WO1999021878A1 - Interaction of alpha-conotoxin peptides with neuronal nicotinic acetylcholine receptors - Google Patents
Interaction of alpha-conotoxin peptides with neuronal nicotinic acetylcholine receptors Download PDFInfo
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- WO1999021878A1 WO1999021878A1 PCT/US1998/022368 US9822368W WO9921878A1 WO 1999021878 A1 WO1999021878 A1 WO 1999021878A1 US 9822368 W US9822368 W US 9822368W WO 9921878 A1 WO9921878 A1 WO 9921878A1
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- 0 CC*C(*)C(C(C(C(C(C(*)C*)N=O)O)(O)OC)O)O Chemical compound CC*C(*)C(C(C(C(C(C(*)C*)N=O)O)(O)OC)O)O 0.000 description 3
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
Definitions
- the present invention relates to derivatives of the alpha conopeptide Mil. an ⁇ -4/7 conotoxin peptide. in which amino acid residues are substituted as described herein while maintaining activity substantially similar to Mil.
- the present invention is further directed to the discovery of the 3-dimensional structure of
- the present invention also relates to computer based programs for the expression of the three-dimensional structure of Mil and peptide analogs, peptide mimetics or non-peptide mimetics thereof.
- the ⁇ -conopeptide Mil has the amino acid sequence: Gly-Cys-Cys-Ser-Asn-Pro-Val-Cys- His-Leu-Glu-His-Ser-Asn-Leu-Cys (SEQ ID NO: l). This peptide contains two disulfide bonds between the first and third cysteine residues and between the second and fourth cysteine residues.
- the C-terminal end may contain a carboxyl or amide group, preferably an amide group.
- the amino acid at position 6 may be proline or 4-trans-hydroxyproline. preferably proline.
- the identification of Mil is described in U.S. Patent No. 5,514.774, inco ⁇ orated herein by reference.
- a major challenge for neurobiology is to define the function of the multiple forms of nicotinic receptors and ion channels that have recently been discovered in the nervous system by molecular techniques.
- the use of gene knock-out organisms is one method for examining such function.
- a complementary approach is to use ligands that specifically inhibit a particular molecular form of receptor or ion channel. Such ligands must be able to discriminate between closely related members of receptor families.
- Nicotinic acetylcholine receptors are ligand-gated ion channels which are key components of nervous systems. The classical role of these receptors was defined at the neuromuscular junction, nicotinic receptors concentrated on the muscle end plate serve as the key macromolecule that detects release of neurotransmitter from the presynaptic terminus of the motor axon. However, in addition to these skeletal muscle nicotinic receptors, many other molecular forms of nicotinic receptors exist; these are generally referred to as neuronal nicotinic receptors. In the central nervous system (CNS), nAChRs play a prominent role in modulating the release of neurotransmitters including dopamine, norepinephrine, acetylcholine, GABA and glutamate.
- CNS central nervous system
- Alpha conopeptide Mil is a small peptide with selective action on neuronal nicotinic receptors. In the autonomic nervous system, Mil was recently used to help pharmacologically dissect the nAChRs which mediate synaptic transmission in the parasympathetic ciliary ganglion. In frog sympathetic ganglia, Mil discriminates between nAChRs in B versus C neurons.
- Mil In the CNS, the specificity of Mil enables identification of subunits of nAChRs which modulate nicotine-stimulated dopamine release. In retina, Mil's selective block was used to confirm the role of nAChRs in the development of visual circuitry.
- SCLC small-cell lung carcinoma
- the use of Mil for detecting the presence and location of small-cell lung carcinoma (SCLC) tumors, treating a patient having SCLC and inhibiting proliferation of SCLC tumors is described in U.S. Patent 5,595,972, incorporated herein by reference.
- the use of Mil for treating disorders resulting from nicotine stimulated dopamine release is described in U.S. Patent 5,780,433, inco ⁇ orated herein by reference.
- the use of Mil as a cardiovascular agent is described in copending provisional application Serial
- the present invention relates to derivatives of the alpha conopeptide Mil, an ⁇ -4/7 conotoxin peptide, in which amino acid residues are substituted as described herein while maintaining activity substantially similar to MIL More specifically, the present invention is directed to the derivatives having the general formula (SEQ ID NO:2):
- Xaa-Cys-Cys-Xaa-Xaa Xaa 2 -Xaa-Cys-Xaa 3 -Xaa-Xaa 4 -Xaa 5 -Xaa-Xaa-Xaa-Cys, wherein Xaa represents an amino acid selected from the group consisting of natural, modified or non-natural amino acids.
- the modifications may be by addition substitution or deletion of one or more amino acid residues.
- the modification may also include the addition or substitution of analogs of the amino acid themselves, such as peptidomimetics, or amino acids with altered side groups, or mimetics, e.g., organic molecules with similar space/structure/function relationships.
- Xaa is any amino acid, preferably Asn or His; Xaa 2 is any amino acid, preferably Pro or hydroxy-Pro; Xaa 3 is any amino acid, preferably His or Asn; Xaa 4 is any amino acid, preferably Glu; and Xaa 5 is any amino acid, preferably His or Asn. It is most preferred that Xaa, is Asn; Xaa, is Pro or hydroxy-Pro; and Xaa 5 is His.
- the C-terminal end may contain a carboxyl or amide group, preferably an amide group.
- a second aspect of the present invention is directed to the discovery of the 3 -dimensional structure of Mil and the relationship of its structure to its specificity for the ⁇ 3 ⁇ 2 subtype of the neuronal nicotinic acetylcholine receptor (nAChR).
- nAChR neuronal nicotinic acetylcholine receptor
- the discovery of the 3-dimensional structure enables the design of ⁇ -4/7 conotoxin peptide analog, peptide mimetics and non-peptide mimetics which demonstrate specificity to different subtypes of neuronal nAChRs
- compounds are developed which demonstrate higher specificity to the ⁇ 3 ⁇ 2 subtype of neuronal nAChR than the ⁇ 3 ⁇ 4 subtype, such as evidence by MIL
- compounds are developed which demonstrate higher specificity to the ⁇ 3 ⁇ 4 subtype of neuronal nAChR than the ⁇ 3 ⁇ 2 subtype, such as evidence by several of the Mil derivatives described herein.
- the derivatives, peptide analogs, peptide mimetics and non-peptide mimetics of the present invention are useful as cardiovascular agents, gastric motility agents, urinary incontinence agents, anti-smoking agents and for treating or detecting small-cell lung carcinoma (SCLC) .
- SCLC small-cell lung carcinoma
- a third aspect of the present invention is directed to the synthesis of mimetics, e.g., organic molecules based on the three-dimensional structure of Mil which demonstrates the same or different specificity to neuronal nAChRs.
- mimetics e.g., organic molecules based on the three-dimensional structure of Mil which demonstrates the same or different specificity to neuronal nAChRs.
- a fourth aspect of the present invention is directed to the synthesis of peptide analogs and peptide mimetics of Mil with altered nicotinic AChR subtype specificity based on the identification of peptide fragments which define the activity and selectivity of the conopeptide Mil and on its three-dimensional structure.
- a fifth aspect of the present invention is directed to synthesis of mimetics, e.g., organic molecules based upon the overall structural activity information relating to MIL
- a sixth aspect of the present invention also provides for computer programs for the expression (such as visual display) of the three-dimensional structure of Mil or a peptide analog or peptide mimetic thereof, and further, a computer program which expresses the identity of each constituent of Mil and the precise location within the overall structure of that constituent, down to the atomic level.
- computer programs for the expression of the three-dimensional structure of a molecule There are many currently available computer programs for the expression of the three-dimensional structure of a molecule. Generally, these programs provide for inputting of the coordinates for the three-dimensional structure of a molecule; means to express (such as visually display) such coordinates, means to alter such coordinates and means to express an image of a molecule having such altered coordinates.
- NMR coordinates of the location of the atoms of an Mil molecule in three dimension space wherein such coordinates have been obtained from NMR analysis of said Mil molecule
- CAVEAT is also provided, therefore, is a computer program for the expression of the three-dimensional structure of a peptide analog or peptide mimetic of MIL Preferred.
- Figure 1 shows the effect of alanine substitutions in the conopeptide Mil on the ability of the analogs to block acetylcholine-gated currents in voltage-clamped Xenopus oocytes expressing cloned rat ⁇ 3 and ⁇ 2 subunits.
- Figure 2 shows the NOESY spectrum (250-ms mixing time) of ⁇ -CT x Mil in H 2 O illustrating sequential NOE connectives between neighboring ⁇ and amide protons.
- the d aN NOE cross peaks are connected by lines and are labeled by residue number. Two traces between residues
- Figure 3 shows an amino acid sequence of ⁇ -CT x Mil with its disulfide bridges drawn to show its disulfide bridge pattern and a summary of short- to medium-range sequential NOE cross peaks as well as experimentally measured 3 J NH.C ⁇ H coupling constants. Arrows pointing down are those with 3 J constants ⁇ 5.0 Hz, and those pointing up are those with 3 J constants >8.0 Hz. Filled bars represent the NOE cross peaks with the thickness classifying NOE intensities from strong (thick) to weak (thin) and the width indicating short- to medium-range separation along the linear sequence. *** represents overlapped NOE cross peaks.
- Figure 4 shows ⁇ proton chemical shift differences (ppm), between observed values from ⁇ -CT x Mil in H 2 0 and random-coil values, plotted versus the residue.
- Figures 5A-B show superimposition of the 14 lowest energy structures of ⁇ -CT x Mil calculated from simulated annealing.
- Figure 6A-C show the angular order parameters for the backbone obtained from the 14 minimum energy structures, plotted versus the residue number.
- Figure 6A shows parameters for ⁇ , Figure 6B for ⁇ ; and Figure 6C for ⁇ .
- Figure 7 shows surface (A,B) and backbone (C, D, E) representations of ⁇ -CT xs Mil, PnIA, and GI.
- Space-filling models of the X-ray crystal structure of ⁇ -CT x PnIA (A) and the lowest energy structure of ⁇ -CT Mil (B) are displayed in pu ⁇ le for hydrophobic residues, in yellow for polar side chains and in red (positive) and blue (negative) for charged side chains.
- Backbone conformations of ⁇ -CT x PnIA (C), ⁇ -CT x Mil (D), and ⁇ -CT GI (E) are displayed in ribbons and surface distributions in dots with the same color codes to illustrate their similarity and difference.
- Figure 8 lists the coordinates of ⁇ -conotoxin MIL These coordinates have been deposited in the Brookhaven Protein Data Bank, Upton, NY 19973, under accession code lm2c.
- Figure 9 shows selective block by ⁇ -Conotoxin Mil of ⁇ 3 ⁇ 2 nAChRs.
- Oocytes expressing various nAChR subunit combinations were voltage-clamped and the response to ACh was measured at various ⁇ -Conotoxin Mil concentrations.
- Data represent the mean ⁇ S.E. for at least three oocytes at each concentration.
- Figure 10 shows that block of ⁇ 3 ⁇ 2 nAChRs by ⁇ -Conotoxin Mil is independent of the membrane potential. Oocytes expressing ⁇ 3 ⁇ 2 nAChRs were voltage-clamped and the response to
- ACh was measured over a range of membrane potentials. Peak-amplitude currents in the absence (o) and presence (* ⁇ ) of 500 pM ⁇ -Conotoxin Mil are plotted against the membrane potential.
- Figure 11A-C show that the competitive antagonist, Dihydro- ⁇ -Erythroidine (DH ⁇ E), protects ⁇ 3 ⁇ 2 nAChRs from blockade by ⁇ -Conotoxin MIL Oocytes expressing ⁇ 3 ⁇ 2 nAChRs were voltage-clamped and the response to ACh was measured.
- DH ⁇ E Dihydro- ⁇ -Erythroidine
- Figure 11 A 500 ⁇ M DH ⁇ E was applied for 5 min. The oocyte was then continuously perfused with buffer without DH ⁇ E.
- Figure 1 20 nM ⁇ -Conotoxin Mil was applied for 2 min. The oocyte was then continuously perfused with buffer without toxin.
- Figure 12A and B show the kinetics of block of ⁇ 3 ⁇ 2 and ⁇ 3 ⁇ 4 nAChRs by ⁇ -Conotoxin
- FIG. 12A shows the response of voltage-clamped oocytes expressing ⁇ 3 ⁇ 2 nAChRs to ACh measured during application of 500 pM ⁇ -Conotoxin Mil (first graph) and following washout from toxin (second graph).
- Figure 12B shows the response of voltage-clamped oocytes expressing ⁇ 3 ⁇ 4 nAChRs to ACh measured during application of 1 ⁇ M ⁇ -Conotoxin Mil (first graph) and following washout from toxin (second graph).
- Figure 13A and B show the recovery kinetics of ⁇ 3 ⁇ 2 and ⁇ 3 ⁇ 4 nAChRs from block by saturating concentrations of ⁇ -Conotoxin MIL
- Figure 13A shows voltage-clamped oocytes expressing ⁇ 3 ⁇ 2 nAChRs and their response to ACh. 2 ⁇ M ⁇ -conotoxin Mil was applied for 5 min. The oocyte was then continuously perfused with buffer without toxin.
- Figure 13B shows oocytes expressing ⁇ 3 ⁇ 4 nAChRs, which were voltage-clamped and their response to ACh. 2.5 mM ⁇ -
- Conotoxin Mil was applied for 5 min. The oocyte was then continuously perfused with buffer without toxin. Data were fit to a 2-site model (solid lines). Dashed lines represent the expected single-exponential recovery kinetics.
- FIGS 14A-C show the Mil "Dock & Lock" model of ligand/receptor interation.
- Mil binds at the interface between the ⁇ and ⁇ subunits.
- Mil interacts with the ⁇ subunit with a very fast k on and k otf and thus moderate affinity.
- Mil interacts with the ⁇ subunit with a very slow k on and k ott and thus moderate affinity.
- Fig. 14A and 14B Mil approaches the receptor and very rapidly binds to the ⁇ subunit (docking interaction). However, this rapid binding to the ⁇ subunit in close proximity to the ⁇ subunit binding site leads to enhancement of the rate of Mil's interaction with the ⁇ subunit and subsequent blockage of the receptor.
- Fig. 14C shows that once Mil is bound to the ⁇ subunit, it has a very slow k otf (locking interaction).
- Mil is able to convert two relatively moderate binding interactions into a very potent and specific interaction with one molecular form of neuronal nAChR.
- SEQ ID NO: 1 is the amino acid sequence for ⁇ -CT x Mil peptide.
- SEQ ID NO:2 is the amino acid sequence for derivatives of the alpha conopeptide MIL
- SEQ ID NO:3 is the amino acid sequence for the FATN chimera of MIL
- the present invention relates to derivatives of Mil, an ⁇ -4/7 conotoxinpeptide, in which amino acid residues are substituted as described herein while maintaining the basic activity of MIL More specifically, the present invention is directed to the derivatives having the general formula (SEQ ID NO:2):
- the modifications may be by addition substitution or deletion of one or more amino acid residues.
- the modification may also include the addition or substitution of analogs of the amino acid themselves, such as peptidomimetics, or amino acids with altered side groups, or mimetics, e.g., organic molecules with similar space/structure/function relationships.
- Xaa is any amino acid, preferably Asn or His:
- Xaa 2 is any amino acid, preferably Pro or hydroxy-Pro;
- Xaa 3 is any amino acid, preferably His or Asn;
- Xaa 4 is any amino acid, preferably Glu; and
- Xaa 5 is preferably His or Asn. It is most preferred that Xaa, is Asn; Xaa-, is Pro or hydroxy-Pro; and Xaa 5 is His.
- the derivatives contain two disulfide bonds between the first and third cysteine residues and between the second and fourth cysteine residues.
- the C-terminal end may contain a carboxyl or amide group, preferably an amide group.
- the amino acid is an ⁇ -amino acid, which includes natural amino acids, including unusual amino acids such as ⁇ -carboxyglutamic acid, as well as modified or non-natural amino acids, such as those described in, for example, Roberts et al. (1983).
- the derivatives of the present invention are highly selective for either the ⁇ 3 ⁇ 2 or ⁇ 3 ⁇ 4 subtype of neuronal nAChRs which are present in the autonomic and central nervous systems. Thus, they are useful as cardiovascular agents, gastric motility agents, urinary incontinence agents and anti-smoking agents, as well as being useful against SCLC.
- the conopeptides can be produced by recombinant DNA techniques well known in the art. Also, these conopeptide derivatives are sufficiently small to be chemically synthesized. General chemical syntheses for preparing the foregoing conopeptide derivatives are described hereinafter, along with specific chemical synthesis of conopeptide derivatives and indications of biological activities of these synthetic products.
- the conopeptides of the present invention may be synthesized and/or substantially pure. By “substantially pure” is meant that the peptide is present in the substantial absence of other biological molecules of the same type; it is preferably present in an amount of at least about 85% purity and preferably at least about 95% purity.
- the conopeptides of the present invention can be produced by recombinant DNA techniques well known in the art , such as those described in, for example, Sambrook et al. (1979).
- the peptides produced in this manner are isolated, reduced if necessary, and oxidized to form the correct disulfide bonds, if present in the final molecule.
- One method of forming disulfide bonds in the conopeptides of the present invention is the air oxidation of the linear peptides for prolonged periods under cold room temperatures or at room temperature. This procedure results in the creation of a substantial amount of the bioactive, disulfide-linked peptides.
- the oxidized peptides are fractionated using reverse-phase high performance liquid chromatography (HPLC) or the like, to separate peptides having different linked configurations. Thereafter, either by comparing these fractions with the elution of the native material or by using a simple assay, the particular fraction having the correct linkage for maximum biological potency is easily determined. It is also found that the linear peptide.
- the oxidized product having more than one fraction can sometimes be used for in vivo administration because the cross-linking and/or rearrangement which occurs in vivo has been found to create the biologically potent conopeptide molecule.
- a somewhat higher dosage may be required.
- peptides can be chemically synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
- the peptide chain can be prepared by a series of coupling reactions in which constituent amino acids are added to the growing peptide chain in the desired sequence.
- various coupling reagents e.g., dicyclohexylcarbodiimide or diisopropylcarbonyldimidazole
- various active esters e.g., esters of N-hydroxyphthalimide or N- hydroxy-succinimide
- the various cleavage reagents to carry out reaction in solution, with subsequent isolation and purification of intermediates, is well known classical peptide methodology.
- an intermediate compound which includes each of the amino acid residues located in its desired sequence in the peptide chain with appropriate side-chain protecting groups linked to various ones of the residues having labile side chains.
- a side chain amino protecting group As far as the selection of a side chain amino protecting group is concerned, generally one is chosen which is not removed during deprotection of the a-amino groups during the synthesis. However, for some amino acids, e.g., His, protection is not generally necessary. In selecting a particular side chain protecting group to be used in the synthesis of the peptides.
- the protecting group preferably retains its protecting properties and is not split off under coupling conditions
- the protecting group should be stable under the reaction conditions selected for removing the a-amino protecting group at each step of the synthesis, and ⁇ the side chain protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.
- peptides are preferably prepared using the Merrifield solid-phase synthesis, although other equivalent chemical syntheses known in the art can also be used as previously mentioned.
- Solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected a-amino acid to a suitable resin.
- a suitable resin can be prepared by attaching an a-amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine (BHA) resin or para- methylbenzhydrylamine (MBHA) resin.
- BHA benzhydrylamine
- MBHA para- methylbenzhydrylamine
- BHA and MBHA resin supports are commercially available, and are generally used when the desired polypeptide being synthesized has an unsubstituted amide at the C-terminus.
- solid resin supports may be any of those known in the art, such as one having the formulae -O-CH 2 -resin support, -NH BHA resin support, or -NH-MBHA resin support.
- unsubstituted amide is desired, use of a BHA or MBHA resin is preferred, because cleavage directly gives the amide.
- N-methyl amide In case the N-methyl amide is desired, it can be generated from an N-methyl BHA resin. Should other substituted amides be desired, the teaching of U.S. Patent No. 4,569,967 (Kornheim et al., 1986) can be used, or should still other groups than the free acid be desired at the C-terminus, it may be preferable to synthesize the peptide using classical methods as set forth in the Houben-Weyl text (1974). The C-terminal amino acid, protected by Boc or Fmoc and by a side-chain protecting group, if appropriate, can be first coupled to a chloromethylated resin according to the procedure set forth in Horiki et al.
- the remaining a-amino- and side chain- protected amino acids are coupled step-wise in the desired order to obtain the intermediate compound defined hereinbefore, or as an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor.
- Selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as a coupling reagent is N,N'-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU in the presence of HoBt or HoAt).
- activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art.
- suitable activating reagents are carbodiimides, such as N,N'- diisopropylcarbodiimide and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide.
- Other activating reagents and their use in peptide coupling are described by Schroder and Lubke (1965) and Kapoor
- Each protected amino acid or amino acid sequence is introduced into the solid-phase reactor in about a twofold or more excess, and the coupling may be carried out in a medium of dimethylformarnide (DMF):CH 2 C1 2 (1 :1) or in DMF or CH 2 C1 2 alone.
- DMF dimethylformarnide
- the coupling procedure is repeated before removal of the a-amino protecting group prior to the coupling of the next amino acid.
- the success of the coupling reaction at each stage of the synthesis if performed manually, is preferably monitored by the ninhydrin reaction, as described by Kaiser et al. (1970).
- Coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al.
- the intermediate peptide can be removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride or TFA (if using Fmoc chemistry), which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups and also the a-amino protecting group at the N-terminus if it was not previously removed to obtain the peptide in the form of the free acid.
- a reagent such as liquid hydrogen fluoride or TFA (if using Fmoc chemistry)
- TFA trifluoroacetic acid
- one or more scavengers such as anisole, cresol, dimethyl sulfide and methylethyl sulfide are included in the reaction vessel.
- Cyclization of the linear peptide is preferably affected, as opposed to cyclizing the peptide while a part of the peptido-resin, to create bonds between Cys residues.
- fully protected peptide can be cleaved from a hydroxymethylated resin or a chloromethylated resin support by ammonolysis, as is well known in the art, to yield the fully protected amide intermediate, which is thereafter suitably cyclized and deprotected. Alternatively, deprotection.
- the conopeptide derivatives of the present invention may possess biological activity different from Mil or may demonstrate the same or varying degrees of the same biological activity as Mil which is known to block native ⁇ 3 ⁇ 2-containing nAChRs and with lower potencies, ⁇ 3 ⁇ 4 containing nAChRs (U.S. Patent 5,780,433 inco ⁇ orated herein by reference), as well as other activities described herein.
- Another aspect of the present invention is directed to the discovery of the 3 -dimensional structure of Mil and the relationship of its structure to its specificity for the ⁇ 3 ⁇ 2 subtype of the neuronal nicotinic acetylcholine receptor (nAChR).
- the discovery of the 3-dimensional structure enables the design of ⁇ -4/7 conotoxin peptide analogs and peptide mimetics which will demonstrate the same specificity to neuronal nAChR.
- Such analogs and peptide mimetics are useful as cardiovascular agents and for treating or detecting small-cell lung carcinoma (SCLC) .
- Mil has the following amino acid sequence (SEQ ID NO:l): Gly-Cys-Cys-Ser-Asn-Pro-Val-Cys-His-Leu-Glu-His-Ser-Asn-Leu-Cys.
- the C-terminus may contain a carboxyl or amide group, preferably an amide group.
- the amino acid at position 6 may be proline or 4-trans-hydroxyproline, preferably proline.
- Mil for: (a) treating a patient having small-cell lung carcinoma (SCLC); (b) inhibiting SCLC proliferation; (c) detecting the presence of SCLC tumors, and (d) detecting the location of SCLC tumors is described in application U.S. Patent 5,595,972, inco ⁇ orated herein by reference. The use of Mil to treat CNS disorders is described in U.S. Patent 5,595,972, inco ⁇ orated herein by reference. The use of Mil to treat CNS disorders is described in U.S. Patent
- the primary structure is obtained by biochemical methods, either by direct determination of the amino acid sequence from the protein, or from the nucleotide sequence of the corresponding gene or cDNA.
- NMR methods may be employed to obtain the secondary and tertiary structure, which requires detailed information about the arrangement of atoms within a protein.
- NMR methods use the magnetic properties of atomic nuclei.
- Certain atomic nuclei such as 'H, l3 C, N, and 31 P have a magnetic moment or spin.
- the chemical environment of such nuclei can be probed by nuclear magnetic resonance, NMR, and this technique can be exploited to give information on the distances between atoms in a molecule. These distances can then be used to derive a three-dimensional model of the molecule.
- Most structure determinations of protein molecules by NMR have used the spin of 'H. since hydrogen atoms are abundant in proteins. When protein molecules are placed in a strong magnetic field, the spin of their hydrogen atoms aligns along the field. This equilibrium alignment can be changed to an excited state by applying radio frequency (RF) pulses to the sample.
- RF radio frequency
- the nuclei of the protein molecule When the nuclei of the protein molecule revert to their equilibrium state, they emit RF radiation that can be measured.
- the exact frequency of the emitted radiation from each nucleus depends on the molecular environment of the nucleus and is different for each atom, unless they are chemically equivalent and have the same molecular environment. These different frequencies are obtained relative to a reference signal and are called chemical shifts.
- the nature, duration and combination of applied RF pulses can be varied enormously and different molecular properties of the sample can be probed by selecting the appropriate combination of pulses. In principle, it is possible to obtain a unique signal (chemical shift) for each hydrogen atom in a protein molecule, except those that are chemically equivalent, for example, the protons on the
- Two-dimensional experiments consist of discreet elements: a preparation period; an evolution period where spins are "labeled” as they process in the xy plane according to their chemical shift; a mixing period, during which correlations are made with other spins; and a detection period, where a free induction decay is recorded.
- Nuclear overhauser effect spectroscopy gives peaks between pairs of hydrogen atoms that are close together in space even if they are from amino acid residues that are quite distant in the primary sequence.
- TOCSY total correlation spectroscopy
- Two dimensional NMR spectra of proteins are inte ⁇ reted by the method of sequential assignment.
- two-dimensional NOE spectra by specifying which groups are close together in space, contain three-dimensional information about the protein molecule. It is far from trivial, however, to assign the observed peaks in the spectra to hydrogen atoms in specific residues along the polypeptide chain because the order of peaks along the diagonal has no simple relation to the order of amino acids along the polypeptide chain. This problem has in principle been solved in the laboratory of Kurt W ⁇ thrich in the E.T.H., Zurich, where the method of sequential assignment was invented.
- Sequential assignment is based on the differences in the number of hydrogen atoms and their covalent connectivity in the different amino acid residues.
- Each type of amino acid has a specific set of covalently connected hydrogen atoms that will give a specific combination of cross-peaks, a "finge ⁇ rint.” in a COSY spectrum. From the COSY spectrum, it is therefore possible to identify the H atoms that belong to each amino acid residue and, in addition, determine the nature of the side chain of that residue. However, the order of these finge ⁇ rints along the diagonal has no relation to the amino acid sequence of the protein.
- NOE spectra that record signals from H atoms that are close together in space. In addition to the interactions between H atoms that are far apart in the sequent, these spectra also record interactions between H atoms from sequentially adjacent residues. These signals in the NOE spectra therefore in principle make it possible to determine which finge ⁇ rint in the COSY spectrum comes from a residue adjacent to the one previously identified.
- NMR spectroscopy identifies H atoms in the protein that are close together in space, and this information is then used to derive, indirectly, a three-dimensional model of the protein.
- Distance constraints can be used to derive possible structures of a protein molecule.
- the result of the sequence-specific assignment of NMR signals is a list of distance constraints from specific hydrogen atoms in one residue to hydrogen atoms in a second residue.
- the list contains a large number of such distances, which are usually divided into three intervals within the region 1.8 A to 5 A, depending on the intensity of the NOE peak.
- This list immediately identifies the secondary structure elements of the peptide or protein molecules because both ⁇ helices and ⁇ sheets have very specific sets of interactions of less than 5 ⁇ between their hydrogen atoms. It is also possible to derive models of the three-dimensional structure of the molecule.
- ⁇ -CT x Mil is a small peptide made of only 16 amino acid residues, it has a well-defined three-dimensional solution structure.
- two disulfide bridges which form a hydrophobic Cys knot there are three helical regions in the structure which contribute to form a very tight conformation.
- the presence of such stable secondary structures and disulfide bridges allows the multidimensional NMR method to be effective in obtaining a very high resolution structure of the peptide.
- the function of His may be to bring Cys 3 and Cys 16 close together to form the second disulfide bridge.
- the space-filling model of ⁇ -CT x Mil shown in Figure 7B is oriented to explore the surface distribution of hydrophobic and hydrophilic side chain groups of the molecule.
- Hydrophobic residues are colored pu ⁇ le to distinguish them from polar residues which are yellow, or charged residues which are red (positive) and blue (negative).
- a flat surface located on top of the molecule in pu ⁇ le is a distinct structural feature representing the cluster of hydrophobic residues exposed to solvent. This hydrophobic surface is formed largely by Gly 1 (excluding the N-terminal amino group), Cys 2 , Cys 3 , Leu 15 , Cys 16 and the disulfide bond between Cys 3 and Cys 16 .
- This flat surface may be important for ligand-receptor binding through hydrophobic interactions.
- NMR data from an ⁇ -4/7 conotoxin Mil mutant suggests that the mutation causes the central helix to further extend beyond the j+3 position towards the C-terminal end and thereby causing its helical pitch to change. This structural change is associated with the loss of activity by three orders of magnitude.
- N- and C-caps Two turns at both the N and C terminal ends (N- and C-caps) of the central helix are also unique structural features found in this class of conotoxins. They are intimately linked with the central helix to dictate the three dimensional folding motif and their positions are i+2 for the N-cap where is the second cysteine residue and ; " +4 for the C-cap where y is the third cysteine. Based on the primary sequence comparison among the sequenced ⁇ -4/7 conotoxins, there appears to be some preference for both N- and C-cap positions. These are asparagine, which is a good hydrogen bonding partner, and histidine and tyrosine, which are sterically bulky aromatic residues.
- Proline residue at position /+3 where . is the second cysteine residue is conserved among all ⁇ -4/7 conotoxins. Its structural role is mostly likely to prevent continuation of the central helix in the amino terminal direction due to its sterically hindering ring structure.
- peptide docking and the hydrophobic face is particularly important for peptide off-rate (peptide locking).
- the use of two physically distinct peptide surfaces to interact with adjacent ⁇ and ⁇ receptor subunits represents a specific strategy for designing ligands selective for receptors with different combinations of ⁇ and ⁇ subunits.
- Mil achieves its remarkable subtype specificity through double-faced interacting with the receptor. Mil's rank order of potency suggests that Mil interacts with both ⁇ and non- ⁇ subunits of nAChRs. Block by Mil is consistent with competitive inhibition as evidenced by voltage- independence of Mil's activity and the ability of competitive antagonist dihydro- ⁇ -erythroidine to interfere with Mil's ability to block the receptor. The shape of the time course recovery curve following block of ⁇ 3 ⁇ 2 and ⁇ 3 ⁇ 4 receptors by high concentrations of Mil indicates that each of these receptors has two binding sites for Mil, and occupancy of either site by toxin blocks receptor function.
- Mil is a roughly wedge-shaped peptide with a hydrophilic edge and a hydrophobic edge.
- the four amino-acids of ⁇ -conotoxin Mil (HLEH) which were substituted to FATN are all located on the hydrophilic edge of the peptide.
- the differential on and off rates for Mil can be used to develop an assay to screen compounds for nAChR antagonistic activity and receptor subtype specificity.
- the on and off rates for the binding of Mil and a compound to be screened to the ⁇ 3 ⁇ 2 and ⁇ 3 ⁇ 4 subunits are determined and compared. If the on rate for the compound is greater than or equal to the on rate for Mil with respect to the ⁇ 3 ⁇ 2 subtype, the on rate for the compound is less than or equal to the on rate for Mil with respect to the ⁇ 3 ⁇ 4 subtype and the off rates for the compound are greater than or equal to the off rates for Mil , then the compound is a nAChR antagonist and specific for the ⁇ 3 ⁇ 2 subtype.
- the on rate for the compound is less than or equal to the on rate for Mil with respect to the ⁇ 3 ⁇ 2 subtype, the on rate for the compound is greater than or equal to the on rate for Mil with respect to the ⁇ 3 ⁇ 4 subtype and the off rates for the compound are greater than or equal to the off rates for Mil, then the compound is a nAChR antagonist and specific for the ⁇ 3 ⁇ 4 subtype.
- compounds can be developed which modulate the activity of neuronal nAChRs having ⁇ 3 ⁇ 2 or ⁇ 3 ⁇ 4 subtype specificity based on the three dimensional structure of Mil as disclosed herein, the structure/biological activity of Mil disclosed herein and molecular modeling analysis.
- compounds are developed which modulate the activity of neuronal nAChRs and which have subtype specificity for either the ⁇ 3 ⁇ 2 or ⁇ 3 ⁇ 4 subtype.
- the goal of rational drug design is to produce structural analogs of biologically active polypeptides or compounds with which they interact (agonists, antagonists, inhibitors, binding partners, etc.). By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules.
- drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules.
- Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
- a substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature.
- Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use.
- the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., pure peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
- Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property.
- the pharmacophore Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g., stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g., spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modeled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
- the physical properties e.g., stereochemistry, bonding, size and/or charge
- data from a range of sources e.g., spectroscopic techniques, x-ray diffraction data and NMR.
- a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
- the template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
- the mimetic is peptide-based
- further stability can be achieved by cyclizing the peptide, increasing its rigidity.
- the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
- Recent advances in techniques for generating 3D molecular structures for typical drug-sized organic molecules and for searching databases of 3D structures combined with a 3D pharmacophore hypothesis give the medicinal chemist new tools that can assist in discovering novel active molecules.
- a 3D pharmacophore can be used as a query to search a database of molecules for those that are predicted to possess the desired activity if the pharmacophore hypothesis is correct. If a receptor structure or receptor site model is available, another type of 3D searching can be used. For this, molecules are selected based on their steric and chemical match to the receptor structure, without the need to propose which interactions might be most important. The methods are complementary and both have assisted in the discovery of active molecules.
- HLEH fragment of Mil has been investigated and its biological activity and receptor binding has been correlated to its structural, chemical and physical attributes (features). These features are used to help understand the range of biological activity observed in a series of compounds, as well as to help guide the design of new compounds with potentially different affinities for nicotinic nAChRs, e.g., compounds with higher affinity for the ⁇ 3 ⁇ 2 subtype or compounds with higher affinity for the ⁇ 3 ⁇ 4 subtype.
- pharmacophore mapping involves three main aspects: finding the features required for biological activity; determining the molecular conformation required (i.e., the "bioactive" conformation); and developing a supe ⁇ osition or alignment rule for the series of compounds.
- the primary information used in pharmacophore mapping is derived naturally from the compounds synthesized in a series and their measured biological activity. From this, structure-activity relations emerge and rudimentary pharmacophore hypotheses can begin to be formulated.
- Pharmacophore mapping is largely a qualitative exercise. Allied with pharmacophore mapping is the field of three-dimensional quantitative structure-activity relations (3D-QSAR). 3D- QSAR techniques attempt to derive and investigate quantitative models of biological activity; namely, models that fit the potency of the studied compounds and that can be used to predict the potency of compounds outside the study set. In fact, the application of many 3D-QSAR methods requires a proposed pharmacophore model. Pharmacophore mapping has its roots in traditional medicinal chemistry and the structure- activity relations derived from the synthesis and biological testing of series of compounds. From the relative measured potencies of individual members of a series, a picture of the types of functionality, and perhaps their spatial relations that are important for activity, begins to emerge. More detailed molecular-modeling studies can then be used to formulate a more rigorous model but even in their absence proposed pharmacophore hypotheses can begin to take shape.
- 3D-QSAR three-dimensional quantitative structure-activity relations
- Receptor site mapping encompasses a variety of computational procedures that identify energetically favorable binding sites on macromolecules.
- the most straightforward procedures involve "painting" a solvent-accessible surface (or an otherwise generated cast) of the macromolecular target according to empirically determined physical properties, such as electrostatic or lipophilic potential, degree of curvature, and hydrogen-bonding character.
- Such methods for thus characterizing the surface of a macromolecule are inco ⁇ orated in programs such as Grasp (Columbia University), DelPhi (Biosym Technologies), MOLCAD (Tripos. Inc.), and Hint (Virginia Commonwealth University).
- Subsequent molecule design involves identification or design of ligands that possess features complimentary to the identified surface characteristics.
- More advanced algorithms involve the actual calculation of interaction enthalpies between the target and potential ligands or fragments.
- the coordinates of the protein or protein fragment of interest (which may be rotated or otherwise transformed) are stripped of any undesired ligand (or portion thereof) and/or of any undesired solvent molecules.
- the coordinates are then processed to attach molecular mechanics parameters to the atomic positions to provide a processed target for mapping.
- the target may be partitioned into discrete binding sites.
- the target or partitioned sites thereof are flooded with given functional group fragments that are subsequently allowed to relax into desired locations, as in the program MCSS (Molecular Simulations, Inc.), or are encased within a regular lattice of site points on which single fragment probes are positioned sequentially; examples of programs that exploit the site-lattice algorithm include Grin Grid (Molecular Discovery, Ltd.), Ludi (Biosym Technologies), Leapfrog (Tripos, Inc.), and Legend (University of Tokyo). In both techniques, the enthalpic contribution to binding affinity is estimated with a molecular mechanics force-field, and appropriate positions of selected functional groups are determined systematically.
- MCSS Molecular Simulations, Inc.
- a box is defined enclosing a desired portion of the target within a defined lattice.
- the lattice resolution i.e., the distance between lattice points, may be defined by the practitioner or may be set by the computer program.
- the other parameters of points within the lattice such as hydrophobicity or other characteristics, may be similarly defined.
- the model is then subjected to group minimization (i.e., molecular mechanics minimization) calculations to identify points or areas of favorable interaction. Data may be handled as in the lattice approach.
- Receptor site maps provide the seeds for ligand evolution via Database searches, which are described above, and for Grow/link methods for de Novo design of new chemical entities.
- Programs for ligand growth first access extensible fragment dictionaries in order to place appropriate functional groups at site points. A genetic algorithm or a subgraph isomo ⁇ hism protocol is then invoked to connect the fragments with small aliphatic chains or rings.
- Stochastic enhancements may be introduced by modification of internal degrees of freedom as well as translation and rotation of the candidate model within the binding cavity. The resulting sets of molecules are scored and filtered by functions that consider the steric constraints of the binding site, the complimentarity of electrostatic and hydrophobic interactions, and a solvation estimate. Programs of this type that could be applied include Ludi (Biosym Technologies), Leapfrog (Tripos, Inc.), Legend (University of Tokyo), Grow (Upjohn), Builder/Delegate (University of California, San Francisco), and Sprout
- DOCK Universally of California, San Francisco
- similar programs fill a given binding site with the smallest set of atom-sized spheres possible; a database search then attempts to orient ligands such that the atoms superimpose onto the centers (or "nuclei") of the site-filling spheres.
- optimization of ligands may be enhanced using the three dimensional structural of MIL
- Use of receptor site maps or hydropathic profiles of Mil may be used to identify preferred positions for functional group components of ligands, and can be used to filter or constrain conformational searches of ligand structures which would otherwise typically be controlled by minimal steric considerations of the ligand structures themselves.
- the availability of an explicit binding site also permits one to determine the mode of ligand binding to the target protein via methods that utilize force-fields directly in simulated annealing, distance geometry, Metropolis Monte Carlo, or stochastic searches for binding modes. Examples of programs that can be applied to rationalize ligand binding include Autodock (Scripps Clinic), DGEOM (QCPE #590), Sculpturethane, etc.
- An alternative protocol for ligand optimization involves 3D database searching in conjunction with knowledge of the binding site. Modeling can reveal multiple candidates for the bioactive conformation of a given ligand. A probe for the correct conformation can include a 3D search to identify several constrained mimics of each possible conformer. Structure-activity relationships of the unconstrained ligand would suggest which functional groups should be retained in the constrained mimics. Finally, the steric and electrostatic requirements of the binding site could constitute a filter for prioritizing the resultant possibilities.
- the structure of Mil permits accurate model-building of homologous peptides and their subsequent use in drug design.
- the Mil structure may be readily used in the development of a reliable model by either knowledge-based template building methods, or by iterations of directed point mutations followed by local molecular mechanics minimizations. Examples of programs that can be applied in the development of a model of Syk-NC include Composer (Birbeck College), Modeler (MSI), and Homology (Biosym Technologies). The resultant model can then be subjected to any of the CADD techniques described above.
- Structure-based approaches include de Novo molecular design, computer-aided optimization of lead molecules, and computer-based selection of candidate drug structures based on structural criteria.
- New pepidomimetic modules may be developed directly from the structure of the peptide ligand by design or database searches for conformationally- restricted peptide replacements.
- structure-based lead discovery may be accomplished using the target protein structure, e.g. receptor, stripped of its ligand.
- fragment seed linking refers to methods for designing structures that contain "linked” "seeds," i.e. chemical structures comprising two or more of the mapped moieties appropriately spaced to reach the respective sites of favorable interactions.
- Growth refers to the design of structures which extend, based on receptor site mapping or to fill available space, a given molecule or moiety.
- the receptor site mapping data Based on the receptor site mapping data, one may also select potential ligands from databases of chemical structures. Potential ligands, or suboptimal ligands, of whatever source, can be refined by using the receptor site maps to filter multiple ligand conformations and orientations according to energetic preferences.
- the structure of Mil permits one to generate a high- quality model of Mil by either knowledge-based homology template methods or iterative site- mutations followed by minimizations. The generated structure of Mil may then be treated as an additional protein target by the methods outlined above.
- the amino acid sequence of ⁇ -conotoxin Mil has been determined (Cartier, et al.. 1996) and the determinants for specificity (specific amino acid residues) for Mil on ⁇ 3 ⁇ 2 neuronal nicotinic receptors have been identified (Harvey, et al., 1997, inco ⁇ orated herein by reference). Knowledge of the determinants and the binding orientation of a peptide can suggest avenues for conformational restriction and peptide bond replacement. A less biased approach involves computer algorithms for searching databases of three-dimensional structures to identify replacements for one or more portions of the peptide ligand, preferably non-peptidic replacement moieties. By this method, one can generate compounds for which the bioactive conformation is heavily populated, i.e., compounds which are based on particularly biologically relevant conformations of the peptide ligand.
- CAVEAT is a program that uses a database search to assist in the design of novel molecules. It is a special-pu ⁇ ose program that was written to aid in the design of small molecules that could mimic a protein loop important in a protein-protein interaction. The basic assumption of the programs is that protein-protein recognition is based on specific positioning of amino acid side chains and that the backbone is not critical and could be replaced.
- the CAVEAT program attempts to find ring systems that can present the side chains in the desired orientation.
- a CAVEAT database consists of the distance and angle relations of vectors, defined by the exocyclic bonds of ring systems, and an index back to the 3D structure of the ring.
- a typical query might define the C ⁇ -C ⁇ vector relations that would be required to present the necessary side chain analogues.
- CAVEAT is optimized for this task.
- the latest CAVEAT suite of programs includes the ability to cluster the hits, based on size or substructure similarity, and to rank hits by size, number of atoms at ring fusions, and whether the designed molecule would have eclipsing interactions.
- Two novel databases are available for use with CAVEAT: Triad, a computer-generated collection of all tricyclic hydrocarbons, and Iliad, a collection of acyclic molecules.
- Software is also available to create CAVEAT databases from the Cambridge Structural Database.
- the inco ⁇ oration into a ligand structure of hydrogen-bond donating or accepting groups that can displace ordered water molecules usually provides a significant entropic gain that leads to a favorable free energy of binding.
- ordered waters are identifiable from the structure, and other ordered waters may be located during computer simulations of a fully solvated structure.
- Structural coordinates of the peptides of this invention may be stored in a machine-readable form on a machine-readable storage medium, e.g. a computer hard drive, diskette, DAT tape, etc., for display as a three-dimensional shape or for other uses involving computer-assisted manipulation of, or computation based on, the structural coordinates or the three-dimensional structures they define.
- a machine-readable storage medium e.g. a computer hard drive, diskette, DAT tape, etc.
- a nonexclusive list of computer programs for viewing or otherwise manipulating protein or peptide structures include the following: Midas (University of California, San Francisco)
- HyperChem Hapercube
- MD Display Universality of Washington
- PKD National Center for Biotechnology Information
- data defining the three-dimensional structure of an ⁇ -conotoxin peptide, or portions or structurally similar homologs or analogs of Mil may be stored in a machine-readable storage medium and may be displayed as a graphical three-dimensional representation of the peptide structure, typically using a computer capable of reading the data from said storage medium and programmed with instructions for creating the representation from such data.
- This invention thus encompasses a machine, such as a computer, having a memory which contains data representing the structural coordinates of a composition of this invention, e.g. the coordinates set forth in Example
- Such data may be used for a variety of pu ⁇ oses, such as the elucidation of other related structures and drug discovery (WO 97/08300).
- a first set of such machine readable data may be combined with a second set of machine-readable data using a machine programmed with instructions for using the first data set and the second data set to determine at least a portion of the coordinates corresponding to the second set of machine-readable data.
- the first set of data may comprise a Fourier transform of at least a portion of the coordinates for Mil set forth in Figure 8
- the second data set may comprise coordinates for a potential analog of MIL
- another object of the invention is to provide a method for determining the three- dimensional structure of a peptide analog or peptide mimetic of Mil using comparative protein modeling techniques and structural coordinates for a composition of this invention.
- Comparative protein modeling involves constructing a model of an unknown structure using structural coordinates of one or more related peptides. Comparative protein modeling may be conducted by fitting common or homologous portions of the protein or peptide whose three-dimensional structure is to be solved to the three-dimensional structure of known homologous structural elements. Comparative protein modeling can include rebuilding part or all of a three-dimensional structure with replacement of amino acids (or other components) by those of the related structure to be solved. For example, using the structural coordinates of Mil, one may determine the three dimensional structure of a peptide analog of Mil using comparative protein modeling. Those coordinates may be stored, displayed, manipulated and otherwise used in like fashion as the Mil coordinates of Example 8.
- This invention further provides for the use of the structural coordinates of a peptide of this invention, or portions thereof, to identify reactive amino acids, such as cysteine residues, within the three-dimensional structure, preferably within or adjacent to a receptor binding site; to generate and visualize a molecular surface, such as a water-accessible surface or a surface comprising the spacefilling van der Waals surface of all atoms; to calculate and visualize the size and shape of surface features of the peptide; to locate potential H-bond donors and acceptors within the three-dimensional structure, preferably within or adjacent to a receptor binding site and to calculate regions of hydrophobicity and hydrophilicity within the three-dimensional structure, preferably within or adjacent to a receptor binding site.
- reactive amino acids such as cysteine residues
- complementary characteristics e.g., size, shape, charge, hydrophobicity/hydrophilicity, receptor specificity, etc.
- one may also predict or calculate the orientation, binding constant or relative affinity of the peptide to a given receptor subtype in the bound state, and use that information to design or select compounds of improved or altered affinity.
- the structural coordinates of the ⁇ -conotoxin peptide, or portion thereof are entered in machine readable form into a machine programmed with instructions for carrying out the desired operation and containing any necessary additional data, e.g. data defining structural and/or functional characteristics of a potential analog, defining molecular characteristics of the various amino acids, etc.
- Compounds of the structures selected or designed by any of the foregoing means may be tested for their ability to bind to an nAChR, inhibit the binding of an nAChR to a natural or non- natural ligand therefor, and/or inhibit a biological function mediated by an nAChR.
- This invention also provides peptidomimetic and mimetic methods for designing a compound capable of binding to an nAChR.
- One such method involves graphically displaying a three-dimensional representation based on coordinates defining the three-dimensional structure of Mil or a portion thereof bound to an nAChR. Interactions between portions of an Mil and the receptor are characterized in order to identify candidate moieties for replacement.
- One or more portions of Mil which interact with the receptor may be replaced with substitute moieties selected from a knowledge base of one or more candidate substitute moieties, and/or moieties may be added to Mil to permit additional interactions with the receptor.
- the computational approaches and structural insights disclosed herein also permit the design or identification of molecules with reduced capacity, or substantial inability, to bind to an nAChR subtype.
- Such information can be useful in research efforts aimed at identifying antagonists of a single subtype of nAChR.
- Compounds first identified by any such methods are also encompassed by this invention.
- a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, e.g. a computer loaded with one or more programs of the sort identified above, is capable of displaying a graphical three-dimensional representation of any of the molecules or molecular complexes described herein.
- Machine-readable storage media comprising a data storage material include conventional computer hard drives, floppy disks, DAT tape, CD- ROM, and other magnetic, magneto-optical, optical, floptical and other media which may be adapted for use with a computer.
- a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structural coordinates of Mil or portion thereof, and in particular, structural coordinates of Mil set forth in Fig. 8 ⁇ a root mean square deviation from the backbone atoms of the amino acids of such protein of not more than 1.5 A.
- An illustrative embodiment of this aspect of the invention is a conventional 3.5" diskette, DAT tape or hard drive encoded with a data set, preferably in PDB format, comprising the coordinates of Fig. 8.
- the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of the structural coordinates set forth in Appendix I. Appendix II or Appendix III (or again, a derivative thereof), and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the X-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structural coordinates corresponding to the second set of machine readable data. Examples of systems useful for this aspect of the present invention are shown in WO/97/08300.
- Bio Activity is used herein to broadly denote function.
- Derivative refers to an amino acid sequence wherein one or more residue of the natural sequence is substituted.
- Features of a compound include any combination of structural, physical or chemical attributes of the compound which are required to elicit a certain biological activity.
- a peptide "fragment,” “portion” or “segment” is a stretch of amino acid residues of at least about two contiguous amino acids, typically at least about three contiguous amino acids and, most preferably, at least about four or more contiguous amino acids.
- NeuronAChR refers to a natural nicotinic acetylcholine receptor found in the CNS.
- Peptide Analog(s) refers to molecules which have more, fewer, different or modified residues from an ⁇ -conotoxin amino acid sequence. The modifications may be by addition, substitution or deletion of one or more amino acid residues. The modification may include the addition or substitution of analogs of the amino acids themselves, such as peptidomimetics or amino acids with altered moieties such as altered side groups. The analogs may possess functions different from natural ⁇ -conotoxin molecule, or may exhibit the same functions, or varying degrees of the same functions.
- the analogs may be designed to have a higher or lower biological activity, have a longer shelf-life or increased stability or be easier to formulate.
- the present analogs are referred to as proteins or peptides for convenience, but contemplated herein are other types of molecules, such as peptidomimetics or chemically modified peptides.
- a peptide analog may be referred to herein as a peptide for convenience.
- eptide mimetic or mimetic is intended to refer to a substance which has the essential biological activity of an ⁇ -conotoxin peptide.
- a peptide mimetic or mimetic may be a peptide- containing molecule that mimics elements of peptide secondary structure (Johnson et al., 1993).
- the underlying rationale behind the use of peptide mimetics or mimetics is that the peptide backbone exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of ligand and receptor.
- a peptide mimetic is designed to permit molecular interactions similar to the natural molecule.
- a mimetic may not be a peptide at all, but it will retain the essential biological activity of a natural conotoxin peptide.
- Related Composition refers to compositions comprising a conotoxin peptide analog or peptide mimetic as an active ingredient.
- Standard annealing refers to a strategy for superimposing flexible molecules to investigate potential alignments.
- substantially pure refers to a peptide which is present in the substantial absence of other biological molecules of the same type; it is preferably present in an amount of at least about 85% purity and most preferably at least about 95% purity.
- Substantially similar activity refers to the activity of a modified peptide, with reference to a natural ⁇ -conotoxin peptide.
- the modified peptide will have substantially similar activity.
- the modified peptide may have an altered amino acid sequence and/or may contain modified amino acids.
- the modified polypeptide may have other useful properties, such as a longer half-life.
- the similarity of activity of the modified peptide may be higher than the activity of the natural ⁇ -conopeptide.
- the modified peptide is synthesized using conventional techniques or is encoded by a modified nucleic acid and produced using conventional techniques.
- the modified nucleic acid is prepared by conventional techniques. A nucleic acid with an activity substantially similar to the natural ⁇ -conotoxin gene function may be used to produce the modified peptide described above.
- Subtype of nAChR refers to different molecular forms of nicotinic acetylcholine receptors.
- Peptide analogs and peptide mimetics of the present invention specific for the ⁇ 3 ⁇ 2 or ⁇ 3 ⁇ 4 subtypes of the nAChR. are prepared on the basis of procedures described herein, using conventional techniques, such as drug modeling, drug design and combinatorial chemistry. Suitable techniques include, but are not limited to those described in U.S. Patent 5,571,698, U.S. Patent 5,514,774, WO 95/21 193 (Ecker, et al. (1995); Persidis (1997); Johnson et al. (1993); Sun et al. (1993)) and the references cited therein which are all inco ⁇ orated herein by reference.
- peptide analogs and peptide mimetics are prepared using commercially available drug design software, including those set forth in Persidis et al. (1997). These peptide analogs and peptide mimetics have the substantially similar activities as an ⁇ -conotoxin described herein and in the published literature.
- compositions containing a compound of the present invention as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques, such as those in Remington 's Pharmaceutical Sciences (1990).
- an antagonistic amount of the active ingredient will be admixed with a pharmaceutically acceptable carrier.
- the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral or parenteral.
- the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions.
- any of the usual pharmaceutical media may be employed, such as, for example, water, glycols. oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
- tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
- the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
- suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
- the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
- the compounds When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
- the active ingredients which are peptides, can also be administered in a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region.
- a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region.
- Suitable delivery systems are described in U.S. Patent No. 5,550,050 and published PCT Application Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO
- Suitable DNA sequences can be prepared synthetically for each active ingredient on the basis of the developed sequences and the known genetic code.
- the active ingredients of the present invention are administered in an amount sufficient to generate the desired effect.
- the dosage range at which these agents exhibit this effect can vary widely, depending upon the severity of the patient's defect, the patient, the route of administration and the presence of other underlying disease states within the patient.
- the active ingredients exhibit their therapeutic effect at a dosage range from about 0.05 mg/kg to about 250 mg/kg, and preferably from about 0.1 mg/kg to about 100 mg/kg of the active ingredient.
- a suitable dose can be administered in multiple sub-doses per day.
- a dose or sub-dose may contain from about 0.1 mg to about 500 mg of the active ingredient per unit dosage form.
- a more preferred dosage will contain from about 0.5 mg to about 100 mg of active ingredient per unit dosage form. Dosages are generally initiated at lower levels and increased until desired effects are achieved.
- Mil derivatives were chemically synthesized and folded in the similar manner. Initial derivatives were prepared with alanine substitutions for each non-cysteine amino acid residue, (i.e., an alanine walk) in order to determine the effect of substitutions at each residue. Additional derivatives or analogs are prepared in the similar manner by substituting any amino acid for the amino acid residues of Mil except the cysteine and His, 2 amino acid residues.
- oocytes 1-2 days after harvesting were injected with cRNA encoding the ⁇ 3 and ⁇ 2 subunits of rat nAChRs and incubated at 25° C for 1-4 days prior to use. Electrophysiological currents were measured using conventional techniques, such as described in Cartier et al. (1996). Measurements were made for oocytes perfused with acetylcholine as controls and for oocytes incubated with either Mil or the analogs prepared in Example 1.
- Figure 1 shows the effect of alanine substitutions in the conopeptide Mil on the ability of the analogs to block acetylcholine-gated currents in voltage-clamped Xenopus oocytes expressing cloned rat ⁇ 3 and ⁇ 2 subunits. Most substitutions decreased Mil's potency 4-fold or less. The notable exceptions are: Glu, ,: 8-fold decrease; Hist,: 25-fold decrease; His, 2 : >10 4 -fold decrease. In addition, it was seen that Asn 5 (which can be substituted with His) and Pro 6 along with His, 2 (which can be substituted with Asn) were essential for high affinity binding to the ⁇ 3 ⁇ 2 nAChR.
- FIDs were apodized with a window function (90° and 135° shifted sine bell) in both dimensions prior to Fourier transformation. Each Fourier transformed FID was baseline corrected by applying a third-order polynomial, and each dimension was referenced to a chemical shift value of 4.76 ppm at the residual water signal.
- Figure 2 depicts two traces of sequential NOE d a connectivities which extend from Cys 2 to Asn 5 and from Val 7 to Cys 16 .
- NOE cross peak volumes were measured from NOESY data with four different mixing times using FELIX 95.0. NOE buildup curves were then fitted with a second-order polynomial and exact distances were generated for all assigned NOE cross peaks. A distance of 1.8 A was used as an appropriate reference for nonoverlapping geminal C ⁇ proton cross peaks, as well as for lower distance bounds. Several volumes of nonoverlapping geminal C ⁇ proton cross peaks were averaged and used for calibrating measured NOE volumes. A pseudoatom correction of 1 A (Wuthrich, K. (1983)) was added to the upper limits of those NOE cross peaks involving spectroscopically degenerate methyl and methylene protons.
- NOE intensity values for the model structure were used for the model structure.
- a new set of distance restraints was then deduced from this mixed NOE intensity matrix.
- a rotational correlation time of 1.5 ns, estimated from NOE buildup curves of nonoverlapping geminal ⁇ protons, and diagonal leakage rate of 1.0 s were used in each IRMA cycle.
- the structure was subjected to RMD and energy minimization steps using a Lennard- Jones potential with a 12.0 A cutoff. Five cycles of 1,000 steps of RMD of 1.0 fs for a duration of 5 ps was calculated at 700 K followed by cooling over 5 ps at 500 k and another 5 ps at 300 K.
- Figure 2 displays those NOE cross peaks according to their intensities along with 3 J NH .
- C ⁇ proton chemical shifts relative to random-coil values are a good assessments for identifying local helical components in peptides and proteins (Wishart, D.S. et al. (1991); Rothemund, S. et al. (1996)).
- the plot shown in Figure 4 strongly suggests that the peptide contains a large portion of helical conformation throughout the sequence except for residues Asn 5 and His 12 .
- transition from a quartic to a Lennard- Jones nonbonded potential was achieved in a final dynamics phase concomitant with final maturation of the nonbond energy factor.
- a total of 50 rounds of SA were conducted in order to sample as large a conformational space as practical given computer processor and disk limitations.
- a final minimization with a convergence criterion of the large derivative not exceeding 0.01 kcal mol "1 A "1 was conducted.
- An RMS deviation-based family clustering scheme was used which resulted in the grouping of these structures into 30 families spanning the energy range 396-5360 kcal/mol with the two families of lowest energy encompassing 18 of the 50 structures.
- MES minimum energy structure
- NMR violations >0.2A b av no. of violations per structure 16.64 ⁇ 3.13 16.50 ⁇ 3.64 av violation 0.30 ⁇ 0.11 0.31 ⁇ 0.11 dihedral violations >10° l/14 c (Cys 2 ⁇ ,) l/4 d (Cys 2 ⁇ ,) 2/4 d (His 9 ⁇ ,) minimum energy structure
- ⁇ -CT x Mil is a small peptide made of only 16 amino acid residues, it has a well-defined three-dimensional solution structure.
- two disulfide bridges which form a hydrophobic Cys knot there are three helical regions in the structure which contribute to form a very tight conformation.
- the presence of such stable secondary structures and disulfide bridges allows the multidimensional NMR method to be effective in obtaining a very high resolution structure of the peptide.
- These two turns associated with Asn 5 and His 12 are perhaps critical residues for the overall fold of the peptide and the presence of such turns is further supported by Ca proton shift data presented in Figure 4
- the space-filling model of ⁇ -CT x Mil shown in Figure 7B is oriented to explore the surface distribution of hydrophobic and hydrophilic side chain groups of the molecule.
- Hydrophobic residues are colored pu ⁇ le to distinguish them from polar residues which are yellow, or charged residues which are red (positive) and blue (negative).
- a flat surface located on top of the molecule in pu ⁇ le is a distinct structural feature representing the cluster of hydrophobic residues exposed to solvent. This hydrophobic surface is formed largely by Gly' (excluding the N-terminal amino group), Cys 2 , Cys 3 , Leu 15 , Cys 16 and the disulfide bond between Cys 3 and Cys 16 .
- This flat surface may be important for ligand-receptor binding through hydrophobic interactions.
- EXAMPLE 7 Electrophysiology cDNA clones encoding nAChR subunits were provided by S. Heinemann and D. Johnson (Salk Institute, San Diego, CA). cRNA was transcribed using RiboMAXTM large scale RNA production systems (Promega, Madison, WI). Diguanosine triphosphate (Sigma, St. Louis, MO) was used for synthesis of capped cRNA transcripts according to the protocol of the manufacturer. Plasmid constructs of mouse and rat nAChR subunits were as conventionally described: l, ⁇ l, ⁇ , ⁇ ; ⁇ 2; ⁇ 3; ⁇ 4; ⁇ 7; ⁇ 9; ⁇ 2; ⁇ 4.
- cRNA was injected with a Drummond 10 ⁇ l microdispenser (Drummond Scientific, Broomall, PA) using conventional techniques. It was fitted with micropipettes pulled rom glass capillaries provided for the microdispenser. The pipette tips were broken to an OD of 22-25 ⁇ m and back-filled with paraffin before mounting on the microdispenser. cRNA were drawn into the micropipette and 50 nl, containing 5 ng cRNA subunit, was injected into each oocyte. In the case of muscle subunits, 0.5-5 ng of each subunit was injected.
- Oocytes were harvested from Xenopus frogs, cut into clumps of 20-50 oocytes, and placed in a 50 ml polypropylene tube (Sarstedt) containing 580 U/ml type 1 collagenase (Worthington Biochemical, Freehold, NJ) in OR-2 (82.5 mM Nacl, 2.0 MM KC1, 1.0 mM MgCL and 5mM HEPES, pH -7.3). The tube was incubated for 1-2 hr on a rotary shaker rotating at 50 rpm. Halfway through the incubation, the solution was exchanged with fresh collagenase solution.
- Oocytes were injected 1-2 days after harvesting and recordings were made 1-7 days after injection.
- An injected oocyte was placed in a -30 ⁇ l recording chamber consisting of a cylindrical well ( ⁇ 4 mm dia x 2 mm deep) fabricated from Sylgard, and gravity-perfused with either ND96 or ND96 containing 1 ⁇ M atropine (ND96A) at a rate of -1 ml/min. All toxin solutions also containing 0.1 mg/ml bovine serum albumin (BSA) to reduce nonspecific adso ⁇ tion of peptide.
- BSA bovine serum albumin
- the perfusion medium could be switched to one containing peptide or acetylcholine (ACh) by use of a distributor valve (Smart Valve, Cavro Scientific Instruments, Sunnyvale, CA) and a series of three-way solenoid valves (model 161T031, Neptune Research, Northboro, MA).
- ACh-gated currents were obtained with a two-electrode voltage-clamp amplifier (model OC-725B, Warner Instrument Co ⁇ ., Hamden, CT) set for "fast” clamp and with clamp gain at maximum (x 2000).
- A/D conversion and digital control of solenoid valves were performed with a Lab-LC or Lab-NB board (National Instruments, Austin, TX) in a Macintosh (Quadra 630 or Ilex) computer.
- the computer communicated with the distributor valve via its serial port.
- Data acquisition and activities of the distributor and solenoid valves were automatically controlled by a home-made virtual instrument constructed with the graphical programming language LabVIEW (National Instruments, Austin.
- the perfusion fluid was switched to one containing ACh for 1 sec. This was automatically done in intervals of 1 -5 min. The shortest time interval was chosen such that reproducible control responses were obtained with no observable rundown. This time interval was dependent upon the nAChR subtypes being tested.
- the concentration of ACh was
- All ACh pulses contained no toxin, for it was assumed that little, if any, bound toxin would have washed away in the brief time ( ⁇ 2 sec) it takes for the responses to peak.
- the peak amplitudes of the ACh-gated current responses were measured by the virtual instrument. All recordings were made at room temperature (-22° C).
- An injected oocyte was voltage-clamped at a membrane potential of -70 mV and response to ACh was measured every 5 min. After several control responses were determined at -70 mV, the membrane potential was stepped to the test potential for 1 min. beginning 10 sec. before application of ACh. The membrane potential was returned to -70 mV between each test. The test potential was varied, in sequence, from -90 mV to -10 mV in steps of 10 mV followed by a final test at a membrane potential of +10mV. After the test sequence, the perfusion solution was switched to one containing toxin. After equilibrium was achieved, the test sequence was repeated.
- Kinetic traces represent the data from one experiment only. All experiments were repeated on at least three oocytes. Reported kinetic parameters are the average from all experiments performed on that subtype.
- EXAMPLE 8 Subtype specificity of Mil nAChR subunits expressed in Xenopus oocytes were used to quantitate the potency of Mil at various nAChR combinations. Each subtype was inhibited in a dose-dependent manner by Mil but with widely differing potencies (Fig. 9). ⁇ -conotoxin Mil most potently inhibited the ⁇ 3 ⁇ 2 combination with 200-fold to > 10,000-fold less potency at other subtypes (Table 2). TABLE 2 Inhibition of nAChR Subtypes by Alpha-conotoxin Mil
- nAChRs Many small molecules act as non-competitive inhibitors of nAChRs by blocking the open channel. Open channel block of nAChRs is generally dependent upon membrane potential. We examined whether Mil block of the ⁇ 3 ⁇ 2 nAChR was voltage-dependent. To accomplish this, acetylcholine response of ⁇ 3 ⁇ 2 nAChRs to acetylcholine was measured over a range of membrane potentials both in the presence and absence of Mil at its approximate IC 5 ⁇ (500 pM). As shown in Fig. 2, blockade by ⁇ -conotoxin Mil shows no voltage dependence over the range tested (-100 mV to +lO mV).
- DH ⁇ E dihydro- ⁇ - erythroidine
- ⁇ -conotoxin Mil a compound that has been shown to be a competitive antagonist of nAChRs. Blockade of ⁇ 3 ⁇ 2 nAChRs expressed in oocytes by DH ⁇ E is fully reversed after four minutes of washout (Fig. 11 A). In contrast, the ⁇ 3 ⁇ 2 nAChR recovers relatively slowly from Mil block after toxin washout (Fig. 1 IC). To test whether ⁇ -conotoxin Mil was acting at the same site as DH ⁇ E, we pre-applied DH ⁇ E to ⁇ 3 ⁇ 2 nAChRs.
- a chimera of ⁇ -conotoxin Mil was constructed.
- a series of peptides from the venom Conus aulicus was isolated that, unlike ⁇ -conotoxin Mil, prefer the ⁇ 3/ ⁇ 4 interface over that of the ⁇ 3/ ⁇ 2.
- One of these peptides is ⁇ - conotoxin AuIA.
- the presence of either histidine or asparagine at position 12 is highly conserved in all conopeptides.
- a conserved sequence of each of the aulicus peptides was used in the design of the Mil chimera indicated in Table 4 in which a string of four amino acids (9-12) of Mil are replaced by FATN.
- substitution of these four amino acids results in a change in selectivity and affinity for the nicotinic receptors.
- affinity of the chimera for the ⁇ 3/ ⁇ 2 interface was more than 1, 000-fold lower (K d has changed from 330 pM for the wild-type to 500 nM for the chimera) but k of ⁇ was changed by a factor of less than two.
- the four amino acid substitution does not affect toxin residues which determine off-rate (which must be elsewhere on the peptide), but clearly effects residues which determine on-rate.
- Other chimeras were synthesized in which amino acid fragment 9-12 was substituted from one peptide into another.
- cyclic peptides based on this structure were constructed utilizing standard FMOC chemistry. These include: cyclic cys-HLEH-cys (SEQ ID NO:4), cyclic A-HLEH-A (SEQ ID NO:5) connected through an amide bond, and cyclic HLEH (amino acid residues 9-12 of SEQ ID NO: 1) connected through an amide bond. These small peptides are screened for biological activity by the procedures of Example 8.
- the FATN sequence (amino acid residues 9-12 of SEQ ID NO: 6) shown in bold is a conserved feature of at least three different ⁇ 3 ⁇ 4-preferring ⁇ -conotoxins from C. aulicus venom.
- the second phase of toxin interaction involves a different surface of the toxin, the "locking face.”
- This locking face binds to a site on the ⁇ 3 subunit which is referred to as the "locking site.”
- the k on for the "locking face- locking site” interaction may be quite slow.
- the locking phase then proceeds much more rapidly (Figs 14A and 7B).
- the presence of two distinct interaction faces, which lead to distinguishable docking-and- locking interactions as described herein may be a general design feature of many Conus peptides and may represent a novel paradigm for achieving subtype selective interaction with multisubunit receptors.
- the term "Janus-ligands" is suggested to refer to binding molecules that have two distinct interaction faces (from Janus, the two-faced Roman god of beginnings). ⁇ -Conotoxin Mil would be a protype Janus ligand.
- the HLEH fragment of Mil appears essential in determining activity and selectivity of the Mil conopeptide. Computational examinations of the HLEH fragment are undertaken in order to develop an organic scaffold for use in developing small molecules. The spacial arrangement of the HLEH fragment is studied computationally using a vector analysis. Databases are then be searched to find organic fragments that have the proper spatial requirements and geometry to be used as a scaffold from which organic models are synthesized. The presence of two physically distinct peptide surfaces (hydrophilic and hydrophobic) on the Mil conopeptide and their interaction with ⁇ and ⁇ receptor subunits presents a specific strategy for designing ligands selective for receptors with different combinations of ⁇ and ⁇ subunits.
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JP2000517986A JP2001521042A (en) | 1997-10-24 | 1998-10-23 | Interaction of alpha-conotoxin peptide with neuronal nicotinic acetylcholine receptor |
CA002308115A CA2308115A1 (en) | 1997-10-24 | 1998-10-23 | Interaction of alpha-conotoxin peptides with neuronal nicotinic acetylcholine receptors |
EP98953885A EP1032588A4 (en) | 1997-10-24 | 1998-10-23 | Interaction of alpha-conotoxin peptides with neuronal nicotinic acetylcholine receptors |
AU11143/99A AU1114399A (en) | 1997-10-24 | 1998-10-23 | Interaction of alpha-conotoxin peptides with neuronal nicotinic acetylcho line receptors |
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US6278397P | 1997-10-24 | 1997-10-24 | |
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WO2000020443A1 (en) * | 1998-10-02 | 2000-04-13 | The University Of Queensland | Novel peptides |
WO2020081583A1 (en) * | 2018-10-16 | 2020-04-23 | Glo Pharma, LLC | Nicotinic acetylcholine receptor peptide antagonist conotoxin compositions and related methods |
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CN114853864B (en) * | 2022-06-01 | 2023-06-20 | 海南大学 | nAChR receptor alpha 3 beta 2 subtype inhibition inactivation type blocking agent, and preparation method and application thereof |
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US5595972A (en) * | 1993-06-29 | 1997-01-21 | University Of Utah Research Foundation | Conotoxin peptides |
US5703792A (en) * | 1993-05-21 | 1997-12-30 | Arris Pharmaceutical Corporation | Three dimensional measurement of molecular diversity |
WO1998022126A1 (en) * | 1996-11-18 | 1998-05-28 | University Of Utah Research Foundation | USE OF CONOTOXIN PEPTIDES ImI AND MII AS CARDIOVASCULAR AGENTS |
US5780433A (en) * | 1996-12-06 | 1998-07-14 | University Of Utah Research Foundation | Use of α-conotoxin MII to treat disorders resulting from nicotine stimulated dopamine release |
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US5633347A (en) * | 1993-06-29 | 1997-05-27 | University Of Utah Research Foundation | Conotoxin peptides |
US5514774A (en) * | 1993-06-29 | 1996-05-07 | University Of Utah Research Foundation | Conotoxin peptides |
US5589340A (en) * | 1993-06-29 | 1996-12-31 | University Of Utah Research Foundation | Process and primers for identifying nucleic acids encoding A-lineage conotoxin peptides |
-
1998
- 1998-10-23 WO PCT/US1998/022368 patent/WO1999021878A1/en not_active Application Discontinuation
- 1998-10-23 CA CA002308115A patent/CA2308115A1/en not_active Abandoned
- 1998-10-23 JP JP2000517986A patent/JP2001521042A/en active Pending
- 1998-10-23 AU AU11143/99A patent/AU1114399A/en not_active Abandoned
- 1998-10-23 EP EP98953885A patent/EP1032588A4/en not_active Withdrawn
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US4447356A (en) * | 1981-04-17 | 1984-05-08 | Olivera Baldomero M | Conotoxins |
US5418944A (en) * | 1991-01-26 | 1995-05-23 | International Business Machines Corporation | Knowledge-based molecular retrieval system and method using a hierarchy of molecular structures in the knowledge base |
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HU S-H, ET AL.: "THE 1.1 AA RESOLUTION CRYSTAL STRUCTURE OF ¬TYR15¾EPI, A NOVEL ALPHA-CONOTOXIN FROM CONUS EPISCOPATUS, SOLVED BY DIRECT METHODS", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 37, no. 33, 18 August 1998 (1998-08-18), US, pages 11425 - 11433, XP002916222, ISSN: 0006-2960, DOI: 10.1021/bi9806549 * |
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SHON K-J, ET AL.: "THREE-DIMENSIONAL SOLUTION STRUCTURE OF ALPHA-CONOTOXIN MII, AN ALPHA3BETA2 NEURONAL NICOTINIC ACETYLCHOLINE RECEPTOR-TARGETED LIGAND", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 36, no. 50, 16 December 1997 (1997-12-16), US, pages 15693 - 15700, XP002916223, ISSN: 0006-2960, DOI: 10.1021/bi971443r * |
SOCIETY FOR NEUROSCIENCE, WASHINGTON, DC.; 25 October 1997 (1997-10-25), CARTIER G E, ET AL.: "ALPHA-CONOTOXIN MII: STRUCTURE/ACTIVITY STUDIES OF A POTENT AND SELECTIVE PEPTIDE ANTAGONIST OF THE ALPHA3B2 SUBTYPE OF NEURONAL NICOTINIC ACETYLCHOLINE RECEPTOR", XP002916221 * |
Cited By (5)
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WO2000020443A1 (en) * | 1998-10-02 | 2000-04-13 | The University Of Queensland | Novel peptides |
US6849601B1 (en) | 1998-10-02 | 2005-02-01 | The University Of Queensland | Peptides |
WO2020081583A1 (en) * | 2018-10-16 | 2020-04-23 | Glo Pharma, LLC | Nicotinic acetylcholine receptor peptide antagonist conotoxin compositions and related methods |
US11130782B2 (en) | 2018-10-16 | 2021-09-28 | Glo Pharma, Inc. | Nicotinic acetylcholine receptor peptide antagonist conotoxin compositions and related methods |
US11970552B2 (en) | 2018-10-16 | 2024-04-30 | Glo Pharma, Inc. | Nicotinic acetylcholine receptor peptide antagonist conotoxin compositions and related methods |
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