SOS bridged helix dimer mimics and methods of using same

Abstract

The present invention relates to macrostructures (and pharmaceutical formulations comprising them) comprising antiparallel coiled-coils as described in this application, the antiparallel coiled-coils comprising a first coil of formula I and a second coil of formula II: T1-g0-a1-b1-c1-d1-e1-f1-g1-a2-b2-c2-d2-e2-f2-g2-a3-b3-c3-d3-e3-f3-T2 (I), T3-f'0-g'0-a'1-b'1-c'1-d'1-e'1-f'1-g'1-a'2-b'2-c'2-d'2-e'2-f'2-g'2-a'3-b'3-c'3-d'3-e'3-T4 (II). Methods of using these macrostructures are also disclosed. TIFF2024501019000060.tif107128

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 131,103, filed December 28, 2020, the contents of which are incorporated by reference in their entirety herein.

[0002] This invention was made with Government support under Grant No. R35GM130333 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy was created on December 17, 2021, is named "147462.002271_ST25.txt" and is 18,005 bytes in size.

[0004] FIELD OF THE PRESENT APPLICATION The present invention is directed to stabilized coiled-coils that mimic the Sos αH / αI coiled-coil, and methods of using such mimetics. [Background technology]

[0005] 2. Background of the Invention Aberrant Ras signaling is associated with a wide range of hyperproliferative diseases, and components of the signaling pathway, including Ras, are the subject of active ongoing efforts for drug discovery. Because the intracellular activity of Ras is regulated by the association of Ras with the guanine nucleotide exchange factor Son of Sevenless (Sos), the high-resolution crystal structure of the Ras-Sos complex provides the basis for the rational design of orthosteric Ras ligands.

[0006] Oncogenic Ras isoforms are the subject of active research due to the difficulty of targeting these biomedically important but "undruggable" proteins. Recent success in covalent targeting of Ras mutants shows promising avenues for ligand design; however, many mutant forms of Ras do not feature nucleophilic residues, suggesting that strategies for non-covalent association with Ras are required.

[0007] Sos is a Ras-specific guanine nucleotide exchange factor that mediates the conversion of Ras from an inactive GDP-bound form to an active GTP-bound form (PA Konstantinopoulos, MV Karamouzis, AG Papavassiliou, "Post-translational modifications and regulation of the RAS superfamily of GTPases as anticancer targets", Nat. Rev. Drug Discov. 6, 541-555 (2007) (Non-Patent Document 1)). Sos catalyzes nucleotide exchange through the insertion of a critical helical segment (αH) between the dynamic conformational switch I and switch II regions adjacent to the nucleotide-binding pocket of Ras, which leads to the disruption of the water-mediated and direct interactions between the protein and the cofactor (see FIG. 1A) (PA Boriack-Sjodin, SM Margarit, D. Bar-Sagi, J. Kuriyan, "The structural basis of the activation of Ras by Sos", Nature 394, 337-343 (1998)).Given the biomedical importance of the dynamic conformational Sos-binding interface of Ras, several rational design and screening strategies have been attempted to isolate ligands for this interface (AR Moore, SC Rosenberg, F. McCormick, S. Malek, "RAS-targeted therapies: is the undruggable drugged?", Nature Reviews Drug Discovery 19, 533-552 (2020)(Non-Patent Document 3); H. Chen, JB Smaill, T. Liu, K. Ding, X. Lu, "Small-Molecule Inhibitors Directly Targeting KRAS as Anticancer Therapeutics", Journal of Medicinal Chemistry 63, 14404-14424 (2020)(Non-Patent Document 4)).Recent efforts to associate with the G12C isoform of Ras (J. Canon et al., "The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity", Nature 575, 217-223 (2019) (Non-Patent Document 5); JM Ostrem, U. Peters, ML Sos, JA Wells, KM Shokat, "K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions", Nature 503, 548-551 (2013) (Non-Patent Document 6)) and with the G12D isoform of Ras (Z. Zhang et al., "GTP-State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf", ACS Central Science 6, 1753-1761 (2020) (Non-Patent Document 7); K. Sakamoto, T. Masutani, T. Hirokawa, "Generation of KS-58 as the first K-Ras(G12D)-inhibitory peptide presenting anti-cancer activity in vivo", Scientific Reports 10, 21671 (2020) (Non-Patent Document 8)) suggests that targeted screening may yield small molecules and peptide macrocycles as potential leads.

[0008] The structure of the Ras-Sos complex provides a basis for the rational design of Sos helix mimetics that associate with the switch region of Ras. Indeed, conformationally stabilized α-helix mimetics that target Ras-Sos protein-protein interactions (PPIs) have been previously developed (DY Yoo, AD Hauser, ST Joy, D. Bar-Sagi, PS Arora, "Covalent Targeting of Ras G12C by Rationally Designed Peptidomimetics", ACS Chem. Biol. 15, 1604-1612 (2020); A. Patgiri, KK Yadav, PS Arora, D. Bar-Sagi, "An orthosteric inhibitor of the Ras-Sos interaction", Nat. Chem. Biol. 7, 585-587 (2011)). The stabilized α-helix was shown to bind Ras at the orthosteric binding site and inhibit Sos-mediated nucleotide exchange, inhibiting Ras activation and phosphorylation of the downstream effector protein ERK (BN Kholodenko, JF Hancock, W. Kolch, "Signalling ballet in space and time", Nature Reviews Molecular Cell Biology 11, 414-426 (2010)), a well-characterized kinase involved in cell proliferation and differentiation. However, the compound preferentially binds to the nucleotide-free form of Ras, suggesting that a single Sos helix may be insufficient in the nucleotide-bound form of Ras to properly associate with the dynamic Ras interface. Wild-type Ras shuttles between the two nucleotide-bound forms (Figure 1A), whereas the oncogenic form of Ras remains activated in its GTP-bound state.Thus, compounds that preferentially associate with the nucleotide-free form of Ras may have limited biological utility.

[0009] Previous results with Sos helix mimetics highlight the significant challenges in developing minimal mimetics of protein secondary structure. Protein secondary structure mimetics have been proven to be a potent class of protein-protein interaction (PPI) inhibitors (YS Chang et al., "Stapled alpha-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy", Proc. Natl. Acad. Sci. USA 110, E3445-3454 (2013)(Non-Patent Document 12); S. Kushal et al., "Protein domain mimetics as in vivo modulators of hypoxia-inducible factor signaling", Proc. Natl. Acad. Sci. USA 110, 15602-15607 (2013)(Non-Patent Document 13); S. Liu, RW Cheloha, T. Watanabe, TJ Gardella, SH Gellman, "Receptor selectivity from minimal backbone modification of a polypeptide agonist", Proc. Natl. Acad. Sci. USA 110, 15602-15607 (2013)(Non-Patent Document 13). USA 115, 12383-12388 (2018)(Non-Patent Document 14); M. Pelay-Gimeno, A. Glas, O. Koch, TN Grossmann, "Structure-Based Design of Inhibitors of Protein-Protein Interactions: Mimicking Peptide Binding Epitopes", Angew. Chem. Int. Ed. 54, 8896-8927 (2015)(Non-Patent Document 15)), but many protein interfaces feature a complexity of binding epitopes that exceeds what can be obtained by simple secondary structure mimics (WS Horne, TNGrossmann, "Proteomimetics as protein-inspired scaffolds with defined tertiary folding patterns", Nat. Chem. 12, 331-337 (2020)(Non-Patent Document 16); H. Adihou et al., "A protein tertiary structure mimetic modulator of the Hippo signalling pathway", Nat. Commun. 11, 5425 (2020)(Non-Patent Document 17); J. Sadek et al., "Modulation of virus-induced NF-kappaB signaling by NEMO coiled coil mimics", Nat Commun 11, 1786 (2020)(Non-Patent Document 18); AM Watkins, MG Wuo, PS Arora, "Protein-Protein Interactions Mediated by Helical Tertiary Structure Motifs", J. Am. Chem. Soc. 137, 11622-11630 (2015) (Non-Patent Document 19)).

[0010] Thus, there is an unmet need to develop minimal mimetics of the Sos protein secondary structure as potential therapeutic targets. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] "Post-translational modifications and regulation of the RAS superfamily of GTPases as anticancer targets", Nat. Rev. Fr. Drug Discov. 6, 541–555 (2007)

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[0012] Various non-limiting aspects and embodiments of the present invention are described below.

[0013] In one aspect, the present invention provides a macrostructure comprising an antiparallel coiled-coil, wherein the antiparallel coiled coil comprises a first coil of formula I and a second coil of formula II: T1-g0-a1-b1-c1-d1-e1-f1-g1-a2-b2-c2-d2-e2-f2-g2-a3-b3-c3-d3-e3-f3-T2(I), T3-f'0-g'0-a'1-b'1-c'1-d'1-e'1-f'1-g'1-a'2-b'2-c'2-d'2-e'2-f'2-g'2-a'3-b'3-c'3-d'3-e'3-T4(II) Including, where: a 1-3 , b 1-3 , c 1-3 , d 1-3 , e 1-3 , f 1-3 , g 0-2 , a' 1-3 , b' 1-3 , c' 1-3 , d' 1-3 , e' 1-3 , f' 0-2 , and g' 0-2 are each, independently, absent or a residue selected from the group consisting of modified or unmodified amino acid residues and analogs thereof; one or more of the following residue pairs: g0-g'2, g1-g'1, g2-g'0, a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, d3-a'1, e1-e'3, e2-e'2, and e3-e'1 are covalently linked by a linker; T1 and T3 are each, independently, a point of attachment from the terminal nitrogen to one or more (preferably one or two) moieties, each of which is, independently, H, -PG1, -C(O)R, -C(O)NR2, -C(O)NH2, -R, -C(O)OR, an amino acid or analog thereof, a peptide, a targeting moiety, or a tag, PG1 is a protecting group for an amine, and each R is, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, peptide, targeting moiety, or tag; and T2 and T4 are each independently selected from the terminal carbonyl, H, -OPG2, -NPG2, -OR, -OH, -NR2, -NH2, -N(R)C(O)C 1-6 Alkyl, -N(H)C(O)C 1-6 PG2 is a protecting group for a carboxylic acid, and each R is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, peptide, targeting moiety, or tag; And here: The first coil comprises at least 10 consecutive residues, the at least 10 consecutive residues being of the formula X 1- X 2- X 3- X 4- X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14- X 15 -X 16 having; The second coil comprises at least 10 consecutive residues, said at least 10 consecutive residues being of the formula X'1-X'2-X'3-X'4-X'5-X'6-X'7-X'8-X'9-X' 10 -X' 11 -X' 12 -X' 13 -X' 14- X'15 -X' 16 having; and Each of the residues is selected from the group shown below (the superscripts indicate the position of the respective residue in Formula I and Formula II; residues at positions a, a', d, d', e, e', g, and g' can optionally be modified or replaced with a linker to facilitate attachment of the linker; the underlined residues are particularly preferred): TIFF2024501019000002.tif80158TIFF2024501019000003.tif251158TIFF2024501019000004.tif138158

[0014] In one embodiment, the length of any linker between residue pairs g0-g'2, g1-g'1, g2-g'0, e1-e'3, e2-e'2, and e3-e'1 is such that the spatial distance between the Cα positions of each residue in the pairs is 10 to 25 Å; and the length of any linker between residue pairs a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, and d3-a'1 is such that the spatial distance between the Cα positions of each residue in the pairs is 5 to 15 Å.

[0015] In one embodiment, at least g0, a1, b1, c1, d1, e1, f1, g1, a2, b2, c2, d2, e2, f2, g2, and a3 are present in a first coil, and at least d'1, e'1, f'1, g'1, a'2, b'2, c'2, d'2, e'2, f'2, g'2, a'3, b'3, c'3, d'3, and e'3 are present in a second coil.

[0016] In one embodiment, each residue independently has the formula: has TIFF2024501019000005.tif23128, where: R 1a , R 1b , R 1c , and R 1dare each independently hydrogen, an amino acid side chain, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or arylalkyl, and each of the amino acid side chain, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and arylalkyl is H, alkyl, alkenyl, alkynyl, azide, -OR 5 , or -SR 5 and when the linker is covalently attached to the residue, the linker is optionally substituted with R 1a , R 1b , R 1c , and R 1d is combined with or replaces one of the following: R 4 is each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or arylalkyl; and R 5 is each independently selected from the group consisting of H, -PG (where PG is a protecting group), alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and arylalkyl.

[0017] In one embodiment, there is a linker between at least one of the residue pairs g0-g'2, g1-g'1, g2-g'0, e1-e'3, e2-e'2, and e3-e'1.

[0018] In one embodiment, the linker between at least one of the residue pairs g0-g'2, g1-g'1, g2-g'0, e1-e'3, e2-e'2, and e3-e'1 is of the formula -Z nwhere n is a number from 1 to 25, and each Z is independently at each occurrence selected from the group consisting of alkylene, alkenylene, arylene, heteroarylene, triazole-diyl, thiazole-diyl, oxazole-diyl, ether, amide, ester, maleimide, thioether, O, S, and Se.

[0019] In one embodiment, the linker between at least one of the residue pairs g0-g'2, g1-g'1, g2-g'0, e1-e'3, e2-e'2, and e3-e'1 is (a) A group consisting of TIFF2024501019000006.tif48156, (b) Group consisting of TIFF2024501019000007.tif67128 having a formula selected from: where: X in group (a) and group (b) is O, S, CR2, NR, or P (preferably, O, S, CH2, or NR); X in group (a) 1 are each independently O, S, NH, or NR; X in group (b) 1 each is independently O, S, C, CR, N, NH, or NR; Each R in groups (a) and (b) is independently H, alkyl, or aryl; and Y in group (a) and group (b) is S.

[0020] In one embodiment, the linker between at least one of the residue pairs g0-g'2, g1-g'1, g2-g'0, e1-e'3, e2-e'2, and e3-e'1 is TIFF2024501019000008.tif98164.

[0021] In one embodiment, there is a linker between at least one of the residue pairs a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, and d3-a'1.

[0022] In one embodiment, the linker between at least one of the residue pairs a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, and d3-a'1 is a disulfide, diselenide, C 1-8 Alkylene, C 2-8 It is selected from the group consisting of alkenylene, arylene, heteroarylene, triazole-diyl, and thiazole-diyl.

[0023] In one embodiment, the linker between at least one of the residue pairs a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, and d3-a'1 is a disulfide bond derived from a cysteine ​​or homocysteine ​​residue, a diselenide derived from a selenocysteine ​​residue, an alkylene derived from an allylglycine residue, or an arylene linker.

[0024] In one embodiment, the antiparallel coiled coil has the following formula III: It is TIFF2024501019000009.tif52152, where: Each dotted line independently represents an optional linker; and Each residue independently has the formula: I have TIFF2024501019000010.tif23128, where: R 1a , R 1b , R 1c , and R 1dare each independently hydrogen, an amino acid side chain, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or arylalkyl, and each of the amino acid side chain, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and arylalkyl is H, alkyl, alkenyl, alkynyl, azide, -OR 5 , or -SR 5 and when the linker is covalently attached to the residue, the linker is optionally substituted with R 1a , R 1b , R 1c , and R 1d is combined with or replaces one of the following: R 4 is each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or arylalkyl; and R 5 is each independently selected from the group consisting of H, -PG (where PG is a protecting group), alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and arylalkyl.

[0025] In one embodiment, at least one of the following conditions is met: (A) in at least one a, a', d, or d' residue, (i) R 1a and R 1c is a side chain of a modified or unmodified amino acid selected from the group consisting of cysteine, homocysteine, selenocysteine, leucine, isoleucine, hexafluoroleucine, valine, hexafluorovaline, allylglycine, threonine, and analogs of each of the foregoing residues; and (ii) R 1b , R 1d , and R 1a and R 1c each of the other independently represents hydrogen, C 1-3 Alkyl, or C2-3 being alkenyl; (B) at least one e, e', g, or g' residue, 1a and R 1c is an amino acid side chain, and (ii) R 1b , R 1d , and R 1a and R 1c each of the other is independently hydrogen or C 1-3 Being alkyl.

[0026] In one embodiment, the antiparallel coiled coil has the following formula: TIFF2024501019000011.tif107130TIFF2024501019000012.tif21693, Here, each dotted line is independently an optional linker.

[0027] In one embodiment, the macrostructure is CHD-1, CHD-2, CHD-3, CHD-4, or CHD-5.

[0028] In another aspect, the present invention provides a pharmaceutical composition comprising the macrostructure of any one of the above embodiments and a pharma- ceutically acceptable vehicle.

[0029] In yet another aspect, the present invention provides a method of inhibiting Ras signaling in a cell, the method comprising the steps of: Contacting a cell with the macrostructure of any one of the preceding aspects under conditions effective to inhibit Ras signaling in the cell.

[0030] In one embodiment, the cell is a mammalian cell.

[0031] In one embodiment, the cells express a mutant Ras protein.

[0032] In one embodiment, the contacting step is performed in a subject.

[0033] In yet another aspect, the present invention provides a method for treating a disorder mediated by Ras signaling in a subject, the method comprising the steps of: administering to the subject a macrostructure of any one of the above-mentioned aspects, or a pharmaceutical formulation of the above-mentioned aspects, under conditions effective to treat the disorder in the subject.

[0034] In yet another aspect, the present invention provides a method of treating a cell proliferative abnormality, differentiation abnormality, and / or neoplastic condition in a subject in need thereof, the method comprising: administering to the subject a macrostructure of any one of the above-mentioned aspects, or a pharmaceutical formulation of the above-mentioned aspects, under conditions effective to treat the cell proliferative abnormality, differentiation abnormality, and / or neoplastic condition in the subject.

[0035] In one embodiment, the cell proliferative abnormality, differentiation abnormality, and / or neoplastic condition is selected from the group consisting of fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, brain cancer, squamous cell carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, and Kaposi's sarcoma; neoplastic abnormalities of the hematopoietic system; cellular proliferative and / or differentiating abnormalities of the breast; cellular proliferative and / or differentiating abnormalities of the lung; cellular proliferative and / or differentiating abnormalities of the colon; cellular proliferative and / or differentiating abnormalities of the liver; cellular proliferative and / or differentiating abnormalities of the ovary; cancers mediated by mutant Ras proteins; and immunoproliferative abnormalities.

[0036] In one embodiment, the disorder and / or condition is pancreatic ductal adenocarcinoma, rectal adenocarcinoma, plasma cell myeloma, colon adenocarcinoma, cholangiocarcinoma, chronic myelomonocytic leukemia, acute myeloid leukemia, melanoma, lung adenocarcinoma, rhabdomyosarcoma, endometrial cancer, salivary gland cancer, thyroid cancer, bladder cancer, oral cancer, or ovarian cancer.

[0037] In one embodiment, the subject is a mammal, hi one embodiment, the subject is a primate (e.g., a human).

[0038] In one embodiment, the abnormality or pathology is mediated by mutant Ras protein. In one embodiment, the mutant Ras protein is H-Ras isoform. In one embodiment, the mutant Ras protein is K-Ras isoform. In one embodiment, the mutant Ras protein has one or more mutations selected from the group consisting of G12C, G12D, G12S, G12V, G13D, G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13R, G13S, G13D, Q61E, Q61H, Q61L, Q61K, Q61P and Q61R.

[0039] These and other aspects of the present invention will become apparent to those of ordinary skill in the art after reading the following detailed description of the invention, including the appended claims. [Brief description of the drawings]

[0040] [Figure 1]Figure 1 provides a schematic of the Ras activation cycle and a blueprint for Sos-based proteomimetics. The intracellular activity of Ras is tightly controlled as part of a balanced feedback loop. Oncogenic mutations upset this equilibrium and increase the intracellular concentration of Ras-GTP, triggering downstream aberrant signaling. Figure 1A shows a molecular model depicting Ras (gray ribbon) in complex with its guanine exchange factor, Sos. Sos mediates nucleotide exchange by inserting a helical hairpin (helix) into the nucleotide-binding pocket of Ras. The nucleotide-binding pocket of Ras is highlighted. To highlight the interaction of the helical hairpin with Ras, a segment of Sos is not shown (PDB code 1NVW). Figure 2B shows a molecular model depicting the key Sos helices and the design of constrained Sos proteomimetics as Ras inhibitors. GDP = guanosine 5'-diphosphate; GTP = guanosine 5'-triphosphate; GAP = GTPase-activating protein; GEF = guanine nucleotide exchange factor. [Diagram 2] Figure 2 shows the rational design of Sos proteomimetics as Ras ligands. Antiparallel helical wheel diagrams depicting the native Sos helical hairpin (top) and the optimized constrained helix dimer mimic (bottom). Figure 2A shows the αH and αI helical domains of Sos that make direct contact with Sos, and many of the energetically important residues that contact Ras, called hotspot residues, are located on the αH helix. Figure 2B shows the designed and synthesized constrained Sos mimic, which has hydrophobic interfaces and non-native residues in both helices to enhance the binding interaction with Ras. To enhance the conformational stability, a dibenzyl ether crosslinker is placed at the "e" position of each helix. The binding affinity of the Sos derivatives to Ras was measured by fluorescence polarization (FP) assay; the binding affinity of the lead derivative, CHDSos-5, and the alanine control, CHDSos-3, was further confirmed by microscale thermophoresis (MST). RH = L-homoarginine. [Figure 3A] FIG. 3 shows the biophysical characterization of CHDSos-5, its stability against proteolysis, and analysis of its Ras binding site. FIG. 3A is a circular dichroism spectrum of CHDSos-5. CD studies were performed at a peptide concentration of 20 μM in 50 mM potassium fluoride aqueous buffer (pH 7.5). FIG. 3B shows the stability of CHDSos-5 against proteolysis in 25% fetal bovine serum analyzed in an HPLC assay discussed in Example 23. Error bars are the mean ± SD of biological replicates. FIG. 3C shows a 1H-15N HSQC titration spectrum of wild-type H-Ras uniformly labeled with 15N and loaded with GDP. Examples of specific Ras residues that shift when titrated with increasing equivalents (1, 2.5, and 5) of CHDSos-5. Figure 3D is a bar graph showing the average chemical shift changes observed for 15N-labeled H-Ras when titrated with increasing amounts of CHDSos-5. Figure 3E shows the binding site for CHDSos-5 on Ras, which was further confirmed by proximity-induced cross-linking. DZ1-CHDSos-5 contains a photoactivatable diazirine group that reacts with a nearby residue on Ras. A fragment with a mass corresponding to DZ1-CHDSos-5 cross-linked to the switch I loop of Ras was identified. The identified fragment corresponds to the switch I region and is shown as a ribbon in the molecular model. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 4A]Figure 4 shows the cellular internalization and efficacy of Sos proteomimetics altered by oncogenic Ras mutations. Figure 4A shows live cell fluorescence imaging at 40X magnification (scale bar = 5 μm) of T24 and H358 cells carrying Ras mutants incubated with fluorescently labeled CHDSos-5 or DMSO for 4 hours and Hoechst stained. Figure 4B shows flow cytometry analysis of fluorescently labeled CHDSos-5 (1 μM) in T24, H358, SW780, BxPC3, and HeLa cells after 1 hour of treatment. Figure 4C shows fluorescence polarization and microscale thermophoresis analysis performed to determine the binding affinity of CHDSos-5 to GDP-loaded wild-type and mutant isoforms of H-Ras. Figure 4D shows the cytotoxicity of CHDSos-5 analyzed in MTT cell viability assay. The bar graph shows the viability of Ras wild-type and mutant cell lines treated with increasing concentrations of CHDSos-5. The results of the MTT assay were also supported by the CellTiter-Glo luminescent cell viability assay (Example 26). Figure 4E is a graph with two y-axes showing the correlation between cellular uptake and toxicity of CHDSos-5. The results of the cellular uptake test using 1 μM of the fluorescent analog (left axis) and the results of the MTT cell viability assay at a concentration of 10 μM (right axis) are shown. Figure 4F is a representative Western blot showing the phosphorylation level of Erk in H358 cells treated with 0 μM, 1 μM, 5 μM, and 10 μM of CHDSos-5. Figure 4G is a Western blot showing the phosphorylation level of ERK in HeLa and H358 cells treated with CHDSos-5 or the negative control CHDSos-3. Figure 4A shows a bar graph comparing ERK phosphorylation in HeLa and H358 cells after treatment with CHDSos-3 and CHDSos-5. Error bars are the mean ± SD of biological duplicates. [Figure 4B] See legend to Figure 4A. [Figure 4C]See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 4G] See legend to Figure 4A. [Figure 4H] See legend to Figure 4A. [Figure 5A] Figure 5 shows the analysis of intracellular interaction partners of the Sos proteomimetic by MS-based quantitative proteomics. Figure 5A is a schematic showing proximity-driven photocrosslinking of DZ2-CHDSos-5 with cellular proteins. [Figure 5B] Figure 5 shows the analysis of intracellular interaction partners of the Sos proteomimetic by MS-based quantitative proteomics. Figure 5B is a volcano plot revealing statistically significant (p < 0.01) enriched proteins in H358 cells upon treatment with DZ2-CHDSos-5 (10 μM) for 4 hours. Enriched protein targets that are members of the Ras GTPase superfamily are named. [Figure 5C] Figure 5 shows the analysis of intracellular interaction partners of Sos proteomimetics by MS-based quantitative proteomics. Figure 5C shows pie charts summarizing the functional diversity (left) and cellular localization (right) of enriched protein targets. [Figure 6] Figure 6 shows alanine scanning mutagenesis of the complex between Ras (light grey) and Sos (dark grey), revealing key Ras binding residues (globules). While the energetically important binding residues are dispersed throughout the broad Sos surface, the αH helix (dark) contains a cluster of those residues, thus providing a lead helical domain for the rational design of Ras ligands. [Figure 7]Figure 7 shows the fluorescence polarization curves for the binding of fluorescently labeled CHDs to wild-type H-Ras bound to GDP. The calculated dissociation constants (Kd) for each CHD are listed in the table. Error bars are the mean ± SD of triplicates. [Figure 8-1] FIG. 8 shows microscale thermophoresis analysis of binding of wild-type and mutant H-Ras labeled with AFDye 647 to CHD. FIG. 8A shows dose-response curves for the interaction between CHDSos-5 and CHDSos-3 and wild-type H-Ras-GDP. FIG. 8B shows dose-response curves for the interaction between CHDSos-5 and CHDSos-3 and G12V H-Ras-GDP. The change in TRIC signal observed from microscale thermophoresis is plotted as Fnorm versus ligand concentration. FIG. 8C shows thermographs of wild-type H-Ras-GDP binding to CHDSos-5 and FIG. 8D shows thermographs of binding to CHDSos-3. Aggregation was observed with CHDSos-3 at concentrations higher than 3 μM, and this caused a disturbance in the binding isotherm. Thermographs of G12V H-Ras-GDP binding to CHDSos-5 (Figure 8E) and to CHDSos-3 (Figure 8F). The low temperature zone is set at 0 seconds and the high temperature zone at 3 seconds is used. Error bars are the mean ± SD from triplicates. [Figure 8-2] See description of Figure 8-1. [Figure 9] Figure 9 shows circular dichroism spectra of crosslinked and linear peptides. CD spectra of individual unlinked αH and αI peptides, an equimolar mixture of αH and αI peptides, and CHDSos-2. CD experiments were performed in 50 mM potassium fluoride aqueous buffer (pH 7.5) with a peptide concentration of 20 μM. [Figure 10] Figure 10 shows titration HSQC NMR spectroscopy revealing the binding site of CHDSos-5 / Ras. 1H-15N HSQC overlay spectra of H-Ras alone, H-Ras:CHDSos-5 (1:2.5), and H-Ras:CHDSos-5 (1:5). [Figure 11] Figure 11 shows that chemical cross-linking of diazirine-modified CHDSos-5 establishes the binding site of Ras. Figure 11A shows a gel shift assay with H-Ras incubated with and without DZ1-CHDSos-5. Figure 11B shows a MALDI-TOF spectrum of trypsin-digested DZ1-CHDSos-5 cross-linked to the switch I loop of Ras, depicting the masses of the identified fragments. No corresponding labeled masses were observed in the unlabeled Ras sample. [Figure 12] Figure 12 shows fluorescence microscopy and flow cytometry studies to analyze the cellular uptake of CHD. Figure 12A shows live cell fluorescence imaging at 40X magnification (scale bar = 5 μm) of Hoechst-stained cells of Ras mutant-bearing T24, H358, and BxPC3 cells incubated with fluorescently labeled peptide for 4 hours and DMSO (negative control) for 4 hours. Cells were exposed to 500 nM CHD and Tat. Figure 12B shows the median fluorescence intensity in T24 and H358 cells after treatment with 1 μM CHDSos-2, CHDSos-3, and CHDSos-5, and Tat for 1 hour. Figures 12C and 12D show flow cytometry analysis of fluorescently labeled CHDSos-5 (1 μM) after 1 hour treatment in the indicated cell lines. Error bars are the mean ± SD of biological duplicates. [Figure 13]Figure 13 shows MTT and CellTiter-Glo assays to determine cell viability in the presence of CHDs. Cell viability by MTT method of the indicated Ras mutant cell lines and control cell lines treated with increasing concentrations of CHDSos-2 (Figure 13A) and CHDSos-3 (Figure 13B). Cell viability tests were performed in the presence of serum. Figure 13C shows cell viability assessed by CellTiter-Glo assay of H358 and HeLa cells treated with increasing concentrations of CHDSos-5. Error bars are the mean ± SD of biological triplicates. [Figure 14] Representative Western blots to investigate the effect of CHDSos-5 on the levels of active / GTP-bound Ras are shown in Figure 14. The levels of Ras-GTP were determined by immunoblotting with a Ras-GTP-specific antibody after treating H358 cells with 10 μM CHDSos-5 for 6 h. [Figure 15] Figure 15 shows a representative Western blot for analyzing ERK phosphorylation. CHD-induced inhibition of ERK phosphorylation was examined using the probe in H358 and HeLa cells after treatment with (A) CHDSos-5 and (B) CHDSos-3 (DMSO, 1, 5, 10 μM). Error bars are the mean ± SD of biological duplicates. [Figure 16] FIG. 16 shows analytical HPLC traces for purified linear and CHD peptides. [Figure 17-1] FIG. 17 shows the chemical structure of the Sos CHD. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 17-3] This is a continuation of Figure 17-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description of the Invention The following detailed description is provided to enable those skilled in the art to make and use the technology of the present invention. For the purpose of explanation, technical terms are described in order to provide a complete understanding of the present invention. However, it will be apparent to those skilled in the art that these details are not required to practice the technology of the present invention. Descriptions of specific applications are provided only as representative examples. The technology of the present invention is not intended to be limited to the embodiments shown, but is to be accorded the broadest possible scope consistent with the principles and features disclosed herein. Preferences and options with respect to any given aspect, feature, or parameter of the present invention should be considered as disclosing any and all preferences and options with respect to all other aspects, features, and parameters of the present invention, unless the context indicates otherwise.

[0042] The present invention provides synthetic Sos protein mimetics that associate with wild-type and oncogenic forms of Ras bound to nucleotides and change downstream kinase signaling. The Sos mimetics are designed to acquire the conformation of the helix-loop-helix motif of Sos, which makes important contact with Ras in the switch region of Ras. Chemoproteomic studies show that the proteomimetics associate with Ras and other intracellular GTPases. In some embodiments, the synthetic proteomimetics are resistant to proteolytic degradation and enter cells by macropinocytosis. Therefore, it has selective toxicity to cancer cells in which macropinocytosis is upregulated, including cancer cells characterized by oncogenic Ras mutations.

[0043] The present invention provides the design of a structure-defined proteomimetic, which reproduces the important binding surface of Sos, which is a well-characterized effector of Ras.The proteomimetic binds to wild-type Ras and its various mutant forms, and downregulates Ras signaling.Importantly and surprisingly, the compound shows enhanced migration into the interior of cancer cells in which macropinocytosis is upregulated.

[0044] In the present invention, we hypothesize that the introduction of additional contact residues from Sos may allow for association with nucleotide-bound Ras (see FIG. 1B). Sos inserts αH into the switch region of Ras, but analysis of the complex indicates that several other residues from Sos also interact with Ras (FIG. 6). The conformation of the αH helix itself is controlled by the αI domain as part of the assembly of the hairpin helix. The αI helix makes important electrostatic contacts with the effector loop of Ras in the switch I region.

[0045] The present invention develops a tertiary structure mimic of Sos that encompasses key binding residues from the helix-loop-helix motif to determine whether increased contacts would allow association with nucleotide-bound Ras (see FIG. 1B). A recently described synthetic approach was utilized to obtain the conformation of the Sos αH and αI helices. In previous efforts, it was observed that helical dimers could be stabilized by judicious replacement of surface salt bridges with covalent bonds and by appropriately altering the surface of the dimer to adopt knob-into-hole helical packing (MG Wuo, SH Hong, A. Singh, PS Arora, "Synthetic Control of Tertiary Helical Structures in Short Peptides", J. Am. Chem. Soc. 140, 16284-16290 (2018); MG Wuo, AB Mahon, PS Arora, "An Effective Strategy for Stabilizing Minimal Coiled Coil Mimetics", J. Am. Chem. Soc. 137, 11618-11621 (2015)). These stabilized proteomimetics are called cross-linked helical dimers, or CHDs. The present invention shows that the Sos CHD is stable to proteolysis and selectively cell membrane permeable, and demonstrates that the Sos CHD associates with high specificity with the Sos-binding surface of nucleotide-bound Ras in biochemical and intracellular environments.The present invention utilizes a combination of rational design principles and computational modeling to exploit previously unexplored and underutilized pockets on target surfaces (F. Lauck, CA Smith, GF Friedland, EL Humphris, T. Kortemme, "RosettaBackrub--a web server for flexible backbone protein structure modeling and design", Nucleic Acids Res. 38, W569-575 (2010); D. Rooklin et al., "Targeting Unoccupied Surfaces on Protein-Protein Interfaces", J. Am. Chem. Soc. 139, 15560-15563 (2017)). The optimized proteomimetics bind to wild-type and mutant Ras with nanomolar to low micromolar affinity, alter nucleotide exchange, associate with Ras and other GTPases of the Ras subfamily as demonstrated by chemoproteomic assays, inhibit downstream activation of Ras-mediated signaling cascades, and have selective toxicity against cancer cells harboring oncogenic Ras mutations.

[0046] Ras remains a challenging target for conventional drug discovery. Numerous strategies for developing lead compounds have been described (R. Spencer-Smith et al., "Inhibition of RAS function through targeting an allosteric regulatory site", Nat. Chem. Biol. 13, 62-68 (2017); ME Welsch et al., "Multivalent Small-Molecule Pan-RAS Inhibitors", Cell 168, 878-889.e829 (2017); T. Maurer et al., "Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity", Proc. Natl. Acad. Sci. USA 109, 5299-5304 (2012); Q. Sun et al., "Discovery of Small Molecules that Bind to K-Ras and Inhibit Sos-Mediated Activation", Angew. Chem. Int. Ed. 51, 6140-6143 (2012); JM Ostrem, KM Shokat, "Direct small-molecule inhibitors of KRAS: from structural insights to mechanism-based design", Nat. Rev. Drug Discov. 15, 771-785 (2016); J. Downward, "Targeting RAS signaling pathways in cancer therapy", Nat. Rev. Cancer 3, 11-22 (2003); AG Stephen, D. Esposito, RK Bagni, F.McCormick, "Dragging ras back in the ring", Cancer Cell 25, 272-281 (2014)), but with the exception of the covalent G12C inhibitor, these leads never progressed to clinical trials (BA Lanman et al., "Discovery of a Covalent Inhibitor of KRAS(G12C) (AMG 510) for the Treatment of Solid Tumors", J. Med. Chem. 63, 52-65 (2020)). Compounds targeting Ras should ideally be able to associate with specific mutant isoforms, because pan-Ras inhibitors are expected to cause unwanted side effects.

[0047] The present invention provides a Sos proteomimetic that binds to the nucleotide-binding pocket of wild-type and mutant Ras, and has selective toxicity against oncogenic Ras cells because the translocation of the proteomimetic into the cell interior is controlled by macropinocytosis and because macropinocytosis is upregulated in mutant Ras cells.

[0048] The synthetic derivatives mimic the Sos helical hairpin that mediates nucleotide exchange and activation of Ras. The design and synthesis of the proteomimetics are based on a strategy to develop minimal mimics of the helical tertiary structure with defined conformation. The strategy results in cross-linked helix dimers (CHDs), which require optimal knob-into-hole helical packing and appropriately positioned covalent cross-linkers for their conformational stability. Computational modeling and rational design principles were utilized to incorporate non-canonical residues to enhance the binding interaction of the Sos derivatives with Ras. Several iterations of design and sequencing were required to develop lead CHDs with the requisite solubility in water and low micromolar binding affinity for wild-type and mutant Ras isoforms. Titration 1 H- 15 N-HSQC NMR and photoaffinity labeling studies confirmed the presence of the lead derivative, CHD Sos -5 has been defined, which associates with Ras at the nucleotide-binding surface of Ras.

[0049] C.H.D. Sos -5 was demonstrated to be cell membrane permeable and showed similar uptake as Tat peptide, a well-characterized cell membrane permeable peptide. Importantly, CHD Sos-5 showed greater intracellular uptake in Ras mutant cells compared to cells harboring only the wild-type variant. This finding of superior internalization in mutant Ras cells is consistent with the recent characterization of the uptake pathway of peptidomimetics - macropinocytosis was recently discovered to be an important mechanism utilized by medium-sized peptidic compounds. Even though the binding affinity of Sos proteomimetics is slightly better to wild-type Ras in biochemical experiments, the upregulation of macropinocytosis in cell lines harboring mutations in Ras acts as a selectivity filter for the specific association of Sos proteomimetics with oncogenic Ras.

[0050] Altering the phosphorylation of ERK, a well-studied downstream kinase affected by Ras activation, CHD Sos -5 potency was assayed. Sos -5 is IC 50 It was demonstrated that CHD1 is a potent ERK inhibitor with a potency of <1 μM in cell lines expressing wild-type Ras, but not in cell lines expressing wild-type Ras. Importantly, the alanine control, CHD1 Sos -3 did not alter ERK phosphorylation at the concentrations tested, suggesting that the efficacy of the lead proteomimetic is sequence-specific. Finally, photoaffinity labeling combined with MS-based quantitative proteomics was utilized to identify CHD in oncogenic Ras cell lines. Sos Interactors with -5 have been investigated, and K-Ras, as well as other members of the Ras family, have been identified. Sos The fact that -5 associates with proteins other than Ras may reflect unknown natural partners of Sos when it is not recruited to the membrane.

[0051] Although there have been many successful proteomic studies utilizing small molecules and peptides to investigate protein-protein interactions (RE Kleiner, LE Hang, KR Molloy, BT Chait, TM Kapoor, "A Chemical Proteomics Approach to Reveal Direct Protein-Protein Interactions in Living Cells", Cell Chemical Biology 25, 110-120.e113 (2018); DP Murale, SC Hong, MM Haque, J.-S. Lee, "Photo-affinity labeling (PAL) in chemical proteomics: a handy tool to investigate protein-protein interactions (PPIs)", Proteome Science 15, 14 (2017)), the use of protein tertiary mimetics to investigate binding partners of therapeutically important protein domains has not been described previously. The biological relevance of targeting a previously unknown binding partner of the Sos helical hairpin remains unclear. Briefly, the present invention provides a pan-Ras ligand that has selective toxicity against cells expressing mutant Ras isoforms in which macropinocytosis as a nutrient uptake pathway is upregulated. The selective uptake of Sos proteomimetics suggests a therapeutic strategy for targeting oncogenic Ras without the need for ligands that specifically associate with various Ras mutations.

[0052] One aspect of the present invention relates to a macrostructure comprising an antiparallel coiled coil, wherein the antiparallel coiled coil comprises a first coil of formula I and a second coil of formula II: T1-g0-a1-b1-c1-d1-e1-f1-g1-a2-b2-c2-d2-e2-f2-g2-a3-b3-c3-d3-e3-f3-T2(I), T3-f'0-g'0-a'1-b'1-c'1-d'1-e'1-f'1-g'1-a'2-b'2-c'2-d'2-e'2-f'2-g'2-a'3-b'3-c'3-d'3-e'3-T4(II) Including, where: a 1-3 , b 1-3 , c 1-3 , d 1-3 , e 1-3 , f 1-3 , g 0-2 , a' 1-3 , b' 1-3 , c' 1-3 , d' 1-3 , e' 1-3 , f' 0-2 , and g' 0-2 are each, independently, absent or a residue selected from the group consisting of modified or unmodified amino acid residues and analogs thereof; one or more of the following residue pairs: g0-g'2, g1-g'1, g2-g'0, a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, d3-a'1, e1-e'3, e2-e'2, and e3-e'1 are covalently linked by a linker; T1 and T3 are each, independently, a point of attachment from the terminal nitrogen to one or more (preferably one or two) moieties, each of which is, independently, H, -PG1, -C(O)R, -C(O)NR2, -C(O)NH2, -R, -C(O)OR, an amino acid or analog thereof, a peptide, a targeting moiety, or a tag, PG1 is a protecting group for an amine, and each R is, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, peptide, targeting moiety, or tag; and T2 and T4 are each independently selected from the terminal carbonyl, H, -OPG2, -NPG2, -OR, -OH, -NR2, -NH2, -N(R)C(O)C 1-6 Alkyl, -N(H)C(O)C 1-6 PG2 is a protecting group for a carboxylic acid, and each R is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, peptide, targeting moiety, or tag; And here: The first coil comprises at least 10 consecutive residues, the at least 10 consecutive residues being of the formula X 1- X 2- X 3- X 4- X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14- X 15 -X 16 having; The second coil comprises at least 10 consecutive residues, said at least 10 consecutive residues being of the formula X'1-X'2-X'3-X'4-X'5-X'6-X'7-X'8-X'9-X' 10 -X' 11 -X' 12 -X' 13 -X' 14- X' 15 -X' 16 having; and Each of the residues is selected from the group shown below (the superscripts indicate the position of the respective residue in Formula I and Formula II; residues at positions a, a', d, d', e, e', g, and g' can optionally be modified or replaced with a linker to facilitate attachment of the linker; the underlined residues are particularly preferred): TIFF2024501019000013.tif75158TIFF2024501019000014.tif202158TIFF2024501019000015.tif192158

[0053] An antiparallel coiled-coil structure has a first amino acid strand (or first coil) and a second amino acid strand (or second coil). As will be immediately apparent to those skilled in the art, the following designations are commonly used to characterize coiled-coil structures and are used throughout this application: the designation "A / B" or " x A y / x' B y' " is used to identify (either specifically or generically) the sequence of each strand, where A is the sequence of the first strand (X1-X2-X3...) and B is the sequence of the second strand (X1'-X2'-X3'...), x, x', y, and y' designate the start (x, x') and end (y, y') positions of the corresponding sequences in each strand relative to the heptad, and " / " distinguishes one sequence from the other. Usually, both sequences A and B are written from left to right, N to C. However, it will be immediately clear to the skilled artisan that, for example, in a plan view perpendicular to the axis of the antiparallel coiled-coil, the strands in the antiparallel coiled-coil structure are spatially aligned opposite each other, with the N-terminus of the first strand at the top and the C-terminus of the second strand at the top. As will be readily apparent to those skilled in the art, in the compounds of the present invention, at least one covalent linker is also present between the residues in the first chain and the residues in the second chain. The position and structure of the linker is sometimes specified in Sequence A and Sequence B by using "Z" and "Z'" instead of X and X', respectively. Alternatively, the position and structure of the linker is specified by further description (e.g., "residue X n and residue X n 'There is a disulfide linker between', etc.

[0054] In at least one embodiment, the number of residues in the first coil and second coil is between 10 and 30 (e.g., within a range having a lower value of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 and an upper value of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, in any combination).

[0055] As will be immediately apparent to one of skill in the art, the helical wheel view herein shows the spatial location of each coil in the antiparallel coiled-coil structure, while the two-dimensional view shows the connections between residues.

[0056] As used above and throughout the present description, the following terms shall be understood to have the following meanings unless otherwise specified. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the event that there are multiple definitions for a term herein, the definition in this section shall prevail unless otherwise specified.

[0057] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon group which may be straight-chained or branched and has about 1 to about 8 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8) carbon atoms in the chain. Branched means that one or more lower alkyl groups, such as methyl, ethyl, or propyl, are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and 3-pentyl.

[0058] The term "alkenyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond, and which may be straight-chained or branched, having about 2 to about 8 (e.g., 2-3, 2-4, 2-5, 2-6, 2-7, 2-8) carbon atoms in the chain. Preferred alkenyl groups have 2 to about 4 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, and i-butenyl.

[0059] The term "alkynyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond, and which may be straight or branched, having about 2 to about 8 (e.g., 2-3, 2-4, 2-5, 2-6, 2-7, 2-8) carbon atoms in the chain. Preferred alkynyl groups have 2 to about 4 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, and n-pentynyl.

[0060] The term "amide isostere" refers to an analog that preserves the hydrogen bonding properties of amide but does not have its metabolic susceptibility. The side chains of asparagine and glutamine residues can be modified into 1,2,3-triazole, 1,2,4-triazole, oxazole, oxadiazole, imidazole, thiazole, thiadiazole, tetrazole, pyrazole, indole, pyrrole, pyridine, pyrazine, diketopiperazine, urea, thiourea, carbamate, and sulfonamide. Suitable amide isosteres include those described in Kumari et al., "Amide Bond Bioisosteres: Strategies, Synthesis, and Successes", J. Med. Chem. 63: 12290-358 (2020), which is incorporated herein by reference in its entirety.

[0061] The term "carboxylic acid isostere" refers to phosphoric acid, sulfonic acid, sulfonamide, sulfonylurea, hydroxamic acid, acylurea, tetrazole, thiazolidinedione, phenol, squaric acid, thiozolidinedione, oxazolidinedione, oxadiazol-5(4H)-one, thiadiazol-5(4H)-one, oxadiazol-5(4H)-thione, oxathiadiazol-2-oxide, isoxazole, tetramic acid, and cyclopentane 1,3-dione.Suitable carboxylic acid derivatives include those described in Lassalas et al., "Structure Property Relationships of Carboxylic Acid Isosteres", J. Med. Chem. 59:3183-203 (2016), which is incorporated herein by reference in its entirety.

[0062] As used herein, the term "cycloalkyl" refers to a non-aromatic, saturated or unsaturated, monocyclic or polycyclic ring system that may contain 3-8 (3, 4, 5, 6, 7, 8, 3-4, 3-5, 3-6, 3-7, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, 7-8) carbon atoms and may contain at least one double bond. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, anti-bicyclopropane, or syn-bicyclopropane.

[0063] As used herein, the term “alkane” refers to an alkane having the formula C, which may be linear or branched. n H 2n+2and having from about 1 to about 8 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8) carbon atoms in the chain. Branched means that one or more lower alkyl groups, such as methyl, ethyl, or propyl, are attached to a linear alkyl chain. Exemplary alkanes include methane, ethane, n-propane, i-propane, n-butane, t-butane, n-pentane, and 3-pentane. The term "alkylene" refers to a divalent group formed by removing two hydrogen atoms from an alkane. Exemplary alkylene groups include, but are not limited to, divalent groups derived from the alkanes listed above.

[0064] As used herein, the term “alkene” refers to an alkene having the formula C, which may be linear or branched. n H 2n and having from about 2 to about 8 (e.g., 2-3, 2-4, 2-5, 2-6, 2-7, 2-8) carbon atoms in the chain. Exemplary alkenes include ethylene, propylene, n-butylene, and i-butylene. The term "alkenylene" refers to a divalent group formed by removing two hydrogen atoms from an alkene. Alkenylene contains a carbon-carbon double bond and has the formula -(C n H 2n-2 Exemplary alkenylene groups include, but are not limited to, divalent groups derived from the alkenes mentioned above.

[0065] As used herein, the term “alkyne” refers to an alkyne having the formula C, which may be linear or branched. n H 2n-2and having from about 2 to about 8 (e.g., 2-3, 2-4, 2-5, 2-6, 2-7, 2-8) carbon atoms in the chain. Exemplary alkynes include acetylene, propyne, butyne, and pentyne. The term "alkynylene" refers to a divalent group formed by removing two hydrogen atoms from an alkyne. Alkynylene contains a carbon-carbon triple bond and has the formula -(C n H 2n-4 Exemplary alkynylene groups include, but are not limited to, divalent groups derived from the alkynes described above.

[0066] The aromatic and heteroaromatic rings can be any single ring, multiple ring, or fused ring structure. For example, aromatic and heteroaromatic rings include: 5- or 6-membered aromatic rings, or 5- or 6-membered heteroaromatic rings containing 0-3 (0, 1, 2, or 3) heteroatoms selected from O, N, and S; bicyclic 9- or 10-membered aromatic ring systems, or 0-3 (0, 1, 2, or 3) heteroatoms selected from O, N, and S; or tricyclic 13- or 14-membered aromatic ring systems, or 0-3 (0, 1, 2, or 3) heteroatoms selected from O, N, and S. Aromatic 5- to 14-membered carbocyclic rings (5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered carbocyclic rings) include, for example, cyclopenta-1,3-diene, benzene, naphthalene, indane, tetralin, and anthracene. 5-membered aromatic heterocycles to 10-membered aromatic heterocycles (5-membered aromatic heterocycles, 6-membered aromatic heterocycles, 7-membered aromatic heterocycles, 8-membered aromatic heterocycles, 9-membered aromatic heterocycles, or 10-membered aromatic heterocycles) include, for example, imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole, pyrazole, benzimidazole, pyridazine, pyrrole, imidazole, oxazole, isoxazole, indazole, isoindole, imidazole, purine, triazine, quinazoline, cinnoline, benzoxazole, acridine, benzisoxazole, and benzothiazole. The term "arylene" refers to a divalent group derived from an aromatic ring by removing one hydrogen atom from each of the two ring carbon atoms. Exemplary arylene groups include, but are not limited to, the divalent groups derived from the aromatic rings described above. The term "heteroarylene" refers to a divalent group derived from a heteroaromatic ring. Exemplary heteroarylene groups include, but are not limited to, the divalent groups derived from the heteroaromatic rings described above.

[0067] The term "ether" refers to a group having the formula -ROR-, where each R is independently a bond, 1-8 Alkylene, C 2-8 The ether may be selected from the group consisting of alkenylene, arylene, and heteroarylene. Exemplary ethers include -C 1-8 Alkylene-OC 1-8 Alkylene- (e.g., -(CH2)2-O-(CH2)2-), -C 2-8 Alkenylene-OC 2-8 Alkenylene-, -arylene-O-arylene-, -heteroarylene-O-heteroarylene-, and -C 1-8 Includes, but is not limited to, alkylene-O-heteroarylene-.

[0068] The term "thioether" refers to a group having the formula -RSR-, where each R is independently a bond, 1-8 Alkylene, C 2-8 The aryl group may be selected from the group consisting of alkenylene, arylene, and heteroarylene. Exemplary thioethers include -C 1-8 Alkylene-SC 1-8 Alkylene- (e.g., -(CH2)2-S-(CH2)2-), -C 2-8 Alkenylene-SC 2-8 Alkenylene-, -arylene-S-arylene-, -heteroarylene-S-heteroarylene-, and -C 1-8 Includes, but is not limited to, alkylene-S-heteroarylene-.

[0069] The term "amide" refers to a compound of the formula -C(O)N(R 1 )(R 1 ) or the formula -C(O)N(R 1 Amide refers to a group having, for example, —C(O)N(R 1 )R-, -RC(O)N(R 1 )R-, -CHR 1 -C(O)N(R 1 )R-, and -C(R 1 )(R 1)-C(O)N(R 1 )R-, where each R is independently a bond, C 1-8 Alkylene, C 2-8 may be selected from the group consisting of alkenylene, arylene, and heteroarylene, and R 1 are each independently hydrogen, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, C 3-8 Exemplary amides include -C 1-8 Alkylene-C(O)N(aryl)-, -C 2-8 Alkenylene-C(O)N(aryl)-, and -C 1-8 Alkylene-C(O)N(C 1-8 alkyl)- (e.g., -(CH2)2-C(O)N(CH3)-).

[0070] The term "ester" refers to a group having the formula -C(O)O-. Esters include, for example, -RC(O)OR-, -CHR 1 -C(O)OR- and -C(R 1 )(R 1 Each R is independently a bond, C 1-8 Alkylene, C 2-8 may be selected from the group consisting of alkenylene, arylene, and heteroarylene, and R 1 are each independently hydrogen, C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, C 3-8 Exemplary esters include -C are selected from the group consisting of cycloalkyl, aryl, heteroaryl, heterocyclyl, and arylalkyl. 1-8 Alkylene-C(O)O-arylene-, -C 2-8 Alkenylene-C(O)O-arylene-, -C 1-8 Alkylene-C(O)O-heteroarylene-, -C 1-8Alkylene-C(O)OC 1-8 Alkylene- (e.g., -(CH2)2-C(O)O-(CH2)2-), and -C 1-8 Includes, but is not limited to, alkylene -C(O)O- (e.g., -(CH2)2-C(O)O-).

[0071] As used herein, the term "heterocyclyl" refers to a stable 3- to 18-membered ring system (a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, or 18-membered ring system) consisting of carbon atoms and 1 to 5 (1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 2-3, 2-4, 2-5, 3-4, 3-5, 4-5) heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Heterocyclyls may be monocyclic or polycyclic ring systems, which may include fused, bridged, or spiro ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocyclyl may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the ring may be partially or fully saturated. Representative monocyclic heterocyclyls include piperidine, piperazine, pyrimidine, morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, pyran, tetrahydropyran, oxetane, and the like. Representative polycyclic heterocyclyls include indole, isoindole, indolizine, quinoline, isoquinoline, purine, carbazole, dibenzofuran, chromene, xanthene, and the like.

[0072] As used herein, the term "aryl" refers to any group having 6 to 19 amino acids (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 6 to 11, 6 to 12, 6 to 13, 6 to 14, 6 to 15, 6 to 16, 1 to 17, 6 to 18, 7 to 8, 7 to 9, 7 to 10, 7 to 11, 7 to 12, 7 to 1 3, 7-14, 7-15, 7-16, 7-18, 7-19, 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 10-11, 10-12, 10-13, 10-14, 10~15, 10~16, 10~17, 10~18, 10~19, 11~12, 11~13, 11~14, 11~15, 11~16, 11~17, 11~18, 11~19, 12~13, 12~14, 12~15, 12~16, 12~17, 12~18, 12~19, 13~14, 13~15, 13~16, 13~17, 13~18, 13~19 , 14-15, 14-16, 14-17, 14-18, 14-19, 15-16, 15-17, 15-18, 15-19, 16-17, 16-18, 16-19, 17-18, 17-19, 18-19) carbon atoms, which may be optionally substituted. Aryl groups for the present invention include, but are not limited to, groups such as phenyl, naphthyl, azulenyl, phenanthrenyl, anthracenyl, fluorenyl, pyrenyl, triphenylenyl, chrysenyl, and naphthacenyl.

[0073] As used herein, "heteroaryl" refers to an aromatic ring group consisting of carbon atoms and one to five heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, furyl, thiophenyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thienopyrrolyl, furopyrrolyl, indolyl, azaindolyl, isoindolyl, indolinyl, indolizinyl, indazolyl, benzimidazolyl, imidazopyridinyl, benzotriazolyl, benzoxazolyl. , benzoxadiazolyl, benzothiazolyl, pyrazolopyridinyl, triazolopyridinyl, thienopyridinyl, benzothiadiazolyl, benzofuyl, benzothiophenyl, quinolinyl, isoquinolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, cinnolinyl, quinazolinyl, quinolizilinyl, phthalazinyl, benzotriazinyl, chromenyl, naphthyridinyl, acridinyl, phenanzinyl, phenothiazinyl, phenoxazinyl, pteridinyl, and purinyl. Further heteroaryls are described in "COMPREHENSIVE HETEROCYCLIC CHEMISTRY: THE STRUCTURE, REACTIONS, SYNTHESIS AND USE OF HETEROCYCLIC COMPOUNDS" (Katritzky et al. eds., 1984), which is incorporated herein by reference in its entirety.

[0074] The term "arylalkyl" refers to a group of the formula -R a R b where R a is alkyl or cycloalkyl as defined above, and R b refers to a moiety in which is aryl or heteroaryl as defined above.

[0075] The compounds described herein may contain one or more asymmetric centers, and thus may give rise to enantiomers, diastereomers, and other types of stereoisomers. Each chiral center may be defined in terms of absolute configuration as (R)- or (S)-. It is intended that the technology of the present invention includes all such suitable isomers, including racemic and optically pure forms, and mixtures thereof. Optically active (R)- and (S)-isomers, (-)- and (+)-isomers, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. In the case where the compounds described herein contain olefinic double bonds, or other asymmetric centers of geometric isomerism, and unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers. Similarly, all tautomeric forms are also intended to be included.

[0076] The term "monocyclic" as used herein refers to a molecular structure having one ring.

[0077] The terms "polycyclic" or "heterocyclic" as used herein refer to a molecular structure having two or more rings, including, but not limited to, fused, bridged, or spiro rings.

[0078] The term "optionally substituted" is used to indicate that a group may have a substitution moiety at each substitutable atom of the group (including multiple substitution moieties at one atom), provided that the normal valence of the designated atom is not excessive and the identity of each substitution moiety is independent of the others. In each residue, up to three H atoms are replaced with the following: alkyl, halogen, haloalkyl, hydroxy, lower alkoxy, carboxy, carboalkoxy (also called alkoxycarbonyl), carboxamido (also called alkylaminocarbonyl), cyano, carbonyl, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, or heteroaryloxy. An "unsubstituted" atom carries all of the hydrogen atoms, the number of which is determined by its valence. If the substitution moiety is keto (i.e., =O), two hydrogens on the atom are replaced. Combining substitute and / or variable components is permissible only if such combinations result in stable compounds; by "stable compound" or "stable structure" is meant a compound that is sufficiently robust to survive isolation from a reaction mixture to a useful degree of purity, and to survive formulation as an efficacious therapeutic agent.

[0079] The term "halogen" means fluorine, chlorine, bromine, or iodine.

[0080] A "peptide," as used herein, is any oligomer of two or more natural or unnatural amino acids, including α-amino acids, β-amino acids, γ-amino acids, L-amino acids, D-amino acids, and combinations thereof. In preferred embodiments, the peptide is about 2 to about 30 amino acids (e.g., about 2 to about 5, about 2 to about 10, about 5 to about 10, about 2 to about 17, about 5 to about 17, about 10 to about 17, about 5 to about 30, about 10 to about 30, or about 18 to about 30). Typically, the peptide is 10 to 17 amino acids in length. In at least one embodiment, the peptide comprises a mixture of α-amino acids and β-amino acids, preferably in the pattern α3 / β1.

[0081] An amino acid, as used herein, may be any natural or unnatural amino acid, including α-amino acids, β-amino acids, γ-amino acids, L-amino acids, and D-amino acids, and an amino acid side chain may be any amino acid side chain of such an amino acid.

[0082] The amino acid in the present invention also includes natural or unnatural amino acid analogs.Amino acid analogs are α-amino acids with unnatural side chains consisting of alkyl, cycloalkyl, aryl, cycloaryl, alkenyl or alkynyl; or β3-amino acids with side chains consisting of alkyl, cycloalkyl, aryl, cycloaryl, alkenyl or alkynyl.As used herein, amino acid analogs also refer to natural or unnatural amino acids that can be substituted for amino acid residues in coiled coil without losing function compared to natural coiled coil sequence. Suitable amino acid analogs / substitutions include the natural amino acid substitutions described in Betts & Russell, "Amino Acid Properties and Consequences of Substitutions", Bioinformatics for Geneticists 289-316 (Michael R. Barnes & Ian C. Gray eds. 2003), which is incorporated herein by reference in its entirety, as well as the non-natural substitutions described below (all available from Sigma Aldrich), and in Gfeller et al., "SwissSidechain: A Molecular and Structural Database of Non-Natural Sidechains", Nucl. Acids Res. 41:D327-D332 (2013), which is incorporated herein by reference in its entirety. As one of skill in the art will appreciate, analogs in the table below that are listed as having a protecting group at the N-terminus and / or C-terminus may be deprotected during conjugation to the adjacent residue. TIFF2024501019000016.tif96157TIFF2024501019000017.tif247157TIFF2024501019000018.tif249157TIFF2024501019000019.tif126157

[0083] Non-limiting examples of substitutions for some amino acid residues include, but are not limited to, those shown below. TIFF2024501019000020.tif32157

[0084] The amino acids of the present invention may also be optionally modified, including, for example, phosphorylation (e.g., phosphoserine, phosphotyrosine, phosphothreonine), halogenation (especially with 3-9 halogens) (preferably with fluorine, e.g., hexafluoroleucine, hexafluorovaline), methylation (e.g., aspartic acid methyl ester, glutamic acid methyl ester, methyllysine, dimethyllysine, trimethyllysine, dimethylarginine, methylarginine, methyltryptophan), and acetylation (e.g., acetyllysine).

[0085] In at least one embodiment of the invention, at least g0, a1, b1, c1, d1, e1, f1, g1, a2, b2, c2, d2, e2, f2, g2, and a3 are present in a first coil, and at least d'1, e'1, f'1, g'1, a'2, b'2, c'2, d'2, e'2, f'2, g'2, a'3, b'3, c'3, d'3, and e'3 are present in a second coil.

[0086] In at least one embodiment of the invention, each residue independently has the formula: TIFF2024501019000021.tif23128, where: R 1a , R 1b , R 1c , and R 1d are each independently hydrogen, an amino acid side chain, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or arylalkyl, and each of the amino acid side chain, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and arylalkyl is H, alkyl, alkenyl, alkynyl, azide, -OR5 , or -SR 5 and when the linker is covalently attached to the residue, the linker is optionally substituted with R 1a , R 1b , R 1c , and R 1d is combined with or replaces one of the following: R 4 is each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or arylalkyl; and R 5 is each independently selected from the group consisting of H, -PG (where PG is a protecting group), alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and arylalkyl.

[0087] As will be readily apparent to those skilled in the art, the linker of the present invention creates a covalent bridge between an amino acid residue / analog in one coil of the coiled-coil structure and an amino acid residue / analog in the other coil of the coiled-coil structure. As will be readily apparent to those skilled in the art, virtually any covalent linker can be used, provided that the appropriate spatial distance between the two residues being linked is maintained. Spatial distance, as used herein, refers to the distance between atoms in the coiled-coil structure as determined in the solid state using a static molecular modeling program (e.g., UCSF Chimera) and / or by evaluating macrocycle crystal structures.For the linkers between residue pairs g0-g'2, g1-g'1, g2-g'0, e1-e'3, e2-e'2, and e3-e'1, the appropriate spatial distance is 10-25 Å (10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 12-13, 12- 14, 12~15, 12~16, 12~17, 12~18, 12~19, 12~20, 12~21, 12~22, 12~23, 12~24, 12~25, 13~14, 13~15, 13~16, 13~17, 13~18, 13~19, 13~20, 13~21, 13~22, 13~23, 13~24, 13~25, 14~15, 14~16, 14~17, 14~18, 14~19, 14~20, 14~ 21, 14-22, 14-23, 14-24, 14-25, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 16-17, 16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 17-18, 17-19, 17-20, 17-21, 17-22, 17-23, 17 24, 17-25, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 20-21, 20-22, 20-23, 20-24, 20-25, 21-22, 21-23, 21-24, 21-25, 22-23, 22-24, 22-25, 23-24, 23-25, or 24-25 Å). In at least one embodiment, the spatial distance is 11-17 Å. In at least one embodiment, the spatial distance is 15-20 Å.For the linkers between residue pairs a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, and d3-a'1, the appropriate spatial distance is 5-15 Å (5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 8-9, 8-10, 8-11, 8- 12, 8-13, 8-14, 8-15, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 10-11, 10-12, 10-13, 10-14, 10-15, 11-12, 11-13, 11-14, 11-15, 12-13, 12-14, 12-15, 13-14, 13-15, or 14-15 Å). In at least one embodiment, the spatial distance is 6-8 Å. In at least one embodiment, the spatial distance is 5-10 Å. Methods for modifying amino acid residues to facilitate attachment of an appropriate linker, including replacing an amino acid side chain with a linker, will also be apparent to one of skill in the art.

[0088] In at least one embodiment of the present invention, the length of any linker between residue pairs g0-g'2, g1-g'1, g2-g'0, e1-e'3, e2-e'2, and e3-e'1 is such that the spatial distance between the Cα positions of each residue in the pairs is 10 to 25 Å; and the length of any linker between residue pairs a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, and d3-a'1 is such that the spatial distance between the Cα positions of each residue in the pairs is 5 to 15 Å.

[0089] In a preferred embodiment, the two amino acids / analogs can be covalently linked to each other using alkylene, alkenylene, arylene, heteroarylene, ether, thioether, amide, maleimide, ester, disulfide, diselenide, -O-, -S-, -Se-, and any combination thereof. As will be apparent to one of skill in the art, the linker can be symmetrical or asymmetrical.

[0090] Suitable examples of linkers between residue pairs g0-g'2, g1-g'1, g2-g'0, e1-e'3, e2-e'2, and e3-e'1 include, but are not limited to, the following: n-, wherein n is a number from 1 to 25 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or any range of 1-25, including, for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1- ... 4, 1-25, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-21, 3-22, 3-23, 3-24, 3-25, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 4-21, 4-22, 4-23, 4-24, 4-25, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-21, 5-22, 5-23, 5-24, 5-25, 6-10, 6-15, 6-20, 6-25, 7-10, 7-15, 7-20, 7-2 These include 5, 8-10, 8-15, 8-20, 8-25, 9-10, 9-15, 9-20, 9-25, 10-15, 10-20, 10-25, 11-15, 11-20, 11-25, 12-15, 12-20, 12-25, 13-15, 13-20, 13-25, 14-15, 14-20, 14-25, 15-20, 15-25, 16-20, 16-25, 17-20, 17-25, 18-20, 18-25, 19-20, 19-25, 20-25, 21-25, 22-25, 23-25, and 24-25;In at least one embodiment, n is 5 to 25) and each Z is independently, at each occurrence, selected from the group consisting of alkylene, alkenylene, arylene, heteroarylene (particularly triazole-diyl, thiazole-diyl, oxazole-diyl), ether, amide, ester, maleimide, thioether, O, S, and Se. Suitable examples of symmetrical linkers include, but are not limited to:; TIFF2024501019000022.tif98164, where TIFF2024501019000023.tif8128 are linkers showing the attachment points to the Cα carbons in the linked residues / analogs, respectively.

[0091] In at least one embodiment of the invention, the linker between at least one of the residue pairs g0-g'2, g1-g'1, g2-g'0, e1-e'3, e2-e'2, and e3-e'1 is (a) A group consisting of TIFF2024501019000024.tif48156, (b) Group consisting of TIFF2024501019000025.tif68128 having a formula selected from: where: X in group (a) and group (b) is O, S, CR2, NR, or P (preferably, O, S, CH2, or NR); X in group (a) 1 are each independently O, S, NH, or NR; X in group (b) 1 each is independently O, S, C, CR, N, NH, or NR; Each R in groups (a) and (b) is independently H, alkyl, or aryl; and Y in group (a) and group (b) is S; and Where: TIFF2024501019000026.tif8128 shows the attachment points to the Cα carbons in each linked residue / analog.

[0092] As will be immediately apparent to those skilled in the art, each antiparallel coiled-coil structure of the present invention may contain any number of linkers ranging from only one to all. In at least one preferred embodiment, there is only one linker. In at least one embodiment of the antiparallel coiled-coil structure, there is at least one linker between g-g' pairs or between e-e' pairs, and there is at least one linker between a-d' pairs or between d-a' pairs. Typically, the coiled-coil structure contains the minimum number of linkers necessary to stabilize the helicity of the coiled-coil. As will be apparent to those skilled in the art, this number varies depending on the overall stability of the unlinked coiled-coil. In one preferred embodiment, there is only one linker. In one preferred embodiment, the only linker is the linker between e-e' pairs. In another preferred embodiment, there are only two linkers.

[0093] In at least one embodiment of the invention, there is a linker between at least one of the residue pairs g0-g'2, g1-g'1, g2-g'0, e1-e'3, e2-e'2, and e3-e'1.

[0094] In at least one embodiment of the invention, there is a linker between at least one of the residue pairs a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, and d3-a'1.

[0095] In at least one embodiment of the present invention, the linker between at least one of the residue pairs a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, and d3-a'1 is a disulfide, diselenide, C 1-8 Alkylene, C 2-8It is selected from the group consisting of alkenylene, arylene, heteroarylene, triazole-diyl, and thiazole-diyl.

[0096] In at least one embodiment of the present invention, the linker between at least one of the residue pairs a1-d'3, a2-d'2, a3-d'1, d1-a'3, d2-a'2, and d3-a'1 is a disulfide bond derived from a cysteine ​​or homocysteine ​​residue, a diselenide derived from a selenocysteine ​​residue, an alkylene derived from an allylglycine residue, or an arylene linker.

[0097] In at least one embodiment, X6 and X' 16 There is a linker between

[0098] In at least one embodiment of the invention, the antiparallel coiled coil has the following formula III: It is TIFF2024501019000027.tif52151, where: Each dotted line independently represents an optional linker; and Each residue independently has the formula: TIFF2024501019000028.tif23128, where: R 1a , R 1b , R 1c , and R 1d are each independently hydrogen, an amino acid side chain, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or arylalkyl, and each of the amino acid side chain, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and arylalkyl is H, alkyl, alkenyl, alkynyl, azide, -OR 5 , or -SR 5 and when the linker is covalently attached to the residue, the linker is optionally substituted with R 1a , R1b , R 1c , and R 1d is combined with or replaces one of the following: R 4 is each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or arylalkyl; and R 5 is each independently selected from the group consisting of H, -PG (where PG is a protecting group), alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and arylalkyl.

[0099] In at least one embodiment of the present invention, at least one of the following conditions is met: (A) in at least one a, a', d, or d' residue, (i) R 1a and R 1c is a side chain of a modified or unmodified amino acid selected from the group consisting of cysteine, homocysteine, selenocysteine, leucine, isoleucine, hexafluoroleucine, valine, hexafluorovaline, allylglycine, threonine, and analogs of each of the foregoing residues; and (ii) R 1b , R 1d , and R 1a and R 1c each of the other independently represents hydrogen, C 1-3 Alkyl, or C 2-3 being alkenyl; (B) at least one e, e', g, or g' residue, 1a and R 1c is an amino acid side chain, and (ii) R 1b , R 1d , and R 1a and R 1c each of the other is independently hydrogen or C 1-3 Being alkyl.

[0100] In at least one embodiment of the invention, the antiparallel coiled coil has the following formula: TIFF2024501019000029.tif107130TIFF2024501019000030.tif21693, Here, each dotted line is independently an optional linker.

[0101] In at least one embodiment of the invention, the macrostructure is CHD-1 (e.g., CHD Sos -1), CHD-2 (for example, CHD Sos -2), CHD-3 (for example, CHD Sos -3), CHD-4 (for example, CHD Sos -4), or CHD-5 (e.g. CHD Sos In at least one embodiment of the present invention, the macrostructure is CHD-2 (CHD-5). Sos -2) or CHD-5 (CHD Sos -5).

[0102] Protective groups primarily function to protect or mask the reactivity of functional groups. Suitable protective groups for protecting amine groups are well known in the art and include, but are not limited to, carbamates, amides, N-alkylamines and N-arylamines, imine derivatives, enamine derivatives, and N-heteroatom derivatives, as described in THEODORA W. GREENE & PETER GM WUTS, "PROTECTIVE GROUPS IN ORGANIC SYNTHESIS", 494-615 (1999), which is incorporated herein by reference in its entirety. Suitable protective groups for this and all aspects of the invention include, for example, tert-butyloxycarbonyl ("Boc"), 9-fluorenylmethyloxycarbonyl ("Fmoc"), carbobenzyloxy ("Cbz"), and trityl. Suitable protective groups for protecting alcohols are also well known in the art. Suitable protecting groups for alcohol include, but are not limited to, silyl ether, ester, and alkyl ether / aryl ether. Suitable protecting groups for protecting thiol groups are also well known in the art. Suitable protecting groups for thiols include, but are not limited to, aryl thioether / alkyl thioether and disulfide. As will be clear to those skilled in the art, when carrying out the methods described herein, the amino acid side chains of Asn, Asp, Gln, Glu, Cys, Ser, His, Lys, Arg, Trp, or Thr will typically need to be protected. Suitable protecting groups for protecting these amino acid side chains are also well known in the art. Methods for protecting and deprotecting functional groups vary depending on the protecting group chosen; however, such methods are well known in the art and are described in THEODORA W. GREENE & PETER GM WUTS, "PROTECTIVE GROUPS IN ORGANIC SYNTHESIS", 372-450 and 494-615 (1999), which is incorporated herein by reference in its entirety.

[0103] As used herein, a "tag" includes any labeling moiety that facilitates detection, quantification, separation, and / or purification of the compounds of the invention. Suitable tags include purification tags, radioactive or fluorescent labels, and enzymatic tags.

[0104] Purification tags, such as polyhistidine (His6-), glutathione-S-transferase (GST-), or maltose binding protein (MBP-), can aid in the purification or separation of a compound, but can also be removed later, i.e., cleaved from the compound after recovery. To facilitate removal of the purification tag, a protease-specific cleavage site can be used. To remove the cleaved purification tag, the desired product can be further purified.

[0105] Other suitable tags include radioactive labels, e.g. 125 I, 131 I, 111 In, or 99 TC and the like. Methods for radiolabeling compounds are known in the art and are described in U.S. Patent No. 5,830,431 to Srinivasan et al., which is incorporated herein by reference in its entirety. Radioactivity is detected and quantified using a scintillation counter or autoradiography. Alternatively, the compound can be conjugated with a fluorescent tag. Suitable fluorescent tags include, but are not limited to, chelates (europium chelates), fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, and Texas Red. Fluorescent labels can be conjugated with compounds using techniques disclosed in "CURRENT PROTOCOLS IN IMMUNOLOGY" (Coligen et al. eds., 1991), which is incorporated herein by reference in its entirety. Fluorescence can be detected and quantified using a fluorometer.

[0106] Enzyme tags generally catalyze a chemical change in a chromogenic substrate that can be measured using a variety of techniques. For example, the enzyme may catalyze a color change in the substrate that can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Examples of suitable enzyme tags include luciferases (e.g., firefly luciferase and bacterial luciferase; see, e.g., U.S. Pat. No. 4,737,456 to Weng et al., incorporated herein by reference in its entirety), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases (e.g., horseradish peroxidase), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating proteins and peptides to enzymes are described in O'Sullivan et al., "Methods for the Preparation of Enzyme - Antibody Conjugates for Use in Enzyme Immunoassay", METHODS IN ENZYMOLOGY 147-66 (Langone et al. eds., 1981), which is incorporated herein by reference in its entirety.

[0107] Targeting moieties in the present invention function to (i) facilitate cellular uptake of a compound, (ii) direct a compound to a particular cell or tissue type (e.g., a signal peptide sequence), or (iii) direct a compound to a particular subcellular localization after cellular uptake (e.g., a transport peptide sequence).

[0108] To facilitate cellular uptake of the compounds of the present invention, the targeting moiety can be a cell-penetrating peptide (CPP). CPPs translocate across the plasma membrane of eukaryotic cells by a pathway that appears to be energy-independent, and have been successfully used for intracellular delivery of macromolecules with molecular weights several times larger than themselves, including antibodies, peptides, proteins, and nucleic acids. Some commonly used CPPs include polyarginine, transportant, protamine, maurocalcine, and M918, which are suitable targeting moieties for use in the present invention and are well known in the art (see Stewart et al., "Cell-Penetrating Peptides as Delivery Vehicles for Biology and Medicine", Organic Biomolecular Chem. 6:2242-55 (2008), which is incorporated herein by reference in its entirety). Additionally, methods of making CPPs are described in US Patent Application Publication No. 20080234183 to Hallbrink et al., which is incorporated by reference in its entirety.

[0109] Another suitable targeting moiety useful for enhancing cellular uptake of compounds is an "importation competent" signal peptide, such as those disclosed in U.S. Patent No. 6,043,339 to Lin et al., which is incorporated herein by reference in its entirety. Importation competent signal peptides are generally about 10 to about 50 amino acid residues in length, typically hydrophobic residues, that allow a compound to penetrate the cell membrane from the outside of the cell to the inside of the cell. Exemplary importation competent signal peptides include the signal peptide from Kaposi fibroblast growth factor (see U.S. Patent No. 6,043,339 to Lin et al., which is incorporated herein by reference in its entirety). Other suitable peptide sequences can be selected from the SIGPEP database (see von Heijne G., "SIGPEP: A Sequence Database for Secretory Signal Peptides", Protein Seq. Data Anal. 1(1):41-42 (1987), which is incorporated herein by reference in its entirety).

[0110] Another suitable targeting moiety is a signal peptide sequence that allows the compound of the present invention to be directed to a certain tissue or cell type. The signal peptide may include at least a portion of a ligand-binding protein. Suitable ligand-binding proteins include high-affinity antibody fragments (e.g., Fab, Fab' and F(ab')2, single-chain Fv antibody fragments), nanobodies or nanobody fragments, fluorobodies, or aptamers. Other ligand-binding proteins include biotin-binding proteins, lipid-binding proteins, periplasmic binding proteins, lectins, serum albumins, enzymes, phosphate- and sulfate-binding proteins, immunophilins, metallothioneins, or various other receptors. For cell-specific targeting, the signal peptide is preferably the ligand-binding domain of a cell-specific membrane receptor. Thus, when the modified compound is delivered intravenously or introduced into the blood or lymph, the compound is adsorbed to the target cell, and the target cell internalizes the compound. For example, if the target cell is a cancer cell, the compound may be conjugated to an anti-C3B(I) antibody, as disclosed in U.S. Patent No. 6,572,856 to Taylor et al., which is incorporated herein by reference in its entirety. Alternatively, the compound may be conjugated to an alpha-fetoprotein receptor, as disclosed in U.S. Patent No. 6,514,685 to Moro, which is incorporated herein by reference in its entirety, or the compound may be conjugated to a monoclonal GAH antibody, as disclosed in U.S. Patent No. 5,837,845 to Hosokawa, which is incorporated herein by reference in its entirety.To target the compound to cardiac cells, the compound can be conjugated with an antibody that recognizes elastin microfibril interfacer (EMILIN2) (Van Hoof et al., "Identification of Cell Surface for Antibody-Based Selection of Human Embryonic Stem Cell-Derived Cardiomyocytes", J. Proteom. Res. 9:1610-18 (2010), which is incorporated herein by reference in its entirety), cardiac troponin I, connexin 43, or any cardiac cell surface membrane receptor known in the art. To target the compound to liver cells, the signal peptide can include a ligand domain that is specific for the liver cell-specific asialoglycoprotein receptor. Methods for preparing such chimeric proteins and chimeric peptides are described in U.S. Pat. No. 5,817,789 to Heartlein et al., which is incorporated herein by reference in its entirety.

[0111] Another suitable targeting moiety is a transport peptide, which directs the intracellular localization of the compound upon internalization of a target cell or tissue. For example, for transport to the endoplasmic reticulum (ER), the compound can be conjugated to an ER transport peptide sequence. Many such signal peptides are known in the art, including signal peptides such as TIFF2024501019000031.tif4128. Other suitable ER signal peptides include an endoplasmic reticulum targeting sequence at the N-terminus, which is the endoplasmic reticulum targeting sequence of the enzyme 17β-hydroxysteroid dehydrogenase type 11 (Horiguchi et al., "Identification and Characterization of the ER / Lipid Droplet-Targeting Sequence in 17β-hydroxysteroid Dehydrogenase Type 11", Arch. Biochem. Biophys. 479(2):121-30 (2008), which is incorporated herein by reference in its entirety), or any of the ER signal peptides disclosed in U.S. Patent Application Publication No. 20080250515 to Reed et al., which is incorporated herein by reference in its entirety, including the nucleic acid sequence encoding the ER signal peptide. Additionally, the compounds of the present invention may include an ER retention signal, such as, for example, the ER retention signal KEDL (SEQ ID NO:32). The method of modifying the compounds of the present invention to incorporate a transport peptide for localizing the compound to the ER can be carried out as described in US Patent Publication No. 20080250515 to Reed et al., which is incorporated herein by reference in its entirety. For transport to the nucleus, the compounds of the present invention can include a nuclear localization transport signal. Suitable nuclear transport peptide sequences are known in the art, including the nuclear transport peptide PPKKKRKV (SEQ ID NO:33). Other nuclear localization transport signals include, for example, the nuclear localization sequence of acidic fibroblast growth factor, and the nuclear localization sequence of transcription factor NF-KB p50, as disclosed in US Patent No. 6,043,339 to Lin et al., which is incorporated herein by reference in its entirety. Other nuclear localization peptide sequences known in the art are also suitable for use in the compounds of the present invention.

[0112] Suitable transit peptide sequences for targeting to mitochondria include: TIFF2024501019000032.tif4128. Other suitable transit peptide sequences suitable for selectively targeting the compounds of the invention to mitochondria are disclosed in U.S. Patent Application Publication No. 20070161544 to Wipf, which is incorporated by reference in its entirety.

[0113] In at least some embodiments of the compounds of the invention, PG, at each occurrence, is independently selected from the group consisting of a protecting group for protecting an amine, a protecting group for protecting a thiol, and a protecting group for protecting a carboxylic acid.

[0114] Another aspect of the present invention relates to a pharmaceutical composition comprising any of the macrostructures described herein and a pharma- ceutically acceptable vehicle. Acceptable pharmaceutical vehicles include solutions, suspensions, emulsions, excipients, powders, or stabilizers. The carrier should be appropriate for the desired mode of delivery.

[0115] In addition, the pharmaceutical composition of the present invention may further contain one or more of the following pharma- ceutically acceptable substances depending on the mode of administration and the nature of the dosage form: diluents, adjuvants, excipients, or vehicles, such as preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavorings, fragrances, antibacterial agents, antifungal agents, lubricants, and dispensing agents. Examples of suspending agents include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable dosage forms can be brought about by using agents that delay absorption, such as aluminum monosterate and gelatin. Examples of suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate. Examples of disintegrating agents include starch, alginic acid, and certain complex silicic acids. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, as well as high molecular weight polyethylene glycols.

[0116] Yet another aspect of the present invention relates to a method of inhibiting Ras signaling in a cell, the method involving contacting the cell with a macrostructure described herein under conditions effective to inhibit Ras signaling in the cell.

[0117] The terms "inhibit" or "inhibiting" as applied to inhibition of Ras signaling means suppressing, decreasing, reducing, or decreasing signaling. Inhibition may be partial or complete. In at least one embodiment, inhibition includes preventing or delaying binding of GTP to Ras.

[0118] Suitable cells include those described below.

[0119] In at least one embodiment, the inhibition is performed in a subject, and the contacting comprises administering a compound to the subject, as described below. Suitable subjects include those described below.

[0120] Another aspect of the present invention is a method for treating a disorder mediated by Ras signaling in a subject, comprising administering to the subject a macrostructure or pharmaceutical formulation as described herein under conditions effective to treat the disorder in the subject.

[0121] The abnormality mediated by Ras signaling is an abnormality caused, at least in part, by enhanced Ras activation. In at least one embodiment, enhanced Ras activation is due to increased and / or prolonged binding of GTP to Ras. In at least one embodiment, enhanced Ras activation is due to more rapid endogenous conversion of GTP to GDP (i.e., conversion that is independent of guanosine nucleotide exchange factors). Suitable abnormalities include cell proliferation abnormalities, differentiation abnormalities, and neoplastic pathologies, as described below. In at least one embodiment, the abnormality is mediated by mutant Ras protein.

[0122] The term "treatment" or "treating" refers to any manner in which one or more symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. Treatment also includes any pharmaceutical use of the compositions herein.

[0123] Another aspect of the present invention relates to methods of using the macrostructures described herein to treat cancer and neoplastic conditions. As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells that have the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapid cell proliferation. Hyperproliferative and neoplastic conditions may be classified as pathological, i.e., as characterizing or constituting a pathological condition, or hyperproliferative and neoplastic conditions may be classified as non-pathological, i.e., as a deviation from normal but not associated with a pathological condition. The terms are intended to include all types of cancerous growths or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or invasive stage. Metastatic tumors can arise from a number of primary tumor types, including, but not limited to, those of breast, lung, liver, colon, and ovarian origin. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include cell proliferation associated with wound repair. Examples of cell proliferative and / or differentiation abnormalities include cancer, such as carcinoma, sarcoma, or metastatic abnormalities. In some embodiments, the compounds are novel therapeutic agents for controlling breast cancer, ovarian cancer, colon cancer, pancreatic cancer, bladder cancer, lung cancer, and metastasis of such cancers.

[0124] Suitable targets include those described below.

[0125] Examples of cancer or neoplastic conditions include, but are not limited to, fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer (including rectal adenocarcinoma), pancreatic cancer, endometrial cancer, salivary gland cancer, oral cancer, ovarian cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, and brain cancer. Carcinoma, squamous cell carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, and Kaposi's sarcoma.

[0126] Examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorders" includes disorders associated with hyperplastic / neoplastic cells of hematopoietic origin, such as disorders associated with hyperplastic / neoplastic cells arising from myeloid, lymphoid, or erythroid lineages, or from their precursors. Preferably, the disorders arise from poorly differentiated acute leukemias, such as erythroblastic leukemia and acute megakaryoblastic leukemia. Further exemplary myeloid disorders include, but are not limited to, plasma cell myeloma, acute myeloid leukemia (AML), acute promyelocytic leukemia (APML), acute myelogenous leukemia (AL), and chronic myelogenous leukemia (CL) (reviewed in Vaickus et al., "Immune Markers in Hematologic Malignancies", Crit. Rev. Oncol. Hemotol. 11:267-97 (1991), which is incorporated by reference in its entirety). Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-lineage and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Additional types of malignant lymphomas include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Sternberg disease. Additional hematopoietic neoplastic disorders include chronic myelomonocytic leukemia (CMML).

[0127] Examples of breast cellular proliferative and / or differentiative abnormalities include, but are not limited to, proliferative breast diseases, including, for example, epithelial hyperplasia, sclerosing adenosis, and small duct papilloma; tumors, including, for example, stromal tumors, such as fibroadenoma, phyllodes tumor, and sarcoma, and epithelial tumors, such as large duct papilloma; breast cancers, including carcinoma in situ (non-invasive cancer), including ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive carcinomas, including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid carcinoma (mucinous carcinoma), tubular carcinoma, and invasive papillary carcinoma, and other malignant neoplasms. Abnormalities in the male breast include, but are not limited to, gynecomastia and carcinoma.

[0128] Examples of pulmonary cell proliferative and / or differentiative abnormalities include, but are not limited to, lung adenocarcinoma, bronchogenic carcinoma including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors such as bronchial carcinoids, other tumors, and metastatic tumors; pleural lesions, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibromas) and malignant mesothelioma.

[0129] Examples of colonic cell proliferative and / or differentiative abnormalities include, but are not limited to, non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinomas, colon cancers, and carcinoid tumors.

[0130] Examples of cellular proliferative and / or differentiative abnormalities of the liver include, but are not limited to, nodular hyperplasia, adenoma, and malignant tumors, including primary carcinomas and metastatic tumors of the liver.

[0131] Examples of ovarian cellular proliferative and / or differentiative abnormalities include, but are not limited to, ovarian tumors, such as coelomic epithelial tumors, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinomas, cystadenofibromas, Brenner tumors, superficial epithelial tumors, and the like; germ cell tumors, such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminomas, endodermal sinus tumors, choriocarcinomas, and the like; sex cord stromal tumors, such as granulosa-theca cell tumors, theca cell tumors, fibromas, androblastomas, hill cell tumors, and gonadoblastomas; and metastatic tumors, such as Krukenberg tumors, and the like.

[0132] In some embodiments, the compounds described herein are used to treat cancer mediated by mutant Ras protein.Cancers that are known to be frequently associated with such mutations include, but are not limited to: non-small cell lung cancer (adenocarcinoma), colon cancer, pancreatic cancer, thyroid cancer (e.g., follicular carcinoma, undifferentiated papillary carcinoma, or papillary carcinoma; e.g., thyroid carcinoma), seminoma, melanoma, bladder cancer, liver cancer, kidney cancer, myelodysplastic syndrome, and acute myeloid leukemia.

[0133] Breast cancer In some embodiments, the present invention provides a method of treating breast cancer by administering a compound described herein. Breast cancer includes invasive breast cancer, including, for example, invasive ductal carcinoma, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, mucinous carcinoma and other tumors with high mucinous content, cystadenocarcinoma, columnar cell mucinous carcinoma, signet ring cell carcinoma, neuroendocrine tumors (including solid neuroendocrine carcinoma, atypical carcinoid tumor, small cell / oat cell carcinoma, or large cell neuroendocrine carcinoma), invasive papillary carcinoma, invasive micropapillary carcinoma, apocrine carcinoma, metaplastic carcinoma, pure epithelial metaplastic carcinoma, mixed epithelial-mesenchymal metaplastic carcinoma, lipid-rich carcinoma, secretory carcinoma, oncocytic carcinoma, adenoid cyst, and cystic carcinoma. carcinoma, acinic cell carcinoma, glycogenic clear cell carcinoma, sebaceous gland carcinoma, inflammatory carcinoma, or bilateral breast carcinoma; mesenchymal tumors, such as hemangioma, hemangiomatosis, hemangiopericytoma, pseudohemangiomatoid stromal hyperplasia, myofibroblastoma, fibromatosis (invasive), inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, or leiomyosarcoma; myoepithelial lesions, such as myoepitheliosis, adenomyoepithelial adenosis, epithelial adenosis), adenomyoepithelioma, or malignant myoepithelioma; fibroepithelial tumors, such as fibroadenoma, phyllodes tumor, low-grade periductal stromal sarcoma, or mammary hamartoma; and tumors of the nipple, such as nipple adenoma, syringoma-like adenoma, or Paget's disease of the nipple.

[0134] Treatment of breast cancer can be performed in combination with any additional therapy, such as a therapy that is part of standard treatment. A surgical procedure, such as a lumpectomy or mastectomy, can be performed before, during, or after treatment with the compound described herein. Alternatively, radiation therapy can be used to treat breast cancer in conjunction with the compound described herein. In another example, the compound described herein is administered in combination with a second therapeutic agent. Such a therapeutic agent can be a chemotherapeutic agent, such as a single agent or a combination of agents and therapies. For example, chemotherapy may be adjuvant therapy with chemotherapy, such as CMF (cyclophosphamide, methotrexate, and 5-fluorouracil); FAC or CAF (5-fluorouracil, doxorubicin, cyclophosphamide); AC or CA (doxorubicin and cyclophosphamide); AC-taxol (AC followed by paclitaxel); TAC (docetaxel, doxorubicin, and cyclophosphamide); FEC (5-fluorouracil, epirubicin, and cyclophosphamide); FECD (FEC followed by docetaxel); TC (docetaxel and cyclophosphamide), and the like. Depending on the tumor characteristics (i.e., HER2 / neu status) and risk of recurrence, trastuzumab may also be added to the regimen in addition to chemotherapy. Hormonal therapy may also be appropriate before, during, or after chemotherapy. For example, tamoxifen can be administered, or the compound in the category of aromatase inhibitor can be administered, where said compound includes but is not limited to aminoglutethimide, anastrozole, exemestane, formestane, letrozole, or vorozole.In other embodiments, angiogenesis inhibitor can be used in combination therapy to treat breast cancer.Angiogenesis inhibitor can be anti-VEGF agent, including but not limited to bevacizumab.

[0135] Ovarian cancer In some embodiments, the compounds described herein can be used to treat ovarian cancer.Ovarian cancer includes ovarian tumors, such as coelomic epithelial tumors, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinomas, cystadenofibromas, Brenner tumors, superficial epithelial tumors, etc.;Germ cell tumors, such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminomas, endodermal sinus tumors, choriocarcinomas, etc.;Sex cord stromal tumors, such as granulosa-theca cell tumors, theca cell tumors, fibromas, androblastomas, hill cell tumors, and gonadoblastomas, etc.;And metastatic tumors, such as Ukenberg tumors, etc.

[0136] The compound of the present invention can be administered in conjunction with a second therapy, such as a therapy that is part of standard treatment.Surgery, immunotherapy, chemotherapy, hormone therapy, radiation therapy, or a combination thereof are some suitable treatments that are available for ovarian cancer.Some suitable surgical procedures include tumor reduction, and unilateral or bilateral oophorectomy, and / or unilateral or bilateral salpingectomy.

[0137] Anticancer drugs that may be used include cyclophosphamide, etoposide, altretamine, and ifosfamide. Hormonal therapy with the drug tamoxifen may be used to shrink ovarian tumors. Radiation therapy may be external beam radiation therapy and / or brachytherapy.

[0138] Prostate cancer In some embodiments, the compounds described herein can be used to treat prostate cancer. Prostate cancer includes adenocarcinoma and metastatic adenocarcinoma. The compounds described herein can be administered in conjunction with a second therapy, such as a therapy that is part of standard treatment. Treatment of prostate cancer can include surgery, radiation therapy, high intensity focused ultrasound (HIFU) therapy, chemotherapy, cryosurgery, hormone therapy, or any combination thereof. Surgery can include prostatectomy, perineal radical prostatectomy, laparoscopic radical prostatectomy, transurethral resection of the prostate, or orchiectomy. Radiation therapy can include external beam radiation therapy and / or brachytherapy. Hormonal therapy may include: orchiectomy, administration of antiandrogens such as flutamide, bicalutamide, nilutamide, or cyproterone acetate; administration of medications that inhibit adrenal androgen production, such as DHEA, such as ketoconazole and aminoglutethimide; and administration of GnRH antagonists or agonists such as abarelix (Plenaxis®), cetrorelix (Cetrotide®), ganirelix (Antagon®), leuprolide, goserelin, triptorelin, or buserelin. Treatment with antiandrogens, drugs that block androgen activity in the body, is another available therapy. Such drugs include flutamide, bicalutamide, and nilutamide. This therapy is typically combined with administration of LHRH analogues or orchiectomy, and is called combined androgen deprivation therapy (CAB). Chemotherapy includes, but is not limited to, administration of docetaxel together with corticosteroids, such as prednisone. Anticancer drugs, such as doxorubicin, estramustine, etoposide, mitoxantrone, vinblastine, paclitaxel, carboplatin, etc., can also be administered to slow the growth of prostate cancer, to alleviate symptoms, and to improve quality of life. Additional compounds, such as bisphosphonates, can also be administered.

[0139] Kidney cancer In some embodiments, the compounds described herein can be used to treat kidney cancer. Kidney cancer includes, but is not limited to, renal cell carcinoma, metastasis from primary neoplasms other than the kidney, renal lymphoma, squamous cell carcinoma, juxtaglomerular tumor (renoma), transitional cell carcinoma, angiomyolipoma, oncocytoma, and Wilms' tumor. The compounds described herein can be administered in conjunction with a second therapy, such as a therapy that is part of standard treatment. Treatment for kidney cancer can include surgery, percutaneous therapy, radiation therapy, chemotherapy, vaccine, or other therapy. Surgical procedures useful for treating kidney cancer in combination with the compounds described herein include nephrectomy, which can include removal of the adrenal gland, removal of retroperitoneal lymph nodes, and removal of any other surrounding tissues affected by tumor infiltration. Percutaneous therapy can include, for example, image-guided therapy, which can involve imaging of the tumor followed by targeting and destroying the tumor by radiofrequency ablation or cryotherapy. In some examples, other chemotherapeutic agents or medications useful for treating kidney cancer can be alpha-interferon, interleukin-2, bevacizumab, sorafenib, sunitib, temsirolimus, or other kinase inhibitors.

[0140] Pancreatic cancer In some embodiments, the present invention provides a method for treating pancreatic cancer by administering a compound described herein, for example, pancreatic cancer selected from epithelioid carcinoma in pancreatic duct and adenocarcinoma in pancreatic duct. The most common type of pancreatic cancer is adenocarcinoma, which occurs in the lining of the pancreatic duct. Suitable treatments available for pancreatic cancer include surgery, immunotherapy, radiation therapy, and chemotherapy. Suitable options for surgical treatment include distal or total pancreatectomy and pancreaticoduodenectomy (Whipple procedure). Radiation therapy, especially external beam radiation, which focuses radiation on tumors with a device outside the body, can be an option for pancreatic cancer patients. Another option is intraoperative electron beam radiation, which is administered during surgery.

[0141] Chemotherapy can also be used to treat pancreatic cancer patients.Suitable anticancer drugs include, but are not limited to, 5-fluorouracil (5-FU), mitomycin, ifosfamide, doxorubicin, streptozocin, chlorozotocin, and combinations thereof.The method provided by the present invention can provide beneficial effects for pancreatic cancer patients by administering the polypeptide of the present invention, or by combining the administration of the compound with surgery, radiation therapy, or chemotherapy.

[0142] Colon cancer In some embodiments, the compounds described herein can be used to treat colon cancer, including, but not limited to, non-neoplastic polyps, adenomas, familial syndromes, colon cancer, colon adenocarcinoma, and carcinoid tumors. Suitable treatments available for colon cancer that can be used in conjunction with the compounds described herein include surgery, chemotherapy, radiation therapy, or targeted drug therapy.

[0143] Radiation therapy may include external beam radiation therapy and / or brachytherapy.

[0144] Chemotherapy can be used to reduce tumor size or slow tumor growth, to reduce the likelihood of metastasis.Chemotherapy is often administered after surgery (adjuvant therapy), before surgery (neoadjuvant therapy), or as primary therapy when surgery is not indicated (palliative therapy).For example, an exemplary regimen of adjuvant chemotherapy includes the infusion of a combination of 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX). First-line chemotherapy regimens may include infusions of 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX) in combination with targeted agents such as bevacizumab, cetuximab, or panitumumab, or infusions of 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI) in combination with targeted agents such as bevacizumab, cetuximab, or panitumumab. Other chemotherapeutic agents that may be useful in combination with the compounds described herein to treat colon cancer are bortezomib (Velcade®), oblimersen (Genasense®, G3139), gefitinib and erlotinib (Tarceva®), and topotecan (Hycamtin®).

[0145] lung cancer Some embodiments provide the method of treating lung cancer with the compound described herein.The example of lung cell proliferation abnormality and / or differentiation abnormality includes but is not limited to: bronchogenic carcinoma, including paraneoplastic syndrome, bronchioloalveolar carcinoma, neuroendocrine tumor, such as bronchial carcinoid, other tumors, and metastatic tumor; pleural lesion, including inflammatory pleural effusion, non-inflammatory pleural effusion, pneumothorax, and pleural tumor, including solitary fibrous tumor (pleural fibroma) and malignant mesothelioma.

[0146] The most common type of lung cancer is non-small cell lung cancer (SCLC), which accounts for approximately 80-85% of lung cancers, and which is divided into squamous cell carcinoma, adenocarcinoma, and undifferentiated large cell carcinoma. Small cell lung cancer, such as small cell lung cancer, accounts for 15-20% of lung cancers. Treatment options for lung cancer include surgery, immunotherapy, radiation therapy, chemotherapy, photodynamic therapy, or a combination thereof. Some suitable options of surgery for treating lung cancer are segmental or wedge resection, lobectomy, or pneumonectomy. Radiation therapy can be external beam radiation therapy or brachytherapy. Some anticancer drugs that can be used in chemotherapy to treat lung cancer in combination with the compounds described herein include cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, irinotecan, etoposide, vinblastine, gefitinib, ifosfamide, methotrexate, or combinations thereof.Photodynamic therapy (PDT) can also be used to treat lung cancer patients.The methods described herein can provide beneficial effects for lung cancer patients by administering the compound or by combining the administration of the compound with surgery, radiation therapy, chemotherapy, photodynamic therapy, or combinations thereof.

[0147] Liver abnormalities Examples of cellular proliferative and / or differentiative abnormalities of the liver include, but are not limited to, nodular hyperplasia, adenoma, and malignant tumors, including primary carcinomas and metastatic tumors of the liver.

[0148] Immunoproliferative disorders Immunoproliferative disorders (also known as "immunoproliferative diseases" or "immunoproliferative neoplasms") are disorders of the immune system characterized by abnormal proliferation of key cells of the immune system, including B cells, T cells, and natural killer (K) cells, or by excessive production of immunoglobulins (also known as antibodies). Such disorders include the general categories of lymphoproliferative disorders, hypergammaglobulinemia, and paraproteinemia. Examples of such disorders include, but are not limited to, X-linked lymphoproliferative disorders, autosomal lymphoproliferative disorders, hyper-Ig syndrome, heavy chain disease, and cryoglobulinemia. Other immunoproliferative disorders may be: graft versus host disease (GVHD); psoriasis; immune disorders associated with transplant rejection of grafts; T-cell lymphoma; T-cell acute lymphoblastic leukemia; testicular angiocentric T cell lymphoma; benign lymphocytic vasculitis; and autoimmune diseases, such as lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin-dependent diabetes mellitus, Goodpasture's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, and the like. hepatitis), ulcerative colitis, Sjogren's syndrome, rheumatoid arthritis, polymyositis, scleroderma, and mixed connective tissue disease.

[0149] Combination treatment In one embodiment, the compounds described herein can be used to treat cancer in conjunction with alkylating and alkylating-like agents. Such agents include, for example, nitrogen mustards, such as chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan; nitrosoureas, such as carmustine, fotemustine, lomustine, and streptozocin; platinum therapeutics, such as carboplatin, cisplatin, oxaliplatin, BBR3464, and satraplatin; or other agents, including but not limited to busulfan, dacarbazine, procarbazine, temozolomide, thiotepa, treosulfan, or uramustine.

[0150] In another embodiment, the compounds described herein can be used in conjunction with antineoplastic agents that are antimetabolites.For example, such antineoplastic agents can be folic acid, such as aminopterin, methotrexate, pemetrexed, or raltitrexed.Alternatively, antineoplastic agents can be purines, such as but not limited to cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, and thioguanine.In a further embodiment, antineoplastic agents can be pyrimidines, such as capecitabine, cytarabine, fluorouracil, floxuridine, and gemcitabine.

[0151] In yet another embodiment, the compounds described herein can be used in combination with anti-neoplastic agents that are spindle poisons / mitotic inhibitors.Agents in this category include taxanes, such as docetaxel and paclitaxel; and also include vinca alkaloids, such as vinblastine, vincristine, vindesine, and vinorelbine.In yet another embodiment, the compounds described herein can be used in combination with anti-neoplastic agents, such as: cytotoxic / antitumor antibiotics from the anthracycline family, such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, or valrubicin; antibiotics from the Streptomyces family, such as actinomycin, bleomycin, mitomycin, or plicamycin; or hydroxyurea. Alternatively, agents used in combination therapy may include a topoisomerase inhibitor, including, but not limited to, camptothecin, topotecan, irinotecan, etoposide, or teniposide.

[0152] Alternatively, the anti-neoplastic agent can be an antibody or an antibody-derived agent. For example, an antibody that targets receptor tyrosine kinase can be used, such as cetuximab, panitumumab, or trastuzumab. Alternatively, the antibody can be an anti-CD20 antibody, such as rituximab or tositumomab, or the antibody can be any other suitable antibody, including but not limited to alemtuzumab, bevacizumab, and gemtuzumab. In other embodiments, the anti-neoplastic agent is a photosensitizer, such as aminolevulinic acid, aminolevulinic acid methyl, porfimer sodium, or verteporfm. In still other embodiments, the anti-neoplastic agent is a tyrosine kinase inhibitor, such as, for example, dediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, or vandetanib. Other anti-neoplastic agents suitable for use in the present invention include, for example, alitretinoin, tretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase (pegaspargase), bexarotene, bortezomib, denileukin diftitox, estramustine, ixabepilone, masoprocol, or mitotane.

[0153] In other or further embodiments, the compounds described herein are used to treat conditions characterized by excessively active cell death or by cell death resulting from physiological damage, etc. Some examples of conditions characterized by cell death or unwanted cell death of immature cells, or unwanted or excessive cell proliferation, include, but are not limited to, hypocellular / hypoplastic abnormalities, acellular / aplastic abnormalities, or hypercellular / hyperplastic abnormalities. Some examples include hematological abnormalities, including, but are not limited to, Fanconi anemia, aplastic anemia, thalassemia, congenital neutropenia, and myelodysplasia.

[0154] In other or further embodiments, the compounds described herein that act to reduce apoptosis are used to treat disorders associated with undesirable levels of cell death. Thus, in some embodiments, the anti-apoptotic compounds described herein are used to treat disorders such as those that cause cell death associated with, for example, viral infections, such as those associated with human immunodeficiency virus (HIV) infection. A wide variety of neurological disorders are characterized by the gradual loss of certain sets of neurons, and the anti-apoptotic compounds described herein are used in some embodiments in the treatment of those disorders. Such disorders include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa, spinal muscular atrophy, and various forms of cerebellar degeneration. Since the loss of cells in these disorders does not induce an inflammatory response, the mechanism of cell death appears to be apoptosis. In addition, some blood disorders are associated with a decrease in blood cell production. These disorders include anemia associated with chronic disease, aplastic anemia, chronic neutropenia, and myelodysplastic syndromes. Abnormalities in blood cell production, such as myelodysplastic syndromes and some types of aplastic anemia, are associated with increased apoptotic cell death in bone marrow. These abnormalities may be due to the activation of genes that promote apoptosis, may be due to acquired deficiencies in stromal cells or hematopoietic survival factors, or may be due to the direct effects of toxins and mediators of immune response. Two common abnormalities associated with cell death are myocardial infarction and stroke. In both of these abnormalities, cells in the central area of ​​ischemia, which occurs when blood flow is suddenly lost, appear to die quickly as a result of necrosis. However, outside the central zone of ischemia, cells die over a longer period of time, and morphologically, they appear to die by apoptosis.

[0155] Administration / contact / cells / subjects In all aspects of the invention relating to administering the macrostructures described herein, the compounds may be administered orally, parenterally, such as subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as the mucous membranes of the nose, throat, and bronchi. The compounds may be administered alone or with a suitable pharmaceutical carrier, and may be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.

[0156] The active compounds of the present invention may be administered orally, for example, with an inert diluent or with an edible carrier that is absorbed, or the active compounds may be enclosed in hard or soft shell capsules, or the active compounds may be compressed into tablets, or the active compounds may be directly incorporated into dietary foods. For oral therapeutic administration, these active compounds may be combined with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of the active compound. The percentage of the compound in these compositions may, of course, vary, and may conveniently be from about 2% to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage is obtained. Preferred compositions of the present invention are prepared so that an oral dosage unit contains about 1 to 250 mg of active compound.

[0157] Tablets, capsules and the like may also contain: binders such as, for example, tragacanth, arabic, corn starch, or gelatin; excipients such as, for example, dicalcium phosphate; disintegrants such as, for example, corn starch, potato starch, alginic acid, and the like; lubricants such as, for example, magnesium stearate, and sweeteners such as, for example, sucrose, lactose, or saccharin. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a fatty oil.

[0158] Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar, or both. A syrup may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and a flavoring, such as cherry or orange flavor.

[0159] These active compounds can also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water, suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oil. Exemplary oils are those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous solutions of dextrose and related sugars, and glycols, such as propylene glycol or polyethylene glycol, are particularly preferred liquid carriers for injectable solutions. These preparations contain a preservative to prevent the growth of microorganisms under ordinary conditions of storage and use.

[0160] Suitable dosage forms for injectable use include sterile aqueous solutions or sterile aqueous dispersions, and also sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.In any case, the dosage form must be sterile and fluid to the extent that it has smooth puncture.The dosage form must be stable under the conditions of manufacture and storage, and must be protected against the contaminating action of microorganisms, such as bacteria and fungi.The carrier can be a solvent or dispersion medium, which includes, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils.

[0161] The compound of the present invention can also be administered directly to airway in the form of aerosol.For use as aerosol, the compound of the present invention in solution or suspension can be packaged with conventional adjuvants and suitable propellant into a pressurized aerosol container, such as propellant of hydrocarbon, such as propane, butane, isobutane, etc.The substance of the present invention can also be administered in non-pressurized form, such as nebulizer or atomizer.

[0162] In all aspects of the invention involving contacting a cell, suitable cells include, but are not limited to, mammalian cells (e.g., primate cells, including human cells, feline cells, canine cells, equine cells, bovine cells, caprine cells, ovine cells, porcine cells, murine cells, rat cells, etc.) In at least one preferred embodiment, the cell expresses a mutant Ras protein.

[0163] In all aspects of the invention relating to subjects, suitable subjects include mammals (e.g., primates, such as humans, cats, dogs, horses, cows, goats, sheep, pigs, mice, rats, etc.) In at least one preferred embodiment, the subject has a disorder mediated by a mutant Ras protein.

[0164] In all methods related to mutant Ras protein, suitable examples include H-Ras isoform, N-Ras isoform, and K-Ras isoform.In at least one embodiment, mutant Ras protein is H-Ras isoform.In at least one embodiment, mutant Ras protein is K-Ras isoform.In at least one embodiment, mutant Ras protein has one or more mutations selected from the group consisting of G12C, G12D, G12S, G12V, G13D, G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13R, G13S, G13D, Q61E, Q61H, Q61L, Q61K, Q61P, and Q61R.In at least one preferred embodiment, mutant Ras protein has G12C mutation or G12D mutation.

[0165] In all aspects of the invention directed to methods involving contacting a cell with one or more compounds, the contacting can be performed using methods that would be apparent to one of skill in the art, and the contacting can be performed in vitro or in vivo. In at least one embodiment of the invention, the contacting stabilizes Ras in an inactive form.

[0166] One approach for delivering agents into cells involves the use of liposomes, which basically involves providing a liposome containing the agent to be delivered and then contacting the targeted cell, tissue, or organ with the liposome under conditions effective to deliver the agent to the cell, tissue, or organ.

[0167] The liposomal delivery systems can also be directed to accumulate in targeted organs, tissues, or cells through active targeting (e.g., by incorporating antibodies or hormones onto the surface of the liposomal vehicle), which can be accomplished by known techniques.

[0168] Another approach for delivering agents, including proteins or polypeptides, involves conjugating the desired agent to a polymer that is stabilized to prevent enzymatic degradation of the conjugated protein or polypeptide. This type of conjugated protein or polypeptide is described in U.S. Patent No. 5,681,811 to Ekwuribe, which is incorporated herein by reference in its entirety.

[0169] Yet another approach for delivering agents involves the preparation of chimeric agents, as described in U.S. Patent No. 5,817,789 to Heartlein et al., which is incorporated herein by reference in its entirety. The chimeric agents may include a ligand domain and an agent (e.g., a compound of the present invention). The ligand domain is specific for a receptor present on a target cell. Thus, when the chimeric agent is delivered intravenously or introduced into the blood or lymph, it adsorbs to the target cell, and the target cell internalizes the chimeric agent.

[0170] The compounds of the invention may be delivered directly to the targeted cells / tissues / organs.

[0171] Additionally and / or alternatively, a compound may be administered to a non-target area along with one or more agents that facilitate the movement of the compound to (and / or uptake of the compound by) the target tissue, organ, or cell. As will be apparent to one of skill in the art, the compound itself can be modified to facilitate transport of the compound to the target tissue, organ, or cell and / or to facilitate uptake of the compound by the target cell (e.g., transport of the compound across a cell membrane).

[0172] These aspects of the present technology are further illustrated by the following examples. All references cited throughout this application are hereby incorporated by reference in their entirety, including those cited in the figures and examples. EXAMPLES

[0173] The following examples are provided to illustrate aspects of the present technology and are not intended to limit the scope of the present technology in any way.

[0174] Solubility was one of the most difficult challenges to overcome, and was also important for carrying out most of the experiments described herein: careful placement of charged and polar residues was necessary to enhance solubility under aqueous conditions without sacrificing affinity.

[0175] Example 1: Synthesis of Bridged Helix Dimer (CHD) Peptides Unless otherwise specified, peptide synthesis was performed under standard Fmoc solid-phase peptide synthesis methods on Rink amide resin. The completed peptides were subjected to final Fmoc deprotection and N-terminal acetyl protection using a solution containing 0.5 M acetic anhydride and 5% diisopropylethylamine (DIEA) in NMP for 30 min, followed by cleavage from the resin. The two desired peptide chains were cleaved from the resin, purified using RP-HPLC, and characterized by MALDI-TOF spectroscopy. The two types of chains are crosslinked as previously described and shown in Scheme 1 below (J. Sadek et al., "Modulation of virus-induced NF-kappaB signaling by NEMO coiled coil mimics", Nat Commun 11, 1786 (2020); MG Wuo, SH Hong, A. Singh, PS Arora, "Synthetic Control of Tertiary Helical Structures in Short Peptides", J. Am. Chem. Soc. 140, 16284-16290 (2018); MG Wuo, AB Mahon, PS Arora, "An Effective Strategy for Stabilizing Minimal Coiled Coil Mimetics", J. Am. Chem. Soc. 137, 11618-11621 (2015)). TIFF2024501019000033.tif96128 Scheme 1. General synthesis scheme for CHD peptides

[0176] Each strand contains one cysteine ​​residue, which is strategically positioned for the addition of a dibenzyl ether crosslinker. The crosslinker was synthesized according to a previously published protocol (MG Wuo, SH Hong, A. Singh, PS Arora, "Synthetic Control of Tertiary Helical Structures in Short Peptides", J. Am. Chem. Soc. 140, 16284-16290 (2018)). The purified strands were first dissolved in 20 mM NH4CO3 buffer (pH = 8.1) and subsequently added to a dibenzyl ether crosslinker solution (5-10 eq.) in acetonitrile. The reaction mixture was stirred at 20 °C for 90 min, followed by C 18 Purification by RP-HPLC on a reversed-phase column (25-65% acetonitrile gradient in water containing 0.1% TFA over 40 min) followed. The purified product was then lyophilized. The lyophilized monocysteine ​​alkylated peptide was then dissolved in 1:1 acetonitrile:20 mM aqueous NH4CO3 (pH 8.1). An excess of the second chain with a free cysteine ​​(1.5 eq.) was added and the resulting mixture was stirred at 20° C. for 1 h. The reaction mixture was incubated at 37° C. for 1 h ... 18 The product was subjected to RP-HPLC on a reversed-phase column (25-65% acetonitrile gradient in water containing 0.1% TFA over 40 min) and characterized by analytical HPLC and MALDI-TOF spectroscopy.

[0177] The HPLC trace of the Sos CHD is provided in Figure 16. The chemical structure of the Sos CHD is provided in Figure 17. Characterization of the CHD peptides by mass spectrometry is summarized in Table 1 below, where R H = L-homoarginine; A β = L-β-alanine; DZ = diazirine photocrosslinker; FITC is 5-fluorescein isothiocyanate linked to the N-terminal amine via a thiourea bond.

[0178] Table 1. Characterization of CHD peptides by mass spectrometry TIFF2024501019000034.tif255152* Mimetics of the αH or αI sequences of Sos with designed changes

[0179] Example 2: Synthesis of Fluorescein-Labeled Peptides The peptide sequences listed in Table 1 above were synthesized as previously described, with the addition of β-alanine at the N-terminus to act as a linker, followed by coupling with fluorescein isothiocyanate (3 eq.) and DIEA (3 eq.) overnight and protected from light. The fluorophore-conjugated peptides were then cleaved from the resin, purified using RP-HPLC, and characterized using MALDI-TOF spectroscopy.

[0180] Example 3: Synthesis of Diazirine Photocrosslinker Peptide The peptide sequences listed (see FIG. 17 and Table 1) were synthesized as previously described with the addition of L-glycine at the N-terminus as a linker followed by coupling with 3-(3-(but-3-ynyl)-3H-diazirin-3-yl)propanoic acid (Sigma-Aldrich) under standard peptide coupling conditions overnight and protected from light. The diazirine-conjugated peptides were then cleaved from the resin, purified using RP-HPLC, and characterized using analytical LCMS.

[0181] Example 4: Circular Dichroism Circular dichroism (CD) experiments were performed at 298 K on a Jasco J-1500 circular dichroism spectrometer equipped with a temperature controller, using a 1 mm long cell and a scan rate of 4.0 nm / min. Spectra generated were baseline subtracted and averaged over a total of five scans. Raw values ​​were normalized to the molar residue ellipticity (MRE). Unless otherwise stated, samples were prepared in a buffer containing 50 mM potassium fluoride in water (pH 7.4) to a final peptide concentration of 20 μM. The concentration of each sample was monitored via the UV absorbance at 280 nm of tryptophan residues.

[0182] Example 5: Protein purification Wild-type and mutant N-terminal His6-H-Ras (residues 1-166) in the pProEx HTb expression vector were expressed in Escherichia coli (BL21). Cells were grown at 37°C until the absorbance at 600 nm was 0.7. Protein expression was induced with 500 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 hours at 30°C or overnight at 18°C. Bacterial cells were pelleted by centrifugation at 4500 rpm for 15 minutes. Bacterial pellets were resuspended in lysis buffer containing: 20 mM Tris pH 7.4, 300 mM NaCl, 2 mM 2-mercaptoethanol, and cOmplete, EDTA-free protease inhibitor cocktail (Roche). 2.5 mM MgCl2 was also added to the lysis, elution, and dialysis buffers. The resuspended pellet was sonicated using a Branson Cell Disrupter 200. A cleared lysate was formed upon centrifugation at 13,000 rpm for 20 min at 4°C, and this was incubated with charged Ni-NTA resin (Invitrogen) for 1 h at 4°C. The resin beads were washed five times with resuspension buffer containing 5 mM imidazole. His6-tagged proteins were eluted by gravity flow with a buffer containing 200 mM imidazole in a buffer containing 20 mM Tris, 300 mM NaCl, pH 7.4. To obtain H-Ras, the eluted proteins were dialyzed twice against a buffer containing 20 mM Tris, 300 mM NaCl, pH 7.4, 2.5 mM MgCl2. For HSQC and photoaffinity labeling studies, the polyhistidine tag was cleaved using Tobacco Etch Virus (TEV) protease prior to nucleotide loading. The concentrated eluate was subjected to nucleotide loading and size exclusion chromatography (GE Life Sciences) at 4°C using a buffer of 25 mM Tris and 50 mM NaCl, pH 7.5, and the desired monomer peak was collected.The eluted protein samples were concentrated in dialysis buffer containing 10% glycerol (v / v) using Amicon centrifugal columns with a molecular weight cutoff of 3000 Da (Millipore), then flash frozen in liquid N2 and stored at −80°C until further use.

[0183] Example 6: Nucleotide Loading Nucleotide loading for fluorescence polarization, HSQC, and nucleotide substitution studies was performed as previously described in SM Margarit et al., "Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS", Cell 112, 685-695 (2003). Purified H-Ras protein was incubated with GDP or GTP (10 eq.) in loading buffer (20 mM Tris, 300 mM NaCl, 5 mM EDTA, pH 7.4) for 90 min on ice. The reaction was then quenched with 12 mM MgCl2 and incubated on ice for 30 min. Depending on the subsequent assay, the protein was subjected to size exclusion chromatography by fast protein liquid chromatography (FPLC) or a pre-equilibrated NAP-5 (GE Life Sciences) column for removal of excess free nucleotides and further purification.

[0184] Example 7: Fluorescence Polarization Binding Assay The relative binding affinities of CHD to wild-type and mutant His6-tagged H-Ras (positions 1-166) preloaded with GDP were determined using a direct fluorescence polarization assay with fluorescein-tagged CHD peptides (Flu-αHBS, Flu-α3βHBS). These experiments were performed using a DTX 880 multimode detector (Beckman) at 25° with excitation and emission wavelengths set at 485 nm and 525 nm, respectively. Addition of increasing concentrations of His6-H-Ras-GDP to a 15 nM solution of fluorescein-labeled CHD in 50 mM Tris, 100 mM NaCl, 0.1% pluronic acid, pH 7.4, was performed in black U-bottom 96-well plates (Brand). Binding affinities (K D ) values ​​are based on biological triplicates and were determined by fitting to a sigmoidal dose-response nonlinear regression model in Graphpad Prism 6. K D1 = (R T *(1-F SB ) + L ST *F SB 2 ) / F SB -L ST (SJ1) where: R T = total concentration of H-Ras (positions 1 to 166), L ST = total concentration of fluorescent peptide, F SB = percentage of fluorescent peptide bound.

[0185] Example 8: Microscale Thermophoresis (MST) Fluorescent labeling of H-Ras protein was performed using Monolith protein labeling kit RED-MALEIMIDE 2nd Generation (MO-L014) from NanoTemper or AFDye 647 maleimide (1122-1) from Fluoroprobes. Briefly, 20 nM H-Ras protein was labeled with 1.5 equivalents of cysteine-reactive dye in 1x PBS for 90 min in the dark. The labeled Ras protein was then purified using a kit-provided column, and the degree of labeling (DOL) was calculated by using the absorbance of the dye and the native absorbance of the protein. The DOL values ​​observed after labeling were 0.43 for wild-type H-Ras-GDP and 0.98 for G12V H-Ras-GDP.

[0186] MST binding assays were performed using a Monolith NT.115 Pico from NanoTemper. To avoid random adsorption, assay conditions were optimized using NanoTemper premium coated capillary tubes. Measurements were performed for 20 seconds at 25 degrees with 10-15% excitation laser power. Assay buffer consisted of 50 mM Tris, 100 mM NaCl, and 0.1% pluronic acid. Dye-labeled 5-10 nM protein was exposed to serial dilutions of the indicated CHDs and incubated at room temperature for 90 minutes before measurement. Error bars are generated from triplicate replicates. Binding affinities were obtained from the MO analysis software in the NT.115 Pico using the following formula: TIFF2024501019000035.tif7128, where Unbd = the response value in the unbound state, Bd = the combined response value, TC = final concentration of fluorescent molecule.

[0187] Example 9: Serum stability assay CHD for protein degradation SosThe stability of -5 was evaluated in 25% serum by using fetal bovine serum (FBS, Innovative Research) in RPMI-1640 medium. For each sample, a 45 μL mixture with 33% FBS (v / v) in serum-free medium was prepared. For each experiment, the addition of 15 μL of the indicated peptide in serum-free medium (final concentration of FBS was 25%) was recorded as the starting time point. Time points of 5 min, 6 h, 12 h, and 24 h were analyzed in triplicate. Each experiment was quenched by adding 30 μL of 100% EtOH prepared at -70 degrees at the determined time points. After cooling on ice for 10 min, each sample was pelleted at 14,000 x g for 5 min. 30 μL of supernatant was separated, and 3 μL of 500 μM L-tryptophan was titrated in the samples to be used as an internal standard. The resulting mixture was then immediately subjected to RP-HPLC using a 3.5 μm, 2.1 x 150 mm C18 analytical column and monitored at 280 nm. Elution peaks were collected and the mass of each peak was determined using MALDI-TOF spectroscopy. The integrated peak areas of intact peptides were used to determine the percent undegraded in each given condition.

[0188] Example 10: Photoaffinity labeling and visualization of conjugated CHD-Ras complexes For each experiment, a sample of 250 μM diazirine-labeled CHD peptide was incubated with 50 μM purified H-Ras in binding buffer (50 mM Tris, 100 mM NaCl, 0.1% pluronic acid, pH 7.4) for 90 min at room temperature (total volume 100 μL). The incubated sample was then photoirradiated at 24°C for 5 min. The photoconjugated sample was then mixed with 1x SDS-PAGE loading buffer containing 5% 2-mercaptoethanol (final volume 200 μL) and heated at 95°C for 5 min. The heated sample was then resolved by SDS-PAGE gel electrophoresis in a 1.0 mm 15% polyacrylamide gel. The resulting gel was stained with Coomassie Brilliant dye and subsequently destained (40% MeOH / 10% acetic acid / 50% water).

[0189] Example 11: In-solution tryptic digestion mass spectrometry In-gel trypsin digestion was performed using an in-gel trypsin digestion kit (Thermo Scientific) according to the manufacturer's instructions. Photoaffinity label-conjugated bands were excised as 1 x 1 mm segments for in-gel trypsin digestion. The excised gel bands were then incubated with 200 μL of Coomassie destaining solution (25 mM ammonium bicarbonate in 1:1 ultrapure water:acetonitrile) at 37°C for 30 min. After incubation, the destaining buffer was removed and the destaining step was repeated two more times. The destained gel bands were treated with reducing buffer (25 mM ammonium bicarbonate, 50 mM TCEP) at 60°C for 10 min. After removing the reducing buffer, the bands were treated with 200 μL of iodoacetamide alkylating solution (25 mM ammonium bicarbonate, 100 mM iodoacetamide) at 37°C for 30 min with shaking in the dark. After alkylation, the gel pieces were washed again twice with 100 μL of destaining buffer, followed by treatment with 100 μL of acetonitrile for 15 min at room temperature. The gel bands were then air-dried for 15 min after removing the acetonitrile. The trypsin solution consisted of 5 μg of trypsin protease (Pierce, MS grade) dissolved in 100 μL of 25 mM ammonium bicarbonate buffer, pH 8.0. The dried gel was treated with 100 μL of trypsin solution with shaking at 30° overnight. After digestion, the buffer was removed, and the digested peptides were extracted from the gel with 25 μL of 1% trifluoroacetic acid. The extracted samples in 1% trifluoroacetic acid were then diluted with 25 μL of ultrapure water and subjected to MALDI-TOF MS analysis.

[0190] Example 12: Heteronuclear Single Quantum Spectroscopy (HSQC) The purification procedure was nearly identical to the previously described protocol, with a few exceptions. E. coli (BL21) cells (4 L) containing the His6-H-Ras construct were grown at 37°C in LB broth fully supplemented with nutrients to an OD of 0.8. Cells were pelleted and resuspended in M9 minimal medium (1 L), followed by incubation in 10% M9 medium supplemented with 20% glucose and 10% ethanol as the sole nitrogen source. 15 NH4Cl was added (Y. Ito et al., "Regional polysterism in the GTP-bound form of the human c-Ha-Ras protein", Biochemistry 36, 9109-9119 (1997)). Protein expression was induced with 1 mM IPTG at OD 0.8 overnight at 18°C. Protein purification and subsequent concentration were performed as described above. The His6 tag was cleaved upon overnight incubation at 4°C with recombinant His6-tagged Tobacco Etch Virus (TEV) protease (Invitrogen) according to the manufacturer's protocol. The resulting protein mixture was loaded onto a charged Ni-NTA column (Invitrogen), and untagged protein was collected in the flow-through fraction. 15 N-labeled H-Ras was then preloaded with GDP according to the protocol mentioned above. 15 H-Ras-GDP, uniformly labeled with N, was buffer exchanged into NMR buffer (20 mM Na2HPO4-NaH2PO4, pH 5.5, 150 mM NaCl, 10 mM MgCl2), concentrated using Amicon Ultra centrifugal filters (Millipore), and analyzed after adding 10% D2O. Data collection was performed on a 600 MHz Bruker 4-channel NMR system equipped with a 5 mm TCI cryogenic probe using standard pulse sequences at 25° and analyzed by TopSpin 4.0.6 (Bruker). 15 N-labeled H-Ras-GDP (100 μM) alone 15 N-1 H-HSQC spectra were collected and peaks were assigned based on published data. For peptide titration experiments, 2.5 and 5 equivalents of CHD Sos -5 is dissolved in the matching buffer, and 15 N- 1 were incubated with H-Ras (100 μM). 1 H nuclei and 15 The average chemical shift differences (ΔδNH) observed for the various resonances of the N nucleus were calculated according to the following formula, with α = 0.14: TIFF2024501019000036.tif10128

[0191] Example 13: Cell Culture T24, HCT-116, and HeLa cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Corning Cellgro) supplemented with 10% fetal bovine serum (FBS, Innovative Research), 1X penicillin / streptomycin (EMD Millipore), and 10 mM HEPES buffer (Sigma-Aldrich). H358, Panc 10.05, A549, and BxPC3 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS (Innovative Research), 1X penicillin / streptomycin, 10 mM HEPES buffer, and 1X sodium pyruvate (Sigma-Aldrich). All cells were maintained in a humidified incubator at 37 degrees and 5% CO2.

[0192] Example 14: Fluorescence microscopy of live cells The cell lines described were cultured at 5 x 10 5Cells / well were seeded on poly-D-lysine coated 35 mm plates (MatTek) and incubated overnight. Growth medium was aspirated and washed with serum-free medium. Cells were then incubated with fluorescein-conjugated peptides at 1 μM (final concentration) dissolved in serum-free medium (0.4% DMSO, v / v) for 4 h at 37°C, protected from light. All compounds were dissolved as concentrated stocks in DMSO. After the designated incubation time, each plate was aspirated and treated with Hoechst dye solution (Hoechst 33342, ThermoFisher Scientific) for 10 min to stain cell nuclei. A stock solution of Hoechst dye (10 mg / mL) was diluted 1:2000 in PBS to prepare the working mixture. The dye solution was removed and the plates were gently washed three times with PBS. DMEM (high glucose, HEPES, phenol red-free, 20% FBS) was added to each plate and used as the imaging solution. Fluorescence images were acquired on an Eclipse Ti fluorescence microscope (Nikon) equipped with the imaging software NIS-Elements and using a 40x objective.

[0193] Example 15: Flow Cytometry The cell lines described were cultured at 1 x 10 5Cells / well were seeded into clear polystyrene 24-well plates (Corning) and incubated overnight. The initial growth medium was replaced with serum-free medium and incubated at 37°C for 2 hours. After aspirating, the cells were incubated in serum-free medium containing 0.4% DMSO (v / v) for 1 hour. The cells were then treated with fluorescein-conjugated peptides at 1 μM (final concentration) in serum-free medium for an additional hour protected from light. All compounds were dissolved as concentrated stocks in DMSO. Each well was aspirated and treated with 1× trypsin (0.25% trypsin, 2.21 mM EDTA, Corning Cellgro) for 10 minutes at 37°C. After trypsinization, the resulting solution was mixed with cold serum-free medium and collected. Samples were centrifuged at 500 rpm for 5 minutes at 4°C. The supernatant was removed and the cell pellet was resuspended in cold PBS and then placed on ice. Each sample was treated with 10% trypan blue (v / v) and immediately analyzed by flow cytometry on a Becton Dickinson Accuri flow cytometer. Data shown consist of median fluorescence intensity for at least 10,000 cells / sample and processed using FlowJo (Tree Star Inc.).

[0194] Example 16: Cell viability assay Cell viability was monitored using MTT (Sigma-Aldrich, M2128) or the CellTiter-Glo luminescent cell viability assay (Promega).

[0195] Example 16A: MTT cell viability assay Cell viability was monitored using a luminescent cell viability assay with MTT (Sigma-Aldrich, M2128). Cells were plated in clear 96-well plates at 2000 cells / well and allowed to adhere overnight at 37°C. Each well was gently aspirated and washed with serum-free medium, after which peptide inhibitors dissolved in complete medium containing 0.5% DMSO (v / v, final concentration) were introduced to the desired concentration (90 μL / well). Cells were incubated in the presence of peptides for 72 hours at 37°C. The MTT reagent solution consisted of thiazolyl blue tetrazolium bromide dissolved in Dulbecco's phosphate-buffered saline (DPBS) at pH 7.4 to a concentration of 5 mg / mL, and was subsequently sterile filtered into a light-proof container. MTT reagent was added to each well (0.45 mg / mL / well, final concentration). Cells were incubated for an additional 4 hours at 37°C. After carefully removing the supernatant, 150 μL of DMSO (lysis solution) is added to each well and mixed to ensure that the insoluble purple formazan precipitate is completely dissolved and released into solution. Absorbance values ​​are recorded at 570 nm using a Synergy HT multidetection microplate reader (BioTek).

[0196] Example 16B: CellTiter-Glo Luminescent Cell Viability Assay Cell viability was monitored using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Cells were plated at 2000 cells / well in opalescent 96-well plates and allowed to adhere overnight at 37°C. Each well was gently aspirated and washed with serum-free medium, followed by complete medium (CHD SosPeptide inhibitors dissolved in DMSO (no DMSO is needed to dissolve -5) are introduced to the desired concentration (90 μL / well). Cells are incubated in the presence of peptides for 72 hours at 37 degrees. The plate and its contents are then equilibrated to room temperature for approximately 30 minutes after treatment with peptides. Well supernatants are gently removed, and then 50 μL of 1X PBS is added to each well. An equal volume (50 μL) of CellTiter-Glo 2.0 reagent is then added to each well, and the resulting contents are mixed for 2 minutes on an orbital shaker to induce cell lysis. Plates are incubated for 10 minutes at room temperature, after which luminescence signals are recorded (0.25-1 second, integration time) using a DTX 880 multimode detector (Beckman). Each plate contained triplicate wells of a killing positive control (cells in 10% DMSO), a negative control (cells in 0.05% DMSO), and a blank (0.05% DMSO, no cells).

[0197] Example 17: Ras activation assay H358 cells were first seeded in 6-well plates (1x10 6 Cells were incubated at 37°C for 6 h at 37°C for 1 h at 4°C for 1 h. Cells were treated with the indicated peptides dissolved in complete medium at the indicated concentrations for 6 h at 37°C. Ras activity was determined by an Active Ras Pulldown and Detection Kit (Thermo Scientific, Cat. No. 16117) according to the manufacturer's instructions. Briefly, cells were lysed with 250 μL of lysis buffer and scraped; the resulting lysate was centrifuged at 13,000 rpm for 10 min at 4°C. The previously cleared lysate was then added with 80 μg of GST-tagged RBD and prewashed glutathione agarose beads and shaken continuously for 1 h at 4°C. The beads were then pelleted, washed three times with buffer, and eluted with 50 μL of 2X reducing sample buffer for western blotting.

[0198] Example 18: ERK activation assay H358 or HeLa cells were first seeded in 6-well plates (1x10 6Cells were incubated at 37°C for 6 hours with the peptides described, dissolved in complete medium at the concentrations indicated. Cells were washed twice with ice-cold PBS and then lysed in cold RIPA buffer (200 μL / well) containing: 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium doexycholic acid, 0.1% SDS, Roche cOmplete protease inhibitor cocktail (1X, Sigma-Aldrich, P2714), and Roche PhosSTOP phosphatase inhibitor cocktail tablets (1X, Sigma-Aldrich, 4906845001). Cells were kept on ice for 5 minutes with occasional rotation to ensure even spreading. Cell lysates were made using a cell scraper, transferred to a microcentrifuge tube, and centrifuged at 13,000 rpm for 10 min at 4°C. The cleared supernatants were collected and total protein concentrations were measured by Pierce BCA protein assay. Lysates were subjected to SDS-PAGE (loading at 10 μg / lane) and Western blot analysis by immunoblotting. Primary antibodies included phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (Cell Signaling, 4370), p44 / 42 MAPK (Erk1 / 2) (Cell Signaling, 4695), Ras (Abcam, ab108602), and α-tubulin (Cell Signaling, 2144). Secondary antibodies included anti-rabbit IgG (Cell Signaling, 7074P2, 1:2000), an HRP-linked antibody. After incubation with primary antibodies, blotted membranes were probed with secondary antibodies and SignalFire ECL reagent (Cell Signaling, 6883P3) and imaged using a ChemiDoc imaging system (Bio-Rad). Densitometry of comparative blots was performed using ImageJ (NIH) and normalized to tubulin expression.

[0199] Example 19: Preparation and enrichment of sample lysates for proteomics H358 cells were grown in 10 cm plates in RPMI-1640 supplemented with FBS until 80-95% confluence. Growth medium was aspirated and cells were washed with Dulbecco's phosphate-buffered saline (DPBS). Cells were then incubated at 37°C in a 5% CO2 atmosphere for 4 hours with the probe DZ2-CHD. Sos Cells were incubated with serum-free medium containing -5 (20 μM in RPMI-1640 containing 0.4% DMSO, 2 replicates) or with the control probe CP-2-66 (20 μM in RPMI-1640 containing 0.4% DMSO, 4 replicates) and then irradiated under UV light (365 nm, 30 min, 4°C). Cells were washed with DPBS, scraped, and transferred to 1.5 mL Eppendorf tubes, pelleted, and stored at -80°C until the next step.

[0200] Cell pellets were resuspended in 500 μL of DPBS and lysed by sonication (15 ms on, 40 ms off, 15% amplitude, 1 sec total on, 2 times). Protein concentrations were normalized using the Lowry protein assay (Pierce) (1-2 mg / mL; 500 μL final volume with DPBS). To each sample, a solution of tris((1-benzyl-4-triazolyl)methyl)amine (30 μl, 1.7 mM in 1:4 DMSO / t-BuOH v / v), tris(2-carboxyethyl)phosphine (10 μl, 50 mM), biotin-PEG3-azide (5 μl, 100 μM), and CuSO4 (10 μl, 50 mM) was added, and the samples were shaken for 1 h at room temperature. A cold MeOH / CHCl3 mixture (2.5 mL, 4:1, v / v) was added to each lysate, followed by 1 mL of cold DPBS. The resulting mixture was vortexed and centrifuged (4,700 x g, 10 min, 4°C). The organic and aqueous layers were aspirated, and the remaining protein disks were further washed by sonication in cold MeOH / CHCl3 solution (2 mL, 4:1) and pelleted by centrifugation (4,700 x g, 10 min, 4°C). The protein pellet was aspirated and combined with freshly prepared urea solution (500 μL, 6 M in DPBS) and SDS solution (10 μL of 10% w / v was used) by sonication. A freshly prepared solution (50 μL) of 1:1 TCEP (200 mM in DPBS) and K2CO3 (600 mM in DPBS) was added and the mixture was incubated at 37°C for 30 min with shaking. A freshly prepared iodoacetamide solution (70 μL, 400 mM in DPBS) was added and the mixture was incubated at RT, protected from light. Each sample was combined with SDS solution (130 μL, 10% in DPBS, w / v) followed by DPBS (5.5 mL) and incubated with pre-equilibrated streptavidin-agarose beads (100 μL of 50% slurry; Pierce) for 1.5 h at RT with rotation.The streptavidin beads were pelleted by centrifugation (750 g, 2 min, 4°C) and washed successively with SDS solution (1 x 5 mL, 0.2% in DPBS), DPBS (2 x 5 mL), and triethylammonium bicarbonate buffer (1 x 5 mL, TEAB, 100 mM, pH 8.4). The beads were resuspended in TEAB (0.5 mL, 100 mM, pH 8.5) and transferred to a LoBind microcentrifuge tube. To ensure complete transfer of the beads, the tube was washed once more with TEAB (0.5 mL, 100 mM, pH 8.5), the beads were then pelleted by centrifugation (750 x g, 2 min, 4°C), and the supernatant was aspirated. Beads from each sample were combined with CaCl2 solution (2 μL, 100 mM) and a solution of sequencing grade porcine trypsin (2 μg, Promega, 100 mM in 200 μL TEAB, pH 8.4) and incubated at 37°C for 14 h with shaking. Beads were pelleted by centrifugation (750 x g, 2 min, 4°C) and the supernatant was transferred to a new LoBind microcentrifuge tube. Each digested sample was labeled with TMT 10plex (Thermo Fisher Scientific): for each sample, a stock solution of TMT reagent (8 μL, 20 μg / μL) was added along with MS grade dehydrated acetonitrile (final acetonitrile concentration was 30%, v / v), followed by 1 h incubation at RT. The reaction was quenched by adding hydroxylamine (6 μL) and left for 15 minutes, followed by the addition of formic acid (5 μL). Each TMT-labeled sample was dried using a vacuum centrifuge, and the samples were combined by redissolving one sample in a solution of trifluoroacetic acid (TFA, 200 μL, 0.1%, in water) and transferring the solution to each sample tube until all samples were redissolved. The process was repeated with an additional amount of TFA solution (100 μL, in water, final volume 300 μL), and the combined samples were dried using a vacuum centrifuge.Samples were fractionated using a fractionation kit (Pierce High pH Reverse Phase Peptide Fractionation Kit) according to the manufacturer's instructions. Briefly, peptide fractions were eluted from the reverse phase spin column using successive solutions of 0.1% triethylamine combined with MeCN (5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 30%, 50%, 95% MeCN). Fractions were combined in pairs (fraction 1 and fraction 7, fraction 2 and fraction 8, etc.), dried using a vacuum centrifuge, and stored at -80 degrees until ready for injection.

[0201] Example 20: LC-MS analysis for proteomics TMT samples were analyzed using a Thermo Fisher Scientific Orbitrap Fusion Lumos mass spectrometer equipped with an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) according to previously reported procedures (Y. Wang et al., "Expedited mapping of the ligandable proteome using fully functionalized enantiomeric probe pairs", Nat. Chem. 11, 1113-1123 (2019); E. Joeh et al., "Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling", Proc. Natl. Acad. Sci. USA 117, 27329-27338 (2020)).

[0202] The dissolved sample (20 μl; 0.1% formic acid in water, v / v) was injected (3 μl / injection) onto an Acclaim PepMap RSLC analytical column (75 μm x 15 cm) equipped with an Acclaim PepMap 100 precolumn (75 μm x 2 mm) and eluted using the following gradient (300 μl / min, column temperature 35°C): 2% Buffer B (0.1% formic acid in acetonitrile) and 98% Buffer A (0.1% formic acid in water) for 10 min; Buffer B was increased to 30% over 192 min, then increased to 60% over 6 min, followed by an increase to 95% over 1 min and held constant for 5 min; Buffer B was decreased to 2% over 1 min and held there for 6 min. The voltage applied to the nanoLC electrospray source was 2.0 kV, and the MS 1 The spectrum is 1 x 10 at a resolution of 120,000. 6 The ion was acquired using automatic gain control (AGC) target values ​​and a maximum injection time of 50 ms. A data-dependent acquisition mode was used with a scan range of 375–1,500 m / z (repeat count 1, duration 20 s). Collision-induced dissociation (CID) was performed using MS 2 Peptide fragmentation (quadrupole ion trap analysis, AGC is 1.8x10 4 , CID collision energy was 30%, maximum injection time was 120 ms, and isolation window was 1.6), and MS 3 The precursors were fragmented by high-energy collision-induced dissociation (HCD, collision energy 65%). MS was detected using an Orbitrap by synchronous precursor selection (SPS). 3 Up to 10 MS in a spectrum 2 Fragment ions can now be included (resolution of 50,000, 1.5 x 10 5 AGC target value of 1, maximum injection time of 120 ms).

[0203] Example 21: Analysis of proteomic data Data processing was performed as previously reported (E. Joeh et al., "Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling", Proc. Natl. Acad. Sci. USA 117, 27329-27338 (2020)). The software package Proteome Discoverer 2.4 (Thermo Fisher Scientific) was used to determine peptide sequences using the Homo sapiens proteome database (42,358 sequences) with the SEQUEST HT algorithm (precursor mass tolerance 10 ppm, fragment ion mass tolerance 0.6 Da, one residual truncation allowed, targeted false discovery rate 1% (Percolator)). Static modifications were set for arbamidomethyl (C, +57.02146) and TMT tags (K and N-terminus, +229.1629), while oxidation (M, +15.994915) was set as a variable modification. Quantification of peptides was performed using MS 3 The analysis was performed at the 100% level with a mass tolerance of reporter ions set at 20 ppm. Subsequent statistical analysis was performed in Python 3.6.5. The abundances and percentages of TMTs obtained from Proteome Discoverer were transformed using log2(x) and p-values ​​were obtained using a t-test with two biological replicates. In addition, identified proteins were required to have a minimum of three unique peptides. Quantitative data are listed in Table 2 below. The mass spectrometry proteomics dataset has been deposited in the MassIVE repository under the dataset identifier MSV000086972.

[0204] Table 2 DZ2-CHD in H358 cells Sos Enriched proteins by MS-based proteomics using -5 photoaffinity crosslinking TIFF2024501019000037.tif221162TIFF2024501019000038.tif250161TIFF2024501019000039.tif250161TIFF2024501019000040.tif35161 a The structure of CP-266 = 3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)-N-methylpropanamide is: The file is TIFF2024501019000041.tif16128.

[0205] Example 22: Design of Sos tertiary helix mimetics The high-resolution structure of the Ras / Sos complex (PDB: 1NVW) shows the catalytic region of Sos binding to Ras as a helical hairpin composed of the αH and αI domains (see FIG. 2A). Computational alanine scanning mutagenesis suggests that the key Ras contacting, or hotspot, residues are present in the αH helix of the Sos hairpin (F929, T935, E942, and N944) and that the αI helix has the potential to associate with the ionic patch, as shown in Table 3 below (A. Patgiri, KK Yadav, PS Arora, D. Bar-Sagi, "An orthosteric inhibitor of the Ras-Sos interaction", Nat. Chem. Biol. 7, 585-587 (2011); T. Kortemme, DE Kim, D. Baker, "Computational alanine scanning of protein-protein interfaces", Sci. Signal. 2004, pl2 (2004)).

[0206] Table 3. Computational alanine scanning to determine hotspot residues in the Ras-Sos complex (PDB: 1NVW and 1BKD). Sos αH helix (929-944): TIFF2024501019000042.tif4128TIFF2024501019000043.tif81128

[0207] CHDs have been developed that have acquired the three-dimensional structure of the helix-loop-helix tertiary structures of αH and αI. The synthesis of CHDs requires the proper placement of crosslinkers at the solvent-exposed surfaces of the two helical segments in addition to enhancing intramolecular contacts at the dimer interface. To mimic the αH / αI helix dimer, the crosslinker is placed at the "e" position of the antiparallel helical construct. In a previous effort (MG Wuo, SH Hong, A. Singh, PS Arora, "Synthetic Control of Tertiary Helical Structures in Short Peptides", J. Am. Chem. Soc. 140, 16284-16290 (2018)), the synthesis of CHDs was optimized by placing a cysteine ​​residue at the "e" position followed by alkylation of the thiol group using a dibenzyl ether crosslinker (see Figure 2B).The native αH / αI interface features suboptimal knobs-into-holes packing; aromatic residues at the "a" and "d" positions were mutated to enhance conformational stability in minimal coiled-coil mimetics (JD Steinkruger et al., "The d'--d--d' vertical triad is less discriminating than the a'--a--a' vertical triad in the antiparallel coiled-coil dimer motif", J. Am. Chem. Soc. 134, 2626-2633 (2012); EB Hadley, OD Testa, DN Woolfson, SH Gellman, "Preferred side-chain constellations at antiparallel coiled-coil interfaces", Proc. Natl. Acad. Sci. USA 105, 530-535 (2008); JM Fletcher et al., "A basis set of de novo Coiled-coil peptide oligomers for rational protein design and synthetic biology", ACS Synth Biol 1, 240-250 (2012)). Other non-interacting residues were also mutated to increase intra- and inter-chain salt bridge interactions and to increase the conformational stability of the helical dimer (P. Lavigne, FD Sonnichsen, CM Kay, RS Hodges, "Interhelical salt bridges, coiled-coil stability, and specificity of dimerization", Science 271, 1136-1137 (1996)). The designed sequences are listed in Figure 2B and Figure 17 and Table 2.

[0208] Sos derivatives were rationally designed with the aid of two Rosetta-based computational web servers: Robetta (T. Kortemme, DE Kim, D. Baker, "Computational alanine scanning of protein-protein interfaces", Sci. Signal. 2004, pl2 (2004)) and Rosetta Backrub (F. Lauck, CA Smith, GF Friedland, EL Humphris, T. Kortemme, "RosettaBackrub--a web server for flexible backbone protein structure modeling and design", Nucleic Acids Res. 38, W569-575 (2010)). The Robetta server provides alanine scanning mutagenesis analysis using a static model of the protein surface, and Backrub suggests residue substitutions with potential to improve scores. These computer web servers were then followed by the visualization software Chimera (EF Pettersen et al., "UCSF Chimera - A visualization system for exploratory research and analysis", Journal of Computational Chemistry 25, 1605-1612 (2004)) to predict non-natural side chains, particularly those that associate with ionic patches on the protein surface. Modeling of the crystal structure of the Ras-Sos complex and apoRas suggests a potential ionic interaction between the K963 residue of the αI helix and the negatively charged patch on the switch I loop of Ras; the lysine residue of Sos is sandwiched between E31 and D33 of Ras. It was now hypothesized that the guanidine functionality of the arginine group could associate with both ionic residues of Ras, which is superior to the primary amine group of the lysine side chain.Based on this hypothesis, a K963R substitution was inserted into the designed CHD.

[0209] C.H.D. Sos Sos-1 most closely mimics the natural Sos hairpin sequence; fluorescently labeled derivatives of this proteomimetic have been shown to inhibit K d Sos binds to H-Ras with a binding affinity of β = 32 ± 6 μM, as measured in a fluorescence polarization (FP) assay using fluorescein-derivatized CHD (see FIG. 7). In the intracellular environment, the binding affinity of the Sos protein for Ras is highly dependent on the membrane localization of the two proteins. The reported K for the solution interaction of nucleotide-bound Ras with the catalytic domain of Sos is β = 32 ± 6 μM. d The value is 14.5 μM (H. Sondermann et al., "Structural analysis of autoinhibition in the Ras activator Son of sevenless", Cell 119, 393-405 (2004)). Sos The binding affinity of -1 is within the range of that predicted for a Sos fragment in a biochemical environment. Computational analysis performed by Rosetta Backrub showed that two contact residues (F929, E942) are not optimally associated with the Ras surface within their respective subpockets. The analysis suggested the substitutions F929W and E942N. CHD Sos Substitution of these two residues in -2 resulted in a 10-fold increase in binding affinity (K d = 2.8 ± 0.5 μM). Sos -3 (F929A, N944A) was designed as a negative control by replacing computationally predicted key αH binding residues with alanines. As predicted, the alanine double mutant bound with significantly reduced affinity (K d > 100 μM).

[0210] To analyze the importance of the ionic interaction between αI and the switch I loop of Ras, we Sos -4 was designed, in which the CHD Sos One cationic arginine residue in K-2 has been replaced by alanine (K963R). The replacement of the arginine group increases the binding affinity by 1.5%. d = 40 ± 20 μM, the binding analysis suggests that ionic interactions between αI and Ras are important for overall complex formation. Finally, L938 in αH was found to be located close to the negatively charged groove in Ras; Sos In -5, the arginine residue was mutated to a non-canonical homoarginine residue that may make ionic contacts. Sos -5 binds H-Ras with slightly improved affinity and inhibits CHD Sos Above -2, K d = 2.0 ± 0.3 μM. Sos This additional cationic residue at -5 also binds to the CHD Sos Its aqueous solubility is also improved compared to -2. Sos -6 is CHD Sos Affinity (K d = 8.6 ± 6.0 μM), indicating that a longer homoarginine side chain is required at this position.

[0211] Lead proteomimetic CHD Sos -5, and the negative control, CHD Sos The binding affinity of CHD-3 was further analyzed using microscale thermophoresis (MST). Sos -5 binds to fluorescently labeled Ras with nanomolar binding affinity (K d = 0.3 ± 0.1 μM, see Figure 2B, Figure 8; as with the polarization assay, the alanine mutant, CHD Sos -3 binds poorly to protein targets (Kd > 100 μM). The improved binding affinity observed with MST suggests that dye placement may not be optimal in fluorescence polarization assays (NJ Moerke, "Fluorescence Polarization (FP) Assays for Monitoring Peptide-Protein or Nucleic Acid-Protein Binding", Curr. Protoc. Chem. Biol. 1, 1-15 (2009)).

[0212] Example 23: CHD is a Sos proteomimetic Sos -5 is conformationally and proteolytically stable and binds to Ras at the dynamic switch region of Ras CHD is a Sos proteomimetic Sos The conformational stability of -5 was assessed using circular dichroism (CD) spectroscopy. CD provides a unique signature for α-helices, which have a maximum at 195 nm and minima at 208 and 222 nm (NR Kallenbach, PC Lyu, HX Zhou, "CD spectroscopy and the helix-coil transition in peptides and polypeptides", Circular Dichroism and the Conformational Analysis of Biomolecules, GD Fasman, Ed. (Plenum Press, New York, 1996); MC Manning, RW Woody, "Theoretical Cd Studies of Polypeptide Helices - Examination of Important Electronic and Geometric Factors", Biopolymers 31, 569-586 (1991)). CD experiments revealed that CHD SosThe results show that Sos-5 has clear features of a helix (Fig. 3A). In contrast, the individual peptides corresponding to the αH and αI of Sos, respectively, display a random coil-like signature (Fig. 9). Titration of equimolar unlinked peptides reveals the absence of a well-defined structure, whereas the cross-linked CHD Sos -2 showed prominent features of a helix.

[0213] Enzymatic proteolysis is a key factor limiting the potential of peptide therapeutics. Cross-linked helix dimers are resistant to proteolytic degradation due to their conformational stability (J. Sadek et al., "Modulation of virus-induced NF-kappaB signaling by NEMO coiled coil mimics", Nat Commun 11, 1786 (2020)). Sos The stability of -5 was analyzed in serum. Sos The rate of proteolysis of CHD-5 was determined in an HPLC-based time-course assay under ex vivo conditions. Sos -5 exhibits considerable resistance to proteolysis by serum proteases, which is 1 / 2 It is calculated that approximately 60% of the proteomimetics remain intact after 24 hours (FIG. 3B).

[0214] Combined MST and fluorescence polarization data indicate that CHD Sos It is shown that -5 binds to Ras with high nanomolar to low micromolar binding affinity. To determine whether the proteomimetic associates with Ras at the Sos-binding region of Ras as designed, titration heteronuclear single quantum coherence NMR spectroscopy (HSQC) experiments were performed, which monitor the changes in chemical shifts of specific protein residues upon ligand binding or conformational changes. 15Increasing concentrations (2.5 eq. and 5 eq.) of CHD against N-labeled H-Ras (100 μM). Sos -5 resulted in changes in peak shifts corresponding to residues within the switch I and switch II regions of Ras (Fig. 3C, Fig. 10). Sos The chemical shift changes observed after titration at -5 (5 eq.) are shown as bar graphs.

[0215] To further confirm the occupancy of the binding site, CHD Sos H-Ras was engineered to carry a diazirine moiety as a phototriggerable crosslinker at the N-terminus of the αI helix (Figure 3E). It was hypothesized that if association occurs at the Sos site, then upon irradiation of the diazirine, the resulting reactive carbene would covalently label the hinge region of Ras. H-Ras was synthesized using 5 eq. of DZ1-CHD Sos The complex was incubated with -5 and exposed to UV light. Agarose gel electrophoresis revealed a new, distinct protein band that was labeled at one site. Trypsinized proteins were analyzed by mass spectrometry to reveal cross-linked Ras fragments. Photocrosslinked and digested DZ1-CHD with the hinge region of Ras. Sos A fragment mass of -5 was observed (FIG. 3E, FIG. S11). Both the titration HSQC results and the chemical cross-linking results indicate that the designed proteomimetic binds to the Sos-binding surface of Ras.

[0216] Example 24: CHD Sos -5 exhibits selective cell membrane permeability in mutant Ras cancer cells In vitro results suggest that the engineered CHDs can alter Ras signaling. To induce effective intracellular changes, efficient uptake of the compounds into cells and efficient localization of the compounds in the cytoplasm within the cells are required. The mechanisms of delivery of peptidomimetics into cancer cells have recently been comprehensively analyzed. Efficient uptake of conformationally constrained peptidomimetics was observed to directly correlate with macropinocytosis activity in each cell line, regardless of the size, charge, and conformation of the peptidomimetic (DY Yoo et al., "Macropinocytosis as a Key Determinant of Peptidomimetic Uptake in Cancer Cells", J. Am. Chem. Soc. 142, 14461-14471 (2020); C. Commisso et al., "Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells", Nature 497, 633-637 (2013)).

[0217] Notably, despite its high molecular weight, CHD showed high levels of intracellular uptake and endosomal escape into the cytoplasm in macropinocytic cells - uptake of CHD was similar to that of Tat, a polycationic cell membrane penetrating peptide known to be highly trafficked into the cell interior (P. Lonn et al., "Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics", Scientific Reports 6, 32301 (2016); P. Lonn, SF Dowdy, "Cationic PTD / CPP-mediated macromolecular delivery: charging into the cell", Expert Opinion on Drug Delivery 12, 1627-1636 (2015)).

[0218] Macropinocytosis can be upregulated in cells with activating mutations in Ras or in cells with other mutations specific to the Ras pathway. Consistent with these previous analyses, live-cell fluorescence microscopy demonstrated the upregulation of fluorescein-labeled CHD into the cytosol of T24 bladder cancer cells (H-Ras G12V) and H358 lung cancer cells (K-Ras G12C) harboring Ras mutants. Sos Significant cellular uptake of CHD-5 was demonstrated (Figure 4A). Sos The intracellular intensity of -5 is similar to that of the fluorescently labeled Tat peptide (Figure 12A). To corroborate the microscopy studies, peptide uptake was quantified using flow cytometry analysis and CHD Sos Similar results were observed for -5 and Tat. Sos -2 and CHD Sos -3 inhibits CHD, possibly due to the removal of an arginine residue. SosThe low-temperature treated T24 and H358 cells showed reduced uptake of the fluorescently tagged CHD peptide compared to cells incubated at 37°C (Figure 4B, Figure 12B). As expected, reduced intracellular uptake of the peptide was observed in HeLa and BxPC3 cells, both of which have low macropinocytosis activity. Compared to cells incubated at 37°C, low-temperature treated T24 and H358 cells showed reduced uptake of the fluorescently tagged CHD peptide compared to cells incubated at 37°C (Figure 4B, Figure 12B). Sos -2 and CHD Sos Although we observed a >90% reduction in the uptake of α-5 (Figures 12C-12D), energy-dependent uptake pathways, including macropinocytosis, must be disrupted in these cells. Enhanced macropinocytosis activity is not restricted to cells with Ras mutations, and several other mutations are also known to upregulate this activity. For example, SW780 cells, which contain an oncogenic FGFR3 fusion, also upregulate CHD. Sos -5 is permeable (Figure 4B). Overall, the dependency of some cancer mutations on cell membrane permeability suggests that some cancers may be more amenable to therapeutic proteomimetics.

[0219] Example 25: CHD Sos-5 binds to wild-type and mutant isoforms of Ras CHD in cancer cells with mutant Ras Sos The enhanced cellular uptake of -5 suggests that Sos proteomimetics may have selective toxicity for these cancer cells. Based on this premise, we investigated the association of H-Ras with mutant forms, particularly those with G12 mutated to cysteine, aspartic acid, and valine (G12C, G12D, and G12V), which inhibits CHD. SosThe potential of -5 was investigated. Substitution of glycine residues with polar and β-branched residues alters the conformation of the switch region of Ras, which is dynamic between "open" and "closed", and these mutations are frequently observed in oncogenic Ras isoforms (S. Lu, H. Jang, R. Nussinov, J. Zhang, "The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B", Scientific Reports 6, 21949 (2016)). For in vitro binding analysis, the mutants were expressed as H-Ras constructs. Although activating mutations are more frequently found in K-Ras isoforms in human cancers, the Sos-binding surfaces of H-Ras and K-Ras are completely conserved, with differences mostly localized in the C-terminal hypervariable region involved in membrane anchoring of the Ras protein (E. Castellano, E. Santos, "Functional Specificity of Ras Isoforms: So Similar but So Different", Genes & Cancer 2, 216-231 (2011)). Fluorescein-derivatized CHD of wild-type and G12X mutant H-Ras proteins. Sos Titration of H-Ras-5 reveals that the proteomimetic binds to all Ras with similar affinities in the low micromolar range, albeit with a preference for wild-type (Figure 4C). Sos The binding affinity of -5 is predicted to apply to wild-type and mutant forms of K-Ras.

[0220] Example 26: CHD Sos -5 has selective toxicity against mutant Ras cancer cells by downregulating Ras signaling In vitro results indicate that CHD SosThis shows that -5 can bind to wild-type and mutant Ras with micromolar affinity and is a potent inhibitor of CHD. Sos The results of our previous study, which showed that Sos-5 has a high degree of selective permeability in Ras mutant cell lines, prompted us to evaluate the potential of Sos proteomimetics to inhibit Ras signaling in cells. Toxicity of Sos proteomimetics was evaluated using the MTT cell viability assay. In preliminary studies, the toxicity of Sos proteomimetics was evaluated in patients with CHD. Sos It was observed that -5 was able to function in the presence of serum. This result is important because peptides may be retained in the medium by hydrophobic components present in serum, and therefore cells often need to be subjected to treatment with peptides under artificial serum-free conditions to evaluate their potential in cells. As expected from the cell membrane permeability results, there was no significant increase in CHD against cell lines containing oncogenic Ras mutations compared to control HeLa cell lines containing wild-type Ras. Sos -2 and CHD Sos -5 showed concentration-dependent toxicity (Figure 4D, Figure 13A). Cell viability was shown to be inversely correlated with the level of intrinsic macropinocytic uptake, which differed between cell lines (Figure 4E). The results suggest that exploiting upregulated macropinocytosis offers a potential, previously untapped advantage for delivering therapeutics to mutant cancer cells. Importantly, the alanine control, CHD Sos -3 had little or no effect on the viability of the cell lines tested, suggesting that targeting of Ras results in cytotoxicity (Figure S13B). The results of the MTT assay were validated using a second cell viability assay based on the CellTiter-Glo luminescence system, which also showed no CHD inhibition against the mutant Ras cell line (H358) compared to the wild-type Ras cell line control (HeLa). Sos The selective toxicity of -5 was confirmed (Figure 13C).

[0221] C.H.D.Sos To demonstrate that the cytotoxicity of Sos-5 correlates with the downregulation of Ras signaling, the effect of the Sos proteomimetic on the intracellular concentrations of GTP-bound Ras and phosphorylated ERK was examined using probes. Sos It was determined whether treatment of cells with CHD-5 down-regulated Ras activation. Intracellular levels of Ras-GTP were assessed using a pull-down assay with the Ras-binding domain (RBD) of Raf1 (SJ Taylor, RJ Resnick, D. Shalloway, "Nonradioactive determination of Ras-GTP levels using activated ras interaction assay", Methods in Enzymology (Academic Press, 2001), vol. 333, pp. 333-342). The extent of Ras phosphorylation was measured in H358 cells by treatment with 10 μM CHD-5. Sos Ras-GTP levels were significantly decreased in the presence of -5 (Figure 14). Ras is a key mediator of multiple signaling pathways, and ERK activation is a well-studied node in the Ras effector pathway (R. Garcia-Gomez, XR Bustelo, P. Crespo, "Protein-Protein Interactions: Emerging Oncotargets in the RAS-ERK Pathway", Trends Cancer 4, 616-633 (2018)). Sos To determine whether the action of -5 results in the intended decrease in ERK phosphorylation, H358 and HeLa cells in complete medium were treated with increasing concentrations of CHD. Sos -3 or CHD Sos CHD cells were treated with 0.01% ethanol at -5. The resulting lysates were blotted for phosphorylated ERK. Sos-5 significantly reduced the activation level of ERK in a concentration-dependent manner in K-Ras G12C mutant cell lines (AA Samatar, PI Poulikakos, "Targeting RAS-ERK signalling in cancer: promises and challenges", Nat. Rev. Drug Discov. 13, 928-942 (2014)), and IC 50 Although CHD had a potent agonist activity of 1000 mg / kg / day, it had little effect in HeLa cells, whereas CHD had a potent agonist activity of 1000 mg / kg / day, whereas CHD had little effect in HeLa cells. This differential activity is consistent with the difference in cell membrane permeability of the proteomimetics between the two cell lines (Figures 4F-4H, Figure 15A). As expected, the alanine control, CHD Sos CHD-3 did not suppress ERK phosphorylation in any of the cell lines (Figures 4G-4H, and Figure 15B). Sos The observed downregulation of activated ERK upon treatment with -5 is consistent with the association of the proteomimetic with Ras in cells.

[0222] Example 27: Chemoproteomic analysis of CHD Sos -5's intracellular target revealed The Ras superfamily of small GTPases consists of more than 150 members and includes the subfamilies Ras, Rho, Rab, Arf, and Ran (D. Vigil, J. Cherfils, KL Rossman, CJ Der, "Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy?", Nat. Rev. Cancer 10, 842-857 (2010)). The superfamily has a highly conserved structure and sequence of the GTP / GDP nucleotide-binding domain, which associates with a variety of structurally conserved GEFs. The formation of the Ras-Sos complex is promoted by membrane localization of both proteins, but it is unclear how many Ras family members could associate with Sos if membrane recruitment was not a determining factor. A designed Sos proteomimetic, CHD Sos -5 mimics part of the nucleotide-binding domain of Sos but does not contain the membrane anchor. Sos It was hypothesized that -5 would have multiple intracellular partners and that chemoproteomic analysis would reveal its primary targets.

[0223] C.H.D. Sos To identify putative binding partners of -5, H358 lung cancer cells were subjected to photoaffinity labeling followed by enrichment and characterization of interactors by a well-established mass spectrometry-based proteomics protocol (CG Parker, MR Pratt, "Click Chemistry in Proteomic Investigations", Cell 180, 605-632 (2020)). Sos This was achieved using a variant of -5 with a photolabelable diazirine crosslinker attached in place of the unnatural homoarginine residue (DZ2-CHD Sos -5, Figure 5A).

[0224] For separate cell populations, DZ2-CHD Sos -5 (10 μM) in parallel with a negative control diazirine probe (CP-2-66, Table 2 above) as previously described (CG Parker et al., "Ligand and Target Discovery by Fragment-Based Screening in Human Cells", Cell 168, 527-541.e529 (2017)), and then exposing these cell populations to UV light (365 nm) to elicit DZ2-CHD Sos-5 induced photocrosslinking of bound protein targets. Cells were lysed, and probe-labeled proteins were conjugated with biotin azide via CuAAC, enriched using streptavidin-coated resin, and trypsinized as previously described in Y. Wang et al., "Expedited mapping of the ligandable proteome using fully functionalized enantiomeric probe pairs", Nat. Chem. 11, 1113-1123 (2019). Tryptic peptides from each population were subsequently labeled with isobaric tandem mass tags (TMTs) to allow for quantitative comparison (GC McAlister et al., "MultiNotch MS3 Enables Accurate, Sensitive, and Multiplexed Detection of Differential Expression across Cancer Cell Line Proteomes", Anal. Chem. 86, 7150-7158 (2014)) and analyzed by mass spectrometry (MS)-based proteomics. Proteins were considered targets if they were enriched >4-fold on average across biological duplicate experiments (p < 0.01; Table 2 above). Overall, 143 protein targets were identified, among which K-Ras, together with seven other members of the Ras GTPase superfamily; GTP-binding nuclear protein (RAN), and several Ras-related proteins (RAB13, RAB10, RAB14, RAB18, RAB5C, RAP1B), mediates the DZ2-CHD Sos -5 (Figure 5B).

[0225] Table 4A. Analysis of sequence similarity among enriched Ras GTPase proteins compared to K-Ras TIFF2024501019000044.tif67153

[0226] (Table 4B) Analysis of sequence similarity among enriched non-Ras G proteins compared to K-Ras. TIFF2024501019000045.tif56153

[0227] Tables 4A and 4B compare the sequences of Ras-related GTPases and other G proteins enriched in the proteomic analysis to that of human K-Ras. Each sequence was aligned and compared to K-Ras within the G domain (aa 1-166) and within the Sos αH / αI hairpin binding region (aa 10-40, 56-75). Sequence identity assesses the degree of completely conserved residues within the described region, while sequence similarity refers to residue substitutions that are similar and variable in chemical properties according to the Gonnet PAM 250 matrix (G. Gonnet, M. Cohen, S. Benner, "Exhaustive matching of the entire protein sequence database", Science 256, 1443-1445 (1992)).

[0228] The analysis suggests that there is a high degree of sequence similarity (50-70%) between the enriched Ras-related proteins and other enriched G proteins in the Sos helix hairpin binding region (J. Colicelli, "Human RAS Superfamily Proteins and Related GTPases", Sci. Signal. 2004, re13-re13 (2004)).

[0229] The enriched targets identified by photoaffinity labeling include proteins with a wide range of functions (Figure 5C). The majority of these interactors are localized within the intracellular compartment of cells, suggesting that upon internalization, CHD undergoes endosomal capture. SosThis supports the hypothesis that Sos-5 evades Ras GTPases (Figure 5C). The biological effects of using Sos proteomimetics to target other GTPases besides Ras, as well as proteins outside the Ras family, remain to be determined.

[0230] While preferred embodiments have been shown and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like, can be made therein without departing from the spirit of the invention, and therefore are deemed to be within the scope of the invention as defined in the appended claims.

[0231] Since various modifications may be made to the above without departing from the scope and spirit of the present invention, it is intended that all content contained in the above description or defined in the appended claims be construed as describing and illustrating the present invention. Many modifications and variations of the present invention are possible in light of the above teachings. Accordingly, the description of the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the scope of the appended claims.

[0232] All patents, patent applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety, as if they were physically presented herein.

[0233] References TIFF2024501019000046.tif218165TIFF2024501019000047.tif248165TIFF2024501019000048.tif199165

Claims

1. A macrostructure comprising an antiparallel coiled coil, wherein the antiparallel coiled coil comprises a first coil of formula I and a second coil of formula II: T 1 -g 0 -a 1 -b 1 -c 1 -d 1 -e 1 -f 1 -g 1 -a 2 -b 2 -c 2 -d 2 -e 2 -f 2 -g 2 -a 3 -b 3 -c 3 -d 3 -e 3 -f 3 -T 2 (I)、 T 3 -f' 0 -g' 0 -a' 1 -b' 1 -c' 1 -d' 1 -e' 1 -f' 1 -g' 1 -a' 2 -b' 2 -c' 2 -d' 2 -e' 2 -f' 2 -g' 2 -a' 3 -b' 3 -c' 3 -d' 3 -e' 3 -T 4 (II) Including, Where: a 1-3 , b 1-3 , c 1-3 , d 1-3 , e 1-3 , f 1-3 , g 0-2 , a' 1-3 , b' 1-3 , c' 1-3 , d' 1-3 , e' 1-3 , f' 0-2 , and g' 0-2 are each, independently, absent or a residue selected from the group consisting of modified or unmodified amino acid residues and analogs thereof; The following residue pairs: g 0 -g' 2 , g 1 -g' 1 , g 2 -g' 0 , a 1 -d' 3 , a 2 -d' 2 , a 3 -d' 1 , d 1 -a' 3 , d 2 -a' 2 , d 3 -a' 1 , e 1 -e' 3 , e 2 -e' 2 , and e 3 -e' 1 are covalently linked by a linker; T 1 and T 3 is each independently a point of attachment from the terminal nitrogen to one or more (preferably one or two) moieties, each moiety being independently H, -PG 1 , -C(O)R, -C(O)NR 2 , −C(O)NH 2 , -R, -C(O)OR, an amino acid or analog thereof, a peptide, a targeting moiety, or a tag; 1 is an amine protecting group, and each R is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, peptide, targeting moiety, or tag; and T 2 and T 4 are each independently from the terminal carbonyl, H, -OPG 2 , -NPG 2 , -OR, -OH, -NR 2 , -NH 2 , -N(R)C(O)C 1-6 Alkyl, -N(H)C(O)C 1-6 an attachment point to an alkyl, an amino acid or analog thereof, a peptide, a targeting moiety, or a tag; 2 is a protecting group for a carboxylic acid, and each R is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, peptide, targeting moiety, or tag; And here: The first coil comprises at least 10 consecutive residues, the at least 10 consecutive residues being of the formula X 1- X 2- X 3- X 4- X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14- X 15 -X 16 having The second coil comprises at least 10 consecutive residues, the at least 10 consecutive residues being of the formula X' 1 -X' 2 -X' 3 -X' 4 -X' 5 -X' 6 -X' 7 -X' 8 -X' 9 -X' 10 -X' 11 -X' 12 -X' 13 -X' 14- X' 15 -X' 16 and The residues are each from the group shown below (superscripts indicate the position of the respective residue in Formula I and Formula II; residues at positions a, a', d, d', e, e', g, and g' can optionally be modified or replaced with a linker to facilitate attachment of the linker; underlined residues are particularly preferred): Selected from: Macro structure.

2. Residue pair g 0 -g' 2 , g 1 -g' 1 , g 2 -g' 0 , e 1 -e' 3 , e 2 -e' 2 , and e 3 -e' 1 is such that the spatial distance between the Cα positions of each residue in the pair is 10-25 Å; and Residue pair a 1 -d' 3 , a 2 -d' 2 , a 3 -d' 1 , d 1 -a' 3 , d 2 -a' 2 , and d 3 -a' 1 is such that the spatial distance between the Cα positions of each residue in the pair is 5-15 Å; The macrostructure of claim 1.

3. At least g 0 , a 1 , b 1 , c 1 , d 1 , e 1 , f 1 , g 1 , a 2 , b 2 , c 2 , d 2 , e 2 , f 2 , g 2 , and a 3 is present in the first coil, and at least d' 1 , e' 1 , f' 1 , g' 1 , a' 2 , b' 2 , c' 2 , d' 2 , e' 2 , f' 2 , g' 2 , a' 3 , b' 3 , c' 3 , d' 3 , and e' 3 The macrostructure of claim 1 or claim 2, wherein the second coil is provided with a first coil.

4. Each residue independently has the formula: having Where: R 1a , R 1b , R 1c , and R 1d are each independently hydrogen, an amino acid side chain, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a heterocyclyl, an aryl, a heteroaryl, or an arylalkyl, and each of the amino acid side chain, the alkyl, the alkenyl, the alkynyl, the cycloalkyl, the heterocyclyl, the aryl, the heteroaryl, and the arylalkyl is each independently H, an alkyl, an alkenyl, an alkynyl, an azide, -OR 5 , or -SR 5 and when the linker is covalently attached to the residue, the linker is 1a , R 1b , R 1c , and R 1d or is substituted for one of the following: R 4 is each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or arylalkyl; and R 5 are each independently selected from the group consisting of H, -PG (wherein PG is a protecting group), alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and arylalkyl; The macrostructure according to any one of claims 1 to 3.

5. Residue pair g 0 -g' 2 , g 1 -g' 1 , g 2 -g' 0 , e 1 -e' 3 , e 2 -e' 2 , and e 3 -e' 1 The macrostructure according to any one of claims 1 to 4, wherein there is a linker between at least one of

6. Residue pair g 0 -g' 2 , g 1 -g' 1 , g 2 -g' 0 , e 1 -e' 3 , e 2 -e' 2 , and e 3 -e' 1 and wherein the linker between at least one of the n -, wherein n is a number from 1 to 25 and each Z is independently at each occurrence selected from the group consisting of alkylene, alkenylene, arylene, heteroarylene, triazole-diyl, thiazole-diyl, oxazole-diyl, ether, amide, ester, maleimide, thioether, O, S, and Se.

7. Residue pair g 0 -g' 2 , g 1 -g' 1 , g 2 -g' 0 , e 1 -e' 3 , e 2 -e' 2 , and e 3 -e' 1 and a linker between at least one of (a) The group consisting of (b) A group consisting of having a formula selected from: Where: In group (a) and group (b), X is O, S, CR 2 , NR, or P (preferably O, S, CH 2 or NR); X in group (a) 1 is each independently O, S, NH, or NR; X in group (b) 1 is each independently O, S, C, CR, N, NH, or NR; R in groups (a) and (b) is each independently H, alkyl, or aryl; and Y in group (a) and group (b) is S. The macrostructure according to any one of claims 1 to 6.

8. Residue pair g 0 -g' 2 , g 1 -g' 1 , g 2 -g' 0 , e 1 -e' 3 , e 2 -e' 2 , and e 3 -e' 1 and a linker between at least one of Selected from the group consisting of: The macrostructure according to any one of claims 1 to 7.

9. Residue pair a 1 -d' 3 , a 2 -d' 2 , a 3 -d' 1 , d 1 -a' 3 , d 2 -a' 2 , and d 3 -a' 1 The macrostructure according to any one of claims 1 to 8, wherein there is a linker between at least one of

10. Residue pair a 1 -d' 3 , a 2 -d' 2 , a 3 -d' 1 , d 1 -a' 3 , d 2 -a' 2 , and d 3 -a' 1 At least one of the linkers between the 1-8 Alkylene, C 2-8 The macrostructure of any one of claims 1 to 9, selected from the group consisting of alkenylene, arylene, heteroarylene, triazole-diyl, and thiazole-diyl.

11. Residue pair a 1 -d' 3 , a 2 -d' 2 , a 3 -d' 1 , d 1 -a' 3 , d 2 -a' 2 , and d 3 -a' 1 The macrostructure according to any one of claims 1 to 10, wherein the linker between at least one of is a disulfide bond derived from a cysteine ​​or homocysteine ​​residue, a diselenide derived from a selenocysteine ​​residue, an alkylene derived from an allylglycine residue, or an arylene linker.

12. The antiparallel coiled coil has the following formula III: The Where: each dotted line independently represents an optional linker; and Each residue independently has the formula: having Where: R 1a , R 1b , R 1c , and R 1d are each independently hydrogen, an amino acid side chain, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a heterocyclyl, an aryl, a heteroaryl, or an arylalkyl, and each of the amino acid side chains, alkyl, alkenyl, alkynyl, cycloalkyl, a heterocyclyl, an aryl, a heteroaryl, and an arylalkyl is H, alkyl, alkenyl, alkynyl, azide, -OR 5 , or -SR 5 and when the linker is covalently attached to the residue, the linker is 1a , R 1b , R 1c , and R 1d or is substituted for one of the following: R 4 is each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or arylalkyl; and R 5 are each independently selected from the group consisting of H, -PG (wherein PG is a protecting group), alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and arylalkyl; The macrostructure according to any one of claims 1 to 11.

13. The following conditions: (A) in at least one a, a', d, or d' residue, (i) R 1a and R 1c is a side chain of a modified or unmodified amino acid selected from the group consisting of cysteine, homocysteine, selenocysteine, leucine, isoleucine, hexafluoroleucine, valine, hexafluorovaline, allylglycine, threonine, and analogs of each of the foregoing residues; and (ii) R 1b , R 1d , and R 1a and R 1c each of the other independently represents hydrogen, C 1-3 Alkyl, or C 2-3 being alkenyl; (B) at least one e, e', g, or g' residue, 1a and R 1c is an amino acid side chain, and (ii) R 1b , R 1d , and R 1a and R 1c each independently represents hydrogen or C 1-3 Being alkyl At least one of the following is satisfied: The macrostructure of claim 12.

14. The antiparallel coiled coil has the formula: having wherein each dotted line is independently an optional linker; The macrostructure according to any one of claims 1 to 13.

15. The macrostructure according to any one of claims 1 to 14, which is CHD-1, CHD-2, CHD-3, CHD-4 or CHD-5.

16. A pharmaceutical composition comprising the macrostructure according to any one of claims 1 to 15 and a pharma- ceutically acceptable vehicle.

17. A pharmaceutical composition for use in a method for inhibiting Ras signaling in a cell, comprising the macrostructure according to any one of claims 1 to 15, The method includes contacting a cell with the macrostructure under conditions effective to inhibit Ras signaling in the cell. Pharmaceutical compositions.

18. 18. The pharmaceutical composition of claim 17, wherein the cell is a mammalian cell.

19. 19. The pharmaceutical composition of claim 17 or claim 18, wherein the cells express a mutant Ras protein.

20. The pharmaceutical composition of any one of claims 17 to 19, wherein the contacting step is performed in a subject.

21. A pharmaceutical composition for treating a disorder mediated by Ras signaling in a subject, comprising the macrostructure described in any one of claims 1 to 15.

22. A pharmaceutical composition for treating a cell proliferative disorder, a differentiation disorder, and / or a neoplastic condition in a subject in need thereof, comprising the macrostructure of any one of claims 1 to 15.

23. The cell proliferative, differentiation, and / or neoplastic pathology is selected from the group consisting of fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, brain cancer, squamous cell carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, and epithelial carcinoma.

23. The pharmaceutical composition of claim 22, wherein the therapeutic agent is selected from the group consisting of: glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, and Kaposi's sarcoma; neoplastic abnormalities of the hematopoietic system; cell proliferative and / or differentiating abnormalities of the breast; cell proliferative and / or differentiating abnormalities of the lung; cell proliferative and / or differentiating abnormalities of the colon; cell proliferative and / or differentiating abnormalities of the liver; cell proliferative and / or differentiating abnormalities of the ovary; cancers mediated by mutant Ras proteins; and immunoproliferative abnormalities.

24. 24. The pharmaceutical composition of any one of claims 21 to 23, wherein the disorder and / or condition is pancreatic ductal adenocarcinoma, rectal adenocarcinoma, plasma cell myeloma, colon adenocarcinoma, cholangiocarcinoma, chronic myelomonocytic leukemia, acute myeloid leukemia, melanoma, lung adenocarcinoma, rhabdomyosarcoma, endometrial cancer, salivary gland cancer, thyroid cancer, bladder cancer, oral cancer, or ovarian cancer.

25. The pharmaceutical composition according to any one of claims 20 to 24, wherein the subject is a mammal.

26. The pharmaceutical composition of any one of claims 20 to 25, wherein the subject is a primate (e.g., a human).

27. The pharmaceutical composition of any one of claims 21 to 26, wherein the disorder or pathology is mediated by a mutant Ras protein.

28. 28. The pharmaceutical composition of claim 19 or claim 27, wherein the mutant Ras protein is an H-Ras isoform.

29. 28. The pharmaceutical composition of claim 19 or claim 27, wherein the mutant Ras protein is a K-Ras isoform.

30. 30. The pharmaceutical composition of any one of claims 19 and 27-29, wherein the mutant Ras protein has one or more mutations selected from the group consisting of G12C, G12D, G12S, G12V, G13D, G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13R, G13S, G13D, Q61E, Q61H, Q61L, Q61K, Q61P, and Q61R.

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