pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject.
  • Suitable excipients include, but are not limited to, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • such a mixture is optionally ground and auxiliaries are optionally added.
  • pharmaceutical compositions are formed to obtain tablets or dragee cores.
  • disintegrating agents e.g., cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate are added.
  • dragee cores are provided with coatings.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to tablets or dragee coatings.
  • compositions for oral administration are push-fit capsules made of gelatin.
  • Certain of such push-fit capsules comprise one or more pharmaceutical agents of the present invention in admixture with one or more filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • pharmaceutical compositions for oral administration are soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • one or more pharmaceutical agents of the present invention are be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • compositions are prepared for buccal administration. Certain of such pharmaceutical compositions are tablets or lozenges formulated in conventional manner.
  • a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.).
  • a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
  • injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like.
  • compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
  • a pharmaceutical composition is prepared for transmucosal administration.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • a pharmaceutical composition is prepared for administration by inhalation.
  • Certain of such pharmaceutical compositions for inhalation are prepared in the form of an aerosol spray in a pressurized pack or a nebulizer.
  • Certain of such pharmaceutical compositions comprise a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined with a valve that delivers a metered amount.
  • capsules and cartridges for use in an inhaler or insufflator may be formulated.
  • Certain of such formulations comprise a powder mixture of a pharmaceutical agent of the invention and a suitable powder base such as lactose or starch.
  • a pharmaceutical composition is prepared for rectal administration, such as a suppository or retention enema.
  • Certain of such pharmaceutical compositions comprise known ingredients, such as cocoa butter and/or other glycerides.
  • a pharmaceutical composition is prepared for topical administration.
  • Certain of such pharmaceutical compositions comprise bland moisturizing bases, such as ointments or creams.
  • ointments or creams include, but are not limited to, petrolatum, petrolatum plus volatile silicones, and lanolin and water in oil emulsions.
  • suitable cream bases include, but are not limited to, cold cream and hydrophilic ointment.
  • the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • one or more compounds of the present invention are formulated as a prodrug.
  • a prodrug upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form.
  • prodrugs are useful because they are easier to administer than the corresponding active form.
  • a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form.
  • a prodrug may have improved solubility compared to the corresponding active form.
  • prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility.
  • a prodrug is an ester.
  • the ester is metabolically hydrolyzed to carboxylic acid upon administration.
  • the carboxylic acid containing compound is the corresponding active form.
  • a prodrug comprises a short peptide (polyamino acid) bound to an acid group.
  • the peptide is cleaved upon administration to form the corresponding active form.
  • a prodrug is produced by modifying a pharmaceutically active compound such that the active compound will be regenerated upon in vivo administration.
  • the prodrug can be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug.
  • ABP Activity-Based Probe
  • ABP consists of a ubiquitin moiety with an epitope tag (e.g. HA tag) at the N-terminus, and a reactive group at the C-terminus.
  • the activity of Parkin-RBR (w/o the R0 inhibitory domain) is significantly higher than the activity of Parkin-R0RBR or the activity of full-length Parkin.
  • TR-FRET Time Resolved Fluorescence Resonance Energy Transfer
  • N-terminal His 6 -tag enabling TR-FRET-assay ⁇ use of the purified protein that still have the N-terminal His 6 -SUMO-tags on.
  • Optimize assay e.g. in terms of concentrations of assay components, buffer, additives, order of addition of reagents, and incubation temperature
  • a Ubiquitin vinyl sulfone probe can be used that irreversibly binds to the active site cysteine of Parkin ligase. Covalent attachment of the probe to the Parkin can be monitored by TR-FRET.
  • Candidate activator compounds can be identified by increasing the activity of Parkin ligase due to an increase in TR-FRET signal. Screening for activating compounds can be distinguished from the controls as follows:
  • 100% activation signal Heat activated Parkin+100 nM control activator in DMSO.
  • 0% activation signal Heat activated Parkin+DMSO only.
  • Parkin activators can be identified by an increase of the 0% activation signal TR-FRET signal.
  • Activator/DMSO 2 ⁇ Activator candidates/2% DMSO Reaction time: 60 minutes
  • the Data can be read in CSV files. There are two tables in those CSV files, which are the values of 655 nm (channel 1) and 615 nm (channel 2) wavelengths respectively.
  • the % Activation of compound titration can then be used to find activation EC50 or highest % activation if less than 75% activation is seen for the candidate compound.
  • the Activity-Based Probe Assay was performed as in Example 2 above with various compounds in Table 1 and/or Table 2. At least two compounds indicated increasing Parkin activity with the activity-based probe Ubiquitin-vinyl sulfone. As demonstrated in FIG. 1 , compound N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide, a chelator compound (AH001) increases the Parkin Ligase reaction with the Activity-based Ubituitin vinyl sulfone probe.
  • AH001 a chelator compound
  • an electrophile and chelator compound increases the Parkin Ligase reaction with the Activity-based Ubituitin vinyl sulfone probe.
  • This example indicates that both Chelators and Electrophiles can Both Regulate and/or Increase Parkin Ligase Activity.
  • a Parkin pUB Auto-ubiquitinylation Assay is used to evaluate a candidate compound's potency to activate Parkin's ability to Auto-ubiquitinylate itself.
  • the principle of this assay is that the E3 Ligase Parkin catalyzes the transfer of Ubiquitin to target proteins, but also has the ability to auto-ubiquitinylate itself.
  • the phospho-Ubiquition (pUb) added to the assay alters the Parkin to a state where small molecule activators can enable the Parkin to auto-ubiquitinylate though the E1-E2 cascade reaction.
  • the use of a Eu cryptate Ubiquition and anti 6His-d2 that binds to the His tagged Parkin will give a signal when the Eu cryptate Ubiquition is auto-ubiquitinylate onto the Parkin which can be monitored by TR-FRET.
  • the Data can be read in CSV files. There are two tables in those CSV files, which are the values of 655 nm (channel 1) and 615 nm (channel 2) wavelengths respectively.
  • the % Activation of compound titration can then be used to find activation EC50 or highest % activation if less than 75% activation is seen for the candidate compound.
  • the Parkin pUB auto-ubiquitinylation Assay was performed as in Example 4 above with various compounds in Table 1 and/or Table 2. At least two compounds indicated increasing Parkin activity with the activity-based probe Ubiquitin-vinyl sulfone. As demonstrated in FIG. 3 , compound N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide, a chelator compound (AH001) increases Parkin activity in an auto-ubiquitination assay. Furthermore, as indicated in FIG.
  • an electrophile compound increases Parkin activity in an auto-ubiquitination assay.
  • both chelators and electrophiles can both regulate and/or increase Parkin ligase activity in an auto-ubiquitination assay.
  • Parkin ligase was incubated with AH007 compound, and the mixture was then subject to tandem mass spectometry analysis after proteolytic digestion to produce fragments of Parkin ligase. The goal was to identify specific fragments of Parkin that contain compound bound AH007, revealing the specific binding residues of Parkin for the compound. Compound An2H07 is also fragmented when analyzed by mass spectrometry. Therefore, characteristic pieces of the compound AH007 that are attached to specific residues of Parkin can also be identified.
  • the mass spectrometry data identified three specific fragments of AH007 at two specific residues of Parkin ligase.
  • the data identified a fragment of 253.08-256.09 of AH007 compound attached to cysteine residue 377 of Parkin ligase (C377) and a fragment of 343.14-346.14 of AH007 compound attached to C377.
  • the data also identified a fragment of 253.08-256.09 of AH007 compound attached to cysteine residue 59 of Parkin ligase (C59).
  • the mass spectrometry data of Parkin ligase incubated with AH007 compound thus indicates that the compound binds and/or attaches to two specific sites in Parkin ligase: C59 and C377.
  • Residues C59 and C377 were the only two consistent sites observed, even when the concentration of AH007 compound was dramatically increased in the mixture with Parkin ligase, suggesting specificity for these sites over numerous other sites of potential attachment.
  • at least C377 is included in ZnF domains of human Parkin Ligase, and thus accords with the theory that cysteine residues in the flexible Parkin ligase ZnF domains are vulnerable for attachment and/or interruption by small molecule candidates.
  • Peptide fragments of Parkin comprising C59 and/or C377 will be useful to design further binding assays and selection of additional modulating agents.

Abstract

The present invention is directed to methods and compositions for activating a Parkin ligase by administering to a subject in need thereof a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger. The present invention is also directed to methods of treating and/or reducing the incidence of diseases or conditions related to the activation of Parkin ligase.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of U.S. application Ser. No. 14/961,285, filed Dec. 7, 2015, which claims the priority benefit of U.S. Provisional Application No. 62/237,400, filed Oct. 5, 2015, U.S. Provisional Application No. 62/222,008, filed Sep. 22, 2015, and U.S. Provisional Application No. 62/087,972, filed Dec. 5, 2014, the disclosures of which are incorporated by reference herein in their entirety.
    • REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
  • This application incorporates by reference a Sequence Listing submitted with this application in computer readable form (CRF) as a text file entitled “ANDI_001_04US_SeqList_ST25” created on Nov. 16, 2018 and having a size of 4 KB.
    • FIELD OF THE INVENTION
  • The present invention relates to methods of activating Parkin ligase by disrupting zinc finger domains for therapeutic benefit.
    • BACKGROUND OF THE INVENTION
  • Ubiquitin-Proteasome Pathway System (UPS) is a critical pathway that regulates key regulator proteins and degrades misfolded or abnormal proteins. UPS is central to multiple cellular processes, and if defective or imbalanced, it leads to pathogenesis of a variety of diseases. Posttranslational modification of proteins by ubiquitin is a fundamental cellular mechanism that regulates protein stability and activity and underlies a multitude of functions, from almost every aspect of biology. The covalent attachment of ubiquitin to specific protein substrates is achieved through the action of E3 ubiquitin ligases. These ligases comprise over 500 different proteins and are categorized into multiple classes defined by the structural element of their E3 functional activity. Specifically, both HECT and RING ligases transfer an activated ubiquitin from a thioester to the e-amino acid group of a lysine residue on a substrate; however, HECT ligases have an active site cysteine that forms an intermediate thioester bond with ubiquitin, while RING ligases function as a scaffold to allow direct ubiquitin transfer from the E2 to substrate. Recent evidence suggests that a subfamily of RING ligases, the RING-between-RING (RBR) family, may contain a catalytic cysteine residue 1,2 in addition to a canonical RING domain. (Riley et al. 2013. Nat Commun. 4:1982, “Riley et al.”), which is herein incorporated by reference in its entirety.
  • Deubiquitinating proteins and ubiquitin-specific proteases (DUBs and USPs) and E3 Ligases play a vital role in the UPS. These proteins are supported by flexible Zinc Finger (ZnF) domains which stabilize the binding of ubiquitin (Ub) for specialized functions.
  • Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection. The individual RING domains for Parkin have been the subject of much debate, in regards to the specific residues that coordinate Zn ions, as well as their relationship to canonical RING crossbrace structures defining classical E2-binding domains. R0 is a novel domain structure, but is more similar to Zn-finger domains than to E3 RING domains (Riley et al. 2013. Nat Commun. 4:1982)
  • While many drug discovery programs focus on the UPS, few have been successful due to the lack of selectivity and direct access to enzymatic protein active sites. The present invention is directed towards a novel approach of disrupting Zn-finger domains that provide a therapeutic benefit for various diseases and disorders, including oncology and neurology disorders.
    • SUMMARY OF THE INVENTION
  • The present invention relates to modulating the structures and/or functions of ligases in the UPS by binding to zinc ions and/or cysteine residues in their ZnF domains, for therapeutic benefit. This mechanism is distinct from binding to the active sites of ligases, which receive the tail of Ub. The present invention is directed to a method for activating Parkin ligase by coordination of small molecules to zinc ions in Parkin ZnF domains, or by chemical reactions of small molecules with cysteine residues in Parkin ZnF domains. The coordination of small molecules to zinc ions may or may not remove the zinc ions from the ZnF domains. The chemical reactions of small molecules with cysteine residues may be reversible or irreversible.
  • Specific embodiments of the present invention include methods of activating a Parkin ligase. In a specific embodiment, the Parkin ligase may be activated by administering to a subject a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger. In another specific embodiment, the compound can coordinate with a Zn ion, and/or bind or react with a cysteine. In a specific embodiment, the compound may react with the thiol group in the cysteine.
  • In another embodiment, the activated Parkin ligase suppresses one or more tumors. In another specific embodiment, the activated Parkin ligase provides dopamine neuron protection.
  • Compounds that can coordinate to a Zn ion include, but are not limited to, a monodentate, bidentate, or tridentate ligand. Compounds that can react with the thiol group in the cysteine residue include, but are not limited to an alkylator, oxidant, Michael acceptor, another unsaturated structure, or a disulfide.
  • In certain embodiments, the compound eliminates damaged mitochondria, increases cell viability during cellular stress, decreases tumor transformation and/or mitigates alpha-synuclein in cells. In certain embodiments, the subject has been diagnosed with cancer. In certain embodiments, cancer is glioblastoma, small cell lung carcinoma, breast cancer or prostate cancer.
  • In some embodiments, the patient has been diagnosed with a neuro-degenerative disease.
  • In particular embodiments, the neurodegenerative disease is Parkinson's disease, dementia, Amyotrophic lateral sclerosis (ALS) or Huntington's disease. In further embodiments, the dementia is dementia with Lewy bodies (DLB), multiple system atrophy (MSA) or Progressive supranuclear palsy (PSP).
  • In other embodiments of the present invention, the compound substantially disrupts the structure of at least one zinc finger in the Parkin ligase. In certain embodiments, at least one zinc finger is selected from one or more of the group consisting of the domains defined by R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465. In a specific embodiment, the amino acid residues of at least one zinc finger corresponds to or aligns within one or more domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase. In further embodiments, the zinc finger comprises four cysteine residues. In a specific embodiment, the compound may be a zinc chelator.
  • In another specific embodiment, the compound can bind or react with one or more cysteine residues. In another specific embodiment, the compound can bind or react with one or more cysteine residues selected from the group consisting of C59 and C377 of human Parkin Ligase.
  • In one embodiment, the compound may substantially disrupt a structure of at least one zinc finger in the Parkin ligase. In another embodiment, the zinc finger in the Parkin ligase may be located within one or more domains selected from the group consisting of R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase. In another specific embodiment the zinc finger that is substantially disrupted is located in IBR amino acids 328-377 of the Parkin ligase.
  • In another embodiment, the compound may act synergistically with Phospho Ubiquitin (pUB) in activating the Parkin ligase.
  • In certain embodiments, Parkin ligase activation alters ubiquitination.
  • In another embodiment, activating the Parkin ligase treats or reduces the incidence of one or more diseases or ailments selected from the group consisting of Alzheimer's Dementia, Parkinson's disease, Huntington Disease, Amyotrophic Lateral Sclerosis (ALS), Freidreich's ataxia, Spinocerebellar Ataxia, Multiple Systems Atrophy, PSP, Tauopathy, Diffuse Lewy Body Disease, Lewy Body dementia, any disorder characterized by abnormal accumulation of α-synuclein, disorders of the aging process, stroke, bacterial infection, viral infection, Mitochondrial related disease, mental retardation, deafness, blindness, diabetes, obesity, cardiovascular disease, multiple sclerosis, Sjogrens syndrome, lupus, glaucoma, including pseudoexfoliation glaucoma, Leber's Hereditary Optic Neuropathy, and rheumatoid arthritis.
  • In a specific embodiment, the bacterial infection is Mycobacterium infection. In another specific embodiment, the viral infection is Hepatitis C infection. In another specific embodiment, the Mitochondrial related disease is selected from one or more of the group consisting of Alpers Disease, Barth Syndrome/LIC (Lethal Infantile Cardiomyopathy), Beta-oxidation Defects, Carnitine-Acyl-Carnitine Deficiency, Carnitine Deficiency, Creatine Deficiency Syndromes, Co-Enzyme Q10 Deficiency, Complex I Deficiency, Complex II Deficiency, Complex III Deficiency, Complex IV Deficiency/COX Deficiency, Complex V Deficiency, CPEO, CPT I Deficiency, CPT II Deficiency, KSS, Lactic Acidosis, LBSL—Leukodystrophy, LCAD, LCHAD, Leigh Disease or Syndrome, Luft Disease, MAD/Glutaric Aciduria Type II, MCAD, MELAS, MERRF, MIRAS, Mitochondrial Cytopathy, Mitochondrial DNA Depletion, Mitochondrial Encephalopathy, Mitochondrial Myopathy, MNGIE, NARP, Pearson Syndrome, Pyruvate Carboxylase Deficiency, Pyruvate Dehydrogenase Deficiency, POLG Mutations, Respiratory Chain related disease, SCAD, SCHAD, and VLCAD.
  • Another embodiment of the invention includes methods of treating and/or reducing the incidence of cancer. A specific embodiment includes administering to a subject in need thereof a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger and induces Parkin ligase activity, wherein the compound can coordinate with a Zn ion and/or react with a thiol group in a cysteine. In another specific embodiment, activating the Parkin ligase suppresses one or more tumors. In another embodiment, the compound eliminates damaged mitochondria, increases cell viability during cellular stress, decreases tumor transformation and/or mitigates α-synuclein in cells. In another embodiment, the cancer is glioblastoma, small cell lung carcinoma, breast cancer or prostate cancer.
  • Another embodiment of the present invention includes methods for treating and/or reducing the incidence of Parkinson's disease. A specific embodiment for treating and/or reducing the incidence of Parkinson's disease includes administering to a subject in need thereof a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger and induces Parkin ligase activity, wherein the compound can coordinate with a Zn ion and/or react with a thiol group in a cysteine.
  • In some embodiments, Parkin ligase activation alters ubiquitination, as defined by the ability of Parkin to modify a substrate protein by covalent attachment of ubiquitin, a substrate protein being Parkin itself, or another protein such as Mitofusion 1 or 2, FBW7, or other publicly reported substrates of Parkin ligase.
  • Another embodiment of the present invention includes pharmaceutical formulations. In a specific embodiment, the pharmaceutical formulations activate Parkin ligase. In another specific embodiment, the pharmaceutical formulations may comprise an effective amount of a compound or its salt thereof that disrupts at least one Parkin ligase zinc finger, and a pharmaceutically acceptable carrier, wherein the compound or its salt thereof can coordinate a Zn ion, and/or react with the thiol group in a cysteine. In another specific embodiment, the compound can bind or react with one or more cysteine residues selected from the group consisting of C59 and C377 of human Parkin Ligase. In a specific embodiment, the pharmaceutical composition is in a formulation selected from the group consisting of a solid, powder, liquid and a gel.
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 indicates that N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide, a chelator compound (identified as AH001 or compound 76 in Table 2) increases the Parkin Ligase reaction with the Activity-based Ubiquitin vinyl sulfone probe.
  • FIG. 2 indicates that 6-benzyl-2,5-dimethyl-3-phenylpyrazolo[1,5-a]pyrimidine-7-thiol, an electrophile and chelator compound (identified as AH007 or compound 77 in Table 2) increases the Parkin Ligase reaction with the Activity-based Ubituitin vinyl sulfone probe.
  • FIG. 3 indicates that compound N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide, a chelator compound (AH001) increases Parkin activity in an auto-ubiquitination assay.
  • FIGS. 4A and 4B indicate that N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide (AH001) with pUB synergistically increases parkin activation in an auto-ubiquitination assay and allows for a lower concentration of pUB to activate parkin.
  • FIG. 5 indicates that 6-benzyl-2,5-dimethyl-3-phenylpyrazolo[1,5-a]pyrimidine-7-thiol, an electrophile compound (AH007) increases Parkin activity in an auto-ubiquitination assay.
    •  DETAILED DESCRIPTION
  • All publications, patents and patent applications, including any drawings and appendices therein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, drawing, or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
    • Definitions
  • Pharmaceutically acceptable salts include those obtained by reacting the active compound functioning as a base, with an inorganic or organic acid to form a salt, for example, salts of hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, camphorsulfonic acid, oxalic acid, maleic acid, succinic acid, citric acid, formic acid, hydrobromic acid, benzoic acid, tartaric acid, fumaric acid, salicylic acid, mandelic acid, carbonic acid, etc. Those skilled in the art will further recognize that acid addition salts may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods.
  • The term “treating” means one or more of relieving, alleviating, delaying, reducing, reversing, improving, or managing at least one symptom of a condition in a subject. The term “treating” may also mean one or more of arresting, delaying the onset (i.e., the period prior to clinical manifestation of the condition) or reducing the risk of developing or worsening a condition.
  • An “effective amount” means the amount of a formulation according to the invention that, when administered to a patient for treating a state, disorder or condition is sufficient to effect such treatment. The “effective amount” will vary depending on the active ingredient, the state, disorder, or condition to be treated and its severity, and the age, weight, physical condition and responsiveness of the mammal to be treated.
  • The term “therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical formulation that is sufficient to result in a desired clinical benefit after administration to a patient in need thereof.
  • All weight percentages (i.e., “% by weight” and “wt. %” and w/w) referenced herein, unless otherwise indicated, are measured relative to the total weight of the pharmaceutical composition.
  • As used herein, “substantially” or “substantial” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is “substantially” enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking, the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained. The use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of action, characteristic, property, state, structure, item, or result. For example, a composition that is “substantially free of” other active agents would either completely lack other active agents, or so nearly completely lack other active agents that the effect would be the same as if it completely lacked other active agents. In other words, a composition that is “substantially free of” an ingredient or element or another active agent may still contain such an item as long as there is no measurable effect thereof
  • As used herein, the “alignment” of two or more protein/amino acid sequences may be performed using the alignment program ClustalW2, available at the ebi website. The following default parameters may be used for Pairwise alignment: Protein Weight Matrix=Gonnet; Gap Open=10; Gap Extension=0.1.
  • “Ubiquitin Proteasome Pathway System (UPS)” as used herein relates to the ubiquitin proteasome pathway, conserved from yeast to mammals, and is required for the targeted degradation of most short-lived proteins in the eukaryotic cell. Targets include cell cycle regulatory proteins, whose timely destruction is vital for controlled cell division, as well as proteins unable to fold properly within the endoplasmic reticulum. Ubiquitin modification is an ATP-dependent process carried out by three classes of enzymes. A “ubiquitin activating enzyme” (E1) forms a thio-ester bond with ubiquitin, a highly conserved 76-amino acid protein. This reaction allows subsequent binding of ubiquitin to a “ubiquitin conjugating enzyme” (E2), followed by the formation of an isopeptide bond between the carboxy-terminus of ubiquitin and a lysine residue on the substrate protein. The latter reaction requires a “ubiquitin ligase” (E3). E3 ligases can be single- or multi-subunit enzymes. In some cases, the ubiquitin-binding and substrate binding domains reside on separate polypeptides brought together by adaptor proteins or culling. Numerous E3 ligases provide specificity in that each can modify only a subset of substrate proteins. Further specificity is achieved by post-translational modification of substrate proteins, including, but not limited to, phosphorylation. Effects of monoubiquitination include changes in subcellular localization. However, multiple ubiquitination cycles resulting in a polyubiquitin chain are required for targeting a protein to the proteasome for degradation. The multisubunit 26S proteasome recognizes, unfolds, and degrades polyubiquitinated substrates into small peptides. The reaction occurs within the cylindrical core of the proteasome complex, and peptide bond hydrolysis employs a core threonine residue as the catalytic nucleophile. It has been shown that an additional layer of complexity, in the form of multiubiquitin chain receptors, may lie between the polyubiquitination and degradation steps. These receptors react with a subset of polyubiquitinated substrates, aiding in their recognition by the 26S proteasome, and thereby promoting their degradation. This pathway is not only important in cellular homeostasis, but also in human disease. Because ubiquitin/proteasome-dependent degradation is often employed in control of the cell division cycle and cell growth, researchers have found that proteasome inhibitors hold some promise of being developed into potential cancer therapeutic agents.
  • Protein degradation through the ubiquitin-proteasome system is the major pathway of non-lysosomal proteolysis of intracellular proteins. It plays important roles in a variety of fundamental cellular processes such as regulation of cell cycle progression, division, development and differentiation, apoptosis, cell trafficking, and modulation of the immune and inflammatory responses. The central element of this system is the covalent linkage of ubiquitin to targeted proteins, which are then recognized by the 26S proteasome, an adenosine triphosphate-dependent, multi-catalytic protease. Damaged, oxidized, or misfolded proteins as well as regulatory proteins that control many critical cellular functions are among the targets of this degradation process. Aberration of this system leads to the dysregulation of cellular homeostasis and the development of multiple diseases (Wang et al. Cell Mol Immunol. 2006 August; 3(4):255-61).
  • “Parkin Ligase” or “Parkin” as used herein relates to a protein which in humans is encoded by the PARK2 gene. (Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, Yokochi M, Mizuno Y, Shimizu N (April 1998). “Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism”. Nature 392 (6676): 605-608. doi:10.1038/33416. PMID 9560156. Matsumine H, Yamamura Y, Hattori N, Kobayashi T, Kitada T, Yoritaka A, Mizuno Y (April 1998). “A microdeletion of D6S305 in a family of autosomal recessive juvenile parkinsonism (PARK2)”. Genomics 49 (1): 143-146. doi:10.1006/geno.1997.5196. PMID 9570960. The protein is a component of a multiprotein E3 ubiquitin ligase complex which in turn is part of the ubiquitin-proteasome system that mediates the targeting of proteins for degradation. Mutations in the PARK2 gene are known to cause a familial form of Parkinson's disease known as autosomal recessive juvenile Parkinson's disease (AR-JP).
  • “Ligase” as used herein, is an enzyme that can catalyze the joining of two or more compounds or biomolecules by bonding them together with a new chemical bond. The “ligation” of the two usually with accompanying hydrolysis of a small chemical group dependent to one of the larger compounds or biomolecules, or the enzyme catalyzing the linking together of two compounds, e.g., enzymes that catalyze joining of groups C—O, C—S, C—N, etc. Ubiquitin-protein (E3) ligases are a large family of highly diverse enzymes selecting proteins for ubiquitination.
  • “Ub Ligases” are involved in disease pathogenesis for oncology, inflammation & infectious disease. E3 ligase belonging to the RING-between-RING (RBR) family of E3 ligases containing both canonical RING domains and a catalytic cysteine residue usually restricted to HECT E3 ligases; termed ‘RING/HECT hybrid’ enzymes. Mutations in Parkin linked to Parkinson's disease, cancer and mycobacterial infection. Parkin is recognized as a neuroprotective protein with a role in mitochondrial integrity. Human genetic data implicate loss of Parkin activity as a mechanism for pathogenesis of Parkinson's Disease (PD).
  • “Zinc Finger (ZnF) Domain” as used herein relates to a protein structure characterized by coordinating zinc ions to stabilize the functional activity. ZnF stabilize the binding of Ub, Deubiquitinating Enzymes (DUBs), and Ligases (E3) in the UPS.
  • “Ligands” as used herein bind to metal via one or more atoms in the ligand, and are often termed as chelating ligands. A ligand that binds through two sites is classified as bidentate, and three sites as tridentate. The “bite angle” refers to the angle between the two bonds of a bidentate chelate. Chelating ligands are commonly formed by linking donor groups via organic linkers. A classic bidentate ligand is ethylenediamine, which is derived by the linking of two ammonia groups with an ethylene (—CH2CH2-) linker. A classic example of a polydentate ligand is the hexadentate chelating agent EDTA, which is able to bond through six sites, completely surrounding some metals. The binding affinity of a chelating system depends on the chelating angle or bite angle. Many ligands are capable of binding metal ions through multiple sites, usually because the ligands have lone pairs on more than one atom. Some ligands can bond to a metal center through the same atom but with a different number of lone pairs. The bond order of the metal ligand bond can be in part distinguished through the metal ligand bond angle (M-X-R). This bond angle is often referred to as being linear or bent with further discussion concerning the degree to which the angle is bent. For example, an imido ligand in the ionic form has three lone pairs. One lone pair is used as a sigma X donor, the other two lone pairs are available as L type pi donors. If both lone pairs are used in pi bonds then the M-N-R geometry is linear. However, if one or both these lone pairs is non-bonding then the M-N-R bond is bent and the extent of the bend speaks to how much pi bonding there may be. It was found that few heteroatoms, such as nitrogen, oxygen, and sulfur atoms, interacted with zinc, ideal distances between the zinc and these heteroatoms were identified. Whereas carboxylates bound to the zinc via both monodentate and bidentate interactions, the hydroxamates bound dominantly in a bidentate manner. These results aid in the design of new inhibitors with the potential to interact with zinc in the target protein. Virtually every molecule and every ion can serve as a ligand for (or “coordinate to”) metals. Monodentate ligands include virtually all anions and all simple Lewis bases. Thus, the halides and pseudohalides are important anionic ligands whereas ammonia, carbon monoxide, and water are particularly common charge-neutral ligands. Simple organic species are also very common, be they anionic (RO− and RCO2 −) or neutral (R2O, R2S, R3-xNHx, and R3P). Complexes of polydentate ligands are called chelate complexes. They tend to be more stable than complexes derived from monodentate ligands. This enhanced stability, the chelate effect, is usually attributed to effects of entropy, which favors the displacement of many ligands by one polydentate ligand. When the chelating ligand forms a large ring that at least partially surrounds the central atom and bonds to it, leaving the central atom at the center of a large ring. The more rigid and the higher its denticity, the more inert will be the macrocyclic complex.
  • “Chelator” as used herein relates to a binding agent that suppresses chemical activity by forming a chelate (a coordination compound in which a metal atom or ion is bound to a ligand at two or more points on the ligand, so as to form, for example, a heterocyclic ring containing a metal atom).
  • “Chelation” as used herein relates to a particular way that ions and molecules bind metal ions. According to the International Union of Pure and Applied Chemistry (IUPAC), chelation involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central atom. Usually these ligands are organic compounds, and are called chelants, chelators, chelating agents, or sequestering agents.
  • “Electrophile” as used herein relates to species that is attracted to an electron rich center. In chemistry, an electrophile is a reagent attracted to electrons. It participates in a chemical reaction by accepting an electron pair in order to bond to a nucleophile. Because electrophiles accept electrons, they are Lewis acids. Most electrophiles are positively charged, have an atom that carries a partial positive charge, or have an atom that does not have an octet of electrons.
  • The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.
  • Ubiquitin-protein (E3) ligases are a large family of enzymes that select various proteins for ubiquitination. These ubiquitin ligases, called “Ub ligases” are known to have a role in various diseases and conditions, including but not limited to, cancer, inflammation and infectious diseases.
  • One specific Ub ligase is Parkin ligase. Parkin ligase is a component of a multiprotein “E3” ubiquitin ligase complex, which in turn is part of the ubiquitin-proteasome system that mediates the targeting of proteins for degradation. Although the specific function of Parkin ligase is not known, mutations in Parkin ligase are linked to various diseases, such as Parkinson's disease, cancer and mycobacterial infection. Parkin ligase is thus an attractive target for therapeutic intervention.
  • Further, there are various known methods for regulating ligases known in the art. Many ligases, particularly ligases involved in the Ubiquitin-Proteasome Pathway System (UPS), are known to have Zinc Finger (ZnF) domains that stabilize critical protein binding regions in that ligase.
  • ZnF domains coordinate zinc ions and this coordination stabilizes functional activity of the protein. The functional activity provided by proteins with ZnF domains can include the regulation of important cellular signaling pathways, such as recognizing ubiquitins, regulation of DNA, such as transcription and repair, and acting as cellular redox sensors. The binding of zinc to ZnF domains, or simply just regulating how zinc interacts with the ZnF domains, are essential to ligases involved in the UPS.
  • Parkin ligase is known to have one or more ZnF domains. The present invention focuses on two different strategies for modulating ZnF domains in Parkin ligase. One strategy of the present invention includes using chelating compounds that bind to the ZnF domains and thus disallow the binding of zinc, or cause the dissociation of zinc, such as Zn, or Zn2+, from the ZnF domain.
  • Another strategy of the present invention includes using compounds that bind or react with a cysteine amino acid residue in the ZnF domain. One or more cysteine residues (and sometimes with the assistance of histidine residues) are essential in ZnF domains for binding to and/or coordinating to the zinc ion. The zinc ion (usually Zn2+) can coordinate with multiple cysteine or histidine residues. The more cysteine residues there are in the domain, the more flexible is the ZnF domain. Ligases, such as Parkin ligase are thought to have multiple cysteine residues coordinated with zinc in their ZnF domains. This flexibility in the ZnF domains of Parkin ligase is thought allow the domain to be reversible, and is thus is one possible mechanism for regulating Parkin ligase.
  • The present invention thus relates to the use of one or more agents or one or more compounds with electrophilic, chelation or both electrophilic and chelation properties that can interact with the zinc ion and/or the cysteine residue(s) in a Parkin ligase and thus modulate Parkin ligase's activity. Specifically, it is believed that not allowing a zinc ion to coordinate in at least one of Parkin ligase's ZnF domains induces its activity. The present invention is thus directed to a method for activating Parkin ligase by the chelation of Zn followed by its removal from the ZnF domain, or through electrophilic attack at the cysteine amino acid(s) that holds the Zn in place.
  • Accordingly, in one embodiment of the present invention, the methods of activating a Parkin ligase include administering to a subject in need thereof a therapeutically effective amount of one or more compounds that disrupt at least one Parkin ligase zinc finger. In another embodiment, the methods of activating a Parkin ligase include administering to a subject two or more compounds that disrupt at least one Parkin ligase zinc finger.
  • In a specific embodiment, the compounds of the present invention may be an electrophile or a chelator. In another embodiment, the compounds of the present invention may be able to function as both an electrophile and as a chelator. For example, the compounds of the present invention can include multiple functional groups wherein a functional group has chelating properties and a functional group that has electrophilic properties.
  • In another specific embodiment, the compound is selected from one or more of the group consisting of the compounds in Table 1 and Table 2 or a salt or ester thereof. Although not bound to a specific theory, it is believed that at least some of the compounds in Table 1 or Table 2 may be chelators, electrophiles or both. For example, it is believed that compounds 76 and 97 from Table 2 act as a chelator, but compound 113 of Table 2 acts as a thiol-reactive electrophile. In another example, compounds from Table 1 or Table 2 can act as both an electrophile and as a nucleophile. It is believed, for example, that compounds 91 and 107 of Table 2 are both chelators, but can possibly also act as thiol-reactive electrophiles. Accordingly, in another specific embodiment, the compounds of the present invention are an electrophile, chelator or both an electrophile and a chelator selected from one or more of the group consisting of the compounds in Table 1 and Table 2 or a salt or ester thereof.
  • In another embodiment, the compounds in Table 1 and Table 2 or a salt or ester thereof bind and active Parkin ligase. For example, compounds N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide (AH001), a chelator compound and 6-benzyl-2,5-dimethyl-3-phenylpyrazolo[1,5-a]pyrimidine-7-thiol (AH007), an electrophile and chelator compound, both independently increase the Parkin Ligase reaction with the Activity-based Ubiquitin vinyl sulfone probe. See Examples 2-3. Therefore, both chelators and/or electrophiles can bind and activate Parkin ligase.
  • In another embodiment, the compound can be 2-(4-benzylpiperazin-1-yl)-N-[(2-hydroxy-3-prop-2-enyl-phenyl)methylideneamino]acetamide (also referred to as Pac-1) or a salt or ester thereof. Although not bound to a specific theory, the structure of Pac-1, provided as compound 114 in Table 2 below, is believed to be a chelator and may also have the ability to increase the Parkin Ligase reaction. In another embodiment, Pac-1, or a salt or thereof, may also be an electrophile and/or be both a chelator and an electrophile.
  • In another embodiment, the compound may be a derivative or analogue of Pac-1. In a specific embodiment, the compound may be a compound as described in PCT Application Publication Nos. WO2010/091382, WO2013/131089, WO2013/124407, WO2014/022858, U.S. Application Publication Nos. US2015/0210659, US2015/0231132, US2014/0073609, US20150017264, US2015/0099759, U.S. Pat. Nos. 8,916,705, 9,102,661, 8,592,584, and 8,778,945, the disclosures of which are incorporated by reference herein in their entirety.
  • In a specific example, Activity-based probe assays and mass spectrometry analysis indicate that some candidate compounds in Table 1 and/or Table 2 can bind or react with multiple cysteine residues in human Parkin ligase. For example, mass spectrometry analysis shows that AH007 binds to at least cysteine residue 59 (C59) and cysteine residue 377 (C377) of Parkin ligase.
  • Accordingly, in a specific embodiment, methods of activating a Parkin ligase include administering to a subject in need thereof a therapeutically effective amount of one or more compounds that disrupt at least one Parkin ligase zinc finger. In a specific embodiment, the one or more compounds are selected from Table 1 and/or Table 2, or a salt or ester thereof. In another embodiment the compound may be a chelator, an electrophile or both a chelator and an electrophile.
  • In another embodiment, the one or more compounds can coordinate with a Zn ion, and/or bind or react with one or more cysteine residues. In a specific embodiment the Zn ion may be either a Zn+ or a Zn2+ ion. In another embodiment, the compound can coordinate to a Zn ion is a monodentate, bidentate, or tridentate ligand.
  • In another embodiment, the compound can bind and/or react with a thiol group in more than one cysteine residues. In another embodiment, the compound can bind and/or react with a thiol group in two cysteine residues. In another embodiment, the compound can bind and/or react with a thiol group in three cysteine residues. In another embodiment, the compound can bind and/or react with a thiol group in four cysteine residues. In another specific embodiment, the compound can bind or react with one or more cysteine residues in one or more domains selected from the group consisting amino acids 141-225, amino acids 238-293, amino acids 313-377, and amino acids 418-449 of human Parkin Ligase. See 060260 at the uniport website.
  • In another specific embodiment, the compound can bind or react with one or more cysteine residues selected from the group consisting of C182, C258 and C377 of human Parkin Ligase. In another specific embodiment, the compound can bind or react with one or more cysteine residues selected from the group consisting of C59 and C377 of human Parkin Ligase. In a specific embodiment, the compound can react with C377 of human Parkin Ligase. In another specific embodiment, the compound is AH007.
  • In another embodiment, the compound can bind or react with one or more cysteine residues selected from one or more residues of a parkin ligase, parkin ligase derivative, or parkin ligase homologue that correspond to or align with C182, C258 and/or C377 of human Parkin Ligase. In another embodiment, the compound can bind or react with one or more cysteine residues selected from one or more residues of a parkin ligase, parkin ligase derivative, or parkin ligase homologue that correspond to or align with C59 and/or C377 of human Parkin Ligase. In another specific embodiment, the compound is AH007.
  • It is also believed that the IBR domain may play a key role in regulating Parkin activity. It is believed that the RO domain includes at least one ZnF domain that as discussed above, could be involved in one possible mechanism for regulating Parkin ligase. Accordingly, in a specific embodiment, the structure of at least one ZnF domain located in the IBR domain (amino acids 328-377) may be substantially disrupted by the administration of a compound to a subject in need thereof. In another embodiment, one or more compounds selected from Table 1 and/or Table 2, or a salt or ester thereof, may substantially disrupt the structure of at least one ZnF domain located in the IBR domain (amino acids 328-377).
  • In another embodiment, the compound can bind and/or react with a cysteine residue, including any histidine residue(s) in or near the ZnF domain.
  • In another embodiment, the compounds may substantially disrupt the structure of at least one zinc finger (or ZnF domain) in the Parkin ligase. In another embodiment, the compounds of the present invention may disrupt one or more ZnF domains in Parkin ligase.
  • Riley et al. describes a human Parkin ligase of 465 amino acids that includes multiple functional areas with Zn coordination residues (amino acid sequence provided in Table 3 (SEQ ID NO:1) and identified in the ncbi website (See sequence NM_004562.2)). Riley et al. discusses 4 domains designated as R0, R1, IBR and R2. R0, R1 and R2 which were previously designated as RING domains. Riley et al., however questions whether the R0, IBR and R2 domains are actual, or traditional RING domains: “R0 is a novel domain structure, but is more similar to Zn-finger domains than to E3 RING domains” Riley et al. also states that neither IBR or the R2 domains resemble the canonical RING domain motif, as they do not have a cross-brace structure as normally associated with RING domains. Furthermore, analysis of the R0, IBR and R2 domains indicates possible vulnerabilities in their zinc centers. Thus, the R0, IBR and R2 domains look like ideal domain candidates for regulating the activity of Parkin Ligase. The R0, IBR and R2 domains refer to amino acids 141-216, amino acids 328-377, and amino acids 415-465 of human Parkin Ligase, respectively.
  • Accordingly, in a specific embodiment, the at least one zinc finger that may be substantially disrupted correspond to or align with one or more domains selected from the group consisting amino acids 141-216, amino acids 328-377, and amino acids 415-465 of human Parkin Ligase. In another specific embodiment, the amino acids from the at least one zinc finger may overlap in an alignment with one or more of the R0, IBR and R2 domains from human Parkin Ligase.
  • In another specific embodiment, the at least one zinc finger that may be substantially disrupted correspond to or align with one or more domains selected from the group consisting amino acids 141-225, amino acids 238-293, amino acids 313-377, and amino acids 418-449 of human Parkin Ligase. See O60260 at the uniport website.
  • In a specific embodiment, at least one of the zinc fingers in the Parkin ligase comprises, one, two, three or four cysteine residues. In another embodiment, the disruption of at least one zinc finger induces the activity of the Parkin ligase. In another embodiment, at least one of the zinc fingers in the Parkin ligase comprises, one, two, three or four cysteine residues from amino acids 141-225, amino acids 238-293, amino acids 313-377, and amino acids 418-449 of human Parkin Ligase. For example, in a specific embodiment, a compound can react and thus disrupt one or more zinc fingers by binding or reacting to one or more cysteine residues selected from the group consisting of C182, C258 and C377 of human Parkin Ligase.
  • The methods of the present invention also include activating auto-ubiquitinization of a Parkin ligase by administering to a subject in need thereof a therapeutically effective amount of one or more compounds. In a specific embodiment, the one or more compounds disrupt at least one Parkin ligase zinc finger. In another example, the compounds in Table 1 and/or Table 2 may be used to activate auto-ubiquitinization of Parkin ligase. In another embodiment, the compounds in Table 1 and/or Table 2 may be used in addition with other compounds to activate auto-ubiquitinization of Parkin ligase. For example, Phospho Ubiquitin (pUB), an endogenous cellular regulator of Parkin, can be added to Parkin ligase which can activate Parkin ligase and its auto-ubiquitinization. In one embodiment, one or more compounds in Table 1 and/or Table 2, or salts and esters thereof, may be administered to a subject in need thereof that acts synergistically with Phospho Ubiquitin (pUB) in activating the Parkin ligase. See, e.g., Example 5. In another embodiment, one or more compounds may be administered with pUB to synergistically increase the activation of Parkin ligase and/or its auto-ubiquitinization. In another embodiment the compound may be a chelator and/or an electrophile. In a specific embodiment, the one or more compounds are selected from Table 1 and/or Table 2, or a salt or ester thereof.
  • In another specific embodiment, the activation of the Parkin ligase treats or reduces the incidence of one or more diseases or ailments selected from the group consisting of Alzheimer's Dementia, Parkinson's disease, Huntington Disease, Amyotrophic Lateral Sclerosis (ALS), Freidreich's ataxia, Spinocerebellar Ataxia, Multiple Systems Atrophy, PSP, Tauopathy, Diffuse Lewy Body Disease, Lewy Body dementia, any disorder characterized by abnormal accumulation of α-synuclein, disorders of the aging process, stroke, bacterial infection, viral infection, Mitochondrial related disease, mental retardation, deafness, blindness, diabetes, obesity, cardiovascular disease, multiple sclerosis, Sjogrens syndrome, lupus, glaucoma, including pseudoexfoliation glaucoma, Leber's Hereditary Optic Neuropathy, and rheumatoid arthritis.
  • In a specific embodiment, the bacterial infection is Mycobacterium infection. In another specific embodiment the viral infection is HIV, Hepatitis B infection or Hepatitis C infection. Another embodiment of the present invention includes methods of treating and/or reducing the incidence of cancer, specifically comprising administering to a subject in need thereof a therapeutically effective amount of one or more compounds that disrupt at least one Parkin ligase zinc finger and induces Parkin ligase activity. In a specific embodiment, the activated Parkin ligase suppresses the growth of one or more tumors and/or prevents metastasis of one or more tumors.
  • In another embodiment the cancer may be selected from one or more of the group consisting of Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma, Lymphoma, Anal Cancer, Appendix Cancer, Astrocytomas, Childhood Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Skin Cancer (Nonmelanoma), Childhood Bile Duct Cancer, Extrahepatic Bladder Cancer, Bone Cancer, Ewing Sarcoma Family of Tumors, Osteosarcoma and Malignant Fibrous Histiocytoma, Brain Stem Glioma, Brain Tumors, Embryonal Tumors, Germ Cell Tumors, Craniopharyngioma, Ependymoma, Bronchial Tumors, Burkitt Lymphoma (Non-Hodgkin Lymphoma), Carcinoid Tumor, Gastrointestinal Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Lymphoma, Primary, Cervical Cancer, Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Chronic Myeloproliferative Neoplasms Colon Cancer, Colorectal Cancer, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Intraocular Melanoma, Retinoblastoma, Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors, Extragonadal Cancer, Ovarian Cancer, Testicular Cancer, Gestational Trophoblastic Disease, Glioma, Brain Stem Cancer, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis, Langerhans Cell Cancer, Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma. Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma, Kidney Cancer, Renal Cell Cancer, Wilms Tumor and Other Childhood Kidney Tumors, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Chronic Lymphocytic Cancer, Chronic Myelogenous Cancer, Hairy Cell Cancer, Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer, Non-Small Cell Cancer, Small Cell Cancer, Lymphoma, Cutaneous T-Cell (Mycosis Fungoides and Sézary Syndrome), Hodgkin Cancer, Non-Hodgkin Cancer, Macroglobulinemia, Waldenström, Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Intraocular (Eye) Cancer, Merkel Cell Carcinoma, Mesothelioma, Malignant, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia, Chronic, Myeloid Leukemia, Acute, Myeloma Multiple, Chronic Myeloproliferative Neoplasms, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer, Lip and Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Epithelial Cancer, Low Malignant Potential Tumor, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Primary Central Nervous System Lymphoma, Rectal Cancer, Renal Cell (Kidney) Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Ewing Cancer, Kaposi Cancer, Osteosarcoma (Bone Cancer), Soft Tissue Cancer, Uterine Cancer, Sézary Syndrome, Skin Cancer, Childhood Melanoma, Merkel Cell Carcinoma, Nonmelanoma, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Skin Cancer (Nonmelanoma), Childhood Squamous Neck Cancer with Occult Primary, Metastatic Cancer, Stomach (Gastric) Cancer, T-Cell Lymphoma, Cutaneous Cancer, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Unknown Primary, Carcinoma of Childhood, Unusual Cancers of Childhood, Urethral Cancer, Uterine Cancer, Endometrial Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenström Macroglobulinemia. Wilms Tumor, and Women's Cancers.
  • In a specific embodiment, the cancer is glioblastoma, small cell lung carcinoma, breast cancer and/or prostate cancer. In another embodiment, the administration of the Parkin ligase suppresses one or more tumors in the subject.
  • In another specific embodiment, the compound eliminates damaged mitochondria, increases cell viability during cellular stress, decreases tumor transformation and/or mitigates alpha-synuclein in cells.
  • In another embodiment, the methods of the present invention include treating and/or reducing the incidence of Parkinson's disease, specifically by administering to a subject in need thereof a therapeutically effective amount of one or more compounds that disrupt at least one Parkin ligase zinc finger and induces Parkin ligase activity, wherein the compound can coordinate with a Zn ion and/or react with a thiol group in a cysteine(s). In another embodiment, the one or more compounds eliminate damaged mitochondria, increases cell viability during cellular stress and/or mitigates alpha-synuclein in cells. “Somatic Mutations of the Parkinson's disease-associated gene PARK2 in glioblastoma and other human malignancies” (Nature Genetics January 2010 42(1)77-82).
  • In another embodiment, the Parkin ligase activation alters ubiquitination. Specifically, the alteration of ubiquitination is caused by the ability of Parkin to modify a substrate protein by covalent attachment of Ubiquitin, a substrate protein being Parkin itself, or another protein such as Mitofusion 1 or 2, FBW7, or other publicly reported substrates of Parkin ligase.
  • In a specific embodiment, the methods of the present invention include treating and/or reducing the incidence of cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger and induces Parkin ligase activity, wherein the compound can coordinate with a zinc ion and/or bind or react with a cysteine. In a specific embodiment, the compound is from Table 1 or Table 2 or a salt or ester thereof. In a specific embodiment, the Parkin ligase suppresses the growth of one or more tumors and/or prevents metastasis of one or more tumors. In another embodiment, the compound eliminates damaged mitochondria, increases cell viability during cellular stress, decreases tumor transformation and/or mitigates alpha-synuclein in cells. In another embodiment, the cancer is glioblastoma, small cell lung carcinoma, breast cancer or prostate cancer.
  • In another embodiment, the compound for treating and/or reducing the incidence of cancer can coordinate to a Zn ion as a monodentate, bidentate, or tridentate ligand. In another embodiment, the compound for treating and/or reducing the incidence of cancer can coordinate to a Zn ion substantially which disrupts the structure of at least one zinc finger in the Parkin ligase. In another embodiment, the amino acid residues of at least one zinc finger corresponds to or aligns with one or more amino acid domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase. In another embodiment, the compound substantially disrupts the structure of at least one zinc finger located in the IBR domain (amino acids 328-377). In another embodiment, the zinc finger comprises four cysteine residues. In another embodiment, the Parkin ligase activation alters ubiquitination. In another embodiment, the compound binds or reacts with the thiol group in a cysteine. In another embodiment, the cysteine is selected from one or more of the group consisting of C59 and C377 of human Parkin ligase. In another embodiment, the cysteine is C377 of human Parkin ligase. In another embodiment, the compound the compound is AH001 and/or AH007.
  • In another embodiment, the compound for treating and/or reducing the incidence of cancer is an alkylator, oxidant, Michael acceptor, another unsaturated structure, and/or has a disulfide. In a specific embodiment, this compound also substantially disrupts the structure of at least one zinc finger in the Parkin ligase. In another embodiment, the amino acid residues of the at least one zinc finger corresponds to or aligns with one or more amino acid domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase. In another embodiment, the zinc finger comprises four cysteine residues.
  • In a specific embodiment, the methods of the present invention include treating and/or reducing the incidence of Parkinson's disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound that disrupts at least one Parkin ligase zinc finger and induces Parkin ligase activity, wherein the compound can coordinate with a zinc ion and/or bind or react with a cysteine. In a specific embodiment, the compound is from Table 1 or Table 2 or a salt or ester thereof. In a specific embodiment, the compound eliminates damaged mitochondria, increases cell viability during cellular stress and/or mitigates alpha-synuclein in cells. In another embodiment, the compound that can coordinate to a zinc ion is a monodentate, bidentate, or tridentate ligand. In another embodiment, the amino acid residues of at least one zinc finger corresponds to or aligns with one or more amino acid domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase. In another embodiment, the compound substantially disrupts the structure of at least one zinc finger located in the IBR domain (amino acids 328-377). In another embodiment, the zinc finger comprises four cysteine residues. In another embodiment, the Parkin ligase activation alters ubiquitination. In another embodiment, the compound binds or reacts with the thiol group in a cysteine. In another embodiment, the cysteine is selected from one or more of the group consisting of C59 and C377 of human Parkin ligase. In another embodiment, the cysteine is C377 of human Parkin ligase. In another embodiment, the compound the compound is AH001 and/or AH007.
  • In another embodiment, the Parkin ligase activation alters ubiquitination wherein the alteration of ubiquitination is caused by the ability of Parkin to modify a substrate protein by covalent attachment of Ubiquitin, a substrate protein being Parkin itself, or another protein such as Mitofusion 1 or 2, FBW7, or other publicly reported substrates of Parkin ligase. In a specific embodiment, a compound induces Parkin ligase activation. In another embodiment, the compound is from Table 1 or Table 2 or a salt or ester thereof. In another embodiment, the compound is an alkylator, oxidant, Michael acceptor, another unsaturated structure, or has a disulfide. In another embodiment, the compound substantially disrupts the structure of at least one zinc finger in the Parkin ligase. In another embodiment, the amino acid residues of at least one zinc finger corresponds to or aligns with one or more amino acid domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase. In another embodiment, the compound substantially disrupts the structure of at least one zinc finger located in the IBR domain (amino acids 328-377). In another embodiment, the zinc finger comprises four cysteine residues. In another embodiment, the Parkin ligase activation alters ubiquitination. In another embodiment, the compound binds or reacts with the thiol group in a cysteine. In another embodiment, the cysteine is selected from one or more of the group consisting of C59 and C377 of human Parkin ligase. In another embodiment, the cysteine is C377 of human Parkin ligase. In another embodiment, the compound the compound is AH001 and/or AH007.
  • While proteins are built-up to cater for the structural and biochemical requirements of the cell, they are also broken-down in a highly-regulated process serving more purposes than just destruction and space management. Proteins have different half-lives, determined by the nature of the amino acids present at their N-termini. Some will be long-lived, while other will rapidly be degraded. Proteolysis not only enables the cell to dispose of misfolded or damaged proteins, but also to fine-tune the concentration of essential proteins within the cell, such as the proteins involved in the cell cycle. This rapid, highly specific degradation can be achieved through the addition of one to several ubiquitin molecules to a target protein. The process is called ubiquitination.
  • Ubiquitination is crucial for a plethora of physiological processes, including cell survival and differentiation and innate and adaptive immunity. In recent years, considerable progress has been made in the understanding of the molecular action of ubiquitin in signaling pathways and how alterations in the ubiquitin system lead to the development of distinct human diseases. It has been shown that ubiquitination plays a role in the onset and progression of cancer, metabolic syndromes, neurodegenerative diseases, autoimmunity, inflammatory disorders, infection and muscle dystrophies (Popovic et al. Nature Medicine 20, 1242-1253 (2014)).
  • Some embodiments of the present invention relate to methods of treating, preventing, or ameliorating one or more symptoms of diseases or disorders associated with but not limited to solid tumors, such as glioma (oligodendrogliomas, mixed gliomas and glioblastomas), lung cancer, breast cancer, prostate cancer, ovarian cancer, and Warburg effect in tumors (restoration of Parkin activity to prevent Warburg effect). Human genetic and pathology data support Parkin protein as a high value target. If there is not enough activated Parkin, then cell death and loss of dopamine neurons occurs. (“Familial Parkinson Disease Gene Product, Parkin, Is a Ubiquitin-Protein Ligase” Nature Genetics 25, 302-305, 1 Jul. 2000).
  • Further embodiments of the present invention relate to methods of treating, preventing, or ameliorating one or more symptoms associated with neurological diseases or disorders including but not limited to Alzheimer's Dementia, Parkinson's disease, Huntington Disease, Amyotrophic Lateral Sclerosis (ALS), Freidreich's ataxia, Spinocerebellar Ataxia, Multiple Systems Atrophy, PSP, Tauopathy, Diffuse Lewy Body Disease, Lewy Body dementia, any disorder characterized by abnormal accumulation of α-synuclein, disorders of the aging process, and stroke.
  • Other embodiments of the present invention relate to methods of treating, preventing, or ameliorating one or more symptoms associated with but not limited to mental retardation, deafness, blindness, diabetes, obesity, cardiovascular disease, and autoimmune diseases such as multiple sclerosis, Sjogrens syndrome, lupus, glaucoma, including pseudoexfoliation glaucoma, Leber's Hereditary Optic Neuropathy, and rheumatoid arthritis.
  • Further embodiments of the present invention of the present invention relate to methods of treating, preventing, or ameliorating one or more symptoms associated with but not limited to Mitochondrial Related Diseases or Capsules as follows:
      • Alpers Disease
      • Barth Syndrome/LIC (Lethal Infantile Cardiomyopathy)
      • Beta-oxidation Defects
      • Carnitine-Acyl-Carnitine Deficiency
      • Carnitine Deficiency
      • Creatine Deficiency Syndromes
      • Co-Enzyme Q10 Deficiency
      • Complex I Deficiency
      • Complex II Deficiency
      • Complex III Deficiency
      • Complex IV Deficiency/COX Deficiency
      • Complex V Deficiency
      • CPEO
      • CPT I Deficiency
      • CPT II Deficiency
      • KSS
      • Lactic Acidosis
      • LBSL—Leukodystrophy
      • LCAD
      • LCHAD
      • Leigh Disease or Syndrome
      • Luft Disease
      • MAD/Glutaric Aciduria Type II
      • MCAD
      • MELAS
      • MERRF
      • MIRAS
      • Mitochondrial Cytopathy
      • Mitochondrial DNA Depletion
      • Mitochondrial Encephalopathy
      • Mitochondrial Myopathy
      • MNGIE
      • NARP
      • Pearson Syndrome
      • Pyruvate Carboxylase Deficiency
      • Pyruvate Dehydrogenase Deficiency
      • POLG Mutations
      • Respiratory Chain
      • SCAD
      • SCHAD
      • VLCAD.
  • Increased Parkin activity eliminates damaged mitochondria (red) and increases cell viability during cellular stress, decreasing tumor transformation and mitigating α-synuclein in cells. Multiple diverse Parkin activating compounds were identified that remove zinc from Parkin. Activation after ZnF unfolding is similar to other known ZnF proteins.
  • The present invention also includes pharmaceutical compositions for activating a Parkin ligase in a subject. In a specific embodiment, the pharmaceutical compositions comprise one or more compounds or the salts thereof that disrupt at least one Parkin ligase zinc finger. In a specific embodiment, the one or more compounds or the salts thereof can coordinate with a Zn ion, and/or react with at least one thiol group in a cysteine. In a specific embodiment, the pharmaceutical compositions may comprise one or more compounds selected from the group consisting of the compounds in Table 1 and Table 2.
  • In another embodiment, the compounds, methods and pharmaceutical compositions in the present invention may be from one or more of following drug classes: 8-hydroxyquinolines; alpha-hydroxyketone; aminomethyl benzimidazoles; aminomethyl indoles; barbiturates; benzisothiazolones, carboxylate, dithiobisbenzamides, dithiocarbamates, formamides, hydrazides, hydroxamates, hydroxypyridinones/hydroxypyranones, hydroxysulfonamides, imidazoles, ketone hydrates, N-acyl ortho-phenylenediamines, N-hydroxyureas, O-substituted phosphamates, phosphamates, phosphones, sulfamates, sulfamides, sulfodiimines, sulfonamides, thiadiazines, thiadiazolothiones, and thiols. For example, the following list in Table 1 is representative, but not an exhaustive list of potential compounds that may be used in the present invention.
  • TABLE 1
    Examples of potential compounds
    Common
    No Class Structure IUPAC names Name(s) References
    1 8-hydroxyquinolines
    Figure US20200087648A1-20200319-C00001
         		5-chloro-7- iodoquinolin-8-ol clioquinol 5-chloro-7-iodo-8- hydroxyquinoline Biochem. J. 2009, 417, 133.
    2 8-hydroxyquinolines
    Figure US20200087648A1-20200319-C00002
         		Quinolin-8-ol 8-hydroxyquinoline Drug Design, Development and Therapy 2013, 7, 1157.
    3 8-hydroxyquinolines
    Figure US20200087648A1-20200319-C00003
         		5-((4-(prop-2-yn-1- yl)piperazin-1- yl)methyl) quinolin-8-ol HLA-20 Drug Design, Development and Therapy 2013, 7, 1157.
    4 8-hydroxyquinolines
    Figure US20200087648A1-20200319-C00004
         		5-((methyl(prop-2-yn-1- yl)amino)methyl) quinolin-8-ol M30 Drug Design, Development and Therapy 2013, 7, 1157.
    5 8-hydroxyquinolines
    Figure US20200087648A1-20200319-C00005
         		5-((4-(2- hydroxyethyl)piperazin- 1-yl)methyl) quinolin-8-ol VK-28 Drug Design, Development and Therapy 2013, 7, 1157.
    6 8-hydroxyquinolines
    Figure US20200087648A1-20200319-C00006
         		5,7-dichloro-2- ((dimethylamino) methyl)quinolin-8-ol PBT2 Drug Design, Development and Therapy 2013, 7, 1157.
    7 8-hydroxyquinolines
    Figure US20200087648A1-20200319-C00007
         		(3S,4S,5S,6R)-2-(5- chloro-7-iodoquinolin-8- yl)-6-(hydroxymethyl)- 1,2l3-dioxane-3,4,5-triol GluCQ Drug Design, Development and Therapy 2013, 7, 1157.
    8 8-hydroxyquinolines
    Figure US20200087648A1-20200319-C00008
         		4-(((8- hydroxyquinolin-5- yl)methyl)amino) benzenesulfonamide HQMABS Drug Design, Development and Therapy 2013, 7, 1157.
    9 8-hydroxyquinolines
    Figure US20200087648A1-20200319-C00009
         		2-methylquinolin- 8-ol 8- hydroxy- quinaldine Drug Design, Development and Therapy 2013, 7, 1157.
    10 alpha-hydroxyketone
    Figure US20200087648A1-20200319-C00010
         		(2r)-2-benzyl-5-hydroxy- 4-oxopentanoic acid Chin. Chem. Lett. 2010, 21, 159
    11 aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00011
         		(1H-benzo[d]imidazol-2- yl)methanamine Bioorg.Med.Chem. Lett. 2002, 12, 2201
    12 aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00012
         		2-(aminomethyl)- N-(2-(benzyloxy) benzyl)-1H-benzo[d] imidazol-6-amine Bioorg.Med.Chem. Lett. 2002, 12, 2201
    13 aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00013
         		2-(aminomethyl)-N-(2- (phenylthio)benzyl)-1H- benzo[d]imidazol-6- amine Bioorg.Med.Chem. Lett. 2002, 12, 2201
    14 aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00014
         		2-(aminomethyl)-N- (cyclohex-3-en-1- ylmethyl)-1H- benzo[d]imidazol-6- amine Bioorg.Med.Chem. Lett. 2002, 12, 2201
    15 aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00015
         		N-(2-(aminomethyl)-1H- benzo[d]imidazol- 6-yl)-4- benzoylbenzamide Bioorg.Med.Chem. Lett. 2002, 12, 2201
    16 aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00016
         		(E)-N-(2-(aminomethyl)- 1H-benzo[d]imidazol-6- yl)-3-(pyridin-3- yl)acrylamide Bioorg.Med.Chem. Lett. 2002, 12, 2201
    aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00017
         		(E)-N-(2-(aminomethyl)- 1H-benzo[d]imidazol-6- yl)-3-(2,6-difluorophenyl) acrylamide Bioorg.Med.Chem. Lett. 2002, 12, 2201
    18 aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00018
         		2-(aminomethyl)-N- benzyl-1H-benzo[d] imidazol-6-amine Bioorg.Med.Chem. Lett. 2002, 12, 2201
    19 aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00019
         		2-(aminomethyl)- N-(4-(benzyloxy) benzyl)-1H- benzo[d]imidazol- 6-amine Bioorg.Med.Chem. Lett. 2002, 12, 2201
    20 aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00020
         		N-(2-(aminomethyl)-1H- benzo[d]imidazol- 6-yl)-2-(benzyloxy) benzamide Bioorg.Med.Chem. Lett. 2002, 12, 2201
    21 aminomethyl benzimidazoles
    Figure US20200087648A1-20200319-C00021
         		N-(2-(aminomethyl)-1H- benzo[d]imidazol-6-yl)- N-(2-(benzyloxy) benzyl)acetamide Bioorg.Med.Chem. Lett. 2002, 12, 2201
    22 aminomethyl indoles
    Figure US20200087648A1-20200319-C00022
         		2-(aminomethyl)- N-(2-(benzyloxy) benzyl)-1H- indol-6-amine Bioorg.Med.Chem. Lett. 2002, 12, 2201
    23 barbiturates
    Figure US20200087648A1-20200319-C00023
         		5-methylpyrimidine- 2,4,6(1H,3H,5H)- trione J. Biol. Chem. 2001, 276, 17405. Protein Sci. 2001, 10, 923. Curr. Pharm. Des. 2005, 11, 295.
    24 barbiturates
    Figure US20200087648A1-20200319-C00024
         		2-hydroxy-5-[4-(2- hydroxy-ethyl)-piperidin- 1-yl]-5-phenyl-1h- pyrimidine-4,6-dione J. Biol. Chem. 2001, 276, 17405
    25 benzisothiazolones
    Figure US20200087648A1-20200319-C00025
         		N-(4-(N-(4-((4-{3- oxobenzo[d]isothiazol- 2(3H)-yl)phenyl) sulfonyl)phenyl) sulfamoyl) phenyl)acetamide BITA Nature Medicine 2004, 10(1), 40.
    26 carboxylate
    Figure US20200087648A1-20200319-C00026
         		N-[(1r)-1-carboxy-3- phenylpropyl]-I-leucyl-I- tryptophan Biochemistry 1984, 23, 5724
    27 dithiobisbenzamides
    Figure US20200087648A1-20200319-C00027
         		2,2′-disulfanediylbis(N- (4-sulfamoylphenyl) benzamide DIBA NSC 654077 J. Med. Chem. 1996, 39, 3606. Nature Medicine 2004, 10, 40.
    28 dithiocarbamates
    Figure US20200087648A1-20200319-C00028
         		Pyrrolidine-1-carbodithioic acid 1-Pyrrolidine- carbodithioc acid PyDT
    29 dithiocarbamates
    Figure US20200087648A1-20200319-C00029
         		Phenylcarbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    30 dithiocarbamates
    Figure US20200087648A1-20200319-C00030
         		Morpholino- carbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    31 dithiocarbamates
    Figure US20200087648A1-20200319-C00031
         		(4-methyl-piperazin-1- yl)carbamodithioic acid N-(4-methyl-1- piperazinyl) carbamo- dithioic acid J. Med. Chem. 2012, 55, 1721.
    32 dithiocarbamates
    Figure US20200087648A1-20200319-C00032
         		(2-morpholinoethyl) carbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    33 dithiocarbamates
    Figure US20200087648A1-20200319-C00033
         		Benzylcarbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    34 dithiocarbamates
    Figure US20200087648A1-20200319-C00034
         		(Pyridin-4-ylmethyl) carbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    35 dithiocarbamates
    Figure US20200087648A1-20200319-C00035
         		(2-(piperidin-1-yl)ethyl) carbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    36 dithiocarbamates
    Figure US20200087648A1-20200319-C00036
         		Thiazol-2-ylcarba- modithioic acid J. Med. Chem. 2012, 55, 1721.
    37 dithiocarbamates
    Figure US20200087648A1-20200319-C00037
         		(2-(1H-imidazol-1- yl)ethyl)carbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    38 dithiocarbamates
    Figure US20200087648A1-20200319-C00038
         		Dimethylcarbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    39 dithiocarbamates
    Figure US20200087648A1-20200319-C00039
         		Diethylcarbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    40 dithiocarbamates
    Figure US20200087648A1-20200319-C00040
         		Pentylcarbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    41 dithiocarbamates
    Figure US20200087648A1-20200319-C00041
         		Piperidine-1- carbodithioic acid J. Med. Chem. 2012, 55, 1721.
    42 dithiocarbamates
    Figure US20200087648A1-20200319-C00042
         		Diisobutylcarbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    43 dithiocarbamates
    Figure US20200087648A1-20200319-C00043
         		Dipropylcarba- modithioic acid J. Med. Chem. 2012, 55, 1721.
    44 dithiocarbamates
    Figure US20200087648A1-20200319-C00044
         		Dibutylcarbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    45 dithiocarbamates
    Figure US20200087648A1-20200319-C00045
         		Dihexylcarbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    46 dithiocarbamates
    Figure US20200087648A1-20200319-C00046
         		Ethyl(hexyl) carbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    47 dithiocarbamates
    Figure US20200087648A1-20200319-C00047
         		Bis(2-hydroxyethyl) carbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    48 dithiocarbamates
    Figure US20200087648A1-20200319-C00048
         		Methyl(phenyl) carbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    49 dithiocarbamates
    Figure US20200087648A1-20200319-C00049
         		Benzyl(methyl) carbamodithioic acid J. Med. Chem. 2012, 55, 1721.
    50 dithiocarbamates
    Figure US20200087648A1-20200319-C00050
         		Morpholine-4- carbodithioic acid J. Med. Chem. 2012, 55, 1721.
    51 dithiocarbamates
    Figure US20200087648A1-20200319-C00051
         		Piperazine-1,4- bis(carbodithioic) acid J. Med. Chem. 2012, 55, 1721.
    52 dithiocarbamates
    Figure US20200087648A1-20200319-C00052
         		4-cyano-4-phenyl- piperidine-1- carbodithioic acid J. Med. Chem. 2012, 55, 1721.
    53 formamide
    Figure US20200087648A1-20200319-C00053
         		Cyclohexylformamide Biochemistry 1997, 36, 3552
    54 hydrazides
    Figure US20200087648A1-20200319-C00054
         		(R)-N-((S)-3-(1H-indol-3- yl)-1-(methylamino)-1- oxopropan-2-yl)-2-(2-(2- ((4′-bromo-[1,1′- biphenyl]-4- yl)sulfonyl)hydrazinyl)-2- oxoethyl)-4- methylpentanamide Bourguet, E. et. al. (2012). Pharmacomodula- tion of Broad Spectrum Matrix Metalloproteinase Inhibitors Towards Regulation of Gelatinases, Enzyme Inhibition and Bioapplications, Sharma, R. (Ed.), pp. 57-84
    55 hydroxamates
    Figure US20200087648A1-20200319-C00055
         		N-hydroxyacetamide acetohydroxamic acid Curr.Pharma.Des. 2005, 11, 295
    56 hydroxamates
    Figure US20200087648A1-20200319-C00056
         		1-hydroxypyridine-2(1H)- thione J. Biol. Inorg. Chem. 2006, 11, 131. Inorg. Chem. 2003, 42, 3423
    57 hydroxamates
    Figure US20200087648A1-20200319-C00057
         		N-hydroxy-N- methylacetamide Curr.Pharma.Des. 2005, 11, 295
    58 hydroxamates
    Figure US20200087648A1-20200319-C00058
         		(R)-N1-((S)- 3-(1H-indol-3- yl)-1-(methylamino)-1- oxopropan-2-yl)-N4- hydroxy-2- isobutylsuccinamide Galardin
    59 hydroxamates
    Figure US20200087648A1-20200319-C00059
         		(2R,3S)-N1-((S)-3,3- dimethyl-1- (methylamino)-1- oxobutan-2-yl)-N4,3- dihydroxy-2- isobutylsuccinamide marimastat
    60 hydroxamates
    Figure US20200087648A1-20200319-C00060
         		N-hydroxy-1- naphthamide naphthyl hydroxamate J. Med. Chem. 2002, 45, 5628.
    61 hydroxamates
    Figure US20200087648A1-20200319-C00061
         		(2S,3R)-N1-hydroxy-3- isobutyl-N4-((S)-1- (methylamino)- 1-oxo-3- phenylpropan-2-yl)-2- ((thiophen-2- ylthio)methyl) succinamide batimastat
    62 hydroxypyridinones/ hydroxypyranones
    Figure US20200087648A1-20200319-C00062
         		3-hydroxy-1- methylpyridin-2(1H)-one J. Biol. Inorg. Chem. 2006, 11, 131. Inorg. Chem. 2003, 42, 3423
    63 hydroxypyridinones/ hydroxypyranones
    Figure US20200087648A1-20200319-C00063
         		3-hydroxy-1,2- dimethylpyridin- 4(1H)-one J. Biol. Inorg. Chem. 2006, 11, 131. Inorg. Chem. 2003, 42, 3423
    64 hydroxypyridinones/ hydroxypyranones
    Figure US20200087648A1-20200319-C00064
         		3-hydroxy-4H-pyran-4- one J. Biol. Inorg. Chem. 2006, 11, 131. Inorg. Chem. 2003, 42, 3423
    65 hydroxypyridinones/ hydroxypyranones
    Figure US20200087648A1-20200319-C00065
         		3-hydroxy-2-methyl-4H- pyran-4-one J. Biol. Inorg. Chem. 2006, 11, 131. Inorg. Chem. 2003, 42, 3423
    66 hydroxypyridinones/ hydroxypyranones
    Figure US20200087648A1-20200319-C00066
         		3-hydroxypyridin-2(1H)- one J. Biol. Inorg. Chem. 2006, 11, 131. Inorg. Chem. 2003, 42, 3423
    67 hydroxypyridinones/ hydroxypyranones
    Figure US20200087648A1-20200319-C00067
         		3-hydroxy-1- methylpyridine-2(1H)- thione J. Biol. Inorg. Chem. 2006, 11, 131. Inorg. Chem. 2003, 42, 3423
    68 hydroxypyridinones/ hydroxypyranones
    Figure US20200087648A1-20200319-C00068
         		3-hydroxy-1,2- dimethylpyridine-4(1H)- thione J. Biol. Inorg. Chem. 2006, 11, 131. Inorg. Chem. 2003, 42, 3423
    69 hydroxypyridinones/ hydroxypyranones
    Figure US20200087648A1-20200319-C00069
         		3-hydroxy-4H-pyran-4- thione J. Biol. Inorg. Chem. 2006, 11, 131. Inorg. Chem. 2003, 42, 3423
    70 hydroxypyridinones/ hydroxypyranones
    Figure US20200087648A1-20200319-C00070
         		3-hydroxy-2-methyl-4H- pyran-4-thione J. Biol. Inorg. Chem. 2006, 11, 131. Inorg. Chem. 2003, 42, 3423
    71 hydroxysulfonamides
    Figure US20200087648A1-20200319-C00071
         		N-hydroxymethane- sulfonamide J. Med. Chem. 2000, 43, 3677.
    72 imidazoles
    Figure US20200087648A1-20200319-C00072
         		Histamine Biochemistry 1997, 36, 10384
    73 ketone hydrates
    Figure US20200087648A1-20200319-C00073
         		(2r)-4,4-dihydroxy-5- nitro-2- (phenylmethyl) pentanoic acid Bioorg. Med. Chem. Lett. 2009, 19, 5009
    74 ketone hydrates
    Figure US20200087648A1-20200319-C00074
         		N-{(5s)-4,4-dihydroxy-6- phenyl-5- [(phenylcarbonyl)amino] hexanoyl}-I-tryptophan Biochemistry 2008, 47, 5942
    75 ketone hydrates
    Figure US20200087648A1-20200319-C00075
         		2,2,2-trifluoro-1-{5-[(3- phenyl-5,6- dihydroimidazo[1,2- a]pyrazin-7(8h)- yl)carbonyl]thiophen-2- yl}ethane-1,1-diol J. Biol. Chem. 2008, 283, 26694
    76 N-acyl ortho- phenylenediamines
    Figure US20200087648A1-20200319-C00076
         		N-(4-aminobiphenyl-3- yl)benzamide Bioorg. Med. Chem. Lett. 2010, 20, 3149
    77 N-hydroxyureas
    Figure US20200087648A1-20200319-C00077
         		D[(n- hydroxyamino)carbonyl] phenylalanine Bioorg. Med. Chem. Lett. 2002, 10, 2015
    78 O-substituted phosphamates
    Figure US20200087648A1-20200319-C00078
         		N-alpha-I- rhamnopyranosyloxy (hydroxyphosphinyl)-I- leucyl-I-tryptophan Eur. J. Biochem. 1986, 157, 261
    79 O-substituted phosphonates
    Figure US20200087648A1-20200319-C00079
         		O-(((1r)-((n-(phenyl- methoxy-carbonyl)- alanyl)- amino)methyl) hydroxyphosphinyl)3-I- phenyllactate Biochemistry 1991, 30, 8171
    80 phosphamates
    Figure US20200087648A1-20200319-C00080
         		N2-phosphono-I- leucinamide Eur. J. Biochem. 1986, 157, 261
    81 phosphones
    Figure US20200087648A1-20200319-C00081
         		N-({(1s,2r)-2-[(s)-[(1r)-1- {[(benzyloxy)carbonyl] amino}-2- phenylethyl]hydroxy) phosporyl]cyclopentyl} carbonyl)-I-tryptophan J. Mol. Biol. 2010, 400, 502
    82 sulfamates
    Figure US20200087648A1-20200319-C00082
         		6-oxo-8,9,10,11- tetrahydro-7H- cyclohepta[c][1]benzo- pyran-3-o-sulfamide Biochem. J. 2005, 385, 715
    83 sulfamides
    Figure US20200087648A1-20200319-C00083
         		Phenylalanine-n- sulfonamide J. Med. Chem. 2002, 45, 5295
    84 sulfodiimines
    Figure US20200087648A1-20200319-C00084
         		Dimethyl-λ6- sulfanediimine Biochemistry 1995, 34, 6602
    85 sulfodiimines
    Figure US20200087648A1-20200319-C00085
         		S-(2-carboxy-3- phenylpropyl) thiodiimine-s-methane J. Biol. Chem. 1992, 267, 19192
    86 sulfonamides
    Figure US20200087648A1-20200319-C00086
         		3,,4-dihydro-2-(3- methoxyphenyl)-2h- thieno-[3,2-e]-1,2- thiazine-6-sulfonamide- 1,1-dioxide Protein Sci. 1998, 7, 2483
    87 sulfonamides
    Figure US20200087648A1-20200319-C00087
         		3-(1h-indol-3-yl)-2-[4-(4- phenyl-piperidin-1-yl)- benzenesulfonylamino]- propionic acid Protein Sci. 1999, 8, 1455
    88 thiadiazines
    Figure US20200087648A1-20200319-C00088
         		(Z)-N,5-dimethyl-3,6- dihydro-2H-1,3,4- thiadiazin-2-imine J. Med. Chem. 2001, 44, 3231.
    89 thiadiazolothiones
    Figure US20200087648A1-20200319-C00089
         		[2-(5-mercapto- [1,3,4]thiadiazol-2- ylcarbamoyl)-1-phenyl- ethyl]-carbamic acid benzyl ester Protein Sci. 1998, 7, 2118
    90 thiols
    Figure US20200087648A1-20200319-C00090
         		(2s,3r)-2-benzyl-3- sulfanylbutanoic acid Chem. Biol. Drug Des. 2010, 75, 29
  • TABLE 2
    Examples of potential compounds
    No. COMPOUND STRUCTURE NAME
    1
    Figure US20200087648A1-20200319-C00091
         		2,2′-(ethane-1,2-diyl)bis (isoindoline-1,3-dione)
    2
    Figure US20200087648A1-20200319-C00092
         		N′-(4-hydroxy-1- phenyl-1H-pyrazole-3- carbonyl)-4-oxo-3,4- dihydrophthalazine-1- carbohydrazide
    3
    Figure US20200087648A1-20200319-C00093
         		N-(5-(pentan-3-yl)- 1,3,4-thiadiazol-2-yl)-1- (pyridin-2-yl)-5-(trifluoromethyl)- 1H-pyrazole- 4-carboxamide
    4
    Figure US20200087648A1-20200319-C00094
         		(3-phenyl-1,2,4-oxadiazol- 5-yl)methyl 3-methyl- 5-(methylamino)isothiazole- 4-carboxylate
    5
    Figure US20200087648A1-20200319-C00095
         		methyl 3-(3-(6-bromo-4- oxoquinazolin-3(4H)- yl)propanamido)thiophene- 2-carboxylate
    6
    Figure US20200087648A1-20200319-C00096
         		4-chloro-5-((2-(2,4- dimethylphenoxy)ethyl) amino)pyridazin-3(2H)- one
    7
    Figure US20200087648A1-20200319-C00097
         		2-(benzo[d]oxazol-2-yl)-4-(3-(tert- butyl)phenoxy)-3- oxopentanenitrile
    8
    Figure US20200087648A1-20200319-C00098
         		7-fluoro-3-methyl-N-(5- (pyridin-2-yl)-4H-1,2,4- triazol-3-yl)benzofuran- 2-carboxamide
    9
    Figure US20200087648A1-20200319-C00099
         		N-(2-methylbenzo[d] oxazol-5-yl)-1-(3- nitrophenyl)-1H-pyrazole- 3-carboxamide
    10
    Figure US20200087648A1-20200319-C00100
         		N-(4-methyl-2,5-dioxo-4- phenethylimidazolidin- 1-yl)pyrazine-2-carboxamide
    11
    Figure US20200087648A1-20200319-C00101
         		N-(3-cyanothiophen- 2-yl)-3-(5-(2- fluorophenyl)oxazol- 2-yl)propanamide
    12
    Figure US20200087648A1-20200319-C00102
         		N′-(5-ethyl-4-methylthiophene- 2-carbonyl)-2- methyl-7,8,9,10-tetrahydro-6H- pyrido[3′,2′:4,5]imidazo [1,2-a]azepine-4- carbohydrazide
    13
    Figure US20200087648A1-20200319-C00103
         		2-phenoxyethyl quinoxaline-2-carboxylate
    14
    Figure US20200087648A1-20200319-C00104
         		2-(benzo[d]oxazol- 2-yl)-3-oxo-3-(1- phenylcyclobutyl) propanenitrile
    15
    Figure US20200087648A1-20200319-C00105
         		N-(1-benzyl-1H-imidazol- 2-yl)-4-oxo~2,3,4,5- tetrahydrobenzo[b] [1,4]thiazepine-7- carboxamide
    16
    Figure US20200087648A1-20200319-C00106
         		N-(4-chlorophenethyl)- 4-methyl-2-(4H-1,2,4- triazol-3-yl)thiazole-5- carboxamide
    17
    Figure US20200087648A1-20200319-C00107
         		N-(8-methylquinolin- 5-yl)-2-(pyrazin-2- yl)thiazole-4-carboxamide
    18
    Figure US20200087648A1-20200319-C00108
         		2-(4-([1,2,4]triazolo[4,3-b] pyridazin-6-yl)-3- methylpiperazin-1- yl)nicotinomtrile
    19
    Figure US20200087648A1-20200319-C00109
         		N′-(6-ethyl-2-(pyridin-2- yl)pyrimidin-4-yl)-5- methylthiophene-2-carbohydrazide
    20
    Figure US20200087648A1-20200319-C00110
         		2-(benzo[d]oxazol-2-yl)-5- (2,3-difluorophenyl)- 3-oxopentanenitrile
    21
    Figure US20200087648A1-20200319-C00111
         		4-cyano-N′-(6-ethyl-2- (pyridin-2-yl)pyrimidin~4- yl)benzohydrazide
    22
    Figure US20200087648A1-20200319-C00112
         		N′-(6-ethyl-2-(pyridin-2- yl)pyrimidin-4-yl)-2,3- dimethylbenzohydrazide
    23
    Figure US20200087648A1-20200319-C00113
         		2-(benzo[d]oxazol-2-yl)- 5-(4-fluorophenyl)-3- oxopentanenitrile
    24
    Figure US20200087648A1-20200319-C00114
         		3-((2-(4-cyanophenoxy) ethyl)amino)pyrazine-2- carbonitrile
    25
    Figure US20200087648A1-20200319-C00115
         		N-(1,1-dioxido-2,3- dihydrobenzo[b]thiophen-6- yl)-2-(4-methoxyphenyl) thiazole-4-carboxamide
    26
    Figure US20200087648A1-20200319-C00116
         		5-oxo-N-(pyrazolo[1,5-a] pyrimidin-7-yl)-1-(p- tolyl)-2,5-dihydro-1H- pyrazole-3-carboxamide
    27
    Figure US20200087648A1-20200319-C00117
         		(3-(1H-imidazol-1-yl) piperidin-1-yl)(3-methyl- 2H-pyrazolo[3,4-b]pyridin- 5-yl)methanone
    28
    Figure US20200087648A1-20200319-C00118
         		5-isobutyl-2-(4H-1,2,4- triazol-3-yl)thieno[2,3- d]pyrimidin-4(3H)-one
    29
    Figure US20200087648A1-20200319-C00119
         		N-([1,2,4]triazolo[4,3-a] pyridin-3-yl)-6-(1- phenylethoxy)nicotinamide
    30
    Figure US20200087648A1-20200319-C00120
         		N-(3-(N-(3,4-dihydro- 2H-pyrrol-5- yl)sulfamoyl)phenyl)-2,3- dimethylquinoxaline-6- carboxamide
    31
    Figure US20200087648A1-20200319-C00121
         		N′-(2-(1H-benzo[d]imidazol-1- yl)acetyl)quinoline- 2-carbohydrazide
    32
    Figure US20200087648A1-20200319-C00122
         		2-(2-ethylphenoxy)-N- (5-(pyridin-2-yl)-4H- 1,2,4-triazol-3-yl)acetamide
    33
    Figure US20200087648A1-20200319-C00123
         		4-chloro-N-(3-hydroxypyridin- 2-yl)picolinamide
    34
    Figure US20200087648A1-20200319-C00124
         		4,5-dichloro-2-((5-(4- chlorophenyl)-1,3,4- oxadiazol-2-yl)methyl) pyridazin-3(2H)-one
    35
    Figure US20200087648A1-20200319-C00125
         		N'-(3-(ethoxymethyl) benzofuran-2- carbonyl)picolinohydrazide
    36
    Figure US20200087648A1-20200319-C00126
         		N-(2-methyl-5-(2-(quinoline-2- carbonyl)hydrazine-1- carbonyl)phenyl) methanesulfonamide
    37
    Figure US20200087648A1-20200319-C00127
         		N-(3-(2-(quinoline-2- carbonyl)hydrazine-1- carbonyl)phenyl)propane- 1-sulfonamide
    38
    Figure US20200087648A1-20200319-C00128
         		4,5-dichloro-2-((5- (thiophen-2-yl)isoxazol-3- yl)methyl)pyridazin-3(2H)- one
    39
    Figure US20200087648A1-20200319-C00129
         		4-(4,5-dichloro-6- oxopyridazin-1(6H)-yl)-N-(2- fluoro-4-methylphenyl) benzenesulfonamide
    40
    Figure US20200087648A1-20200319-C00130
         		2-(((5-bromothiophen-2- yl)methyl)(methyl) amino)-N-(thiazol-2- yl)acetamide
    41
    Figure US20200087648A1-20200319-C00131
         		N′-(5,6-dihydro-4H- cyclopenta[b]thiophene-2- carbonyl)isoquinoline-1- carbohydrazide
    42
    Figure US20200087648A1-20200319-C00132
         		N-(spiro[chromane- 2,1′-cyclobutan]-4- yl)tetrazolo[1,5-b] pyridazin-6-amine
    43
    Figure US20200087648A1-20200319-C00133
         		6,6-dimethyl-2,4-bis (methylthio)-5,8-dihydro- 6H-pyrano[4′,3′:4,5]furo [2,3-d]pyrimidine
    44
    Figure US20200087648A1-20200319-C00134
         		5-(isopropoxymethyl) quinolin-8-ol
    45
    Figure US20200087648A1-20200319-C00135
         		5-((pentyloxy)methyl) quinolin-8-ol
    46
    Figure US20200087648A1-20200319-C00136
         		5-chloro-7-((4-(4- fluorophenyl)piperazin-1- yl)methyl)quinolin-8-ol
    47
    Figure US20200087648A1-20200319-C00137
         		N-(2-methoxyphenyl)- 3-(quinolin-8- ylthio)propanamide
    48
    Figure US20200087648A1-20200319-C00138
         		2-((methyl(1-(pyridin-2- yl)propyl)amino)methyl) nicotinic acid
    49
    Figure US20200087648A1-20200319-C00139
         		N-methyl-N-((2- methyl-1H-imidazol-4- yl)methyl)-1-(pyridin- 2-yl)ethan-1-amine
    50
    Figure US20200087648A1-20200319-C00140
         		N-((2-ethyl-3,5-dimethyl- 1H-indol-7-yl)methyl)- 2-(4H-1,2,4~triazol-4- yl)isonicotinamide
    51
    Figure US20200087648A1-20200319-C00141
         		(7-fluoro-2,3-dihydro- [5,5′-bibenzofuran]~2- yl)methanamine
    52
    Figure US20200087648A1-20200319-C00142
         		2-methyl-7-(((4- methylpyridin-2-yl)amino)(2- nitrophenyl)methyl) quinolin-8-ol
    53
    Figure US20200087648A1-20200319-C00143
         		6-fluoro-1-hexyl-7- morpholino-4-oxo-1,4- dihydroquinoline-3- carboxylic acid
    54
    Figure US20200087648A1-20200319-C00144
         		(Z)-2-(benzo[d] thiazol-2-yl)-3-((2,3- dihydrobenzo[b][1,4]dioxin-6- yl)amino)acrylonitrile
    55
    Figure US20200087648A1-20200319-C00145
         		2-(isobutylamino)-N-(5- (methoxymethyl)-1,3,4- thiadiazol-2-yl)thiazole- 4-carboxamide
    56
    Figure US20200087648A1-20200319-C00146
         		1-(1-(2~chlorobenzyl)- 1H~indol-3-yl)-2- morpholinoethane-1,2-dione
    57
    Figure US20200087648A1-20200319-C00147
         		2-(benzo[d]thiazol- 2-ylamino)-6- pbenylpyrimidine-4- carboxylic acid
    58
    Figure US20200087648A1-20200319-C00148
         		N-(2,4-dimethylphenyl)- 1H-benzo[d]imidazole- 2-carbothioamide
    59
    Figure US20200087648A1-20200319-C00149
         		N-((2-fluorophenyl) (8-hydroxyquinolin-7- yl)methyl)acetamide
    60
    Figure US20200087648A1-20200319-C00150
         		N-(4-(4-chlorophenyl) thiazol-2-yl)-1H-1,2,4- triazole-3-carboxamide
    61
    Figure US20200087648A1-20200319-C00151
         		N-(4-(4-(5-(azetidin- 1-ylmethyl)pyridin-2- yl)piperazine-1-carbonyl) phenyl)acetamide
    62
    Figure US20200087648A1-20200319-C00152
         		2~(2-(((1H-imidazol-2- yl)methyl)amino)ethyl)- N-(2-chlorophenyl)thiazole- 4-carboxamide
    63
    Figure US20200087648A1-20200319-C00153
         		1-(4-(((5-methylpyrazin- 2-yl)methyl)amino)-2- (pyridin-2-yl)-5,8- dihydropyrido[3,4- d]pyrimidin-7(6H)- yl)ethan-1-one
    64
    Figure US20200087648A1-20200319-C00154
         		2-(4-(1′-methyl-1H,1′H- [2,2′-biimidazol]-1- yl)phenyl)-1H-benzo[d] imidazole
    65
    Figure US20200087648A1-20200319-C00155
         		4-(3-(2-(1H-imidazol-1- yl)ethoxy)phenyl)-1H- pyrrolo[2,3-b]pyridine
    66
    Figure US20200087648A1-20200319-C00156
         		N-(2-hydroxyethyl)-3- (5-methylfuran-2-yl)-N- (thiazol-2-ylmethyl)benzamide
    67
    Figure US20200087648A1-20200319-C00157
         		N-((1-isopropyl-1H- benzo[d]imidazol-2- yl)methyl)-N-methyl-1- (pyridin-2-yl)ethan-1- amine
    68
    Figure US20200087648A1-20200319-C00158
         		5-((1H-imidazol-1- yl)methyl)-N-((2-ethyl-3- methyl~1H-indol-5- yl)methyl)furan-2- carboxamide
    69
    Figure US20200087648A1-20200319-C00159
         		N-(2-(2,3-dihydrobenzo [b][1,4]dioxin-2- yl)ethyl)-6-(1H-pyrrol- 2-yl)nicotinamide
    70
    Figure US20200087648A1-20200319-C00160
         		2-((4-(2-methoxyquinolin-3- yl)phenyl)thio)acetamide
    71
    Figure US20200087648A1-20200319-C00161
         		(2-(4-fluorophenyl)morpholino)(8- hydroxyquinolin-7-yl)methanone
    72
    Figure US20200087648A1-20200319-C00162
         		N-(2-ethyl-2H-1,2,3- triazol-4-yl)-2-(5-(5- methylfuran-2-yl)-1H- indazol-1-yl)acetamide
    73
    Figure US20200087648A1-20200319-C00163
         		N-((4-ethyl-2-methylthiazol- 5-yl)methyl)-2- (pyridin-2-yl)-5,6,7,8- tetrahydropyrido[3,4- d]pyrimidin-4-amine
    74
    Figure US20200087648A1-20200319-C00164
         		N-(7-acetyl-2,3- dihydrobenzo[b][1,4]dioxin-6- yl)-2-iodobenzamide
    75
    Figure US20200087648A1-20200319-C00165
         		2-(5-bromo-2- hydroxybenzoyl)-N-(3- (trifluoromethyl)phenyl) hydrazine-1- carbothioamide
    76
    Figure US20200087648A1-20200319-C00166
         		N,N′-(1-phenyl-1H- 1,2,4-triazole-3,5- diyl)dibenzamide
    77
    Figure US20200087648A1-20200319-C00167
         		6-benzyl-2,5-dimethyl- 3-phenylpyrazolo[1,5- a]pyrimidine-7-thiol
    78
    Figure US20200087648A1-20200319-C00168
         		(Z)-2-(1H-benzo[d]imidazol- 2-yl)-3-(3-iodo-4,5- dimethoxyphenyl)acrylonitrile
    79
    Figure US20200087648A1-20200319-C00169
         		5-((2-(5-(trifluoromethyl)- 1H-imidazol-2- yl)pyridin-4-yl)oxy)-N-(4- (trifluoromethyl)phenyl)- 1H-benzo[d]imidazol- 2-amine
    80
    Figure US20200087648A1-20200319-C00170
    81
    Figure US20200087648A1-20200319-C00171
         		N4-(6-chloro-2- methoxyacridin-9-yl)- N1,N1-diethylpentane- 1,4-diamine
    82
    Figure US20200087648A1-20200319-C00172
         		5H-dibenzo[b,f] azepine-5-carboxamide
    83
    Figure US20200087648A1-20200319-C00173
         		10-(3-(4-methylpiperazin- 1-yl)propyl)-2- (trifluoromethyl)- 10H-phenothiazine
    84
    Figure US20200087648A1-20200319-C00174
         		8-(4,4-bis(4- fluorophenyl)butyl)-1- phenyl-1,3,8-triazaspiro [4.5]decan-4-one
    85
    Figure US20200087648A1-20200319-C00175
         		(2-butylbenzofuran-3-yl)(4-(2- (diethylamino)ethoxy)-3,5- diiodophenyl)methanone
    86
    Figure US20200087648A1-20200319-C00176
         		10-(2-(1-methylpiperidin- 2-yl)ethyl)-2- (methylthio)-10H- phenothiazine
    87
    Figure US20200087648A1-20200319-C00177
         		4-(4-(4-chlorophenyl)-4- hydroxypiperidin-1- yl)-N,N-dimethyl- 2,2-diphenylbutanamide
    88
    Figure US20200087648A1-20200319-C00178
         		3-(2-chloro-10H- phenothiazin-10-yl)- N,N-dimethylpropan- 1-amine
    89
    Figure US20200087648A1-20200319-C00179
         		1-(1-(4,4-bis(4- fluorophenyl)butyl) piperidin-4-yl)-1,3- dihydro-2H-benzo[d] imidazol-2-one
    90
    Figure US20200087648A1-20200319-C00180
         		5-((3,4- dimethoxyphenethyl) (methyl)amino)-2- (3,4-dimethoxyphenyl)-2- isopropylpentanenitrile
    91
    Figure US20200087648A1-20200319-C00181
         		(Z)-2-((2-(5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-4- yl)hydrazono)methyl)phenol
    92
    Figure US20200087648A1-20200319-C00182
         		N,N-dimethyl-1-(10H- phenothiazin-10- yl)propan-2-amine
    93
    Figure US20200087648A1-20200319-C00183
         		3-(10,11-dihydro-5H- dibenzo[a,d][7]annulen- 5-ylidene)-N- methylpropan-1-amine
    94
    Figure US20200087648A1-20200319-C00184
         		N-(1-butyl-1H-benzo [d]imidazol-2- yl)benzainide
    95
    Figure US20200087648A1-20200319-C00185
         		3-amino-3H-spiro [benzo[h]quinazoline- 5,1′-cyclopentan]-4(6H)-one
    96
    Figure US20200087648A1-20200319-C00186
         		5-phenyl-2-(2-phenyl-1-(4- phenylbutanamido) ethyl)thiazole-4- carboxylic acid
    97
    Figure US20200087648A1-20200319-C00187
         		5-chloro-N-(2-chloro- 4-nitrophenyl)-2- hydroxybenzamide
    98
    Figure US20200087648A1-20200319-C00188
         		(E)-5-(4-hydroxystyryl) benzene-1,3-diol
    99
    Figure US20200087648A1-20200319-C00189
         		N,N-dimethyl-3-(10H- phenothiazin-10- yl)propan-1-amine
    100
    Figure US20200087648A1-20200319-C00190
         		N,N-dimethyl-3- (2-(trifluoromethyl)- 10H-phenothiazin- 10-yl)propan-1- aminen
    101
    Figure US20200087648A1-20200319-C00191
         		3-((1,2,3,4-tetrahydroacridin-9- yl)amino)propan-1-olo
    102
    Figure US20200087648A1-20200319-C00192
         		10H-phenothiazine
    103
    Figure US20200087648A1-20200319-C00193
         		1-isopropyl-3-(4-methyl- 2-(pyrrolidin-1- yl)quinolin-6-yl)thiourea
    104
    Figure US20200087648A1-20200319-C00194
         		1-(furan-2-ylmethyl)- 3-(4-methyl-2- morpholinoquinolin- 6-yl)thiourea
    105
    Figure US20200087648A1-20200319-C00195
         		10H-phenoxazine
    106
    Figure US20200087648A1-20200319-C00196
         		N,N′-(1H-1,2,4-triazole-3,5- diyl)dibenzamide
    107
    Figure US20200087648A1-20200319-C00197
         		(E)-2-((2-(5,6,7,8- tetrahydropyrido [4′,3′:4,5]thieno[2,3- d]pyrimidin-4- yl)hydrazono)methyl)phenol
    108
    Figure US20200087648A1-20200319-C00198
         		3-(4-morpholinopyrido [3′,2′:4,5]furo[3,2- d]pyrimidin-2-yl)phenol
    109
    Figure US20200087648A1-20200319-C00199
         		6-(4-(2-(piperidin-1- yl)ethoxy)phenyl)-3- (pyridin-4-yl)pyrazolo [1,5-a]pyrimidine
    110
    Figure US20200087648A1-20200319-C00200
         		6-(2-(piperidin-1-yl) ethoxy)-3-(pyridin- 4-yl)pyrazolo[1,5-a]pyrimidine
    111
    Figure US20200087648A1-20200319-C00201
         		N-allyl-6- bromoquinazolin-4-amine
    112
    Figure US20200087648A1-20200319-C00202
         		4-(2-chloro-10H- phenoxazin-10-yl)-N,N- diethylbutan-1-amine
    113
    Figure US20200087648A1-20200319-C00203
         		benzyl (4-methyl- 1-oxo-1-((1-oxohexan- 2-yl)amino)pentan- 2-yl)carbamate
    114
    Figure US20200087648A1-20200319-C00204
         		2-(4-benzylpiperazin-1-yl)-N-[(2- hydroxy-3-prop-2-enyl- phenyl)methylideneamino] acetamide
  • TABLE 3
    Amino Acid Sequence of Human Parkin Ligase 
    (SEQ IQ NO: 1)
    MIVFVRFNSSHGFPVEVDSDTSIFQLKEVVAKRQGVPADQLRVIFAGKEL
    RNDWTVQNCDLDQQSIVHIVQRPWRKGQEMNATGGDDPRNAAGGCEREPQ
    SLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAGSPAGRSIYNSFYVYC
    KGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGECQSPH
    CPGTSAEFFFKCGAHPTSDKETSVALHLIATNSRNITCITCTDVRSPVLV
    FQCNSRHVICLDCFHLYCVTRLNDRQFVHDPQLGYSLPCVAGCPNSLIKE
    LHHFRILGEEQYNRYQQYGAEECVLQMGGVLCPRPGCGAGLLPEPDQRKV
    TCEGGNGLGCGFAFCRECKEAYHEGECSAVFEASGTTTQAYRVDERAAEQ
    ARWEAASKETIKKTTKPCPRCHVPVEKNGGCMHMKCPQPQCRLEWCWNCG
    CEWNRVCMGDHWFDV
  • The present invention also relates to the pharmaceutical formulations for activating Parkin ligase in a subject comprising an effective does of an agent that disrupts at least one Parkin ligase zinc finger, wherein the agent or the compound that can coordinate a zinc ion, or the agent or the compound that can react with the thiol group in a cysteine.
  • Further embodiments of the present invention are directed to the pharmaceutical formulations further comprising a pharmaceutically acceptable excipient or adjuvant.
  • The methods of the present invention include any clinically-acceptable route of administration of the composition to the subject. In various aspects, the route of administration is systemic, e.g., oral or by injection. The agents or compounds, or pharmaceutically acceptable salts or derivatives thereof, are administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperintoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally, intraportally, and parenterally. Alternatively or in addition, the route of administration is local, e.g., topical, intra-tumor and peri-tumor. In some embodiments, the compound is administered orally.
  • In other embodiments the agents disclosed herein are administered by the intravenous route. In further embodiments, the parenteral administration may be provided in a bolus or by infusion.
  • The manner in which the composition is administered is dependent, in part, upon the cause and/or location. One skilled in the art will recognize the advantages of certain routes of administration.
  • The method includes administering an effective amount of the agent or compound (or composition comprising the agent or compound) to achieve a desired biological response, e.g., an amount effective to alleviate, ameliorate, or prevent, in whole or in part, a symptom of a condition to be treated, e.g., oncology and neurology disorders.
  • In various aspects, the amount of the compound of any one of structural formulas shown in Table 1 and or Table 2, or salt or ester thereof administered is about 0.001 mg/kg to about 100 mg/kg body weight (e.g., about 0.01 mg/kg to about 10 mg/kg or about 0.1 mg/kg to about 5 mg/kg).
  • The concentration of a disclosed compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic characteristics of the compound(s) employed, and the route of administration. The agent may be administered in a single dose or in repeat doses. The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. Treatments may be administered daily or more frequently depending upon a number of factors, including the overall health of a patient, and the formulation and route of administration of the selected compound(s). An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • The compound or composition of the invention may be manufactured and/or administered in single or multiple unit dose forms.
  • Further embodiments of the present invention relate to the composition comprising the compound of any one of structural formulas shown in Table 1, and or Table 2 and a pharmaceutically-acceptable carrier, e.g., a pharmaceutically-acceptable excipient, carrier, binder, and/or diluent.
  • Techniques for formulation and administration of the disclosed compounds of the invention can be found in Remington: the Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, Pa. (1995).
  • In other embodiments, the compounds described herein, and the pharmaceutically acceptable salts, solvates, hydrates, and prodrugs thereof, are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. Optionally, the composition comprises one or more additional therapeutic agents. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.
  • In certain embodiments, the compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.
  • In certain embodiments, pharmaceutical compositions of the present invention comprise one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
  • In certain embodiments, a pharmaceutical composition of the present invention is prepared using known techniques, including, but not limited to mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.
  • Additional embodiments relate to the pharmaceutical formulations wherein the formulation is selected from the group consisting of a solid, powder, liquid and a gel.
  • In certain embodiments, a pharmaceutical composition of the present invention is a liquid (e.g., a suspension, elixir and/or solution). In certain of such embodiments, a liquid pharmaceutical composition is prepared using ingredients known in the art, including, but not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.
  • In certain embodiments, a pharmaceutical composition of the present invention is a solid (e.g., a powder, tablet, and/or capsule). In certain of such embodiments, a solid pharmaceutical composition comprising one or more ingredients known in the art, including, but not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents.
  • In certain embodiments, a pharmaceutical composition of the present invention is formulated as a depot preparation. Certain such depot preparations are typically longer acting than non-depot preparations. In certain embodiments, such preparations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. In certain embodiments, depot preparations are prepared using suitable polymeric or hydrophobic materials (for example an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • In certain embodiments, a pharmaceutical composition of the present invention comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
  • In certain embodiments, a pharmaceutical composition of the present invention comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80 and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • In certain embodiments, a pharmaceutical composition of the present invention comprises a sustained-release system. A non-limiting example of such a sustained-release system is a semi-permeable matrix of solid hydrophobic polymers. In certain embodiments, sustained-release systems may, depending on their chemical nature, release pharmaceutical agents over a period of hours, days, weeks or months.
  • In certain embodiments, a pharmaceutical composition of the present invention is prepared for oral administration. In certain of such embodiments, a pharmaceutical composition is formulated by combining one or more agents and pharmaceutically acceptable carriers. Certain of such carriers enable pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject. Suitable excipients include, but are not limited to, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In certain embodiments, such a mixture is optionally ground and auxiliaries are optionally added. In certain embodiments, pharmaceutical compositions are formed to obtain tablets or dragee cores. In certain embodiments, disintegrating agents (e.g., cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate) are added.
  • In certain embodiments, dragee cores are provided with coatings. In certain such embodiments, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to tablets or dragee coatings.
  • In certain embodiments, pharmaceutical compositions for oral administration are push-fit capsules made of gelatin. Certain of such push-fit capsules comprise one or more pharmaceutical agents of the present invention in admixture with one or more filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In certain embodiments, pharmaceutical compositions for oral administration are soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In certain soft capsules, one or more pharmaceutical agents of the present invention are be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
  • In certain embodiments, pharmaceutical compositions are prepared for buccal administration. Certain of such pharmaceutical compositions are tablets or lozenges formulated in conventional manner.
  • In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
  • In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • In certain embodiments, a pharmaceutical composition is prepared for administration by inhalation. Certain of such pharmaceutical compositions for inhalation are prepared in the form of an aerosol spray in a pressurized pack or a nebulizer. Certain of such pharmaceutical compositions comprise a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In certain embodiments using a pressurized aerosol, the dosage unit may be determined with a valve that delivers a metered amount. In certain embodiments, capsules and cartridges for use in an inhaler or insufflator may be formulated. Certain of such formulations comprise a powder mixture of a pharmaceutical agent of the invention and a suitable powder base such as lactose or starch.
  • In certain embodiments, a pharmaceutical composition is prepared for rectal administration, such as a suppository or retention enema. Certain of such pharmaceutical compositions comprise known ingredients, such as cocoa butter and/or other glycerides.
  • In certain embodiments, a pharmaceutical composition is prepared for topical administration. Certain of such pharmaceutical compositions comprise bland moisturizing bases, such as ointments or creams. Exemplary suitable ointment bases include, but are not limited to, petrolatum, petrolatum plus volatile silicones, and lanolin and water in oil emulsions. Exemplary suitable cream bases include, but are not limited to, cold cream and hydrophilic ointment.
  • In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • In certain embodiments, one or more compounds of the present invention are formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyamino acid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.
  • In certain embodiments, a prodrug is produced by modifying a pharmaceutically active compound such that the active compound will be regenerated upon in vivo administration. The prodrug can be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392).
  • Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.
    • EXAMPLES Example 1: Identification of Parkin Activators Assay Principle:
  • The assay based on the irreversible reaction of an Activity-Based Probe (ABP) with the active site cysteine in the enzyme. ABP consists of a ubiquitin moiety with an epitope tag (e.g. HA tag) at the N-terminus, and a reactive group at the C-terminus. The activity of Parkin-RBR (w/o the R0 inhibitory domain) is significantly higher than the activity of Parkin-R0RBR or the activity of full-length Parkin. The covalent attachment of ABP to Parkin can be monitored by Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
      • Parkin-R0RBR, full-length Parkin→low TR-FRET signal (negative control)
      • Parkin RBR→high TR-FRET signal (positive control)
        Compounds increasing the activity of Parkin-R0RBR or the activity of full-length—Parkin can be identified by an increase in TR-FRET signal.
        Strategy: use of N-terminal His-SUMO tagged constructs of Parkin-R0RBR, full-length Parkin and Parkin-RBR. (from Evotec Slides; Based on Riley et al. 2013. Nat Commun. 4:1982 & on information provided by E3x Bio; grant Application)
    Constructs:
  • Full-length Parkin (1-465), R0RBR (141-465) and RBR (238-465) expression with N-terminal His6-SUMO-tag (can potentially be removed during purification using SENP1 protease) in E. coli as described by Riley et al.
  • N-terminal His6-tag enabling TR-FRET-assay→use of the purified protein that still have the N-terminal His6-SUMO-tags on.
  • Small scale tests are conducted for all constructs to evaluate which construct, full-length Parkin or R0RBR, give better yield to facilitate an HTS-assay.
    • Phase 1: Protein Production
  • Initiate gene synthesis through third party for full-length Parkin with N-terminal His6-SUMO, His6-SUMO-R0RBR and His6-SUMO-RBR, codon-optimized for expression in E. coli and subcloning into a suitable expression vector
  • Small scale test expression evaluated by Western Blotting to estimate the yield of soluble protein
  • Transform the RBR construct as well as either the full-length Parkin construct or the R0RBR construct into BL21(DE3) and express as outlined in Riley et al., in the scale of 6-24 L (depending on outcome of small scale test expression)
  • Purification of ˜10 mg of the RBR construct as well as either the full-length Parkin construct or the R0RBR construct as described by Riley et al.*, i.e. IMAC, MonoQ and size exclusion.
    • Phase 2: Assay Development
  • Goal: Set-up robust primary screening assays in 1,536-well assay plate format
  • Establish assays in 384-well format with a reasonable dynamic range (e.g. using Parkin +/− the R0 inhibitory domain)
  • Optimize assay (e.g. in terms of concentrations of assay components, buffer, additives, order of addition of reagents, and incubation temperature)
  • Run time course experiments to define optimal incubation times
  • Demonstrate assay robustness (goal: Z′>0.5)
  • Demonstrate readout stability
  • Test DMSO tolerance
  • Demonstrate specificity of the assay signal obtained using the Parkin RBR domain (w/o the R0 inhibitory domain) by titration of Ub (competing with ABP)
  • Transfer assay from 384- to final 1,536-well screening plate format; adapt the assay to the EVOscreen™ Mark III HTS platform
  • If necessary, fine-tune the assay conditions in order to optimize assay robustness in this high density plate format (goal: Z′>0.5) and to demonstrate assay suitability for high-throughput screening (HTS)
  • Confirm stability of assay reagents under screening conditions over time
  • Demonstrate plate-to-plate and day-to-day assay robustness
  • Estimate and procure the amounts of all assay reagents required for screening and hit profiling.
    • Phase 3: Screening
  • Marker Library Screen (MLS):
  • Pre-screening of a diverse marker library of approximately 2.5 k representative lead-like compounds against the primary screening assay at two concentrations in triplicate
  • Statistical analysis of the MLS and hit definition using the 3-sigma-method (plate-based, based on the scatter of compound-free DMSO wells)
  • Selection of the optimal compound concentration for primary screening
    • Primary Screen (PS):
  • Screening of approximately 75,000 lead-like compounds against the primary screening assay at one uniform compound concentration (n=1); re-screening of compound plates that do not meet an agreed re-screen criterion (e.g. Z′>0.5)
  • Hit definition for the primary screen using the 3-sigma-method (plate-based, based on the scatter of compound-free DMSO wells)
  • Statistical analysis of the primary screen→Primary Hit Compounds (Parkin activators)
    • Hit Confirmation (HC):
  • Selection of a set of up to approximately 750 primary hits for Hit Confirmation
  • Cherry picking of the selected compounds and reformatting for testing
  • Retesting of the selected cpds against the primary screening assay at the compound screening concentration (n=3)
  • Statistical analysis of the Hit Confirmation campaign→Identification of confirmed small molecule Parkin activators.
    • Phase 4: HitProfiling (HP):
  • Selection of a set of up to approximately 250 confirmed hit compounds for Hit Profiling
  • Cherry picking of the selected compounds and reformatting for concentration-response testing
  • Concentration-response testing as 11-point compound dilution series against the primary screening assay (n=2)
  • Automated data fitting of the concentration response curves and calculation of the resulting IC50 values
  • LC/MS inspection of the hit compounds to confirm compound identity and purity
  • Structure-activity relationship analysis (SAR) of the active hit compounds
  • Confirmed & profiled small molecule Parkin activators.
    • Example 2: Activity-Based Probe Assay Using an Ubiquitin Vinyl Sulfone Probe
  • A Ubiquitin vinyl sulfone probe can be used that irreversibly binds to the active site cysteine of Parkin ligase. Covalent attachment of the probe to the Parkin can be monitored by TR-FRET. Candidate activator compounds can be identified by increasing the activity of Parkin ligase due to an increase in TR-FRET signal. Screening for activating compounds can be distinguished from the controls as follows:

  • 100% activation signal=Heat activated Parkin+100 nM control activator in DMSO. 0% activation signal=Heat activated Parkin+DMSO only. Parkin activators can be identified by an increase of the 0% activation signal TR-FRET signal.
  • Assay Conditions:
    • Materials:
    • Assay Plate: White 384 well plate (Corning 3572)
    • Enzyme: Parkin-His tagged 203 μM (10.5 mg/ml)
    • Probe: Ubiquitin vinyl-sulfone (HA-Ub-VS Boston Biochem U-212)
    • DMSO: DMSO (Sigma cat # D4540-100ML)
    • Reaction Buffer: 50 mM HEPES (pH 8.5), 150 mM NaCl, 0.01% Tween 20, 0.1% BSA
    • Detection Buffer: 50 mM HEPES (pH 8.5), 150 mM NaCl, 0.01% Tween 20, 0.1% BSA, 800 mM KF
    • Detection Reagent A: 2.6 nM Anti-6HIS-Eu cryptate and 40 nM Anti-HA-XL665 in detection buffer
    • Eu cryptate: Anti-6HIS-Eu cryptate (CisBio 61HISKLA)
    • XL665: Anti-HA-XL665 (CisBio 610HAXLA)
      Enzyme Reaction (15 min pre incubation Parkin with activator only)
    Parkin: 40 nM HA-Ub-VS Probe: 70 nM
  • Activator/DMSO: 2× Activator candidates/2% DMSO
    Reaction time: 60 minutes
    • Temperature: 22° C.
  • Total volume: 10 μl reaction
    • Detection Reaction
  • Take 10 μl of Enzyme Reaction above and add 10 μl detection Reagent A under the following conditions:
  • Reaction time: 60 minutes
  • Temperature: 22° C.
  • Total volume: 20 μl
  • Assay Procedure:
      • 1) Heat activate Parkin in Enzyme reaction buffer (500 μl/1.5 ml tube: Eppendorf Thermomixer 5 minutes, 400 rpm at 58° C. and put on ice until needed).
      • 2) Load assay plate wells with 4.8 μl 84.5 nM Parkin in reaction buffer by use of Bravo.
      • 3) Deliver 0.2 μl 200× activator candidates in DMSO by use of HP D-300 compound dispenser. Highest 200× concentration=20 μm and then twofold dilutions.
      • 4) Spin 1000 rpm, 2 minutes, at room temp.
      • 5) Incubate plate for 15 minutes at room temp.
      • 6) Add 5 μl 140 nM HA-Ub-VS Probe in reaction buffer by use of Bravo.
      • 7) Spin 1000 rpm, 2 minutes, at room temp.
      • 8) Incubate plate for 60 minutes at room temp.
      • 9) Add 10 μl 2.6 nM Anti-6HIS-Eu cryptate and 40 nM Anti-HA-XL665 in detection buffer.
      • 10) Spin 1000 rpm, 2 minutes, at room temp.
      • 11) Incubate plate for 60 minutes at room temp.
      • 12) Read plates on Perkin Elmer Envision instrument.
  • Data Analysis: The Data can be read in CSV files. There are two tables in those CSV files, which are the values of 655 nm (channel 1) and 615 nm (channel 2) wavelengths respectively. The data is converted to an HTRF Ratio=(Channel 1/Channel 2)*10,000
  • The average of all the 0 uM controls (DMSO only)=BKGD (Background−0% activation). Subtract BKGD from each HTRF Ratio value=HTRF−BKGD. The average of all the 100 uM 100 nM control activator in DMSO controls=Max (100% activation). The following equation is then used to calculate % Activation for each well/candidate as follows: % Activation=(HTRF−BKGD/Max)*100.
  • The % Activation of compound titration can then be used to find activation EC50 or highest % activation if less than 75% activation is seen for the candidate compound.
    • Example 3: Activity-Based Probe Assay with Candidate Electrophile and Chelator Compounds
  • The Activity-Based Probe Assay was performed as in Example 2 above with various compounds in Table 1 and/or Table 2. At least two compounds indicated increasing Parkin activity with the activity-based probe Ubiquitin-vinyl sulfone. As demonstrated in FIG. 1, compound N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide, a chelator compound (AH001) increases the Parkin Ligase reaction with the Activity-based Ubituitin vinyl sulfone probe.
  • Similarly, as indicated in FIG. 2, 6-benzyl-2,5-dimethyl-3-phenylpyrazolo[1,5-a]pyrimidine-7-thiol, an electrophile and chelator compound (AH007) increases the Parkin Ligase reaction with the Activity-based Ubituitin vinyl sulfone probe. This example indicates that both Chelators and Electrophiles can Both Regulate and/or Increase Parkin Ligase Activity.
    • Example 4: Parkin pUB Auto-ubiquitinylation Assay
  • A Parkin pUB Auto-ubiquitinylation Assay is used to evaluate a candidate compound's potency to activate Parkin's ability to Auto-ubiquitinylate itself.
  • The principle of this assay is that the E3 Ligase Parkin catalyzes the transfer of Ubiquitin to target proteins, but also has the ability to auto-ubiquitinylate itself. The phospho-Ubiquition (pUb) added to the assay alters the Parkin to a state where small molecule activators can enable the Parkin to auto-ubiquitinylate though the E1-E2 cascade reaction. The use of a Eu cryptate Ubiquition and anti 6His-d2 that binds to the His tagged Parkin will give a signal when the Eu cryptate Ubiquition is auto-ubiquitinylate onto the Parkin which can be monitored by TR-FRET.
  • Similar to the Activity-based probe assay in Examples 2 and 3, screening for activating compounds can be distinguished from the controls as follows:

  • 100% activation signal=pUb activated Parkin+40 nM control activator in DMSO.

  • 0% activation signal=pUb activated Parkin+DMSO only.
      • Parkin activators can be identified by an increase of the 0% activation signal TR-FRET signal.
    Materials:
    • Assay Plate: White 384 well plate (Corning 3572)
    • Enzyme 1: 5 μM E1 (Ubiquitin-activating enzyme/UBE1 Boston Biochem E-305)
    • Enzyme 2: 25 μM E2 (UBcH7/Ube2L3 Boston Biochem E2-640)
    • Enzyme 3: Parkin-His tagged 203 μM (10.5 mg/ml) Supplied by An2H
    • pUb: 230 μM (2 mg/ml) Phospho-Ubiquitin (S65) (Boston Biochem U-102) Supplied by An2H.
    • Eu Cryptate Reagent: 1.77 μM Ubiquitin Eu (CisBio 61UBIKLA) Reconstitute with 250 μl distilled water.
    • DMSO: DMSO (Sigma cat # D4540-100ML)
    • PF-127: Pluronic F-127 (Fisher Scientific 50-310-494)
    • Reaction Buffer: 50 mM HEPES, 50 mM NaCl, 1 mM MgCl2, 0.005% Tween20, 0.1% PF-127, pH 8.5
    • Detection Buffer: 50 mM HEPES, 50 mM NaCl, 800 mM KF, 5 mM EDTA, 0.005% Tween20, 0.1% PF-127, pH 8.5
    • Detection Reagent Z: 13.4 nM Anti-6His-d2 in detection buffer
    • d2 Reagent: 2.67 μM Anti-6His-d2 (CisBio 61HISDLA) Reconstitute with 2501l distilled water.
  • Assay Conditions:
  • 10 μl Enzyme Reaction (15 min pre incubation Parkin, pUb and activator only)
  • Parkin: 196 nM
  • pUb: 392 nM
  • Activator/DMSO: IX Activator/1% DMSO
  • E1: 5 nM
  • E2: 50 nM
  • Ubiquitin Eu: 8.8 nM
  • Reaction time: 120 minutes
  • Temperature: 22° C.
  • Total volume: 10 μl reaction
  • Detection Reaction
  • Take 10 μl of Enzyme Reaction above and add 10 μl detection Reagent Z under the following conditions:
    Reaction time: 60 minutes
    • Temperature: 22° C.
  • Total volume: 20 μl
  • Assay Procedure:
      • 1) Load assay plate wells with 4.9 μl 400.0 nM Parkin, 800 nM pUb in reaction buffer by use of Bravo.
      • 2) Deliver 0.1 μl 100× activator candidates in DMSO by use of HP D-300 compound dispenser. Highest 100× concentration=100 μm and then twofold dilutions. Add each compound and control in duplicate wells.
      • 3) Spin 1000 rpm, 2 minutes, at room temp.
      • 4) Incubate plate for 15 minutes at room temp.
      • 5) Add 5 μl 10 nM E1, 100 nM E2, 17.6 nM Ubiquitin Eu and 2 mM ATP in Reaction Buffer by use of Bravo.
      • 6) Spin 1000 rpm, 2 minutes, at room temp.
      • 7) Incubate plate for 120 minutes at room temp.
      • 8) Add 10 μl 13.4 nM anti his d2 in detection buffer by use of Bravo.
      • 9) Spin 1000 rpm, 2 minutes, at room temp.
      • 10) Incubate plate for 60 minutes at room temp.
      • 11) Read plates on Perkin Elmer Envision instrument.
  • Data Analysis: The Data can be read in CSV files. There are two tables in those CSV files, which are the values of 655 nm (channel 1) and 615 nm (channel 2) wavelengths respectively. The data is converted to an HTRF Ratio=(Channel 1/Channel 2)*10,000
  • The average of all the 0 uM controls (DMSO only)=BKGD (Background−0% activation). Subtract BKGD from each HTRF Ratio value=HTRF−BKGD. The average of all the 100 uM control activator in DMSO controls=Max (100% activation). The following equation is then used to calculate % Activation for each well/candidate as follows: % Activation=(HTRF-BKGD/Max)*100.
  • The % Activation of compound titration can then be used to find activation EC50 or highest % activation if less than 75% activation is seen for the candidate compound.
    • Example 5: Parkin pUB Auto-Ubiquitinylation Assay with Candidate Electrophile and Chelator Compounds
  • The Parkin pUB auto-ubiquitinylation Assay was performed as in Example 4 above with various compounds in Table 1 and/or Table 2. At least two compounds indicated increasing Parkin activity with the activity-based probe Ubiquitin-vinyl sulfone. As demonstrated in FIG. 3, compound N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide, a chelator compound (AH001) increases Parkin activity in an auto-ubiquitination assay. Furthermore, as indicated in FIG. 4A, N,N′-(1-phenyl-1H-1,2,4-triazole-3,5-diyl)dibenzamide (AH001) with pUB synergistically increases parkin activation in an auto-ubiquitination assay.
  • Similarly, as indicated in FIG. 5, 6-benzyl-2,5-dimethyl-3-phenylpyrazolo[1,5-a]pyrimidine-7-thiol, an electrophile compound (AH007) increases Parkin activity in an auto-ubiquitination assay. This example indicates that both chelators and electrophiles can both regulate and/or increase Parkin ligase activity in an auto-ubiquitination assay.
    • Example 6: Residues C59 and C377 are Critical for Modulator Binding to Parkin
  • Parkin ligase was incubated with AH007 compound, and the mixture was then subject to tandem mass spectometry analysis after proteolytic digestion to produce fragments of Parkin ligase. The goal was to identify specific fragments of Parkin that contain compound bound AH007, revealing the specific binding residues of Parkin for the compound. Compound An2H07 is also fragmented when analyzed by mass spectrometry. Therefore, characteristic pieces of the compound AH007 that are attached to specific residues of Parkin can also be identified.
  • These fragments were characterized for alterations in fragment size indicative of bound small molecule of the predicted molecular weight of AH007. The mass spectrometry data identified three specific fragments of AH007 at two specific residues of Parkin ligase. The data identified a fragment of 253.08-256.09 of AH007 compound attached to cysteine residue 377 of Parkin ligase (C377) and a fragment of 343.14-346.14 of AH007 compound attached to C377. The data also identified a fragment of 253.08-256.09 of AH007 compound attached to cysteine residue 59 of Parkin ligase (C59). The mass spectrometry data of Parkin ligase incubated with AH007 compound thus indicates that the compound binds and/or attaches to two specific sites in Parkin ligase: C59 and C377. Residues C59 and C377 were the only two consistent sites observed, even when the concentration of AH007 compound was dramatically increased in the mixture with Parkin ligase, suggesting specificity for these sites over numerous other sites of potential attachment. It is also believed that at least C377 is included in ZnF domains of human Parkin Ligase, and thus accords with the theory that cysteine residues in the flexible Parkin ligase ZnF domains are vulnerable for attachment and/or interruption by small molecule candidates. Peptide fragments of Parkin comprising C59 and/or C377 will be useful to design further binding assays and selection of additional modulating agents.
  • The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
  • While the invention has been described in connection with proposed specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims (25)

1.-37. (canceled)
38. A method of activating a Parkin ligase to treat a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the compound
Figure US20200087648A1-20200319-C00205
or a pharmaceutically acceptable salt thereof.
39. The method of claim 38, wherein the compound disrupts at least one Parkin ligase zinc finger, wherein the compound can coordinate with a zinc ion, and/or bind or react with a cysteine.
40. The method of claim 38, wherein activating the Parkin ligase treats or reduces the incidence of one or more diseases or ailments selected from the group consisting of Alzheimer's Dementia, Parkinson's disease, Huntington Disease, Amyotrophic Lateral Sclerosis (ALS), Freidreich's ataxia, Spinocerebellar Ataxia, Multiple Systems Atrophy, PSP, Tauopathy, Diffuse Lewy Body Disease, Lewy Body dementia, any disorder characterized by abnormal accumulation of α-synuclein, disorders of the aging process, stroke, bacterial infection, viral infection, Mitochondrial related disease, mental retardation, deafness, blindness, diabetes, obesity, cardiovascular disease, multiple sclerosis, Sjogrens syndrome, lupus, glaucoma, including pseudoexfoliation glaucoma, Leber's Hereditary Optic Neuropathy, and rheumatoid arthritis.
41. The method of claim 38, wherein activating the Parkin ligase treats or reduces the incidence of cancer.
42. The method of claim 38, wherein the compound substantially disrupts the structure of at least one zinc finger in a Parkin ligase.
43. The method of claim 38, wherein Parkin ligase activation alters ubiquitination.
44. The method of claim 38, wherein the patient has been diagnosed with a neuro-degenerative disease.
45. The method of claim 44, wherein the neuro-degenerative disease is Parkinson's disease, dementia, Amyotrophic lateral sclerosis (ALS) and Huntington's disease.
46. The method of claim 45, wherein the dementia is dementia with Lewy bodies (DLB), multiple system atrophy (MSA) or Progressive supranuclear palsy (PSP).
47. The method of claim 39, wherein the compound that can coordinate to a zinc ion is a monodentate, bidentate, or tridentate ligand.
48. The method of claim 39, wherein the amino acid residues of at least one zinc finger corresponds to or aligns within one or more domains selected from the group consisting R0 amino acids 141-216, IBR amino acids 328-377, and R2 amino acids 415-465 of human Parkin Ligase.
49. The method of claim 48, wherein at least one zinc finger comprises four cysteine residues.
50. The method of claim 48, wherein the compound substantially disrupts the structure of at least one zinc finger located in the IBR domain (amino acids 328-377).
51. The method of claim 38, wherein the activated Parkin ligase suppresses the growth of one or more tumors and/or prevents metastasis of one or more tumors.
52. A method of treating cancer in a patient in thereof, comprising administering to the patient an effective amount of the compound
Figure US20200087648A1-20200319-C00206
or a pharmaceutically acceptable salt thereof.
53. The method of claim 52, wherein the cancer is selected from the group consisting of colon, lung and ovarian cancer.
54. The method of claim 52, wherein the cancer is glioblastoma, small cell lung carcinoma, breast cancer or prostate cancer.
55. The method of claim 52, wherein the cancer is s selected from one or more of the group consisting of Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma, Lymphoma, Anal Cancer, Appendix Cancer, Astrocytomas, Childhood Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Skin Cancer (Nonmelanoma), Childhood Bile Duct Cancer, Extrahepatic Bladder Cancer, Bone Cancer, Ewing Sarcoma Family of Tumors, Osteosarcoma and Malignant Fibrous Histiocytoma, Brain Stem Glioma, Brain Tumors, Embryonal Tumors, Germ Cell Tumors, Craniopharyngioma, Ependymoma, Bronchial Tumors, Burkitt Lymphoma (Non-Hodgkin Lymphoma), Carcinoid Tumor, Gastrointestinal Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Lymphoma, Primary, Cervical Cancer, Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Chronic Myeloproliferative Neoplasms Colon Cancer, Colorectal Cancer, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Intraocular Melanoma, Retinoblastoma, Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors, Extragonadal Cancer, Ovarian Cancer, Testicular Cancer, Gestational Trophoblastic Disease, Glioma, Brain Stem Cancer, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis, Langerhans Cell Cancer, Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma, Kidney Cancer, Renal Cell Cancer, Wilms Tumor and Other Childhood Kidney Tumors, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Chronic Lymphocytic Cancer, Chronic Myelogenous Cancer, Hairy Cell Cancer, Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer, Non-Small Cell Cancer, Small Cell Cancer, Lymphoma, Cutaneous T-Cell (Mycosis Fungoides and Sézary Syndrome), Hodgkin Cancer, Non-Hodgkin Cancer, Macroglobulinemia, Waldenström, Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Intraocular (Eye) Cancer, Merkel Cell Carcinoma, Mesothelioma, Malignant, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia, Chronic, Myeloid Leukemia, Acute, Myeloma Multiple, Chronic Myeloproliferative Neoplasms, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer, Lip and Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Epithelial Cancer, Low Malignant Potential Tumor, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Primary Central Nervous System Lymphoma, Rectal Cancer, Renal Cell (Kidney) Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Ewing Cancer, Kaposi Cancer, Osteosarcoma (Bone Cancer), Soft Tissue Cancer, Uterine Cancer, Sézary Syndrome, Skin Cancer, Childhood Melanoma, Merkel Cell Carcinoma, Nonmelanoma, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Skin Cancer (Nonmelanoma), Childhood Squamous Neck Cancer with Occult Primary, Metastatic Cancer, Stomach (Gastric) Cancer, T-Cell Lymphoma, Cutaneous Cancer, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Unknown Primary, Carcinoma of Childhood, Unusual Cancers of Childhood, Urethral Cancer, Uterine Cancer, Endometrial Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenström Macroglobulinemia, Wilms Tumor, and Women's Cancers.
56. A method of treating cancer or a neuro-degenerative disease in a patient in thereof, comprising administering to the patient an effective amount of the compound
Figure US20200087648A1-20200319-C00207
or a pharmaceutically acceptable salt thereof.
57. The method of claim 56, wherein the cancer is selected from the group consisting of colon, lung and ovarian cancer.
58. The method of claim 56, wherein the cancer is selected from one or more of the group consisting of Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma, Lymphoma, Anal Cancer, Appendix Cancer, Astrocytomas, Childhood Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Skin Cancer (Nonmelanoma), Childhood Bile Duct Cancer, Extrahepatic Bladder Cancer, Bone Cancer, Ewing Sarcoma Family of Tumors, Osteosarcoma and Malignant Fibrous Histiocytoma, Brain Stem Glioma, Brain Tumors, Embryonal Tumors, Germ Cell Tumors, Craniopharyngioma, Ependymoma, Bronchial Tumors, Burkitt Lymphoma (Non-Hodgkin Lymphoma), Carcinoid Tumor, Gastrointestinal Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Lymphoma, Primary, Cervical Cancer, Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Chronic Myeloproliferative Neoplasms Colon Cancer, Colorectal Cancer, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Intraocular Melanoma, Retinoblastoma, Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors, Extragonadal Cancer, Ovarian Cancer, Testicular Cancer, Gestational Trophoblastic Disease, Glioma, Brain Stem Cancer, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis, Langerhans Cell Cancer, Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma, Kidney Cancer, Renal Cell Cancer, Wilms Tumor and Other Childhood Kidney Tumors, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Chronic Lymphocytic Cancer, Chronic Myelogenous Cancer, Hairy Cell Cancer, Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer, Non-Small Cell Cancer, Small Cell Cancer, Lymphoma, Cutaneous T-Cell (Mycosis Fungoides and Sézary Syndrome), Hodgkin Cancer, Non-Hodgkin Cancer, Macroglobulinemia, Waldenström, Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Intraocular (Eye) Cancer, Merkel Cell Carcinoma, Mesothelioma, Malignant, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia, Chronic, Myeloid Leukemia, Acute, Myeloma Multiple, Chronic Myeloproliferative Neoplasms, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer, Lip and Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Epithelial Cancer, Low Malignant Potential Tumor, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Primary Central Nervous System Lymphoma, Rectal Cancer, Renal Cell (Kidney) Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Ewing Cancer, Kaposi Cancer, Osteosarcoma (Bone Cancer), Soft Tissue Cancer, Uterine Cancer, Sézary Syndrome, Skin Cancer, Childhood Melanoma, Merkel Cell Carcinoma, Nonmelanoma, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Skin Cancer (Nonmelanoma), Childhood Squamous Neck Cancer with Occult Primary, Metastatic Cancer, Stomach (Gastric) Cancer, T-Cell Lymphoma, Cutaneous Cancer, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Unknown Primary, Carcinoma of Childhood, Unusual Cancers of Childhood, Urethral Cancer, Uterine Cancer, Endometrial Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenström Macroglobulinemia, Wilms Tumor, and Women's Cancers.
59. (canceled)
60. The method of claim 56, wherein the neuro-degenerative disease is Parkinson's disease, dementia, Amyotrophic lateral sclerosis (ALS) and Huntington's disease.
61. The method of claim 60, wherein the dementia is dementia with Lewy bodies (DLB), multiple system atrophy (MSA) or Progressive supranuclear palsy (PSP).
US16/193,184 2014-12-05 2018-11-16 Parkin ligase activation methods and compositions Abandoned US20200087648A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/193,184 US20200087648A1 (en) 2014-12-05 2018-11-16 Parkin ligase activation methods and compositions

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US201462087972P 2014-12-05 2014-12-05
US201562222008P 2015-09-22 2015-09-22
US201562237400P 2015-10-05 2015-10-05
US14/961,285 US10155936B2 (en) 2014-12-05 2015-12-07 Parkin ligase activation methods and compositions
US16/193,184 US20200087648A1 (en) 2014-12-05 2018-11-16 Parkin ligase activation methods and compositions

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US14/961,285 Continuation US10155936B2 (en) 2014-12-05 2015-12-07 Parkin ligase activation methods and compositions

Publications (1)

Publication Number Publication Date
US20200087648A1 true US20200087648A1 (en) 2020-03-19

Family

ID=56092683

Family Applications (2)

Application Number Title Priority Date Filing Date
US14/961,285 Expired - Fee Related US10155936B2 (en) 2014-12-05 2015-12-07 Parkin ligase activation methods and compositions
US16/193,184 Abandoned US20200087648A1 (en) 2014-12-05 2018-11-16 Parkin ligase activation methods and compositions

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US14/961,285 Expired - Fee Related US10155936B2 (en) 2014-12-05 2015-12-07 Parkin ligase activation methods and compositions

Country Status (11)

Country Link
US (2) US10155936B2 (en)
EP (1) EP3227437A4 (en)
JP (1) JP2017538699A (en)
KR (1) KR20170092634A (en)
CN (1) CN107249580A (en)
AU (1) AU2015357487A1 (en)
BR (1) BR112017011644A2 (en)
CA (1) CA2968094A1 (en)
IL (1) IL252425A0 (en)
MX (1) MX2017007095A (en)
WO (1) WO2016090371A2 (en)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016090371A2 (en) 2014-12-05 2016-06-09 An2H Discovery Limited Parkin ligase activation methods and compositions
WO2016196955A1 (en) 2015-06-04 2016-12-08 Drexel University Inhibitors of RAD52 Recombination Protein and Methods Using Same
GB201514015D0 (en) * 2015-08-07 2015-09-23 Arnér Elias S J And Dept Of Health And Human Services Novel pyridazinones and their use in the treatment of cancer
CN109922802A (en) * 2016-06-03 2019-06-21 An2H探索有限公司 Triazole heterocyclic carbamate derivatives and its compositions related and treatment method
US11401255B2 (en) 2016-07-28 2022-08-02 Mayo Foundation For Medical Education And Research Small molecule activators of Parkin enzyme function
WO2018178237A1 (en) * 2017-03-30 2018-10-04 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for the treatment of mitochondrial genetic diseases
CN109745324A (en) * 2017-11-06 2019-05-14 中国科学院上海生命科学研究院 The micromolecular inhibitor of non-classical NF-kB access and its application
WO2019100106A1 (en) * 2017-11-24 2019-05-31 The University Of Sydney Antibacterial compounds and methods of use thereof
US10889553B2 (en) 2017-12-01 2021-01-12 Nysnobio Ireland Dac Asymmetric triazole benzamide derivatives and the compositions and methods of treatment regarding the same
CN109988157B (en) * 2018-01-03 2022-12-27 四川大学 Novel small molecular compound, preparation method and application in preparation of drug-resistant mycobacterium tuberculosis and other mycobacteria drugs
WO2019169158A1 (en) * 2018-02-28 2019-09-06 Crestone, Inc. Novel antimycobacterial heterocyclic amides
CA3097886A1 (en) * 2018-04-20 2019-10-24 Nysnobio Ireland Dac Methods for treating patients with cancer having defects in cyclin d regulation
EP4094754A1 (en) * 2018-06-19 2022-11-30 Universidad Pablo de Olavide Compositions for treating and/or preventing protein-aggregation diseases
KR102066842B1 (en) * 2018-08-09 2020-01-17 대한민국 Pharmaceutical composition for preventing or treating tuberculosis comprising 10-DEBC or pharmaceutically acceptable salts thereof
KR102091336B1 (en) * 2018-10-30 2020-03-19 건국대학교 산학협력단 Compound for inhibiting peptide deformylase and composition containing the same
IL264854A (en) * 2019-02-14 2020-08-31 Bahat Anat Spt5 inhibitors and methods of use thereof
AU2020357957A1 (en) * 2019-10-01 2022-05-12 Drexel University Quinoline inhibitors of RAD52 and methods of use
KR20210054242A (en) * 2019-11-05 2021-05-13 아주대학교산학협력단 Tumor necrosis factor alpha(TNF-α) small-molecule inhibitor

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9313866D0 (en) * 1993-07-05 1993-08-18 Diomed Dev Ltd Compositions for the treatment of inflammatory proliferative skin disease and their use
US8143299B2 (en) 2004-10-25 2012-03-27 University Of Medicine And Dentistry Of New Jersey Anti-mitotic anti-proliferative compounds
WO2008134474A2 (en) 2007-04-27 2008-11-06 The Board Of Trustees Of The University Of Illinois Compositions and methods including cell death inducers and procaspase activation
US8062839B2 (en) * 2007-05-21 2011-11-22 Elan Pharma International Limited Parkin substrate and assay
CA2690653A1 (en) 2007-06-15 2008-12-24 Irm Llc Protein kinase inhibitors and methods for using thereof
KR101014351B1 (en) * 2008-08-04 2011-02-15 한국생명공학연구원 Composition for prevention or treatment of cancer or cardiovascular disease containing triazolyl-benzamide derivatives or pharmaceutically acceptable salts thereof inhibiting ERK2 as an active ingredient
BRPI1008651B1 (en) 2009-02-09 2020-01-21 Univ Illinois procaspase activating compounds, a drug that comprises them and their use
CA2784765A1 (en) 2010-01-15 2011-07-21 Janssen Pharmaceuticals, Inc. Novel substituted bicyclic triazole derivatives as gamma secretase modulators
EP2621926B1 (en) 2010-09-30 2017-08-30 Merck Sharp & Dohme Corp. Pyrazolopyrimidine pde10 inhibitors
US8603994B2 (en) 2010-11-11 2013-12-10 Valted, Llc Transcriptional repression leading to Parkinson's disease
US8916705B2 (en) 2011-10-14 2014-12-23 The Board of Trustees of The University of Illilnois Procaspase-activating compounds and compositions
FR2987537B1 (en) 2012-02-23 2015-12-25 Cliris METHOD AND SYSTEM FOR SUPERVISION OF A SCENE, IN PARTICULAR IN A SALE SITE
AU2013225682B2 (en) 2012-03-02 2016-04-14 The Board Of Trustees Of The University Of Illinois Potent anticancer activity via dual compound activation
CA2866021C (en) 2012-03-06 2020-09-22 The Board Of Trustees Of The University Of Illinois Procaspace 3 activation by pac-1 combination therapy
BR112015002357B1 (en) 2012-08-03 2022-03-15 The Board Of Trustees Of The University Of Illinois Enzyme activating compounds and compositions
WO2014041125A1 (en) 2012-09-13 2014-03-20 Baden-Württemberg Stiftung Gmbh Specific inhibitors of protein p21 as therapeutic agents
JP6713982B2 (en) 2014-07-24 2020-06-24 ファイザー・インク Pyrazolopyrimidine compounds
WO2016090371A2 (en) 2014-12-05 2016-06-09 An2H Discovery Limited Parkin ligase activation methods and compositions

Also Published As

Publication number Publication date
US20160160205A1 (en) 2016-06-09
CA2968094A1 (en) 2016-06-09
KR20170092634A (en) 2017-08-11
BR112017011644A2 (en) 2018-02-14
WO2016090371A3 (en) 2016-07-28
JP2017538699A (en) 2017-12-28
CN107249580A (en) 2017-10-13
WO2016090371A2 (en) 2016-06-09
MX2017007095A (en) 2017-11-17
IL252425A0 (en) 2017-07-31
AU2015357487A1 (en) 2017-06-08
EP3227437A4 (en) 2018-08-22
EP3227437A2 (en) 2017-10-11
US10155936B2 (en) 2018-12-18

Similar Documents

Publication Publication Date Title
US20200087648A1 (en) Parkin ligase activation methods and compositions
Ghosh et al. Developing β‐secretase inhibitors for treatment of Alzheimer’s disease
De Simone et al. Glycogen synthase kinase 3β: a new gold rush in anti-Alzheimer’s disease Multitarget Drug Discovery? Miniperspective
Krishna et al. Design, synthesis and 3D-QSAR studies of new diphenylamine containing 1, 2, 4-triazoles as potential antitubercular agents
US20180125848A1 (en) Preparation and methods of use for ortho-aryl 5-membered heteroaryl-carboxamide containing multi-targeted kinase inhibitors
US9808448B2 (en) Pharmaceutical compounds and use of same in cancer and tauopathies
US9926306B2 (en) Inhibition of MCL-1 and/or BFL-1/A1
US10633369B2 (en) Method of inhibiting DYRK1B
Nemr et al. Design, synthesis and mechanistic study of new benzenesulfonamide derivatives as anticancer and antimicrobial agents via carbonic anhydrase IX inhibition
US11299467B2 (en) Chemical compounds
US10844023B2 (en) Small molecule regulators of mitochondrial fusion and methods of use thereof
US20240043445A1 (en) Thienopyranones and Furanopyranones as Kinase, Bromodomain, and Checkpoint Inhibitors
US11472814B2 (en) Thienopyranones and furanopyranones as checkpoint inhibitors and modulators of anti-tumor immunity
CA3049010A1 (en) Methods for the treatment of neurological disorders
US20230134821A1 (en) Myc-max inhibitor compound therapeutics for cancer treatment, methods and uses associated therewith
US20160002273A1 (en) Thieno[3,2-d]pyrimidine-6-carboxamides and analogues as sirtuin modulators
McKinzie et al. Discovery and early clinical development of LY3202626, a low-dose, CNS-penetrant BACE inhibitor
US10918647B2 (en) Selective bromodomain inhibition of fungal Bdf1
Gajić et al. Synthesis and analysis of 4-oxothiazolidines as potential dual inhibitors of deoxyribonuclease I and xanthine oxidase
US9261497B2 (en) Method of treating cancer with modulators of SCFSkp2
US20200317674A1 (en) Pyrazolopyrimidine derivatives and the compositions and methods of treatment regarding the same
US20090318711A1 (en) Non-Covalent Inhibitors of AmpC ß-Lactamase
US20170057955A1 (en) Pyridopyrimidinone Compounds for Modulating the Catalytic Activity of Histone Lysine Demethylases (KDMs)
US20230406853A1 (en) Covalent cdk2-binding compounds for therapeutic purposes
US20230158025A1 (en) Galactokinase inhibitors

Legal Events

Date Code Title Description
AS Assignment

Owner name: AN2H DISCOVERY LIMITED, IRELAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:JOHNSTON, JENNIFER;REEL/FRAME:048427/0268

Effective date: 20160122

AS Assignment

Owner name: NYSNOBIO IRELAND DAC, IRELAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:AN2H DISCOVERY LIMITED;REEL/FRAME:052670/0446

Effective date: 20200511

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION