EP1978988A2 - Regulation de jonctions communicantes a l aide de peptides - Google Patents

Regulation de jonctions communicantes a l aide de peptides

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Publication number
EP1978988A2
EP1978988A2 EP07718080A EP07718080A EP1978988A2 EP 1978988 A2 EP1978988 A2 EP 1978988A2 EP 07718080 A EP07718080 A EP 07718080A EP 07718080 A EP07718080 A EP 07718080A EP 1978988 A2 EP1978988 A2 EP 1978988A2
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EP
European Patent Office
Prior art keywords
seq
gap junction
polypeptide
protein
cx43ct
Prior art date
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EP07718080A
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German (de)
English (en)
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EP1978988A4 (fr
Inventor
Mario Delmar
Steven M. Taffet
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Research Foundation of State University of New York
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Research Foundation of State University of New York
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Publication of EP1978988A2 publication Critical patent/EP1978988A2/fr
Publication of EP1978988A4 publication Critical patent/EP1978988A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the peptide-based regulation of gap junctions.
  • Connexins are integral membrane proteins that oligomerize to form intercellular channels called gap junctions.
  • the most abundant gap junction protein in a number of mammalian systems is connexin43 ("Cx43").
  • Gap junction channels are responsible for direct cell-to-cell communication. These channels are dynamic pores that are regulated in response to changes in the cellular environment and by protein interactions. In the heart, gap junction channels arc a critical mechanism for the passage of electrical impulses (Lerner et al., "Accelerated Onset and Increased Incidence of Ventricular Arrhythmias Induced by Ischemia in Cx43-def ⁇ cient Mice," Circ.
  • the individual subunit of the connexon is the molecule connexin.
  • This molecule resides in the membrane, with its N-terminal ("NT"), cytoplasmic loop ("CL”) and C-terminal ("CT") domains in the cytoplasmic space, as illustrated in Figure 1.
  • NT N-terminal
  • CL cytoplasmic loop
  • CT C-terminal
  • There arc at least 20 different connexin isotypes in the mouse genome and 21 in the human genome (Willecke et al., "Structural and Functional Diversity of Connexin Genes in. the Mouse and Human Genome,” Biol. Chem. 383(5):725-737 (2002)).
  • Gap junctions allow the passage of ions and small molecules between cells and are regulated by a variety of chemical interactions between the connexin molecule and the microenvironment. As such, gap junctions act as active filters to control the passage of intercellular messages to modulate function.
  • Flufenamic acid has been shown to be an effective inhibitor of gap junctions but the mechanism is thought to be indirect and not specific to any connexin protein (Srinivas & Spray, "Closure of Gap Junction Channels by Arylaminobenzoates,” MoI. Pharmacol. 63(6):1389— 1397 (2003)). Perhaps among the more specific inhibitors are the glycyrrhetinic acids (Davidson et al., "Reversible Inhibition of Intercellular Junctional Communication by Glycyrrhetinic Acid," Biochem. & Biophys. Res. Commun.
  • Antiarrhythmic peptide 10 alters gap junctional communication (Muller et al., "Actions of the Antiarrhythmic Peptide AAPlO on Intercellular Coupling,” Naunyn Schmiedebergs Arch. Pharmacol. 356(l):76-82 (1997); Dhcin ct al., "Effects of the New Antiarrhythmic Peptide ZP 123 on Epicardial Activation and Repolarization Pattern," Cell Commun. Adhes.
  • the present invention is directed to overcoming these deficiencies in the art.
  • a first aspect of the present invention relates to a protein or polypeptide comprising the formula A — [W — A] n where A is a peptide of formula XXXXRXPXXXX where X is any amino acid, R is arginine, and P is proline. W is a linker, and n is any number from 1 through 5.
  • a second aspect of the present invention relates to a protein or polypeptide that has an amino acid sequence consisting essentially of the formula X n RXPX m where each X is any amino acid, R is arginine, P is proline, n is any number from zero to fifteen, m is any number from zero to fifteen, and the sum of n and m is between ten and fifteen.
  • a third aspect of the present invention relates to a method of screening for compounds that modulate Cx43CT. This method involves contacting one or more candidate compounds with a polypeptide comprising an RXP-binding domain of Cx43CT, and identifying the candidate compounds that bind to the polypeptide as compounds that modulate Cx43CT.
  • a fourth aspect of the present invention relates to a method for measuring Cx43CT-binding affinity of a compound that binds to Cx43CT. This method involves contacting the compound with a polypeptide comprising an RXP- binding domain of Cx43CT, under conditions effective to permit binding and determining the dissociation constant for the interaction between the polypeptide and the compound.
  • a fifth aspect of the present invention relates to a method for identifying the location of an RXP-binding domain of Cx43CT.
  • This method involves contacting a protein or polypeptide with Cx43CT under conditions effective to permit binding between the protein or polypeptide and an RXP-binding domain of Cx43CT, where the protein or polypeptide ; measuring resonance of one or more amino acids of the Cx43CT; (i) has the formula A — [W — A] n where A is a peptide of formula XXXXRXPXXX where X is any amino acid, R is arginine, and P is proline; W is a linker; and n is any number from 1 through 5; or (ii) consists essentially of the formula X n RXPXm where each X is any amino acid, R is arginine, P is proline, n is any number from zero to fifteen, m is any number from zero to fifteen, and the sum of n and m is between ten and fifteen; and determining the location of any of the one or more amino acids whose resonance changes in the presence of the protein or polypeptide, where
  • a sixth aspect of the present invention relates to a method for modulating a Cx43 gap junction channel. This method involves contacting the Cx43 gap junction channel with a compound that binds to an RXP-binding domain of the Cx43 gap junction channel under conditions effective to modulate the Cx43 gap junction channel.
  • the present invention supplies a feasible, peptide-based strategy to manipulate Cx43 regulation in native tissues.
  • the proteins and polypeptides of the present invention can also be used as tools to characterize the specific role of gap junction regulation in health and disease.
  • Modulation of gap junction intercellular communication (“GJIC”) has potential as a pharmaceutical intervention in several diseases.
  • Figure 1 is a schematic diagram showing the topology of a Cx43 subunit. 'TSfT" is the amino terminal. "CL” is the cytoplasmic loop. “CT” is the carboxyl terminal domain. “L2” represents the portion of the CL from residues 119— 144.
  • Figures 2 A— D are graphs illustrating the balance of basic (“B”) and acidic ("A") residues in peptides chosen at random from the RXP library ( Figures 2A— C) or sequenced after selection for Cx43CT binding ( Figure 2D).
  • the number of acidic residues (asp or glu) was substracted from the number of basic residues (lys, arg or his) within the sequence.
  • Thirty "doublets” were randomly assigned from the library ( Figures 2A-C) and compared to 30 Cx43CT-bound peptides. A preponderance of 4 basic residues was found from the bound peptides ( Figure 2D).
  • Figures 3A-B are Sensograms showing the results of SPR studies comparing the binding of RXP-4 and RXP-E to Cx43CT.
  • Figure 3 A illustrates a Sensogram obtained from the binding of Cx43CT to RXP-4 (250 ⁇ M). The rapid and complete dissociation upon washout indicates weak intermolecular binding.
  • Figure 3B illustrates Sensograms showing concentration-dependent binding of RXP-E to Cx43CT. The increased amplitude and slower dissociation kinetics when compared to RXP-4 is apparent.
  • Dissociation constant (K D ) was measured from the rate of association and dissociation, based on first rate order kinetics, and estimated to be 3.9 ⁇ M.
  • FIGS 4A— B are schematic diagrams showing resonance peaks corresponding to Cx43CT residues as recorded by an NMR 15 N-HSQC protocol. Each insert depicts the contour of individual amino acids (noted on the top of the insert box) as recorded in the absence or the presence of either peptide RXP-4 ( Figure 4A) or RXP-E ( Figure 4B).
  • the top inserts are examples of amino acids whose position in the HSQC map was unaffected by the peptides; amino acids whose position in space was modified by the peptides are shown in the middle and bottom inserts. Both peptides caused a shift in the position of residues 376, 378 and 379, as well as residues 343-346.
  • Figures 5A-B are graphs of junctional current (Ij) traces obtained from
  • N IO) of 0.1 mM RXP-E in the internal pipette solution.
  • Time zero corresponds to the onset of octanol superfusion.
  • Uncoupling refers to the complete loss of junctional current under the voltage clamp protocol illustrated in Figures 5A-B and described in Example 12.
  • Figure 6A illustrates the percent of pairs that remained coupled at the end of each minute after onset of octanol.
  • Figure 6B shows the average junctional conductance (Gj) as a function of time after onset of octanol. For each cell pair, Gj was measured relative to the value recorded before octanol exposure.
  • FIG. 7 A shows the percent of pairs that remained coupled at the end of each minute after onset of octanol.
  • Figure 7B shows the average of junctional conductance (G,) as a function of time after onset of octanol. For each cell pair, G j was measured relative to the value recorded before octanol exposure. RXP-E failed to prevent uncoupling in cells expressing the truncated form of Cx43.
  • Figure 8 is a graph illustrating the time course of changes in junctional conductance (Gj) relative to control, as a function of time after patch break.
  • Figures 9 A— C are a schematic diagram ( Figure 9A) and graphs ( Figures 9B-C) relating to single channel data obtained from Cx43 -expressing N2a cells exposed to RXP-E (0.1 mM).
  • Figure 9A shows the original traces and all-point histograms (right of trace). Transjunctional voltage was +60 mV. Voltage pulses were held for 10 seconds. Traces were obtained from cell pairs showing a very low level of coupling (no uncoupling agents used).
  • the pipette solution was pH 7.2 (upper trace) or pH 6.2 (middle and lower traces).
  • the histogram was best fit by a single Gaussian, centered at 100.5 pS. A peak corresponding to transitions in or out of the residual state is absent.
  • Figures 10A- B are junctional current recordings from N2a cells transfected with M257. Octanol was used to uncouple cells to demonstrate single channels in Figure 1OA. The recordings presented in Figure 1OB (representative of at least 5 traces) were obtained in the presence of RXP-E. The results indicate that the increase in the open time for wild-type Cx43 channels in the presence of RXP-E is not seen in M257 channels. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention relates to a protein or polypeptide having the formula A-[W-A] n where A is a peptide of formula XXXXRXPXXXX where X is any amino acid, R is arginine, and P is proline. W is a linker, and n is any number from 1 through 5.
  • Human Cx43 is a 382 aa protein that has the following amino acid sequence: 1 MGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWG 47 DEQSAFRCNTQQPGCENVCYDKSFPISHVRFWVLQIIFVS VPTLLYLAH 96 VFYVMRKEEKLNKJKEEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEH 143 GKVKMRGGLLRTYIISILFKSIFEVAFLLIQWYTYGFSLSAVYTCKRDPC 193 PHQVDCFLSRPTEKTIFIIFMLWSLVSLALNIIELFYVFFKGVKDRVKG 243 KSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVT 291 GDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFD 337 FPDDNQNSKKLAAGHELQPLA
  • CT carboxy-terminal
  • residues 255—382 of SEQ ID NO:1 presents itself as a potential target of chemical (Duffy et al., "pH-Dependent Intramolecular Binding and Structure Involving Cx43 Cytoplasmic Domains," J. Biol. Chem. 277(39):36706-36714 (2002); Morley et al., “Intramolecular Interactions Mediate pH Regulation of Connexin43 Channels,” Biophys. J.
  • the proteins and polypeptides of the present invention may be produced or isolated using methods known in the art. Suitable production methods include, for example, recombinant genetic engineering, chemical synthesis, and cell- free translation.
  • a nucleic acid molecule encoding a polypeptide or protein of the present invention can be introduced into an expression system of choice using conventional recombinant technology.
  • the nucleic acid molecule is incorporated into a vector-expression system of choice to prepare a nucleic acid construct using standard cloning procedures known in the art, such as those described in JOSEPH SAMBROOK & DAVID W. RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (3d ed.
  • the nucleic acid molecule may be inserted into a vector in the sense (i.e., 5'— >3') direction, such that the open reading frame is properly oriented for the expression of the encoded protein under the control of a promoter of choice.
  • Single or multiple nucleic acids may be ligated into an appropriate vector in this way, under the control of a suitable promoter, to prepare a nucleic acid construct for expressing a protein or polypeptide of the present invention.
  • the vector should also contain the necessary elements for the transcription and translation of the inserted protein- or polypeptide-coding sequences.
  • nucleic acid molecule encoding the protein or polypeptide Once the isolated nucleic acid molecule encoding the protein or polypeptide has been cloned into an expression system, it is ready to be incorporated into a host cell. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, lipofection, protoplast fusion, mobilization, particle bombardment, clcctroporation (Neumann ct al., "Gene Transfer into Mouse Lyoma Cells by Electroporation in High Electric Fields," EMBO J.
  • nucleic acid molecules are cloned into the host cell using standard cloning procedures known in the art.
  • Suitable hosts include, but are not limited to, bacteria, virus, yeast, fungi, mammalian cells, insect cells, plant cells, and the like. [0037] The host cell is then cultured in a suitable medium, and under conditions suitable for expression of the protein or polypeptide of interest. After cultivation, the cell is disrupted by physical or chemical means, and the protein or polypeptide purified from the resultant crude extract.
  • cultivation may include conditions in which the protein or polypeptide is secreted into the growth medium of the recombinant host cell, and the protein or polypeptide is isolated from the growth medium.
  • Alternative methods may be used as suitable.
  • the proteins and polypeptides of the present invention can also be synthesized in a cell-free protein synthesis system.
  • the above expression vector DNA is transcribed in vitro, and the resultant rnRNA is added to a cell-free translation system to synthesize the protein.
  • the cell- free translation system is prepared from an extract of a eukaryotic cell or a bacterial cell, or a portion thereof.
  • Such cell-free translation systems include those prepared from a rabbit reticulocyte, from a wheat germ, and fromii. coli S30 extract.
  • proteins and polypeptides of the present invention may be purified by methods that will be apparent to one of skill in the art. [0041] Mutations or variants of the above polypeptides or proteins are encompassed by the present invention. Variants may be modified by, for example, the deletion or addition of amino acids that have minimal influence on the properties and secondary structure of the desired protein or polypeptide.
  • a protein or polypeptide of the present invention may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein or polypeptide that co-translationally or post-translationally directs transfer of the protein or polypeptide.
  • the protein or polypeptide may also be conjugated to another sequence for ease of synthesis, purification, or identification of the protein or polypeptide.
  • the linker in the protein or polypeptide according to this aspect of the present invention is preferably about 1 to about 15 amino acids long, more preferably about 1 to about 13 amino acids long, and most preferably about 8 to about 13 amino acids long.
  • Suitable linkers include the formula Gly-Gly-Gly-Scr (SEQ ID NO:2), AHARVPFYSHS (SEQ ID NO:3), AETHARVPFYSHS (SEQ ID NO:4), and AVPFYSHS (SEQ ID NO:5).
  • n is any number from 1 through 4, most preferably 1 or 2.
  • Exemplary proteins or polypeptides according to this aspect of the present invention include those having an amino acid sequence of DVPGRDPGYIKGGGSAHARVPFYSHSLNRNRKPSLYQ (SEQ ID NO:6), EIQPRSPLMFSGGGSAHARVPFYSHSAKEARWPRAHR (SEQ ID NO:7), GIAAREPNSHDGGGSAHARVPFYSHSRDLWRKPAKSL (SEQ ID NO:8), WEEPRRPFTMSGGGSAETHARVPFYSHSPMRHRLPGVHL (SEQ ID NO:9), or SDDLRSPQLHNGGGSAVPFYSHSHMVRRKPRNPR (SEQ ID NO.-10).
  • the protein or polypeptide has at least 4 more basic amino acid residues than acidic amino acid residues.
  • the present invention also relates to a protein or polypeptide that has an amino acid sequence consisting essentially of the formula X n RXPX m where each X is any amino acid, R is arginine, P is proline, n is any number from zero to fifteen, m is any number from zero to fifteen, and the sum of n and m is between ten and fifteen.
  • n is any number from one to eight.
  • m is any number from one to eight.
  • n and m are between 10 and 12. In another preferred embodiment, n is any number from one to eight, m is any number from one to eight, and the sum of n and m is between 10 and 12.
  • Exemplary proteins or polypeptides according to this aspect of the present invention include those having an amino acid sequence of
  • GHLHLRVPTLKM (SEQ ID NO:11), EFIRSPHS VD WL (SEQ ID NO: 12), SQSRNPPMPPPR (SEQ ID NO: 13), RRPPYRVPPKLF (SEQ ID NO: 14), SLYERHPASTYP (SEQ ID NO: 15), HTVSRRPLPSSG (SEQ ID NO: 16), RHTHGNLLRFPP (SEQ ID NO: 17), RNNLNQTYPERR (SEQ ID NO: 18), YSLLPVRPVALT (SEQ ID NO: 19), RKPTQSLPTRLV (SEQ ID NO:20), TRRPHKMRSDPL (SEQ ID NO:21), TLTWHTKTPVRP (SEQ ID NO:22), SRQFLHSLDRU?
  • Another aspect of the present invention relates to a method of screening for compounds that modulate Cx43CT. This method involves contacting one or more candidate compounds with a polypeptide comprising an RXP -binding domain of Cx43CT, and identifying the candidate compounds that bind to the polypeptide as compounds that modulate Cx43CT.
  • the RXP-binding domain preferably includes amino acid residues 343-346 of SEQ ID NO : 1 , amino acid residues 375-379 of SEQ ID NO:1, or combinations thereof.
  • the method screens for candidate compounds that exhibit pH-dependent binding to Cx43CT.
  • a polypeptide comprising an RXP-binding domain of Cx43CT is contacted with a candidate compound that binds to the polypeptide, at a first pH under conditions effective to permit binding, and binding between the polypeptide and the compound is detected.
  • the candidate compound is contacted with the polypeptide at a second pH under substantially similar conditions and binding between the polypeptide and the candidate compound is detected.
  • the binding levels measured at the first and second pHs are compared, where a difference in binding level indicates that the candidate compound exhibits pH-dependent binding to Cx43CT.
  • Binding between the polypeptide and a candidate compound may be detected by a variety of mechanisms.
  • One useful test is the use of Surface Plasmon Resonance (SPR) (Duffy et al., "Functional Demonstration of Connexin-protein Binding Using Surface Plasmon Resonance," CellAdhes. Commun.
  • Nuclear magnetic resonance (NMR) is also a technique that can determine whether two proteins interact, as well as determine where within the protein that binding takes place. This technique is described in detail below, and in Sorgen et al., "Sequence-specific Resonance Assignment of the Carboxyl Terminal Domain of Connexin43," J. Biomol. NMR 23(3):245-246 (2002); Sorgen et al., "pH- Dependent Dimerization of the Carboxyl Terminal Domain of Cx43," Biophys. J. 87(1):574-581 (2004); and Sorgen et al., "Structural Changes in the Carboxyl Terminus of the Gap Junction Protein Connexin43 Indicates Signaling between
  • Binding Domains for c-Src and Zonula Occludens-1 J. Biol. Chem. 279(2):54695— 54701 (2004), which are hereby incorporated by reference in their entirety.
  • Another technique that may be used in the detection of protein binding is cross-linking (Sorgen et al., "pH-Dependent Dimerization of the Carboxyl Terminal Domain of Cx43,” Biophys. J. 87(1):574-581 (2004), which is hereby incorporated by reference in its entirety). This technique involves the association of two proteins followed by treatment with a chemical crosslinker to covalently link the two peptides.
  • the method screens for candidate compounds that modulate the structure of Cx43CT.
  • a candidate compound is contacted with a polypeptide comprising an RXP- binding domain of Cx43CT under conditions effective to permit binding between the candidate compound and the polypeptide.
  • Resonance of one or more amino acids of the polypeptide is measured.
  • the resonance of the one or more amino acids of the polypeptide with and without the candidate compound is compared, where a difference in resonance of the one or more amino acids with and without the candidate compound indicates that the candidate compound modulates the structure of Cx43CT.
  • the resonance of the one or more amino acids measured in the presence of the test compound can be compared to the previously-assigned resonance peaks.
  • resonance of the one or more amino acids can be measured in the absence of the candidate compound to establish a reference for comparing resonance in the presence of the candidate compound.
  • Suitable methods for measuring resonance of amino acids include, for example, gradient-enhanced two-dimensional 1H- 35 N HSQC experiments (Kay et al., "Pure Absorption Gradient Enhanced Heteronuclear Single Quantum Correlation Spectroscopy with Improved Sensitivity," J. Am. Chem. Soc. 114:10663-10665 (1992), which is hereby incorporated by reference in its entirety).
  • the method screens for candidate compounds that modulate the activity of a Cx43 gap junction channel.
  • gap junction intercellular communication GJIC of a Cx43 gap junction channel is measured.
  • a Cx43 gap junction channel is contacted with a candidate compound and the GJIC of the channel is measured under substantially similar conditions.
  • the GJIC levels measured with and without the candidate compound are compared, where a difference in GJIC indicates that the candidate compound modulates the activity of the channel.
  • Increase in GJIC indicates that the candidate compound increases the number of channels that form, increases the number of channels that open, and/or increases the duration of opening of a channel (i.e. the open state of the channel is maintained).
  • Decrease in GJIC indicates that the candidate compound decreases the number of available channels, prevents opening of a channel, or reduces the duration of opening of a channel.
  • GJIC may be measured using methods known in the art.
  • Suitable methods include measuring conductance of the gap junction channel, using, for example, dual- whole-cell voltage clamp assay (Seki et al., "Modifications in the Biophysical Properties of Connexin43 Channels by a Peptide of the Cytoplasmic Loop Region,” Circ. Res. 95(4):e22 ⁇ e28 (2004), which is hereby incorporated by reference in its entirety).
  • Dye transfer assays may also be used to measure GJIC, which involve following the transfer of gap junction permeable dyes to neighboring cells.
  • the basic transfer protocol involves the use of a mechanical scrape to open cells to a dye such as Lucifer yellow. This dye can pass through a gap junction and then be visualized in adjacent cells (el-Fouly et al., "Scrape-loading and Dye Transfer. A Rapid and Simple Technique to Study Gap Junctional Intercellular Communication," Exp. Cell Res. 168(2):422-430 (1987), which is hereby incorporated by reference in its entirety).
  • this dye may be microinjected into a single cell and transfer followed into neighboring cells (Lampe et al., "Phosphorylation of Connexin43 on Serine368 by Protein Kinase C Regulates Gap Junctional Communication," J. Cell Biol. 149(7): 1503-1512 (2000)).
  • the method screens for candidate compounds that modulate octanol- induced uncoupling of a gap junction channel.
  • two or more cells expressing one or more Cx43 gap junction channels are treated with octanol and the GJIC of the channel(s) is measured.
  • two or more cells expressing one or more Cx43 gap junction channels arc treated with octanol, a candidate compound is contacted with the Cx43 gap junction channel(s), and the GJIC of the channel(s) is measured.
  • the GJIC levels measured with and without the candidate compound are compared, where a difference in GJIC indicates that the candidate compound modulates octanol-induced uncoupling of a gap junction channel.
  • the method screens for candidate compounds that modulate pH-dependent uncoupling of a gap junction channel.
  • two or more cells expressing one or more Cx43 gap junction channels arc treated at a first pH and the GJIC of the channcl(s) is measured.
  • the pH of the extracellular fluid is changed to a second pH and the GJIC of the channel(s) is measured.
  • the GJIC levels measured at the first and second pHs are compared, where a difference in GJIC indicates that there is pH-dependent uncoupling of a gap junction channel.
  • two or more cells expressing one or more Cx43 gap junction channels are treated at a first pH, a candidate compound is contacted with one or more of the channels), and the GJIC of the channel(s) is measured.
  • the pH of the extracellular fluid is changed to a second pH, a candidate compound is contacted with one or more of the channel(s), the GJIC of the channel(s) is measured, and the GJIC levels measured at the first and second pHs are compared.
  • the difference in GJIC measured in the first experiment is compared to the difference in GJIC measured in the second experiment, where change in the difference in GJIC indicates that the candidate compound modulates pH-dependent uncoupling of a gap junction channel.
  • a smaller difference in GJIC measured in the second experiment i.e. in the presence of the candidate compound
  • the difference in GJlC measured in the first experiment i.e. without the candidate compound
  • a larger difference in GJIC measured in the second experiment i.e. in the presence of the candidate compound
  • the difference in GJIC measured in the first experiment i.e. without the candidate compound indicates that the compound enhances pH-dcpcndcnt uncoupling of the channel.
  • the method screens for candidate compounds that modulate uncoupling of a Cx43 gap junction channel in a CT- dependent manner.
  • two or more cells expressing one or more Cx43 gap junction channels are treated with octanol, a candidate compound is contacted with the Cx43 gap junction channel(s) and the GJIC of the channel(s) is measured.
  • two or more cells expressing one or more Cx43 gap junction channels that lack the CT domain are treated with octanol, the candidate compound is contacted with the Cx43 gap junction channel(s), and the GJIC of the channel(s) is measured.
  • the GJIC levels measured with the Cx43 gap junction channel(s) and the channe(s) that lack the CT domain are compared, where higher or lower GJIC in the second experiment relative to the first experiment indicates that the candidate compound modulates uncoupling of a Cx43 gap junction channel in a CT-dependent manner. (Substantially the same GJIC indicates that the candidate compound either does not modulate uncoupling of the channel, or modulates uncoupling of the channel in a CT- independent manner. Whether the compound modulates octanol- induced uncoupling of the channel may be determined using the methods described above.) [0064] In another preferred embodiment, the method screens for candidate compounds that stabilize a Cx43 gap junction channel in an open state.
  • mean open time of a Cx43 gap junction channel is measured.
  • a candidate compound is exposed to a Cx43 gap junction channel and the mean open time of the channel is measured under substantially similar conditions.
  • the mean open times measured with and without the candidate compound are compared, where a difference in mean open time indicates that the candidate compound stabilizes the open state of a Cx43 gap junction channel.
  • An increase in mean open time indicates that the candidate compound stabilizes a Cx43 gap junction channel in an open state.
  • a decrease in mean open time indicates that the candidate compound does not stabilize a Cx43 gap junction channel in an open state.
  • Mean open time of the gap junction channel may be measured, for example, as described in Examples 6 and 15 of the present application, and as described in Moreno et al., "Role of the Carboxyl Terminal of Connexin43 in Transjunctional Fast Voltage Gating," Circ. Res. 90(4):450-457 (2002), 92(l):c30 (2003) (erratum), which is hereby incorporated by reference in its entirety.
  • Another aspect of the present invention relates to a method for measuring Cx43CT ⁇ binding affinity of a compound that binds to Cx43CT.
  • This method involves contacting the compound with a polypeptide comprising an RXP- binding domain of Cx43CT, under conditions effective to permit binding and determining the dissociation constant for the interaction between the polypeptide and the compound.
  • the Cx43CT-binding affinity of a compound that binds to Cx43CT may be measured, for example, using the methods described in Salamon et al., "Surface Plasmon Resonance Spectroscopy as a Tool for Investigating the Biochemical and Biophysical Properties of Membrane Protein Systems. II: Applications to Biological Systems," Biochim. Biophys. Acta 1331(2):131-152
  • Another aspect of the present invention relates to a method for identifying the location of an RXP-binding domain of Cx43CT.
  • This method involves contacting a protein or polypeptide according to the present invention with Cx43CT under conditions effective to permit binding between the protein or polypeptide and an RXP-binding domain of Cx43CT.
  • Resonance of one or more amino acids of the Cx43CT is measured, and the location of any of the one or more amino acids whose resonance changes in the presence of the protein or polypeptide is determined.
  • the location of the amino acids whose resonance changes in the presence of the protein or polypeptide indicates that the amino acid is located within an RXP-binding domain. Resonance may be measured and compared as described above.
  • Suitable proteins and polypeptides include (i) those having the formula A — [W — A] n where A is a peptide of formula XXXXRXPXXX where X is any amino acid, R is arginine, and P is proline; W is a linker; and n is any number from 1 through 5; and (ii) those that have an amino acid sequence consisting essentially of the formula X n RXPXm where each X is any amino acid, R is arginine, P is proline, n is any number from zero to fifteen, m is any number from zero to fifteen, and the sum of n and m is between ten and fifteen.
  • Another aspect of the present invention relates to a method for modulating a Cx43 gap junction channel. This method involves contacting the Cx43 gap junction channel with a compound that binds to an RXP -binding domain of the Cx43 gap junction channel under conditions effective to modulate the Cx43 gap junction channel. [0071] This aspect of the present invention may be carried out in vitro or in vivo.
  • the compounds that bind to an RXP -binding domain of the Cx43 gap junction channel may be administered to a subject under conditions effective to contact the Cx43 gap junction channel with the compound.
  • These compounds can be administered orally, parenterally, for example, intradermally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as that of the nose, throat, and bronchial tubes. They may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.
  • the compounds may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • these compounds may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compound in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; cxcipicnts such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin.
  • a liquid carrier such as a fatty oil.
  • Various other materials may be present as coatings or to modify the physical form of the dosage unit.
  • tablets may be coated with shellac, sugar, or both.
  • a syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
  • These compounds may also be administered parenterally. Solutions or suspensions of these compounds can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils.
  • Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
  • liquid carriers particularly for injectable solutions.
  • these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • the compounds may also be administered directly to the airways in the form of an aerosol.
  • the compounds in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutanc with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutanc with conventional adjuvants.
  • the compounds also may be administered in a non- pressurized form such as in a nebulizer or atomizer.
  • the compounds may be administered directly to the targeted tissue.
  • the compounds may be administered to a non- targeted area along with one or more tissue-specific agents that facilitate migration of the compounds to the targeted tissue.
  • Opening of the gap junction channel may be modulated by contacting the channel with a compound that, for example, prevents opening of the channel, decreases the degree to which the channel opens, promotes opening of the channel, increases the degree to which the channel opens, or stabilizes the channel in an open state.
  • Closing of the gap junction channel may be modulated by contacting the channel with a compound that, for example, prevents closing of the channel, decreases the degree to which the channel closes, promotes closing of the channel, increases the degree to which the channel closes, or stabilizes the channel in a closed state.
  • Suitable proteins and polypeptides according to this aspect of the present invention include, for example, (i) those having the formula A — [W — A] n where A is a peptide of formula XXXXRXPXXX where X is any amino acid, R is arginine, and P is proline; W is a linker; and n is any number from 1 through 5; and (ii) those that have an amino acid sequence consisting essentially of the formula X n RXPX m where each X is any amino acid, R is arginine, P is proline, n is any number from zero to fifteen, m is any number from zero to fifteen, and the sum of n and m is between ten and fifteen. Specific examples of such proteins and polypeptides are identified above.
  • Suitable compounds according to this aspect of the present invention include, for example, compounds that: exhibit pH-depending binding to Cx43CT; modulate structure of Cx43CT; modulate activity of the Cx43 gap junction channel; modulate octanol-induced uncoupling of the Cx43 gap junction channel; modulate pH-dependent uncoupling of the Cx43 gap junction channel; modulate CT-dependent uncoupling of the Cx43 gap junction channel; or stabilize the Cx43 gap junction channel in an open state.
  • Compounds that have a combination of these effects are also contemplated. Suitable compounds may be identified according to the methods disclosed herein.
  • a "library" of bacteriophage, displaying ⁇ 55 copies of ' 2.7 x 10 9 random 12-mer peptides (Ph.D.- 12TM Phage display peptide library kit; New England BioLabs Inc.) was utilized.
  • Wells in 24-well plates were coated with 15 ⁇ g of recombinant CT and blocked with 5 mg/ml BSA. After removing the unbound viruses by washing with Tris buffered saline containing 0.5% tween-20 (TBS-T), low affinity binders were eluted with a solution of TBS-containing Cx43CT at a concentration of 100 ⁇ g/ml.
  • High affinity binders were recovered by overlaying the well with a culture of E. coli, to allow the tightly bound phage to infect the bacteria.
  • TMs culture was amplified and the virus was precipitated with PEG. The amplified product was used for the subsequent round of panning. All steps were conducted at pH 6.5 unless otherwise indicated.
  • the phage recovered from the last round of binding were grown on a lawn of E. coli for plaque purification. Each plaque, representing a single clone, was picked and amplified. The phage were isolated and analyzed by DNA sequencing. The peptide sequences displayed were deduced from these sequences.
  • phage display library of sequence XXXXRXPXXXX, where X is any amino acid flanking an arginine and a proline with an additional random residue at the center.
  • the randomized peptides were followed by a Gly-Gly-Gly-Ser (SEQ ID NO:2) spacer.
  • the library was cloned into M 13 phage. These phage were analyzed for titer and sequence prior to screening for Cx43CT binding as described above.
  • SPR Surface Plasmon Resonance
  • Example 6 Electrophysiological Analysis.
  • M257 (a mutant of Cx43 truncated at amino acid 257 (Morley et al., “Intramolecular Interactions Mediate pH Regulation of Connexin43 Channels,” Biophys. J. 70(3):1294-1302 (1996), which is hereby incorporated by reference in its entirety)) was transiently expressed in N2a cells also using an eGFP- containing IRES plasmid.
  • Example 7- Non-biased Phage Display [0092] Initial control experiments were conducted to standardize the phage display assay. The library was presented to purified streptavidin, a protein known to bind preferentially to peptides containing an HPQ consensus motif (Devlin et al., "Random Peptide Libraries: A Source of Specific Protein Binding Molecules," Science 249(4967) :404-406 (1990); Blasko et al., “Mechanistic Studies with Potent and Selective Inducible Nitric-oxide Synthase Dimerization Inhibitors," J. Biol.
  • the library was presented to Cx43CT. After three rounds of selection and amplification, DNA of 156 phage plaques was purified and sequenced. Of the estimated 2.7x10 9 different sequences in the library, 48 unique sequences were recovered. One particular sequence (PRPTMGNLPDVL) (SEQ ID NO:27) was recovered from 45 different plaques. This particular peptide showed strong homology (using the GAP routine of the Wisconsin package, GCG software) with a 10 amino acid segment (RATLLNVPDL) (SEQ ID NO:28) of the second PDZ domain of the tight junction protein, zonula ocludens-1 (ZO-I).
  • Example 8 The "RXP” Motif and Biased Phage Display.
  • Example 7 revealed that 16 out of 48 unique sequences shared a motif "RXP" (where X represents any amino acid).
  • the motif was shown in 11 peptides in the N- to C- terminal orientation and in 5 peptides in the reverse direction. Specific sequences are presented in Table 1.
  • the motif occurred in different positions within the 12-mer peptide, thus preventing proper alignments to determine the frequency of other amino acids at specific positions relative to the RXP.
  • a biased phage display library was generated where the RXP motif was forced to the center of the sequence, flanked at each side by 4 randomized amino acids.
  • Sixty clones containing an insert (out of an estimated 3-4 xlO 4 total clones in the library) were chosen at random for sequencing prior to exposure of the library to Cx43CT.
  • the binding step was carried out at pH 6.5 in two of the runs (run 1 and 2) and at pH 7.4 in one additional run (run 3).
  • the total number of phage plaques containing a full coding insert that were recovered after the binding step were 120, 119 and 163 for runs 1 , 2 and 3, respectively.
  • the results showed that 89% of all sequences corresponded to "doublets," that is, peptides where two RXP 11-mers had been inserted in tandem. These inserts may be consequent to incomplete restriction enzyme digestion as well as possible residual activity of the Klenow polymerase used in second strand production. The combination of these two factors would result in blunt ends that subsequently ligated. Analysis of the doublet sequences indicated that one or both of these processes occurred in each doublet. Furthermore, five specific peptides were recovered from all three runs and represented a large fraction of the repeats observed within the same run (see Table 2).
  • Table 4 Summary of sequences obtained from modified phage display procedure.
  • control peptides Sixty (60) control peptides (thirty "pairs") were used and compared to the 30 doublets recovered from all screenings. Each unique doublet was counted as one, regardless of the frequency with which it appeared after Cx43CT binding. As shown in Figures 2A— C, Gaussians were centered near 2.0 for the control peptides. In other words, regardless of the randomization, the control peptides had a balance of two basic residues (consistent with the fact that the library was biased to express one arginine in each 11-mer). In contrast, the frequency histogram obtained from the doublets peptides that bound Cx43CT had an abundance of 4 basic over acidic residues as shown in Figure 2D. These results indicate that Cx43CT preferentially bound peptides with a higher number of basic residues, perhaps consequent to the positive balance of charge within the peptide.
  • Phage display allows for the rapid screening of thousands of peptides, but the characteristics of binding cannot be properly defined. Furthermore, the peptides are part of a capsid protein, which may affect the ability of the peptide to properly interact with the target protein. Therefore, some of the peptides identified by the phage screening were selected to, using SPR, farther characterize their ability to bind Cx43CT. Recombinant Cx43CT was covalently bound to a carboxymethyl dextran matrix and synthetic peptides presented for binding.
  • RXP-A SEQ ID NO:6
  • RXP-D SEQ ID MO:9
  • peptide RXP-E SEQ ID NO: 10
  • FIG. 3B The transitions were well-fit by exponential functions and the rates of association and dissociation were used as described in Example 4 to estimate the kinetic parameters.
  • a full range of concentrations was tested in three separate occasions and at two different pHs of the solvent (6.5 and 7.4). No differences were observed as a function of pH.
  • K D dissociation constant
  • Example 11 Nuclear Magnetic Resonance (NMR).
  • Peptides RXPl, RXP4 and RXP-E were tested for their ability to modify the structure of Cx43CT.
  • the peptides were diluted in PBS (pH 5.8) containing 15 N-Cx43CT, and 15 N-HSQC spectra were acquired.
  • Results for RXP4 and RXP-E are shown in Figures 4A (RXP4) and 4B (RXP-E).
  • the specific resonance peaks that correspond to each amino acid in the Cx43CT sequence have been assigned (Sorgen et al., "Sequence-specific Resonance Assignment of the
  • Example 12 Effect of RXP-E on Cx43 Channels.
  • the ability of RXP-E to bind to Cx43CT suggests that this peptide may also alter the behavior of Cx43 channels.
  • Gap junction currents were recorded from N2a cells transfected with Cx43. To reduce macroscopic currents and allow for detection of single channel events, cell pairs were superfused with octanol (Anumonwo et al., "The Carboxyl Terminal Domain Regulates the Unitary Conductance and Voltage Dependence of Connexin40 Gap Junction Channels," Circ. Res.
  • FIG. 5 A depicts junctional current traces obtained from a Cx43 -expressing cell pair under control conditions (no RXP-E).
  • Transjunctional voltage (Vj) was +60 mV.
  • Octanol was added to the superfusate at the point indicated by the arrow. After a short delay, junctional current abruptly decreased, reaching zero within three minutes after onset of octanol.
  • the ability of octanol to uncouple gap junctions with a high degree of efficiency has been extensively reported in the literature (Johnston & Ramon, "Electrotonic Coupling in Internally Perfused Crayfish Segmented Axons," J. Physiol. 317:509-518 (1981), which is hereby incorporated by reference in its entirety).
  • Figure 5B The traces shown in Figure 5B were obtained from a different cell pair.
  • RXP-E was diluted in the internal pipette solution.
  • Cumulative data are presented in Figures 6 A— B.
  • Figure 6 A depicts the percent of cell pairs that remained coupled following the onset of octanol superfusion (uncoupling defined as zero junctional current elicited by a 60-mV transjunctional voltage pulse).
  • Ten different cell pairs were recorded using patch pipettes filled with a solution containing RXP-E. Seven pairs were tested without RXP-E.
  • Example 14 RXP-E Partially Prevents Acidification-induced Uncoupling.
  • RXP-E can interfere with Cx43 regulation by factors with some possible pathophysiological relevance.
  • pHj reduced intracellular pH
  • Ij junctional current
  • the progression of uncoupling was interrupted and Gj decreased only to 57.0 ⁇ 5.8% of control.
  • the presence of a control peptide (open triangles) did not alter pHi mediated uncoupling.
  • the data show that RXP-E partially prevented the closure of Cx43 channels consequent to a reduction in pHi.
  • Cx43 channels can reside in at least three distinct states: closed, open and residual (see Harris, “Emerging Issues of Connexin Channels: Biophysics Fills the Gap,” Q. Rev. Biophys. 34(3):325-472 (2001), 35(1): 109 (2002) (erratum), which is hereby incorporated by reference in its entirety).
  • Open-to- residual transitions are considered responsible for "fast Vj gating" (i.e., the rapid, voltage-dependent component of junctional current inactivation seen in Cx43 and other connexin channels) (Moreno et al., "Role of the Carboxyl Terminal of Connexin43 in Transjunctional Fast Voltage Gating," Circ. Res.
  • Figure 9A was recorded from another pair, using patch pipettes containing an internal solution buffered to pH 6.2. Single channel events were seen immediately after patch break. Overall, the events recorded showed a unitary conductance similar to that in the absence of RXP-E, though open times were greatly prolonged and the residual state was noticeably absent. Cumulative data are shown in Figures 9B-C.
  • Figure 9B shows a histogram of the measured values of unitary conductance. The Gaussian distribution centered at 100.5 pS, not different from what has been reported for wild- type Cx43 channels (Moreno et al., "Role of the Carboxyl Terminal of Connexin43 in Transjunctional Fast Voltage Gating," Circ. Res.
  • Dual patch clamp was used in a whole-cell configuration to deliver
  • Cx43CT had a preference for peptides with an excess of basic residues, which may be associated with their positive charge.
  • the pKa of histidine is near the pHs used for binding in the present study, the probability of the imidazole ring of histidine being protonated at the pHs tested is certainly higher than that of any other amino acid, with the exception of lysine and arginine.
  • a preference on the position of the basic residues within the primary sequence of the peptide was not observed. However, it remains to be tested whether there is a conservation of secondary structure not apparent from the primary sequence. Further NMR studies will be directed at determining the structure of RXP peptides and identifying relevant structures that may facilitate the binding to Cx43CT.
  • a single stranded oligonucleotide containing the randomized sequence must be replicated using the Klenow fragment of DNA polymerase. This forms a double stranded DNA fragment with blunt ends. Restriction digestion puts "sticky ends” on this fragment for its insertion into the phage DNA. In rare cases, one of these "sticky ends” was either not produced due to incomplete digestion, or filled after digestion due to continued activity of the DNA polymerase. This led to blunt ends capable of ligation, rather than mismatched "sticky ends.” These events were rare enough as to not be detected in the sampling of the library sequences performed prior to screening.
  • both peptides also modified the position of residues within the second alpha helical domain of Cx43CT (Delmar et al., "Structural Bases for the Chemical Regulation of Connexin43 Channels,” Cardiovasc. Res. 62(2):268-275 (2004), which is hereby incorporated by reference in its entirety), which is involved in pH-dependent dimerization of the protein (Sorgen et al., "pH-Dependent Dimerization of the
  • RXP peptides may interfere with both intra- and inter-molecular interactions that could regulate the function of a gap junction channel.
  • RXP-E may use the CT as a "scaffolding,” from which it can also interact with pore-forming or pore- vestibular regions (including the CL domain; see Duffy et al., "pH-Dependent Intramolecular Binding and Structure Involving Cx43 Cytoplasmic Domains," J. Biol. Chem.
  • RXP-E By interfering with gap junction regulation, RXP-E (or future derivatives of it) may serve as tools to dissect the specific role that gap junction regulation plays in determining the electrophysiological profile of the ischemic heart.
  • Peptides or pcptidc-dcrivcd molecules have been used in the past in an attempt to regulate Cx43. A number of those sequences have been derived from Cx43 itself (Calero et al., "A lVmer Peptide Interferes with Acidification-induced Uncoupling of Connexin43," Circ. Res.

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Abstract

La présente invention concerne des protéines et des polypeptides qui (i) répondent à la formule A-[W-A]n dans laquelle A représente un peptide répondant à la formule XXXXRXPXXXX, dans laquelle X représente un acide aminé, R représente une arginine et P une proline ; W représente un agent de liaison ; et n représente un quelconque nombre compris entre 1 et 5 ; ou qui (ii) répondent essentiellement à la formule XnRXPX3n dans laquelle chaque X représente un quelconque acide aminé, R représente une arginine, P représente une proline, n est un quelconque nombre compris entre zéro et quinze, m est un quelconque nombre compris entre zéro et quinze, la somme de n et m étant comprise entre dix et quinze. L’invention concerne également des procédés dans lesquels ces protéines et polypeptides servent à : cribler des composés modulant Cx43CT, mesurer l’affinité de liaison à Cx43CT d’un composé se liant à Cx43CT et à identifier l’emplacement d’un domaine de liaison à RXP de Cx43CT.
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DUFFY HEATHER S ET AL: "pH-dependent intramolecular binding and structure involving Cx43 cytoplasmic domains" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM, US, vol. 277, no. 39, 27 September 2002 (2002-09-27), pages 36706-36714, XP009108338 ISSN: 0021-9258 *
KWAK BRENDA R ET AL: "Selective inhibition of gap junction channel activity by synthetic peptides" JOURNAL OF PHYSIOLOGY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 516, no. 3, 1 May 1999 (1999-05-01), pages 679-685, XP009108312 ISSN: 0022-3751 *
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