WO2013010218A1 - Inhibition of clathrin - Google Patents

Inhibition of clathrin Download PDF

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WO2013010218A1
WO2013010218A1 PCT/AU2012/000858 AU2012000858W WO2013010218A1 WO 2013010218 A1 WO2013010218 A1 WO 2013010218A1 AU 2012000858 W AU2012000858 W AU 2012000858W WO 2013010218 A1 WO2013010218 A1 WO 2013010218A1
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clathrin
inhibitor
binding
compound according
alkyl
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PCT/AU2012/000858
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French (fr)
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WO2013010218A9 (en
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Haucke VOLKER
Phillip Robinson
Adam Mccluskey
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Freie Universität Berlin
Newcastle Innovation Limited
Children's Medical Research Institute
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Publication of WO2013010218A1 publication Critical patent/WO2013010218A1/en
Publication of WO2013010218A9 publication Critical patent/WO2013010218A9/en

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    • 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
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/34—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/36—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/40—Unsubstituted amino or imino radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/42—Amino or imino radicals substituted by hydrocarbon or substituted hydrocarbon radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/50—Nitrogen atoms bound to hetero atoms
    • C07D277/52—Nitrogen atoms bound to hetero atoms to sulfur atoms, e.g. sulfonamides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/06—Peri-condensed systems

Definitions

  • the present invention relates to the inhibition of clathrin and in particular though not exclusively, to the inhibition of clathrin-mediated endocytosis (CME) and cell division (mitosis).
  • CME clathrin-mediated endocytosis
  • mitosis mitosis
  • the inhibition of clathrin has application to the prophylaxis or treatment of various diseases and physiological conditions.
  • Clathrin-mediated endocytosis is a major process by which cargo is internalized into intracellular vesicles, thereby regulating the cell surface levels and endocytic uptake of important plasma membrane proteins. These include nutrient and growth factor receptors, ion channels, adhesion proteins, and synaptic vesicle (SV) membrane proteins in the nervous system.
  • the clathrin based endocytic machinery is also hijacked by pathogens such as bacteria and viruses to get access to the cell interior.
  • clathrin plays a function in traffic between the trans-Golgi network and the endo-lysosmal system, a pathway related to lysosomal storage disorders among other diseases.
  • CCPs clathrin-coated pits
  • CCPs clathrin-coated pits
  • These factors serve as scaffolds for the co-assembly of heterotetrameric AP-2 complexes, which coordinate recognition of transmembrane cargo with the recruitment of endocytic accessory proteins.
  • Early endocytic structures are presumably stabilized by an assembling clathrin coat built from soluble clathrin triskelia comprising three heavy and three light chains.
  • the central building block of triskelia is the 190 kDa clathrin heavy chain, which forms an extended three-legged structure.
  • the N-terminal ⁇ -propeller domain (referred to as the terminal domain or TD) at the distal end of the leg adopts a WD40- like fold (ter Haar et al., 2000), while the C terminus is near the vertex of the triskelion (Brodsky et al., 2001).
  • the clathrin cage has been postulated to serve at least two major functions in the endocytic process: (i) stabilizing deformed membrane domains, thereby facilitating the transition to invaginated pits, and (ii) providing an interaction hub for the recruitment of accessory factors that regulate progression of endocytosis.
  • stabilizing deformed membrane domains thereby facilitating the transition to invaginated pits
  • providing an interaction hub for the recruitment of accessory factors that regulate progression of endocytosis Indeed structural and proteomic studies have revealed a surprisingly simple architecture of accessory protein- clathrin interactions.
  • clathrin box motifs simple degenerate peptides that bind to a structurally well-defined site on the TD of clathrin heavy chain (ter Haar et al., 2000), consistent with the clathrin TD functioning as a central protein-protein interaction hub within the endocytic network.
  • Clathrin has a central role in many cellular processes. Besides CME, clathrin has involvment in mitosis. Mitosis is the stage of cell division where the chromosomes segregate and two new daughter cells form. Segregation is achieved via kinetochores which mediate attachment of chromosomes to spindle microtubules (MTs) known as the mitotic spindle. In its triskelia form clathrin binds the mitotic spindle, providing a structural lattice (Royle et al, 2005). The localisation of clathrin to the mitotic spindle is dependent on Aurora A, TACC3, and ch-TOG. Inhibitors of Aurora A target the mitotic spindle and are currently in pre-clinical or clinical studies as cancer treatments.
  • Mitosis is the stage of cell division where the chromosomes segregate and two new daughter cells form. Segregation is achieved via kinetochores which mediate attachment
  • the inhibition of clathrin thus has application to the prophylaxis or treatment of a wide range of diseases and conditions.
  • Inhibitors of clathrin would not only be important tools for unravelling the role of clathrin during mitosis but in at least some forms , have application as anti-mitotic compounds useful for the treatment of cancer and cell proliferative conditions.
  • small molecule inhibitors of dynamin (a protein with a central role in endocytosis) have been reported, it is believed that no specific inhibitors of clathrin have previously been described in the literature.
  • the invention stems from the surprising finding that the clathrin terminal domain (TD) has an unexpected central role in clathrin-coated pit (CCP) dynamics.
  • the invention relates, to the identification and characterization of a specific binding site on the clathrin TD enabling the inhibition of clathrin-mediated endocytosis (CME) in cells and more generally, inhibition of clathrin function.
  • CME clathrin-mediated endocytosis
  • the identification of the binding site facilitates the rational design of inhibitors for targeted binding to the clathrin TD for the inhibition of clathrin.
  • a clathrin inhibitor wherein the inhibitor binding to a binding site of the terminal domain (TD) of clathrin forms a complex with the clathrin TD, the binding site of the clathrin TD being defined by at least one or more of amino acids He 52, He 62, He 80, Phe 91 and/or He 93 of SEQ ID No. 1.
  • a complex of a clathrin inhibitor and the terminal domain (TD) of clathrin or a fragment of the clathrin TD the inhibitor binding to a binding site of the clathrin TD, and the binding site being defined by at least one or more of amino acids He 52, He 62, He 80, Phe 91 and/or He 93 of SEQ ID No. 1.
  • the binding of the clathrin inhibitor to the clathrin TD is provided by binding interactions of the inhibitor with at least some of amino acids He 52, lie 62, He 80, Phe 91 and/or He 93 of SEQ. No. 1.
  • the clathrin inhibitor interacts with all of amino acids He 52, He 62, He 80, Phe 91 , and He 93 of SEQ ID No.1.
  • the binding site of the clathrin TD for the clathrin inhibitor is defined by one or more of further amino acids He 66, Arg 64, Leu 82 and/or Lys 96 of SEQ ID No. 1 , and the binding of the inhibitor is provided by binding interactions of the inhibitor with the one or more further amino acids.
  • the clathrin inhibitor interacts with all of amino He 66, Arg 64, Leu 82 and Lys 96 of SEQ ID. No. 1.
  • the binding site can be further defined by amino acid Val 50 of SEQ ID No. 1 and the binding of the inhibitor is provided by binding a interaction of the inhibitor with that amino acid.
  • the binding interactions between the clathrin inhibitor with the clathrin TD can comprise direct contact of the inhibitor with at least some of the amino acids defining the binding site of the clathrin TD for the inhibitor.
  • the TD involve direct contact between the inhibitor and respective of the amino acids defining the binding site of the clathrin TD.
  • the binding of the inhibitor to the clathrin TD is stabilised by one or more hydrogen bonds between the inhibitor and the clathrin TD.
  • the hydrogen bonds can be direct hydrogen bonds between the inhibitor and the clathrin TD and/or hydrogen bonds mediated by water.
  • a method for inhibiting clathrin comprising contacting clathrin with an effective amount of at least one clathrin inhibitor for forming a complex with the terminal domain (TD) of clathrin in
  • a complex in accordance with the invention can be formed in vitro or in vivo, and in some forms, may be isolated or at least partially purified.
  • animal cells or tissues can be treated with the inhibitor in vitro or in vivo for the inhibition of clathrin, and the invention expressly extends to the prophylaxis or treatment of diseases or conditions responsive to the inhibition of clathrin, or for which the inhibition of clathrin may otherwise be beneficial.
  • a method for inhibiting clathrin in a mammal comprising administering to the mammal an effective amount of at least one clathrin inhibitor for forming a complex with the terminal domain (TD) of clathrin in accordance with one or more embodiments of the invention.
  • the clathrin inhitor(s) are administered for the inhibition of CME and/or inhibition of cell division.
  • a method for providing a clathrin inhibitor comprising:
  • the inhibitor to bind to a binding site for the inhibitor on the terminal domain (TD) of clathrin;
  • the inhibitor forms a complex with the binding site of the clathrin TD in accordance with one or more embodiments of the invention on the basis of the modelling, the formation of the complex being indicative of capacity of the inhibitor to inhibit clathrin.
  • the designing of the inhibitor can involve fitting the inhibitor into the binding site of the clathrin TD.
  • a range of inhibitors that bind to the clathrin TD binding site and inhibit clathrin function have been identified by the inventors.
  • the identification and characterisation of the clathrin TD binding site as described herein facilitates the rational design of inhibitors for use in embodiments of the invention.
  • a pharmaceutical composition comprising at least one compound of Formula I to Formula V, or a prodrug or pharmaceutically acceptable salt of the compound, together with a pharmaceutically acceptable carrier.
  • a method for inhibiting clathrin comprising contacting clathrin with an effective amount of a compound of Formula 1 to Formula V, or a prodrug or physiologically acceptable salt of the compound.
  • a method for inhibiting clathrin in an animal comprising administering to the mammal an effective amount of a compound of Formula I or Formula V to the mammal, or a prodrug or pharmaceutically acceptable salt thereof.
  • a clathrin inhibitor embodied by the inventionfor use in inhibiting clathrin in a mammal, or a prodrug or physiologically acceptable salt of the inhibitor.
  • a clathrin inhibitor embodied by the invention in the manufacture of a medicament for inhibiting clathrin in a mammal in need thereof, or a prodrug or physiologically acceptable salt of the inhibitor.
  • terminal domain of clathrin and variations thereof such as “clathrin TD” and “clathrin terminal domain” as used herein is meant the N-terminal ⁇ - propeller region of clathrin heavy chain 1 , such as defined by amino acids 2-479 of SEQ ID No. 1 (Genbank Accession No. NM-004859.3, National Center for
  • clathrin inhibitor as used herein and variations thereof such as “inhibitor of clathrin”, is meant a compound that interacts with the clathrin TD in accordance with the invention to at least partly inhibit the functional activity of clathrin, examples of which are the inhibition of clathrin in clathrin-mediated endocytosis (CME) as may be expressed by a reduction in CME and/or the level of CME in cells, and the inhibition of cell growth and proliferation by inhibiting mitosis.
  • CME clathrin-mediated endocytosis
  • the inhibition of clathrin in accordance with the invention can be total or partial inhibition of the functional activity of clathrin.
  • agent as used herein is meant a clathrin inhibitor as described herein, or a composition or preparation comprising a clathrin inhibitor.
  • binding in the context of binding of the clathrin inhibitor with the binding site of the clathrin terminal domain, is meant association of the clathrin inhibitor with the binding site through binding interactions of the inhibitor with at least some the amino acids defining the clathrin TD binding site.
  • a binding interaction of the inhibitor with the clathrin TD can, for example, comprise direct contact of the inhibitor with amino acid(s) of the clathrin TD that define the binding site, the formation of one of more direct hydrogen bonds between the inhibitor and the clathrin TD, and/or the formation of one or more hydrogen bonds between the inhibitor and the clathrin TD mediated by water.
  • the identification of the clathrin TD binding site and provision of inhibitors of clathrin in accordance with one or more embodiments of the invention not only facilitates the dissection of the mechanisms of endocytic pathways and CME and CCP dynamics in research, but has application to modulation of cell signalling, inhibition of entry of viruses and other pathogenic agents into cells, inhibition of synaptic
  • clathrin for the prophylaxis or treatment of a wide variety of diseases or physiological conditions.
  • these inhibition of clathrin has application to inhibiting mitosis, and hence in the prophylaxis or treatment of cancer and more generally, cell proliferative conditions.
  • inhibitors, compounds and methods embodied by the invention have application to use in research into clathrin function.
  • Figure 1 Amino acid sequence for Homo sapiens clathrin heavy chain 1 (SEQ ID No. 1 ; Genbank Accession No. NM-004859.3 (GI: 11527063), National Centre for Biotechnology Information, National Institutes of Health, Rockville Pike, Bethesda, Maryland, USA).
  • the clathrin TD of Homo sapiens clathrin heavy chain 1 is defined by amino acid residues 2-479 of SEQ ID. No 1.
  • Amino acid lof SEQ ID No. 1 is an initiator methionine residue.
  • Figure 2 A ligplot showing interaction of the clathrin inhibitor pitstop 1 with the clathrin TD.
  • Figure 3 A ligplot showing interaction of the clathrin inhibitor pitstop 2 with the clathrin TD.
  • FIG. 4 Pitstops compete with clathrin box ligands for a common site on the clathrin TD.
  • A, C Ribbon representations of the clathrin TD in top view. The blades of the TD- -propeller are numbered from 1 to 7. Both inhibitors bind specifically to the clathrin-box binding site (between blades 1 and 2 of the clathrin TD).
  • B Close-up view of the binding site for pitstop 1. The inhibitor and amino acids of the binding groove are shown in ball-and-stick mode. Hydrogen bonds formed at both ends of pitstop 1 are indicated by broken lines. The non-polar portion of pitstop 1 is sandwiched between Phe91 and the Arg64 side chains.
  • the aromatic ring of Phe91 is located in the same position as in the inhibitor-free form of the clathrin TD and stacks against the edge of the bromobenzene of pitstop 2.
  • E, F pitstops (surface representations) are superimposed with the clathrin box-containing AP-3 p3-hinge peptide (sequence AVSLLDLDA) (SEQ ID No. 2).
  • Pitstop 1 and pitstop 2 are shown in solid form. Binding of the inhibitors to the clathrin TD blocks the association of the 3-hinge peptide and similar clathrin-box ligands.
  • Figure 5 Pitstop 2 reversibly inhibits clathrin-mediated endocytosis.
  • (B) Reversibility and dose-dependence of pitstop 2-mediated inhibition of Tf uptake. Tf internalization after washout is not significantly different from uptake in the DMSO control (SEM; n 3 independent experiments; *p ⁇ 0.05, ***p ⁇ 0.0001).
  • Figure 6 Ultrastructural analysis of intermediates observed in pitstop 2-treated cells.
  • A Representative examples of clathrin-coated structures observed at the plasma membrane of Cos7 cells treated with 0.1 % DMSO or 30 ⁇ pitstop 2. Scale bar, 200 nm. Morphological groups were: shallow CCPs (stage 1), non-constricted u-shaped CCPs (stage 2), constricted ⁇ -shaped CCPs (stage 3), or structures containing complete clathrin coats (stage 4).
  • reticulospinal synapses (A, B) Electron micrographs of reticulospinal synapses microinjected with pitstop 1 and stimulated at 5 Hz for 20 min. Note the depletion of SVs (sv) at sites of release and large membrane expansions and pockets (asterisks), which in some sections (e.g., in B) surrounded the active zone (marked with thick arrows). Boxed area containing clathrin-coated pits (CCPs) is shown as inset at higher magnification in A.
  • C EM-image of a control non-injected synapse from the same spinal cord preparation.
  • FIG. 6A Bar diagram showing the relative abundance of different stages of coated endocytic intermediates (see above description for Fig. 6A) in control synapses stimulated at 5 Hz (white bars) and stimulated synapses microinjected with pitstop 1 (black bars). Scale bars for (A-C, and F), 0.5 ⁇ and (D, E), 100 nra.
  • FIG. 8 Treatment with the pitstops 1 and 2 causes mitotic failure.
  • pitstop 1 more than triples the mitotic index from 1% in untreated controls and 2% in DMSO treated to 7%, and at 10 ⁇ , pitstop 2 increases the mitotic index to 5%, while clathrin depletion with siRNA increased the mitotic index to 11%.
  • FIG. 9 Pitstops 1 and 2 affect the mitotic spindle.
  • A The triskelial clathrin molecule provides a structural lattice for the mitotic spindle. In the absence of clathrin the spindle collapses becoming narrow. The graph shows the percentage of cells with narrow spindles when treated with pitstops. The inhibition of clathrin by the pitstop compounds gave the same narrow spindle phenotype as clathrin depleted cells.
  • B Graph quantifying spindle width as a ratio of the total cell width shows that upon pitstop treatment the spindles significantly narrow.
  • C Images from fluorescence microscopy showing the narrow spindle phenotype caused by treatment with pitstops.
  • FIG 10 The pitstop compounds affect K fibre organisation during metaphase. Graph showing percentage of cells with disrupted HURP localisation. HURP is a marker for fibres, which are a unique subset of the spindle microtubules that bind to the kinetochores of chromosomes during metaphase. Depletion or inhibition of clathrin by siRNA or the pitstop compounds caused disruption to HURP staining.
  • Figure 11 The pitstops activate the metaphase checkpoint.
  • HeLa cells where synchronised at the G2/M boarder by the cdkl inhibitor RO-3306, then released in the presence of the indicated drug or control for 90 min, then fixed and stained for CENPB (red), MAD2 (green) and DNA (DAPI, blue). Captured images were decovoled and the channels combined.
  • B The number of co-localisations between CENPB and MAD2 were scored for n>30 cells per sample, and are shown on the dot graph; the median is represented by the line. Inhibition by the pitstop compounds increased the number of active checkpoints per cell.
  • C HeLa cells were depleted of either clathrin heavy chain (CHC) or epsin by siRNA and scored as in (B). Depletion of CHC increased the number of active checkpoints. N>30 cells per sample, and are shown on the dot graph; the median is represented by the line.
  • Figure 12 Pitstops induce multinucleation. The level of multinucleation (indicative of failed cytokinesis) more than doubled in cell populations treated with pitstop 1 or pitstop 2, or in cells depleted of clathrin by treatment by specific siRNA.
  • Figure 13 Crystal data for the terminal domain of clathrin complexed with pitstop 1.
  • Figure 15 Crystal data for amino acids in pocket of the terminal domain of clathrin defining the binding site of clathrin for pitstop 1.
  • Figure 16 Crystal data for amino acids in pocket of the terminal domain of clathrin defining the binding site of clathrin for pitstop 2.
  • alkyl used either alone or in a compound word such as alkylaryl refers to a straight chain, branched or mono- or polycyclic alkyl.
  • straight chain and branched alkyl include methyl, ethyl, propyl, wo-propyl, butyl, and the like.
  • cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
  • alkenyl refers to a straight chain, branched or cyclic alkenyl with one or more double bonds. Examples of alkenyl ethenyl, propenyl, and butenyl.
  • the alkyl or alkenyl will be a linear alkyl of linear alkenyl substituent. Most typically, the alkyl or alkenyl will have 10 carbon atoms or less in its longest chain and most typically, will be a lower alkyl or lower alkenyl.
  • a lower alkyl or lower alkenyl as described herein is to be taken to have 6 carbon atoms or less in the longest chain of the alkyl or alkenyl group.
  • a lower alkyl or lower alkenyl as described herein may have 5, 4, 3, 2 or 1 carbon atom(s) in its longest chain.
  • alkoxy refers to an alkyl group with an oxygen radical substituent. Examples include methoxy, ethoxy and n-propoxy.
  • lower alkoxy refers to an alkoxy with 6 atoms or less in the longest alkyl chain of the alkoxy group (e.g., 5, 4, 3, 2 or 1 carbon atoms).
  • An alkoxy substituent as described herein will typically be a methoxy group.
  • aryl used either alone or in compound words such as “alkylaryl”, refers to a single, polynuclear, conjugated or fused aromatic hydrocarbon or aromatic heterocyclic ring system. Examples of aryl groups include phenyl and naphthyl.
  • the aromatic heterocyclic ring system can contain one or more heteroatoms independently selected from N, O and S and will normally contain 5 or 6 ring members.
  • an aryl group of a compound embodied by the invention or utilised in a method embodied by the invention will be selected from the group consisting of phenyl, naphthyl and benzyl.
  • halo refers to a halogen atom.
  • the halogen atom is typically selected from the group consisting of F, CI, Br and I.
  • heteroaryl refers to a heterocyclic aromatic ring system.
  • Heterocyclic, heteroaryl, aryl and other ring systems can have from 5 to 7 ring atoms unless specified otherwise, and may contain one or more double bonds. Moreover, the ring or rings may independently be saturated and/or optionally, have one or more substituents.
  • terminally substituted as used herein in the context of an alkyl or alkenyl is meant that the end carbon atom of the longest chain of the alkyl or alkenyl that is remote from the group substituted by the alkyl or alkenyl is substituted.
  • Such computer assisted modelling can involve the provision of an intial structural representation of an inhibitor and electronically modelling it into a representation of the clathrin TD binding site, such as an electron density map of the binding site to determine whether the fit of the putative inhibitor forms the desired complex with the binding site or whether optimisation of the structure of the putative inhibitor is desirable.
  • the designing of an inhibitior of clathrin may also involve modelling the inhibitor to at least partially fit or conform to the structure of a pharmacore or other clathrin inhibitor as described herein.
  • the design and optimisation of the structural features of the putative inhibitor can involve modification of the backbone scaffold of the inhibitor and/or modification (e.g., replacement), exclusion or addition of scaffold substituents to alter the interaction of the inhibitor with the binding site.
  • modification e.g., replacement
  • exclusion or addition of scaffold substituents to alter the interaction of the inhibitor with the binding site.
  • This can take into account steric, lipophilic, and/or charge considerations (e.g., attraction and/or repulsion) to provide for formation of a complex with the clathrin TD in accordance with embodiments of the invention and/or to alter (i.e., increase or decrease) the affinity with which the inhibitor binds to the clathrin TD binding site.
  • the design and modelling of the inhibitor can also take into account the formation of hydrogen bonds between the inhibitor and the clathrin TD wherein the hydrogen bond(s) can be formed directly between the inhibitor and the clathrin TD or be mediated by water, or be a mixture of those two possibilities.
  • the design of compounds and modelling of ligand binding is for instance described in United States Patent Application Publication No. 2004/0219653, US 2009/0275047, US 2005/0170431 and US 2010/0247569, the entire contents of all of which are incorporated herein in their entirety by cross-reference.
  • TD heavy chain of rat clathrin consisting of amino acid residues 1-494 and the methodology for obtaining same has previously been reported (ter Haar et al, 1998), as has the crystal structure for a fragment comprising amino acid residues 1 -363 of the terminal domain TD of clathrin heavy chain respectively complexed with a peptide derived from ⁇ -arrestin 2 or the ⁇ -subunit of the AP-3 complex (the crystal structural data for which is available under ID codes 1C9L and 1C9I from the RBSC Protein Data Bank, Rutgers, the State University of New Jersey, Taylor Road, Piscataway, NJ, USA) (ter Haar, 1998; ter Haar, 2000), reference to which can also be had for the design and provision of clathrin inhibitors in accordance with the invention, and the contents of all of which are also expressly incorporated herein in their entirety by cross-reference.
  • a compound for use in a method of the invention may, for example, be a compound of Formula I, or a prodrug or pharmaceutically acceptable salt thereof, wherein:
  • Ri is alkyl, alkenyl, alkylaryl, aryl, or a ring having from 5 to 7 ring atoms including from 0 to 3 heteroatoms typically selected from O, N or S, wherein the alkyl, alkenyl, alkylaryl, aryl, and ring group are optionally substituted;
  • R 2 is O, S or NH
  • Ri , X, and R 2 form a ring A, the ring having from 5 to 7 ring atoms including from 0 to 3 heteroatoms typically selected from O, N or S and being optionally substituted; and
  • X is N or a carbon atom
  • Y is O, S, or NH
  • ring B and ring C each independently have 5 to 7 ring atoms including from 0 to 3 heteroatoms typically selected from O, N or S; and
  • R 3 , R4, and Rs are each independently H or an optional substituent.
  • Ri is a ring substituent having from 5 to 7 ring atoms
  • the ring may include zero or one or more double bonds.
  • the ring will have 6 ring atoms.
  • the ring will not include any hetero ring atoms.
  • Ri is a lower alkyl, lower alkylaryl, heteroaryl, or aryl group, wherein the lower alkyl, lower alkylaryl or aryl group is optionally substituted.
  • the aryl group may be a heteroaryl group.
  • Ri may be a C ⁇ -Cs alkyl group and typically, a d-C3 alkyl, C2-C3 alkyl, or C1-C2 alkyl.
  • TheC Cs alkyl group when substituted, will typically be terminally substituted.
  • Ri may be a lower alkyl terminally substituted with a ring having from 5 to 7 ring atoms including from 0 to 3 heteroatoms typically selected from 0, N and S. This ring may include zero or one or more double bonds. Typically, this ring has 6 ring atoms.
  • the lower alkylaryl can be a C]-C2 alkylaryl and typically, is a Cj-C 2 alkylphenyl.
  • the C1-C2 alkylphenyl can be unsubstituted.
  • the phenyl ring of the Ci-C 2 alkylphenyl is typically selected from the group consisting of 2-substituted phenyl, 3- substituted phenyl, and 4-substituted phenyl groups. Most typically, the C1-C2
  • alkylphenyl is a benzyl group.
  • Ri is an aryl group
  • the aryl is typically phenyl.
  • the phenyl can be selected from the group consisting of 2-substituted phenyl,
  • Substituents of Ri may, for instance, be independently selected from the group consisting of hydroxyl, sulfhydryl, amino, nitro, halo, cyano, lower alkoxy, lower alkyl, N(CH 3 ) 2) C0 2 H, SO3H, PO4H.
  • Rj may be a C1-C3 alkoxy, C1.C3 alkanol, or a C1-C3 alkylthiol and more usually, a Ci-C 2 alkoxy, C2-C3 alkanol, or C2-C3 alkylthio.
  • R 2 in a compound of Formula I is O.
  • ring A of a compound of Formula I may be substituted with one or more substituents selected from alkyl, alkenyl, alkylaryl and aryl, wherein the alkyl, alkenyl, alkylaryl and aryl substituents are optionally substituted.
  • Each of ring A and ring B of a compound of Formula I may each independently be an aryl group, or a heteroaryl group having from 1 to 3 hetero ring atoms typically selected from O, N and S.
  • rings B and C of Formula I each have 6 ring atoms. Most typically, rings B and C form a naphthyl group wherein the naphthyl group is optionally substituted.
  • a compound of Formula I has the structure of Formula Ila as follows:
  • a compound of Formula I has the structure of Formula lib as follows:
  • Formula lib wherein X, Y, Rj, R 2 , R 3 , R4 and R 5 are as for Formula I.
  • X in a compound of Formula I,IIa or lib is N.
  • Y in a compound of Formula I, Ila or lib is O or S and preferably, is
  • R3 and R» of a compound of Formula I, Ila or lib are independently selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkoxy, alkyl, alkylaryl, aryl, NHR', NR' 2 , C0 2 H, SO3H and PO4H.
  • R3 and R4 of a compound of Formula I, Ila or lib are independently selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkyl, alkylaryl, aryl, NHR', NR' 2 , C0 2 H, S0 3 H and PO4H.
  • R 5 of a compound of Formula lib is selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkoxy, alkyl, alkylaryl, aryl, NHR', NR' 2 , C0 2 H, SO3H and PO4H.
  • R5 of a compound of Formula lib is selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkyl, alkylaryl, aryl, NHR', NR' 2) C0 2 H, - SO3H and PC H.
  • each R' of a compound of Formula I, Ila or lib is independently H, alkyl, alkenyl, aryl, or alkylaryl, wherein the alkyl, alkenyl, aryl or alkylaryl is optionally substituted.
  • At least one of R 3 and R of a compound of Formula I, Ila or lib is other than H.
  • R5 of a compound of Formula Ila is 3 ⁇ 4 aryl, napthyl, benzyl or tetralin, wherein the aryl, napthyl,benzyl and tetralin are optionally substituted.
  • R5 is aryl
  • the aryl group is typically phenyl or napthyl.
  • ring A of a compound of Formula I, Ila and lib has 5 or 6 ring atoms and most typically, 5 ring atoms. Most typically, ring A is an imadazolyl group.
  • ring A has an aryl group substituent, the aryl group being fused with ring A.
  • the aryl group fused with ring A will normally have 6 ring atoms and most typically is a phenyl group.
  • the phenyl group is optionally substituted.
  • ring A when substituted with phenyl forms a benzimidazol group.
  • R5 of a compound of Formula I or Ila is a substituent of Formula III, as follows:
  • G is a bond with ring C
  • ring D has from 5 to 7 ring atoms optionally including from 0 to 3 heteroatoms selected from N, O and S;
  • each W is independently H, halo, nitro, amino, hydroxyl, sulfhydryl, C0 2 H, S0 3 H, or P0 4 H.
  • ring D is a carbocyclic or aryl group. Most typically, ring D has 6 ring atoms.
  • At least one W substituted of Formula III is other than H.
  • R 5 of compound of Formula I or Ila is a substituent group of Formula IV as follows:
  • each W is independently H, C0 2 H, S0 3 H, or P0 4 H.
  • a compound for use in a method embodied by the invention may be a compound of Formula V, or a prodrug or pharmaceutically acceptable salt thereof, wherein:
  • X is O, S, or NH
  • Y is O, N or a carbon atom
  • Z is O, S or NH
  • Ri is H, C0 2 H, S0 3 H, P0 H, alkyl, alkenyl, alkylcarboxy, or alkylaryl;
  • R' is H, alkyl, alkenyl, alkylaryl, aryl, the alkyl, alkenyl, alkylaryl and aryl group being optionally substituted;
  • R 3 is aryl or a polycyclic group having at least 2 fused rings each independently having from 5 to 7 ring atoms, the aryl and the polycyclic group being optionally substituted.
  • Rj in a compound of Formula V is H or a lower alkyl or lower alkenyl, wherein the lower alkyl or lower alkenyl group is optionally substituted and generally, terminally substituted.
  • the lower alkyl is typically a C1-C3 alkyl and more preferably, a C1-C2 alkyl.
  • the lower alkyl is terminally substituted, preferably with a carboxy or thiocarboxy group (e.g., a lower alkylcarboxy or lower alkylthiocarboxy).
  • Substituents of R ⁇ in a compound of Formula V may for instance, be selected from the group consisting of hydroxyl, sulfhydryl, amino, nitro, halo, cyano, C0 2 H, S0 3 H and P0 4 H.
  • R' is alkyl, alkenyl or aryl, wherein the alkyl, alkenyl or aryl is optionally substituted.
  • the lower alkyl and alkenyl are lower alkyl and lower alkenyl, respectively.
  • R 3 of Formula V is phenyl or an aryl group with
  • R 3 of Formula V is a polyclyclic group
  • R 3 will typically have 2 fused rings at least one of which is an aryl group having 6 ring atoms.
  • each ring of the aryl group has 6 ring atoms.
  • R 3 of Formula V may, for example, be optionally substituted and selected from the group consisting of phenyl, napthyl, benzopyranyl, quinolinyl, quinozalinyl, quinozolinyl, cinnolinyl, benzofuranyl, isobenzofuranyl, indolyl, isoindolyl,
  • benzothiophenyl benso[c]thiophenyl, benzimidazolyl, purinyl, indazolyl, benzoxazolyl, benzoisoxazolyl, and benzothiazolyl.
  • the R 3 group is selected from phenyl, napthyl, and benzopyranyl (e.g., benzopyran-4-one).
  • R 3 is phenyl
  • the phenyl in a compound of Formula V, the phenyl may be mono, di, tri or tetra-substituted (e.g., trihydroxy phenyl or tetrahydroxy phenyl).
  • Suitable such substituents may, for example, be selected from the group of substituents as defined for M in Formula Va below.
  • a compound of Formula V has the structure of Formula Va as follows:
  • each M is independently selected from H, halo, hydroxy, sulfhydryl, nitro, amino, cyano, C0 2 H, S0 3 H, P0 4 H, OR', NHR', NR' 2 , S0 2 R ⁇ S0 2 NH 2 , wherein R' is as defined as Formula II.
  • at least one M substituent is other than hydrogen.
  • R' is a lower alkyl or lower alkenyl, the lower alkyl or alkenyl being optionally substituted.
  • the alkyl or alkenyl group is optionally terminally substituted.
  • the terminal substituent of R' may, for example, be selected from the group consisting of amino, nitro, halo, cyano, hydroxy, sulfhydryl, and typically, is amino.
  • the phenyl group shown in Formula Va is selected from the group consisting of 4-substituted phenyl, and 3, 5 -substituted phenyl groups.
  • a compound of Formula Va may have the structure of Formula Vb as follows:
  • M in a compound of Formula Vb is halo, nitro or -OR'NH 2 , wherein R' is a lower alkyl or lower alkenyl.
  • R' is typically a C1-C4 alkyl and preferably, a C3 alkyl.
  • a compound of Formula V may have the structure of Formula V- la as follows:
  • G is O or S
  • T is O, S, N or a carbon atom
  • each M is independently as defined for Formula Va.
  • At least one M substituent of a compound of Formula V-la is other than hydrogen.
  • At least one M in a compound of Formula V-la may be halo, nitro or -OR'NFb, wherein R' is a lower alkyl or lower alkenyl, and -OR'NH 2 is typically a C 1 -C4 alkyl ⁇ and preferably, a C3 alkyl.
  • a compound of Formula Vl-a has the structure of Formula V-lb as follows:
  • T in a compound of Formula V-la or V-lb is O, S or N.
  • the dashed line(s) in Formula I to Formula V-lb above indicate a bond depending on the applicable atom(s) and/or substituent(s) involved.
  • Clathrin inhibitory activity of compounds may be determined utilising methodology and assays described herein.
  • compounds useful as clathrin inhibitors in embodiments of the invention can be identified utilising compound library screening strategies, such strategies being well known to a person skilled in the art.
  • the compounds indentified as LI and L2 in Table 1 below were identified utilising this technique and were validated by assaying for selective inhibition of clathrin TD- amphiphysin association as described herein
  • Reagents and conditions (a) HNO3, H 2 S0 4 , 17 hr; (b) (i) Fuming sulfuric acid (30 % S0 3 ), 90 °C, 30 min; (ii) Saturated KC1, 4 °C, 18 hr; (c) Cone.
  • H 2 S0 4 (95-98 %), cone HNO3 (70 %), 60 °C, 90 min;
  • Fuming sulfuric acid (oleum), 40-50°C, 3 hr; b. Kcl (sat), 4°C, 12 hr; (iii) RNH2, EtOH, 18 hr.
  • Clathrin inhibitors according to the invention may be prepared by any conventional techniques known to a person skilled in the art, including by organic synthesis strategies, solid phase-assisted techniques or by commercially available automated synthesizers. Alternatively, conventional recombinant techniques alone or in combination with conventional synthetic approaches can be utilised.
  • the clathrin inhibitors as described herein may be used for in vitro or in vivo diagnostic, prophylactic or therapeutic purposes.
  • Clathrin-mediated endocytosis is important for many cell trafficking and cell signaling pathways, and the inhibition of clathrin function has application in the prophylaxis or treatment of diverse diseases and conditions. It is known, for example, that endocytosis is a major contributor or direct cause of diverse human diseases and a list of vesicle trafficking-specific diseases has been published, see for example Aridor and Hannan (2000)-, and Aridor and Hannan (2002), the contents of which are incorporated herein by reference in their entirety. In particular, clathrin is involved in trafficking from the cell surface, and also plays a role in trafficking or fission events from the Golgi apparatus and endosomes.
  • Alzheimer's disease In the brain, diseases and conditions in which endocytosis plays a role include Alzheimer's disease.
  • AD Alzheimer's disease
  • APP ⁇ -amyloid precursor protein
  • the endosome is the first compartment along the CME pathway after internalization of APP or ApoE, and endosomal alterations are evident in pyramidal neurons in Alzheimer brain. Endocytic pathway activation is prominent in APP processing and ⁇ -amyloid formation and is an early feature of neurons in vulnerable regions of the brain in sporadic Alzheimer's disease.
  • Another example of disease involving CME relates to presynaptic synuclein protein which is a prime candidate for contributing to Lewy body diseases, including Parkinson's disease, Lewy body dementia and a Lewy body variant of AD. Exogenous synuclein causes neuronal cell death due to its endocytosis and formation of
  • polycystic kidney disease is yet another example of a disease involving endocytosis of ClC-5 chloride channel and endocytosis blockers are known to prevent its internalisation. Indeed, the prophylaxis or treatment of any physiological disorder amendable to the inhibition of CME as described herein is expressly encompassed by the invention. It is known, for instance, that human polyomavirus JCV is the etiologic agent of progressive multifocal leukoencephalopathy, a fatal central nervous system (CNS) demyelinating disease and its entry to neurons is blocked by endocytosis inhibitors such as chlorpromazine.
  • CNS central nervous system
  • the nerve terminal is an electrically excitable structure which maintains a net negative membrane potential so that depolarization can activate voltage-sensitive Ca 2+ channels to trigger exocytosis. Epilepsy results in the sustained and uncontrolled exocytosis from these terminals.
  • AEDs have been discovered using similar traditional methodologies - screening of compounds in animal models of acute provoked seizures. Possibly as a result, most AEDs have similar principal mechanisms of action, such as inhibiting voltage gated sodium channels and/or enhancing GABAergic activity.
  • a rare example of a new AED acting via a novel mechanism is levetiracetam ( eppraTM) which inhibits the putative SV refilling protein SV2A and inhibits exocytosis.
  • SVE synaptic vesicle endocytosis
  • Some SV cycle genes cause epilepsy in humans (e.g., synapsin), or epileptic-like seizures when genetically knocked-out in animals (e.g., SV2, amphiphysin or synaptojanin).
  • a ubiquitous mechanism to stop a seizure is to block synaptic mechanisms that are associated with pathologically sustained neuronal burst firing.
  • a reduction in synaptic transmission is the common feature of all anti-epileptic drugs (AEDs). For most AEDs the mechanistic basis of this reduction is uncertain.
  • AEDs anti-epileptic drugs
  • inhibition of clathrin mediated SVE by blocking clathrin may lead to an activity-dependent run-down in synaptic transmission reducing the availability of SVs to sustain or propagate a seizure.
  • SVE is the rate limiting step in synaptic
  • Targeting clathrin in accordance with an embodiment of the invention may therefore provide a number of advantages over standard therapy.
  • conventional AEDs reduce synaptic transmission at all times, but a clathrin inhibitor may only exert effect at high frequency or after sustained stimulation that is, under conditions associated with a seizure.
  • Inhibiting clathrin mediated SVE may also limit the effect to overactive neurons and thus have reduced side-effects by allowing physiological neurotransmission to occur unimpeded.
  • CME mediated pathways are also utilized by many pathogenic agents such as viruses, toxins and symbiotic microorganisms to gain entry into cells.
  • pathogenic agents such as viruses, toxins and symbiotic microorganisms to gain entry into cells.
  • botulism neurotoxins and tetanus neurotoxin are bacterial proteins that inhibit transmitter release at distinct synapses and cause two severe neuroparalytic diseases, tetanus and botulism. Their action is dependent on their internalisation via endocytosis into nerve terminals.
  • SAC spindle assembly checkpoint
  • Kinetochores are a large protein assembly that mediates the attachment of chromosomes to spindle microtubules (MTs). These microtubules are known as kinetochore fibres (K-fibres). Clathrin in its triskelia form binds to the mitotic spindle to provide a structural lattice.
  • Blocking CME prevents cell proliferation in many of these examples and provides further evidence of the anti-cancer and ant-proliferative activity of clathrin inhibitors.
  • Inhibition of CME may also have application to the prophylaxis or treatment of pain.
  • neuropathic pain typically develops when peripheral nerves are damaged through surgery (including spine surgery), bone compression in cancer, diabetes, viral infection (including shingles or HIV infection), AIDS, alcoholism, amputation, chemotherapy, facial nerve problems, or multiple sclerosis, and is a major factor causing impaired quality of life for millions of people worldwide.
  • Anticonvulsant drugs such as phenytoin and gabapentin are highly efficacious in treating neuropathic pain. These drugs act through modulation of synaptic vesicle transmission indicating the potential of the inhibition of clathrin mediated SVE in the treatment of this debilitating condition. That is, inhibition of synaptic vesicle endocytosis may halt or limit pain signalling and thereby reduce or ameliorate the sensation of pain experienced.
  • examples of specific diseases and conditions for which methods of the invention find application for the prophylaxis or treatment of include, but are not limited to, cell proliferative diseases and conditions, multifocal leukoencephalopathy, polycystic kidney disease, ⁇ -amyloid associated diseases, Alzheimer's disease, neurodegenerative disorders, neuropsychiatric disorders, psychotic disorders, psychosis, bipolar disorders, schizophrenia, aberrant up-regulated neuronal excitation, seizures, neuropathic pain, migraine, mucolipidosis, cell proliferative diseases and conditions, neurological, diseases responsive to inhibition of clathrin, and diseases and conditions mediated or otherwise associated with synaptic signal transmission, CME or SVE (e.g., such as epilepsy), or cell vesicle trafficking.
  • CME or SVE e.g., such as epilepsy
  • Cell proliferative diseases and conditions that may be treated in accordance with embodiments of the invention include cancer, skin conditions such as psoriasis and scleroderma, benign growths, and cardiovascular diseases and conditions including atherosclerosis.
  • the cancer may, for instance, be selected from the group consisting of carcinomas, sarcomas, lymphomas, leukaemias, and cancer of the liver, tongue, mouth, oropharynx, nasopharynx, gastrointestinal tract, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin (e.g., melanoma and basal cell carcinoma) amongst others.
  • the inhibition of CME in accordance with one or more embodiments of the invention may also be useful in inhibiting entry of a range of pathogenic agents into cells and so be useful in the prophylaxis or treatment of diseases or conditions associateded with this, non-limiting examples of which include nerve viral infection, botulinum neurotoxin A (BoNT/A) and C2 toxin from Clostridium botulinum (botulism), Toxin A (TcdA) and B (TcdB) from Clostridium difficile (antibiotic- associated diarrhoea), a-toxin (TcnA) from Clostridium novyi (necrosis, oedema), Lethal toxin (TcsL) from Clostridium sordellii, (toxic shock syndrome, sepsis), Tetanus neurotoxin (TeNT) from Clostridium Tetani (tetanus), diphtheria toxin from
  • Corynebacterium diphtheriae diptheria
  • leukotoxin LLT
  • DNT Dermonecrotic toxin
  • Chlamydia psittaci Chlamydia trachomatis
  • Anthrax toxin from Bacillus anthracis (Anthrax), Listeria monocytogenes (Listeriosis), Yersinia pseudotuberculosis (Pseudotuberculosis (yersinia)), Staphylococcus aureus (Golden staph infections), Candida albicans (candidiasis), Porphyromonas gingivalis (periodontitis), Uropathogenic Escherichia coli (urinary tract infection), Adeno- associated virus, Adenovirus (Respiratory infection
  • papillomavirus BP VI Carcinomas/ haemangio-endotheliomas of the bladder
  • Human papillomavirus 31 HPV31
  • 16 HPV16
  • Canine parvovirus Poliovirus
  • HRV Human rhinovirus 2
  • Rotavirus gastroenteritis
  • Semliki forest virus SFV
  • Semliki forest virus SFV
  • Simian virus 40 SV-40
  • VSV Vesicular stomatitis virus
  • VSV Vesicular stomatitis virus
  • the above list is to be taken as only indicative only of the pathogenic agents for which compounds may be administered in accordance with embodiments of the invention. More generally, further examples include for instance, so-called "zippering bacteria" which bind to cellular receptors and invade cells in a clathrin- dependent manner such as enteropathogenic E. coli bacteria, and reovirus.
  • Suitable pharmaceutically acceptable salts include acid and amino acid addition salts, base addition salts, esters and amides that are within a reasonable benefit/risk ratio, pharmacologically effective and appropriate for contact with mammalian tissues without undue toxicity, irritation or allergic response.
  • Representative acid addition salts include hydrochloride, sulfate, bisulfate, maleate, fumarate, succinate, tartrate, tosylate, citrate, lactate, phosphate, oxalate and borate salts.
  • Representative base addition salts include those derived from ammonium, potassium, sodium and quaternary ammonium hydroxides.
  • Suitable salts also include alkali metal and alkali earth cation salts such a sodium, calcium, magnesium, and potassium salts, as well as ammonium and amine cation salts.
  • alkali metal and alkali earth cation salts such as sodium, calcium, magnesium, and potassium salts, as well as ammonium and amine cation salts.
  • Suitable pharmaceutical salts are for example exemplified in S. M Berge et al, J. Pharmaceutical Sciences ( 1997), 66 : 1 - 19, the contents of which is incorporated herein in its entirety by cross-reference.
  • Prodrugs of compounds in embodiments of the invention include those in which groups selected from carbonates, carbamates, amides and alkyl esters have been covalently linked to free amino, amido, hydroxy or carboxylic groups of the compounds. Suitable prodrugs also include phosphate derivatives such as acids, salts of acids, or esters, joined through a phosphorus-oxygen bond to a free hydroxl or other appropriate group.
  • a prodrug can for example be inactive when administered but undergo in vivo modification into the active compound that binds to clathrin such that the function of clathrin is inhibited as a result of cleavage or hydrolysis of bonds or other form of bond modification of the compound post administration.
  • the prodrug form of the active compound can have greater cell membrane permeability than the active compound thereby enhancing potency of the active compound.
  • a prodrug can also be designed to minimise premature in vivo hydrolysis of the prodrug external of the cell such that the cell membrane permeability characteristics of the prodrug are maintained or enhanced for optimum availability to cells and for systemic use of the compound.
  • Esterified prodrugs may for instance be provided by stirring a compound embodied by the invention with an appropriate anhydride or acid chloride (in molar excess) in a pyridine/N,N-dimethylformamide (DMF) solution in the presence of a suitable catalyst such as dimethylaminopyridine (DM AP).
  • a suitable catalyst such as dimethylaminopyridine (DM AP).
  • the solution may need to be refluxed to drive the reaction to completion.
  • the esterified product is purified by either recrystallization or by
  • esters include Q-C7 alkyl, phenyl and phenyl(Ci-6) alkyl esters. Preferred esters include methyl esters.
  • a clathrin inhibitor can be administered to a mammal in alone or be co-administered with one or more other therapeutic compounds or drugs conventionally used for treating the applicable disease or disorders.
  • co-administered is meant simultaneous administration in the same formulation or in two different formulations by the same or different routes, or sequential administration by the same or different routes, wherein the administered drugs have overlapping therapeutic windows.
  • sequential administration is meant one is administered after the other.
  • the terminal domain (TD) of clathrin is highly conserved between mammalian species.
  • the amino acid sequence is conserved 100% (i.e. all amino acids are identical) between the terminal domain of bovine, human, rat, mouse, pig, and dog clathrin.
  • methods and clathrin inhibitors in accordance with the invention have particular application to inhibiting the activity of human clathrin, the invention is not limited thereto and extends to inhibiting clathrin activity of other mammals including but not limited to, bovine, ovine, porcine, rodent (e.g., mouse, rat, guinea pig), canine, feline, and primate clathrin.
  • a clathrin inhibitor as described herein will generally be formulated into a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions include sterile aqueous solutions suitable for injection and sterile powders for the extemporaneous preparation of injectable solutions. Such injectable compositions will be fluid to the extent that syringability exists. Injectable solutions will typically be prepared by incorporating the active(s) in the selected carrier prior to sterilising the solution by filtration. In the case of sterile powders, preferred methods of preparation are vacuum drying and freeze- drying techniques which yield a powder of the active and any additional desired ingredient from previously sterile filtered solutions thereof.
  • a pharmaceutical composition in accordance with the invention can be provided in a form wherein the components of the composition are admixed with one another.
  • the clathrin inhibitor(s) can be provided partially or totally seperately for combination with other components to form the composition, such as in the form of a kit.
  • the clathrin inhibitor can be formulated into any orally acceptable carrier deemed suitable.
  • the inhibitor can be formulated with an inert diluent, an assimilable edible carrier or it may be enclosed in a hard or soft shell gelatin capsule.
  • a clathrin inhibitor can be provided in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions or syrups.
  • Clathrin inhibitors as described herein can also be formulated into topically acceptable preparations including creams, lotions or ointments for internal or external application. Topically acceptable compositions can be applied directly to the site of treatment including by way of dressings and the like impregnated with the preparation.
  • a pharmaceutical composition embodied by the invention can also incorporate one or more preservatives such as parabens, chlorobutanol, phenol, and sorbic acid.
  • prolonged absorption of the composition may be brought about by the inclusion of agents for delaying absorption such as aluminium monosterate.
  • Tablets, troches; pills, capsules and like can also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatine, a disintegrating agent such as corn starch, potato starch or alginic acid, a lubricant such as magnesium sterate a sweetening agent such as sucrose, lactose or saccharin; and a flavouring agent.
  • Pharmaceutically acceptable carriers include any suitable conventionally known physiologically acceptable solvents, dispersion media, isotonic preparations and solutions including for instance, physiological saline. Use of such ingredients and media for pharmaceutically active substances is well known. It is particularly preferred to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Clathrin inhibitors as described herein can also be provided in dosage unit form.
  • a dosage unit form as used herein is to be taken to mean physically discrete units, each containing a predetermined quantity of the selected compound(s) calculated to produce a therapeutic or prophylactic effect. When the dosage unit form is a capsule it can contain the active in a liquid carrier.
  • Various other ingredients may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugars or both.
  • compositions embodied by the invention will generally contain clathrin inhibitors in a range of from about at least 0.1% by weight up to about 80% w/w of the composition.
  • the amount of the clathrin inhibitor(s) in the composition will be such that a suitable effective dosage will be delivered to the individual taking into account the proposed mode of administration.
  • Preferred oral compositions will contain between about 0.1 ⁇ g and 4000 mg of a clathrin inhibitor.
  • the dosage of clathrin inhibitor(s) administered will depend on a number of factors including whether the inhibitors are to be administered for prophylactic or therapeutic use, the disease or condition for which the active is intended to be administered, the severity of the condition, the age of the individual, and related factors including weight and general health of the individual as may be determined in accordance with accepted medical principles. For instance, a low dosage may initially be given which is subsequently increased at each administration following evaluation of the individual's response. Similarly, frequency of administration can be determined in the same way that is, by continuously monitoring the individual's response between each dosage and if necessary, increasing the frequency of administration or
  • a clathrin inhibitor as described herein will be administered at a dosage up to about 50 mg/kg body weight and preferably, in a range of from about 5 Mg kg to about 100 Mg/kg body weight.
  • Routes of administration include but are not limited to respiritoraly, intravenously, intraperitonealy, subcutaneously, intramuscularly, by infusion, orally, rectally, topically, by implant, or by other conventionally employed methods of administration.
  • intravenous routes particularly suitable routes are via injection into blood vessels which supply the target tissue to be treated.
  • Suitable pharmaceutically acceptable carriers and formulations useful in compositions of the present invention may for instance be found in handbooks and texts well known to the skilled addressee, such as "Remington: The Science and Practice of Pharmacy (Mack Publishing Co., 1995)", the contents of which is incorporated herein in its entirety by reference.
  • the animal treated by a method embodied by the invention will typically be a mammal such as a member of the bovine, porcine, ovine or equine families, a laboratory test animal such as a mouse, rat, rabbit, guinea pig, cat or dog, or a primate or human being as outlined above.
  • a mammal such as a member of the bovine, porcine, ovine or equine families
  • a laboratory test animal such as a mouse, rat, rabbit, guinea pig, cat or dog, or a primate or human being as outlined above.
  • non-mammalian species may also be treated with clathrin inhibitors as described herein for research or other purposes in accordance with the invention.
  • Patent stop is a word coined for use as a trademark by the Applicants in respect of chemical compounds and its use whether alone or in coined words such as "Pitstop 1" and "Pitstop 2" is expressly reserved for use by the Applicants for chemical compounds.
  • clathrin X22, Affinity Bioreagents
  • AP-2 a-adaptin, Abeam
  • intersectin 1 S750; kindly provided by Dr. Thomas Siidhof, Howard Hughes Medical Institute, Stanford University, Palo Alto, USA
  • FCHo 1/2 kindly provided by Dr. Harvey McMahon, MRC Laboratory of Molecular Biology, Cambridge, U.K.
  • dynamin 1/2/3 Hudy-1, Upstate
  • EEA1 BD transduction
  • Gadkin Schomidt et al., 2009
  • transferrin receptor Zymed
  • TGN46 Seroton-6-phosphate receptor
  • GM130 BD transduction
  • mannose-6-phosphate receptor CI-M6PR
  • Fusion proteins encoding GST-clathrin heavy chain TD (amino acids 1-364), GST-AP-2 alpha-appendage (amino acids 702-938), and GST-AP-1 gamma-appendage (amino acids 690-822) were in pGEX4T-l expression vectors and His 6 -amphiphysin 1 (250-578), Hise-gadkin (amino acids 52-302), and His6-stonin 2 (amino acids 1-242) were in pET28a.
  • Expression plasmids were transformed into Escherichia coli BL21 -Codon PlusTM (DE3)-RP competent cells (Stratagene). His 6 - or GST-tagged fusion proteins were expressed and purified using HIS-SelectTM nickel affinity gel (Sigma) or GST Bind® resin (Novagen) following the manufacturer's instructions.
  • Purified Hise-tagged protein was diluted into screening buffer (20 mM HEPES pH 7.4, 50 mM NaCl, 1 mM DTT, 1 mM PMSF), added to a 384 well ELISA plate (high-binding PS Microplate, Greiner Bio-One) and bound for 1 h at RT. Non-specific binding was prevented by addition of 50 ⁇ blocking buffer (20 mM HEPES pH7.4, 50 mM NaCl, 1 mM DTT, 1 mM PMSF, 2 % BSA, 2.5 % milk) followed by incubation overnight at 4°C.
  • Cells (5xl0 5 ) were grown in 10 cm dishes. Following inhibitor treatment, cells (floating and adherent) were collected and single-cell suspensions were fixed in 80% ice-cold ethanol at -20°C for at least 16 h. Cells were stained with propidium iodide and the cell cycle was analyzed as described previously (Joshi et al., 2010). Cell cycle profiles were acquired with a FACS Canto Flow Cytometer (Becton Dickinson) using FACS Diva software (v5.0.1) at 488 nm; Cell cycle profiles were analyzed using Flow Jo software (v7.1 ).
  • Cells were seeded in 10 cm dishes (lxlO 5 cells/dish). On day 0 (24 h after seeding), cells in triplicate were treated in the presence or absence of pitstops at concentrations of 1 , 3, 10 and 30 ⁇ . After 20 h, the cell number and viability were measured using a Vi-CELL XR cell viability analyzer as previously described (Joshi et al., 2010).
  • LDH lactate dehydrogenase
  • Growth inhibitory assays were carried out using an MTT assay.
  • Cells in logarithmic growth were transferred to 96-well plates (100 ⁇ medium/well) at a density of 2500 cells/well for HeLa, HT29, H460, A431 , and DU 145 cells, 3000 cells/well for SW480, 3500 cells/well for MCF7, BE2-C and SJ-G2, and 2000 cells/well for A2780.
  • cytotoxicity and growth inhibitory effects were evaluated using the MTT (3-[4,5-dimethyltiazol-2-yl] 2,5-diphenyl-tetrazolium bromide) assay.
  • GI50 values were calculated from the MTT dose response curve from three independent experiments, each performed in duplicate. GI50 is the drug concentration at which cell growth is inhibited by 50% based on the difference between the optical density values on day 0 and those at the end of drug exposure. 1.9 TUNEL assay
  • Cos7 cells (80% confluent) were grown in plastic dishes. Growth media were replaced by optiMEM with 0, 1 % DMSO or 30 ⁇ pitstop 2 for 10 min. Cells were fixed with 2% glutaraldehyde (GA) in PBS. After scraping and pelleting, subsequent electron microscopy preparation and morphometric analysis were performed as previously described (Ferguson et al., 2009). Briefly, after epoxy resin embedding and sectioning micrographs were taken along the cell perimeter at x20000. Images were combined in order to reconstruct cell perimeter and numbers of clathrin-coated intermediates were estimated.
  • Rat primary neuronal cultures (P 1 ; DIV 14-21 ) were assayed either in incubation medium (resting conditions) or high potassium solution.
  • Incubation medium contained (concentration in mM, pH 7.4): 170 NaCl/ 3.5 KC1/ 0.4 KH 2 P0 , 20 TES(N- tris[hydroxyl-methyl]-methyl-2-aminoethane-sulfonic acid), 5 NaHC0 3 / 5 glucose/ 1,2 Na 2 S04/ 1.2 MgCl 2 and 1.3 CaCl 2 ; at pH 7,4; high potassium solution was identical to the above but containing 120 mM NaCl plus 50 mM KC1.
  • Reagents were introduced into borosilicate glass injection microelectrodes and microinj ected into the reticulospinal axons with pressure pulses (5 - 15 psi) of 200 ms duration. Microinjections were monitored with a CCD camera (Roper Scientific, Arlington, AZ) or (Princeton Instruments, Trenton, NY) connected to a standard fluorescence upright microscope (Olympus BHX-50) with lOx and 40x water- immersion objectives. Membrane potentials of the impaled axons were monitored throughout injections.
  • the number of synaptic vesicles (SVs) and coated pits and the membrane length of pockets or invaginations (curvature index) in the endocytic zone in injection experiments were determined from middle sections of at least five serially cut synapses.
  • the values for the numbers of SVs and coated pits were normalized to the length of the active zone as the number of SVs in the cluster is proportional to the length of the active zone in the reticulospinal synapse.
  • the length of endocytic zone membrane invaginations and pockets was measured using the NIH ImageJ software.
  • GST-clathrin heavy chain TD was purified by GSH affinity chromatography (Qiagen).
  • the GST-tag was cleaved off by thrombin and separated from TD via GST affinity chromatography.
  • the cleaved TD was further purified by size exclusion chromatography using a Superdex S200 column (Amersham Biosciences) in 10 mM Tris, pH 7.5, 50 mM NaCl, and 4 mM dithiothreitol, concentrated up to 22 mg/ ml (-0.5 mM), and supplied with a 4-fold molar surplus of pitstops 1 or 2.
  • Crystals were grown at 18°C using the sitting drop vapor diffusion method.
  • the reservoir solution contained 20% PEG 3350, 150 mM potassium acetate, and 0.1 M Tris, pH 8.0, and drops were prepared by mixing 2 ⁇ of reservoir and 2 ⁇ of protein solution. Crystals formed within a few minutes and reached their final size in 1-2 days at 18 °C.
  • the crystals were soaked briefly in a cryoprotection solution that was prepared by increasing the PEG 3350 concentration up to 40 % in 150 mM potassium acetate, and 0.1 M Tris, pH 8.0. Crystals were then mounted in nylon loops, and flash- cooled in liquid N 2 .
  • X-ray data were collected at Beamline BL1 at BESSY-II, Berlin, and processed using XDS (Kabsch, 2010) and Xscale.
  • the phase problem was solved by molecular replacement using the CCP4 program MOLREP (Collaborative
  • Cos7 cells seeded on matrigel-coated glass coverslips were preincubated with 30 ⁇ pitstop 2 or DMSO (0.1%) in DMEM/ 0.1 % FCS/ 10 mM HEPES for 15 min, fixed in 4% PFA or methanol, and stained with antibodies as previously described (Schmidt et al., 2009). Cells were imaged using spinning disc confocal or total internal reflection microscopy (TIRFM). Pearson's correlations were obtained using Volocity software (Impro vision).
  • Cos7 cells stably expressing eGFP-clathrin LC
  • FCHo2-eGFP transiently expressing FCHo2-eGFP
  • AP-2o-eGFP transiently expressing FCHo2-eGFP
  • dynamin2-eGFP were used.
  • CCP dynamics were imaged by TIRFM (Visitron) under the control of Slidebook 5 (3i Inc). Time series of 2 min (at 0.5 Hz) were acquired 5 min after addition of 30 ⁇ pitstop 2 or DMSO (0.1%) in HBSS media supplemented with 0.1 % FCS and 10 mM HEPES (pH 7.4) at 37°C. For lifetime analysis events were tracked manually.
  • Fluorescence recovery after photobleaching (FRAP) experiments were performed using a spinning disc confocal microscope (Perkin Elmer) controlled by Volocity (Improvision). Cells were imaged for 12 s, then bleached in a region of 15 x 15 ⁇ , and imaged for an additional 120 s at 0.5 Hz. Fluorescence recovery was analyzed by defining CCPs in the FRAP region before bleaching, followed by measuring the fluorescence intensity in the FRAP region over time. Intensity values were corrected for photobleaching in a non-bleached control area. 1.17. Ligand internalization and surface staining
  • HeLa or Cos7 cells seeded on MatrigelTM-coated glass coverslips were starved (2 h) before incubation with 20 ⁇ g Alexa 488 568 -transferrin or 100 ng Alexa 595/488 -EGF for 15 min at 37°C. Washed cells were fixed in 4 % PFA, 4 % sucrose in PBS, pH 7.4 and processed for epifluorescence microscopy. Fluorescence levels were quantified using a Zeiss Axiovert200M fluorescence microscope under the control of Slide Book5 software (3i Inc., Gottingen, Germany).
  • HeLa cells were seeded on glass cover slips and starved overnight. The cells were washed once with ice-cold PBS and then incubated at 4°C for 30 min with
  • TZM-bl cells (Wei et al., 2002) were plated in 96 well plates and incubated for
  • Crystal structural data for the terminal domain of clathrin respectively complexed with pitstop 1 and pitsop 2 was deposited with the RCSB Protein Data Bank, Rutgers, the State University of New Jersey, Taylor Road, Piscataway, NJ, USA on 25 November 2010 under PDB codes 2xzg (for pitstop 1) and 2xzh (for pitstop 2), reference to which can be had for the design and provision of clathrin inhibitors in accordance with the invention and the contents of all of which is expressly incorporated herein in its entirety by cross-reference.
  • the crystal data for pitstop 1 and pitstop 2 complexed with the terminal domain of clathrin is also set out in Figures 13 and 14. 2. Identification and characterisation of the clathrin inhibitors pitstop 1 and pitstop 2 and their binding to the clathrin terminal domain
  • Clathrin function in mammalian cells has mainly been evaluated via two approaches: knockdown of clathrin or expression of dominant-negatively acting clathrin-binding fragments of endocytic accessory proteins. While both of these strategies have shown that clathrin is essential for CCP formation or stability, the physiological role of endocytic ligand association of its TD remains unclear. This lack of knowledge is surprising as clathrin box-TD interactions form a major hub within the endocytic network. It has been suggested that multiple redundant interactions of clathrin TD with its endocytic ligands may serve to stably recruit clathrin to
  • clathrin assembled at nascent endocytic sites could serve as an organizing scaffold that regulates CCP dynamics by providing spatially defined binding sites on its TD for accessory proteins that drive CCP maturation and disassembly.
  • amphiphysin an endocytic protein harboring a clathrin box motif (Slepnev et al., 2000; Dell' Angelica, 2001)
  • amphiphysin an endocytic protein harboring a clathrin box motif (Slepnev et al., 2000; Dell' Angelica, 2001)
  • clathrin box motif Slepnev et al., 2000; Dell' Angelica, 2001
  • pitstops 1 and 2 Based on subsequent studies and their ability to impair CCP function (see below), these compounds were named pitstops 1 and 2 (see Tables 2 and 3 above for the chemical structures of these compounds). Pitstops 1 and 2 selectively inhibited ligand association of clathrin TD with IC50 values of 18 ⁇ and 12 ⁇ , respectively. Neither
  • the structure of the clathrin TD in complex with pitstops by protein X-ray crystallography was determined to understand the molecular basis of clathrin TD inhibition by pitstops.
  • Clathrin TD-containing crystals diffracted up to a resolution of 1.7 A and the structure was solved by molecular replacement using the clathrin TD as a search model (see Table 4).
  • inhibitor molecules in complex with clathrin TD could be identified and modelled into the determined electron densities
  • hydrophobic contacts mediated by hydrogen bonds and by hydrophobic contacts. Hydrogen bonds are indicated by dashed lines between the atoms involved, while hydrophobic contacts are represented by an arc with spokes radiating towards the atoms of the relevant pitstop compound that they contact. The contacted atoms are shown with spokes radiating back. As shown, pitstop 1 makes direct contact with He 52, He 62, He 66,
  • He 80 lie 93 , Phe 91, Arg 64, Leu 82 and Lys 96 of the clathrin TD, whilst pitstop 2 makes direct contact with He 93, He 80, Val 50, He 52, He 62 and Phe 91.
  • the overall structures of clathrin TD in complex with either pitstops 1 or 2 are shown in Fig. 4A, C.
  • the clathrin TD adopts a WD40-like structure comprised of a seven-bladed ⁇ -propeller each with four antiparallel strands (ter Haar et al., 2000). Both pitstop inhibitors bind the interface between the first and second blades
  • Fig. 4B, D at a site overlapping with that used by clathrin bo -containing accessory proteins (Fig. 4E-F), which bind to the TD with low affinity (Brodsky et al., 2001 ;
  • Pitstop 1 lies in a hydrophobic cavity formed by four isoleucines (52, 62, 80, 93), Leu 82, and Phe 91. Its conformation is stabilized by five hydrogen bonds at both ends of pitstop 1 (see Table 5 below). Only one direct hydrogen bond is formed between the sulfonate group of pitstop 1 and the ⁇ -amino group ( ⁇ 2 atom) of Gln82. All other hydrogen bonds are mediated by water. Comparison of ligand-free clathrin TD with the pitstop 1 -bound form shows that two residues in the clathrin box-binding site undergo major conformational changes upon ligand binding.
  • Alexa -Tf HeLa cells efficiently internalized Tf into perinuclear recycling endosomes.
  • Preincubation of HeLa cells with pitstop 2 led to a dose-dependent inhibition of Tf uptake with an IC50 value (12-15 uM) very similar to that measured for blocking clathrin TD function in vitro (Fig. 5 A, B).
  • IC50 value (12-15 uM) very similar to that measured for blocking clathrin TD function in vitro (Fig. 5 A, B).
  • IC50 value (12-15 uM) very similar to that measured for blocking clathrin TD function in vitro
  • Application of 30 uM pitstop 2 completely blocked Tf endocytosis, similarly to what has been observed in clathrin knockdown cells.
  • Pitstop 2-induced block of Tf endocytosis in HeLa cells was completely reversed within 1-3 h of drug washout.
  • Treatment with similar concentrations of pitstop 2 also blocked Tf uptake in other cell types including Cos7 and BSCl cells, astrocytes or primary neurons.
  • Pitstop 2 also caused a potent inhibition of EGF uptake (Fig. 5C). This is consistent with the previously suggested notion that the majority of EGF is endocytosed via a clathrin- mediated entry route in most cell types. Pitstop 1 was found to display comparably low cell membrane penetration but exhibited qualitatively similar effects, albeit at higher doses (not shown).
  • Shiga toxin is known to enter cells via a clathrin-independent glycosphingolipid- dependent route. HeLa cells were found to rapidly internalize Shiga toxin which accumulated in the cis-Golgi area, irrespective of the presence of pitstop 2 (30 ⁇ ). A slight though statistically insignificant delay in Shiga toxin delivery to the cis-Golgi in pitstop 2-treated cells was observed, indicating that clathrin TD-ligand interactions are not required for retrograde transport of Shiga toxin. This supports the specificity of pitstop 2 for the CME pathway.
  • HIV- 1 entry to cells has been shown to occur
  • Epsl5 or the small molecule dynamin inhibitor dynasore (Miyauchi et al., 2009). This suggests an obligatory role for clathrin in HIV-1 entry.
  • HeLa reporter cell lines were infected with HIV-1 for 2 h in the presence or absence of pitstop 2.
  • HIV-1 infectivity in this reporter cell line by about 70% at a concentration of the compound of 5 ⁇ and >90% at a concentration of 10 ⁇ (Fig. 5F), confirming that
  • CME is the main route of productive entry in this cell line.
  • pitstop 2 is an efficient and selective inhibitor of CME that acts via blocking ligand access to the clathrin TD.
  • the clathrin TD plays a crucial role in regulating coated pit dynamics
  • Clathrin dynamics were studied in more detail in view of the lack of knowledge of the physiological role of ligand binding to the clathrin TD in endocytosis.
  • FRAP fluorescence recovery after photobleaching
  • pitstop 2 treated cells did not show recovery of clathrin LC-eGFP fluorescence within 120 s, confirming that clathrin TD function indeed regulates CCP dynamics. Recovery was so slow that a reliable ⁇ value could not be determined under the imaging conditions utilised. These data indicate that pitstop 2 stalls the maturation and/ or consumption of pre-existing CCPs.
  • clathrin TD function may be required for the de novo assembly of clathrin-coated structures. For this, use was made of 1 -butanol, an alcohol which depletes cellular PI(4,5)P 2 levels causing CCP disassembly and loss of clathrin from the plasma membrane. Clathrin LC-eGFP containing pits rapidly reformed after washout of 1 -butanol in control and pitstop 2-treated cells, indicating that TD-ligand interactions are not required for the de novo recruitment or assembly of clathrin at the plasma membrane. This conclusion was further supported by ultrastructural analysis (see Fig. 6 and Fig. 7).
  • correctly at the light microscopic level may exhibit functional defects upon acute inhibition of complex formation with the clathrin TD.
  • TGN or endosomal proteins including AP-1, EEA1, Gadkin, CD63, TGN46, or the mannose 6-phosphate receptor MPR46 was also determined. None of these factors showed a significant change in localization within 15 min of pitstop 2 application. However, a partial loss of transferrin receptor (TfR) was noted from perinuclear endosomes, which instead appeared to be shifted towards the cell surface of pitstop 2-treated cells, consistent with the observed defects in Tf endocytosis (Fig. 5 A, B).
  • TfR transferrin receptor
  • FCHo proteins are early-acting factors postulated to facilitate CCP formation by linking PI(4,5)P2-rich membrane sites to endocytic AP-2 binding scaffold proteins intersectin and Epsl5/ Epsl5R.
  • TIRFM-based imaging of eGFP-FCHo2 dynamics in Cos7 cells revealed that FCHo2 -containing structures were relatively long- lived, with lifetimes between 26 s and over 120 s.
  • the mean lifetimes of FCHo2-eGFP- containing puncta were dramatically increased following application of pitstop 2 with puncta appearing nearly immobile. Consistent with their increased lifetimes, FCHo2- positive spots failed to recover in FRAP experiments.
  • clathrin TD function is required for the productive consumption of nascent CCPs containing FCHo2.
  • pitstop 2 induced a comparatively minor shift in the mean life span of AP- 2o-eGFP puncta.
  • Dyn2 is a component required for fission of mature CCPs.
  • Dynamin 2-eGFP displayed a lifetime distribution similar to that observed for AP-2 or clathrin.
  • Application of pitstop 2 shifted this distribution to longer life spans with a behavior that was intermediary between the effects seen for clathrin and AP-2. 7.
  • Fig. 6A corresponding to late fission stages, and structures containing a complete clathrin coat
  • These latter intermediates may comprise free clathrin-coated vesicles as well as late intermediates that remain attached to the plasma membrane via a connection not visible in the plane of the section.
  • CME synaptic vesicle
  • pitstops 1 and 2 Two small molecule inhibitors of clathrin TD function termed pitstops 1 and 2 that competitively interfere with the association of endocytic clathrin box ligands were developed.
  • pitstops 1 and 2 Two small molecule inhibitors of clathrin TD function termed pitstops 1 and 2 that competitively interfere with the association of endocytic clathrin box ligands were developed.
  • pitstop 2 Two small molecule inhibitors of clathrin TD function termed pitstop 1 and 2 that competitively interfere with the association of endocytic clathrin box ligands were developed.
  • In vitro experiments in conjunction with structural data based on protein X-ray crystallography as well as experiments in living cells clearly demonstrated that these compounds selectively perturb clathrin TD function.
  • Effects of pitstop 2 on CME are evident within 2-5 min of cell treatment, presumably only being limited by the rate of drug diffusion across the cell membrane. Reversible inhibition of C
  • pitstops 1 and 2 The similarity of phenotypes exhibited by pitstops 1 and 2 in lamprey reticulospinal synapses in situ and in Cos 7 cells in vitro provides evidence that both compounds share a common clathrin TD- based molecular mechanism of action. In the presence of pitstop 2, clathrin- independent internalization pathways and secretory traffic remain unperturbed (data not shown). As such, pitstops represent novel cellular tools to selectively inhibit CME.
  • pitstops in living cells revealed a novel and unexpected role for the clathrin TD in endocytic pit dynamics.
  • the clathrin TD represents the most frequently used interaction hub within the endocytic network, surprisingly little is known regarding the functional role of TD-clathrin box ligand interactions.
  • the use of pitstop 2 to acutely inhibit ligand access to the clathrin TD provides evidence for a central role for clathrin TD function in regulating CCP dynamics in mammalian cells. Mammalian cell endocytosis differs from yeast where clathrin plays a comparably minor role in endocytosis.
  • Proteins that contain clathrin box motifs comprise a diverse set of factors implicated in both early and late stages of endocytosis (Brodsky et al., 2001 ;
  • Blocking ligand access to the TD by pitstops may stall productive maturation of CCPs by preventing dynamic exchange of key endocytic factors driving progression of the pathway or monitoring completion of the previous step.
  • the unexpected finding that pitstop-mediated inhibition of ligand binding to the clathrin TD does not cause clathrin dissociation from membranes nor prevents the de novo assembly of clathrin- containing structures at the plasma membrane suggests that other interactions may be crucial for clathrin recruitment.
  • Clathrin is most highly expressed in the brain where it is concentrated at presynaptic nerve terminals. Evidence from morphological, genetic and knockdown studies has suggested that CME may be a key mechanism for the regeneration of SVs during stimulation induced exo-endocytic cycling.
  • RNAi and genetic approaches A drawback of RNAi and genetic approaches is that changes in clathrin levels occur over several days or even the entire lifespan of the animal, making it difficult to distinguish direct from indirect effects. This is important as synapses can adapt to chronic loss of endocytic proteins (Kim and Ryan, 2009). Using pitstops as reported herein to acutely perturb clathrin TD function provides a direct demonstration for an important role of clathrin in S V recycling in vertebrate neurons under conditions of high activity.
  • the pitstop inhibitors developed herein and their derivatives thereof may serve drugs for blocking the entry of viruses and pathogens which hijack the clathrin machinery, including HIV, hepatitis C virus, ebola virus, and Listeria monocytogenes amongst others.
  • EXAMPLE 2 Inhibition of clathrin function induces aberrant mitotic defects and inhibition of cancer cell division 1. Clathrin function is essential for mitosis
  • the graph in Fig. 9B also shows that in cells treated with pitstop 1 or pitstop 2, the spindles become significantly (P>0.01) narrower with a median ratio of 0.4 (pitstop 1) and 0.35 (pitstop 2) of the total cell width. Depletion of clathrin by siRNA also caused a significant decrease in the median spindle width ratio to 0.36 of the total cell width as compared to untreated control (0.65), DMSO (0.67), and the dynamin (and endocytosis) inhibitor dynole 34-2 (0.63).
  • the action of the pitstop compounds is consistent with targeted inhibition of clathrin.
  • these findings indicate that the pitstop compounds prevent mitotic progression by specifically blocking the onset of chromosome segregation and further, that they appear to activate the spindle assembly checkpoint (SAC) in mitosis.
  • SAC spindle assembly checkpoint
  • microtubules to regrow for 5 min at 37°C following their disassembly by cold treatment (cells were placed on ice for 30 mins).
  • Depletion of clathrin by siRNA prevented MT re-growth from centrosomes in metaphase cells.
  • neither pitstop 1 nor pitstop 2 mediated inhibition of clathrin prevented MT regrowth from the mitotic spindle poles.
  • As expected clathrin no longer decorated the mitotic spindle and spindle poles in cells after clathrin knockdown by siRNA.
  • the pitstop compounds do not affect the mitotic spindle localisation of clathrin (Fig. 8C).
  • TACC3 spindle localization was not affected by pitstop-mediated inhibition as clathrin remained on the spindle under these conditions.
  • TACC3 spindle localisation was significantly reduced by siRNA-mediated depletion of clathrin by >25% as evidenced by quantitation of TACC3 staining and fluorescence intensity.
  • Clathrin inhibitors reduce cell growth in a range of cancer cell lines
  • anti-mitotic compounds have anti-cancer properties by causing cell death following activation of the SAC.
  • Aurora A selective inhibitors such as
  • MLN8237 represent such compounds.
  • pitstop 1 and pitstop 2 also activate the SAC, the question of whether they can inhibit cell proliferation and induce cell death was assessed.
  • Cell growth was assessed by MTT assay in twelve cancer cell lines derived from different tissues: namely, SMA-560 (mouse glioma), SJ-G2 and U87
  • pitstop 1 had no effect on cell growth after 72 hours of continuous exposure.
  • pitstop 2 caused a dose-dependent decline in cell growth in four of the cancer cell lines, namely, HT29, BE2-C and U87 at low concentrations and A431 at a higher concentration (see Table 8).
  • the best (lowest) GI50 value concentration that causes 50% growth inhibition was > 5 ⁇ .
  • clathrin inhibitors can prevent cell proliferation and reduce viability in a range of cancer cell lines, favourable properties for an anti- cancer agent.
  • Table 7 Growth inhibition (GI50) induced by clathrin inhibitors
  • Clathrin has a second role in mitosis at the final stage of mitosis - cytokinesis. Unlike the role of clathrin at the mitotic spindle, its role in cytokinesis is thought to be dependent on its endocytic function. Consistent with this, inhibition of dynamin II (Dyn2), a key endocytic protein that is required for cytokinesis, utilising dynole 34-2 caused an increase in multinucleated cells, an indicator of cytokinesis failure (Fig. 11). Depletion of clathrin using siRNA and pitstop 1 and pitstop 2 -mediated inhibition of clathrin also caused multinucleation (Fig. 12). Hence, the pitstop compounds induce cytokinesis failure consistent with targeted inhibition of clathrin.
  • the endocytic protein clathrin plays two roles important for mitotic progression: 1) at the mitotic spindle to stablise K-fibres for chromosome segregation; and 2) during cytokinesis for membrane abscission to generate to independent daughter cells.
  • the present study shows that the small molecule inhibitors of clathrin, pitstop 1 and pitstop 2, produce mitotic phenotypes consistent with these two mitotic roles for clathrin.
  • these compounds and their analogues represent extremely valuable tools that can be exploited to characterise the molecular mechanism(s) of action of clathrin during mitosis. More globally, the use of these compounds provide for an improved understanding of the molecular mechanisms of mitotic progression in cells, and hence of the fundamental cell division process.
  • chemotherapeutics are being developed that specifically target key mitotic proteins to either activate or inhibit the SAC, such as Aurora kinase and kinesin spindle protein. These targeted inhibitors prevent proliferation of most tumour cells in vitro and reduce tumour volume in vivo by inhibiting growth and/or triggering cell death following SAC activation/inhibition (Taylor and Peters, 2008). Consequently, many targeted inhibitors, such as the Aurora kinase inhibitor MLN8054 (Millennium, USA), are currently in cancer clinical trials (Ma and Adjei, 2008), and are expected to have a more favourable therapeutic window than currently used chemotherapeutic agents such as taxol, as they would spare non- dividing, non-differentiating cells.
  • mitotic inhibitors to be efficacious anti-cancer agents is dependent on their ability to induce apoptosis following mitotic insult as it is predicted to result in tumour regression. However, they do not always result in cell death with varied cellular responses reported which include apoptosis, mitotic catastrophe, mitotic slippage, senescence and reversible mitotic arrest with continued cell growth. Hence, there has been an ongoing need for the
  • Step 1 3-sulfo-l,8-naphthalic anhydride, sodium salt
  • 1,8-Naphthalic anhydride (0.990 g, 5.00 mmol) was dissolved in oleum (6 mL). The resulting solution was stirred at 120 °C for 1 hour (until a drop of the mixture, when added to water, did not precipitate), then cooled to room temperature. The cooled solution was then poured into water (25 mL). Addition of aqueous saturated sodium chloride (20 mL) resulted in precipitation of the product. The white solid was collected by filtration, washed with water (5 mL), then ethanol (10 mL), and dried under vacuum.
  • the lead compound that allowed development of Pitstop 1 is 8-(3-carboxy-7- oxo-7H-benzo[i/e]benzo[4,5]imidazo[2, 1 -a]isoquinolin-4-yl)naphthalene- 1,4,5- tricarboxylic acid (LI) (see Table 1).
  • Reductive retrosynthetic fragmentation of LI revealed the compound is essentially a substituted 1,8-naphthalimide derivative, comprised of a 1,8-naphthalic anhydride and a 1,2-phenylenediamine moiety attached to a naphthalimide core.
  • the naphthalimide core was utilised as a minimal
  • Scheme 4 Scaffold simplification of LI reveals a naphthalimide core, and provides a pharmacophoric entity for the rational design of clathrin-TD inhibitors.
  • Table 8 Compounds identified by an ELISA-based screening strategy as inhibitors of the activity of clathrin. For the purposes of identifying clathrin-selective inhibitors, the dynamin I IC50 values are also provided.
  • Traffic jam a compendium of human diseases that affect intracellular transport processes. Traffic 1-, 836-851.
  • Traffic jams II an update of diseases of intracellular transport. Traffic 3, 781-790.
  • HIV enters cells via endocytosis and dynamin-dependent fusion with endosomes.
  • Pechstein A., Bacetic, J., Vahedi-Faridi, A., Gromova, K., Sundborger, A., Tomlin, N., Krainer, G., Vorontsova, 0., Schafer, J.G., Owe, S.G., et al. (2010).
  • Atomic structure of clathrin A ⁇ propeller terminal domain joins an a zigzag linker. Cell, Vol. 95, 563-573.

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Abstract

Inhibitors are provided for inhibiting the activity of clathrin. In particular, binding site(s) on the clathrin terminal doman (TD) for binding inhibitors have been identified. The binding sites are defined by amino acid(s) in the group Ile 52, Ile 62, Ile 80, Phe 91, and Ile 93 of the clathrin TD (SEQ ID No. 1). In at least some forms, the binding site may be further defined by amino acid(s) in the group Ile 66, Arg 64, Leu 82 andf Lys 96 of SEQ ID No. 1, or by Val 50 of SEQ ID No.1. There are also provided methods for prophylaxis or treatment of disease and conditions responsive to the inhibition of clathrin.

Description

INHIBITION OF CLATHRIN
FIELD OF THE INVENTION
The present invention relates to the inhibition of clathrin and in particular though not exclusively, to the inhibition of clathrin-mediated endocytosis (CME) and cell division (mitosis). The inhibition of clathrin has application to the prophylaxis or treatment of various diseases and physiological conditions.
BACKGROUND OF THE INVENTION
Clathrin-mediated endocytosis (CME) is a major process by which cargo is internalized into intracellular vesicles, thereby regulating the cell surface levels and endocytic uptake of important plasma membrane proteins. These include nutrient and growth factor receptors, ion channels, adhesion proteins, and synaptic vesicle (SV) membrane proteins in the nervous system. The clathrin based endocytic machinery is also hijacked by pathogens such as bacteria and viruses to get access to the cell interior. In addition, clathrin plays a function in traffic between the trans-Golgi network and the endo-lysosmal system, a pathway related to lysosomal storage disorders among other diseases. Assembly of clathrin-coated pits (CCPs) at plasma membrane sites is likely initiated by the recruitment of early-acting endocytic proteins. These factors serve as scaffolds for the co-assembly of heterotetrameric AP-2 complexes, which coordinate recognition of transmembrane cargo with the recruitment of endocytic accessory proteins. Early endocytic structures are presumably stabilized by an assembling clathrin coat built from soluble clathrin triskelia comprising three heavy and three light chains. The central building block of triskelia is the 190 kDa clathrin heavy chain, which forms an extended three-legged structure. The N-terminal β-propeller domain (referred to as the terminal domain or TD) at the distal end of the leg adopts a WD40- like fold (ter Haar et al., 2000), while the C terminus is near the vertex of the triskelion (Brodsky et al., 2001).
The clathrin cage has been postulated to serve at least two major functions in the endocytic process: (i) stabilizing deformed membrane domains, thereby facilitating the transition to invaginated pits, and (ii) providing an interaction hub for the recruitment of accessory factors that regulate progression of endocytosis. Indeed structural and proteomic studies have revealed a surprisingly simple architecture of accessory protein- clathrin interactions. Most of these factors, including amphiphysins, Epsl5/ Epsl5R, OCRL, and arrestins, harbor so-called clathrin box motifs, simple degenerate peptides that bind to a structurally well-defined site on the TD of clathrin heavy chain (ter Haar et al., 2000), consistent with the clathrin TD functioning as a central protein-protein interaction hub within the endocytic network.
Clathrin has a central role in many cellular processes. Besides CME, clathrin has involvment in mitosis. Mitosis is the stage of cell division where the chromosomes segregate and two new daughter cells form. Segregation is achieved via kinetochores which mediate attachment of chromosomes to spindle microtubules (MTs) known as the mitotic spindle. In its triskelia form clathrin binds the mitotic spindle, providing a structural lattice (Royle et al, 2005). The localisation of clathrin to the mitotic spindle is dependent on Aurora A, TACC3, and ch-TOG. Inhibitors of Aurora A target the mitotic spindle and are currently in pre-clinical or clinical studies as cancer treatments.
The inhibition of clathrin thus has application to the prophylaxis or treatment of a wide range of diseases and conditions. Inhibitors of clathrin would not only be important tools for unravelling the role of clathrin during mitosis but in at least some forms , have application as anti-mitotic compounds useful for the treatment of cancer and cell proliferative conditions. There is an ongoing need to identify molecular reagents that can selectively inhibit clathrin function and acutely interfere with CME and or mitosis. Whilst small molecule inhibitors of dynamin (a protein with a central role in endocytosis) have been reported, it is believed that no specific inhibitors of clathrin have previously been described in the literature.
SUMMARY OF THE INVENTION
Broadly stated, the invention stems from the surprising finding that the clathrin terminal domain (TD) has an unexpected central role in clathrin-coated pit (CCP) dynamics. In particular, the invention relates, to the identification and characterization of a specific binding site on the clathrin TD enabling the inhibition of clathrin-mediated endocytosis (CME) in cells and more generally, inhibition of clathrin function. The identification of the binding site facilitates the rational design of inhibitors for targeted binding to the clathrin TD for the inhibition of clathrin.
In an aspect of the invention there is provided a clathrin inhibitor wherein the inhibitor binding to a binding site of the terminal domain (TD) of clathrin forms a complex with the clathrin TD, the binding site of the clathrin TD being defined by at least one or more of amino acids He 52, He 62, He 80, Phe 91 and/or He 93 of SEQ ID No. 1.
In another aspect of the invention there is provided a complex of a clathrin inhibitor and the terminal domain (TD) of clathrin or a fragment of the clathrin TD, the inhibitor binding to a binding site of the clathrin TD, and the binding site being defined by at least one or more of amino acids He 52, He 62, He 80, Phe 91 and/or He 93 of SEQ ID No. 1.
Typically, the binding of the clathrin inhibitor to the clathrin TD is provided by binding interactions of the inhibitor with at least some of amino acids He 52, lie 62, He 80, Phe 91 and/or He 93 of SEQ. No. 1.
Typically, the clathrin inhibitor interacts with all of amino acids He 52, He 62, He 80, Phe 91 , and He 93 of SEQ ID No.1.
In at least some embodiments, the binding site of the clathrin TD for the clathrin inhibitor is defined by one or more of further amino acids He 66, Arg 64, Leu 82 and/or Lys 96 of SEQ ID No. 1 , and the binding of the inhibitor is provided by binding interactions of the inhibitor with the one or more further amino acids.
Typically, in this embodiment, the clathrin inhibitor interacts with all of amino He 66, Arg 64, Leu 82 and Lys 96 of SEQ ID. No. 1.
In another embodiment, the binding site can be further defined by amino acid Val 50 of SEQ ID No. 1 and the binding of the inhibitor is provided by binding a interaction of the inhibitor with that amino acid.
The binding interactions between the clathrin inhibitor with the clathrin TD can comprise direct contact of the inhibitor with at least some of the amino acids defining the binding site of the clathrin TD for the inhibitor.
Typically, the above binding interactions between the inhibitor and the clathrin
TD involve direct contact between the inhibitor and respective of the amino acids defining the binding site of the clathrin TD. Typically, the binding of the inhibitor to the clathrin TD is stabilised by one or more hydrogen bonds between the inhibitor and the clathrin TD. The hydrogen bonds can be direct hydrogen bonds between the inhibitor and the clathrin TD and/or hydrogen bonds mediated by water.
In another aspect of the invention there is provided a method for inhibiting clathrin, comprising contacting clathrin with an effective amount of at least one clathrin inhibitor for forming a complex with the terminal domain (TD) of clathrin in
accordance with one or more embodiments of the invention, or a prodrug or
physiologically acceptable salt of the inhibitor.
A complex in accordance with the invention can be formed in vitro or in vivo, and in some forms, may be isolated or at least partially purified.
As such, animal cells or tissues can be treated with the inhibitor in vitro or in vivo for the inhibition of clathrin, and the invention expressly extends to the prophylaxis or treatment of diseases or conditions responsive to the inhibition of clathrin, or for which the inhibition of clathrin may otherwise be beneficial.
Accordingly, in another aspect there is provided a method for inhibiting clathrin in a mammal, comprising administering to the mammal an effective amount of at least one clathrin inhibitor for forming a complex with the terminal domain (TD) of clathrin in accordance with one or more embodiments of the invention.
Typically, the clathrin inhitor(s) are administered for the inhibition of CME and/or inhibition of cell division.
In another aspect there is provided a method of screening a putative clathrin inhibitor, comprising:
fitting a model of a putative inhibitor of clathrin to a representation of a binding site for the inhibitor on the terminal domain (TD) of clathrin;
modelling interaction of the inhibitor with the binding site to evaluate whether the inhibitor forms a complex with the clathrin TD in accordance with one or more embodiments of the invention; and
evaluating whether the inhibitor may inhibit clathrin on the basis of the modelling, the formation of the complex being indicative of the capacity of the. inhibitor to inhibit clathrin. In still another aspect there is provided a method for providing a clathrin inhibitor, comprising:
designing the inhibitor to bind to a binding site for the inhibitor on the terminal domain (TD) of clathrin;
modelling interaction of the inhibitor with the binding site; and
evaluating whether the inhibitor forms a complex with the binding site of the clathrin TD in accordance with one or more embodiments of the invention on the basis of the modelling, the formation of the complex being indicative of capacity of the inhibitor to inhibit clathrin.
In this embodiment, the designing of the inhibitor can involve fitting the inhibitor into the binding site of the clathrin TD.
A range of inhibitors that bind to the clathrin TD binding site and inhibit clathrin function (e.g., in CME or mitosis) have been identified by the inventors.
Advantageously, the identification and characterisation of the clathrin TD binding site as described herein facilitates the rational design of inhibitors for use in embodiments of the invention.
In yet another aspect of the invention there is provided a clathrin inhibitor compound of Formula I to Formula V as described herein, or a prodrug or
pharmaceutically acceptable salt of the compound.
In another aspect of the invention there is provided a pharmaceutical composition comprising at least one compound of Formula I to Formula V, or a prodrug or pharmaceutically acceptable salt of the compound, together with a pharmaceutically acceptable carrier.
In another aspect of the invention there is provided a method for inhibiting clathrin, comprising contacting clathrin with an effective amount of a compound of Formula 1 to Formula V, or a prodrug or physiologically acceptable salt of the compound.
In another aspect of the invention there is provided a method for inhibiting clathrin in an animal, comprising administering to the mammal an effective amount of a compound of Formula I or Formula V to the mammal, or a prodrug or pharmaceutically acceptable salt thereof. In another aspect there is provided a clathrin inhibitor embodied by the inventionfor use in inhibiting clathrin in a mammal, or a prodrug or physiologically acceptable salt of the inhibitor.
In another aspect of the invention there is provided the use of a clathrin inhibitor embodied by the invention in the manufacture of a medicament for inhibiting clathrin in a mammal in need thereof, or a prodrug or physiologically acceptable salt of the inhibitor.
In another aspect of the invention there is provided the use of a compound of Formula I to Formula V in the manufacture of a medicament for prophylaxis or treatment of a disease or condition as described herein.
By the term "terminal domain of clathrin" and variations thereof such as "clathrin TD" and "clathrin terminal domain" as used herein is meant the N-terminal β- propeller region of clathrin heavy chain 1 , such as defined by amino acids 2-479 of SEQ ID No. 1 (Genbank Accession No. NM-004859.3, National Center for
Biotechnology Information, National Institutes of Health, Rockville Pike, Bethesda, Maryland, USA), and fragments thereof providing the binding site for a clathrin inhibitor in accordance with the invention.
By the term "clathrin inhibitor" as used herein and variations thereof such as "inhibitor of clathrin", is meant a compound that interacts with the clathrin TD in accordance with the invention to at least partly inhibit the functional activity of clathrin, examples of which are the inhibition of clathrin in clathrin-mediated endocytosis (CME) as may be expressed by a reduction in CME and/or the level of CME in cells, and the inhibition of cell growth and proliferation by inhibiting mitosis. The inhibition of clathrin in accordance with the invention can be total or partial inhibition of the functional activity of clathrin.
By the term "agent" as used herein is meant a clathrin inhibitor as described herein, or a composition or preparation comprising a clathrin inhibitor.
By the term "binding" as used herein in the context of binding of the clathrin inhibitor with the binding site of the clathrin terminal domain, is meant association of the clathrin inhibitor with the binding site through binding interactions of the inhibitor with at least some the amino acids defining the clathrin TD binding site. A binding interaction of the inhibitor with the clathrin TD can, for example, comprise direct contact of the inhibitor with amino acid(s) of the clathrin TD that define the binding site, the formation of one of more direct hydrogen bonds between the inhibitor and the clathrin TD, and/or the formation of one or more hydrogen bonds between the inhibitor and the clathrin TD mediated by water.
The identification of the clathrin TD binding site and provision of inhibitors of clathrin in accordance with one or more embodiments of the invention, not only facilitates the dissection of the mechanisms of endocytic pathways and CME and CCP dynamics in research, but has application to modulation of cell signalling, inhibition of entry of viruses and other pathogenic agents into cells, inhibition of synaptic
transmission (such as in epilepsy) and more generally, the targeted inhibition of clathrin for the prophylaxis or treatment of a wide variety of diseases or physiological conditions. In addition to this role in CME, as described above, these inhibition of clathrin has application to inhibiting mitosis, and hence in the prophylaxis or treatment of cancer and more generally, cell proliferative conditions. Moreover, inhibitors, compounds and methods embodied by the invention have application to use in research into clathrin function.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers, integers or steps.
Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the invention as it existed in Australia or elsewhere before the priority date of this application.
The features and advantages of the invention will become further apparent from the following detailed description of exemplary embodiments of the invention together with the accompanying drawings. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1 : Amino acid sequence for Homo sapiens clathrin heavy chain 1 (SEQ ID No. 1 ; Genbank Accession No. NM-004859.3 (GI: 11527063), National Centre for Biotechnology Information, National Institutes of Health, Rockville Pike, Bethesda, Maryland, USA). The clathrin TD of Homo sapiens clathrin heavy chain 1 is defined by amino acid residues 2-479 of SEQ ID. No 1. Amino acid lof SEQ ID No. 1 is an initiator methionine residue.
Figure 2: A ligplot showing interaction of the clathrin inhibitor pitstop 1 with the clathrin TD.
Figure 3: A ligplot showing interaction of the clathrin inhibitor pitstop 2 with the clathrin TD.
Figure 4: Pitstops compete with clathrin box ligands for a common site on the clathrin TD. (A, C) Ribbon representations of the clathrin TD in top view. The blades of the TD- -propeller are numbered from 1 to 7. Both inhibitors bind specifically to the clathrin-box binding site (between blades 1 and 2 of the clathrin TD). (B) Close-up view of the binding site for pitstop 1. The inhibitor and amino acids of the binding groove are shown in ball-and-stick mode. Hydrogen bonds formed at both ends of pitstop 1 are indicated by broken lines. The non-polar portion of pitstop 1 is sandwiched between Phe91 and the Arg64 side chains. On one side of the non-polar portion of pitstop 1 parallel π-π stacking interactions take place with the aromatic ring of Phe91. On the other end polar π-stacking interactions take place between the guanidinium group of Arg64 (δ+ dipole) and the π-electrons of the aromatic rings (δ- dipole) of the inhibitor. (D) Close-up view of the binding site for pitstop 2. The inhibitor and amino acids are shown in ball-and-stick mode. Bidentate hydrogen bonds at 3.0 A distance are formed between the guanidinium group of Arg64 and O and N atoms of the central portion of pitstop 2. The aromatic ring of Phe91 is located in the same position as in the inhibitor-free form of the clathrin TD and stacks against the edge of the bromobenzene of pitstop 2. (E, F) For comparison, pitstops (surface representations) are superimposed with the clathrin box-containing AP-3 p3-hinge peptide (sequence AVSLLDLDA) (SEQ ID No. 2). Pitstop 1 and pitstop 2 are shown in solid form. Binding of the inhibitors to the clathrin TD blocks the association of the 3-hinge peptide and similar clathrin-box ligands. Figure 5: Pitstop 2 reversibly inhibits clathrin-mediated endocytosis.
(A) Pitstop 2 reversibly inhibits Tf uptake. HeLa cells were incubated with Alexa -Tf in the presence of DMSO or 30 μΜ pitstop 2. Tf uptake is seen to resume after washout of the drug for 1 h. Scale bar, 10 μπι. (B) Reversibility and dose-dependence of pitstop 2-mediated inhibition of Tf uptake. Tf internalization after washout is not significantly different from uptake in the DMSO control (SEM; n= 3 independent experiments; *p < 0.05, ***p < 0.0001). (C) Pitstop 2 inhibits EGF uptake. HeLa ceils were incubated with Alexa488-EGF in the presence of DMSO or 30 μΜ pitstop 2. Data represent SEM (n= 3 independent experiments; ***p < 0.0001). (D) Pearson's correlation between Alexa488-EGF and AP-2. Data represent SEM (n= 3 independent experiments; *p < 0.05). (E) Pitstop 2 blocks HIV infection. Shown is the HIV Tat- driven luciferase activity in cells treated with increasing concentrations of pitstop 2 during infection. HIV Tat-driven luciferase activity in mock-treated cells was set as 100 % infection.
Figure 6: Ultrastructural analysis of intermediates observed in pitstop 2-treated cells. (A) Representative examples of clathrin-coated structures observed at the plasma membrane of Cos7 cells treated with 0.1 % DMSO or 30 μΜ pitstop 2. Scale bar, 200 nm. Morphological groups were: shallow CCPs (stage 1), non-constricted u-shaped CCPs (stage 2), constricted Ω-shaped CCPs (stage 3), or structures containing complete clathrin coats (stage 4). (B) Bar diagram displaying the numbers of clathrin-coated endocytic intermediates observed along the perimeter of Cos7 cells treated with 0.1 % DMSO or 30 μΜ pitstop 2.
Figure 7: Pitstop 1 inhibits activity-induced SV recycling in lamprey
reticulospinal synapses. (A, B) Electron micrographs of reticulospinal synapses microinjected with pitstop 1 and stimulated at 5 Hz for 20 min. Note the depletion of SVs (sv) at sites of release and large membrane expansions and pockets (asterisks), which in some sections (e.g., in B) surrounded the active zone (marked with thick arrows). Boxed area containing clathrin-coated pits (CCPs) is shown as inset at higher magnification in A. (C) EM-image of a control non-injected synapse from the same spinal cord preparation. (D, E) Constricted CCPs from a control non-injected axon and a synapse microinjected with pitstop 1. Note the similar shape of CCPs and the presence of clathrin coats in both cases. (F) Electron micrograph of a synapse microinjected with pitstop 1 but kept at rest. (G, H, I) Bar diagrams showing the difference in numbers of SVs (Nsv), curvature index (CI), and the number of clathrin- coated intermediates (Nccp) between control synapses stimulated at 5 Hz (white bars) and pitstop 1-microinjected stimulated synapses (black bars), respectively, ax- axoplasmic matrix; d-dendrite. (J) Bar diagram showing the relative abundance of different stages of coated endocytic intermediates (see above description for Fig. 6A) in control synapses stimulated at 5 Hz (white bars) and stimulated synapses microinjected with pitstop 1 (black bars). Scale bars for (A-C, and F), 0.5 μπι and (D, E), 100 nra.
Figure 8: Treatment with the pitstops 1 and 2 causes mitotic failure.
The percentage of cells trapped in mitosis increased with increasing concentrations of the pitstop compounds. At 30 μΜ pitstop 1 more than triples the mitotic index from 1% in untreated controls and 2% in DMSO treated to 7%, and at 10 μΜ, pitstop 2 increases the mitotic index to 5%, while clathrin depletion with siRNA increased the mitotic index to 11%.
Figure 9: Pitstops 1 and 2 affect the mitotic spindle. (A) The triskelial clathrin molecule provides a structural lattice for the mitotic spindle. In the absence of clathrin the spindle collapses becoming narrow. The graph shows the percentage of cells with narrow spindles when treated with pitstops. The inhibition of clathrin by the pitstop compounds gave the same narrow spindle phenotype as clathrin depleted cells. (B) Graph quantifying spindle width as a ratio of the total cell width shows that upon pitstop treatment the spindles significantly narrow. (C) Images from fluorescence microscopy showing the narrow spindle phenotype caused by treatment with pitstops. Both pitstop 1 and 2 caused the spindle microtubules to collapse as evidenced by tubulin staining. The same type of collapsed spindles is seen in cells depleted of clathrin by siRNA. In untreated control cells, or cells treated with vehicle DMSO, the dynamin (and thereby endocytosis) inhibitor dynole 34-2, or depletion of epsin 1 by siRNA did not cause the spindle to collapse demonstrating the observed effect of tpitstops is not solely due to endocytosis inhibition.
Figure 10: The pitstop compounds affect K fibre organisation during metaphase. Graph showing percentage of cells with disrupted HURP localisation. HURP is a marker for fibres, which are a unique subset of the spindle microtubules that bind to the kinetochores of chromosomes during metaphase. Depletion or inhibition of clathrin by siRNA or the pitstop compounds caused disruption to HURP staining. Figure 11 : The pitstops activate the metaphase checkpoint. (A) Representative images of (A) and (Α') showing the co localisation of CENPB (red), MAD2 (green), and DNA (DAPI, blue), white arrows in zoom shows active checkpoints. HeLa cells where synchronised at the G2/M boarder by the cdkl inhibitor RO-3306, then released in the presence of the indicated drug or control for 90 min, then fixed and stained for CENPB (red), MAD2 (green) and DNA (DAPI, blue). Captured images were decovoled and the channels combined. (B) The number of co-localisations between CENPB and MAD2 were scored for n>30 cells per sample, and are shown on the dot graph; the median is represented by the line. Inhibition by the pitstop compounds increased the number of active checkpoints per cell. (C) HeLa cells were depleted of either clathrin heavy chain (CHC) or epsin by siRNA and scored as in (B). Depletion of CHC increased the number of active checkpoints. N>30 cells per sample, and are shown on the dot graph; the median is represented by the line.
Figure 12: Pitstops induce multinucleation. The level of multinucleation (indicative of failed cytokinesis) more than doubled in cell populations treated with pitstop 1 or pitstop 2, or in cells depleted of clathrin by treatment by specific siRNA.
Figure 13 : Crystal data for the terminal domain of clathrin complexed with pitstop 1.
( Figure 14: Crystal data for the terminal domain of clathrin complexed with pitstop 2.
Figure 15: Crystal data for amino acids in pocket of the terminal domain of clathrin defining the binding site of clathrin for pitstop 1.
Figure 16: Crystal data for amino acids in pocket of the terminal domain of clathrin defining the binding site of clathrin for pitstop 2.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION
In this specification, the term "alkyl" used either alone or in a compound word such as alkylaryl refers to a straight chain, branched or mono- or polycyclic alkyl. Examples of straight chain and branched alkyl include methyl, ethyl, propyl, wo-propyl, butyl, and the like. Examples of cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. The term "alkenyl" refers to a straight chain, branched or cyclic alkenyl with one or more double bonds. Examples of alkenyl ethenyl, propenyl, and butenyl.
Typically, the alkyl or alkenyl will be a linear alkyl of linear alkenyl substituent. Most typically, the alkyl or alkenyl will have 10 carbon atoms or less in its longest chain and most typically, will be a lower alkyl or lower alkenyl. A lower alkyl or lower alkenyl as described herein is to be taken to have 6 carbon atoms or less in the longest chain of the alkyl or alkenyl group. Hence, a lower alkyl or lower alkenyl as described herein may have 5, 4, 3, 2 or 1 carbon atom(s) in its longest chain.
The term "alkoxy" refers to an alkyl group with an oxygen radical substituent. Examples include methoxy, ethoxy and n-propoxy. The term "lower alkoxy" refers to an alkoxy with 6 atoms or less in the longest alkyl chain of the alkoxy group (e.g., 5, 4, 3, 2 or 1 carbon atoms). An alkoxy substituent as described herein will typically be a methoxy group.
The term "aryl" used either alone or in compound words such as "alkylaryl", refers to a single, polynuclear, conjugated or fused aromatic hydrocarbon or aromatic heterocyclic ring system. Examples of aryl groups include phenyl and naphthyl.
When the aryl comprises a heterocyclic aromatic ring system, the aromatic heterocyclic ring system can contain one or more heteroatoms independently selected from N, O and S and will normally contain 5 or 6 ring members. Typically, an aryl group of a compound embodied by the invention or utilised in a method embodied by the invention will be selected from the group consisting of phenyl, naphthyl and benzyl.
The term " halo" as used herein refers to a halogen atom. The halogen atom is typically selected from the group consisting of F, CI, Br and I.
The term "heteroaryl" refers to a heterocyclic aromatic ring system.
Heterocyclic, heteroaryl, aryl and other ring systems (eg., single and fused ring systems) as described herein can have from 5 to 7 ring atoms unless specified otherwise, and may contain one or more double bonds. Moreover, the ring or rings may independently be saturated and/or optionally, have one or more substituents.
By the term terminally substituted as used herein in the context of an alkyl or alkenyl is meant that the end carbon atom of the longest chain of the alkyl or alkenyl that is remote from the group substituted by the alkyl or alkenyl is substituted.
Methodology for screening a putative inhibitor of clathrin and the designing of an inhibitor of clathrin in accordance with embodiments of the invention are well known to a person skilled in the art. Computer programs and systems for this are commercially available, and any conventionally known such methodology and/or modelling software can be utilised (e.g., AutoDock 4.2, AutoDock Vina (The Scripps Research Institute, La Jolla, CA, USA). Whilst the screening or modelling of clathrin inhibitors and their interaction with the clathrin TD binding site may at least in part be accomplished manually with graphical representations and 3 -dimensional modelling, computer based systems are the preferred option. Such computer assisted modelling can involve the provision of an intial structural representation of an inhibitor and electronically modelling it into a representation of the clathrin TD binding site, such as an electron density map of the binding site to determine whether the fit of the putative inhibitor forms the desired complex with the binding site or whether optimisation of the structure of the putative inhibitor is desirable. The designing of an inhibitior of clathrin may also involve modelling the inhibitor to at least partially fit or conform to the structure of a pharmacore or other clathrin inhibitor as described herein.
The design and optimisation of the structural features of the putative inhibitor can involve modification of the backbone scaffold of the inhibitor and/or modification (e.g., replacement), exclusion or addition of scaffold substituents to alter the interaction of the inhibitor with the binding site. This can take into account steric, lipophilic, and/or charge considerations (e.g., attraction and/or repulsion) to provide for formation of a complex with the clathrin TD in accordance with embodiments of the invention and/or to alter (i.e., increase or decrease) the affinity with which the inhibitor binds to the clathrin TD binding site. The design and modelling of the inhibitor can also take into account the formation of hydrogen bonds between the inhibitor and the clathrin TD wherein the hydrogen bond(s) can be formed directly between the inhibitor and the clathrin TD or be mediated by water, or be a mixture of those two possibilities. The design of compounds and modelling of ligand binding is for instance described in United States Patent Application Publication No. 2004/0219653, US 2009/0275047, US 2005/0170431 and US 2010/0247569, the entire contents of all of which are incorporated herein in their entirety by cross-reference.
Further, the crystal structure of a 55 kD fragment of the the terminal domain
(TD) of the heavy chain of rat clathrin consisting of amino acid residues 1-494 and the methodology for obtaining same has previously been reported (ter Haar et al, 1998), as has the crystal structure for a fragment comprising amino acid residues 1 -363 of the terminal domain TD of clathrin heavy chain respectively complexed with a peptide derived from β-arrestin 2 or the β-subunit of the AP-3 complex (the crystal structural data for which is available under ID codes 1C9L and 1C9I from the RBSC Protein Data Bank, Rutgers, the State University of New Jersey, Taylor Road, Piscataway, NJ, USA) (ter Haar, 1998; ter Haar, 2000), reference to which can also be had for the design and provision of clathrin inhibitors in accordance with the invention, and the contents of all of which are also expressly incorporated herein in their entirety by cross-reference.
A compound for use in a method of the invention may, for example, be a compound of Formula I, or a prodrug or pharmaceutically acceptable salt thereof, wherein:
Figure imgf000016_0001
Formula I
and wherein:
Ri is alkyl, alkenyl, alkylaryl, aryl, or a ring having from 5 to 7 ring atoms including from 0 to 3 heteroatoms typically selected from O, N or S, wherein the alkyl, alkenyl, alkylaryl, aryl, and ring group are optionally substituted; and
R2 is O, S or NH; or
Ri , X, and R2 form a ring A, the ring having from 5 to 7 ring atoms including from 0 to 3 heteroatoms typically selected from O, N or S and being optionally substituted; and
X is N or a carbon atom;
Y is O, S, or NH;
ring B and ring C each independently have 5 to 7 ring atoms including from 0 to 3 heteroatoms typically selected from O, N or S; and
R3, R4, and Rs are each independently H or an optional substituent.
In instances Ri is a ring substituent having from 5 to 7 ring atoms, the ring may include zero or one or more double bonds. Generally, the ring will have 6 ring atoms. Typically, the ring will not include any hetero ring atoms. Typically, Ri is a lower alkyl, lower alkylaryl, heteroaryl, or aryl group, wherein the lower alkyl, lower alkylaryl or aryl group is optionally substituted. The aryl group may be a heteroaryl group.
In some embodiments, Ri may be a C\-Cs alkyl group and typically, a d-C3 alkyl, C2-C3 alkyl, or C1-C2 alkyl. TheC Cs alkyl group when substituted, will typically be terminally substituted.
In one or more embodiments, Ri may be a lower alkyl terminally substituted with a ring having from 5 to 7 ring atoms including from 0 to 3 heteroatoms typically selected from 0, N and S. This ring may include zero or one or more double bonds. Typically, this ring has 6 ring atoms.
In at least some embodiments in which Ri is a lower alkylaryl, the lower alkylaryl can be a C]-C2 alkylaryl and typically, is a Cj-C2 alkylphenyl. The C1-C2 alkylphenyl can be unsubstituted. When substituted, the phenyl ring of the Ci-C2 alkylphenyl is typically selected from the group consisting of 2-substituted phenyl, 3- substituted phenyl, and 4-substituted phenyl groups. Most typically, the C1-C2
alkylphenyl is a benzyl group.
When Ri is an aryl group, the aryl is typically phenyl. When substituted, the phenyl can be selected from the group consisting of 2-substituted phenyl,
2,4-substituted phenyl, and 2,5-substituted phenyl groups.
Substituents of Ri may, for instance, be independently selected from the group consisting of hydroxyl, sulfhydryl, amino, nitro, halo, cyano, lower alkoxy, lower alkyl, N(CH3)2) C02H, SO3H, PO4H. For example, Rj may be a C1-C3 alkoxy, C1.C3 alkanol, or a C1-C3 alkylthiol and more usually, a Ci-C2 alkoxy, C2-C3 alkanol, or C2-C3 alkylthio.Typically, R2 in a compound of Formula I is O.In at least some embodiments, ring A of a compound of Formula I may be substituted with one or more substituents selected from alkyl, alkenyl, alkylaryl and aryl, wherein the alkyl, alkenyl, alkylaryl and aryl substituents are optionally substituted.
Each of ring A and ring B of a compound of Formula I may each independently be an aryl group, or a heteroaryl group having from 1 to 3 hetero ring atoms typically selected from O, N and S.
Typically, rings B and C of Formula I each have 6 ring atoms. Most typically, rings B and C form a naphthyl group wherein the naphthyl group is optionally substituted. In at least some embodiments, a compound of Formula I has the structure of Formula Ila as follows:
Figure imgf000018_0001
Formula Ila wherein, X, Y, R\, R2, R3, R and R5 are as for Formula I.
In at least some embodiments, a compound of Formula I has the structure of Formula lib as follows:
Figure imgf000018_0002
Formula lib wherein X, Y, Rj, R2, R3, R4 and R5 are as for Formula I.Typically, X in a compound of Formula I,IIa or lib is N.
Typically, Y in a compound of Formula I, Ila or lib is O or S and preferably, is
O.
Typically, R3 and R» of a compound of Formula I, Ila or lib are independently selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkoxy, alkyl, alkylaryl, aryl, NHR', NR'2, C02H, SO3H and PO4H.
Most typically, R3 and R4 of a compound of Formula I, Ila or lib are independently selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkyl, alkylaryl, aryl, NHR', NR'2, C02H, S03H and PO4H.
In some embodiments, R5 of a compound of Formula lib is selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkoxy, alkyl, alkylaryl, aryl, NHR', NR'2, C02H, SO3H and PO4H. Most typically, R5 of a compound of Formula lib is selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkyl, alkylaryl, aryl, NHR', NR'2) C02H, - SO3H and PC H.Typically, each R' of a compound of Formula I, Ila or lib is independently H, alkyl, alkenyl, aryl, or alkylaryl, wherein the alkyl, alkenyl, aryl or alkylaryl is optionally substituted.
Typically, at least one of R3 and R of a compound of Formula I, Ila or lib is other than H.
In some embodiments, R5 of a compound of Formula Ila is ¾ aryl, napthyl, benzyl or tetralin, wherein the aryl, napthyl,benzyl and tetralin are optionally substituted.
When R5 is aryl, the aryl group is typically phenyl or napthyl.
Typically, ring A of a compound of Formula I, Ila and lib has 5 or 6 ring atoms and most typically, 5 ring atoms. Most typically, ring A is an imadazolyl group.
Typically, in compounds of Formula I, Ila or lib in which Ri, X and R2 form ring A, R2 is NR$ and R* forms ring A with R] and X.
Typically, ring A has an aryl group substituent, the aryl group being fused with ring A. The aryl group fused with ring A will normally have 6 ring atoms and most typically is a phenyl group. The phenyl group is optionally substituted. Typically, ring A when substituted with phenyl forms a benzimidazol group.
In at least some embodiments, R5 of a compound of Formula I or Ila is a substituent of Formula III, as follows:
Figure imgf000019_0001
Formula III wherein:
G is a bond with ring C;
ring D has from 5 to 7 ring atoms optionally including from 0 to 3 heteroatoms selected from N, O and S; and
each W is independently H, halo, nitro, amino, hydroxyl, sulfhydryl, C02H, S03H, or P04H. Typically, ring D is a carbocyclic or aryl group. Most typically, ring D has 6 ring atoms.
Typically, at least one W substituted of Formula III is other than H.
Typically, R5 of compound of Formula I or Ila is a substituent group of Formula IV as follows:
Figure imgf000020_0001
wherein each W is independently H, C02H, S03H, or P04H.
In another form, a compound for use in a method embodied by the invention may be a compound of Formula V, or a prodrug or pharmaceutically acceptable salt thereof, wherein:
Figure imgf000020_0002
Formula V
and wherein:
X is O, S, or NH;
Y is O, N or a carbon atom;
Z is O, S or NH;
Ri is H, C02H, S03H, P0 H, alkyl, alkenyl, alkylcarboxy, or alkylaryl;
R2 is O, S, NH, NHR\ NHS(=0)2R\ S(=0)2R\ sulfonyl, NHC(=0)NH, NHC(=S)NH, or NHC(=0);
R' is H, alkyl, alkenyl, alkylaryl, aryl, the alkyl, alkenyl, alkylaryl and aryl group being optionally substituted; and
R3 is aryl or a polycyclic group having at least 2 fused rings each independently having from 5 to 7 ring atoms, the aryl and the polycyclic group being optionally substituted. Typically, Rj in a compound of Formula V is H or a lower alkyl or lower alkenyl, wherein the lower alkyl or lower alkenyl group is optionally substituted and generally, terminally substituted.
When R\ of Formula V is lower alkyl, the lower alkyl is typically a C1-C3 alkyl and more preferably, a C1-C2 alkyl. In at least some embodiments, the lower alkyl is terminally substituted, preferably with a carboxy or thiocarboxy group (e.g., a lower alkylcarboxy or lower alkylthiocarboxy).
Substituents of R\ in a compound of Formula V, may for instance, be selected from the group consisting of hydroxyl, sulfhydryl, amino, nitro, halo, cyano, C02H, S03H and P04H.
Typically, R2 in a compound of Formula V is S, NH, HN-S(=0)2-R' , S(=0)2R' , or NHC(=S)NH, and R' is alkyl, alkenyl or aryl, wherein the alkyl, alkenyl or aryl is optionally substituted. Typically, the lower alkyl and alkenyl are lower alkyl and lower alkenyl, respectively.
Typically R' of HN-S(=0)2-R' is aryl and most typically, phenyl, benzyl naphthyl, or tetralin.
In at least some embodiments, R3 of Formula V is phenyl or an aryl group with
2 fused rings each independently having from 5 to 7 ring atoms including from 0 to 3 heteroatoms selected from O, N and S.
When R3 of Formula V is a polyclyclic group, R3 will typically have 2 fused rings at least one of which is an aryl group having 6 ring atoms. Typically, each ring of the aryl group has 6 ring atoms.
R3 of Formula V may, for example, be optionally substituted and selected from the group consisting of phenyl, napthyl, benzopyranyl, quinolinyl, quinozalinyl, quinozolinyl, cinnolinyl, benzofuranyl, isobenzofuranyl, indolyl, isoindolyl,
benzothiophenyl, benso[c]thiophenyl, benzimidazolyl, purinyl, indazolyl, benzoxazolyl, benzoisoxazolyl, and benzothiazolyl.
Typically, the R3 group is selected from phenyl, napthyl, and benzopyranyl (e.g., benzopyran-4-one). When R3 is phenyl, in a compound of Formula V, the phenyl may be mono, di, tri or tetra-substituted (e.g., trihydroxy phenyl or tetrahydroxy phenyl).
Suitable such substituents may, for example, be selected from the group of substituents as defined for M in Formula Va below. In at least some embodiments, a compound of Formula V has the structure of Formula Va as follows:
Figure imgf000022_0001
Formula Va wherein each M is independently selected from H, halo, hydroxy, sulfhydryl, nitro, amino, cyano, C02H, S03H, P04H, OR', NHR', NR'2, S02R\ S02NH2, wherein R' is as defined as Formula II. Typically, at least one M substituent is other than hydrogen.
Typically, when M is OR', the R' is a lower alkyl or lower alkenyl, the lower alkyl or alkenyl being optionally substituted. When substituted, the alkyl or alkenyl group is optionally terminally substituted. The terminal substituent of R' may, for example, be selected from the group consisting of amino, nitro, halo, cyano, hydroxy, sulfhydryl, and typically, is amino.
Typically, the phenyl group shown in Formula Va is selected from the group consisting of 4-substituted phenyl, and 3, 5 -substituted phenyl groups.
In at least some embodiments, a compound of Formula Va may have the structure of Formula Vb as follows:
Figure imgf000022_0002
Formula Vb
Typically, M in a compound of Formula Vb is halo, nitro or -OR'NH2, wherein R' is a lower alkyl or lower alkenyl.
When an M substituent is -OR'NH2 in a compound of Formula Va or Vb, R' is typically a C1-C4 alkyl and preferably, a C3 alkyl. In other embodiments, a compound of Formula V may have the structure of Formula V- la as follows:
Figure imgf000023_0001
Formula V-la wherein:
G is O or S;
T is O, S, N or a carbon atom; and
each M is independently as defined for Formula Va.
Typically, at least one M substituent of a compound of Formula V-la is other than hydrogen.
At least one M in a compound of Formula V-la may be halo, nitro or -OR'NFb, wherein R' is a lower alkyl or lower alkenyl, and -OR'NH2 is typically a C1-C4 alkyl · and preferably, a C3 alkyl.
Typically, a compound of Formula Vl-a has the structure of Formula V-lb as follows:
Figure imgf000023_0002
Typically, T in a compound of Formula V-la or V-lb is O, S or N.
The dashed line(s) in Formula I to Formula V-lb above indicate a bond depending on the applicable atom(s) and/or substituent(s) involved.
It will be understood that all combinations and permutations of teh compounds provided for by Formula I to Formula V-lb as defined above are expressly
encompassed, including compounds not specifically exemplified herein. Moreover, compounds of the invention in which substitutents of Ri of a compound of Formula I or II are replaced with a bioisostere may be provided and utilised in a method described herein. This is also the case with substituents R3, R4 and W of compounds of Formula I and II, and M of compounds of Formula V, and all such compounds and their use is expressly is expressly encompassed. Bioisosteres are, for example, described in Lima and Barreiro, 2005, the contents of which is encompassed herein in its entirety by cross- reference.
Clathrin inhibitory activity of compounds may be determined utilising methodology and assays described herein. In addition, compounds useful as clathrin inhibitors in embodiments of the invention can be identified utilising compound library screening strategies, such strategies being well known to a person skilled in the art. The compounds indentified as LI and L2 in Table 1 below were identified utilising this technique and were validated by assaying for selective inhibition of clathrin TD- amphiphysin association as described herein
Examination of lead compounds LI and L2 (see Table 1 below) led to the development of minimalist pharmacores. From these pharmacores synthetic
methodologies were developed that gave rise to focused compound libraries that are exemplified in Table 2 and Table 3.
Table 1 : Initial compound inhibitors identified by compound library screening
Figure imgf000024_0001
Table 2: Naphthalamide library compounds
Figure imgf000025_0001
Figure imgf000026_0001
Table 3: ' Rhodanine compound library
Figure imgf000026_0002
Figure imgf000027_0001
Examples of synthetic routes for the synthesis of compounds of Tables 2-3 are shown below.
Figure imgf000028_0001
Scheme 1. Reaction scheme for the synthesis of a range of substituted 1 ,8- naphthalic anhydrides from the commercially available 1 ,8-naphthalic anhydride.
Reagents and conditions: (a) HNO3, H2S04, 17 hr; (b) (i) Fuming sulfuric acid (30 % S03), 90 °C, 30 min; (ii) Saturated KC1, 4 °C, 18 hr; (c) Cone. H2S04 (95-98 %), cone HNO3 (70 %), 60 °C, 90 min; (d) SnCl2, HCl, ethanol, 80 °C, 2 hr; (e) KOH, H20, 220 °C, 1 hr; (f) (i) NaN02, HCl; (ii) H2S04, H20, heat; (g) (CH3)2SO, K2C03, acetone, 80 °C, 18 hr. Dashed arrows indicate that whilst synthetic preparation is possible, commercial availability removed the need to synthesise these compounds.
Figure imgf000028_0002
Scheme 2. Reagents and Conditions: (i) SnCl2, Hcl, EtOH, 80°C, 2 hr; (ii) a.
Fuming sulfuric acid (oleum), 40-50°C, 3 hr; b. Kcl (sat), 4°C, 12 hr; (iii) RNH2, EtOH, 18 hr.
Figure imgf000029_0001
Scheme 3. Reagents and Conditions: (i) catalytic piperidine, EtOH, μΑλ^νε
Clathrin inhibitors according to the invention may be prepared by any conventional techniques known to a person skilled in the art, including by organic synthesis strategies, solid phase-assisted techniques or by commercially available automated synthesizers. Alternatively, conventional recombinant techniques alone or in combination with conventional synthetic approaches can be utilised.
Moreover, the clathrin inhibitors as described herein may be used for in vitro or in vivo diagnostic, prophylactic or therapeutic purposes.
Clathrin-mediated endocytosis (CME) is important for many cell trafficking and cell signaling pathways, and the inhibition of clathrin function has application in the prophylaxis or treatment of diverse diseases and conditions. It is known, for example, that endocytosis is a major contributor or direct cause of diverse human diseases and a list of vesicle trafficking-specific diseases has been published, see for example Aridor and Hannan (2000)-, and Aridor and Hannan (2002), the contents of which are incorporated herein by reference in their entirety. In particular, clathrin is involved in trafficking from the cell surface, and also plays a role in trafficking or fission events from the Golgi apparatus and endosomes. In the brain, diseases and conditions in which endocytosis plays a role include Alzheimer's disease. In Alzheimer's disease (AD) β-amyloid precursor protein (APP) is internalized from axonal cell surfaces in clathrin-coated vesicles, sorted away from recycling synaptic vesicles, and transported to endosomes and the cell soma. Mutations within the genes encoding API 80 and CALM, two clathrin binding proteins involved in CME that are displaced by the inhibitors described herein are directly implicated in in Alzheimer's disease (Harold et al., 2009; Yao et al., 1999). The endosome is the first compartment along the CME pathway after internalization of APP or ApoE, and endosomal alterations are evident in pyramidal neurons in Alzheimer brain. Endocytic pathway activation is prominent in APP processing and β-amyloid formation and is an early feature of neurons in vulnerable regions of the brain in sporadic Alzheimer's disease. Another example of disease involving CME relates to presynaptic synuclein protein which is a prime candidate for contributing to Lewy body diseases, including Parkinson's disease, Lewy body dementia and a Lewy body variant of AD. Exogenous synuclein causes neuronal cell death due to its endocytosis and formation of
intracytoplasmic inclusions. Cell death and a-synuclein aggregates are direct consequences of its endocytosis in human neuroblastoma cells. Dent's disease
(polycystic kidney disease) is yet another example of a disease involving endocytosis of ClC-5 chloride channel and endocytosis blockers are known to prevent its internalisation. Indeed, the prophylaxis or treatment of any physiological disorder amendable to the inhibition of CME as described herein is expressly encompassed by the invention. It is known, for instance, that human polyomavirus JCV is the etiologic agent of progressive multifocal leukoencephalopathy, a fatal central nervous system (CNS) demyelinating disease and its entry to neurons is blocked by endocytosis inhibitors such as chlorpromazine.
In addition, endocytosis has been implicated in epilepsy and CME plays a key role in synaptic vesicle (SV) recycling. Human epilepsies are heterogeneous conditions, broadly subdivided as genetic or acquired. Whatever the origin, seizures are associated with a massive burst of synaptic transmission. The genes identified to cause epilepsies mostly encode ion channels gated by voltage or neurotransmitters that are highly enriched in nerve terminals and controls their excitability. All these genes directly or indirectly regulate synaptic transmission. One common feature of all anti- epileptic drugs (AEDs) is that they reduce synaptic transmission. Synaptic
transmission is. the release of neurotransmitter by exocytosis from their storage compartments, synaptic vesicles, in presynaptic nerve terminals. It is controlled by the electrical properties of neurons and chemical transmission across the synapse. The nerve terminal is an electrically excitable structure which maintains a net negative membrane potential so that depolarization can activate voltage-sensitive Ca2+ channels to trigger exocytosis. Epilepsy results in the sustained and uncontrolled exocytosis from these terminals.
Currently available epilepsy therapy is unsatisfactory in many patients.
Approximately 50% of patients fail to have their seizures controlled by the first medication prescribed and ~30% will continue to have seizures despite trials of multiple drugs. Most AEDs have been discovered using similar traditional methodologies - screening of compounds in animal models of acute provoked seizures. Possibly as a result, most AEDs have similar principal mechanisms of action, such as inhibiting voltage gated sodium channels and/or enhancing GABAergic activity. A rare example of a new AED acting via a novel mechanism is levetiracetam ( eppra™) which inhibits the putative SV refilling protein SV2A and inhibits exocytosis.
The massive burst of exocytosis that occurs during an epileptic seizure cannot be sustained for more than 1 minute without compensation by clathrin mediated synaptic vesicle endocytosis (SVE). SVE occurs after exocytosis and retrieves the empty SVs for reuse. Thus, SVE is the mechanism used to replenish SV supply. There are only ~200 SVs in a nerve terminal, enough storage for less than half a minute of maximal synaptic transmission. As such, sustained synaptic transmission (as occurs in a seizure) is a cycle of exocytosis and endocytosis of SVs - the S V cycle. Considerable evidence links most elements of the SV cycle directly or indirectly to epilepsy. Some SV cycle genes cause epilepsy in humans (e.g., synapsin), or epileptic-like seizures when genetically knocked-out in animals (e.g., SV2, amphiphysin or synaptojanin). A ubiquitous mechanism to stop a seizure is to block synaptic mechanisms that are associated with pathologically sustained neuronal burst firing. A reduction in synaptic transmission is the common feature of all anti-epileptic drugs (AEDs). For most AEDs the mechanistic basis of this reduction is uncertain. However, inhibition of clathrin mediated SVE by blocking clathrin may lead to an activity-dependent run-down in synaptic transmission reducing the availability of SVs to sustain or propagate a seizure. ' Indeed, a recent report has shown that SVE is the rate limiting step in synaptic
transmission after a prolonged stimulation time Targeting clathrin in accordance with an embodiment of the invention may therefore provide a number of advantages over standard therapy. In particular, conventional AEDs reduce synaptic transmission at all times, but a clathrin inhibitor may only exert effect at high frequency or after sustained stimulation that is, under conditions associated with a seizure. Inhibiting clathrin mediated SVE may also limit the effect to overactive neurons and thus have reduced side-effects by allowing physiological neurotransmission to occur unimpeded.
CME mediated pathways are also utilized by many pathogenic agents such as viruses, toxins and symbiotic microorganisms to gain entry into cells. For instance, botulism neurotoxins and tetanus neurotoxin are bacterial proteins that inhibit transmitter release at distinct synapses and cause two severe neuroparalytic diseases, tetanus and botulism. Their action is dependent on their internalisation via endocytosis into nerve terminals.
Cell division (mitosis) results in equal segregation of duplicated chromosomes into two independent daughter cells. Progression through mitosis is highly regulated and is thought to involve membrane trafficking proteins, such as those required for endocytosis (Schweitzer et al, 2005). Premature chromosome segregation (metaphase- anaphase transition) results in aneuploidy, which is a hallmark of many human cancers. This adverse situation is avoided by activation of the spindle assembly checkpoint (SAC), a cellular mechanism that monitors proper assembly of the mitotic spindle. The SAC delays anaphase onset until all chromosomes are stably attached to kinetochores (Musacchio and Salmon, 2007). The SAC is thus an active signal produced by improperly attached kinetochores, which is conserved in all eukaryotes. The SAC stops the cell cycle by negatively regulating CDC20, thereby preventing the activation of the polyubiquitylation activities of anaphase promoting complex. Kinetochores are a large protein assembly that mediates the attachment of chromosomes to spindle microtubules (MTs). These microtubules are known as kinetochore fibres (K-fibres). Clathrin in its triskelia form binds to the mitotic spindle to provide a structural lattice. Here it complexes with TACC3 and ch-TOG to stabilise the K-fibres to allow effective chromosome binding (Royle et al, 2005). Mitotic spindle proteins are recruited during metaphase in a temporal manner. Aurora A-mediated phosphorylation of TACC3 at Ser-558 is required to target TACC3 to the spindle. This in turn recruits ch-TOG and clathrin. Thus, the mitotic spindle localisation of clathrin is dependent on Aurora A and TACC3, and possibly ch-TOG.
Several prior art small molecule inhibitors targeting mitotic proteins are currently in pre-clinical or clinical studies as cancer treatments. Inhibitors of Aurora A are an example of such compounds as they activate the SAC leading to cell death. Clathrin spindle localisation is abolished in cells treated with Aurora A inhibitors (Booth et al, 2011). As such, the the anti-mitotic effects of these inhibitors may in part be due to indirectly blocking the mitotic function of clathrin. Hence, small molecule inhibitors of clathrin are not only useful molecular tools to unravel the role of clathrin during mitosis but in at least some embodiments, have application as anti-mitotic compounds for the treatment of cancer and cell proliferative conditions. Many growth factor receptors (e.g., EGF-R) also require CME for
internalisation and maintenance of cellular activities from signalling to cell growth. Blocking CME prevents cell proliferation in many of these examples and provides further evidence of the anti-cancer and ant-proliferative activity of clathrin inhibitors.
Inhibition of CME may also have application to the prophylaxis or treatment of pain. For instance, neuropathic pain typically develops when peripheral nerves are damaged through surgery (including spine surgery), bone compression in cancer, diabetes, viral infection (including shingles or HIV infection), AIDS, alcoholism, amputation, chemotherapy, facial nerve problems, or multiple sclerosis, and is a major factor causing impaired quality of life for millions of people worldwide. Anticonvulsant drugs such as phenytoin and gabapentin are highly efficacious in treating neuropathic pain. These drugs act through modulation of synaptic vesicle transmission indicating the potential of the inhibition of clathrin mediated SVE in the treatment of this debilitating condition. That is, inhibition of synaptic vesicle endocytosis may halt or limit pain signalling and thereby reduce or ameliorate the sensation of pain experienced.
As such, examples of specific diseases and conditions for which methods of the invention find application for the prophylaxis or treatment of include, but are not limited to, cell proliferative diseases and conditions, multifocal leukoencephalopathy, polycystic kidney disease, β-amyloid associated diseases, Alzheimer's disease, neurodegenerative disorders, neuropsychiatric disorders, psychotic disorders, psychosis, bipolar disorders, schizophrenia, aberrant up-regulated neuronal excitation, seizures, neuropathic pain, migraine, mucolipidosis, cell proliferative diseases and conditions, neurological, diseases responsive to inhibition of clathrin, and diseases and conditions mediated or otherwise associated with synaptic signal transmission, CME or SVE (e.g., such as epilepsy), or cell vesicle trafficking.
Cell proliferative diseases and conditions that may be treated in accordance with embodiments of the invention include cancer, skin conditions such as psoriasis and scleroderma, benign growths, and cardiovascular diseases and conditions including atherosclerosis. The cancer may, for instance, be selected from the group consisting of carcinomas, sarcomas, lymphomas, leukaemias, and cancer of the liver, tongue, mouth, oropharynx, nasopharynx, gastrointestinal tract, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin (e.g., melanoma and basal cell carcinoma) amongst others.
As indicated above, the inhibition of CME in accordance with one or more embodiments of the invention may also be useful in inhibiting entry of a range of pathogenic agents into cells and so be useful in the prophylaxis or treatment of diseases or conditions asociated with this, non-limiting examples of which include nerve viral infection, botulinum neurotoxin A (BoNT/A) and C2 toxin from Clostridium botulinum (botulism), Toxin A (TcdA) and B (TcdB) from Clostridium difficile (antibiotic- associated diarrhoea), a-toxin (TcnA) from Clostridium novyi (necrosis, oedema), Lethal toxin (TcsL) from Clostridium sordellii, (toxic shock syndrome, sepsis), Tetanus neurotoxin (TeNT) from Clostridium Tetani (tetanus), diphtheria toxin from
Corynebacterium diphtheriae (diptheria), leukotoxin (LKT) from Mannheimia haemolytica (bovine respiratory disease), Dermonecrotic toxin (DNT) from Bordetella (swine atrophic rhinitis), Chlamydia psittaci and Chlamydia trachomatis (chlamdyia), Anthrax toxin from Bacillus anthracis (Anthrax), Listeria monocytogenes (Listeriosis), Yersinia pseudotuberculosis (Pseudotuberculosis (yersinia)), Staphylococcus aureus (Golden staph infections), Candida albicans (candidiasis), Porphyromonas gingivalis (periodontitis), Uropathogenic Escherichia coli (urinary tract infection), Adeno- associated virus, Adenovirus (Respiratory infection), African swine fever virus (African swine fever), Bluetongue virus- 1 (Bluetongue disease/ catarrhal fever), Chikungunya virus (CHIKV) (Chikungunya disease), Coxsackievirus B3 (CVB3) and A9 (CVA9) (Viral myocarditis/ CNS infection/ respiratory disease), Dengue virus 1 & 2 (DENV) (Dengue fever), Zaire Ebolavirus (Ebola hemorrhagic fever), Echovirus (EV1) from Picornaviridae (Echovirus disease), Equine infectious anemia virus (EIAV) (Equine infectious anemia), Feline infectious peritonitis virus (FIPV) (Feline infectious peritonitis), Hantaan virus (Korean hemorrhagic fever), Hepatitis B virus (HBV) (Hepatitis B), Hepatitis C virus (HCV) (Hepatitis C), Herpes Simplex virus (Herpes simplex), Kaposi's sarcoma-associated herpes-virus (KSHV) (Kaposi's sarcoma), Human immunodeficiency virus (HIV) (Acquired-immunodeficiency syndrome (AIDS)), Influenza virus (influenza), Jaagsiekte sheep retrovirus (JSRV) (Jaagsiekte/ ovine pulmonary adenocarcinoma), Junin arenavirus (JUNV) (Argentine hemorrhagic fever), Murine norovirus-1 (MNV-1) (murine norovirus disease), Bovine
papillomavirus BP VI (Carcinomas/ haemangio-endotheliomas of the bladder), Human papillomavirus 31 (HPV31) and 16 (HPV16) (genital warts/ cervical cancer), Canine parvovirus (Parvo), Poliovirus (poliomyelitis), Human rhinovirus 2 (HRV) (common cold), Rotavirus (gastroenteritis), Semliki forest virus (SFV) (Semliki forest virus disease), Simian virus 40 (SV-40), Vesicular stomatitis virus (VSV) (vesicular stomatitis). The above list is to be taken as only indicative only of the pathogenic agents for which compounds may be administered in accordance with embodiments of the invention. More generally, further examples include for instance, so-called "zippering bacteria" which bind to cellular receptors and invade cells in a clathrin- dependent manner such as enteropathogenic E. coli bacteria, and reovirus.
Suitable pharmaceutically acceptable salts include acid and amino acid addition salts, base addition salts, esters and amides that are within a reasonable benefit/risk ratio, pharmacologically effective and appropriate for contact with mammalian tissues without undue toxicity, irritation or allergic response. Representative acid addition salts include hydrochloride, sulfate, bisulfate, maleate, fumarate, succinate, tartrate, tosylate, citrate, lactate, phosphate, oxalate and borate salts. Representative base addition salts include those derived from ammonium, potassium, sodium and quaternary ammonium hydroxides. Suitable salts also include alkali metal and alkali earth cation salts such a sodium, calcium, magnesium, and potassium salts, as well as ammonium and amine cation salts. The provision of such salts is well known to the skilled addressee. Suitable pharmaceutical salts are for example exemplified in S. M Berge et al, J. Pharmaceutical Sciences ( 1997), 66 : 1 - 19, the contents of which is incorporated herein in its entirety by cross-reference.
Prodrugs of compounds in embodiments of the invention include those in which groups selected from carbonates, carbamates, amides and alkyl esters have been covalently linked to free amino, amido, hydroxy or carboxylic groups of the compounds. Suitable prodrugs also include phosphate derivatives such as acids, salts of acids, or esters, joined through a phosphorus-oxygen bond to a free hydroxl or other appropriate group. A prodrug can for example be inactive when administered but undergo in vivo modification into the active compound that binds to clathrin such that the function of clathrin is inhibited as a result of cleavage or hydrolysis of bonds or other form of bond modification of the compound post administration. The prodrug form of the active compound can have greater cell membrane permeability than the active compound thereby enhancing potency of the active compound. A prodrug can also be designed to minimise premature in vivo hydrolysis of the prodrug external of the cell such that the cell membrane permeability characteristics of the prodrug are maintained or enhanced for optimum availability to cells and for systemic use of the compound.
Esterified prodrugs may for instance be provided by stirring a compound embodied by the invention with an appropriate anhydride or acid chloride (in molar excess) in a pyridine/N,N-dimethylformamide (DMF) solution in the presence of a suitable catalyst such as dimethylaminopyridine (DM AP). In some cases, the solution may need to be refluxed to drive the reaction to completion. On completion of the reaction, the esterified product is purified by either recrystallization or by
chromatography. Representative esters include Q-C7 alkyl, phenyl and phenyl(Ci-6) alkyl esters. Preferred esters include methyl esters.A clathrin inhibitor can be administered to a mammal in alone or be co-administered with one or more other therapeutic compounds or drugs conventionally used for treating the applicable disease or disorders. By "co-administered" is meant simultaneous administration in the same formulation or in two different formulations by the same or different routes, or sequential administration by the same or different routes, wherein the administered drugs have overlapping therapeutic windows. By "sequential" administration is meant one is administered after the other.
The terminal domain (TD) of clathrin is highly conserved between mammalian species. For example, the amino acid sequence is conserved 100% (i.e. all amino acids are identical) between the terminal domain of bovine, human, rat, mouse, pig, and dog clathrin. Whilst methods and clathrin inhibitors in accordance with the invention have particular application to inhibiting the activity of human clathrin, the invention is not limited thereto and extends to inhibiting clathrin activity of other mammals including but not limited to, bovine, ovine, porcine, rodent (e.g., mouse, rat, guinea pig), canine, feline, and primate clathrin.
A clathrin inhibitor as described herein will generally be formulated into a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. Pharmaceutical compositions include sterile aqueous solutions suitable for injection and sterile powders for the extemporaneous preparation of injectable solutions. Such injectable compositions will be fluid to the extent that syringability exists. Injectable solutions will typically be prepared by incorporating the active(s) in the selected carrier prior to sterilising the solution by filtration. In the case of sterile powders, preferred methods of preparation are vacuum drying and freeze- drying techniques which yield a powder of the active and any additional desired ingredient from previously sterile filtered solutions thereof. As will be understood, a pharmaceutical composition in accordance with the invention can be provided in a form wherein the components of the composition are admixed with one another.
Alternatively, in another embodiment, the clathrin inhibitor(s) can be provided partially or totally seperately for combination with other components to form the composition, such as in the form of a kit.
For oral administration, the clathrin inhibitor can be formulated into any orally acceptable carrier deemed suitable. In particular, the inhibitor can be formulated with an inert diluent, an assimilable edible carrier or it may be enclosed in a hard or soft shell gelatin capsule. Moreover, a clathrin inhibitor can be provided in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions or syrups. Clathrin inhibitors as described herein can also be formulated into topically acceptable preparations including creams, lotions or ointments for internal or external application. Topically acceptable compositions can be applied directly to the site of treatment including by way of dressings and the like impregnated with the preparation.
A pharmaceutical composition embodied by the invention can also incorporate one or more preservatives such as parabens, chlorobutanol, phenol, and sorbic acid. In addition, prolonged absorption of the composition may be brought about by the inclusion of agents for delaying absorption such as aluminium monosterate. Tablets, troches; pills, capsules and like can also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatine, a disintegrating agent such as corn starch, potato starch or alginic acid, a lubricant such as magnesium sterate a sweetening agent such as sucrose, lactose or saccharin; and a flavouring agent.
Pharmaceutically acceptable carriers include any suitable conventionally known physiologically acceptable solvents, dispersion media, isotonic preparations and solutions including for instance, physiological saline. Use of such ingredients and media for pharmaceutically active substances is well known. It is particularly preferred to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Clathrin inhibitors as described herein can also be provided in dosage unit form. A dosage unit form as used herein is to be taken to mean physically discrete units, each containing a predetermined quantity of the selected compound(s) calculated to produce a therapeutic or prophylactic effect. When the dosage unit form is a capsule it can contain the active in a liquid carrier. Various other ingredients may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugars or both.
Pharmaceutical compositions embodied by the invention will generally contain clathrin inhibitors in a range of from about at least 0.1% by weight up to about 80% w/w of the composition. The amount of the clathrin inhibitor(s) in the composition will be such that a suitable effective dosage will be delivered to the individual taking into account the proposed mode of administration. Preferred oral compositions will contain between about 0.1 μg and 4000 mg of a clathrin inhibitor.
The dosage of clathrin inhibitor(s) administered will depend on a number of factors including whether the inhibitors are to be administered for prophylactic or therapeutic use, the disease or condition for which the active is intended to be administered, the severity of the condition, the age of the individual, and related factors including weight and general health of the individual as may be determined in accordance with accepted medical principles. For instance, a low dosage may initially be given which is subsequently increased at each administration following evaluation of the individual's response. Similarly, frequency of administration can be determined in the same way that is, by continuously monitoring the individual's response between each dosage and if necessary, increasing the frequency of administration or
alternatively, reducing the frequency of administration.
Typically, a clathrin inhibitor as described herein will be administered at a dosage up to about 50 mg/kg body weight and preferably, in a range of from about 5 Mg kg to about 100 Mg/kg body weight.
Routes of administration include but are not limited to respiritoraly, intravenously, intraperitonealy, subcutaneously, intramuscularly, by infusion, orally, rectally, topically, by implant, or by other conventionally employed methods of administration. With respect to intravenous routes, particularly suitable routes are via injection into blood vessels which supply the target tissue to be treated. Suitable pharmaceutically acceptable carriers and formulations useful in compositions of the present invention may for instance be found in handbooks and texts well known to the skilled addressee, such as "Remington: The Science and Practice of Pharmacy (Mack Publishing Co., 1995)", the contents of which is incorporated herein in its entirety by reference.
The animal treated by a method embodied by the invention will typically be a mammal such as a member of the bovine, porcine, ovine or equine families, a laboratory test animal such as a mouse, rat, rabbit, guinea pig, cat or dog, or a primate or human being as outlined above. However, it will be understood non-mammalian species may also be treated with clathrin inhibitors as described herein for research or other purposes in accordance with the invention.
"Pitstop" is a word coined for use as a trademark by the Applicants in respect of chemical compounds and its use whether alone or in coined words such as "Pitstop 1" and "Pitstop 2" is expressly reserved for use by the Applicants for chemical compounds.
The invention will be further described herein after with reference to a number of non-limiting Examples.
EXAMPLE 1 : Pitstop inhibitors of clathrin function
1. Methodology and reagents Protocols and reagents utilised in the present study are described below unless indicated to the contrary.
1.1 Antibodies
The following antibodies were used in this study: clathrin (X22, Affinity Bioreagents), AP-2 (a-adaptin, Abeam), intersectin 1 (S750; kindly provided by Dr. Thomas Siidhof, Howard Hughes Medical Institute, Stanford University, Palo Alto, USA), FCHo 1/2 (kindly provided by Dr. Harvey McMahon, MRC Laboratory of Molecular Biology, Cambridge, U.K.), dynamin 1/2/3 (Hudy-1, Upstate), EEA1 (BD transduction), Gadkin (Schmidt et al., 2009), transferrin receptor (Zymed), TGN46 (Serotec), GM130 (BD transduction), mannose-6-phosphate receptor (CI-M6PR,
Affinity Bioreagents), AP-1 (al-adaptin, Sigma), CD63 (RFAC4, MiUipore), and GST (peroxidase conjugate, Sigma). 1.2 DNA constructs
Fusion proteins encoding GST-clathrin heavy chain TD (amino acids 1-364), GST-AP-2 alpha-appendage (amino acids 702-938), and GST-AP-1 gamma-appendage (amino acids 690-822) were in pGEX4T-l expression vectors and His6-amphiphysin 1 (250-578), Hise-gadkin (amino acids 52-302), and His6-stonin 2 (amino acids 1-242) were in pET28a.
1.3 Protein purification
Expression plasmids were transformed into Escherichia coli BL21 -Codon Plus™ (DE3)-RP competent cells (Stratagene). His6- or GST-tagged fusion proteins were expressed and purified using HIS-Select™ nickel affinity gel (Sigma) or GST Bind® resin (Novagen) following the manufacturer's instructions.
1.4 ELISA-based binding assay
Purified Hise-tagged protein was diluted into screening buffer (20 mM HEPES pH 7.4, 50 mM NaCl, 1 mM DTT, 1 mM PMSF), added to a 384 well ELISA plate (high-binding PS Microplate, Greiner Bio-One) and bound for 1 h at RT. Non-specific binding was prevented by addition of 50 μΐ blocking buffer (20 mM HEPES pH7.4, 50 mM NaCl, 1 mM DTT, 1 mM PMSF, 2 % BSA, 2.5 % milk) followed by incubation overnight at 4°C. Following extensive washes (with 20 mM HEPES pH 7.4, 50 mM NaCl, 0.05 % Tween 20), chemical compounds diluted in DMSO (10 μΐ) were added and incubated together with GST-tagged protein for 1 h at RT in screening buffer. After 3 washes, HRP-coupled anti-GST antibodies were added in screening buffer and the plate was incubated for 15 min at RT. Following additional washes, 50 μΐ TMB substrate (Pierce Biotechnology) was added and the plate was incubated for 20 min before the reaction was terminated by adding 50 μΐ IN sulphuric acid. The amount of bound protein was determined by photometric measurement in a plate reader. Relative binding was calculated as % of DMSO control. 1.5 Cell cycle analysis by flow cytometry
Cells (5xl05) were grown in 10 cm dishes. Following inhibitor treatment, cells (floating and adherent) were collected and single-cell suspensions were fixed in 80% ice-cold ethanol at -20°C for at least 16 h. Cells were stained with propidium iodide and the cell cycle was analyzed as described previously (Joshi et al., 2010). Cell cycle profiles were acquired with a FACS Canto Flow Cytometer (Becton Dickinson) using FACS Diva software (v5.0.1) at 488 nm; Cell cycle profiles were analyzed using Flow Jo software (v7.1 ).
1.6 Trypan blue exclusion assay
Cells were seeded in 10 cm dishes (lxlO5 cells/dish). On day 0 (24 h after seeding), cells in triplicate were treated in the presence or absence of pitstops at concentrations of 1 , 3, 10 and 30 μΜ. After 20 h, the cell number and viability were measured using a Vi-CELL XR cell viability analyzer as previously described (Joshi et al., 2010).
1.7 Lactate dehydrogenase (LDH) toxicity assay
Toxicity was assayed by determination of lactate dehydrogenase (LDH) activity. HeLa cells were seeded in 96 well plates. Asynchronously growing cells were treated in the presence or absence of pitstop 1 or pitstop 2 at the indicated concentration for 8h. The supernatant (50 μΐ) was added to 100 μΐ of LDH assay reagent (Sigma- Aldrich) and the reaction was allowed to develop for 20 min. Absorbance was measured at 490 nm and 690 nm (plate background absorbance). Values were normalized to drug media background value and toxicity was calculated as a % of a 100% lysed cell control.
1.8 MTT Assay
Growth inhibitory assays were carried out using an MTT assay. Cells in logarithmic growth were transferred to 96-well plates (100 μΐ medium/well) at a density of 2500 cells/well for HeLa, HT29, H460, A431 , and DU 145 cells, 3000 cells/well for SW480, 3500 cells/well for MCF7, BE2-C and SJ-G2, and 2000 cells/well for A2780. On day 0, (24 h after plating) cells in duplicate were treated with or without pitstops. After 72 h drug exposure, cytotoxicity and growth inhibitory effects were evaluated using the MTT (3-[4,5-dimethyltiazol-2-yl] 2,5-diphenyl-tetrazolium bromide) assay. GI50 values were calculated from the MTT dose response curve from three independent experiments, each performed in duplicate. GI50 is the drug concentration at which cell growth is inhibited by 50% based on the difference between the optical density values on day 0 and those at the end of drug exposure. 1.9 TUNEL assay
A TUNEL Apoptosis Detection Kit for Adherent Cells (FITC-labeled POD) from GenScript was used according to the provided manual. 1.10 Electron microscopy analysis of Cos7 cells and primary hippocampal neurons
Cos7 cells (80% confluent) were grown in plastic dishes. Growth media were replaced by optiMEM with 0, 1 % DMSO or 30 μΜ pitstop 2 for 10 min. Cells were fixed with 2% glutaraldehyde (GA) in PBS. After scraping and pelleting, subsequent electron microscopy preparation and morphometric analysis were performed as previously described (Ferguson et al., 2009). Briefly, after epoxy resin embedding and sectioning micrographs were taken along the cell perimeter at x20000. Images were combined in order to reconstruct cell perimeter and numbers of clathrin-coated intermediates were estimated.
Rat primary neuronal cultures (P 1 ; DIV 14-21 ) were assayed either in incubation medium (resting conditions) or high potassium solution. Incubation medium contained (concentration in mM, pH 7.4): 170 NaCl/ 3.5 KC1/ 0.4 KH2P0 , 20 TES(N- tris[hydroxyl-methyl]-methyl-2-aminoethane-sulfonic acid), 5 NaHC03/ 5 glucose/ 1,2 Na2S04/ 1.2 MgCl2 and 1.3 CaCl2; at pH 7,4; high potassium solution was identical to the above but containing 120 mM NaCl plus 50 mM KC1. All media contained glutamate receptor blockers CNQX (20 μΜ) and AP5 (50 μΜ). Neurons were preincubated with 0.1 % DMSO or 30 μΜ pitstop 2 for 5 min at RT. Cultures were then either left for an additional 10 min in the same solution before fixation or were stimulated with high potassium solution for 90 s and rinsed with incubation medium for 7 min to assess vesicle recovery in presence of 30 μΜ pitstop 2 or 0.1% of DMSO as a control. After fixation with 2% GA, neurons were post-fixed in osmium
tetroxide 1%, incubated with 1% aqueous uranyl acetate, dehydrated and flat-embedded into epoxy resin. Glass was removed with the help of liquid nitrogen. Ultrathin sections were viewed with a Zeiss TEM 912 and images were taken at x30000 magnifications. 30 synaptic boutons per condition were analyzed. 1.11 Microinjection of compounds into lamprey reticulospinal axons and electron microscopic analysis of the preparation
Spinal cord preparations from adult male and female lampreys (Lampetra fluviatilis) were used in this study. Lampreys were kept in an aerated fresh water aquarium at 4°C. Animals were treated according to the Swedish Animal Welfare Act (SFS 1988:534), as approved by the Local Animal Research Committee of Stockholm. All efforts were made to minimize animal suffering and to reduce the number of animals used. Following anesthetization by immersion in 0.1 g/1 tricaine
methanesulphonate (MS-222, Sigma) and decapitation, trunk segments of the spinal cord were dissected out and transferred to a cooled (7-9°C) bath containing oxygenated lamprey Ringer's solution. All dissection steps, injections, and physiological recordings were performed at 7-9°C in Ringer's solution containing: 2 mM HEPES, 109 mM NaCl, 2.1 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, 4 mM glucose, 0.5 mM L- glutamine. Reagents were introduced into borosilicate glass injection microelectrodes and microinj ected into the reticulospinal axons with pressure pulses (5 - 15 psi) of 200 ms duration. Microinjections were monitored with a CCD camera (Roper Scientific, Tucson, AZ) or (Princeton Instruments, Trenton, NY) connected to a standard fluorescence upright microscope (Olympus BHX-50) with lOx and 40x water- immersion objectives. Membrane potentials of the impaled axons were monitored throughout injections.
Spinal cords were fixed during stimulation in 3% glutaraldehyde, 4% tannic acid in 0.1 M cacodylate buffer, pH 7.4, for 1 h, followed by incubation in the same fixative without tannic acid overnight. After postfixation in 1% osmium tetroxide for 1 h and dehydration in graded alcohol series, the specimens were embedded in Durcupan ACM (Fluka, Saint Quentin Fallavier, France).
Serial ultrathin sections were cut with a diamond knife (Diatome, Switzerland) on a Leica Ultracut UTC ultramicrotome (Leica, Sweden), mounted on formvar-coated copper slot grids, counterstained with uranyl acetate and lead citrate, and examined at 80 kV in a Tecnai 12 transmission electron microscope (FEI, Sweden). Effects of microinjections were analyzed in synapses cut at various distances from microinjection sites (i.e., different concentrations). For quantitative analysis, the number of synaptic vesicles (SVs) and coated pits and the membrane length of pockets or invaginations (curvature index) in the endocytic zone in injection experiments were determined from middle sections of at least five serially cut synapses. The values for the numbers of SVs and coated pits were normalized to the length of the active zone as the number of SVs in the cluster is proportional to the length of the active zone in the reticulospinal synapse. The length of endocytic zone membrane invaginations and pockets was measured using the NIH ImageJ software.
1.14. Protein crystallography
GST-clathrin heavy chain TD was purified by GSH affinity chromatography (Qiagen). The GST-tag was cleaved off by thrombin and separated from TD via GST affinity chromatography. The cleaved TD was further purified by size exclusion chromatography using a Superdex S200 column (Amersham Biosciences) in 10 mM Tris, pH 7.5, 50 mM NaCl, and 4 mM dithiothreitol, concentrated up to 22 mg/ ml (-0.5 mM), and supplied with a 4-fold molar surplus of pitstops 1 or 2.
Crystals were grown at 18°C using the sitting drop vapor diffusion method. The reservoir solution contained 20% PEG 3350, 150 mM potassium acetate, and 0.1 M Tris, pH 8.0, and drops were prepared by mixing 2 μΐ of reservoir and 2 μΐ of protein solution. Crystals formed within a few minutes and reached their final size in 1-2 days at 18 °C. The crystals were soaked briefly in a cryoprotection solution that was prepared by increasing the PEG 3350 concentration up to 40 % in 150 mM potassium acetate, and 0.1 M Tris, pH 8.0. Crystals were then mounted in nylon loops, and flash- cooled in liquid N2. X-ray data were collected at Beamline BL1 at BESSY-II, Berlin, and processed using XDS (Kabsch, 2010) and Xscale. The phase problem was solved by molecular replacement using the CCP4 program MOLREP (Collaborative
Computational Project, 1994) using the 2.7 A structure of clathrin TD (Protein Data Bank code 1C9I) as a model (ter Haar., 1998; ter Haar et al., 2000). All water molecules and ligand atoms were omitted from the starting model. Subsequent cycles of refinement to 1.7 A resolution were performed using Refmac5 (Murshudov et al., 1997). Pitstops 1 and 2 were built using the Dundee PRODRG2 server and fitted in the electron density (Schuttelkopf and van Aalten, 2004). Fig. 3A-F were produced with PyMOL software (see: http://www.pymol.org/). 1.15 Fluorescence microscopy of immunostained samples
Cos7 cells seeded on matrigel-coated glass coverslips were preincubated with 30 μΜ pitstop 2 or DMSO (0.1%) in DMEM/ 0.1 % FCS/ 10 mM HEPES for 15 min, fixed in 4% PFA or methanol, and stained with antibodies as previously described (Schmidt et al., 2009). Cells were imaged using spinning disc confocal or total internal reflection microscopy (TIRFM). Pearson's correlations were obtained using Volocity software (Impro vision).
1.16 Confocal and TIRF-based live imaging
For live cell microscopy, Cos7 cells stably expressing eGFP-clathrin LC
(Gaidarov et al., 1999), or transiently expressing FCHo2-eGFP, AP-2o-eGFP (Gaidarov et al., 1999), and dynamin2-eGFP were used. CCP dynamics were imaged by TIRFM (Visitron) under the control of Slidebook 5 (3i Inc). Time series of 2 min (at 0.5 Hz) were acquired 5 min after addition of 30 μΜ pitstop 2 or DMSO (0.1%) in HBSS media supplemented with 0.1 % FCS and 10 mM HEPES (pH 7.4) at 37°C. For lifetime analysis events were tracked manually.
Fluorescence recovery after photobleaching (FRAP) experiments were performed using a spinning disc confocal microscope (Perkin Elmer) controlled by Volocity (Improvision). Cells were imaged for 12 s, then bleached in a region of 15 x 15 μπι , and imaged for an additional 120 s at 0.5 Hz. Fluorescence recovery was analyzed by defining CCPs in the FRAP region before bleaching, followed by measuring the fluorescence intensity in the FRAP region over time. Intensity values were corrected for photobleaching in a non-bleached control area. 1.17. Ligand internalization and surface staining
HeLa or Cos7 cells seeded on Matrigel™-coated glass coverslips were starved (2 h) before incubation with 20 μg Alexa488 568-transferrin or 100 ng Alexa595/488-EGF for 15 min at 37°C. Washed cells were fixed in 4 % PFA, 4 % sucrose in PBS, pH 7.4 and processed for epifluorescence microscopy. Fluorescence levels were quantified using a Zeiss Axiovert200M fluorescence microscope under the control of Slide Book5 software (3i Inc., Gottingen, Germany). To analyze ligand sequestration into CCPs cells were incubated with labeled EGF in the presence or absence of 30 μΜ pitstop 2 for 30 min at 8°C. Washed cells were fixed and stained for AP-2a. Cells were imaged using TIRFM and analyzed by Slidebook5 software. Colocalization was assessed by determining Pearson's correlation coefficients.
1.18 Shiga toxin internalization and retrograde traffic
HeLa cells were seeded on glass cover slips and starved overnight. The cells were washed once with ice-cold PBS and then incubated at 4°C for 30 min with
1 μg/ml Cy3-StxB (kindly provided by Dr. Ludger Johannes, Institute Curie, Paris, France) in DMEM/ 0.2% BSA/ 20 mM HEPES pH 7.4. After two washes cells were fixed immediately or incubated for 15 to 60 min in growth medium at 37°C before fixation. Fixed cells were processed for immunofluorescence microscopy using antibodies against GM130. For quantifications, the total fluorescence of Cy3-StxB per cell was analyzed and the fraction localized to the Golgi was determined by quantifying the overlap with the GM130-positive area. 1.19 HIV infection
TZM-bl cells (Wei et al., 2002) were plated in 96 well plates and incubated for
2 h in serum-free medium prior to the experiment. Cells were preincubated with pitstop 2 or solvent for 30 min, and infections were carried out by adding different amounts of HrV-1 strain NL4-3 obtained from infected MT-4 cells. Virus-containing medium was removed after 2 h and fresh medium containing 100 ng/ ml of the HIV coreceptor antagonist AMD3100 was added to prevent further entry events. At 48 h post infection, cells were lysed, and HIV Tat-driven luciferase activity was measured using the Steady- Glo assay (Promega) according to the manufacturer's recommendations. 1.20 Data deposition
Crystal structural data for the terminal domain of clathrin respectively complexed with pitstop 1 and pitsop 2 was deposited with the RCSB Protein Data Bank, Rutgers, the State University of New Jersey, Taylor Road, Piscataway, NJ, USA on 25 November 2010 under PDB codes 2xzg (for pitstop 1) and 2xzh (for pitstop 2), reference to which can be had for the design and provision of clathrin inhibitors in accordance with the invention and the contents of all of which is expressly incorporated herein in its entirety by cross-reference. The crystal data for pitstop 1 and pitstop 2 complexed with the terminal domain of clathrin is also set out in Figures 13 and 14. 2. Identification and characterisation of the clathrin inhibitors pitstop 1 and pitstop 2 and their binding to the clathrin terminal domain
Clathrin function in mammalian cells has mainly been evaluated via two approaches: knockdown of clathrin or expression of dominant-negatively acting clathrin-binding fragments of endocytic accessory proteins. While both of these strategies have shown that clathrin is essential for CCP formation or stability, the physiological role of endocytic ligand association of its TD remains unclear. This lack of knowledge is surprising as clathrin box-TD interactions form a major hub within the endocytic network. It has been suggested that multiple redundant interactions of clathrin TD with its endocytic ligands may serve to stably recruit clathrin to
membranes, thereby facilitating clathrin assembly through local enrichment.
Alternatively, clathrin assembled at nascent endocytic sites could serve as an organizing scaffold that regulates CCP dynamics by providing spatially defined binding sites on its TD for accessory proteins that drive CCP maturation and disassembly.
To distinguish between these two models, a chemical biology approach designed to identify inhibitors that selectively interfere with TD complex
formation with its endocytic ligands was employed. Specifically, an ELISA- based screen for small molecules that can displace the B/C domain of
amphiphysin (an endocytic protein harboring a clathrin box motif (Slepnev et al., 2000; Dell' Angelica, 2001)) from recombinant clathrin TD was carried out. From a library of about 17,000 small molecules two lead compounds that exhibited selective inhibition of clathrin TD-amphiphysin association were identified and validated. Retrosynthetic analysis identified simple approaches to in-house
synthesis of two focused compound libraries based on the original leads. This led to the identification of two molecules of distinct chemical scaffolds which
specifically disrupt ligand association with the clathrin TD. Based on subsequent studies and their ability to impair CCP function (see below), these compounds were named pitstops 1 and 2 (see Tables 2 and 3 above for the chemical structures of these compounds). Pitstops 1 and 2 selectively inhibited ligand association of clathrin TD with IC50 values of 18 μΜ and 12 μΜ, respectively. Neither
compound affected complex formation between the ear domain of AP-2a and amphiphysin (a top-site ligand) or stonin 2 (a side-site ligand), or between the AP- 1 γ-ear and its accessory binding partner gadkin (Schmidt et al., 2009). The pitstops also did not influence the in vitro GTPase activity of full-length native sheep brain dynamin 1 (not shown). GI50 (μΜ) values for pitstop 2 were
determined for the following of cancer cell lines by MTT assay: HT29 (Colon) cells (6.2 ± 2.0 μΜ); A431 (Skin) cells (38 ± 9 μΜ); BE2-C (Neuroblastoma) cells (5.7 ± 0.9 μΜ); U87MG (Gliablastoma) cells (5.3 ± 0.9 μΜ). The GI50 value is the concentration of the clathrin inhibitor that inhibits cell growth by 50 % (i.e., the lower the value the greater the growth inhibition). Errors represent SEM (n=3 independent experiments). 3. X-ray crystallography of pitstops in complex with the clathrin TD
The structure of the clathrin TD in complex with pitstops by protein X-ray crystallography was determined to understand the molecular basis of clathrin TD inhibition by pitstops. Clathrin TD-containing crystals diffracted up to a resolution of 1.7 A and the structure was solved by molecular replacement using the clathrin TD as a search model (see Table 4).
Table 4: X-ray data collection and refinement statistics
Clathrin TD + Clathrin TD +
Pitstop 1 Pitstop 2
Data collection
Space group P212121 P212121
Cell dimensions
a, b, c (A) 71.34, 73.92, 84.38 70.90, 74.02, 82.12
«, β, γ θ 90.00, 90.00, 90.00 90.00, 90.00, 90.00
Resolution (A) 33.85- 1.70 33.7-1.69
•^sym ΟΓ Rmerge 0.048 (0.243)3 0.072 (0.562)3
I / si 24.34 (4.19) 19.10 (3.94)"
Completeness (%) 95.1 (70.1)a 100 (100)a
Redundancy 5.9 (2.6)a 7.2 (7.1)a Refinement
Resolution (A) 33.85- 1.70 33.7-1.69
No. reflections 44946 46615
^work / -^free 15.9 / 20.0 17.1 / 21.4
No. atoms 3341 3431
Protein 2832 2887
Ligand/ion 27 28
Water 458 429
-5-factors
Protein 15.3 17.0
Ligand/ion 26.0 21.6
Water 29.8 29.5
R.m.s. deviations
Bond lengths (A) 0.024 0.025
Bond angles (°) 1.892 1.968 a Highest resolution shell is shown in parentheses
Following refinement, inhibitor molecules in complex with clathrin TD could be identified and modelled into the determined electron densities The was achieved as follows. The first refinement cycles were run without pitstops (pdb file contained exclusively amino acids from clathrin TD), and the pitstops were then fitted into the electron density map. Further refinement cycles were then run with a pdb file that contained clathrin TD complexed with pitstop 1 or 2.
Ligplots showing the binding interactions of of pitstop 1 and pitsop 2 with the clathrin TD are shown in Fig.2 and Fig. 3. The interactions shown are
mediated by hydrogen bonds and by hydrophobic contacts. Hydrogen bonds are indicated by dashed lines between the atoms involved, while hydrophobic contacts are represented by an arc with spokes radiating towards the atoms of the relevant pitstop compound that they contact. The contacted atoms are shown with spokes radiating back. As shown, pitstop 1 makes direct contact with He 52, He 62, He 66,
He 80, lie 93 , Phe 91, Arg 64, Leu 82 and Lys 96 of the clathrin TD, whilst pitstop 2 makes direct contact with He 93, He 80, Val 50, He 52, He 62 and Phe 91.
The overall structures of clathrin TD in complex with either pitstops 1 or 2 are shown in Fig. 4A, C. The clathrin TD adopts a WD40-like structure comprised of a seven-bladed β-propeller each with four antiparallel strands (ter Haar et al., 2000). Both pitstop inhibitors bind the interface between the first and second blades
Fig. 4B, D) at a site overlapping with that used by clathrin bo -containing accessory proteins (Fig. 4E-F), which bind to the TD with low affinity (Brodsky et al., 2001 ;
Edeling et al., 2006b).
Pitstop 1 lies in a hydrophobic cavity formed by four isoleucines (52, 62, 80, 93), Leu 82, and Phe 91. Its conformation is stabilized by five hydrogen bonds at both ends of pitstop 1 (see Table 5 below). Only one direct hydrogen bond is formed between the sulfonate group of pitstop 1 and the ε-amino group (Νε2 atom) of Gln82. All other hydrogen bonds are mediated by water. Comparison of ligand-free clathrin TD with the pitstop 1 -bound form shows that two residues in the clathrin box-binding site undergo major conformational changes upon ligand binding. While the phenyl ring of Phe 91 rotates by 180° around the Ca-Cp bond and stacks against the non-polar portion of the compound on one side, the guanidinium group of the Arg 64 side chain stacks on pitstop 1 on the opposite side.
Pitstop 2 occupies the identical hydrophobic cavity used by pitstop 1. However, differences in the binding modes of both molecules (Fig. 4D) were noted. In particular, the side chain of Arg 64 is positioned along the central part of pitstop 2 and forms bidentate hydrogen bonds with O and N atoms of the thiazol-4(5H)-one ring. The conformational change of Arg 64 allows enough space for the non-polar portion of pitstop 2 to insert deeply into the hydrophobic cavity, towards the first blade. The phenyl ring of Phe 91. stacks against the non-polar edge of the bromobenzene group of pitstop 2 (Fig. 4D), and the conformation is stabilised by 2 two direct hydrogen bonds with Arg 64 (see Table 6). Since the solvent-exposed bromobenzene moiety lies on the second blade of the clathrin TD, the first and the second blades are blocked by pitstop 2. Table 5: Interaction of pitstop 1 with clathrin TD via hydrogen bonds
Figure imgf000051_0001
Table 6: Binding interaction of pitstop 2 with clathrin TD - Hydrogen bonds
Figure imgf000051_0002
The analysis reveals that the two pitstops fit into the targeted TD groove in two different poses and induce distinct structural changes in clathrin TD. This provides a molecular basis for the selective displacement of clathrin box ligands from the TD and accounts for the in vitro and in vivo activities of the pitstops. 4. Pitstop 2 selectively inhibits clathrin-mediated endocytosis and HIV entry
To examine whether pitstop 2 can act in living cells the effect of the pitstop on the internalization of transferrin (Tf) and epidermal growth factor (EGF) was analayzed. Receptor-mediated-internalization of both of these ligands is known to largely depend on clathrin function in most cell types (Brodsky et al., 2001). When exposed to
Alexa -Tf HeLa cells efficiently internalized Tf into perinuclear recycling endosomes. Preincubation of HeLa cells with pitstop 2 led to a dose-dependent inhibition of Tf uptake with an IC50 value (12-15 uM) very similar to that measured for blocking clathrin TD function in vitro (Fig. 5 A, B). Application of 30 uM pitstop 2 completely blocked Tf endocytosis, similarly to what has been observed in clathrin knockdown cells. Pitstop 2-induced block of Tf endocytosis in HeLa cells was completely reversed within 1-3 h of drug washout. In another cell line, U20S, the IC50 for Tf uptake was 9.7+1.5 μΜ (n=5 independent experiments, from 3 distinct synthetic batches of pitstop 2). Treatment with similar concentrations of pitstop 2 also blocked Tf uptake in other cell types including Cos7 and BSCl cells, astrocytes or primary neurons. Pitstop 2 also caused a potent inhibition of EGF uptake (Fig. 5C). This is consistent with the previously suggested notion that the majority of EGF is endocytosed via a clathrin- mediated entry route in most cell types. Pitstop 1 was found to display comparably low cell membrane penetration but exhibited qualitatively similar effects, albeit at higher doses (not shown).
To better understand the mechanism by which pitstop 2 inhibits receptor- mediated endocytosis, Cos7 cells were incubated with Alexa488-EGF at 4°C to accumulate ligand-receptor complexes in CCPs (Krauss et al., 2006). A similar although slightly enhanced accumulation of EGF was observed in pitstop 2-treated cells (Fig. 5D, E) suggesting that pitstop 2 does not interfere with cargo recognition or sequestration into CCPs, a step mediated by endocytic adaptors such as AP-2. These data argue against non-specific effects of pitstop 2 on plasma membrane organization and suggests that pitstop 2 blocks CME at a step subsequent to cargo sequestration.
Shiga toxin is known to enter cells via a clathrin-independent glycosphingolipid- dependent route. HeLa cells were found to rapidly internalize Shiga toxin which accumulated in the cis-Golgi area, irrespective of the presence of pitstop 2 (30 μΜ). A slight though statistically insignificant delay in Shiga toxin delivery to the cis-Golgi in pitstop 2-treated cells was observed, indicating that clathrin TD-ligand interactions are not required for retrograde transport of Shiga toxin. This supports the specificity of pitstop 2 for the CME pathway.
Many viruses and pathogens exploit the clathrin-dependent endocytic machinery for cell entry. For example, HIV- 1 entry to cells has been shown to occur
predominantly or exclusively via CME in studies using dominant negative dynamin or
Epsl5, or the small molecule dynamin inhibitor dynasore (Miyauchi et al., 2009). This suggests an obligatory role for clathrin in HIV-1 entry. To evaluate this, HeLa reporter cell lines were infected with HIV-1 for 2 h in the presence or absence of pitstop 2.
Subsequent fusion events were blocked with an HIV entry inhibitor and infection was scored by luciferase readout after 48 h. Pitstop 2 potently and specifically reduced
HIV-1 infectivity in this reporter cell line by about 70% at a concentration of the compound of 5 μΜ and >90% at a concentration of 10 μΜ (Fig. 5F), confirming that
CME is the main route of productive entry in this cell line.
The results demonstrate that pitstop 2 is an efficient and selective inhibitor of CME that acts via blocking ligand access to the clathrin TD.
5. The clathrin TD plays a crucial role in regulating coated pit dynamics
Clathrin dynamics were studied in more detail in view of the lack of knowledge of the physiological role of ligand binding to the clathrin TD in endocytosis. Live Cos? cells stably expressing eGFP-clathrin light chains (LC) were monitored by total internal reflection microscopy (TIRFM) at 37°C. Cells were treated with DMSO (0.1 %) or 30 μΜ pitstop 2. Consistent with previously reported data, clathrin LC-containing CCPs formed and disappeared at the plasma membrane with most events exhibiting lifetimes between 26 and 89 s. A smaller fraction of long-lived clathrin-coated structures were detected with lifetimes of more than 90 s. However, a low level of short-lived events were also observed that could not be quantitatively tracked by the imaging software utilised and were therefore excluded from analysis. Treatment of cells with pitstop 2 led to a dramatic shift towards long-lived clathrin-coated structures, many of which exhibited lifetimes well beyond the usual imaging time (limited by the eventual bleaching of clathrin LC-eGFP). Increased CCP lifetimes were paired with a minor decrease in the mean clathrin LC-eGFP intensity per object in TIRFM. A slightly increased dimming of CCPs in confocal was noticed when compared to TIRF imaging, presumably due to increased bleaching of stalled clathrin puncta. Thus, surprisingly, pitstop 2-mediated interference with clathrin TD-ligand association does not have a strong effect on the stability of membrane-associated clathrin, but dramatically perturbs CCP dynamics.
To corroborate the role of clathrin TD function in CCP dynamics, fluorescence recovery after photobleaching (FRAP) was paired with live cell spinning disc confocal microscopy imaging. Cells were imaged after the addition of 30 μΜ pitstop 3 of DMSO (0.1%). Frames were taken at 0.5 Hz, starting 10s before FRAP, followed by imaging for another 2 minutes after photobleaching. Following the bleaching laser pulse, clathrin LC-eGFP fluorescence rapidly recovered to about 80 % of its initial value with a τ of approximately 30 s in control cells, consistent with the mean CCP lifetimes measured by TIRFM-based particle tracking. However, pitstop 2 treated cells did not show recovery of clathrin LC-eGFP fluorescence within 120 s, confirming that clathrin TD function indeed regulates CCP dynamics. Recovery was so slow that a reliable τ value could not be determined under the imaging conditions utilised. These data indicate that pitstop 2 stalls the maturation and/ or consumption of pre-existing CCPs.
The question of whether clathrin TD function may be required for the de novo assembly of clathrin-coated structures was then evaluated. For this, use was made of 1 -butanol, an alcohol which depletes cellular PI(4,5)P2 levels causing CCP disassembly and loss of clathrin from the plasma membrane. Clathrin LC-eGFP containing pits rapidly reformed after washout of 1 -butanol in control and pitstop 2-treated cells, indicating that TD-ligand interactions are not required for the de novo recruitment or assembly of clathrin at the plasma membrane. This conclusion was further supported by ultrastructural analysis (see Fig. 6 and Fig. 7).
If pitstop 2 mainly interferes with CCP dynamics but not with clathrin recruitment relatively subtle effects on the distribution of endogenous endocytic proteins would be expected. No significant changes were observed in the colocalization of clathrin, FCHo 1/2, intersectin, AP-2, or dynamin 2 (Dyn2) by either qualitative or quantitative analysis. A different behavior was seen for endogenous amphiphysin 1, which was partially lost from the plasma membrane of pitstop 2-treated cells. Thus, amphiphysin and possibly other clathrin box ligands require association with the clathrin TD for function, while other components retain their plasma membrane localization, likely via binding to AP-2, to membrane PI(4,5)P2, or both. However, it cannot be ruled out that even those endocytic proteins that appear to be localized i ■ '
correctly at the light microscopic level may exhibit functional defects upon acute inhibition of complex formation with the clathrin TD.
The distribution of various TGN or endosomal proteins including AP-1, EEA1, Gadkin, CD63, TGN46, or the mannose 6-phosphate receptor MPR46 was also determined. None of these factors showed a significant change in localization within 15 min of pitstop 2 application. However, a partial loss of transferrin receptor (TfR) was noted from perinuclear endosomes, which instead appeared to be shifted towards the cell surface of pitstop 2-treated cells, consistent with the observed defects in Tf endocytosis (Fig. 5 A, B).
6. Dynamics of FCHo2, AP-2, and dynamin in pitstop 2-treated cells
As pitstop 2 was found to impair clathrin dynamics, the behavior of other key endocytic proteins which act either prior to or concomitant with clathrin assembly was evaluated. FCHo proteins are early-acting factors postulated to facilitate CCP formation by linking PI(4,5)P2-rich membrane sites to endocytic AP-2 binding scaffold proteins intersectin and Epsl5/ Epsl5R. TIRFM-based imaging of eGFP-FCHo2 dynamics in Cos7 cells revealed that FCHo2 -containing structures were relatively long- lived, with lifetimes between 26 s and over 120 s. The mean lifetimes of FCHo2-eGFP- containing puncta were dramatically increased following application of pitstop 2 with puncta appearing nearly immobile. Consistent with their increased lifetimes, FCHo2- positive spots failed to recover in FRAP experiments. Hence, clathrin TD function is required for the productive consumption of nascent CCPs containing FCHo2.
Surprisingly, pitstop 2 induced a comparatively minor shift in the mean life span of AP- 2o-eGFP puncta. These results are consistent with our observation that pitstop 2-treated cells remain able to sequester cargo into CCPs, a process largely mediated by AP-2.
Finally, the life span of dynamin.2 (Dyn2) was investigated. Dyn2 is a component required for fission of mature CCPs. Dynamin 2-eGFP displayed a lifetime distribution similar to that observed for AP-2 or clathrin. Application of pitstop 2 shifted this distribution to longer life spans with a behavior that was intermediary between the effects seen for clathrin and AP-2. 7. Morphological analysis of pitstop 2-treated cells
The live imaging data reported herein collectively provides evidence that pitstop 2-mediated inhibition of CME results from defects in endocytic protein dynamics, most strikingly seen for FCHo2 and clathrin, but not in clathrin recruitment. As CME is a constitutive process in non-neuronal cells with CCPs forming and disassembling continuously, it would be expected that acute perturbation of endocytic protein dynamics by pitstop 2-mediated inhibition of clathrin TD function results in acute "trapping" of clathrin-coated intermediates representing multiple stages of the pathway. To analyze whether this is the case, control or pitstop 2-treated Cos7 cells were subjected to electron microscopic and morphometric analyses. Clathrin-coated endocytic intermediates were classified according to their morphological profiles as shallow CCPs, non-constricted u-shaped CCPs, constricted Ω-shaped CCPs
corresponding to late fission stages, and structures containing a complete clathrin coat (Fig. 6A). These latter intermediates may comprise free clathrin-coated vesicles as well as late intermediates that remain attached to the plasma membrane via a connection not visible in the plane of the section. mAs predicted, endocytic profiles of control cells and those incubated with pitstop 2 appeared very similar. No major changes in the total number of CCPs (Fig. 6B) or in the representation of various endocytic stages were observed (Fig. 6C). It was, therefore, concluded that pitstop-mediated inhibition of clathrin TD function acutely arrests CME at multiple stages.
8. Pitstops inhibit endocytic recycling of synaptic vesicles in neurons
One of the most prominent functions of CME in vertebrates is the exo-endocytic recycling of synaptic vesicle (SV) membranes at nerve terminals. Unlike CME in non- neuronal cells analyzed thus far, endocytic reformation of SVs in neurons is strictly coupled to stimulation-dependent exocytosis resulting in a wave of endocytic activity. The effects of pitstop 2-mediated clathrin inhibition on SV reformation in cultures of primary hippocampal neurons were investigated using electron microscopy. Chemical stimulation of SV exocytosis resulted in a pronounced depletion of SVs from nerve terminals of pitstop 2-treated neurons, suggesting that SV reformation by endocytosis was inhibited. No significant changes in SV numbers were seen in resting control neurons. Loss of S V membranes in pitstop 2-treated neurons appeared to be counterbalanced partly by presynaptic membrane expansion. The poor membrane penetration of pitstop 1 prevented analysis of its effects on endocytosis in living cells. However, its high solubility in aqueous media provided ideal conditions for its intracellular application by microinjection. The effect of pitstop 1 on SV endocytosis in the lamprey reticulospinal synapse was therefore assessed (Pechstein et al., 2010), a model perfectly suited to test whether acute inhibition of ligand association with the clathrin TD inhibits CME but permits clathrin recruitment. Pitstop 1 was injected into lamprey reticulospinal axons and stimulated at 5 Hz for 20 min to induce SV recycling at a physiological rate. Analysis of ultrathin sections revealed a profound loss of SVs at active zones in synapses from these axons (Fig. 7 A, B, G), compared to control non-injected axons (Fig. 7C). No morphological alterations were observed in axons microinjected with pitstop 1 and kept at rest (Fig. 7E). Loss of SVs in stimulated axons was accompanied by a dramatic expansion of the plasma membrane, which due to anatomical constraints formed membrane pockets around the active zone (Fig. 7A, B, H). A significant accumulation of CCPs was observed within the membrane expansions compared to control synapses (Fig. 7-C, I). Consistent with observations in the present study in Cos 7 cells, no dramatic alterations in stage representation of CCPs was observed (Fig. 7J). Analysis of CCPs at high-magnification (Fig. 7D, E) and by tilting in the microscope did not reveal detectable differences in their morphology. It was, therefore, concluded that intra- axonal application of pitstop 1 strongly inhibits SV recycling in situ by inhibiting CME at multiple stages.
9. Discussion
Two small molecule inhibitors of clathrin TD function termed pitstops 1 and 2 that competitively interfere with the association of endocytic clathrin box ligands were developed. In vitro experiments in conjunction with structural data based on protein X-ray crystallography as well as experiments in living cells clearly demonstrated that these compounds selectively perturb clathrin TD function. Effects of pitstop 2 on CME are evident within 2-5 min of cell treatment, presumably only being limited by the rate of drug diffusion across the cell membrane. Reversible inhibition of CME was observed in a broad variety of cells at concentrations closely matching the IC50 values of the in vitro binding experiments. While pitstop 2 readily crosses cell membranes, pitstop 1 has reduced efficacy compared to pitstop 1. The similarity of phenotypes exhibited by pitstops 1 and 2 in lamprey reticulospinal synapses in situ and in Cos 7 cells in vitro provides evidence that both compounds share a common clathrin TD- based molecular mechanism of action. In the presence of pitstop 2, clathrin- independent internalization pathways and secretory traffic remain unperturbed (data not shown). As such, pitstops represent novel cellular tools to selectively inhibit CME.
The use of pitstops in living cells revealed a novel and unexpected role for the clathrin TD in endocytic pit dynamics. Although the clathrin TD represents the most frequently used interaction hub within the endocytic network, surprisingly little is known regarding the functional role of TD-clathrin box ligand interactions. The use of pitstop 2 to acutely inhibit ligand access to the clathrin TD provides evidence for a central role for clathrin TD function in regulating CCP dynamics in mammalian cells. Mammalian cell endocytosis differs from yeast where clathrin plays a comparably minor role in endocytosis. While it might be expected that pitstops interfere with clathrin recruitment to the membrane, live cell imaging revealed specific effects on CCP dynamics instead. These data are explained by a model where pitstops interfere with the progression of CCPs at multiple stages. Consistent with this, the accumulation of distinct endocytic intermediates in pitstop 2-treated cells was not observed by electron microscopy.
Proteins that contain clathrin box motifs comprise a diverse set of factors implicated in both early and late stages of endocytosis (Brodsky et al., 2001 ;
Dell' Angelica, 2001). These include adaptors such as AP180/CALM, Dab2 and β- arrestins (ter Haar et al., 2000), membrane deforming proteins (e.g. epsin and amphiphysin; Slepnev et al., 2000), as well as uncoating factors (i.e. auxilin/GAK or the PI(4,5)P2-hydrolyzing enzymes synaptoj anin-p 170 and OCRL. This functional diversity of clathrin TD ligands may contribute to the observation that pitstop inhibits CME at multiple stages.
Blocking ligand access to the TD by pitstops may stall productive maturation of CCPs by preventing dynamic exchange of key endocytic factors driving progression of the pathway or monitoring completion of the previous step. The unexpected finding that pitstop-mediated inhibition of ligand binding to the clathrin TD does not cause clathrin dissociation from membranes nor prevents the de novo assembly of clathrin- containing structures at the plasma membrane suggests that other interactions may be crucial for clathrin recruitment. Clathrin is most highly expressed in the brain where it is concentrated at presynaptic nerve terminals. Evidence from morphological, genetic and knockdown studies has suggested that CME may be a key mechanism for the regeneration of SVs during stimulation induced exo-endocytic cycling. A drawback of RNAi and genetic approaches is that changes in clathrin levels occur over several days or even the entire lifespan of the animal, making it difficult to distinguish direct from indirect effects. This is important as synapses can adapt to chronic loss of endocytic proteins (Kim and Ryan, 2009). Using pitstops as reported herein to acutely perturb clathrin TD function provides a direct demonstration for an important role of clathrin in S V recycling in vertebrate neurons under conditions of high activity.
The data reported herein also provides strong evidence that HIV enters cells largely via CME. Hence, the pitstop inhibitors developed herein and their derivatives thereof may serve drugs for blocking the entry of viruses and pathogens which hijack the clathrin machinery, including HIV, hepatitis C virus, ebola virus, and Listeria monocytogenes amongst others.
EXAMPLE 2: Inhibition of clathrin function induces aberrant mitotic defects and inhibition of cancer cell division 1. Clathrin function is essential for mitosis
An acute 90 min treatment of HeLa carcinoma cells with pitstop 1 or pitsop 2 caused an increase in mitotic index that is, the percentage of cells in metaphase, in a dose-dependent manner (Fig. 8). The data also suggests that clathrin is required for chromosome segregation. Consistent with this role, clathrin was observed to locate to the mitotic spindle (Fig. 9C). Characterisation of the mitotic spindle revealed that pitstop-treated cells have (1) a thicker metaphase plate, indicating that chromosomes were not aligned properly (Fig. 9C), (2) a reduction in spindle width at the cell equator (Fig. 9A-C), (3) a reduction in spindle microtubules (Fig. 9C), and (4) disrupted HURP staining indicating unattached K-fibres (Fig. 10).
In particular, treatment with pitstops caused the percentage of narrow spindles to increase from 10% and 14% in untreated and DMSO control cells respectively to 24% in pitstop 1 treated cells and 29% in pitstop 2 treated cells. This phenotype was analogous with the phenotype induced in clathrin-depleted cells using specific siRNA, wherein the percentage of cells with narrow spindles increased to 45%. Further, untreated controls, DMSO and dynole 34-2 treated cells had HURP mis-localisation in 1 1%, 10%, and'l l%,of cells respectively. However, pitstop 1 treated cells exhibited HURP mis-localisation in 41% of the cells, whilst in cells treated with pitstop 2, the level was 39% of cells. These levels were comparable to the 42% of cells with HURP mis-localisation seen in clathrin depleted cells after siRNA.
The graph in Fig. 9B also shows that in cells treated with pitstop 1 or pitstop 2, the spindles become significantly (P>0.01) narrower with a median ratio of 0.4 (pitstop 1) and 0.35 (pitstop 2) of the total cell width. Depletion of clathrin by siRNA also caused a significant decrease in the median spindle width ratio to 0.36 of the total cell width as compared to untreated control (0.65), DMSO (0.67), and the dynamin (and endocytosis) inhibitor dynole 34-2 (0.63).
In addition to the aberrant mitotic spindles formed when clathrin is inhibited or depleted from cells, an increase in the percentage of cells harboring multipolar spindles was observed (>2 spindle poles; P>0.01), an effect also seen in siRNA clathrin depleted cells. Centrioles were identified by Centin2 staining. Spindle microtubules (MTs) originate from the centrosome (spindle pole), which consists of two centrioles surrounded by percentriolar matrix (PCM). The fidelity of the PCM is important for completion of metaphase in cells undergoing mitosis. The spindle poles of pitstop- treated cells did not affect PCM organization as indicated by γ-tubulin staining. In contrast, a significant number of spindle poles contained less than two centrioles. This phenotype was again analogous to that induced by clathrin siRNA, and suggests that clathrin may participate in centriole cohesion. Untreated, DMSO treated, and dynole 34-2 treated control cells showed a normal count of 2 centriole centrosomes. Dynole 34-2 (2-Cyano-N-octyl-3-(l-(3-dimethylaminopropyl)-lH-indol-3-yl)-acrylamide (Hill et al., 2009) is a potent inhibitor of dynamin II (Dyn2). Dyn2 plays a central role in endocytosis.
Thus, the action of the pitstop compounds is consistent with targeted inhibition of clathrin. Moreover, these findings indicate that the pitstop compounds prevent mitotic progression by specifically blocking the onset of chromosome segregation and further, that they appear to activate the spindle assembly checkpoint (SAC) in mitosis. 2. Clathrin plays a role in mitotic spindle assembly
Next a MT re- growth assay utilising HeLa cells was employed to determine if clathrin was required for mitotic spindle assembly. This involved allowing
microtubules to regrow for 5 min at 37°C following their disassembly by cold treatment (cells were placed on ice for 30 mins). Depletion of clathrin by siRNA prevented MT re-growth from centrosomes in metaphase cells. In contrast, neither pitstop 1 nor pitstop 2 mediated inhibition of clathrin prevented MT regrowth from the mitotic spindle poles. As expected clathrin no longer decorated the mitotic spindle and spindle poles in cells after clathrin knockdown by siRNA.
In particular, in control cells, DMSO, and dynole 34-2 (30 μΜ) treated cells the
MT regrew to median lengths of 3 μΜ, 3.5 μΜ, and 3.8 μΜ respectively. This recovery was not impeded by treatment with the pitstops 1 or 2 (30 μΜ), with spindle jegrowth reaching a median MT length of 4 μΜ (pitstop 1) or 3.7 μΜ (pitstop 2). The absence of clathrin protein itself by siRNA treatment caused a significant delay in MT recovery with a median length of 1.3 uM. However, the pitstop compounds do not affect the mitotic spindle localisation of clathrin (Fig. 8C).
The data indicate that mitotic spindle pole localization of clathrin is required for mitotic spindle assembly and that clathrin acts as a scaffold for recruitment of key spindle proteins required for spindle growth. Clathrin forms a complex with TACC3 at the mitotic spindle. Consistent with clathrin being required for TACC3 spindle localization, TACC3 spindle localization was not affected by pitstop-mediated inhibition as clathrin remained on the spindle under these conditions. In contrast, TACC3 spindle localisation was significantly reduced by siRNA-mediated depletion of clathrin by >25% as evidenced by quantitation of TACC3 staining and fluorescence intensity.
3. Pitstops activate the spindle assembly checkpoint
To confirm that pitstops activate the SAC the recruitment of a critical SAC component, MAD2, to the kinetochores was observed. In untreated and DMSO-treated control cells MAD2 did not locate to the kinetochores, as indicated by a lack of co- localisation with the centromere component, CENPB (Fig. 11). This indicates that the SAC has not been activated and cells are able to proceed to segregate their chromosomes. In contrast, MAD2 co-localised or located adjacent to CENPB foci in HeLa cells treated with either pitstop compound and in clathrin-depleted HeLa cells (Fig. 11). Depletion of another endocytic protein, epsin, whose function is also associated with metaphase, did not result in recruitment of MAD2 to the kinetochores and thus the SAC was not active. It wasconcluded that the pitstops activate the SAC.
4. Clathrin inhibitors reduce cell growth in a range of cancer cell lines
Several anti-mitotic compounds have anti-cancer properties by causing cell death following activation of the SAC. Aurora A selective inhibitors, such as
MLN8237, represent such compounds. As pitstop 1 and pitstop 2 also activate the SAC, the question of whether they can inhibit cell proliferation and induce cell death was assessed. Cell growth was assessed by MTT assay in twelve cancer cell lines derived from different tissues: namely, SMA-560 (mouse glioma), SJ-G2 and U87
(glial HeLa (cervical), MCF-7 (breast), A2780 (ovary), H460 (lung), A431 (skin), DU145 (prostate), MIA (pancreas), BE(2)-C (neuronal) and HT29 and SW480 (colon). Pitstop 1 had no effect on cell growth after 72 hours of continuous exposure. In contrast, pitstop 2 caused a dose-dependent decline in cell growth in four of the cancer cell lines, namely, HT29, BE2-C and U87 at low concentrations and A431 at a higher concentration (see Table 8). The best (lowest) GI50 value (concentration that causes 50% growth inhibition) was > 5 μΜ. To improve potency and cell permeability, analogues of pitstop 2 were generated. The structures of these analogues are shown in Table 7. All eleven analogues were significantly more potent at inhibiting growth and reducing viability in all cancer cell lines than the parent pitstop 2. KAM-6-H12 and KAM-6-H53 were the most potent in this series and were also verified to retain inhibtion of Amph binding to the clathrin terminal domain (TD). A2780 ovarian cancer cells were the most sensitive cells. Other analogues in the same family as pitstop 1 showed 10 fold more potent inhibition of cancer cell growth at nanomolar
concentrations (data not shown). Thus, clathrin inhibitors can prevent cell proliferation and reduce viability in a range of cancer cell lines, favourable properties for an anti- cancer agent. Table 7: Growth inhibition (GI50) induced by clathrin inhibitors
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
5. Inhibition of clathrin function causes cytokinesis failure
Clathrin has a second role in mitosis at the final stage of mitosis - cytokinesis. Unlike the role of clathrin at the mitotic spindle, its role in cytokinesis is thought to be dependent on its endocytic function. Consistent with this, inhibition of dynamin II (Dyn2), a key endocytic protein that is required for cytokinesis, utilising dynole 34-2 caused an increase in multinucleated cells, an indicator of cytokinesis failure (Fig. 11). Depletion of clathrin using siRNA and pitstop 1 and pitstop 2 -mediated inhibition of clathrin also caused multinucleation (Fig. 12). Hence, the pitstop compounds induce cytokinesis failure consistent with targeted inhibition of clathrin.
6. Discussion
The endocytic protein clathrin plays two roles important for mitotic progression: 1) at the mitotic spindle to stablise K-fibres for chromosome segregation; and 2) during cytokinesis for membrane abscission to generate to independent daughter cells. The present study shows that the small molecule inhibitors of clathrin, pitstop 1 and pitstop 2, produce mitotic phenotypes consistent with these two mitotic roles for clathrin. As such, these compounds and their analogues represent extremely valuable tools that can be exploited to characterise the molecular mechanism(s) of action of clathrin during mitosis. More globally, the use of these compounds provide for an improved understanding of the molecular mechanisms of mitotic progression in cells, and hence of the fundamental cell division process.
New classes of potential chemotherapeutics are being developed that specifically target key mitotic proteins to either activate or inhibit the SAC, such as Aurora kinase and kinesin spindle protein. These targeted inhibitors prevent proliferation of most tumour cells in vitro and reduce tumour volume in vivo by inhibiting growth and/or triggering cell death following SAC activation/inhibition (Taylor and Peters, 2008). Consequently, many targeted inhibitors, such as the Aurora kinase inhibitor MLN8054 (Millennium, USA), are currently in cancer clinical trials (Ma and Adjei, 2008), and are expected to have a more favourable therapeutic window than currently used chemotherapeutic agents such as taxol, as they would spare non- dividing, non-differentiating cells. The ability of these mitotic inhibitors to be efficacious anti-cancer agents is dependent on their ability to induce apoptosis following mitotic insult as it is predicted to result in tumour regression. However, they do not always result in cell death with varied cellular responses reported which include apoptosis, mitotic catastrophe, mitotic slippage, senescence and reversible mitotic arrest with continued cell growth. Hence, there has been an ongoing need for the
identification of new targets and the development of new compounds to broaden the therapeutic index of anti-mitotic agents as anti-cancer chemotherapeutics. The results described herein demonstrate that small molecule inhibitors of clathrin are potent anti- mitotic compounds that activate the SAC and may be exploited as anti-cancer therapeutic agents.
EXAMPLE 3: Synthesis of pitstops 1. Sodium 2-(4-aminobenzyl)-l, 3-dioxo-2,3-dihydro-lH- benzo[de]isoquinoline- 5 -sulfonate (Pitstop 1)
Figure imgf000066_0001
Step 1 : 3-sulfo-l,8-naphthalic anhydride, sodium salt
1,8-Naphthalic anhydride (0.990 g, 5.00 mmol) was dissolved in oleum (6 mL). The resulting solution was stirred at 120 °C for 1 hour (until a drop of the mixture, when added to water, did not precipitate), then cooled to room temperature. The cooled solution was then poured into water (25 mL). Addition of aqueous saturated sodium chloride (20 mL) resulted in precipitation of the product. The white solid was collected by filtration, washed with water (5 mL), then ethanol (10 mL), and dried under vacuum.
Yield 1.462 g (95.6 %), MP > 300 °C.
Ή-NMR (DMSO-ί ί) δ 7.91 (1H, t, J= 7.4 Hz), 8.49 (1H, d, J= 7.5 Hz), 8.62
(lH, d, J= 4.7 Hz), 8.65 (lH, s), 8.73 (lH, s).
13C-NMR (DMSO-^) 119.7, 128.4, 130.0, 130.3, 131.4, 131.6, 133.1, 136.5, 147.7, 161.1, 161.2. Step 2: 2-(4-Aminobenzyl)-l,3-dioxo-2,3-dihydro-lH-benzo[de]isoquinoline-5- sulfonic acid
4-Amino(methyl)aniline (0.092 g, 0.75 mmol), DMF (1 mL) and triethylamine (0.5 mL) were added to a suspension of 3-sulfo-l,8-naphthalic anhydride, sodium salt (0.197 g, 0.66 mmol) in toluene (5 mL). The resulting suspension was refluxed at 130 °C for 18 hours. The reaction mixture was then cooled to room temperature and the precipitated product collected by filtration, washed with toluene, and dried under vacuum. A yellow-orange solid was obtained.
Yield 0.167 g (65 %), MP > 300 °C.
Ή-NMR (DMSO-c¾ δ 4.97 (2H, br s, NH2), 5.07 (2H, s), 6.46 (2H, d, J= 8.4 Hz), 7.07 (2H, d, J= 8.4 Hz), 7.87 (lH, dd, J= 7.5, 8.1 Hz), 8.49 (1H, dd, J= 0.9, 7.5 Hz), 8.55 (1H, dd, J= 0.9, 8.1 Hz), 8.65 (1H, d, J= 1.5 Hz), 8.68 (1H, d, J= 1.5 Hz);
13C-NMR (DMSO-4 ; 42.7, 113.8, 122.1, 122.2, 124.5, 127.3, 127.8, 128.9, 129.2, 130.2, 131.2, 131.3, 135.3, 147.2, 148.0, 163.5, 163.6 ppm. 2. (Z)-N-(5-(4-bromobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)naphth^ sulfonamide (Pitstop 2)
Figure imgf000067_0001
To 1 -naphthalene sulfonyl chloride (420 mg, 1.85 mmol) in dry THF (30 mL) was added pseduothiohydantoin (221 mg, 1.9 mmol) and triethylamine (0.56 mL, 4.0 mmol). The resultant mixture was heated at reflux overnight to produce a brownish
r
precipitate in an orange solution. EtOAc (50 mL) was added and the mixture washed with water (2 x 50 mL), dried (MgSC ) and evaporated in vacuo. An attempt to purify using chromatography failed to yield a clean sample and so the crude material (150 mg,
MS 305 [M-H]') was used directly in the next step. This material was dissolved in
EtOH (50 mL) and -bromobenzaldehyde (120 mg, 0.65 mmol) and piperidine
(2 drops) was then added. After heating at reflux for 3 h the volume of the resultant yellow solution was reduced by half and then cooled. The precipitate formed was filtered off, washed with ether (2 x 10 mL) and dried in vacuo to yield the desired product; (132 mg, 15% overall) mp 230 °C (decomp).
1H NMR (300 MHz, DMSO-4 : δ 8.69 (d, J = 8.4 Hz, lH), 8.17 (d, J = 7.2
Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.61 (m, 3H), 7.46 (d, J = 8.5 Hz, 2H), 7.29 (s, 1H) ppm.
13C NMR (75 MHz, DMSO-rftf): 181.0, 174.8, 138.7, 133.9, 133.5, 132.4, 132.0,
131.8, 131.0, 128.3, 127.9, 127.0, 126.9, 126.3, 125.8, 125.1, 124.3, 121.9 ppm. MS:
471 [M-H]". EXAMPLE 4: Analogues of Sodium 2-(4-aminobenzyl)-l,3-dioxo-2,3-dihydro- lH-benzofdeJisoquinoline-5-sulfonate (Pitstop 1)
The lead compound that allowed development of Pitstop 1 is 8-(3-carboxy-7- oxo-7H-benzo[i/e]benzo[4,5]imidazo[2, 1 -a]isoquinolin-4-yl)naphthalene- 1,4,5- tricarboxylic acid (LI) (see Table 1). Reductive retrosynthetic fragmentation of LI revealed the compound is essentially a substituted 1,8-naphthalimide derivative, comprised of a 1,8-naphthalic anhydride and a 1,2-phenylenediamine moiety attached to a naphthalimide core. The naphthalimide core was utilised as a minimal
pharmoacophoric entity. The chemistry for preparation of the 1,8-naphthalimide scaffold is well-established and so allows for the rapid generation of analogues, and hence the development of a SAR profile. Scaffold simplification of LI for the design of inhibitors of clathrin-TD is shown in Scheme 4 and identified inhibitors of clathrin- TD are set out in Tables 8-10.
Figure imgf000069_0001
Scheme 4: Scaffold simplification of LI reveals a naphthalimide core, and provides a pharmacophoric entity for the rational design of clathrin-TD inhibitors.
Table 8: Compounds identified by an ELISA-based screening strategy as inhibitors of the activity of clathrin. For the purposes of identifying clathrin-selective inhibitors, the dynamin I IC50 values are also provided.
Dynamin l t Compound Structure Clathrin IC50 Selectivity
Figure imgf000069_0002
Figure imgf000070_0001
Selectivity was calculated using [Clathrin ICso]/[Dynamin I IC50]. A higher selectivity value indicates a greater selectivity for clathrin, whilst a selectivity value <1 indicates an inhibitor that is more selective for dynamin I. Table 9: Inhibition of clathrin and dynamin I GTPase by anhydride modified analogues 2, 11-23.
Figure imgf000071_0001
Clathrin IC50 Dynamin IC50
Compound ID R3 4 Rs
(μΜ) (μΜ)
2 S03Na H H 18 -
11 H H H - -
12 H S03K . H - -
13 S03K NH2 H -100 123 ±48
14 H Br H - 106 ±12
15 H NH2 H - 82
16 H N02 H - 157 ±35
17 H CI H - 125 ± 25
18 N02 H H - 129 ±53
19 OH H H - 13 ±6
20 N02 H N02 116 ±60
21 Br H H - 141 ±47
22 NH2 H H - 22 ±4
23 OCH3 H H - 142 ± 38
(-) Not Active - No inhibitory activity observed at 100 μΜ; (~100) ~50 % inhibition at 100 μΜ. Table 10: Inhibition of Clathrin-TD and Dynamin I GTPase by compounds in
24-36.
Figure imgf000072_0001
24 0 H H NH2 - >300
25 1 H H H 6.9 ± 1.6 -
26 1 H H COOH -100 185 ±39
27 1 NH2 H H 10±2 108 ±52
28 1 H H CI 10± 1 111
29 1 H H OCH3 16±3 -
30 1 H H Br 15 ±4 -
31 1 H H N(CH3)2 -100 -
32 1 H H N02 - 49 ± 22
33 1 H NH2 H -100 264
34 1 H H OH 22 ±1 -
35 1 H H CH3 15 ±4 -
36 1 H H F 12 ±2 >300
Clathrin IC50 Dynamin IC50
Compound n R7 Rs R9
(μΜ) (μΜ)
24 0 H H NH2 - >300
25 1 H H H 6.9 ±1.6 -
26 1 H H COOH -100 185 ±39
27 1 NH2 H H 10±2 108 ± 52
28 1 H H CI 10± 1 111
29 1 H H OCH3 16 ± 3 -
30 1 H H Br 15 ± 4 31 1 ,Η H N(CH3)2 -100 - .
32 1 H H N02 - 49 ± 22
33 1 H NH2 H -100 264
34 1 H H OH 22 ± 1 -
35 1 H H CH3 15 ± 4 -
36 1 H H F 12 ± 2 > 300
(-) Not Active - No inhibition observed at 100 uM (clathrin) or 300 μΜ (dynamin);
(-100) Approximately 50 % inhibition at a drug concentration of 100 uM. 1. Synthesis of compounds
Figure imgf000073_0001
Scheme 5: Synthesis of 2 from 1,8-naphthalie anhydride. Reagents and Conditions:
(a) (i) Fuming sulfuric acid (30 % S03), 90 °C, 90 min; (ii) Saturated KCl, 4 °C, 18 hr;
(b) 4-aminobenzylamine (3 equiv.), ethanol, 100 °C, 18 hr.
Figure imgf000073_0002
Scheme 6: Generic reaction scheme for the synthesis of N-4-benzylamino substituted naphthylimides. Reagents and conditions: appropriately substituted naphthylimide, 4-aminobenzylamine (3 equivalents), TEA (3 drops), Ethanol, 100 °C, 18 hr. 1.1 3-Sulfo-N-(4-aminobenzyl)-l , 8-naphthalimide, potassium salt (2)
To a stirring suspension of 3-sulfo-l,8-naphthalic anhydride (potassium salt) (0.302 g, 0.95 mmol) in ethanol ( 15 mL), was added TEA ( 10 drops) and
4-aminobenzylamine (350 μΐ,, 3.09 mmol). An excess of amine was used to minimise formation of the di-imide. The resulting off-white suspension was stirred at 100 °C for 18 hours, then cooled to room temperature with stirring. The cream product was collected by filtration, washed sequentially with ethanol and ether, and dried under vacuum. A cream solid was obtained.
Yield 0.401 g (85 %); MP > 250 °C; 1 H-NMR (DMSO-</tf) δ 4.97 (2H, br s,
NH2), 5.07 (2H, s), 6.46 (2H, d, J= 8.4 Hz), 7.07 (2H, d, J= 8.4 Hz), 7.87 (1H, dd, J= 7.5, 8.1 Hz), 8.49 (1H, dd, J= 0.9, 7.5 Hz), 8.55 (1H, dd, J= 0.9, 8.1 Hz), 8.65 (1H, d, J= 1.5 Hz), 8.68 (1H, d, J= 1.5 Hz); 13C-NMR (100 MHz) (DMSO-</tf) δ 42.9, 114.0 (2C), 122.3 (2C), 124.7, 127.4, 128.1, 128.9, 129.3 (2C), 130.4, 131.3, 131.6, 135.4, 147.0, 148.0, 163.6, 163.7 ppm; MS (ESI-) m/z: 381 (M - K) (100 %); HRMS (ESI-) m/z calculated for Ci9H13KN205S (M - K) 381.0551
1.2 3-Sulfo-N-(5-carboxy-2-hydroxyphenyl)-l, 8-naphthalimide, sodium salt (3) Synthesised using the same general procedure as for analogue 2 above, commencing with 3-sulfo-l,8-naphthalic anhydride (sodium salt) and 3-amino-4- hydroxybenzoic acid. Product was isolated as the triethylamine salt. A pale brown solid was obtained.
Yield 0.142 g (38 %); MP > 250 °C; 1 H-NMR (DMSO-<¾ δ 0.99 (9H, Et3N, t, J = 7.2 Hz), 2.60 (6H, Et3N, q, J= 7.2 Hz), 7.00 (1H, d, J= 8.4 Hz), 7.82 (1H, d, J= 2.1 Hz), 7.86 (1H, dd, J= 2.1, 8.4 Hz), 7.90 (1H, dd, J= 12, 8.4 Hz), 8.48 (1H, dd, J= 0.9, 7.2 Hz), 8.60 (1H, dd, J= 0.9, 8.4 Hz), 8.65 (1H, d, J= 1.5 Hz), 8.70 (1H, d, J= 1.5 Hz); ,3C-NMR (100 MHz) (DMSO-^) δ 10.9 (3C, Et3N), 45.7 (3C, Et3N), 116.3, 122.6, 122.9 (2C), 124.4, 127.8, 127.9, 128.6, 130.1, 131.0, 131.3 (2C), 132.3, 135.2, 147.1, 157.1, 163.4, 163.6, 167.8 ppm; MS (ESI-) m/z: 412 (M - Na) (100 %); HRMS (ESI-) m/z calculated for Ci9Hi0NNaO8S (M - Na) 412.0133. 1.3 4-Amino-3-sulfo-N-benzyl-l,8-naphthalimide, potassium salt (4)
Synthesised using the same general procedure as for analogue 2, commencing with 4-amino-3-sulfo-l,8-naphthalic anhydride (potassium salt) and benzylamine. A yellow solid was obtained.
Yield 0.148 g (60 %); MP > 250 °C; 1H-NMR (DMSO c¾ δ 5.31 (2H, s), 7.24 (5H, m), 7.69 (1H, dd, J= 7.2, 8.4 Hz), 7.87 (2H, br s), 8.43 (1H, d, J= 7.2 Hz), 8.63 (1H, s), 8.71 (1H, d, J= 8.4 Hz); 13C-NMR (DMSO-i¾ δ 42.7, 106.5, 120.8, 121.9, 124.8, 125.2, 127.0, 127.6 (2C), 128.5 (2C), 129.8, 130.2, 131.5, 133.1, 138.1, 148.6, 163.2, 163.9 ppm; MS (ESI-) m/z: 381 (M - ) (100 %); HRMS (ESI-) m/z calculated for C19H13KN2O5S (M - K) 381.0551.
1.4 4-Amino-3-sulfo-N-(2-mercaptoethyl)-l,8-naphthalimide, potassium salt (5) Synthesised using the same general procedure as for analogue 2, commencing with 4-amino-3-sulfo-l,8-naphthalic anhydride (potassium salt) and cysteamine. A yellow solid was obtained.
Yield 0.245 g (82 %); MP > 250 °C; 1H-NMR (DMSO-</tf) δ 2.96 (2H, m), 4.31 (2H, m), 7.69 (1H, dd, J= 7.2, 8.4 Hz), 7.84 (2H, br s), 8.43 (1H, d, J= 7.2 Hz), 8.61 (1H, s), 8.70 (1H, d, J= 8.4 Hz); 13C-NMR (DMSO-ck) δ 35.2, 38.2, 106.5, 120.8, 121.8, 124.8, 125.1, 129.7, 130.1, 131.4, 133.0, 148.5, 163.0, 163.9 ppm; MS (ESI-) m/z: 351 (M - K); HRMS (ESI-) m/z calculated for CnHnKNzC^ (M - K) 351.0115
1.5 4-Amino-3,6-disulfo-N-benzyl-l,8-naphthalimide, dipotassium salt (6)
To a stirred suspension of 4-amino-3,6-sulfo-l ,8-naphthalic anhydride, potassium salt (0.6 mmol), in 1 M Li+/H+ acetate pH 5 buffer (5 mL), was added benzylamine (3.3 mmol). The reaction mixture was stirred at 120 °C for 18 hours, then diluted to 20 mL with hot water. Addition of KC1 (~0.5 g), followed by cooling to 4 °C, resulted in precipitation of the product. The resulting suspension was then stored at 4 °C for 24 hours. The solid product was collected by filtration, washed sequentially with water, ethanol and ether, and dried under vacuum. A yellow-grey solid was obtained. Yield 0.117 g (49 %); MP > 250 °C; 1H-NMR (DMSO-<¾ δ 5.22 (2H, s, H,), 7.25 (5H, m), 7.99 (2H, br s, NH2), 8.60 (IH, s), 8.64 (IH, d, J= 0.9 Hz), 8.94 (IH, d, J = 0.9 Hz); ,3C-NMR (DMSO-c¾ δ 42.7, 106.4, 120.3, 121.6, 125.4, 126.6, 127.1, 127.5 (2C), 128.6 (2C), 129.4, 129.7, 133.3, 138.0, 144.9, 149.3, 163.1, 163.8 ppm; MS (ESI- ) m/z: 499 (M - ), 461 (M - 2 + H), 230 (M - 2 ) (100 %); HRMS (ESI-) m/z calculated for d9H12K2N208S2 (M - K) 498.9678.
1.6 4-Amino-3-sulfo-N-propyl-l,8-naphthalimide, potassium salt (7) Synthesised using the same general procedure as for 6, commencing with
4-amino-3-sulfo-l,8-naphthalic anhydride (potassium salt) and ^-propylamine. A yellow solid was obtained.
Yield 0.104 g (48 %); MP > 250 °C; 1H-NMR (DMSO-^s) δ 0.89 (3H, t, J= 7.5 Hz, H3 ), 1.61 (2H, sextet, J= 7.5 Hz, Η2·), 3.97 (2H, t, J= 7.5 Hz, Hr), 7.68 (IH, dd, J = 7.2, 8.4 Hz, H6), 7.80 (2H, br s, NH2), 8.42 (IH, d, J= 7.2 Hz, H7), 8.61 (IH, s, H2), 8.68 (IH, d, J= 8.4 Hz, H5); ,3C-NMR (DMSO-4 δ 11.6 (C3 , 21.1 (C2-), 41.0 (Cr), 106.8 (CO, 120.8 (C4a), 122.0 (C8), 124.7 (C6), 125.2 (C3), 129.6 (C8a), 129.9 (C5), 131.2 (C7), 132.8 (C2), 148.3 (C4), 163.1 (Cio), 163.9 (C9) ppm; MS (ESI-) m/z: 333 (M - K) (100 %); HRMS (ESI-) m/z calculated for CisH^K^OsS (M - K) 333.0551.
1.7 4-Amino-3-sulfo-N-ethyl-l,8-naphthalimide, potassium salt (8)
Synthesised using the same general procedure as for 6, commencing with 4- amino-3-sulfo-l,8-naphthalic anhydride (potassium salt) and ethylamine. A yellow solid was obtained.
Yield 0.187 g (85 %); MP > 250 °C; 1H-NMR (DMSO-</6) δ 1.17 (3H, t, J= 6.9 Hz, ¾■), 4.04 (2H, q, J= 6.9 Hz, Hr), 7.68 (IH, dd, J= 7.2, 8.4 Hz, H6), 7.80 (2H, br s, NH2), 8.42 (IH, dd, J= 0.6, 7.2 Hz, H7), 8.62 ('IH, s, H2), 8.68 (IH, dd, J= 0.6, 8.4 Hz, H5); 13C-NMR (DMSO-.¾ δ 13.5 (Cr), 34.5 (Cr), 106.8 (Ci), 120.8 (C4a), 122.1 (C8), 124.7 (C6), 125.1 (C3), 129.6 (C8a), 129.9 (C5), 131.2 (C7), 132.8 (C2), 148.3 (C4)s 162.9 (Co), 163.7 (C9) ppm; MS (ESI-) m/z: 319 (M - K) (100 %); HRMS (ESI-) m/z calculated for C,4HnKN205S (M - K) 319.0394. 1.8 3-Nitro-N-(2-carboxy-4-hydroxyphenyl)-l, 8-naphthalimide (9)
Synthesised using the same general procedure as for 2, commencing with 3 -nitro- 1,8-naphthalic anhydride and 2-amino-5-hydroxybenzoic acid. Product was isolated as the triethylamine salt. A yellow-brown solid was obtained.
Yield 0.246 g (64 %); MP 224-226 °C; Ή-NMR (DMSO-i/< δ 1.00 (9H, Et3N, t, J= 7.2 Hz), 2.59 (6H, Et3N, q, J= 7.2 Hz), 7.32 (1H, d, J= 8.1 Hz), 7.47 (lH, dd, J= 1.5, 8.1 Hz), 7.55 (1H, d, J= 1.5 Hz), 8.09 (1H, dd, J= 7.2, 8.4 Hz), 8.70 (1H, dd, J= 0.9, 7.2 Hz), 8.83 (1H, dd, J= 0.9, 8.1 Hz), 8.98 (1H, d, J= 2.4 Hz), 9.53 (1H, d, J= 2.4 Hz); ,3C-NMR (100 MHz) (DMSO-<¾ δ 10.3 (3C, Et3N,), 45.5 (3C, Et3N,), 117.4,
120.0. 123.1, 123.3, 124.7, 125.1, 129.5, 129.8, 130.1, 130.2, 131.2, 134.1, 136.7 (2C),
146.1, 153.3, 162.2, 162.7, 168.3 ppm; MS (ESI-) m/z: 377 (M - H) (100 %), 755 (2M - H); HRMS (ESI-) m/z calculated for C19H10N2O7 (M - H) 377.0415 1.9 3-Nitro-N-(4-carboxy-2-hydroxyphenyl)-l, 8-naphthalimide (10)
Synthesised using the same general procedure as for analogue 2, commencing with 3-nitro- 1,8-naphthalic anhydride and 4-amino-3-hydroxybenzoic acid. Product was isolated as the triethylamine salt. A brown solid was obtained.
Yield 0.254 g (64 %); MP 230-232 °C; 1H-NMR (DMSO-c¾ δ 0.87 (9H, Et3N, t, J= 7.2 Hz), 2.58 (6H, q, Et3N, J= 7.2 Hz), 6.93 (1H, dd, J= 2.7, 8.4 Hz), 7.12 (IH, d, J= 8.4 Hz), 7.44 (1H, d, J= 2.7 Hz), 8.06 (ΪΗ, dd, J= 7.2, 8.4 Hz), 8.64 (1H, dd, J= 0.9, 7.2 Hz), 8.79 (1H, dd, J= 0.9, 8.4 Hz), 8.92 (1H, d, J= 2.4 Hz), 9.49 (1H, d, J= 2.4 Hz), 9.83 (1H, br s, OH); 13C-NMR (100 MHz) (DMSO-fik) δ 9.2 (3C, Et3N), 45.1 (3C, Et3N), 117.5, 117.9, 122.8, 123.5, 125.0, 126.5, 129.4, 129.7, 130.1, 130.9, 131.2, 133.9, 134.9, 136.3, 146.0, 157.3, 162.9, 163.4, 167.5 ppm; MS (ESI-) m/z: 377 (M - H) (100 %), 755 (2M - H); HRMS (ESI-) m/z calculated for C19HioN207 (M - H) 377.0415 1.10 N-(4-Aminobenzyl)-l, 8-naphthalimide (11)
Synthesised using the same general procedure as for analogue 2, commencing with 1,8-naphthalic anhydride and 4-aminobenzylamine. A yellow solid was obtained. Yield 0.359 g (95 %); MP > 250 °C; 1H-NMR (DMSO-^) δ 4.96 (2H, br s), 5.06 (2H, s), 6.46 (2H, d, J= 8.4 Hz), 7.08 (2H, d, J= 8.4 Hz), 7.85 (2H, dd, J= 7.2, 8.4 Hz), 8.43 (2H, dd, J= 1.5, 8.4 Hz), 8.49 (2H, dd, J= 1.5, 7.2 Hz); 13C-NMR (DMSO-4 5 42.7, 113.8 (2C), 122.1 (2C), 124.6, 127.4 (3C), 129.4 (2C), 131.0 (2C), 131.4, 134.5 (2C), 148.0, 163.5 (2C) ppm; MS (ESI+) m/z: 303 (M + H) (calc); HRMS (ESI+) m/z calculated for Ci9H,4N202 (M + H) 303.1128
1.11 4-Sulfo-N-(4-aminobenzyl)-l , 8-naphthalimide, potassium salt (12) Synthesised using the same general procedure as for analogue 2, commencing with 4-sulfo-l ,8-naphthalic anhydride (potassium salt) and 4-aminobenzylamine.
Yield 0.314 g (94 %); MP > 250 °C; 1H-NMR (DMSO-ck) 64.95 (2H, br s), 5.06 (2H, s), 6.46 (2H, d, J= 8.4 Hz), 7.07 (2H, d, J= 8.4 Hz), 7.87 (1H, dd, J= 7.2, 8.4 Hz), 8.21 (1H, d, J= 7.5 Hz), 8.47 (1H, d, J= 7.5 Hz), 8.49 (1H, dd, J= 1.2, 7.2 Hz), 9.25 (1H, dd, J= 1.2, 8.4 Hz); ,3C-NMR (DMSO-¾ 542.7, 113.8 (2C), 122.2, 122.9, 124.5, 125.2, 127.0, 127.7, 128.2, 129.3 (2C), 130.5, 130.8, 134.4, 148.0, 150.1, 163.3, 163.7 ppm; MS (ESI-) m/z 381 (M - K) (100 %); HRMS (ESI-) m/z calculated for C19H13 N2O5S (M - K) 381.0551. 1.12 . 4-Amino-3-sulfo-N-(4-aminobenzyl)-l, 8-naphthalimide, potassium salt (13)
To a stirring su pension of 4-amino-3-sulfo-l,8-naphthalic anhydride (potassium salt) (0.200 g, 0.60 mmol) in ethanol (7 mL), was added TEA (3 drops) and 4- aminobenzylamine (0.221 g, 1.81 mmol). A 3-fold excess of 4-aminobenzylamine was used to minimise formation of the di-imide. The resulting yellow-orange suspension was stirred at 100 °C for 18 hours, then cooled to room temperature with stirring. The yellow product was collected by filtration, washed sequentially with ethanol and ether, and dried under vacuum.
Yield 0.245 g (94 %); MP > 250 °C; 1H-NMR (DMSO-^) δ 4.92 (2H, br s), 5.02 (2H, s), 6.45 (2H, d, J= 8.4 Hz), 7.04 (2H, d, J= 8.4 Hz), 7.68 (1H, dd, J= 7.2, 8.4 Hz), 7.82 (2H, br s), 8.42 (1H, dd, J= 1.2, 7.2 Hz), 8.63 (1H, s), 8.68 (1H, dd, J= 1.2, 8.4 Hz); l3C.-NMR (DMSO-<¾) δ 42.3, 106.8, 113.8 (2C), 120.8, 122.1, 124.8, 125.1, 125.2, 129.2 (2C), 129.6, 130.0, 131.4, 133.0, 147.8, 148.4, 163.2, 163.9 ppm; MS (ESI-) m/z: 396 (M - K) (100 %); HRMS (ESI-) m/z calculated for C^HMKNSOSS (M - K) 396.0660.
1.13 4-Bromo-N-(4-aminobenzyl)-l, 8-naphthalimide (14)
Synthesised using the same general procedure as for analogue 2, commencing with 4-brofno-l,8-naphthalic anhydride and 4-aminobenzylamine.
Yield 0.316 g (91 %); MP 238-240 °C; Ή-NMR
Figure imgf000079_0001
δ 4.97 (2H, br s), 5.04 (2H, s), 6.47 (2H, d, J= 8.4 Hz), 7.08 (2H, d, J= 8.4 Hz), 7.94 (lH, dd, J= 7.2, 8.4 Hz), 8.16 (1H, d, J= 7.8 Hz), 8.30 (1H, d, J= 7.8 Hz), 8.49 (1H, dd, J= 1.2, 8.4 Hz), 8.54 (1H, dd, J= 1.2, 8.4 Hz); 13C-NMR (DMSO-^) δ 42.8, 113.7 (2C), 121.9, 122.7, 124.3, 128.2, 128.8, 129.3, 129.6 (2C), 129.8, 131.1, 131.4, 131.8, 132.7, 148.1, 162.8, 162.9 ppm; MS (ESI+) m/z: 381 (M + H) (calc); HRMS (ESI+) m/z calculated for Ci9H13BrN202 (M + H) 381.0233. 1.14 4-Amino-N-(4-aminobenzyl)-l, 8-naphthalimide (15)
Synthesised using the same general procedure as for analogue 2, commencing with 4-amino- 1 ,8-naphthalic anhydride and 4-aminobenzylamine.
Yield 0.342 g (91 %); MP > 250 °C; Ή-NMR (DMSO-</6) δ 4.92 (2H, br s), 5.01 (2H, s), 6.45 (2H, d, J= 8.4 Hz), 6.83 (1H, d, J= 8.4 Hz), 7.04 (2H, d, J= 8.4 Hz), 7.42 (2H, br s, NH2), 7.63 (1H, dd, J= 7.2, 8.4 Hz), 8.18 (1H, d, J= 8.4 Hz), 8.41 (1H, dd, J= 1.2, 7.2 Hz), 8.59 (1H, dd, J= 1.2, 8.4 Hz); 13C-NMR (DMSO-4 δ 42.2, 107.8, 108.4, 113.7 (2C), 119.6, 122.0, 124.2, 125.3, 129.3 (2C), 129.5, 129.8, 131.3, 134.2, 147.8, 152.9, 163.1, 163.9 ppm; MS (ESI+) m/z: 318 (M + H) (calc); HRMS (ESI+) w/z calculated for Ci9H15N302 (M + H) 318.1237
1.15 4-Nitro-N-(4-aminobenzyl)- 1, 8-naphthalimide (16)
Synthesised using the same general procedure as for analogue 2, commencing with 4-nitro-l,8-naphthalic anhydride and 4-aminobenzylamine. A 2-fold excess of 4-aminobenzylamine was used to minimise formation of the di-imide and suppress displacement of the 4-nitro group. A pink-brown solid was obtained. Yield 0.313 g (87 %); MP 218-220 °C; 1H-NMR (£)MSO-d6) δ 4.89 (2H, br s, NH2), 5.01 (2H, s), 6.47 (2H, d, J= 8.4 Hz), 7.06 (2H, d, J= 8.4 Hz), 7.97 (1H, dd, J= 7.5, 8.7 Hz), 8.42 (1H, d, J= 7.8 Hz), 8.52 (1H, d, J= 7.8 Hz), 8.53 (1H, dd, J= 0.9, 7.5 Hz), 8.58 (1H, dd, J= 0.9, 8.7 Hz); ,3C-NMR (DMSO-4s) 5 43.1, 113.8 (2C), 122.7, 122.8, 124.0, 124.4, 126.5, 128.3, 128.9, 129.5 (2C), 129.9, 130.2, 132.0, 148.2, 149.2, 162.2, 163.0 ppm; MS (ESI+) m/z: 348 (M + H) (calc); HRMS (ESI+) m/z calculated for C9H13N3O4 (M + H) 348.0979.
1.16 4-Chloro-N-(4-aminobenzyl)-l,8-naphthalimide (\l)
Synthesised using the same general procedure as for analogue 2,' commencing with 4-chloro-l,8-naphthalic anhydride and 4-aminobenzylamine. A yellow solid was obtained.
Yield 0.325 g (88 %); MP 228-230 °C; 1H-NMR (DMSO-rf*) 54.97 (2H, br s), 5.04 (2H, s), 6.47 (2H, d, J= 8.4 Hz), 7.08 (2H, d, J= 8.4 Hz), 7.93 (1H, dd, J= 7.5,
8.4 Hz), 7.96 (1H, d, J= 7.8 Hz), 8.38 (1H, d, J= 7.8 Hz), 8.52 (1H, dd, J= 1.2, 7.5 Hz), 8.53 (1H, dd, J= 1.2, 8.4 Hz); l3C-NMR (DMSO-4 δ 42.8, 113.7 (2C), 121.4, 122.7, 124.3, 127.8, 128.3, 128.5, 128.7, 129.5 (2C), 130.2, 131.1, 131.8, 137.7, 148.1, 162.7, 163.0 ppm; MS (ESI+) m/z: 337 (M + H) (calc); HRMS (ESI+) m/z calculated for C9H13CIN2O2 (M + H) 337.0738'
1.17 3-Nitro-N-(4-aminobenzyl)-l,8-naphthalimide (18)
Synthesised using the same general procedure as for analogue 2, commencing with 3-nitro-l,8-naphthalic anhydride and 4-aminobenzylamine. A light orange-brown solid was obtained.
Yield 0.296 g (83 %); MP > 250 °C; Ή-NMR (DMSO-rf5) δ 4.99 (2H, br s),
5.05 (2H, s), 6.47 (2H, d, J= 8.4 Hz), 7.09 (2H, d, J= 8.4 Hz), 7.99 (1H, dd, J= 7.2, 8.4 Hz), 8.63 (1H, dd, J= 1.2, 7.2 Hz), 8.70 (1H, dd, J= 1.2, 8.4 Hz), 8.91 (1H, d, J= 2.4 Hz), 9.41 (1H, d, J= 2.4 Hz); 13C-NMR (DMSO-4 δ 43.0, 113.7 (2C), 122.5,
123.1, 123.9, 124.0, 129.3, 129.4, 129.5 (2C), 129.8, 130.9, 134.2, 136.4, 145.9, 148.2, 162.3, 162.7 ppm; MS (ESI+) m/z 348 (M + H) (calc); HRMS (ESI+) m/z calculated for CI9Hi3N304 (M + H) 348.0979. 1.18 3-Hydroxy-N-(4-aminobenzyl)-l,8-naphthalimide (19)
Synthesised using the same general procedure as for analogue 2, commencing with 3-hydroxy- l,8-naphthalic anhydride and 4-aminobenzylamine. A yellow solid was obtained.
Yield 0.312 g (83 %); MP > 250 °C; Ή-NMR (DMSO-t ) δ 4.96 (2H, br s), 5.03 (2H, s), 6.46 (2H, d, J= 8.4 Hz), 7.06 (2H, d, J= 8.4 Hz), 7.62 (1H, d, J= 2.4 Hz), 7.71 (1H, dd, J= 7.2, 8.4 Hz), 8.01 (lH, d, J= 2.4 Hz), 8.20 (1H, dd, J= 1.2, 8.4 Hz), 8.23 (1H, dd, J= 1.2, 7.2 Hz), 10.45 (lH, br s, OH); 13C-NMR (DMSO-t¾ δ 42J, 113.7 (2C), 116.0, 122.0, 122.1, 122.2, 123.5, 124.6, 127.5, 127.7, 129.3 (2C), 132.8, 133.5, 148.0, 156.4, 163.3, 163.7 ppm; MS (ESI+) m/z: 319 (M + H) (calc); HRMS (ESI+) m/z calculated for C19H14N2O3 (M + H) 319.1077.
1.19 3, 6-Dinitro-N-(4-aminobenzyl)-l, 8-naphthalimide (20)
Synthesised using the same general procedure as for analogue 2, commencing with 3,6-dinitro-l,8-naphthalic anhydride and 4-aminobenzylamine. A dark brown solid was obtained.
Yield 0.244 g (72 %); MP 218-220 °C; Ή-NMR (DMSO-4 δ 4.99 (2H, br s, NH2), 5.07 (2H, s), 6.46 (2H, d, J= 8.4 Hz), 7.09 (2H, d, J= 8.4 Hz), 9÷04 (2H, d, J = 2.1 Hz), 9.71 (2H, d, J= 2.1 Hz); I3C-NMR (100 MHz) (DMSO-i¾ δ 43.4, 113.8 (2C), 123.7, 124.5 (2C), 126.2 (2C), 129.6 (2C), 130.9, 131.6 (2C), 131.7, 147.2 (2C), 148.3, 161.8 (2C) ppm; MS (ESI+) m/z: 393 (M + H) (calc); HRMS (ESI+) m/z calculated for C19H12N4O6 (M + H) 393.0830.
1.20 3-Bromo-N-(4-aminobenzyl)-l, 8-naphthalimide (21)
Synthesised using the same general procedure as for analogue 2, commencing with 3-bromo-l,8-naphthalic anhydride and 4-aminobenzylamine. A pale yellow solid was obtained. ί
Yield 0.316 g (91 %); MP 220-222 °C; 1H-NMR (DMSO-4 δ 4.98 (2H, br s),
5.03 (2H, s), 6.46 (2H, d, J= 8.4 Hz), 7.07 (2H, d, J= 8.4 Hz), 7.86 (1H, dd, J= 7.2,
8.4 Hz), 8.35 (lH, dd, J = 1.2, 8.4 Hz), 8.41 (lH, d, J= 1.8 Hz), 8.47 (1H, dd, J= 1.2, 7.2 Hz), 8.70 (1H, d, J= 1.8 Hz); ,3C-NMR (100 MHz) (DMSO-c¾ δ 43.0, 113.9 (2C), 120.4, 122.5, 124.2, 124.5, 126.2, 128.8, 129.5 (2C), 131.5, 133.0 (2C), 133.8, 136.1, 148.2, 162.7, 163.3 ppm; MS (ESI+) m/z: 381 (M + H) (calc); HRMS (ESI+) m/z calculated for Ci9H13BrN202 (M + H) 381.0233
1.21 3-Amino-N-(4-aminobenzyl)-l,8-naphthalimide (22)
Synthesised using the same general procedure as for analogue 2, commencing with 3-amino-l,8-naphthalic anhydride and 4-aminobenzylamine. A yellow solid was obtained.
Yield 0.320 g (86 %); MP 248-250 °C; 1H-NMR (OMSO-d6) δ 4.95 (2H, br s),
5.03 (2H, s), 5.97 (2H, br s, NH2), 6.46 (2H, d, J= 8.4 Hz), 7.04 (2H, d, J= 8.4 Hz), 7.27 (1H, d, J= 2.4 Hz), 7.60 (1H, dd, J= 7.2, 8.4 Hz), 7.96 (1H, d, J= 2.4 Hz), 8.02 (1H, dd, J= 1.2, 8.4 Hz), 8.07 (1H, dd, J= 1.2, 7.2 Hz); 13C-NMR (DMSO-40 5 42.5, 1 12.0, 1 13.7 (2C), 120.7, 121.9, 122.0, 122.7, 124.7, 125.7, 127.1, 129.2 (2C), 131.7, 133.7, 147.9, 148.1, 163.7, 163.9 ppm; MS (ESI+) m/z 318 (M + H) (calc); HRMS (ESI+) m/z calculated for C19H15N3O2 (M + H) 318.1237
1.22 3-Methoxy-N-(4-aminobenzyl)-l,8-naphthalimide (23)
Synthesised using the same general procedure as for analogue 2, commencing with 3-methoxy-l,8-naphthalic anhydride and 4-aminobenzylamine. A pale yellow solid was obtained.
Yield 0.266 g (73 %); MP 238-240 °C; Ή-NMR (DMSO-c¾ δ 3.97 (3H, s), 4.96 (2H, br s), 5.04 (2H, s), 6.46 (2H, d, J= 8.4 Hz), 7.07 (2H, dd, J= 8.4 Hz), 7.79 (1Ή, dd, J= 7.2, 8.4 Hz), 7.89 (1H, d, J= 2.7 Hz), 8.03 (1H, d, J= 2.7 Hz), 8.32 (2H, m); 13C-NMR (DMSO-i¾) δ 42.7, 56.1, 113.5, 113.7 (2C), 121.9, 122.1, 122.9, 123.7, 124.5, 127.9, 128.4, 129.4 (2C), 133.2, 133.3, 148.0, 157.9, 163.2, 163.6 ppm; MS (ESI+) m/z 333 (M + H) (calc); HRMS (ESI+) m/z calculated for C2oHi6N203 (M + H) 333.1234. 1.23 3-Sulfo-N-(4-aminophenyl)-l , 8-naphthalimide, sodium salt (24)
Synthesised using the same general procedure as for analogue 2, commencing with 3-sulfo-l,8-naphthalic anhydride (sodium salt) and 1,4-phenylenediamine. An off- white solid was obtained.
Yield 0.281 g (86 %); MP > 250 °C; Ή-NMR (DMSO-<¼) δ 5.25 (2H, br s), 6.63 (2H, d, J= 8.7 Hz), 6.94 (2H, d, J= 8.7 Hz), 7.88 (1H, dd, J= 7.2, 8.4 Hz), 8.46 (1H, dd, J = 0.9, 7.2 Hz), 8.57 (1H, dd, J= 0.9, 8.4 Hz), 8.63 (1H, d, J= 1.8 Hz), 8.66 (1H, d, J= 1.8 Hz); ,3C-NMR (DMSO-c^) δ 113.9 (2C), 122.8, 122.9, 124.0, 127.7 (2C), 128.6, 129.4 (2C), 130.0, 131.1, 131.2, 135.1, 147.1, 148.8, 164.0, 164.2 ppm; MS (ESI-) m/z: 367 (M - Na) (100 %); HRMS (ESI-) m/z calculated for
Ci8HuN2Na05S (M - Na) 367.0394.
3-Sulfo-N-(benzyl)-l, 8-naphthalimide, potassium salt (25)
To a stirring suspension of 3-sulfo- 1 ,8-naphthalic anhydride (potassium salt) (0.251 g, 0.79 mmol) in 1M Li+/H+ acetate buffer (pH 5, 5mL) was added benzylamine (173 μΐ-, 1.58 mmol). The resulting cream-brown suspension was then stirred at 130 °C for 18 hours. The reaction mixture was then diluted to 15 mL with water, and KC1 (~0.5 g) was added. After cooling to room temperature with stirring, the resulting cream-brown suspension was allowed to stand at 4 °C overnight, to increase product precipitation. The precipitated product was then collected by filtration, washed sequentially with cold water, ethanol and ether, and dried under vacuum. An off-white solid was obtained. /
Yield 0.267 g (83 %); MP > 250 °C; 1H-NMR (DMSO-<¾ δ 5.26 (2H, s), 7.28
(5H, m), 7.88 (1H, dd, J= 7.2, 8.4 Hz), 8.50 (1H, dd, J= 1.2, 7.2, Hz), 8.57 (1H, dd, J = 1.2, 8.4 Hz), 8.68 (1H, d, J= 1.8 Hz), 8.70 (1H, d, J= 1.8 Hz); 13C-NMR (OMSO-d6) δ 43.1, 122.1 (2C), 127.2, 127.4, 127.6 (2C), 127.9, 128.6 (2C), 129.0, 130.4, 131.2, 131.4, 135.4, 137.5, 147.2, 163.6, 163.7 ppm; MS (ESI-) m/z: 366 (M - K) (100 %); HRMS (ESI-) m/z calculated for C19H12KNO5S (M - K) 366.0442. 1.25 3-Sulfo-N-(4-carboxybenzyl)-l,8rnaphthalimide, potassium salt (26)
Synthesised using the same general procedure as for analogue 25, commencing with 3-sulfo-l,8-naphthalic anhydride (potassium salt) and 4-(aminomethyl)benzoic acid (1.2 equivalents). A pale yellow solid was obtained.
Yield 0.261 g (91 %); MP > 250 °C; 1H-NMR (DMSO-.¾ δ 5.32 (2H, s), 7.46 (2H, d, J= 8.4 Hz), 7 86 (2H, d, J= 8.4 Hz), 7.89 (1H, dd, J= 7.5, 8.1 Hz), 8.50 (1H, dd, J= 1.2, 7.5 Hz), 8.58 (lH, dd, J= 1.2, 8.1 Hz), 8.68 (1H, d, J= 1.8 Hz), 8.70 (IH, d, J= 1.8 Hz); 13C-NMR (DMSO-rftf) 6 43.1, 122.0, 122.1, 127.5 (3C), 127.8, 129.0, 129.6 (2C), 129.7, 130.4, 131.2, 131.4, 135.4, 142.5, 147.2, 163.6, 163.7, 167.3 ppm; MS (ESI-) m/z 410 (M - K); HRMS (ESI-) m/z calculated for C20Hi2KNO7S (M - K) 410.0340.
1.26 3-Sulfo-N-(2-aminobenzyl)-l,8-naphthalimide, potassium salt (27)
Synthesised using the same general procedure as for analogue 2, commencing with 3-sulfo-l,8-naphthalic anhydride (potassium salt) and 2-aminobenzylamine. A yellow solid was obtained.
Yield 0.292 g (86 %); MP > 250 °C; 1H-NMR (DMSO-^) 6 5.02 (2H, s, Hr), 5.15 (2H, br s), 6.43 (1H, m), 6.65 (1H, dd, J= 1.2, 8.1 Hz), 6.87 (1H, dd, J= 1.5, 7.5 Hz), 6.92 (1H, m), 7.89 (1H, dd, J= 7.5, 8.1 Hz), 8.50 (IH, dd, J= 1.2, 7.5 Hz), 8.56 (1H, dd, J= 1.2, 8.1 Hz), 8.68 (IH, d, J= 1.5 Hz), 8.69 (IH, d, J= 1.5 Hz); 13C-NMR (DMSC s) 6 40.3, 1 15.3, 1 16.5, 120.0, 122.1, 122.2, 127.5, 127.6, 128.0, 128.1, 129.1, 130.6, 131.3, 131.8, 135.6, 146.3, 147.0, 163.9, 164.0 ppm; MS (ESI-) m/z: 381 (M - K) (100 %); HRMS (ESI-) m/z calculated for C^HuK zOsS (M - K) 381.0551.
1.27 3-Sulfo-N-(4-chlorobenzyl)-l,8-naphthalimide, potassium salt (28)
To a stirring suspension of 3-sulfo-l,8-naphthalic anhydride (potassium salt) (0.251 g, 0.79 mmol) in 1M Li+/H* acetate buffer (pH 5, 5mL) was added
4-chlorobenzylamine (173 ' uL, 1.58 mmol). The resulting cream-brown suspension was then stirred at 130 °C for 18 hours. The reaction mixture was then diluted to 15 mL with water, and KC1 (-0.5 g) was added. After cooling to room temperature with stirring, the resulting cream-brown suspension was allowed to stand at 4 °C overnight, to increase product precipitation. The precipitated product was then collected by filtration, washed sequentially with cold water, ethanol and ether, and dried under vacuum. An off-white solid was obtained.
Yield 0.057 g (83 %); MP > 250 °C; 1H-NMR (DMSO-^) 6 5.24 (2H, s), 7.34
(2H, d, J= 8.7 Hz), 7.40 (2H, d, J= 8.7 Hz), 7.88 (IH, dd, J= 7.2, 8.4 Hz), 8.50 (IH, dd, J= 1.2, 7.2 Hz), 8.57 (IH, dd, J= 1.2, 8.4 Hz), 8.67 (IH, d, J= 1.5 Hz), 8.69 (IH, d, J= 1.5 Hz); 13C-NMR (DMSO-<¾ 5 42.6, 122.0, 122.1, 127.4, 127.8, 128.5 (2C),
129.0. 129.6 (2C), 130.3, 131.2, 131.4, 131.8, 135.4, 136.5, 147.3, 163.6, 163.7 ppm; MS (ESI-) m/z: 400 (M - K) (100 %); HRMS (ESI-) m/z calculated for C19HnClKN05S
(M - K) 400.0052.
1.28 3-Sulfo-N-(4-methoxybenzyl)-l , 8-naphthalimide, potassium salt (29) Synthesised using the same general procedure as for analogue 28, commencing with 3-sulfo-l,8-naphthalic anhydride (potassium salt) and 4-methoxybenzylamine
(1.05 equivalents). An off-white solid was obtained.
Yield 0.286 g (83 %); MP > 250 °C; 1H-NMR (DMSO-^) δ 3.69 (3H, s), 5.19
(2H, s), 6.84 (2H, d, J= 8.7 Hz), 7.32 (2H, d, J= 8.7 Hz), 7.87 (IH, dd, J= 7.2, 8.1 Hz), 8.49 (lH, dd, J= 1.2, 7.2 Hz), 8.55 (IH, dd, J= 1.2, 8.1 Hz), 8.66 (IH, d, J= 1.8
Hz), 8.69 (IH, d, J= 1.8 Hz); 13C-NMR (DMSO-^) δ 42.5, 55.2, 113.9 (2C), 122.0,
122.1, 127.3, 127.8, 128.9, 129.4 (2C), 129.5, 130.3, 131.2, 131.3, 135.3, 147.3, 158.5, 163.5, 163.6 ppm; MS (ESI-) m/z: 396 (M - K) (100 %); HRMS (ESI-) m/z calculated for C2oHi4 N06S (M - K) 396.0547.
1.29 3-Sulfo-N- (4-bromobenzyl)-l, 8-naphthalimide, potassium salt (30)
Synthesised using the same general procedure as for analogue 28, commencing with 3-sulfo-l,8-naphthalic anhydride (potassium salt) and 4-bromobenzylamine (1.1 equivalents). An off-white solid was obtained.
Yield 0.037 g (77 %); MP > 250 °C; Ή-NMR (DMSO-^) δ 5.22 (2H, s), 7.33 (2H, d, J= 8.4 Hz), 7.48 (2H, d, J= 8.4 Hz) 7.88 (IH, dd, J= 7.2, 8.4 Hz), 8.50 (IH, dd, J= 1.2, 7.2 Hz), 8.57 (IH, dd, J= 1.2, 8.4 Hz), 8.67 (IH, d, J= 1.8 Hz), 8.69 (IH, d, J= 1.8 Hz); 13C-NMR (DMSO-i¾ 6 42.6, 120.3, 122.0, 122.1, 127.4, 127.8, 129.0, 129.9 (2C), 130.3, 131.2, 131.4 (3C), 135.4, 137.0, 147.2, 163.6, 163.7 ppm; MS (ESI-) m/z: 444 (M - K) (100 %); HRMS (ESI-) m/z calculated for Ci9H,iBrKN05S (M - ) 443.9547.
1.30 3-Sulfo-N-(4-(dimethylamino)benzyl)-l,8-naphthalimide, potassium salt (31)
Synthesised using the same general procedure as for analogue 2, commencing with 3-sulfo- 1 ,8-naphthalic anhydride (potassium salt) and 4- (dimethylamino)benzylamine (dihydrochloride salt) (1.2 equivalents). An excess of
TEA was added to react with liberated HC1. A pale yellow solid was obtained.
Yield 0.271 g (95 %); MP > 250 °C; 1H-NMR (DMSO--¾ δ 2.81 (6H, s), 5.14
(2H, s), 6.63 (2H, d, J= 8.7 Hz), 7.23 (2H, d, J= 8.7 Hz), 7.87 (1H, dd, J= 7.5, 8.1
Hz), 8.49 (1H, dd, J= 1.2, 7.5 Hz), 8.55 (1H, dd, J= 1.2, 8.1 Hz), 8.65 (1H, d, J= 1.5 Hz), 8.69 (1H, d, J= 1.5 Hz); 13C-NMR (DMSO--¾ δ 40.3 (2C), 42.6, 112.4 (2C),
122.1 (2C), 125.1, 127.3, 127.8, 128.9, 129.2 (2C3), 130.2, 131.2, 131.3, 135.3, 147.2,
149.9, 163.4, 163.5 ppm; MS (ESI-) m/z: 409 (M - K) (100 %); HRMS (ESI-) m/z calculated for C2]Hi7KN205S (M - K) 409.0864. 1.31 3-Sulfo-N-(4-nitrobenzyl)-l,8-naphthalimide, potassium salt (32)
Synthesised using the same general procedure as for analogue 28, commencing with 3-sulfo- 1,8-naphthalic anhydride (potassium salt) and 4-nitrobenzylamine
(hydrochloride salt) (1.2 equivalents). A light brown solid was obtained.
Yield 0.250 g (88 %); MP > 250 °C; Ή-NMR (DMSO--¾ δ 5.37 (2H, s), 7.63
(2H, d, J= 8.7 Hz), 7.89 (1H, dd, J= 7.2, 8.4 Hz), 8.15 (2H, d, J= 8.7 Hz), 8.50 (1H, dd, J= 1.2, 7.2 Hz), 8.58 (1H, dd, J= 1.2, 8.4 Hz), 8.69 (2H, s); 13C-NMR (DMSO-i¾ δ 43.0, 122.0 (2C), 123.7 (2C), 127.6, 127.9, 128.6 (2C), 129.0, 130.5, 131.2, 131.5, 135.5, 145.4, 146.8, 147.2, 163.6, 163.7 (C9) ppm; MS (ESI-) m/z: 411 (M - K) (100 %); HRMS (ESI-) m/z calculated for Ci9HnK 207S (M - K) 411.0292. 1.32 3-Sulfo-N-(3-aminobenzyl)-l ,8-naphthalimide, potassium salt (33)
Synthesised using the same general procedure as for analogue 2, commencing with 3-sulfo-l,8-naphthalic anhydride (potassium salt) and 3-aminobenzylamine. A pale yellow solid was obtained.
Yield 0.271 g (83 %); MP > 250 °C; 1H-NM (DMSO-ck) δ 4.96 (2H, br s), 5.12 (2H, s), 6.39 (1H, ddd, J= 1.2, 2.1, 7.8 Hz), 6.46 (1H, d, J= 2.1 Hz), 6.49 (1H, dd, J= 1.2, 7.8 Hz), 6.92 (1H, t, J= 7.8 Hz), 7.88 (1H, dd, J= 7.2, 8.1 Hz), 8.50 (1H, dd, J = 1.2, 7.2 Hz), 8.56 (lH, dd, J= 1.2, 8.1 Hz), 8.67 (lH, d, J= 1.8 Hz), 8.70 (1H, d, J= 1.8 Hz); 13C-NMR (DMSO-rfe) δ 43.2, 112.4, 112.8, 115.0, 122.1 (2C), 127.4, 128.0, 129.0 (2C), 130.4, 131.2, 131.5, 135.4, 138.0, 147.1, 149.0, 163.5 (C,0), 163.6 (C9) ppm; MS (ESI-) m/z: 381 (M - K) (100 %); HRMS (ESI-) m/z calculated for
Ci9H13K 205S (M - K) 381.0551. 1.33 3-Sulfo-N-(4-hydroxybenzyl)-l,8-naphthalimide, potassium salt (34)
Synthesised using the same general procedure as for analogue 28, commencing with 3-sulfo-l,8-naphthalic anhydride (potassium salt) and 4-hydroxybenzylamine (1.2 equivalents). A cream-brown solid was obtained.
Yield 0.293 g (88 %); MP > 250 °C; 1H-NMR (DMSO-tf*) δ 5.14 (2H, s), 6.67
(2H, d, J= 8.7 Hz), 7.20 (2H, d, J= 8.7 Hz), 7.87 (1H, dd, J= 7.2, 8.1 Hz), 8.49 (1H, dd, J= 1.2, 7.2 Hz), 8.55 (1H, dd, J= 1.2, 8.1 Hz), 8.65 (1H, d, J= 1.5 Hz), 8.69 (1H, d, J= 1.5 Hz), 9.27 (1H, br s); 13C-NMR (DMSO-ok) δ 42.6, 115.2 (2C), 122.1 (2C), 127.3, 127.8 (2C), 128.9, 129.4 (2C), 130.3, 131.2, 131.3, 135.3, 147.2, 156.6, 163.5, 163.6 ppm; MS (ESI-) m/z 382 (M - K) (100 %); HRMS (ESI-) m/z calculated for Ci9H,2KN06S (M - K) 382.0391.
1.34 3-Sulfo-N-(4-methylbenzyl)-l ,8-naphthalimide, potassium salt (35) Synthesised using the same general procedure as for analogue 28, commencing with 3-sulfo-l,8-naphthalic anhydride (potassium salt) and 4-methylbenzylamine (0.6 equivalents). An off-white solid was obtained. Yield 0.365 g (100 %); MP > 250 °C; 1H-NMR (DMSO-i/«$) δ-2.23 (3H, s), 5.21 (2H, s), 7.09 (2H, d, J= 8.1 Hz), 7.25 (2H, d, J= 8.1 Hz), 7.88 (1H, dd, J = 7.2, 8.4 Hz), 8.49 (1H, dd, J= 1.2, 7.2 Hz), 8.56 (1H, dd, J= 1.2, 8.4 Hz), 8.67 (1H, d, J= 1.8 Hz), 8.69 (1H, d, J= 1.8 Hz); 13C-NMR (DMSC-c¾ δ 20.8, 42.9, 122.0, 122.1, 127.4, 127.7 (2C), 127.8, 128.9, 129.1 (2C), 130.3, 131.2, 131.4, 134.5, 135.4, 136.3, 147.2, 163.5, 163.6 ppm; MS (ESI-) m/z: 380 (M - K) (100 %); HUMS (ESI-) m/z calculated for C20H14 NO5S (M - K) 380.0598.
1.35 3-Sulfo-N-(4-fluorobenzyl)-l,8-naphthalimide, potassium salt (36)
Synthesised using the same general procedure as for analogue 28, commencing with 3-sulfo-l,8-naphthalic anhydride (potassium salt) and 4-fluorobenzylamine (0.1 equivalents). An off-white solid was obtained.
Yield 0.049 g (73 %); MP > 250 °C; 1H-NMR (QMSO-ek) δ 5.23 (2H, s), 7.10 (2H, t, J= 8.7 Hz), 7.42 (2H, dd, J= 5.7, 8.7 Hz), 7.88 (lH, dd, J= 7.2, 8.4 Hz), 8.50 (1H, dd, J= 1.2, 7.2 Hz), 8.57 (1H, dd, J= 1.2, 8.4 Hz), 8.67 (1H, d, J= 1.5 Hz), 8.69 (1H, d, J= 1.5 Hz, H2); 13C-NMR (DMSO-c¾ δ 42.5 (C, , 115.2 (2JCF = 21.3 Hz, 2C4), 122.0, 122.1, 127.4, 127.8, 129.0, 129.9 (3JCF = 8.2 Hz, 2C), 130.3, 131.2, 131.4, 133.7 (4JCF = 2.1 Hz), 135.4, 147.2, 161.4 ('JcF = 242.9 Hz), 163.5, 163.6 ppm; MS (ESI-) m/z: 384 (M - ) (100 %); HRMS (ESI-) m/z calculated for Ci9H,iFKN05S (M - ) 384.0347.
Although the invention has been described with reference to a number of exemplary embodiments, the skilled addressee will understand that numerous variations and modifications may be made without departing from the scope of the invention. Accordingly, the embodiments described above are not to be taken as being limiting. LITERATURE REFERENCES
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Claims

1. A method for inhibiting clathrin, comprising contacting clathrin with an effective amount of a clathrin inhibitor for forming a complex with a binding site on the the terminal domain (TD) of clathrin for the inhibitor, the binding site of the clathrin TD being defined by at least one or more of amino acids He 52, He 62, lie 80, Phe 91 and/or He 93 of SEQ ID No. 1, or a prodrug, or physiologically acceptable salt, of the inhibitor.
2. A method according to claim 1 wherein the binding of the inhibitor to the clathrin TD is stabilised by one or more hydrogen bonds between the inhibitor and the clathrin TD.
3. A method according to claim 1 or 2 wherein the binding of the inhibitor is provided by binding interactions of the inhibitor with at least some of amino acids
He 52, He 62, He 80, Phe 91 and/or lie 93 of SEQ. No. 1.
4. A method according to claim 3 wherein the binding of the inhibitor is provided by binding interactions with all of amino acids He 52, He 62, He 80, Phe 91, and lie 93 of SEQ ID No.l.
5. A method according to claim 3 or 4 wherein the binding site is further defined by one or more of amino acids He 66, Arg 64, Leu 82 and/or Lys 96 of SEQ ID No. 1, and the binding of the inhibitor is provided by binding interactions of the inhibitor with the one or more further amino acids.
6. A method according to claim 4 wherein the inhibitor interacts with all of amino He 66, Arg 64, Leu 82 and Lys 96 of SEQ ID. No. 1.
7. A method according to claim 3 or 4 wherein the binding site is further defined by amino acid Val 50 of SEQ ID No. 1 and the binding of the inhibitor is provided by binding interaction of the inhibitor with that amino acid.
8. A method according to any one of claims 3 to 7 wherein respective of the binding interactions between the inhibitor with the clathrin TD comprise direct contact of the inhibitor with the clathrin TD.
9. A method according to any one of claims 1 to 6 wherein the binding of the inhibitor to the clathrin TD is stabilised by one or more hydrogen bonds between the inhibitor and amino acids selected from the group consisting of Ser 67, Gin 89, Val 50, He 62, and Arg 64 of SEQ ID. No. 1.
10. A method according to claim 9 wherein the binding of the inhibitor to the clathrin TD is stabilised by hydrogen bonds between the inhibitor and amino acids
Ser 67, Gin 89, Val 50, He 62, and Arg 64 of SEQ ID. No. 1.
11. A method according to any one of claims 1 to 4 wherein the binding of the inhibitor to the clathrin TD is stabilised by one or more hydrogen bonds between the inhibitor and Arg 64 of SEQ ID No. 1.
12. A method according to any one of claims 1 to 11 comprising administering an effective amount of the inhibitor to an animal for inhibiting clathrin in the animal, or a prodrug, or physiologically acceptable salt, of the inhibitor.
13. A method according to claim 12 wherein the inhibitor is administered for the inhibition of mitosis or clathrin-mediated endocytosis (CME).
14. A method for prophylaxis or treatment of a disease or condition in the animal comprising administering an effective amount of a clathrin inhibitor for forming a complex with the terminal domain (TD) of clathrin, the inhibitor binding to a binding site of the terminal domain (TD) of clathrin wherein the binding site of the clathrin TD is defined by at least one or more of amino acids He 52, He 62, He 80, Phe 91 and/or He 93 of SEQ ID No. 1, or a prodrug or physiologically acceptable salt of the inhibitor.
15. A method according to claim 14 wherein the binding of the inhibitor to the clathrin TD is stabilised by one or more hydrogen bonds between the inhibitor and the clathrin TD.
16. A method according to claim 14 or 15 wherein the binding of the inhibitor is provided by binding interactions of the inhibitor with at least some of amino acids
He 52, lie 62, He 80, Phe 91 and/or He 93 of SEQ. No. 1.
17. A method according to claim 16 wherein the binding of the inhibitor is provided by binding interactions with all of amino acids He 52, He 62, He 80, Phe 91, and lie 93 of SEQ ID No.l.
18. A method according to any one of claims 14 to 17 wherein the binding site is further defined by one or more of amino acids He 66, Arg 64, Leu 82 and/or Lys 96 of SEQ ID No. 1 , and the binding of the inhibitor is provided by binding interactions of the inhibitor with the one or more further amino acids.
19. A method according to claim 18 wherein the binding of the inhibitor is provided by binding interactions with all of amino He 66, Arg 64, Leu 82 and Lys 96 of
SEQ ID. No. 1.
20. A method according to any one of claims 14 to 17 wherein the binding site is further defined by amino acid Val 50 of SEQ ID No. 1 and the binding of the inhibitor is provided by binding interaction of the inhibitor with that amino acid.
21. A method according to any one of claims 16 to 20 wherein respective of the binding interactions between the inhibitor with the clathrin TD are direct contact of the inhibitor with the clathrin TD.
22. A method according to any one of claims 14 to 18 wherein the binding of the inhibitor to the clathrin TD is stabilised by one or more hydrogen bonds between the inhibitor and amino acids selected from the group consisting of Ser 67, Gin 89, Val 50, He 62, and Arg 64 of SEQ ID. No. 1.
23. A method according to claim 22 wherein the binding of the inhibitor to the clathrin TD is stabilised by hydrogen bonds between the inhibitor and amino acids
Ser 67, Gin 89, Val 50, He 62, and Arg 64 of SEQ ID. No. 1.
24. A method according to claim 20 wherein the binding of the inhibitor to the clathrin TD is stabilised by one or more hydrogen bonds between the inhibitor and Arg 64 of SEQ ID No. 1.
25. A method according to any one of claims 14 to 24 wherein the disease or condition is selected from the group consisting of epilepsy, Alzheimer's disease, Lewy body disease, Lewy body dimentia, Parkinson's disease, β-amyloid associated diseases, Dent's disease, pathogenic infections involving clathrin mediated endocytosis (CME) for infection of cells, cancer, cell proliferative disorders, neuropathic pain, multifocal leukoencephalopathy, psychotic disorders, psychosis, bipolar disorders, schizophrenia, aberrant up-regulated neuronal excitation, seizures, migraine, mucolipidosis, and cell trafficking, neurodegenerative, neuropyschiatric and neurological disease and conditions mediated by, or associated with, synaptic signal transmission or CME.
26. A clathrin inhibitor for use in inhibiting clathrin in a mammal, wherein the inhibitor binding to a binding site of the terminal domain (TD) of clathrin forms a complex with the clathrin TD, the binding site of the clathrin TD being defined by at least one or more of amino acids He 52, He 62, He 80, Phe 91 and/or lie 93 of SEQ ID No. 1.
27. A clathrin inhibitor wherein the inhibitor binding to a binding site of the terminal domain (TD) of clathrin forms a complex with the clathrin TD, the binding site of the clathrin TD being defined by at least one or more of amino acids lie 52, He 62, He 80, Phe 91 and/or He 93 of SEQ ID No. 1.
28. An inhibitor according to claim 26 or 27 wherein the binding of the inhibitor is provided by binding interactions of the inhibitor with at least some of amino acids
He 52, He 62, He 80, Phe 91 and/or He 93 of SEQ. No. 1.
29. An inhibitor according to claim 28 wherein the binding of the inhibitor is provided by binding interactions of the inhibitor with all of amino acids He 52, He 62, He 80, Phe 91, and He 93 of SEQ ID No.l.
30. An inhibitor according to claim 28 or 29 wherein the binding site is further defined by one or more of amino acids He 66, Arg 64, Leu 82 and/or Lys 96 of SEQ ID No. 1, and the binding of the inhibitor is provided by binding interaction of the inhibitor with the one or more further amino acids.
31. An inhibitor according to claim 30 wherein the binding of the inhibitor is provided by binding interactions of the inhibitor with all of amino He 66,
Arg 64, Leu 82 and Lys 96 of SEQ ID. No. 1.
32. Ah inhibitor according to claim 28 or 29 wherein the binding site is further defined by amino acid Val 50 of SEQ ID No. 1 and the binding of the inhibitor is provided by a binding interaction of the inhibitor with that amino acid.
33. An inhibitor according to any one of claims 28 to 32 wherein respective of the binding interactions between the inhibitor with the clathrin TD comprise direct contact of the inhibitor with the clathrin TD.
34. An inhibitor according to any one of claims 28 to 31 wherein the complex is stabilised by one or more hydrogen bonds between the inhibitor and amino acids selected from the group consisting of Ser 67, Gin 89, Val 50, He 62, and Arg 64 of SEQ ID. No. 1.
35. An inhibitor according to claim 34 wherein the binding of the inhibitor to the clathrin TD is stabilised by hydrogen bonds between the inhibitor and amino acids
Ser 67, Gin 89, Val 50, He 62, and Arg 64 of SEQ ID. No. 1.
36. An inhibitor according to claim 32 wherein the binding of the inhibitor to the clathrin TD is stabilised by one or more hydrogen bonds between the inhibitor and Arg 64 of SEQ ID No. 1.
37. A complex of a clathrin inhibitor and the terminal domain (TD) of clathrin or a fragment of the clathrin TD, the inhibitor being as defined in any one of claims 26 to 36 and binding to the binding site of the clathrin TD.
38. A method of screening a putative clathrin inhibitor, comprising:
fitting a model of a putative inhibitor of clathrin to a representation of a binding site for the inhibitor on the terminal domain (TD) of clathrin, the binding site being defined by at least one or more of amino acids He 52, lie 62, He 80, Phe 91 and/or lie 93 of SEQ ID No. 1 ;
modelling interaction of the inhibitor with the binding site to evaluate whether the inhibitor forms a complex with the binding site of the clathrin TD; and
evaluating whether the inhibitor may inhibit clathrin on the basis of the modelling, the formation of the complex being indicative of capacity of the inhibitor to inhibit clathrin.
39. A method according to claim 38 wherein the binding site for the inhibitor is defined by all of amino acids He 52, lie 62, He 80, Phe 91 and He 93 of SEQ ID No. 1;
40. A method according to claim 38 or 39 wherein the binding site is further defined by one or more of amino acids He 66, Arg 64, Leu 82 and/or Lys 96 of SEQ ID No. 1.
41. A method according to claim 38 or 39 wherein the binding site for the inhibitor is further defined by amino acid Val 50 of SEQ ID No. 1
42. A method according to any one of claims 38 to 41 wherein the modelling comprises evaluating whether the complex is formed by binding interactions between the inhibitor and at least some of the amino acids.
43. A method according to claim 42 wherein respective of the binding interactions comprise direct contact of the inhibitor with the amino acids.
44. A method according to any one of claims 38 to 43 wherein the method further comprises determining whether one or more hydrogen bonds are formed between the inhibitor and the clathrin TD to stabilise the complex, the formation of the hydrogen bond(s) being indicative of the inhibitor to inhibit clathrin.
45. A method for providing a clathrin inhibitor, comprising:
designing the inhibitor to bind to a binding site for the inhibitor on the terminal domain
(TD) of clathrin, the binding site of the clathrin TD being defined by at least one or more of amino acids He 52, He 62, lie 80, Phe 91 and/or He 93 of SEQ ID No. 1 ;
modelling interaction of the designed inhibitor with the binding site; and
evaluating whether the designed inhibitor forms a complex with the binding site of the clathrin TD on the basis of the modelling, the formation of the complex being indicative of capacity of the inhibitor to inhibit clathrin.
46. A compound of Formula I, or a prodrug or pharmaceutically acceptable salt thereof, wherein:
Figure imgf000096_0001
Formula I and wherein:
Ri is alkyl, alkenyl, alkylaryl, or aryl or a ring having from 5 to 7 ring atoms including from 0 to 3 heteroatoms selected from O, N or S, wherein the alkyl, alkenyl, alkylaryl or aryl are optionally substituted; and
R2 is O, S or NH; or
Ri , X, and R2 form a ring A, the ring having from 5 to 7 ring atoms including from 0 to 3 heteroatoms selected from O, N or S and being optionally substituted; and
X is N or a carbon atom;
Y is O, S. or NH; Λ
ring B and ring C each independently have 5 to 7 ring atoms including from 0 to 3 heteroatoms selected from 0, N or S; and
R3, R4 and R5 are each independently H or an optional substituent.
47. A compound according to claim 46, wherein:
Ri is alkyl, alkenyl, alkylaryl, or aryl, wherein the alkyl, alkenyl, alkylaryl or aryl is optionally substituted.
48. A compound according to claim 46 or 47, wherein:
R2 is 0; and
rings B and C each have 6 ring atoms.
49. A compound according to any one of claims 46 to 48 wherein Rj is alkyl. ■>
50. A compound according to claim 49 wherein R] is lower alkyl.
51. A compound according to any one of claims 46 to 48 wherein Rj is alkylaryl.
52. A compound according to claim 51 wherein the lower alkylaryl is an alkylphenyl.
53. A compound according to claim 52 wherein the alkylphenyl is a Ci-C2 alkylphenyl.
54. A compound according to claim 53 wherein the phenyl ring of the alkylphenyl is selected from the group consisting of 2-substituted phenyl, 3-substituted phenyl and 4-substituted phenyl groups.
55. A compound according to any one of claims 46 to 48 wherein Ri is aryl.
56. A compound according to claim 55 wherein Ri is phenyl.
57. A compound according to claim 56 wherein the phenyl is selected from the group consisting of 2-substituted phenyl, 2,4-substituted phenyl, and 2,5-substituted phenyl groups.
58. A compound according to any one of claims 46 to 57 wherein Ri is substituted with one or more substituents selected from the group consisting of hydroxyl, sulfhydryl, amino, nitro, halo, cyano, lower alkoxy, C02H, S03H, P04H, phenyl, and aryl.
59. A compound according to claim 58 wherein Ri is substituted with one or more substituents selected from the group consisting of hydroxyl, sulfhydryl, amino, nitro, halo, cyano, C02H, SO3H, PO4H, phenyl, and aryl.
60. A compound according to claim 46 wherein Rj, X and R2 form ring A.
61. A compound according to claim 60, wherein R2 is NR$ and R6 forms ring A with Rj and X.
62. A compound according to claim 60 or 61 wherein ring A has 5 ring atoms.
63. A compound according to claim 60 or 61, wherein ring A is substituted with an aryl group, the aryl group being fused with ring A.
64. A compound according to claim 63 wherein the aryl group fused with ring A is phenyl.
65. A compound according to claim 64 wherein ring A and the phenyl group substituent form a benzimadazol group.
66. A compound according to claim 64 or 65 wherein the phenyl fused with ring A is a 4- substituted phenyl.
67. A compound according to any one of claims 64 to 66 wherein the phenyl fused with ring A has one or more substituents selected from the group consisting of hydroxyl, sulfhydryl, amino, nitro, halo, cyano, alkoxy, C02H, SO3H, PO4H.
68. A compound according to claim 67 wherein the phenyl fused with ring A has one or more substituents selected from the group consisting of hydroxyl, sulfhydryl, amino, nitro, halo, cyano,
Figure imgf000098_0001
69. A compound according to claim any one of claims 43 to 68, wherein rings B and C each have 6 ring atoms.
70. A compound according to any one of claims 46 to 69 being a compound of Formula Ila, wherein:
Figure imgf000098_0002
Formula Ila
and wherein:
R3 and R4 are independently selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkoxy, alkyl, alkylaryl, aryl, NHR', NR'2, C02H, SO3H and P04H; R' is independently H, alkyl, alkenyl, aryl, or alkylaryl, wherein the alkyl, alkenyl, aryl or alkylaryl is optionally substituted; and
R5 is H, aryl, napthyl, benzyl or tetralin, wherein the aryl, napthyl benzyl and tretralin are optionally substituted.
71. A compound according to claim 70 wherein R3 and R4 are independently selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkyl, alkylaryl, aryl, NHR', NR'2, C02H, SO3H and P0 H.
72. A compound according to claim 70 or 71 wherein at least one of R3 and R» is other than H.
73. A compound according to any one of claims 46 to 69 being a compound of Formula lib, wherein:
Figure imgf000099_0001
Formula lib
and wherein:
R3, R4 and R5 are independently selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkoxy, alkyl, alkylaryl, aryl, NHR', NR'2, C02H, S03H and P04H; and
R' is independently H, alkyl, alkenyl, aryl, or alkylaryl, wherein the alkyl, alkenyl, aryl or alkylaryl is optionally substituted.
74. A compound according to claim 731 wherein R3, R4 and R5 are independently selected from H, halo, cyano, hydroxyl, sulfhydryl, nitro, amino, alkyl, alkylaryl, aryl, NHR', NR'2, C02H, SO3H and P04H.
75. A compound according to claim 73 or 74 wherein at least one of R3, R4 and R5 is other than H.
76. A compound according to any one of claims 70 to 72 wherein R5 is a substituent of Formula III, as follows:
Figure imgf000100_0001
Formula III
wherein:
G is a bond with ring C;
ring D has from 5 to 7 ring atoms optionally including from 0 to 3 heteroatoms selected from N, O and S; and
each W is independently H, halo, nitro, amino, hydroxyl, sulfhydryl, CO2H, SO3H, or PO4H.
77. A compound according to claim 76 wherein at least one W substituted is other than H.
78. A compound according to claim 76 or 77 wherein each W is independently H, C02H, SO3H, or P04H.
79. A compound of Formula V, or a prodrug or pharmaceutically acceptable salt thereof, wherein:
Figure imgf000100_0002
Formula V
and wherein:
X is O, S, or NH;
Y is 0, N or a carbon atom;
Z is O, S or NH;
Ri is H, C02H, SO3H, P04H, alkyl, alkenyl, alkylcarboxy, or alkylaryl;
R2 is O, S, NH, NHR', NHS(=0)2R', S(=0)2R\ sulfonyl, NHC(=0)NH, NHC(=S)NH, or NHC(=0);
R' is H, alkyl, alkenyl, alkylaryl, aryl, the alkyl, alkenyl, alkylaryl and aryl group being optionally substituted; and
R3 is aryl or a polycyclic group having at least 2 fused rings each independently having from 5 to 7 ring atoms, the aryl and the polycyclic group being optionally substituted.
80 A compound according to claim 79 wherein Ri is H or a lower alkyl or lower alkenyl, wherein the lower alkyl or lower alkenyl group is optionally substituted.
81. A compound according to claim 80 wherein Ri is a lower alkyl and is terminally substituted.
82. A compound according to claim 81 wherein the lower alkyl is a C C3 alkyl.
83. A compound according to any one of claims 79 to 82 wherein R2 is S, NH, or HN-S(=0)2- R', and R' is alkyl, alkenyl or aryl, wherein the alkyl, alkenyl or aryl is optionally substituted.
84. A compound according to claim 83 wherein R2 is HN-S(=0)2-R' wherein R' is aryl, and preferably, phenyl or napthyi.
85. A compound according to any one of claims 79 to 84 wherein R2 is OR' and R' is a lower alkyl or lower alkenyl, the lower alkyl or alkenyl being optionally terminally substituted with a substituent selected from the group consisting of amino, nitro, halo, cyano, hydroxy, sulfhydryl.
86. A compound according to any one of claims 79 to 85 wherein R3 is phenyl or an aryl group with 2 fused rings each independently having from 5 to 7 ring atoms including from 0 to 3 heteroatoms selected from O, N and S, the phenyl or aryl group being optionally substituted.
87. A compound according to any one of claims 79 to 86 being a compound of Formula Va as follows:
Figure imgf000101_0001
Formula Va wherein each M is independently selected from H, halo, hydroxy, sulfhydryl, nitro, amino, cyano, alkoxy, C02H, S03H, P04H, OR', NHR', NR'2, S02R', S02NH2; and
R' is independently alkyl, alkenyl, aryl, or alkylaryl, wherein the alkyl, alkenyl, aryl or alkylaryl is optionally substituted
88. A compound according to claim 87 wherein each M is independently selected from H, halo, hydroxy, sulfhydryl, nitro, amino, cyano, alkoxy, C02H, SO3H, P04H, OR', NHR', NR'2, S02R', S02NH2. -
89. A compound according to claim 87 or 88 wherein at least one M substituent is other than hydrogen.
90. A compound according to any one of claims 87 to 89 being a compound of Formula Vb as follows:
Figure imgf000101_0002
Formula Vb
91. A compound according to claim 90 wherein M is halo, nitro or -OR'NH2, wherein R' is a lower alkyl or lower alkenyl.
92. A compound according to claim 91 wherein M is -OR'NH2 and R' is a C1-C4 alkyl and preferably, a C3 alkyl. . r
93. A compound according to any one of claims 77 to 86 being a compound of Formula V- 1 a as follows:
Figure imgf000102_0001
Formula V- la wherein:
G is O or S;
T is 0, S, N or a carbon atom; and
each M is is independently selected from H, halo, hydroxy, sulfhydryl, nitro, amino, cyano, C02H, SO3H, PO4H, OR', NHR', NR'2, S02R', S02NH2; and
R' is independently alkyl, alkenyl, aryl, or alkylaryl, wherein the alkyl, alkenyl, aryl or alkylaryl is optionally substituted
94. A compound according to claim 93 wherein at least one M substituent is other than hydrogen.
95. A compound according to claim 93 or 94 wherein at least one M is halo, nitro or -OR'NH2, wherein R' is a lower alkyl or lower alkenyl.
96. A compound according to claim 95 wherein R' of -OR'NH2 is a C1-C4 alkyl.
97. A compound according to claim 95 wherein R' of -OR'NH2 is a C3 alkyl.
98. A compound according to any one of claims 93 to 97 being a compound of Formula V-lb as follows:
Figure imgf000102_0002
99. A pharmaceutical composition comprising a compound of any one of claims 46 to 98, or a prodrug or pharmaceutically acceptable salt of the compound, together with a pharmaceutically acceptable carrier.
100. A method for inhibiting clathrin, comprising contacting clathrin with an effective amount of a compound as defined in any one of claims 46 to 98.
101. A method for inhibiting clathrin in an animal, comprising administering to the mammal an effective amount of a compound as defined in any one of claims 46 to 98 to the mammal, or a prodrug or pharmaceutically acceptable salt of the compound.
102. A compound as defined in any one of claims 46 to 98 for use in inhibiting clathrin in a mammal, or a prodrug, or physiologically acceptable salt, of the compound.
103. A compound according to any one of claims 46 to 98 for use in inhibiting clathrin in a mammal, or a prodrug, or physiologically acceptable salt, of the compound.
104. A compound according to any one of claims 46 to 98 for use in prophylaxis or treatment of a disease or condition selected from the group consisting of epilepsy, Alzheimer's disease, Lewy body disease, Lewy body dimentia, Parkinson's disease, β-amyloid associated diseases, Dent's disease, pathogenic infections involving clathrin mediated endocytosis (CME) for infection of cells, cancer, cell proliferative disorders, neuropathic pain, multifocal leukoencephalopathy, psychotic disorders, psychosis, bipolar disorders, schizophrenia, aberrant up-regulated neuronal excitation, seizures, migraine, mucolipidosis, and cell trafficking, neurodegenerative, neuropyschiatric and neurological disease and conditions mediated by, or associated with, synaptic signal transmission or CME., or a prodrug, or physiologically acceptable salt, of the compound.
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