WO2007139573A1 - Dispositif de visualisation de ribosomes et d'arn de petites molécules biologiquement actives - Google Patents

Dispositif de visualisation de ribosomes et d'arn de petites molécules biologiquement actives Download PDF

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WO2007139573A1
WO2007139573A1 PCT/US2006/037905 US2006037905W WO2007139573A1 WO 2007139573 A1 WO2007139573 A1 WO 2007139573A1 US 2006037905 W US2006037905 W US 2006037905W WO 2007139573 A1 WO2007139573 A1 WO 2007139573A1
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binding partner
ligand binding
interest
amino acid
peptidomimetic
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Lucas Gartenmann Dickson
Virginia Cornish
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The Trustees Of Columbia University In The City Of New York
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link

Definitions

  • the present invention relates to Ribosome and RNA Display of Biologically Active Small Molecules ("RRDBASM”), a technology which utilizes engineered aminoacyl- tRNAs together with diverse mRNA libraries to produce small molecule ligands for biologically interesting molecules.
  • RRDBASM Ribosome and RNA Display of Biologically Active Small Molecules
  • the present invention provides for methods and compositions that may be used to produce biologically active peptidomimetic compounds with enhanced properties relative to their natural peptide counterparts.
  • the erythromycin biosynthetic cluster has been functionally rewired for synthesis of ring-expanded analogs of erythromycin (Jacobsen et al., 1997, Science 277: 367-369), and Walsh's group has exploited the relaxed substrate specificity of the tyrocidine thioesterase macrocyclization catalyst to generate a 168-member cyclic peptide library from peptide precursors synthesized by solid phase methods (Jacobsen et al., 1997, Science 277: 367-369).
  • Peptidomimetics are an important class of natural products (Ripka et al., 1998, Curr. Opin. Chem. Biol. 2j . 441-452). As evidenced by non-ribosomal peptide natural products such as bacitracin and bleomycin, relatively simple side chain substitutions and post-translational modifications can render peptides suitable even as therapeutics (Walsh, 2004, Science 303: 1805-1810). Current approaches to the synthesis of libraries of peptidomimetics largely rely on solid-phase chemical synthesis. For example, solid-phase chemical syntheses have been developed for N-alkyl glycines (“peptoids”)(Simon et al., 1992, Proc. Natl. Acad.
  • the present invention provides for the preparation of peptide and peptomimetic libraries by methods which utilize the Ribosomal Biosynthetic Machinery (“RBM”) and non-natural aminoacyl tRNAs. More than thirty years ago, Chapeville et al. proved the adaptor function of the tRNA molecule by demonstrating that reduction of Cys- tRNACys to Ala-tRNACys with Raney/Ni gave a hybrid tRNA that incorporated Ala in response to a Cys codon (Chapeville et al., 1962, Proc. Natl. Acad. Sci. U.S.A. 48: 1086- 1092).
  • RBM Ribosomal Biosynthetic Machinery
  • modified aminoacyl-tRNAs such as N ⁇ - acetyl Lys-tRNALys, could be used as substrates by the ribosome and associated factors (Johnson et al, 1976, Biochemistry 15; 569-575; Baldini et al, 1988, Biochemistry Th 7951- 7959).
  • Synthetic suppressor tRNAs have been used to incorporate amino acids with altered pKa values, constrained steric conformations, and other properties for studies of enzyme mechanism and protein stability (Cornish et al., 1995, Angew. Chem. Int. Ed. Engl. 34: 621-633; Gilmore et al., 1999, Topics Curr. Chem. 202: 77-99). It has been shown that this approach can be used in Xenopus oocytes, allowing studies of membrane proteins (Nowak et al., 1995, Science 268: 439-442).
  • a suppressor tRNA has been used to incorporate biotin-Lys in ribosome display (Li et al., 2002, J. Am. Chem.
  • Aminoacyl-tRNA synthetases have been engineered that can charge the suppressor tRNA in vivo, making it possible to incorporate the synthetic amino acid in vivo and generate the modified protein in high yield (Wang et al., 2001, Science 292; 498-500; Wang, et al., 2005, Angew. Chem. Int. Ed. 44; 34-66).
  • cells have long been fed unnatural amino acids for tRNA charging and incorporation in place of their natural amino acid counterpart (Link et al., 2003, Curr. Opin. Biotechnol. 14: 603-609).
  • the present invention relates to Ribosome and RNA Display of Biologically Active Small Molecules ("RRDBASM”), a technology which utilizes engineered aminoacyl- tRNAs together with diverse mRNA libraries to produce small molecule ligands for biologically interesting molecules.
  • RRDBASM Ribosome and RNA Display of Biologically Active Small Molecules
  • the present invention provides for the use of amino acid analogs which, incorporated into a peptide or peptide mimic, confer desirable characteristics, such as enhanced activity, stability, bioavailability, immunogenicity, and/or other therapeutically beneficial property.
  • the analogs of the invention undergo, either spontaneously or under particular reaction conditions, a post- translational reaction to produce an inter-residue linkage with enhanced stability.
  • the present invention provides for methods for producing ligands, either directly or as part of a library subjected to screening, for ligand binding partners of interest, including, but not limited to, proteins with SH3 domains, XIAP, components of the Notch pathway, tubulin, and HIV and Rce 1 proteases.
  • ligand binding partners of interest including, but not limited to, proteins with SH3 domains, XIAP, components of the Notch pathway, tubulin, and HIV and Rce 1 proteases.
  • the present invention further provides for compositions comprising novel amino acid analogs.
  • FIGURE 1 The ribosome is nature's machinery for translating mRNA templates into peptides. Only the aminoacyl-tRNA with the cognate anticodon binds to the codon on the mRNA template and subsequently leads to the formation of the new peptide bond. According to the invention, the ribosomal machinery is co-opted for synthesis of peptidomimetic libraries by feeding synthetic acyl-tRNA substrates to a purified translation system. In the pure translation display cycle translation products binding to ligand binding partner may be selected from the size of a library having more than 10 11 members. In the figure, an amino acid analog is shown being incorporated into a peptide. Translation components (here, derived from E.
  • initiation factors IFl, IF2, and IF3 initiation factors IFl, IF2, and IF3; elongation factors EF-Tu, EF-Ts, and EF-G; ribosome and natural aa-tRNAs.
  • SD is a Shine-Delgarno ribosome binding sequence.
  • FIGURE 2 Pure Translation Display, an extension of ribosome display.
  • DNA is in vitro transcribed and translated using the purified translation system.
  • ribosome complexes peptidomimetic translation product, ribosome and associated mRNA
  • active peptidomimetics are selected through binding to an immobilized ligand binding partner, here, the BIR3 domain of XIAP.
  • the mRNA of biologically active translation products is amplified by RT-PCR.
  • FIGURE 3 N-alkyl peptide synthesis using thia-Pro analogs.
  • FIGURE 4 De nuovo designed genetic code, where compounds 1-8 are ⁇ - hydroxy amino acid analogs (referred to herein as compounds (1-8)HA, respectively.)
  • FIGURE 5 Synthetic scheme for the synthesis of aminoacyl-tRNA.
  • Reagents include (a) i)6-Nitroveratrylcarbonyl chloride (NvoCl), Na 2 CO 3 , dioxan/water 1:1 (Robertson et al, 1991, J. Am. Chem. Soc. 113:2722-2729) ii)NvoCL, triethylamine, THF; iii) Chloroacetonitril, triethylamine; (b) pdCpA, DMF, ii) tRNA 'CA , T$ RNA ligase; iii) hv (Ellman et al, 1991, Methods Enzymol. 202:301-336).
  • FIGURE 6A-B Close-up of EF-Tu residues interacting with aa-tRNA.
  • A Left panel: EF-Tu from T. aquaticus with yeast Phe-tRNAPhe and the GTP analog GDPNP (Nissen et al., 1995, Science 270: 1464-1472).
  • EF-Tu is rendered in a blue ribbon, and the aminoacylated tRNA is in CPK colors.
  • the rectangular black bpx encompasses the aminoacylated acceptor stem.
  • B (Right panel): enlargement of the boxed region, after rotation of approximately 90 degrees toward the viewer. The view is centered on the phenylalanine residue carried by the tRNA.
  • the aminoacyl tRNA is in orange, His 67 of EF- Tu is magenta, and other residues that contact the acceptor stem are in blue. Labeled residues are targets for the mutagenic screen summarized in the text.
  • FIGURE 7 N-Alkyl pure ribosome display library design.
  • FIGURE 8 A new genetic code.
  • FIGURE 9 Central biochemical events involved in transducing and modulating Notch signals. Key steps represented in the figure are discussed in the text.
  • FIGURE 10 Incorporation of ⁇ -hydroxy acids.
  • FIGURE 11 Mechanism of intramolecular rearrangement. After acylation of the ⁇ -hydroxy function the nucleophilic attach of the free amino function onto the ester carbonyl is facilitated by formation of a five-membered ring. The posttranslational rearrangement leads to the desired ⁇ -hydroxy- ⁇ -peptide backbone.
  • FIGURE 12 The aspartyl protease catalyzed protein hydrolysis of substrate (structure 11) .
  • FIGURE 13A-D The compounds A-D contain an ⁇ -hydroxy- ⁇ -amino acids substructure that can act as analogs of the transition state T of the aspartyl protease catalyzed protein hydrolysis (FIGURE 12) (De Clercq, 1995, J. Med. Chem. 38: 2491; Boehme et al., 1995, Ann. Rep. Med. Chem.
  • FIGURE 14A-J A is a general structure for 34 related ⁇ -hydroxy- ⁇ -amino acids. Compared to (2S, 3S)-allophenylnorstatine C (FIGURE 13) the ⁇ -hydroxy- ⁇ -amino acids B-D have bigger aromatic substituents.
  • FIGURE 15. ⁇ -hydroxy compounds of the invention.
  • FIGURE 16. ⁇ -thio compounds of the invention.
  • amino acid analog as that term is used herein is a molecule having general formula 1 or 6:
  • R may be a non-naturally occurring side chain with or without reactive functional groups and biological activity. Reactive functional groups may be used for post- translational modifications.
  • X in formula 6 may be either OH ( ⁇ -hydroxy acids) or SH ( ⁇ -thio acids).
  • the present invention provides for the use of particular classes of amino acid analogs, including N-alkyl analogs, proline analogs (including aza-, oxo- and thia-proline analogs) and ⁇ -hydroxy analogs (including ⁇ -hydroxy- ⁇ -amino analogs).
  • N-Alkyl analogs include, but are not limited to, the following, referred to as compounds (21-27)N-Alk, respectively:
  • Proline analogs according to the invention may be represented by the following general formulas 2 and 3 :
  • X may be N (for aza-proline analogs), O (for oxo-proline analogs) or S (for thia-proline analogs).
  • each OfR 1 - R 8 may be, for example and not by way of limitation, H, methyl, dimethyl, (C 1 - C 4 )alkyl, aryl (e.g., phenyl or substituted phenyl), hydroxy(C 1 - C 4 )alkyl, CaTbOXyI(C 1 - C 4 )alkyl, or amino(Ci - C 4 )alkyl.
  • R 1 and R 2 (for formula 2) or R 7 and R 8 (for formula 3) may be the same or different and may be selected from the group consisting of H, methyl, dimethyl, (C 1 - C 4 )alkyl, aryl (e.g., phenyl or substituted phenyl), hydroxy ⁇ - C 4 )alkyl, carboxyl(d - C 4 )alkyl, or amino ⁇ - C 4 )alkyl, where in a non-limiting subset of embodiments at least one of the pair R 1 and R 2 (for formula 2) or the pair R 7 and R 8 (for formula 3) is H.
  • FIGURE 3 A general scheme for N-alkyl peptide synthesis using thia-proline analogs is shown in FIGURE 3.
  • Thia-proline analogs include, but are not limited to, the following specific compounds, referred to as compounds (28-33)Thia-Pro, respectively:
  • the present invention provides for analogs of (28-33) Thia-Pro in which the S in the ring is substituted with either N, to produce analogous compounds (28-33)Aza-Pro, or O, to produce analogous compounds (28-33)Oxo-Pro.
  • Thia-proline analogs incorporated into a peptide or peptoid, may be postranslationally reduced, for example, using Raney/Ni hydrogenation.
  • post-translational rearrangement of ⁇ -hydroxy- ⁇ -amino acids may produce a ⁇ -hydroxy- ⁇ -peptide backbone, having enhanced stability, as shown in FIGURE I l.
  • the amino acid analog comprises an ⁇ -hydroxy acid moiety, e.g., as in formula 6:
  • R group of formula 6 may be varied so that the resulting analog is a ⁇ -hydroxy- ⁇ -amino acid, an ⁇ -hydroxy- ⁇ -amino acid, an ⁇ - thio- ⁇ -amino acid, or an ⁇ -thio- ⁇ -amino acid.
  • R may be aryl(d - C 4 )alkylamino, hydroxy ⁇ !
  • the present invention provides for compounds having the structural formulas 8 ( ⁇ -hydroxy- ⁇ -amino acids), 9 ( ⁇ -hydroxy- ⁇ - amino acids) or 10 (respectively) as follows:
  • R 1 -R 6 can be part of cyclic or acyclic structures.
  • Each OfR 1 - R 6 may be, for example and not by way of limitation, H, methyl, dimethyl, (C 1 - C 4 )alkyl, aryl (e.g., phenyl or substituted phenyl), hydroxyCQ - C 4 )alkyl, CaAoXyI(C 1 - C 4 )alkyl, or amino ⁇ - C 4 )alkyl.
  • the invention provides for the use of hydroxy acids numbered 1-8 in FIGURE 4, referred to hereafter, respectively, as compounds (1 -8)HA, as well as hydroxy acids depicted in FIGURES 13 A-D and 14A-J.
  • the invention provides for the use of alpha hydroxy O-methyl serine.
  • Other specific, non-limiting examples of ⁇ -hydroxy and ⁇ -thio compounds which may be used according to the invention are depicted, respectively, in FIGURES 15 and l6.
  • Artificial aminoacyl tRNAs may be prepared by linking an amino acid analog, as described in the preceding section, to a tRNA using any method known in the art.
  • an artificial tRNA may be prepared as follows, using a chemoenzymatic method as set forth in Heckler, et al., 1984, Biochemistry 23:1468-1473 and later modified by Noren et al., 1989, Science 244, 182-188. This method takes advantage of the fact that all tRNAs end in an invariant CCA-3".
  • a truncated tRNA-CA lacking the terminal pCpA dinucleotide may be prepared by run-off transcription and then purified by precipitation or gel electrophoresis.
  • An aminoacyl-pdCpA dinucleotide may be synthesized and then ligated to the tRNA-CA using T4 RNA ligase.
  • the aminoacyl-pdCpA dinucleotide may then be made by acylating pdCpA with the cyanomethyl active ester of the amino acid.
  • the dinucleotide may be synthesized using standard nucleotide chemistry. Briefly, 6-N, 6-N, 2" -O, 3"-O-tetrabenzoyl adenosine may be prepared by transiently protecting the 5" hydroxyl of adenosine with dimethoxytrityl. The protected adenosine may then be coupled to the 2"-deoxycytidinylphosphoramidite and oxidized under standard conditions.
  • a phosphoramidite may then be added to the 5" hydroxyl group of deoxycytidine and subsequently oxidized. Finally, the benzoyl and cyanoethyl protecting groups are removed under basic conditions, and the pdCpA may be purified by HPLC and "activated" as the tetrabutylammonium salt.
  • a photolabile protecting group may be used for the amino acid because it can be removed after coupling of the aa- pdCpA to the tRNA-CA. The amino acid then may be prepared as the N- nitroveratryloxycarbonyl cyanomethyl active ester.
  • the NVOC protecting group may be installed under standard conditions, and then the cyanomethyl group may be introduced using chloroacetonitrile and triethylamine as the base.
  • the cyanomethyl active ester may then be used to selectively acylate pdCpA at the 2 "/3" hydroxyl group (the two rapidly interconvert at room temperature) using the tetrabutylammonium salt of pdCpA.
  • the aa-pdCpA may be purified using HPLC and then coupled to tRNA-CA enzymatically and photodeprotected. In specific non-limiting embodiments, synthesis of 5mg of an aa-pdCpA may be sufficient for at least 100 translation reactions.
  • the present invention provides for compositions comprising an aminoacyl tRNA, said aminoacyl tRNA further comprising, (and, in a translational sense, "charged with”) one of the amino acid analogs described herein.
  • compositions comprising an aminoacyl tRNA, said aminoacyl tRNA further comprising, (and, in a translational sense, "charged with”) one of the amino acid analogs described herein.
  • FIGURE 5 One specific, non-limiting embodiment of a synthetic scheme for the synthesis of amino-acyl tRNA is shown in FIGURE 5.
  • FIGURE 1 schematically depicts a cell-free translation system. Any suitable cell-free translation system known in the art may be used. It is desirable to control the populations of various tRNAs so that incorporation of a synthetic aminoacyl tRNA carrying an amino acid analog is not substantially inhibited by the presence of acyl-tRNAs competing for the same codon.
  • the cell-free translation system of Forster et al., 2003, Proc. Natl. Acad. Sci. U.S.A. 100:6353 may be used, as follows. This system utilizes ribosomes purified exhaustively to remove measurable contaminating RS charging activities, recombinant translation factors (Forster et al., 2001, Anal. Biochem. 297:60-70), in vitro- synthesized mRNAs, in vzTro-charged native tRNA isoacceptors, and chemoenzymatically synthesized aatRNAs. Briefly, mRNAs and translation mixes may be prepared as described in Forster et al., 2001, Anal. Biochem.
  • initiation factor (IF)2 may be further purified by gelfiltration chromatography, and (iii) the ribosomes may be subjected to additional washing, in that an additional high-speed spin of 1 min may precede the final pelleting of the four- times-washed ribosomes to remove residual insoluble material.
  • Ribosomes and factors desirably are not contaminated with RSs or proteases, as measured by charging of total tRNA (Sigma) with 15 14C-labeled amino acids (New England Nuclear) and by stability of peptides. Macromolecular concentrations in translations may be adjusted slightly to give 0.5 ⁇ M each of IFl, IF2, IF3, EF-G, and EF-Ts, 2.5 ⁇ M EF-Tu, four-times-washed ribosomes at 0.029 A260 unit/ ⁇ l [27 nM estimated to be active], 1 ⁇ M mRNA, 0.2 ⁇ M , and 0.5 ⁇ M for each elongator aa-tRNA.
  • Translations may be performed at 37 0 C for 30 min without preincubation. Translations may then be analyzed, for example by cation-exchange (treatment with alkali, acidification, then minichromatography to separate anionic formylated peptides from unformylated amino acids).
  • cell-free translations as described in United States Patent No. 6,977,150 by Forster orUnited States Patent Application Publication No. 2002/0123101 by Inoue, et al. may be used.
  • the present invention provides for cell-free translation systems in which the amount or type of EF-Tu is modified relatively to conventionally used systems.
  • linear iV-alkyl amino acids are excluded from peptides/peptoids synthesized in purified cell-free translation systems largely due to EF-Tu preferentially binding to natural amino acids.
  • a number of approaches may be used to compensate for any such binding disadvantage manifested by an amino acid analog.
  • the relative concentrations of EF-Tu and aminoacyl-tRNA substrates may be modulated.
  • a simple competitive inhibitor model for analog-tRNA binding to EF-Tu in the presence of a pool of natural aa-tRNA substrates shows that even modest decreases in EF-Tu affinity would significantly affect EF-Tu occupancy.
  • competitive inhibitors show that even modest decreases in EF-Tu affinity would significantly affect EF-Tu occupancy.
  • competition inhibitors shows that even modest decreases in EF-Tu affinity would significantly affect EF-Tu occupancy.
  • EF-Tu and the analog-tRNA and aa-tRNA substrates are all present at ca. 1 ⁇ M, because the KDs of the natural aa-tRNA substrates for EF-Tu » GTP are ca.
  • InM for an analog-tRNA impaired only two orders of magnitude in its KD for EF-Tu-GTP, the effective KD is:
  • This simple competitive inhibition model predicts that analogs impaired only one or two orders of magnitude in EF- Tu binding can be "fixed” simply by using EF-Tu in excess or optimizing the relative concentrations of EF-Tu and the natural and analog-tRNA substrates in the translation mixture based on their relative KDs.
  • the concentration of EF- Tu may be increased by a factor of at least 10, at least 50, at least 100, or at least 1000.
  • EF-Tu may be genetically engineered to promote binding. Both rational design and screening may be used to redesign the substrate specificity of EF-Tu.
  • There are two high-resolution structures of EF-Tu » GTP*aa-tRNA ternary complexes.106, 108 These structures paint a consistent picture of the residues that line the amino acid binding pocket (Fig. 13, Note that the residue numbers are from T. aquaticus, and the corresponding residues from E. coli Tu are: H66 for H67, E215 for E226, F218 for F229, F262 in place of H273, N273 for N285, and V274 for V286.
  • the N285 residue is conserved in greater than 98% of EF-Tu sequences, emphasizing its importance).
  • Cassette mutagenesis may be used to randomize each active-site residue independently. Both for library generation and subsequent characterization, EF-Tu may be purified and activated. Then, pools of EF-Tu mutants may be screened, for example in a 96-well plate, using a fluorescent GTP analog (mant-GTP) to score ribosome-dependent GTPase activation of EF- Tu as a change in fluorescence.115
  • the fluorescence GTPase assay is more readily adapted to a 96-well plate format and eliminates EF-Tu mutants with improved binding, but defective on the ribosome.
  • the present invention provides for a variant of E. coli EF-Tu which is at least 90 percent homologous to NCBI Accession No. AAC76954, comprises a mutation at one or more (e.g., 2, 3, 4, 5 or 6) of the following residues: H66, E215, F218, F262, N273, or V274, and is a functional elongation factor.
  • EF-Tu from another strain, species, or genus may be used.
  • an EF-Tu from a halophilic or thermophilic bacteria may be used, including, but not limited to, Thermus thermophilus, Bacillus stearothermophilus, and Thermus thermophilus HB 8. 5.4 PREPARING COMPOUNDS USING RRDBASM
  • ribosome and RNA display technology may be used. Such techniques are described in, for example, Forster et al., 2003, Proc. Natl. Acad. Sci. U.S.A. 100:6353-6357; Tan et al., 2004, J. Am. Chem. Soc. 126:12752-12753; and United States Patent No. 6,977,150. Additional references may be found in the list of references provided below. Schematic diagrams of ribosome and RNA display technologies are presented in FIGURES 1 and 2.
  • the present invention may be used to synthesize a particular compound of interest.
  • the compound may be a peptide or a peptidomimetic.
  • a peptide as defined herein, is a molecule comprised of natural amino acids where the backbone or main chain of the molecule consists of units joined by peptide bonds.
  • a peptidomimetic as defined herein is a molecule comprised of amino acids and/or amino acid analogs, where an amino acid with an unnatural side chain is incorporated and/or the backbone or main chain of the molecule comprises at least one bond that is not a peptide bond or where the peptide bond is N-substituted.
  • such a bond may be an ester bond.
  • the peptidomimetic may be a polyester (see below).
  • the peptide or peptidomimetic may be between about 2 and 100, or between about 2 and 80, or between about 2 and 60, or between about 2 and 40, or about 2 and 20, or about 2 and 10, or about 2 and 5, residues in length.
  • Ligand refers to a compound that is able to bind to a biologically active molecule, its "ligand binding partner,” where the relationship between ligand and ligand binding partner is such that there is a binding affinity between them; that is to say, the relationship may be enzyme/substrate; hormone/receptor; antigen/antibody; etc.
  • a ligand of interest which may be a naturally occurring or a synthetic molecule, may be used as a model for which small molecule analogs may be developed according to the invention.
  • ligands for which such analogs may be developed include kynostatins, tubulin polymerization inhibitors such as hemiasterlin, talbotulins (e.g., HTI-286), belamide A, or dolastatins.
  • the ligand binding partner may be used as the basis for identifying members of a small molecule library that are suitable modulators (for example, by screening a pure translational display system using ligand binding partner bound to a solid phase (e.g., a substrate, matrix, plate or bead) to select suitable candidates which are later amplified by PCR (directed evolution)).
  • suitable ligand binding partner targets include, but are not limited to, Human Immunodeficiency Virus (HIV) protease, Rce 1 protease, the Crk SH3 Domain, and IAP proteins.
  • HIV Human Immunodeficiency Virus
  • the present invention provides for methods of producing diverse libraries of small molecule ligands for a ligand binding partner of interest.
  • One source of diversity may be the mRNA directing the translational synthesis of the small molecules, such that the coding sequence of said RNA may contain one or more residue which is varied relative to a sequence encoding a ligand of interest.
  • Methods of the invention encompass the use of a single RNA species or a plurality of RNA species (a library), where the latter can increase diversity by orders of magnitude.
  • a second source of diversity may be the use of multiple amino acid analogs.
  • Such multiple amino acid analogs may be linked to a single species of tRNA or to a plurality of species of tRNA.
  • the redundancy of the genetic code (and the availability of more than 20 possible codons) may be utilized to expand the number of possible amino acids which may be incorporated into a translation product.
  • aminoacyl tRNAs bound to the natural amino acids and to various analogs may be utilized in a de nuovo genetic code, as shown in FIGURE 4.
  • FIGURE 2 schematically depicts a rationale by which peptide or peptidomimetic ligands generated according to the invention may be allowed to bind to their ligand binding partner while still associated with the ribosome and encoding mRNA, thereby allowing for the selection of RNAs which encode bindable ligands; said selected RNAs may be amplified by PCR and then allowed to pass through one or more "round(s)" of selection.
  • SH3 domains typically bind peptide ligands that adopt a polyproline type II helical conformation, with proline residues strongly favored at the P-I and P2 positions (FIGURE 7).
  • the peptide recognition surface has two pockets at the P-I and P2 positions that accommodate the structural property of proline residues that is unique among the 20 natural amino acids: N-substitution (Nguyen et al., 1998, Science 282: 2088-2092).
  • the library may contain 16 AsnB tRNAs, with GNN anticodons assigned to 16 different building blocks (see FIGURE 8 for an enlarged genetic code), to read a modified genetic code constructed from the 16 NNC codons.
  • the mRNAs in the library may encode the peptidomimetic sequence MPxPxxPRxx (FIGURE 7), where sites of variation in the library are defined by the x and include the P-I and P2 positions.
  • FIGURE 8 that the 16-letter alphabet may be enriched in N-alkyl amino acids, but not restricted to them, in order to maximize the structural complexity of the library.
  • the alphabet may also be designed to include a mix of hydrophobic, polar, and charged amino acid analogs to ensure peptide solubility.
  • Peptides/peptoids from the library may then be selected by ribosome display (FIGURE 2) for binding to the Crk SH3 domain, immobilized as a GST fusion protein on glutathione-agarose beads (or, alternatively, a biotin-modified Crk SH3 domain-ref 84).137
  • the mRNA associated with the bound peptides may be recovered by addition of EDTA (which dissociates the ribosomal subunits and releases the mRNA), then amplified by RT- PCR. This cycle of translation, selection, recovery and amplification may be repeated for additional cycles until the library converges, with the identity of a set number (e.g., a dozen, 20, etc.) clones determined after each round by DNA sequencing.
  • Drosophila Notch and its homologs in humans and other multicellular animals define a unique class of highly conserved transmembrane receptors that normally regulate cell growth, differentiation, and death in a variety of tissue types.
  • Notch signaling vary as a function of dose and context. Activation of Notch can favor choice of one cell fate over another, promote cell proliferation or cell cycle arrest, cause differentiation or self-renewal, and enhance survival or apoptosis (Artavanis-Tsakonas et al., 1999, Science 284_i 770-6; Weng et al, 2004, Curr Opin Genet Dev 14: 48-54).
  • Notch 1 one of four Notch homologues in mammals, is normally required at several stages in the development and maturation of T-cells.
  • Evidence implicating Notchl in the choice of T cell lineage commitment comes from both gain and loss-of function experiments in mice (Pear et al., 2003, Semin Immunol l_5j . 69-79).
  • Expression of constitutively active hNl in hematopoietic stem cells inhibits normal marrow B cell development and induces the development of CD4+CD8+ double positive (DP) immature T- cells in the bone marrow (Pui et al., 1999, Immunity UJ.299-308).
  • inducible notchl knockout mice fail to develop mature T cells due to a requirement for Notchl during early stages of intrathymic T cell development (Radtke et al., 1999, Immunity Kh 547-58).
  • T-ALL T cell acute lymphocytic leukemia/lymphoma
  • Activation or inactivation of Notch signaling has also been linked to a variety of other cancers, including skin, breast, lung, pancreas, and CNS tumors (Axelson, 2004, Semin Cancer Biol l_4j . 317-9).
  • FIGURE 9 a schematic diagram of Notch-associated cell signals. Normally, a Notch signal is activated when ligand binding induces proteolytic cleavages that release the intracellular portion of Notch (ICN) from the membrane, permitting it to translocate to the nucleus, where it turns on transcription of target genes (Lai, 2004, Development 13JU 965-73; Hansson et al., 2004, Semin Cancer Biol 14: 320-8).
  • ICN intracellular portion of Notch
  • Notch orchestrates transcriptional activation of target genes by forming a nuclear complex that includes the transcription factor CSL, ICN, and a co- activator protein of the mastermind-like family (MAML).
  • MAML mastermind-like family
  • the transcriptional targets of Notch in T-cells are the bHLH protein HES-I, the Notch modifier Deltex-1, and the pre-T cell receptor alpha subunit, but there is considerable debate about what other genes are direct transcriptional targets of Notch 1 in T-cells. Delivery of a dominant-negative form of MAML-I arrests T-ALL tumor cell lines dependent on Notch 1 for growth, indicating that the activity of nuclear Notch 1 complexes is not only important for T-cell development, but also important for tumor cell proliferation.
  • a desirable ligand binding partner to use for selection is the CSL transcription factor, which is the only known effector of activated Notch proteins.
  • CSL transcription factor which is the only known effector of activated Notch proteins.
  • RRQHGQLWFPEGF SEQ ID NO: I
  • a 13-residue peptide derived from the intracellular part of Notch suffices to bind the CSL transcription factor with high affinity (Kovall et al., 2004, Embo J 23_i 3441-51), indicating that selection of a library-encoded peptidomimetic that competes with Notch for CSL binding should be possible.
  • the selected ligand may be a valuable reagent for probing the CSL-dependent consequences of Notch activation.
  • activating mutations of human Notchl are found in more than 50 % of acute T-cell lymphocytic leukemias (T-ALL), and T-ALL cell lines with such activating mutations undergo growth arrest when Notch activity is blocked, indicating that Notch activation plays a central role in the molecular pathogenesis in T-ALL.
  • CSL ligands For selection of CSL ligands, complexes between CSL and DNA duplexes that contain a 5"-biotinylated nucleotide on one strand may be captured onto avidin-coated dishes.
  • a 10-residue library constructed from the 16 NNC codons may be used, with cycles of translation, selection, recovery and amplification repeated for 10 cycles or until the library converges, with the identity of 20 clones determined after each round by DNA sequencing.
  • the ability of the selected ligands to bind to the CSL-DNA complex may then be evaluated by fluorescence polarization or by titration calorimetry.
  • XIAP is the most potent member of the class of inhibitors of apoptosis proteins. Through binding to caspases, XIAP prevents apoptosis but the complete function of XIAP and IAPs in general is not certain. Peptidomimetics binding to IAPs can be used for the investigation of apoptosis and the elucidation of the role of IAPs. XIAP inhibitors may provide leads for the discovery of anti-cancer drugs. X-ray analysis has shown that SMAC N-terminus binds to the BIR3 domain of
  • a peptidomimetic library may be generated to bind to the BIR3 domain.
  • the two positions on both sides of the proline residue may be randomized and only the four amino acids A, V, F and Y may be used for these positions. This would result in a library size of 256 pentamers.
  • a limited alphabetic code assigning the well-established AsnB tRNAs (AS ⁇ BGAU, AsnBoou, AsnBouu, AsnBocu, ASIIBAAC) to the amino acids A, V, F 3 Y and P reading the codons AUC, ACC, AAC, AGC, and GTT may be used.
  • the initial library may be created using PCR.
  • the 5" primer may encompass a fixed upstream non-coding region encoding a Shine-Delgarno sequence followed by a start codon, the library insert consisting Of (ANC) 2 GTT(ANC) 2 (GTT is coding for P) and an 18 nt sequence complementary to the spacer poly(V/T).
  • the 3" primer may consist of an oligonucleotide complementary to the DNA sequence that follows the cloned poly(V/T) insert.
  • mRNA templates may be prepared by runoff transcription, and the libraries may be translated using the purified system as described in Forster et al., 2004, Analyt. Biochem. 333:358-364.
  • the stalled ribosome- mRNA peptide ternary complexes may be as described in Hanes et al., 2000, Meth. Enzymol. 328:404-430.
  • the residues 241-356 of the human XIAP BIR3 protein may be immobilized as a GST fusion protein on glutathione-agarose beads (Nguyen et al, 1998, Science 282:2088-2092) or a biotin-modified (Hanes et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:4937-4942) XIAP BIR3 domain.
  • the mRNA may be recovered by addition of EDTA and amplified by RT-PCR. This cycle of transcription, translation, selection and recovery may be repeated until DNA sequencing that may be carried out after each round indicates that the library size is converging to a predetermined number (e.g., 20 clones). IfAVPFY is not recovered in the selection, whether or not the hits are binding to the XIAP BIR3 domain may be tested, and then whether AVPYF can be isolated, when mixed with much weaker binding AGP YF,may be tested at different molar ratios (e.g., ranging from 1/100 to 1,000,000).
  • molar ratios e.g., ranging from 1/100 to 1,000,000.
  • a stalled ternary complex it may be desirable to switch to a mRNA display method as set forth in Roberts et al. 1997, Proc. Natl. Acad. Sci. U.S.A. 94:12297-12302 and Keefe and Szostak, 2001, Nature 410:715- 718.
  • Members of the library determined to bind to XIAP BIR3 may be synthesized in quantity by solid phase peptide synthesis.
  • a C-terminal QSEK sequence may be attached and the lysine residue may be linked to fluorescein-5-6-carboxyamidocaproid acid N- succimidyl ester (FAM).
  • each fluorescently labeled peptidomimetic for binding to the human XIAP BIR3 domain may be determined using a fluorescence polarization based competitive binding assay (Nikolovska-Coleska et al., 2004, J. Med. Chem. 47:2430-2440). Activity of said peptides may be tested in a caspase-9 activation assay, an analysis of apoptosis and a cell growth inhibition assay (Nikolovska-Coleska et al., 2004, J. Med. Chem. 47:2430-2440).
  • Rce 1 protease may be a ligand binding partner for which small molecules are developed using the invention.
  • the C-term that is typically linked to the RNA would need to be accessed.
  • a pseudo C-term resembling the CAAX box may be generated.
  • the HIV protease is essential for the virus life cycle. It is responsible for the site specific cleavage of the Gag-polyprotein and the Gag/Pol-polyprotein and thereby generates the functional viral proteins and enzymes that are required for the viral maturation (Beaulieu et al., 1997, J. Med. Chem. 40: 2164; Darke and Huff, 1994, Adv. Pharmacol. 25: 399; Darke et al., 1988, Biochem. Biophys. Res. Commun. J_56i 297).
  • the HIV protease is a target for antiviral agents in the treatment of AIDS.
  • the HIV protease is an aspartyl protease that preferentially cleaves between aromatic amino acids and proline or between pairs of hydrophobic and aromatic amino acids (FIGURE 12) (Badelassi et al., 2002, HeIv. Chim. Acta 85: 3090; Tomasselli and Henrikson, 1994, Methods Enzymol. 241: 279; Beck et al., 2000, Virology 274: 391.
  • the potent HIV-I protease inhibitor kynostatins (KNI)-227 A and (KNI)-272 B contain the same o?-hydroxy-/?-amino substructure as (2S, 3S)- allophenylnorstatine C (FIGURE 13).
  • the incorporation efficiency of hydroxy acids was assayed using a mRNA template coding for MVE.
  • the incorporation yield is determined based on the Dowex assay in which the amount of 3 H-labeled glutamic acid that is incorporated into the depsipeptides fM-aHa-E is compared to the formation of the tripeptide fM-V-E.
  • For the positive control natural amino acids on fully modified tRNAs are used while the ⁇ -hydoxy acids are loaded onto the tRNA GA c AsnB - This Asn-based tRNA adaptor was engineered to read the VaI codon GUU.
  • the yields for the hydroxy acids shown in Figure 10 are 98% for aHA, 50-55% for aHOMS and 65% for aHF.
  • the polyester fM-aHA-aHOMS-aHF-E was formed with a yield of 50-60% using the mRNA for MNTVE and the acyl-tRNAs pairings aHOMS- tRNA AsnB GGU, aHA-tRNA AsnB GUU and aHF-tRNA AsnB GAC.
  • ⁇ -hydroxy acid monomers showing high efficiency in single-site incorporation experiments were chosen.
  • PTS purified translation system
  • aHF and aHA were previously incorporated with 65% and 98% yield, respectively.
  • To enhance the solubility of the final oligomer product aHOMS was selected as a third ⁇ -hydroxy acid unit.
  • the incorporation efficiency of hydroxy acids is assayed using a mRNA template coding for MVE.
  • the incorporation yield is determined based on the Dowex assay in which the amount of 3 H-labeled glutamic acid that is incorporated into the depsipeptides fM-aHa-E is compared to the formation of the tripeptide fM-V-E.
  • For the positive control natural amino acids on fully modified tRNAs are used while the ⁇ -hydoxy acids are loaded onto the previously used tRNA 0A c AsnB (PNAS/JACS/MetO5). This Asn-based tRNA adaptor was engineered to read the VaI codon GUU.
  • Racemic aHOMS was synthesized form methyl 2-bromo-3- methoxypropionate.
  • the O-Nvoc protected derivatives of aHOMS were synthesized and ligated onto tRNAs "CA .
  • the resulting aHOMS-tRNAoAc ⁇ " 13 was tested for single-site incorporation into the depsipeptide fM-aHOMS-E. Based on the Dowex assay the incorporation efficiency of aHOMS is 53%.
  • the 6>-Nvoc protected derivatives of the ⁇ -hydroxy acids were synthesized and ligated onto tRNAs.
  • "CA Racemic aHOMS was synthesized form methyl 2-bromo-3- methoxypropionate (SynQuest laboratories, Alachua FL).
  • the carboxylic acid of 4- nitrobenzoic acid was activated with cesium carbonate in DMF for the nucleophilic attack onto the bromide. Saponification with 1 M aqueous lithium hydroxide gave the deprotected aHOMS.
  • the O-Nvoc protection of the hydroxy function was carried out in dry THF using triethylamine as base.
  • the carboxylic acid was activated as cyanomethyl ester using chloroacetonitrile and triethylamine. Under dry conditions five equivalents of the active ester were reacted with one equivalent of pdCpA in DMF as previously reported. The reaction was accelerated by addition of tertbutylammonium acetate.
  • a system was developed by constructing a mRNA with the three adjacent test codons AAC, ACC, and GUU and Asn-based tRNA adaptors engineered to read these codons. This genetic code was reassigned to the three ⁇ -hydroxy acids aHA, aHOMS and aHF. The chemoenzymatic synthesis of aHOMS-tRNA AsnB GGU, aHA-tRNA AsnB GUU and aHF-tRNA AsnB GAC and the mRNA was carried out using methods known in the art.
  • the yield for the ribosomal synthesis of the polyester 1 fJVl-aHA- aHOMS-aHF-E was 57% compared to the formation of the tripeptide fM-V-E.
  • Synthetic genes were cloned to enable in vitro synthesis of tRNA "C ⁇ species for ligation to aHa-pdCpA (aHa is used as abreviaiton for a nonspecific ⁇ -hydroxy acid).
  • the tRNA sequences contained substitutions at their 5' and 3' termini to maintain the secondary structure of the aminoacyl stems while enabling efficient transcription initiation at the first nucleotide with GMP by T7 RNA polymerase.
  • the O-Nvoc-aHa-pdCpA derivatives of aHOMS, aHA and aHF were prepared and ligated to tRNA "CA species by using general methods.
  • Natural aa-tRNAs were prepared from pure isoacceptors or with pure recombinant RSs. The specific activity for the 3 H-labeled glutamic acid was 8,400 dpm/pmol.
  • mRNAs and translation mixes were prepared using published methods. Typically translations were typically performed with 1 pmol of limiting input of fMet-tRNA fM et i (5 ⁇ L) for Dowex analysis, 4 pmol (20 ⁇ L) for HPLC analysis and at a 10 pmol (50 ⁇ L) for mass spectrometric analysis.
  • the concentrations in translations were 0.6 ⁇ M of IFl, 0.5 ⁇ M each of IF2, IF3, EF-G, and EF-Ts, 3.6 ⁇ M EF-Tu, four-times-washed ribosomes at 0.029.4260 unit/ ⁇ l [27 nM estimated to be active], 1 ⁇ M mRNA, 0.2 ⁇ M fMet-tRNAjMe t i, 0.5 ⁇ M 3 H-labeled E-tRNAi 01 ", and 1 ⁇ M for elongator tRNAs (photodeprotected aHa- tRNAs or aa-tRNAs, Val-tRNA Val ) or 1 ⁇ M tRNA GAC AsnB'CA . Translations were performed without preincubation at 37 0 C for 30 min.
  • aHOMS alpha hydroxy O-methyl serine
  • AbF 10 was Nvoc protected and activated as cynomethyl active ester for acylation of pdCpA.
  • the Resulting Nvoc-protected pdCpA was ligated with T4 RNA ligase onto the truncated (- CA) Asn-based tRNA with the anticodon GAC.
  • Prior to translation of the Nvoc protective group was photolytically removed.
  • the yield for single incorporation of abF was determined using the MVE mRNA.
  • the incorporation of 3H-labeled glutamic acid for abF was compared to the incorporation of natural valine on fully modified tRNA with the same anticodon GAC.
  • SM-319777 is Structure 13
  • SM-319777 is structurally closely related to a family of natural products called kynostatins.
  • N-alkyl moiety at the N terminus which may either be of importance for biological activity or used for a post translational cyclization or chemical modification.
  • the removal of the N- terminal fM may also be achieved by chemical methods. Treatment with cyanogen bromide cleaves at methionine or cystein residue natural amino acids and treatment with I 2 cleaves at the unnatural amino acid allylglycine. As shown below, removal of the N-terminal dipeptide fM-allylglycine results in the free N-terminus (in the ellipse) resembling belamide A (structure 15, infra). Cyclization may be achieved using either enzymes such as thioesterases or chemical methods. After removal of the N-terminal fM, the resulting free terminus may be reacted with other functional groups on the side chains. For example:
  • - macrolactonization may be achieved by enzymatic or chemical reaction of a side chain carboxylic acid, ester, or thioester with the amino function of a free N-terminus or of a side chain;
  • alkenes may be used to induce cyclization between two side chain alkenes
  • a side chain carbonyl and an amine may be used to form an imine and reduction of the imine may be used to make a cyclic amine by reductive amination;
  • a side chain carbonyl and a hydroxylamine may be used to form an oxime
  • a halide may be reacted with a thiol, hydroxy, or amino function
  • an amide may be reacted with an acetylene to form a 1,2,3-triazole and a macrocucle;
  • - oxidative coupling of a histidine and a tryptophane residue may be used to form a macrocycle as found in the family of celogentins.
  • the present invention may be used to produce, either directly or by selection from a library of variants, peptide or peptidomimetic compounds resembling the following tubulin inhibitors:

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Abstract

La présente invention concerne la visualisation de Ribosomes et d'ARN de Petites Molécules Biologiquement Actives (« RRDBASM »), une technologie qui utilise des aminoacyl-ARNt synthétisés conjointement avec diverses bibliothèques d'ARNm pour produire des petites molécules ligands pour des molécules biologiquement intéressantes. La présente invention concerne des procédés et des compositions qui peuvent être utilisés pour produire des composés peptidomimétiques biologiquement actifs avec des propriétés améliorées par rapport à leurs homologues peptidiques naturels.
PCT/US2006/037905 2006-05-31 2006-09-27 Dispositif de visualisation de ribosomes et d'arn de petites molécules biologiquement actives WO2007139573A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104059107A (zh) * 2014-06-27 2014-09-24 魏景芬 一种磷酸糖醇类四氢噻唑-4-羧酸化合物及其应用
CN106349087A (zh) * 2015-09-02 2017-01-25 四川瑞希康生物医药有限公司 （r）‑2‑氨基‑3‑（联苯基‑4‑基）‑1‑丙醇的合成
CN108396034A (zh) * 2017-02-06 2018-08-14 武汉臻智生物科技有限公司 一种提高无细胞体系蛋白质合成的方法
AU2019201264B2 (en) * 2013-03-15 2020-07-16 Cancer Research Technology, Llc Methods and compositions for Gamma-glutamyl cycle modulation
US20200248174A1 (en) * 2014-03-05 2020-08-06 National University Corporation Kobe University Genomic sequence modification method for specifically converting nucleic acid bases of targeted dna sequence, and molecular complex for use in same

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040091955A1 (en) * 2001-01-25 2004-05-13 Forster Anthony C. Process and compositions for peptide, protein and peptidomimetic synthesis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040091955A1 (en) * 2001-01-25 2004-05-13 Forster Anthony C. Process and compositions for peptide, protein and peptidomimetic synthesis

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2019201264B2 (en) * 2013-03-15 2020-07-16 Cancer Research Technology, Llc Methods and compositions for Gamma-glutamyl cycle modulation
US20200248174A1 (en) * 2014-03-05 2020-08-06 National University Corporation Kobe University Genomic sequence modification method for specifically converting nucleic acid bases of targeted dna sequence, and molecular complex for use in same
CN104059107A (zh) * 2014-06-27 2014-09-24 魏景芬 一种磷酸糖醇类四氢噻唑-4-羧酸化合物及其应用
CN106349087A (zh) * 2015-09-02 2017-01-25 四川瑞希康生物医药有限公司 （r）‑2‑氨基‑3‑（联苯基‑4‑基）‑1‑丙醇的合成
CN108396034A (zh) * 2017-02-06 2018-08-14 武汉臻智生物科技有限公司 一种提高无细胞体系蛋白质合成的方法

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