Peptides and compounds that bind to elongation initiation factor 4E
阅读说明：本技术 与延伸起始因子4e结合的肽和化合物 (Peptides and compounds that bind to elongation initiation factor 4E ) 是由 S·吴 C·J·布朗 于 2020-03-30 设计创作，主要内容包括：本发明涉及与延伸起始因子4E(eIF4E)结合的肽,其包含氨基酸序列CEX--(1)GX-(2)X-(3)X-(4)X-(5)C(SEQ ID NO:1),其中X--(1)是选自由苏氨酸(T)、甲硫氨酸(M)或亮氨酸(L)组成的组的氨基酸,X-(2)和X-(3)是选自由苯丙氨酸(F)、经修饰的苯丙氨酸和酪氨酸(Y)组成的组的氨基酸,X-(4)和X-(5)是任何氨基酸,其中两个半胱氨酸残基通过二硫键连接。本文限定的肽可以用于治疗与帽依赖性翻译失调有关的疾病状况,例如癌症、传染病、孤独症或脆性X染色体综合征。(The present invention relates to peptides which bind to elongation initiation factor 4E (eIF4E) comprising the amino acid sequence CEX- 1 GX 2 X 3 X 4 X 5 C (SEQ ID NO:1), wherein X- 1 Is an amino acid selected from the group consisting of threonine (T), methionine (M) or leucine (L), X 2 And X 3 Is selected from benzeneAmino acids of the group consisting of alanine (F), modified phenylalanine and tyrosine (Y), X 4 And X 5 Is any amino acid in which two cysteine residues are linked by a disulfide bond. The peptides defined herein may be used to treat disease conditions associated with cap-dependent translational disorders, such as cancer, infectious disease, autism, or fragile X syndrome.)
1. A peptide comprising the amino acid sequence CEX1GX2X3X4X5C(SEQ ID NO:1)，
Wherein X1Is an amino acid selected from the group consisting of threonine (T), methionine (M) or leucine (L);
wherein X2Is an amino acid selected from the group consisting of a hydrophobic amino acid, an aromatic amino acid, a modified hydrophobic amino acid, and a modified aromatic amino acid;
wherein X3Is an amino acid selected from the group consisting of a hydrophobic amino acid, an aromatic amino acid, a modified hydrophobic amino acid, and a modified aromatic amino acid;
wherein X4Is any amino acid;
wherein X5Is any amino acid;
wherein the two cysteine residues are linked by a disulfide bond; and
wherein the peptide binds to elongation initiation factor 4E (eIF 4E).
2. The peptide of claim 1, wherein the peptide binds to eIF4E at an mRNA 5' cap binding site.
3. The peptide of claim 1 or 2, wherein binding of the peptide to eIF4E inhibits eIF4E activity.
4. The peptide of any one of claims 1-3, wherein the peptide binds to eIF4E in an open conformation.
5. The peptide of any one of claims 1-4, wherein X1Is methionine (M).
6. The method of any one of claims 1-5A peptide of which X2And X3Independently selected from the group consisting of phenylalanine (F), tyrosine (Y) and modified phenylalanine.
7. The peptide of any one of claims 1-6, wherein X4And X5Independently glutamine (Q), aspartic acid (D), alanine (a), lysine (K), glycine (G) or leucine (L).
8. The peptide of any one of claims 1-7, wherein X5Is D-aspartic acid or L-aspartic acid.
9. The peptide of any one of claims 1-8, wherein the peptide comprises an amino acid sequence selected from the group consisting of: ACEMGFFQDCG (SEQ ID NO:2), CEMGFFQDCG (SEQ ID NO:3), ACEMGFFADCG (SEQ ID NO:4), ACEMGFFKDCG (SEQ ID NO:5), ACEMGFFLDCG (SEQ ID NO:6), CEMGFFADC (SEQ ID NO:7), ACEMGYFQDCG (SEQ ID NO:28) and ACEMGFYQDCG (SEQ ID NO: 32).
10. The peptide according to any of claims 1-9, wherein the peptide consists of amino acid sequence CEMGFFADC (SEQ ID NO: 7).
11. The peptide of any one of claims 1-10, wherein the peptide is conjugated to one or more additional peptides.
12. The peptide of claim 11, wherein the one or more additional peptides are cell penetrating peptides.
13. The peptide according to any one of claims 1 to 12 for use as a medicament.
14. The peptide according to any of claims 1-12 for use in the treatment of a disease condition associated with cap-dependent translational dysregulation.
15. The peptide of claim 14, wherein the disease condition is cancer, infectious disease, autism, or fragile-X syndrome.
16. A pharmaceutical composition comprising the peptide of any one of claims 1-13 and a pharmaceutically acceptable carrier.
17. Use of the peptide of any one of claims 1-12 or the pharmaceutical composition of claim 16 in the manufacture of a medicament for treating a disease condition associated with cap-dependent translational disorders.
18. The use of claim 17, wherein the disease condition is cancer, infectious disease, autism, or fragile-X syndrome.
Technical Field
The present invention relates to a cell proliferation inhibitory peptide. In particular, the invention relates to peptides that bind to elongation factor 4E (eIF 4E).
Background
The most common cancer mutations are found in the signal transduction pathways into the translation machinery. These include well studied and validated oncogenes such as MYC, RAS and PIK 3C. In addition, it is often found that a wide range of translation initiation factors are amplified or mis-regulated in tumors. eIF4E (elongation initiation factor 4E) is often misregulated and overexpressed in most cancers and is closely associated with the upregulation of a large class of oncogenic-related proteins, such as VEGF and c-MYC.
eIF4E plays an important role in translation initiation by interacting directly with the 5' cap structure of mRNA. All nuclear transcribed mrnas have this 5' cap structure (m7GTP) consisting of a guanosine methylated at position 7 and linked to the first nucleotide of the mRNA by a 5' to 5' triphosphate bridge. Most eukaryotic mrnas, including oncogenes, are translated in an eIF4E cap-dependent manner, and overexpression of eIF4E results in increased translation of these genes. Therefore, it is desirable to control the level of eIF4E activity to prevent uncontrolled proliferation.
One way to modulate eIF4E levels and activity is to modulate eIF4E activity through eIF4E binding protein (4E-BP). The 4E-BP negatively regulates eIF4E and shares a common binding motif with eIF4G, which they use to bind directly to eIF4E and competitively displace eIF4G scaffold protein. The substitution of eIF4G impairs assembly of the eIF4F complex on the 5' cap structure and prevents cap-dependent translation.
Thus, there is a need to identify eIF4E binding agents with mechanisms of action different from those described above to circumvent the emerging resistance mechanisms that have been reported for compounds targeting the eIF4E upstream signaling pathway involved in regulating protein synthesis. There is also a need to develop eIF4E binding agents for use in anticancer therapy.
Summary of The Invention
In one aspect, there is provided a composition comprising the amino acid sequence CEX1GX2X3X4X5C (SEQ ID NO:1), wherein X1Is an amino acid selected from the group consisting of threonine (T), methionine (M) or leucine (L); wherein X2Is an amino acid selected from the group consisting of a hydrophobic amino acid, an aromatic amino acid, a modified hydrophobic amino acid, and a modified aromatic amino acid; wherein X3Is an amino acid selected from the group consisting of a hydrophobic amino acid, an aromatic amino acid, a modified hydrophobic amino acid, and a modified aromatic amino acid; wherein X4Is any amino acid; wherein X5Is any amino acid; wherein the two cysteine residues are linked by a disulfide bond; and wherein the peptide binds to elongation initiation factor 4E (eIF 4E).
In another aspect, there is provided a peptide as described herein for use as a medicament.
In another aspect, there is provided a use of a peptide described herein for treating a disease condition associated with cap-dependent translational dysregulation.
In another aspect, a pharmaceutical composition comprising a peptide described herein and a pharmaceutically acceptable carrier is provided.
In another aspect, there is provided a use of a peptide as described herein or a pharmaceutical composition as described herein in the manufacture of a medicament for treating a disease condition associated with cap-dependent translational dysregulation.
In another aspect, there is provided a method of treating a disease condition associated with cap-dependent translational dysregulation in an individual in need thereof, comprising administering to the individual a peptide described herein or a pharmaceutical composition described herein.
Definition of
As used herein, the term "protein" or "peptide" refers to a polymeric form of an amino acid. A protein is understood to comprise more amino acids than a peptide, and a "protein" typically comprises at least about 60 amino acids, while a peptide typically comprises from 2 to about 60 amino acids. Amino acids in a protein or peptide may exist in either the D or L configuration or a combination of both configurations and may be chemically or biochemically modified. Modification of amino acids can occur at the side chain or peptide bond joining the amino acids. Peptide bonds (-CONH-) can be modified to alter the nature of the peptide bond. Peptides can also be chemically modified by the addition of covalently bound chemical groups such as biotin, thiols, cysteine, amides, carboxyl groups, linear or branched alkyl groups, primary or secondary amines, lipids, phospholipids, fatty acids, cholesterol, and the like. These chemical groups may be added at the N-and/or C-terminus of the peptide or within the peptide. The peptide may also be modified by adding one or more additional bonds, such as disulfide bonds, within the peptide between the N-and C-termini. The amine group at the N-terminus can also be chemically modified, for example by addition of a cap by acetylation.
As used herein, the term "amino acid" refers to a compound containing an amine (-NH-) -2) And carboxyl (-COOH) functional groups and side chains. Amino acids can be encoded by triplet codons (canonical amino acids) or variant codons (non-canonical amino acids) of the nucleic acid. Amino acids can be characterized as alpha-amino acids or beta-amino acids. In the alpha-amino acid, an amine group and a carboxyl group are attached to the first carbon. In beta-amino acids, the amine and carboxyl groups are attached to adjacent carbons. Amino acids can also be characterized as D-amino acids (in which the stereo alpha carbon of the amino group has the D-configuration) or L-amino acids (in which the stereo alpha carbon of the amino group has the L-configuration). Amino acids can also be classified according to the nature of their side chains. For example, amino acids can be classified as acidic, basic, polar, hydrophobic, or aromatic, and amino acids can fall into one or more categories. Aromatic amino acids include phenylalanine, tryptophan, tyrosine, and histidine. Hydrophobic amino acid packetIncluding glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). Polar amino acids include serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), glutamine (Gln), and tyrosine (Tyr). Basic amino acids include arginine (Arg), lysine (Lys) and histidine (His), and acidic amino acids include aspartic acid (aspartic acid) or aspartic acid (Asp) and glutamic acid (glutamic acid) or glutamic acid (glutamate) (Glu).
Amino acids as used herein may be naturally occurring, non-naturally occurring or synthetic. Amino acids used herein may also be modified by substitution of one or more groups on the side chain.
Table 1: amino acids
The terms "modified," "modifying," or grammatical variants thereof, as used herein in the context of modified amino acids, refer to amino acids in which one or more groups on the side chain are substituted. One or more of the following groups may be substituted: alkyl, alkenyl, alkynyl, thioalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, halogen, carboxy, haloalkyl, haloalkynyl, hydroxy, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamino, alkynylamino, acyl, alkenoyl, alkynoyl, acylamino, diacylamino, acyloxy, alkylsulfonyloxy, heterocyclyloxy, heterocyclylamino, haloheterocycloalkyl, alkylsulfinyl, alkylcarbonyloxy, alkylthio, acylthio, phosphorus-containing groups such as phosphono and phosphinyl, aryl, heteroaryl, alkylarylaryl, arylthio, phosphorus-containing groups such as phosphono and phosphinyl, aryl, heteroaryl, alkylarylamino, alkoxycarbonyloxy, alkoxycarbonylAlkylheteroaryl, cyano, cyanate, isocyanate, -C (O) NH (alkyl) and-C (O) N (alkyl)2。
The term "alkyl" as used herein includes within its meaning monovalent ("alkyl") and divalent ("alkylene") straight or branched chain saturated aliphatic groups having from 1 to 10 carbon atoms, for example having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
The term "alkenyl" includes within its meaning monovalent ("alkenyl") and divalent ("alkenylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having 2-10 carbon atoms, for example having 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, and having at least one E, Z, cis or trans stereochemical double bond, as applicable, anywhere in the alkyl chain.
The term "alkynyl" as used herein includes within its meaning monovalent ("alkynyl") and divalent ("alkynylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having 2 to 10 carbon atoms and having at least one triple bond anywhere in the carbon chain.
The term "cycloalkyl" as used herein refers to cyclic saturated aliphatic groups and includes within its meaning monovalent ("cycloalkyl") and divalent ("cycloalkylene") saturated, monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon groups having from 3 to 10 carbon atoms, for example, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
The term "cycloalkenyl" as used herein refers to cyclic unsaturated aliphatic groups and includes within its meaning monovalent (cycloalkenyl) and divalent (cycloalkenylene) monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon groups having 3 to 10 carbon atoms and having at least one E, Z, cis or trans stereochemical double bond, as applicable, anywhere in the alkyl chain. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclopentenyl, cyclohexenyl, and the like.
The term "heterocycloalkyl" as used herein includes within its meaning monovalent ("heterocycloalkyl") and divalent ("heterocycloalkylene") saturated, monocyclic, bicyclic, polycyclic or fused hydrocarbon radicals having 3 to 10 ring atoms, wherein 1 to 5 ring atoms are heteroatoms selected from O, N, NH or S. Examples include pyrrolidinyl, piperidinyl, quinuclidinyl, azetidinyl, morpholinyl, tetrahydrothienyl, tetrahydrofuranyl, tetrahydropyranyl and the like.
The term "heterocycloalkenyl" as used herein includes within its meaning monovalent (heterocycloalkenyl) and divalent (heterocycloalkenylene) saturated, monocyclic, bicyclic, polycyclic or fused hydrocarbon groups having 3 to 10 ring atoms and having at least 1 double bond, wherein 1 to 5 ring atoms are heteroatoms selected from O, N, NH or S.
The term "heteroaromatic group" and variants such as "heteroaryl" or "heteroarylene" as used herein include within its meaning monovalent ("heteroaryl") and divalent ("heteroarylene") mononuclear, polynuclear, conjugated and fused aryl groups having 6-20 atoms, wherein 1-6 atoms are heteroatoms selected from O, N, NH and S.
The term "heteroatom" or variants such as "hetero- (heter-)" as used herein refers to O, N, NH and S.
The term "alkoxy" as used herein refers to a straight or branched chain alkoxy group.
The term "amino" as used herein in the context of substitution refers to-NRaRbA radical of the form (I) in which RaAnd RbEach selected from the group consisting of, but not limited to, hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl.
The term "aromatic group" or variants such as "aryl" or "arylene" as used herein refers to aromatic hydrocarbons having monovalent (aryl) and divalent (arylene) mononuclear, polynuclear, conjugated and fused residues of 6-10 carbon atoms.
The term "aralkyl" as used herein includes within its meaning monovalent ("aryl") and divalent ("arylene") mononuclear, polynuclear, conjugated and fused aromatic hydrocarbon radicals attached to divalent saturated, straight and branched chain alkylene radicals.
The term "heteroaralkyl" as used herein includes within its meaning monovalent ("heteroaryl") and divalent ("heteroarylene") mononuclear, polynuclear, conjugated and fused aromatic hydrocarbon radicals attached to divalent saturated, straight and branched chain alkylene radicals.
The term "halogen" or variants such as "halide" or "halo (halo)" as used herein refers to fluoro, chloro, bromo, iodo and cyano halides.
The term "equilibrium dissociation constant" or "dissociation constant" (K) as used hereind) Is a measure of the strength of binding between two molecules, such as a protein and its ligand. The smaller the dissociation constant, the more tightly two molecules bind and the higher the affinity between the two molecules.
The term "inhibit" as used herein in the context of eIF4E activity refers to the disruption, reduction, or absence of the level of biological activity of eIF4E as compared to the level of activity of eIF4E that is not inhibited.
In the context of peptides or proteins, the terms "macrocycle" and "macrocycle" refer to a cyclic peptide that is a polypeptide chain containing a cyclic sequence of bonds. This can be achieved by: the linkage between the amino-and carboxy-termini of peptides, for example in cyclosporine; linkage between the amino terminus and the side chain, as in bacitracin; carboxy-terminal and side-chain, for example in colistin; or two side chains or more complex arrangements, for example in amanitine.
The term "pharmaceutical composition" as used herein refers to a mixture of one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof with other chemical ingredients, e.g., physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of the compound to an organism.
The term "pharmaceutically acceptable carrier" as used herein refers to a vehicle generally accepted in the art for delivering a biologically active agent (i.e., the disclosed inhibitor alone or in combination with any of the disclosed compounds in the context of the specification) to a mammal, such as a human. Such carriers are generally formulated according to a number of factors determined and considered within the purview of one of ordinary skill in the art. These factors include, but are not limited to, the type and nature of the active agent to be formulated; administering to a subject a drug-containing composition; the intended route of administration of the composition; and the therapeutic indications for which it is intended. Pharmaceutically acceptable carriers include aqueous and non-aqueous liquid media, and various solid and semi-solid dosage forms. Such carriers may include many different ingredients and additives in addition to the active agent, including such additional ingredients in the formulation for a variety of reasons, such as stabilization of the active agent as is well known to those of ordinary skill in the art. Non-limiting examples of pharmaceutically acceptable carriers are hyaluronic acid and its salts and microspheres (including but not limited to poly (D, L) -lactide-co-glycolic acid copolymer (PLGA), poly (L-lactic acid) (PLA), poly (caprolactone) (PCL), and Bovine Serum Albumin (BSA)). The term "cancer" as used herein refers to any of a number of diseases characterized by: uncontrolled abnormal proliferation of cells, the ability of affected cells to spread locally or to other parts of the body through the blood stream and lymphatic system (i.e., metastasis), and any of a number of characteristic structural and/or molecular features. Examples of cancer include, but are not limited to, breast, bronchial, colon, colorectal, liver, lung, prostate, ovarian, brain, pancreatic, head and neck, stomach, and bladder cancer, non-hodgkin lymphoma, leukemia, neuroblastoma, melanoma, glioma, or glioblastoma.
Brief description of the drawings
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
FIG. 1 shows Next Generation Sequencing (NGS) phage sequencing results and fluorescence polarization competition experiments. NGS-enhanced phage display screening against eIF4E was performed using the C7C library (available from NEB). C7C ═ disulfide-constrained cyclic peptides displayed on the pili of M13 bacteriophage. Figure 1A shows that a unique and novel motif that interacts with eIF4E was identified, in contrast to peptide interaction motifs typically isolated from screening linear 12mer libraries. FIG. 1B shows binding sites identified using fluorescence polarization experiments. The above figure shows that the C7C peptide cannot displace a fluorescently labeled peptide containing a peptide interaction motif. This motif is present in the protein interaction partners of eIF4E- >4E-BPs (1, 2 and 3) and eIF4G 1. The lower panel shows that the C7C macrocycle EE-02 can displace fluorescently labeled m7 GTP.
FIG. 2 shows tryptophan quenching experiments. The figure demonstrates how m7GTP and EE-02 have very different characteristics, which highlights that they can bind in different binding conformations. PHAGSOL is a phage-derived peptide identified to interact with the eIF4G binding site on eIF 4E-containing a canonical peptide interaction motif.
Figure 3 shows the structure of the eIF4E cap binding site in the complex with or without m7 GDP. Figure 3A shows a comparison of m7 GDP-bound eIF4E structure (left) with unbound structure (right). When binding m7GDP, the indole side chains of W56 and W102 are inserted into the guanine ring of the nucleotide. E103 of eIF4E forms 2 hydrogen bonds that recognize the guanine ring. In addition, the methyl group present on guanine ring N7 induces the formation of quaternary amines, thereby imparting a delocalized positive charge on the ring system. This results in the formation of a cation-pi interaction with W56 and W102, further stabilizing the binding conformation. The diphosphate tail of m7GDP is recognized and forms electrostatic interactions with R157, K159 and K162. Residue R112 further recognizes the phosphate moiety via several structured water molecules. This conformation is referred to as a "closed" conformation. When eIF4E was not bound, a significant conformational change occurred around the region responsible for binding to the N7 methyl substituted guanine moiety. These changes are rotation of W102, movement of the ring containing W56 and rotation of E103 out of the cap binding site. This conformation is referred to as the "open" state. Figure 3B shows the position of the canonical peptide and cap binding site on eIF 4E. Figure 3C shows surface representations of m7 GDP-bound eIF4E and "apo" eIF4E structures, demonstrating a clear structural difference in the shape and size of the cap binding site between the two states.
FIG. 4 shows a comparison of eIF4E structure binding to EE-02 or m7 GDP. Figure 4A shows a surface representation of EE-02 bound eIF4E, demonstrating that its cap binding site is similar in shape and size to that of "apo" eIF4E, and highlights the difference in cap site for eIF4E bound to m7 GDP. Figure 4B shows a comparison of EE-02 bound eIF4E structure with m7GDP bound structure. The conformation of EE-02 binding eIF4E is very similar to the "apo" structure of eIF 4E. E3 of EE-02 interacts directly with R112, M4 forms a hydrogen bond with S92 of eIF4E behind the cap binding site, while residues F6 and F7 form a hydrophobic interaction. Residues W56 and W102, responsible for recognition of the guanine ring system, are rotated out of the cap binding site. E103, which forms an h-bond with m7GDP, also swings out (swang out) a cap binding site and is not involved in the interaction of EE-02 with eIF 4E. Furthermore, in contrast to m7GDP, EE-02 does not directly interact with residues forming the diphosphate binding pocket (R157, K159 and K162).
Figure 5 shows the key interactions resulting from conserved residues of the interaction motifs identified by NGS phage sequencing in the crystal structure. The key hydrogen bond formed by EE-02 with eIF4E and itself is also highlighted.
All images in the above figure are based on the true XTAL of EE-02 complexed with eIF4ETMAnd (5) structure.
Detailed Description
In a first aspect, the present invention relates to a polypeptide comprising the amino acid sequence CEX1GX2X3X4X5C (SEQ ID NO:1), wherein X1Is an amino acid selected from the group consisting of threonine (T), methionine (M) or leucine (L); wherein X2Is an amino acid selected from the group consisting of a hydrophobic amino acid, an aromatic amino acid, a modified hydrophobic amino acid, and a modified aromatic amino acid; wherein X3Is an amino acid selected from the group consisting of a hydrophobic amino acid, an aromatic amino acid, a modified hydrophobic amino acid, and a modified aromatic amino acid; wherein X4Is any amino acid; wherein X5Is any amino acid; wherein the two cysteine residues are linked by a disulfide bond; and wherein the peptide binds to elongation initiation factor 4E (eIF 4E).
In one embodiment, the peptide comprises the amino acid sequence CEX1GFFX4X5C(SEQ ID NO:13)。
The peptides of the invention can be isolated from the cells or reaction vessels by methods well known in the art. The peptides of the invention may also be purified by methods well known in the art.
In some embodiments, the peptides of the invention may be chemically synthesized and then isolated or purified. For example, the peptides of the invention can be synthesized by fluorenylmethoxycarbonyl protecting group strategy (FMOC) solid phase chemical synthesis. Followed by iodine-mediated oxidation to form disulfide bonds that bind the peptides.
In one embodiment, the peptide binds to eIF4E at the mRNA 5' cap binding site. The peptide binds with high affinity to the mRNA 5' cap binding site of eIF 4E. In one embodiment, the peptide has a dissociation constant (K) of less than about 250nM, less than about 200nM, less than about 150nM, less than about 100nM, less than about 50nMd) Binds to the mRNA 5' cap binding site. In one embodiment, the peptide binds at the 5' cap binding site of the mRNA with a K of less than 100nMdBinding to eIF 4E.
The peptide may interact with the 5 'cap binding site by a mechanism or mode different from the interaction of the cap analog (m7GDP) with the 5' cap binding site.
In one embodiment, the peptide binds to the eIF4E cap binding site in a different conformation than m7 GDP. In another embodiment, binding of the peptide to eIF4E disrupts cap analog binding to the cap binding site. This in turn inhibits the activity of eIF 4E.
In one embodiment, the peptide binds to eIF4E in an open conformation. "open conformation" refers to the conformation of phenylalanine at positions 102 and 56 (W102 and W56) when phenylalanine at positions 102 and 56 (W102 and W56) does not interact with m7GDP and swings out of the binding site. In contrast, "closed conformation" refers to the conformation of W102 and W56 when W102 and W56 are swung back (swang back) into the cap binding site with m7GDP sandwiched between them.
In one embodiment, binding of the peptide to eIF4E inhibits eIF4E activity.
It is generally understood that the disulfide bond between two cysteine residues cyclizes the peptide.
It will be appreciated that the cyclized peptides of the invention interact with eIF4E and that when the disulfide bond is reduced, the interaction is eliminated. The peptides of the invention represent the key motifs and the necessity of constraining peptides responsible for this interaction with eIF 4E.
In one embodiment, the invention comprises the amino acid sequence CEX1GX2X3X4X5C (SEQ ID NO:1) peptideCan be at X1Position is methionine (M). In another embodiment, X2And X3Independently selected from the group consisting of tyrosine (Y), phenylalanine (F) or modified phenylalanine.
Modified phenylalanine refers to phenylalanine in which one or more groups on the aromatic ring are substituted.
In another embodiment, X4And X5Independently glutamine (Q), aspartic acid (D), alanine (a), lysine (K), glycine (G) or leucine (L).
In yet another embodiment, X5Is aspartic acid (D). In yet another embodiment, X5Is D-aspartic acid or L-aspartic acid.
In some embodiments, the peptide may comprise one or more additional amino acid residues at the N-terminus and/or C-terminus. These one or more additional amino acid residues may be from the bacteriophage or a portion of the bacteriophage on which the peptide is displayed. In one embodiment, the peptide comprises an additional alanine residue at the N-terminus and an additional glycine residue at the C-terminus. Alanine and glycine residues are non-random parts of the phage, which are present outside the random parts that make up the phage library.
In one embodiment of the invention, the peptide comprises an amino acid sequence selected from the group consisting of ACEMGFFQDCG (SEQ ID NO:2), CEMGFFQDCG (SEQ ID NO:3), ACEMGFFADCG (SEQ ID NO:4), ACEMGFFKDCG (SEQ ID NO:5), ACEMGFFLDCG (SEQ ID NO:6), CEMGFFADC (SEQ ID NO:7), ACEMGYFQDCG (SEQ ID NO:28), and ACEMGFYQDCG (SEQ ID NO: 32).
In another embodiment, the peptide consists of an amino acid sequence selected from the group consisting of seq id no: ACEMGFFQDCG (SEQ ID NO:2), CEMGFFQDCG (SEQ ID NO:3), ACEMGFFADCG (SEQ ID NO:4), ACEMGFFKDCG (SEQ ID NO:5), ACEMGFFLDCG (SEQ ID NO:6), CEMGFFADC (SEQ ID NO:7), ACEMGYFQDCG (SEQ ID NO:28) and ACEMGFYQDCG (SEQ ID NO: 32).
In another embodiment, the peptide consists of amino acid sequence CEMGFFADC (SEQ ID NO: 7).
The peptides of the invention may be conjugated with one or more additional compounds. In one embodiment, the peptide is conjugated to one or more additional peptides.
In some embodiments, the one or more additional peptides are cell penetrating peptides. Cell Penetrating Peptides (CPPs) are peptides that promote cellular uptake of the peptide. CPPs may be classified as synthetic, chimeric, or protein-derived, or may be classified as cationic, hydrophobic, or amphiphilic. Examples of cell penetrating peptides include, but are not limited to, polyarginine, transactivating Transcriptional Activator (TAT), transporter (transportan), and penetrating protein (penetratin).
The invention also relates to the use of the peptides described herein as medicaments.
In one embodiment, the peptides described herein are used to treat a disease condition associated with cap-dependent translational disorders.
The invention also provides pharmaceutical compositions comprising the peptides described herein.
The invention also provides the use of a peptide or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of a disease condition associated with cap-dependent translational dysregulation.
In one embodiment, the cap-dependent translational disorder is the result of abnormal eIF4E expression or abnormal eIF4E activity.
In some embodiments, the aberrant eIF4E expression is eIF4E overexpression. In other embodiments, the aberrant eIF4E activity is an increase in eIF4E activity above physiological levels. It is generally understood that overexpression of eIF4E means increased expression of eIF4E compared to normal physiological expression levels. It is also generally understood that an increase in eIF4E activity may be mediated by an increase in phosphorylation of 4E-BP 1.
In one embodiment, the disease condition associated with overexpression of eIF4E is cancer.
In another embodiment, the disease condition associated with cap-dependent translational disorders is an infectious disease that enforces control (hijack) of cap-dependent translation. Examples of such diseases include, but are not limited to, influenza and viral diseases caused by coronaviruses.
In yet another embodiment, the disease condition associated with cap-dependent translational dysregulation is fragile X syndrome or autism.
The invention also provides vectors comprising nucleic acid sequences encoding the peptides described herein. The invention also provides a host cell transfected with a vector as described herein, wherein the host cell expresses a peptide as described herein.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", and the like are to be read broadly and not restrictively. Furthermore, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The present invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter therefrom, regardless of whether or not the removed material is specifically recited herein.
Other embodiments are within the following non-limiting examples and the appended claims. Further, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
Experimental part
Non-limiting examples and comparative examples of the present invention are further described in more detail by referring to specific examples, which should not be construed as limiting the scope of the present invention in any way.
Method
Phage display method and fluorescence competition assay
Fluorescence anisotropy measurement and KdMeasurement of
General assay design
Targeting 50nM carboxyfluorescein (FAM) -labeled tracer peptide (KKRYSRDFLLALQK- (FAM)) (SEQ ID NO:8) or 50nM fluorescein-labeled m7GTP-6-FAM titrates purified eIF 4E. Tracer peptide and FAM-labelled m7K of GTPd(dissociation constants) were determined by fitting their respective experimental titrations to the 1:1 binding model using the following equation:
wherein [ P]Is the protein concentration, [ L ]]Is the concentration of the labeled peptide, r is the measured anisotropy, r0Is the anisotropy of the free peptide, rbIs the anisotropy of the eIF 4E-tracer peptide complex, [ L ]]tIs the total concentration of FAM-labeled peptide, [ P ]]tIs the total concentration of eIF 4E. Assay K for the determination of the interaction of tracer peptide with eIF4Ed50.3nM, and m7K of GTP-6-FAMdIt was 149.0 nM. Both of which were later used for subsequent determination of K in competition experimentsd。
The apparent K of the various molecules was then determined by a competitive anisotropy experimentdThe value is obtained.
Binding assessment at the 4E:4G site
Titration was performed with eIF4E at a constant concentration of 200nM and the labeled peptide at a constant concentration of 50 nM. The competitor was then titrated against the complex of FAM-labeled peptide and eIF 4E. Determination of apparent K by fitting the experimental data to the equation shown belowdThe value:
d＝Kd1+Kd2+[L]st+[L]t-[P]t
e＝([L]t-[P]t)Kd1+([L]st-[P]t)Kd2+Kd1Kd 2
f＝-Kd1Kd2[P]t
[L]stand [ L]tIndicating labeled ligand and total unlabeled ligand input concentrations, respectively. Kd2Is the dissociation constant for the interaction between unlabeled ligand and protein. In all types of competition experiments, [ P ] is assumed]t>[L]stOtherwise there will always be a large amount of free labelled ligand and the measurement will be disturbed. Kd1Is the apparent K of the labeled peptide used in the respective experimentsd. The tracer peptide was dissolved in 1mM DMSO and diluted into assay buffer. The readings were performed using an Envision Multi-label Reader (PerkinElmer). The experiment was performed in PBS (2.7mM KCl,137mM NaCl,10mM Na)2HPO4And 2mM KH2PO4(pH 7.4)), 3% DMSO (v/v) and 0.1% Tween20 buffer. All titrations were performed in triplicate. Curve fitting was performed using Prism 4.0 (GraphPad).
Cap analogue binding assessment
Titration was performed with eIF4E constant at 250nM and labeled m7GTP constant at 50 nM. The competitor was then titrated against the FAM-labeled complex of m7GTP and eIF 4E. Determination of apparent K by fitting the experimental data to the equation shown belowdThe value:
d＝Kd1+Kd2+[L]st+[L]t-[P]t
e＝([L]t-[P]t)Kd1+([L]st-[P]t)Kd2+Kd1Kd 2
f＝-Kd1Kd2[P]t
[L]st and [ L ]]t represents the labeled ligand and total unlabeled ligand input concentrations, respectively. Kd2Is the dissociation constant for the interaction between unlabeled ligand and protein. In all types of competition experiments, [ P ] is assumed]t>[L]stOtherwise there will always be a large amount of free labelled ligand and the measurement will be disturbed. Kd1Is the apparent K of the labeled peptide used in the respective experimentsd. FAM-labeled m7GTP was provided at 1mM in Tris-HCL pH7.5(Jena Biosciences) and diluted into the assay buffer. The readings were performed using an Envision Multi-label Reader (PerkinElmer). In PBS (2.7mM KCl,137mM NaCl,10mM Na)2HPO4And 2mM KH2PO4(pH 7.4)), 3% DMSO (v/v) and 0.1% Tween20 buffer. All titrations were performed in triplicate. Curve fitting was performed using Prism 4.0 (GraphPad).
NGS enhanced phage display
Selection of C7C disulfide-bound phage libraries against eIF4E
Phage library
The C7C phage library was purchased from New England Biolabs. The library was a random peptide combinatorial library with disulfide-binding loops fused to the minor capsid protein (pIII) of M13 phage. The displayed peptide was expressed at the N-terminus of pIII in the form of AC-X7-CGGGS (X ═ random amino acid) (SEQ ID NO: 9). The two cysteines spontaneously form disulfide bridges under oxidative conditions to constrain the peptide sequence. The library consisted of approximately 1X 109(i.e., unique) sequence of electroporationAnd (4) forming.
eIF4E fixation and phage selection
A96-well polystyrene plate is coated with streptavidin solution (150. mu.l per well, 20. mu.g/ml, and incubated overnight at 4. 3. the coating solution is then removed. the wells on the 96-well plate are then washed 4 times with 200. mu.l binding buffer (50mM Tris-HCl,150mM NaCl (pH 7.4). after washing, the wells are loaded with 150. mu.l biotin-labeled protein (5-10 mg/ml in binding buffer). streptavidin control wells are loaded with only 150. mu.l binding buffer. the 96-well plate is then incubated at room temperature for 15min, then the contents are decanted. the wells are then washed 4 times with 200. mu.l binding buffer. after washing, the wells are blocked with 200. mu.l blocking buffer ((50mM Tris-HCl,150mM NaCl (pH 7.4.). 0.01% BSA (w/v), 0.1% Tween20(v/v)) at room temperature for 30 min. simultaneously, 1X 10 in blocking buffer12Phage libraries were prepared at pfu/ml and also incubated for 30min at room temperature. The blocking buffer was then decanted from the 96-well plate and 100. mu.l of phage library was distributed between wells, approximately 10 per well11And pfu. The plates were then incubated at room temperature for 1 hour. Unbound phage was then removed by pipetting from each well. The wells were then washed 8 times with 200. mu.l of wash buffer. The bound phage were then incubated with 100. mu.l of elution buffer (0.2M glycine-HCl, pH 2.2+ 0.1% (w/v) BSA) for 9min at room temperature. The supernatants containing the eluted phage were then transferred separately to separate tubes containing 15. mu.l of neutralization buffer (1M Tris-HCl, pH 9.1).
Phage amplification and M13 DNA isolation
The overnight cultured E.coli (ER 2738 strain) was diluted 100-fold and 3ml of the culture was dispensed into each 15ml culture tube. Cultures were incubated at 37C for 4-4.5 hours while shaking at 230 rpm. After incubation, the tubes were centrifuged at 4000rpm for 15min at 4 ℃ to pellet the bacterial cells. To pellet the phage, the supernatant in each tube was poured into a separate 5ml falcon tube containing MP buffer (30. mu.l, 100X) and vortexed. 700 μ l of precipitated phage solution was loaded into a separate Qiagen spin column and centrifuged at 8000rpm for 30 seconds. The flow-through resulting therefrom is discarded (flow-through). After this step, 700. mu.l of buffer PB was applied to each spin column and centrifuged at 8000rpm for 30 s. The flow-through was discarded again and the procedure repeated. After this step was completed, 700. mu.l of buffer PE was added to each spin column. Each column was spin centrifuged at 8000rpm for 30s and the flow through in the collection tube was discarded. The centrifugation step was repeated, but the spin centrifugation was extended to 2min to remove any remaining PE buffer. The spin column was transferred to a fresh 1.5ml Eppendorf tube. The DNA was eluted into each column by adding EB buffer (50. mu.l) and then waiting for 1min followed by centrifugation at 800rpm for 30 s. For more details on the buffers and procedures, see M13QIAprep Spin M13 kit manual (Qiagen, Ltd).
MP buffer formula
1. Citric acid monohydrate 3.3g was dissolved in 3ml of high purity water at room temperature (21 ℃ C.)
2. The solution was incubated for 5min by stirring at 200rpm
3. The solution was filtered through a 0.2 μm sterile filter using a syringe to give a final volume of 6ml of Buffer MP.
Barcode and library amplification
The 15 barcoded reverse primers were designed with adapters compatible with Illumina sequencing. The library DNA was PCR amplified using barcoded primers flanking the variable region. ssM13 Each eluate of phage DNA is uniquely encoded. In short, useBuffer, 200. mu.M of each dNTP, 1.5mM MgCl20.5. mu.M forward primer, 0.5. mu.M barcoded reverse primer and one unitHigh fidelity DNA polymerase, 25. mu.L in total, amplified library DNA (15 samples, 50ng each). PCR was performed using the following thermocycler program: 30s at 95 ℃, then 10s at 95 ℃,15 s at 60.5 ℃ and 30s at 72 ℃, 25 cycles, followed by 5min final extension at 72 ℃ and then held at 4 ℃. Using low molecular weightQuantitative DNA ladder as a standard (NEB, # N3233S), dsDNA fragments from PCR were quantified by running a 2% (w/v) agarose gel in Tris-borate-EDTA buffer at 100 volts for about 45 min. The dsDNA fragments (15 samples, 40ng each) were pooled together and stored inSizeSelectTM 2% agarose gel (Invitrogen, # G6610-02). Referring to ladder, collect the desired band corresponding to 121bp with RNase-free water. The DNA concentration of the purified DNA was determined using PicoGeen (Thermofoisher) according to the manufacturer's instructions.
DNA template preparation and Illumina sequencing (NextSEQ)
Sequencing was performed by AXIL Scientific. The pooled DNA was hybridized to Illumina chips, bridge amplified, and then sequenced using Illumina technology.500Mid Output Kit (150 cycles) (directory number FC-404-500/550Mid Output Kit v2(150 cycles) (Cat. No. FC-404-2001) were used to sequence reads in both the forward (F) and reverse (R) directions. FASTQ files were processed using double-ended analysis using an internally developed python script.
Differential enrichment and sequence identity generation
The FASTQ files are analyzed and parsed to separate the sequences into their respective selections by the associated barcodes. The frequency of each unique peptide sequence encoded by the variable domain is then determined and normalized by the total number of sequences in the selection. This process is repeated for each repeat, e.g., eIF4E repeats 1-3 and streptavidin controls 1-3. Sequences not observed in a particular repeat were designated as having a normalized frequency of zero. The enrichment ratio for each sequence was determined by dividing the average normalized frequency for the particular sequence (from the replicate set for that selection) by the frequency in the control replicate set. Since the denominator cannot be zero when taking the ratio, sequences that do not appear in all three repeats are assigned arbitrary values (e.g., 0.0001) before the ratio is calculated. The significance (p-value) of the ratio was assessed using the unequal variance student's t-test (Welch test). A heat map is then generated for any determined values of p-value and enrichment ratio. Typical values used are p-value <0.1 and ratio > 5.0. Sequences were ordered by their enrichment in the heatmap. The heatmap identifies sequences isolated from a particular selection that are significantly more abundant relative to sequences isolated from a control selection (or a non-relevant selection group). Sequence identifiers were generated from the enriched sequences using WebLogio (https:// webbloo. bergelley. edu /).
Tryptophan quenching experiment
Use ofTryptophan quenching experiments were performed with a multi-plate reader and black matte 96-well plates. Samples (100 μ l) were prepared with 1 μ M purified eIF4E for a range of concentrations of the following compounds: m is7GTP, PHAGESOL (KKRYSRDQLVAL) (SEQ ID NO:10) or EE-02. The concentration range tested was 0.01-20. mu.M. The sample was excited with 290nm UV light to minimize the effect of tyrosine fluorescence. Tryptophan was measured at an emission wavelength of 355 nm.
Crystallization of eIF4E: EE-02
Crystallization of
The eIF4E: EE-02 complex was crystallized by vapor diffusion using the sitting drop method. The crystal drops contained eIF4E and EE-02 at concentrations of 150. mu.M and 300. mu.M, respectively. Sitting drops were placed in 48 well Intelli-Plate (hampton study) and 1 μ l protein sample was mixed with 1 μ l of the master well solution. Crystals were grown in 0.2M ammonium sulfate 0.1M Tris trisodium acetate pH 4.6 and 25% PEG 4000 for one week. To collect X-ray data at 100K, crystals were transferred to an equivalent mother liquor (heat li quor solution) containing 20% (v/v) glycerol and then snap frozen in liquid nitrogen.
Data collection and refinement
Using a CCD detector, in Australia of ray MX1Data was collected on the great lead synchrotron. The crystal is diffracted toAnd integration and scaling using XDS. The initial phase of binary complex crystals of eIF4E was resolved by molecular replacement and program PHASER using the human eIF4E structure (PDB accession code: 4BEA) with water and other binding components removed as a search model. The initial model is subjected to rigid refinement, then an iterative loop of manual model construction is performed in Coot, and constraint refinement is performed in Refmac 6.0.
General procedure
expression, refolding and purification of eIF4E (for Fluorescence Polarization (FP), Surface Plasmon Resonance (SPR), and crystallization Study experiment)
Rossetta pLysS competent bacteria were transformed with pET11d expression plasmid containing the full length eIF4E clone. Both materials are supplied by dunde's cyclel ltd. Cells expressing the full-length eIF4E construct were grown to OD in LB medium at 37 ℃600Approximately 0.6, and induction of eIF4E was started with 1mM IPTG. The cultures were immediately placed in a shaking incubator (shaker-incubator) at 37 ℃ for 3 h. Cells were harvested by centrifugation and the cell pellet was resuspended in 50mM Tris pH 8.0, 10% sucrose and then sonicated.
The sonicated samples were centrifuged at 17,000g for 10min at 4 ℃. The resulting pellet was resuspended in Tris/Triton buffer (50mM Tris pH 8.0, 2mM EDTA, 100mM NaCl, 0.5% Triton X-100). The samples were then centrifuged at 25,000g for 15min at 4 ℃ and the pellet resuspended in Tris/Triton buffer. After re-centrifugation, the remaining pellet was dissolved in 6M guanidine hydrochloride, 50mM Hepes-KOH pH 7.6, 5mM DTT. The protein concentration of the sample was then adjusted to 1 mg/mL.
The denatured proteins were refolded by dilution at 1/10 into refolding buffer consisting of 20mM Hepes-KOH, 100mM KCl and 1mM DTT. The refolded protein was concentrated and desalted into refolding buffer using an Amersham PD10 column. eIF4E protein samples were run on a monoQ column and eluted with a 1M KCl gradient. eIF4E eluted as a spike at about 0.3M KCl.
Surface plasmon resonance (for Structure Activity Relationship (SAR) tables)
For peptide stock solutions, compounds were dissolved in 100% DMSO to a concentration of 10 mM; immediately prior to analysis, the peptide stock solution was further diluted into DMSO and/or running buffer. The running buffer consisted of 10mM Hepes pH 7.6, 0.15M NaCl and 0.1% Tween 20. Peptide stock/DMSO diluted peptide solutions were diluted into 1.03 × running buffer to prepare peptide solutions with a final DMSO concentration of 3%. Working concentrations of peptide were achieved by further diluting the samples into running buffer containing 3% DMSO. Surface plasmon resonance experiments were performed on a Biacore T100 machine.
Pure eIF4E was immobilized on CM5 sensor chip. CM5 chips were conditioned by injection of 100mM HCl 6s, followed by injection of 0.1% SDS 6s, and quenched by injection of 50mM NaOH 6s at a flow rate of 100. mu.l/min. NHS (115mg ml) was used-1) And EDC (750mg ml)-1) In 10. mu.l min-1The sensor chip surface was activated for 7 min. Purified eIF4E was diluted with 10mM sodium acetate buffer (pH 5.0) to a final concentration of 0.5 μ M and M7GTP was present in a 2:1 ratio to stabilize eIF 4E. The amount of eIF4E immobilized on the activation surface was controlled by varying the contact time of the protein solution, approximately 1000 RU. After fixation of the protein, injection of 1M ethanolamine (pH 8.5) for 7min (10. mu.l min)-1) To quench excess active succinimide ester groups.
Six buffer blanks were injected first to fully equilibrate the instrument, followed by a solvent calibration curve, followed by a further 2 blank injections. The solvent calibration curve was set by adding different amounts of 100% DMSO to 1.03x running buffer to generate a series of DMSO solutions (3.8%, 3.6%, 3.4%, 3.2%, 3%, 2.85%, 2.7%, and 2.5%, respectively). Compounds were injected for 60s using a flow rate of 50 μ l/min and dissociation was monitored for 180 s. The data collection rate was 10 Hz. KdsDetermined using BiaEvaluation software (Biacore) and based on the response of eIF 4E-coated CM5 chips at equilibrium and on the dissociation of each peptideAnd calculated in conjunction with the kinetics of the phase data. Both equilibrium and kinetic data were fit to a 1:1 binding model. K was determined for each individual peptide by three separate titrationsd. At least two concentration points are replicated in each titration to ensure stability and robustness of the chip surface.
GST affinity purification for biotinylated eIF4E
Full length eIF4E was ligated into GST fusion expression vector pGEX-4(GE Lifesciences) by double digestion with BAMH1 and NDE 1. BL21 DE3 competent bacteria were then transformed with the GST-tagged eIF4E fusion construct. Cells expressing the GST fusion construct were grown to OD in LB medium at 37 ℃600About 0.6 and was induced at 1mM at room temperature. Cells were harvested by centrifugation and the cell pellet resuspended in 50mM Tris pH 8.0, 10% sucrose and then sonicated. The sonicated samples were centrifuged at 17,000g for 60min at 4 ℃. The supernatant was applied to a 5ml FF GST column (Amersham) pre-equilibrated in wash buffer with 1mM DTT (phosphate buffered saline, 2.7mM KCL and 137mM NaCL, pH 7.4). The column was then further washed with 6 volumes of wash buffer. eIF4E was then purified from the column by cleavage with precipitation (ge lifesciences) protease. 50 units of thrombin protease (in a column volume of PBS with 1mM DTT buffer) were injected onto the column. The cleavage reaction was allowed to proceed overnight at 4 ℃. The cleaved protein is then eluted from the column with a wash buffer. Protein fractions were analyzed using SDS-PAGE gels and concentrated using a Millipore Centricon (10kDa MWCO) concentrator. The eIF4E protein sample was then dialyzed into a buffer solution containing 20mM Hepes-KOH, 100mM KCl, and 1mM DTT. The eIF4E protein sample was then passed through a monoQ column and eluted with a 1M KCl gradient. eIF4E eluted as a spike at about 0.3M KCl. Protein concentration was determined using UV absorbance.
Sortase-mediated biotinylation of eIF4E for immobilization and (n) phage display
The biotin tag was incorporated into GS-eIF4E using sortase-mediated ligation. In ligation buffer (50mM Tris, 1500mM NaCl,10mM TCEP (pH 8.0)), the following reagents were mixed in a volume of 1000. mu.l: 40 μ M GS-eIF4E, 5 μ M SrtA61-206-His6(8M), 200 μ M biotin-KGGGLPET-GG-OHse (Ac) -amide. The samples were incubated at room temperature for 4 hours. The labeling efficiency of 2. mu.l samples was analyzed by SDS-PAGE gels. After analysis, the reaction samples were then purified to remove all reaction components except eIF 4E. The sample was resuspended with 50. mu.l of prewashed His-Tag magnetic beads (Qiagen) and incubated on a roller for 5min at room temperature. 7. The tube was placed on a magnet for 2min and then the supernatant containing the biotinylated protein was transferred to a fresh Eppendorf tube. The biotinylated protein was dialyzed against 2L PBS using Slide-A-Lyzer cassette (10k MWCO) (4 hours), then the buffer was changed to 50mM Tris, 150mM NaCl (pH7.4), and dialyzed overnight. The protein concentration was measured. Protein samples were aliquoted and snap frozen.
Results
Novel macrocyclic peptides that bind eIF4E at cap binding site
The phage display library was panned against N-terminally biotinylated eIF 4E. The phage display library used consisted of 7 random amino acids constrained by a disulfide bond between two cysteine residues (ACXXXXXXXC-GGGS) (SEQ ID NO:9), which were fused via a GGS linker to the minor capsid protein (pIII) of the M13 phage. The library was purchased commercially from New England Biolabs (NEB). eIF4E was biotinylated by peptide ligation, whereby a biotin-labeled peptide (biotin-LPETGG) (SEQ ID NO:37) was fused to the N-terminal glycine on eIF4E using sortase. Purification by using GST tag and removal of the GST tag with thrombin yielded eIF4E with an N-terminal glycine. Biotinylated eIF4E was immobilized on streptavidin coated plates.
Next Generation Sequencing (NGS) enhanced phage display was performed by 3x selection and 3x control selection against eIF4E (against both Mdm2 and eIF 4A). The selection is performed as described in the method. Phage were isolated, amplified and sequenced using Illumina nextsseq technology (see methods). The data was analyzed using an internal python script and a unique set of peptides with a novel eIF4E interaction motif was identified (figure 1).
The identified consensus motif consists of C-E- (T/M/L) -G-F-F-X-X-C (SEQ ID NO: 11). X represents any amino acid. Parallel NGS-enhanced phage selection was performed using a phage library consisting of random 12mer linear peptides (NEB) against eIF 4E. However, this library only identified eIF4E good interaction binding motifs that interact with eIF4g binding sites on eIF 4E. (yxxxl Φ, Φ ═ any hydrophobic amino acids) (SEQ ID NO: 12).
From these initial results, it was predicted that the novel peptide motif might interact with eIF4E at the eIF4G binding site, or cap binding site or elsewhere on the protein through different binding poses. The binding site was initially delineated on eIF4E by using two types of competitive fluorescence polarization experiments, one using FAM-labeled cap analogs and the other using FAM-labeled eIF4G 1-derived peptides (fig. 1). These experiments confirmed that EE-02(ACEMGFFQDCG) (SEQ ID NO:2) (FIG. 1) is the most potent peptide in the identified group and that this novel interaction motif disrupts cap analog binding to eIF 4E.
The structure of EE — 02 is significantly different from that of the cap analog, which consists of a guanine triphosphate molecule with a methyl substitution at the N7 position of the guanine ring. This directly suggests that the macrocyclic peptide can interact with the cap binding site by a very different mechanism.
To confirm this hypothesis, a tryptophan quenching experiment was performed in which EE-02, m7GTP, or eIF4G1 derived peptides were titrated against eIF4E (fig. 2).
PhageSol peptide gave significantly lower amounts of tryptophan quenching compared to other titrants. This result was expected because only one tryptophan residue (W73) was located near the eIF4G binding site, in particular m7GTP/m7GDP binds at the cap binding site and is inserted between the two tryptophan residues (W56 and W102) compared to m7GTP/m7GDP, thus resulting in significantly more quenching. However, EE-02, which also binds at the cap binding site, gives rise to features that are distinctly different from m7GTP/m7 GDP. This difference suggests that the interaction of EE-02 with eIF4E occurs through a different pattern and may be allosteric in how it affects the cap binding site.
To address this issue, structural studies were conducted and successfully addressed the crystal structure of EE-02 in conjunction with eIF4E (fig. 3 and 4).
Structural studies have shown that EE-02 interacts directly with the cap binding site and does not insert between tryptophan rings. This result explains the different curves observed in the tryptophan quenching experiments. In addition, the EE-02 macrocycle binds to eIF4E without mimicking the phosphate tail or cation-pi interaction produced by m7GDP (fig. 3, 4 and 5).
EE-02 interacts with eIF4E through 4 distinct pockets (fig. 5). The macrocycle itself forms a β -hairpin into the cap binding site enabling the residues E3, E4, F6 and F7 to protrude (project) into the corresponding pockets shown in figure 5.
EE-02 was also subjected to alanine scanning mutagenesis to confirm the critical interactions identified in the crystal structure (table 2).
Table 2: alanine scanning mutagenesis of EE-02
Determination of K Using Surface Plasmon Resonance (SPR) as described in the methodsd. Alanine scanning confirmed that residues E3, M4, G5, F6, and F7 were all critical for binding to eIF 4E. The sensitivity of G5 to substitution highlights its importance in allowing the formation of a β -hairpin with the correct geometry in the binding pocket. Substitution of Q8 with a did not affect binding. However, substitution of D9 with a resulted in a two-fold decrease in binding.
Table 3: targeted mutagenesis at E3 and M4 sites of EE-02
E4 could not be replaced by D4, emphasizing the correct orientation of the carboxyl group to interact with R112 of eIF4EImportance of use. Replacement of M4 by shorter and smaller aliphatic groups (e.g., I and L) resulted in loss of binding. M4 was also replaced with the unnatural aliphatic amino acids norleucine and cyclobutylalanine of similar size to methionine. However, K for eIF4E determined for these moleculesdStill reduced, this highlights the importance of the interaction of the sulfur atom with S92.
Table 4: targeted mutagenesis of residue G5
G5 was replaced with different amino acids (β -alanine (bA), sarcosine (Sar), P) to explore the different stereochemical constraints around this position. All of this resulted in a significant reduction in the macrocyclic binding to eIF4E, further highlighting the role of G5 in allowing β -hairpin formation and orienting key interacting residues to their optimal positions for binding eIF 4E.
TABLE 5 Targeted mutagenesis of the F6 and F7 positions in EE-02
By exploring the ring substituents on F6 and F7, binding to eIF4E can be gradually increased. Replacement of F6 with Y6 or F7 with Y7 resulted in two molecules (EE-30 and EE-35, respectively) that bound more tightly than EE-02. These results indicate that these positions can be further explored to generate better binding analogs.
Table 6: target mutagenesis of Q8 in EE-02
Position 8 of EE-02 does not show a strict preference for a particular residue. This is linked to the fact that there is no interaction with eIF4E in the crystal structure. However, a slight decrease in binding of the G mutation suggests that the rigidizing strategy at this position may improve binding through an entropy-reducing mechanism.
Table 7: truncation study of EE-02
These studies were directed to finding the smallest Molecular Weight (MW) macrocycle that can bind eIF 4E. In addition, N-terminal capping of the macrocycle was studied to identify the effect of acetylation on eIF4E binding with free amines. This resulted in EE-44 being identified, non-library phage amino acid positions a1 and G11 could be removed, and acetylation significantly reduced macrocyclic binding. EE-44, due to its small MW and reduced size, is a more suitable starting point for rational design of macrocycles with improved penetration (permeability).
Table 8: summary of the sequence listing
Identity of
The above examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will be evident that many modifications and changes may be made to the specific embodiments of the invention described above and illustrated in the embodiments without departing from the underlying principles thereof. All such modifications and variations are intended to be covered by this application.
Sequence listing
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<223> Xaa can be any naturally occurring amino acid, non-naturally occurring amino acid, or synthetic amino acid
<400> 9
Ala Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Gly Gly Gly Ser
1 5 10
<210> 10
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 10
Lys Lys Arg Tyr Ser Arg Asp Gln Leu Val Ala Leu
1 5 10
<210> 11
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> Xaa can be threonine, methionine or leucine
<220>
<221> MISC_FEATURE
<222> (7)..(8)
<223> Xaa can be any naturally occurring amino acid, non-naturally occurring amino acid, or synthetic amino acid
<400> 11
Cys Glu Xaa Gly Phe Phe Xaa Xaa Cys
1 5
<210> 12
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa can be any naturally occurring amino acid, non-naturally occurring amino acid, or synthetic amino acid
<220>
<221> MISC_FEATURE
<222> (4)..(5)
<223> Xaa can be any naturally occurring amino acid, non-naturally occurring amino acid, or synthetic amino acid
<220>
<221> MISC_FEATURE
<222> (7)..(7)
<223> Xaa can be any hydrophobic amino acid
<400> 12
Tyr Xaa Arg Xaa Xaa Leu Xaa
1 5
<210> 13
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (5)..(8)
<223> Xaa can be any naturally occurring amino acid
<400> 13
Cys Glu Xaa Gly Xaa Xaa Xaa Xaa Cys
1 5
<210> 14
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 14
Ala Cys Ala Met Gly Phe Phe Gln Asp Cys Gly
1 5 10
<210> 15
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 15
Ala Cys Glu Ala Gly Phe Phe Gln Asp Cys Gly
1 5 10
<210> 16
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 16
Ala Cys Glu Met Ala Phe Phe Gln Asp Cys Gly
1 5 10
<210> 17
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 17
Ala Cys Glu Met Gly Ala Phe Gln Asp Cys Gly
1 5 10
<210> 18
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 18
Ala Cys Glu Met Gly Phe Ala Gln Asp Cys Gly
1 5 10
<210> 19
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 19
Ala Cys Glu Met Gly Phe Phe Gln Ala Cys Gly
1 5 10
<210> 20
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 20
Ala Cys Asp Met Gly Phe Phe Gln Asp Cys Gly
1 5 10
<210> 21
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 21
Ala Cys Glu Ile Gly Phe Phe Gln Asp Cys Gly
1 5 10
<210> 22
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 22
Ala Cys Glu Leu Gly Phe Phe Gln Asp Cys Gly
1 5 10
<210> 23
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<220>
<221> MOD_RES
<222> (4)..(4)
<223> Nle
<400> 23
Ala Cys Glu Leu Gly Phe Phe Gln Asp Cys Gly
1 5 10
<210> 24
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Cyclobutylalanine
<400> 24
Ala Cys Glu Ala Gly Phe Phe Gln Asp Cys Gly
1 5 10
<210> 25
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<220>
<221> MOD_RES
<222> (5)..(5)
<223> bAla
<400> 25
Ala Cys Glu Met Ala Phe Phe Gln Asp Cys Gly
1 5 10
<210> 26
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<220>
<221> MOD_RES
<222> (5)..(5)
<223> methylation of Glycine to sarcosine
<400> 26
Ala Cys Glu Met Gly Phe Phe Gln Asp Cys Gly
1 5 10
<210> 27
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 27
Ala Cys Glu Met Pro Phe Phe Gln Asp Cys Gly
1 5 10
<210> 28
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 28
Ala Cys Glu Met Gly Tyr Phe Gln Asp Cys Gly
1 5 10
<210> 29
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 29
Ala Cys Glu Met Gly Trp Phe Gln Asp Cys Gly
1 5 10
<210> 30
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 30
Ala Cys Glu Met Gly Leu Phe Gln Asp Cys Gly
1 5 10
<210> 31
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 31
Ala Cys Glu Met Gly Phe Leu Gln Asp Cys Gly
1 5 10
<210> 32
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 32
Ala Cys Glu Met Gly Phe Tyr Gln Asp Cys Gly
1 5 10
<210> 33
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 33
Ala Cys Glu Met Gly Phe Trp Gln Asp Cys Gly
1 5 10
<210> 34
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 34
Ala Cys Glu Met Gly Tyr Tyr Gln Asp Cys Gly
1 5 10
<210> 35
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 35
Ala Cys Glu Met Gly Leu Leu Gln Asp Cys Gly
1 5 10
<210> 36
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 36
Ala Cys Glu Met Gly Phe Phe Gly Asp Cys Gly
1 5 10
<210> 37
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 37
Leu Pro Glu Thr Gly Gly
1 5
<210> 38
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 38
Cys Glu Met Gly Phe Phe Gln Asp Cys
1 5
<210> 39
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 39
Ala Cys Glu Thr Gly Phe Phe Thr Gly Cys Gly
1 5 10
<210> 40
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 40
Ala Cys Glu Leu Gly Tyr Tyr Asn Asp Cys Gly
1 5 10
<210> 41
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 41
Ala Cys Glu Thr Gly Phe Phe Leu Lys Cys Gly
1 5 10
<210> 42
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 42
Ala Cys Glu Leu Gly Phe Tyr Arg Leu Cys Gly
1 5 10
<210> 43
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 43
Ala Cys Glu Thr Gly Phe Phe Leu Arg Cys Gly
1 5 10
<210> 44
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 44
Ala Cys Glu Thr Gly Tyr Phe Ser Gln Cys Gly
1 5 10
<210> 45
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 45
Ala Cys Ile His Ser Pro Thr Ser Leu Cys Gly
1 5 10
<210> 46
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 46
Ala Cys Glu Thr Gly Phe Tyr Lys Thr Cys Gly
1 5 10
<210> 47
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence
<400> 47
Ala Cys Glu Met Gly Tyr Phe Gly Asn Cys Gly
1 5 10
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