WO2003097664A2 - Inhibiteurs de cysteine proteases a base de propeptides - Google Patents

Inhibiteurs de cysteine proteases a base de propeptides Download PDF

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WO2003097664A2
WO2003097664A2 PCT/CA2003/000730 CA0300730W WO03097664A2 WO 2003097664 A2 WO2003097664 A2 WO 2003097664A2 CA 0300730 W CA0300730 W CA 0300730W WO 03097664 A2 WO03097664 A2 WO 03097664A2
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compound
inhibitor
cathepsin
group
inhibitors
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PCT/CA2003/000730
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WO2003097664A3 (fr
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Traian Sulea
Enrico O. Purisima
Robert Menard
Jing Wang
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National Research Council Of Canada
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic

Definitions

  • the present invention relates to reversible non-covalent cysteine protease inhibitors and more particularly to peptidomimetics designed to mimic the binding mode of cysteine protease propeptides at the active site.
  • cathepsins cysteine proteases of the papain superfamily
  • cathepsins have been found to have important physiological role in bone resorption and remodeling, thyroid hormone liberation and immune response processes (McGrath, M. E. (1999) Annu. Rev. Biophys. Biomol. Struct. 28, 181-204; and McKerrow, J. H. & James, M. N. G., eds. (1996) Perspect. Drug Dis. Des. 6, 1-125).
  • Cysteine proteases are also targets for the development of anti-parasitic therapeutics (McKerrow et al., (1993) Annu. Rev. Microbiol., 47, 821-853). These enzymesare therefore attractive therapeutic targets.
  • cathepsin L is a target for the development of anti-cancer agents.
  • cathepsin L is synthesized as an inactive proenzyme containing an autoinhibitory 96-residue N- terminal propeptide. Inhibition is accomplished by having part of the propeptide span the active site thereby blocking access to it. The propeptide binds to the active site with a backbone direction reverse to that of a normal peptide substrate making it resistant to hydrolysis by the enzyme. Activation of the proenzyme to mature functional cathepsin L requires removal of the propeptide.
  • the independent propeptide fragment is a potent inhibitor of mature cathepsin L.
  • a synthetic peptide (Phe 4p -Gln 90p ) consisting of 87 of the 96 residues of the propeptide sequence has an inhibition constant, Kj, of 0.088 nM against cathepsin L.
  • Kj inhibition constant
  • successive truncation of the propeptide sequence results in a dramatic reduction in potency (Carmona, E., et al., (1996) Biochemistry 35, 8149-8157).
  • the fragments Arg 21p - Tyr 95p and Gly 52p -Tyr 95p have s of 11.5 and 2900 nM, respectively.
  • Those truncation studies suggest that the binding affinity of these propeptides arises from the extensive contact with the enzyme as well as the maintenance of the appropriate three-dimensional structure.
  • One aim of the present invention is to provide low molecular weight molecules that utilize the propeptide inhibition mode.
  • cysteine protease peptidomimetic inhibitors that have the scaffold of blocked tripeptides with a central D-amino acid.
  • the inhibitors of the present invention have a good inhibitory potency, preferably in the nanomolar range.
  • the inhibitors are short peptide-based molecules that mimic the propeptide reverse-binding mode and span the S2' to S3 subsites of cathepsin L. Making use of both the primed and unprimed subsites improves potency and specificity.
  • the reverse-binding mode and its noncovalent nature were confirmed through the determination of a 1.9 A resolution crystal structure of cathepsin L complexed with one of the inhibitors of the present invention.
  • cysteine protease inhibitor having a reverse binding mode relative to a substrate and spanning S' and S subsites of the active site of a cysteine protease, said inhibitor non-covalently binding to the cysteine protease and having the following formula:
  • AAi and AA 3 are backbones of amino acids defined by R1 and R3,
  • AA 2 is glycine or a backbone of a D-amino acid defined by R2, R1 is an amino acid sidechain that binds in the S1' subsite of the protease, R2 is an amino acid sidechain that binds in the S1 subsite of the protease, R3 is an amino acid sidechain that bind in the S2 subsite of the protease, A is an N-terminal capping group that binds in the S2' subsite of the protease, and B is a C-terminal capping group that binds in the S3 subsite of the protease.
  • -A is preferably selected from the group consisting of phenyl acetyl, biphenyl acetyl
  • -B is preferably selected from the group consisting of Ala-NH 2 ,
  • -R1 is preferably selected from the group consisting of:
  • -R2 is preferably selected from the group consisting of:
  • -R3 is preferably selected from the group consisting of:
  • a cysteine protease inhibitor selected from the group consisting of Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11 , Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20, Compound 21 , Compound 22, Compound 23, Compound 24, Compound 25, Compound 26, Compound 27, Compound 28, Compound 29, Compound 30, Compound 31 , Compound 32, Compound 33, Compound 34, Compound 35, Compound 36, Compound 37, Compound 38, Compound 39, Compound 40, Compound 41 , Compound 42, and Compound 43, and more preferably selected from the group consisting of Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 10, Compound 11 , Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 20, Compound 21 , Compound 24, Compound 27, Compound 29, Compound 30, Compound 31 ,
  • Mcy S-methylcysteine
  • Npa 2-naphthylalanine
  • Orn ornithine.
  • nuclear range used in the present application is intended to qualify inhibitors having a Kj of ⁇ 1 ⁇ M.
  • the Compounds of the present invention are inhibitors when their Kj is lower than 100 ⁇ M.
  • Fig. 1 illustrates the design path for the reduction of the full-length propeptide to a capped tripeptide inhibitor in accordance with one embodiment of the present invention
  • Figs. 2A to 2C illustrates modeled structures for the molecules in Fig. 1 ;
  • Figs. 3A to 3C illustrate the crystal structure of Compound 13 (from Table 1 below) in complex with cathepsin L (stereo views);
  • Figs. 4A to 4C illustrate proteolytic stability control assays using HPLC profiles of reaction mixtures with either E-64 alone (Fig 4A), cathepsin L alone (Fig. 4B), or Compound 7 alone (Fig. 4C);
  • Figs. 5A and 5B illustrate HPLC profiles of reaction mixtures containing compound 7 after incubation with cathepsin L for 1 h (Fig. 5A) or for 4 h (Fig. 5B);
  • Figs. 6A and 6B illustrate the crystal structure of inhibitor Compound 13 complexed with cathepsin L (stereo views).
  • Fig. 6A illustrates the detailed intermolecular interactions.
  • the inhibitor dimer is shown as thick capped sticks with carbon atoms colored black for the first molecule and gray for the second.
  • Cathepsin L residues 4.5 A around the inhibitor dimer are displayed in thin ball-and- stick representation. Hydrogen bonds are indicated with thin black lines. Select protein residues are labeled to help orient the viewer.
  • Fig. 6A illustrates the crystal structure of inhibitor Compound 13 complexed with cathepsin L (stereo views).
  • Fig. 6A illustrates the detailed intermolecular interactions.
  • the inhibitor dimer is shown as thick capped sticks with carbon atoms colored black for the first molecule and gray for the second.
  • Cathepsin L residues 4.5 A around the inhibitor dimer are displayed in thin ball-and- stick representation. Hydrogen bonds are indicated with thin black
  • FIG. 6B illustrates a comparison with the crystal structure of the active site-spanning segment (residues Val74p to Gln79p, capped sticks) of procathepsin L propeptide (PDB code 1cs8) after alignment of the corresponding Ca atoms of the protein.
  • the cathepsin L— 13 complex is shown in red and procathepsin L in blue.
  • Peptides were synthesized by Fmoc solid-phase chemistry using manual coupling (Fmoc-amino acid, 4 equiv; 2-(H-Benzotriazole-1-yl)-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate (TBTU), 4 equiv; N,N-Diisopropylethylamine (DIPEA), 6 equiv in N-methylpyrilidone.
  • DIPEA N,N-Diisopropylethylamine
  • cleavage from Wang resin was done by incubation with the respective amines for 3 to 5 days and subsequent deprotection of the cleaved product by the same cleavage cocktail as described above.
  • the peptides were purified by reverse phase HPLC on a semipreparative VydacTM C18 (1x25 cm) column using a 60-min linear gradient of 10-80% acetonitrile (containing 0.1 % TFA) on a Waters Delta Prep 4000 (Waters Ltd., Mississauga, ON). Purity was evaluated by analytical HPLC. The molecular mass of the final products was verified using a SCIEX API IIITM mass spectrometer (PE SCIEX, Thornhill, ON).
  • the substrate Cbz-Phe-Arg-MCA and the irreversible inhibitor E-64 were purchased from Bachem (King of Prussia, PA) and Peptides International (Louisville, KY), respectively.
  • Human cathepsins B, K and L were prepared as described previously (Carmona, E., et al., (1996) Biochemistry 35, 8149-8157; Nagler, D. K., et al., (1997) Biochemistry 36, 12608-12615; and Nagler, D. K., et al., (1999) Biochemistry 38, 4868-4874). All recombinant enzymes were expressed in the yeast Pichia pastoris as a prepro- ⁇ -factor fusion construct using the culture conditions recommended by Invitrogen Corp.
  • the secreted proenzymes were autocatalytically activated, purified and stored either at 4°C (cathepsins B and L) or -80°C (cathepsin K) inhibited by MMTS or HgCI 2 (Carmona, E., et al., (1996) Biochemistry 35, 8149-8157; Nagler, D. K., et al., (1997) Biochemistry 36, 12608-12615; and Nagler, D. K., et al., (1999) Biochemistry 38, 4868-4874).
  • procathepsin L The crystal structure of procathepsin L (PDB code 1cs8) was used as the starting point for the modeling study. Structure manipulation and visualization were done in SYBYLTM 6.6 (Tripos, Inc., St. Louis, MO). The proregion residues were removed except for the fragment Met 75p -Asn-Gly-Phe-Gln 79p . Crystallographic water molecules buried in the mature enzyme were retained. Both N- and C- termini of the protein were modeled in the ionized state. The peptide fragment was capped at the N- and C-terminal ends with acetyl and NH 2 groups, respectively. All histidine residues were protonated and the catalytic cysteine was modeled as a thiolate.
  • Bound conformations of the inhibitors were modeled by docking them according to the template followed by a conformational search using a Monte Carlo- minimization (MCM) procedure.
  • MCM Monte Carlo- minimization
  • Starting structures for each cycle of minimization were obtained by randomly perturbing one or more dihedral angles in the inhibitor.
  • the perturbations involved random changes in the side chain dihedral angles as well as crankshaft rotations of peptide units.
  • Selected residues of the protein around the active site were allowed to relax during the minimization.
  • the set of mobile protein residues defined as extending 8 A around the docked template ligand was used for all the inhibitors.
  • a total of 1000 MCM cycles was carried out for each inhibitor.
  • the AMBER force field was supplemented with parameters for unnatural amino acids and blocking groups.
  • the cathepsin L - Compound 13 complex (see Table 1 hereinafter for the structure of Compound 13) was prepared by incubating the protein with the inhibitor in the presence of 2 mM DTT.
  • the protein was kept in a buffer containing 50 mM sodium acetate pH 6.0, 100 mM NaCI. Due to the limited solubility of the inhibitor, 0.01 mM concentrations of protein and inhibitor were used to prepare the initial mixture that was then concentrated up to 8.7 mg/ml with a final ratio of 1 :1.2 M (protein : inhibitor).
  • the crystal was grown by using the hanging drop vapor diffusion method with a reservoir solution of 18% (w/v) polyethylene glycol 8000, 200 mM sodium citrate pH 4.2, 200 mM Li 2 SO 4 , 8% isopropanal at 18°C.
  • the crystallization drop contained 2 ml of inhibitor complex and 2 ml of the reservoir solution.
  • rod-shaped crystals appeared and were grown for up to two weeks. Diffraction data was collected on an Raxis IIC area detector mounted on a RU300 rotating anode generator with the reservoir solution supplemented with 18% glycerol as a cryoprotectant.
  • the mature cathepsin L molecule taken from the procathepsin L structure (PDB code 1cjl) was used as a starting model for molecular replacement solution using the Amore program.
  • the rigid body refinement of the cathepsin L model in Amore results in a correlation factor of 66.3 and R cryst of 33.7.
  • Further minimization in with the CNS software reduced the R-factor to 0.31.
  • the calculated difference map clearly showed the presence of the inhibitor as well as part of a second molecule.
  • the inhibitor was modeled into the map. Model building and refinement were done in O and CNS, respectively. Appropriate entries were added to the dictionaries of both programs to accommodate the nonstandard groups of the inhibitor.
  • the crystal structure of the human procathepsin L was used as starting point for inhibitor design (Fig. 1 ).
  • the focus was put on the five-residue stretch of the propeptide, Met 75p -Asn-Gly-Phe-Gln 79p that spans subsites S2' to S3 of the mature enzyme in the reverse substrate-binding mode.
  • the capped pentapeptide, compound 1 was synthesized based on this native sequence and no inhibition (Ki > 100 ⁇ M) of mature cathepsin L was found. This result was anticipated. Without the anchoring residues that flank this fragment in the full-length propeptide, peptide 1 may not have sufficiently strong interactions with the protein to lock it into the reverse-binding mode.
  • the C-terminal Gin was replaced with Phe, which fits well into the S3 subsite of the enzyme.
  • the third modification was the substitution of Gly in the middle of the peptide with a D-Arg.
  • a D- rather than L-amino acid was selected for this position because the designed reverse-binding mode of our inhibitory peptide alters the orientation of the C ⁇ carbon making a D-amino acid sterically favored over an L- amino acid.
  • D-Arg is being hereby given as a non- limitative example of one amino acid that work best.
  • incorporation of a D- amino acid at this position has the added advantage of potentially improving proteolytic stability. Docking of the peptide in the substrate-binding mode results in a collision of a large side chain of a D-amino acid with the protein atoms, regardless whether the D-amino acid occupies the S2, S1 , or S1' subsite. Hence, the D-Arg substitution would make any of the three C-terminal peptide bonds resistant to cleavage. The remaining putative cleavage site before Asn would not be favored since in the substrate-like binding mode, the S2 subsite of the enzyme, the most important determinant of binding affinity and specificity, would not be occupied by the peptide. Furthermore, a biphenylacetyl group in S1 would not be well accommodated. Compound 2 (see Table 1 below) was synthesized and inhibits cathepsin L with a Ki of 38 ⁇ M.
  • AAi to AA 3 are amino acids defined by R1 , R2, and R3, with AA 2 in the D- configuration.
  • A, R1 , R2, R3 and B bind in the S2', SV, S1 , S2 and S3 subsites of the protease, respectively.
  • A is preferably a biphenylacetyl N-terminal blocking group, or a derivative thereof that do not destroy the activity of the inhibitor.
  • A is preferably a biphenylacetyl N-terminal blocking group, or a derivative thereof that do not destroy the activity of the inhibitor.
  • One skilled in the art will have no difficulty in determining if a derivative of the blocking group negates the activity of the inhibitor by assaying the activity using routine testing. Removing a phenyl ring from the biphenyllacetyl group negate the activity of the inhibitors of the present invention.
  • other modifications may be made to the blocking group without negating the activity of the inhibitors while still benefiting from the teaching of
  • the choice of groups targeted to the S subsites of the protease are determined by the substrate specificity of the target protease.
  • the S1 and S2 subsites are the most characterized.
  • cathepsin B accepts arginine, lysine, ⁇ /-benzyloxycarbonyllysine, guanidine-phenylalanine, homophenylalanine or norleucine at the S1 subsite (therefore R2 is a sidechain of one of the aforementioned amino acids or amino acid derivatives) and phenylalanine, tyrosine, 3,5-diiodotyrosine, ⁇ -(2-naphthyl)alanine, arginine, guanidinophenylalanine and citrulline at the S2 subsite (therefore R3 is a sidechain of one of the aforementioned amino acids or amino acid derivatives).
  • Cathepsin L and cruzain accept arginine, lysine, homophenylalanine, guanidinophenylalanine, citrulline or norleucine at the S1 subsite (therefore R2 is a sidechain of one of the aforementioned amino acids or amino acid derivatives) and phenylalanine, tyrosine or ⁇ -(2-naphthyl)alanine at the S2 subsite (therefore R3 is a sidechain of one of the aforementioned amino acids or amino acid derivatives).
  • Cathepsin S accepts an argine, lysine, homophenylalanine, guanidinophenylalanine, citrulline or norleucine at the S1 subsite (therefore R2 is a sidechain of one of the aforementioned amino acids or amino acid derivatives) and phenylalanine, tyrosine, ⁇ -(2-naphthyl)alanine, valine, leucine, norleucine, isoleucine or alanine at the S2 subsite (therefore R3 is a sidechain of one of the aforementioned amino acids or amino acid derivatives).
  • Dipeptidylpeptidase-1 accepts a phenylalnine or tyrosine at the S1 subsite (therefore R2 is a sidechain of one of the aforementioned amino acids) and a glycine or alanine at the S2 subsite (therefore R3 is a sidechain of one of the aforementioned amino acids).
  • Calpain accepts phenylalanine, tyrosine, methionine, ⁇ -sulfonylmethylalanine or valine at the S1 position (therefore R2 is a sidechain of one of the aforementioned amino acids or amino acid derivatives) and valine, leucine, norleucine or isoleucine at the S2 position(therefore R3 is a sidechain of one of the aforementioned amino acids or amino acid derivatives).
  • the S3 subsite is less specific but typically accepts phenylalanine or leucine (therefore B is one of the aforementioned amino acids).
  • the S1 ' subsite accepts small amino acids such as alanine, serine, cysteine and asparagine (therefore R1 is a sidechain of one of the aforementioned amino acids or amino acid derivatives). Longer sidechains can make use of multiple S' binding sites.
  • Figs. 2A and 2B The modeled structures of Compounds 1 and 2 docked into the cathepsin L binding site are shown in Figs. 2A and 2B.
  • Fig. 2A illustrates Compound 2 in the active site of mature cathepsin L displayed as a Connolly surface. The full length propeptide is depicted as a yellow tube.
  • Fig. 2B is an expanded view of Compounds 1 (red) and 2 (blue) in the active site.
  • Fig. 2C illustrates the binding mode of the inhibitor compound 7.
  • the side chains of these reverse substrate-binding mode peptides are oriented to potentially utilize the canonical binding subsites of the enzyme, consistent with available data from the proenzyme structures (Cygler, M. & Mort, J. S. (1997) Biochimie 79, 645-652).
  • the backbone conformation of the inhibitors (interacting with subsites S2 to S1 ') preserves the intermolecular hydrogen bonding interactions established by the template propeptide.
  • the aliphatic portion of the side chain of a D-Arg residue at the central position of the tripeptide core is well-accommodated in the S1 subsite, and has its guanidinium group largely solvent-exposed.
  • Figs. 4A TO 4C The HPLC profiles of the buffer with either E-64, cathepsin L or compound 7 alone were recorded as controls (Figs. 4A TO 4C).
  • Figs. 5A and 5B show the HPLC profile of the aliquots (plus E-64) taken after 1 h and 4 h incubation time, respectively. There is no sign of cleavage of the inhibitor by the enzyme after 4 h of incubation confirming the inability of this inhibitor to bind in a productive substrate-like mode.
  • the same experiments were carried out with compounds 13 and 14 with similar results.
  • cathepsins K and B In parenthesis for cathepsins K and B is the ratio of K ⁇ relative to that of cathepsin L for a given inhibitor.
  • Compound 7 shows a 310- and 210-fold selectivity for cathepsin L over cathepsins K and B, respectively.
  • This selectivity for cathepsin L over K is remarkable considering that, by comparison, the full-length propeptide of cathepsin L shows only two-fold selectivity for cathepsin L over K (Guay, J., et al., (2000) Eur. J. Biochem. 267, 6311-6318).
  • the low-molecular weight peptides of the present invention exhibit both potency and selectivity.
  • the crystal structure of mature human cathepsin L in complex with Compound 13 was determined at 1.9 A resolution.
  • the asymmetric unit of the crystal contains two cathepsin L - Compound 13 complexes which adopt very similar conformations.
  • cathepsin L is very similar to that of the mature enzyme part as observed in the procathepsin L crystal structure at 1.8 A resolution (PDB code 1cs8) with an rmsd between equivalent C atoms of 0.45 A.
  • PDB code 1cs8 the procathepsin L coordinates used as a basis for the present molecular modeling calculations provided a good approximation of the mature cathepsin L structure.
  • Fig. 3A shows that the Fo-Fc simulated annealing omit map for the active site region of the complex between cathepsin L and Compound 13.
  • the crystal structure demonstrates that Compound 13 inhibits human cathepsin L by binding directly into the substrate-binding site of the mature enzyme, in a binding mode that matches remarkably the one predicted by molecular modeling (see below). That is, the present experimental structure shows unambiguously that the designed blocked tripeptide Compound 13 occupies both primed and non-primed susbites of cathepsin L in the reverse substrate-binding mode. Also, there is no evidence for covalent bond formation between the enzyme and inhibitor molecules.
  • the electron density around the active site Cys is consistent with its sulfur oxidized to sulfinate (-SO ⁇ ). It is worth noting that the catalytic Cys in the procathepsin L structure was also oxidized, to sulfonate (-SO 3 -) in that case.
  • Fig. 3A the electron density is contoured at a level of 2s. All atoms within a 3 A radius were omitted prior to the refinement and map calculation.
  • Fig. 3C illustrates a comparison of the crystallographic (red) and modeled (blue) cathepsin
  • Fig. 3B shows how it fits into the active site groove.
  • Fig. 3B steric fit of the inhibitor in the active site groove of the enzyme displayed is illustrated as a Connolly surface.
  • the carbon atoms of the inhibitor dimer are black for the first molecule and gray for the second.
  • the D-Arg and Phe residues, and the N-(2- phenylethyl) amide blocking group of the inhibitor bind to the enzyme subsites S1 , S2, and S3, respectively, whereas the Cys residue and the 4-biphenylacetyl blocking group of Compound 13 are accommodated into S1 ' and S2' subsites, respectively.
  • the backbone of the inhibitor establishes five direct intermolecular hydrogen bonds with the enzyme (Figs. 6A and 6B). These include two hydrogen bonds between Gly68 and the N-(2-phenylethyl) amide NH group and D-Arg backbone carbonyl of the inhibitor, two hydrogen bonds between Asp162 main chain carbonyl and inhibitor's D-Arg and Tyr backbone NH groups, and one hydrogen bond between the Trp189 indole NH group and the 4-biphenylacetyl carbonyl of the inhibitor.
  • the phenyl ring of the inhibitor's N-(2-phenylethyl) amide blocking group fits well into the relatively shallow S3 subsite and interacts with the side chains of Glu63, Leu69, Tyr72, with the CH2 of Gly68 and the carbonyl of Gly61. It appears that the 2-carbon aliphatic linker optimally positions the aromatic ring of the blocking group into S3 subsite, while the linker itself does not contact the protein.
  • the Tyr side chain of the inhibitor interacts extensively with the deep, hydrophobic S2 pocket of cathepsin L, contacting the side chains of Leu69, Met70, Ala135 and Ala214.
  • the D-Arg side chain of Compound 13 is situated in the less- delineated S1 pocket with the guanidinium group being largely solvent exposed.
  • the inhibitor's Cys side chain interacts with the side chains of the S1 ' residues Ala138, Asp162, His163, and Trp189. Its sulfur atom is engaged in a disulfide bond with a second inhibitor molecule only half of which has clear electron density (see below).
  • the 4-biphenylacetyl rings of the inhibitor pack against the S2' subsite, and more specifically, they interact with Gln21 , Gly23 and the main chain atoms of Cys22. This conformation and binding mode are made possible by the flexibility afforded by the methylene group between the biphenyl moiety and the carbonyl group.
  • the electron density allows fitting of part of a second inhibitor molecule into the primed region of the cathepsin L binding site (Figs. 3A and 3B).
  • This second inhibitor molecule is covalently attached through a disulfide bond to the first inhibitor molecule described above.
  • the C-terminal part of the second inhibitor molecule comprising of the Tyr residue and the N-(2-phenylethyl) amide blocking group could not be built in the observed electron density map. From the visible part, the Cys and D-Arg residues have the highest temperature factors when compared to the atoms of the protein and the first inhibitor molecule.

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Abstract

La présente invention concerne de nouveaux inhibiteurs non covalents de cystéine protéases à base de propeptides, qui ont été mis au point pour imiter le mode d'auto-inhibition des propeptides de ces enzymes. Tout comme le propeptides, ces inhibiteurs à base de peptides indiquent un mode de liaison inverse relativement à un substrat et s'étendent à la fois sur les sites secondaires S' et S du site actif de l'enzyme. A la différence d'études antérieures dans lesquelles même une troncature modérée du propeptide pleine longueur a conduit à une réduction rapide de l'efficacité, ces inhibiteurs calibrés par des tripeptides bloqués maintiennent une efficacité nanomolaire. De plus, dans le cas de la cathepsine L, ces peptides courts présentent une sélectivité supérieure (jusqu'à 310 fois) pour inhiber la cathepsine L sur K contrairement à seulement une double sélectivité du propeptide du résidu 96 de la cathepsine L. Une structure crystallographique par rayons X de 1,9 Å du complexe de la cathepsine L avec un des inhibiteurs confirme le mode de liaison inverse indiqué de l'inhibiteur, ainsi que sa nature non covalente. Une analyse enzymatique montre également que les inhibiteurs sont résistants à l'hydrolyse à des concentrations élevées de l'enzyme.
PCT/CA2003/000730 2002-05-15 2003-05-15 Inhibiteurs de cysteine proteases a base de propeptides WO2003097664A2 (fr)

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Cited By (5)

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WO2007099348A2 (fr) * 2006-03-02 2007-09-07 Fusion Antibodies Limited Peptide et ses utilisations
WO2013063656A1 (fr) * 2011-11-04 2013-05-10 Oral Health Australia Pty Ltd Propetides inhibiteurs gingipains
WO2013127981A1 (fr) 2012-03-01 2013-09-06 Veterinärmedizinische Universität Wien Inhibiteurs de protéases pour le traitement des infections par trichomonas gallinae
US8598114B2 (en) 2007-12-20 2013-12-03 Lytix Biopharma As Antimicrobial compounds
US9556223B2 (en) 2008-10-02 2017-01-31 Lytix Biopharma As Antimicrobial compounds

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007099348A2 (fr) * 2006-03-02 2007-09-07 Fusion Antibodies Limited Peptide et ses utilisations
WO2007099348A3 (fr) * 2006-03-02 2007-11-08 Fusion Antibodies Ltd Peptide et ses utilisations
US8598114B2 (en) 2007-12-20 2013-12-03 Lytix Biopharma As Antimicrobial compounds
US9556223B2 (en) 2008-10-02 2017-01-31 Lytix Biopharma As Antimicrobial compounds
US11548912B2 (en) 2008-10-02 2023-01-10 Peptide Patents AS Antimicrobial compounds
WO2013063656A1 (fr) * 2011-11-04 2013-05-10 Oral Health Australia Pty Ltd Propetides inhibiteurs gingipains
WO2013127981A1 (fr) 2012-03-01 2013-09-06 Veterinärmedizinische Universität Wien Inhibiteurs de protéases pour le traitement des infections par trichomonas gallinae

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