BRAG2 inhibitors and their applications

Abstract

The present invention relates to a compound having the following chemical structure (I): The present invention relates to molecules having the formula: The present invention particularly relates to molecules active as BRAG2 inhibitors and their applications. In particular, the present invention relates to BRAG2 inhibitors in the treatment of cancer or angiogenesis.

Description

[Technical Field]

[0001] This invention relates in particular to molecules active as BRAG2 inhibitors and their applications. In particular, this invention relates to BRAG2 inhibitors in the treatment of cancer or angiogenesis. [Background technology]

[0002] Conventional technology Cells respond to changes in their environment by assembling dynamic signaling complexes on their membrane surfaces that receive signals and transmit information. Such signaling platforms are often dysfunctional in disease, either due to mutations affecting their regulation or because they are used by pathological pathways. This makes drugs that modulate their activity highly sought after, yet signaling nodes remain challenging targets for conventional antagonistic inhibitors due to their structural flexibility (typically involving large conformational changes, their extensive protein-protein interfaces, and their diverse protein-lipid interactions). Consequently, current drug development efforts focus on developing novel strategies that utilize the structural features of membrane-bound signaling complexes.

[0003] Low molecular weight GTPases and their regulators belong to the category of superficial membrane proteins involved in pathological conditions. The large family of low molecular weight GTPases are major components of the membrane surface signaling platform, playing crucial roles in signal transduction, cell motility, membrane translocation, and coordination between these pathways. Due to their importance in maintaining homeostasis in normal cells, low molecular weight GTPase function is distorted or hijacked in various pathological conditions, such as cancer, cardiovascular disease, and bacterial or viral infections. Therefore, inhibiting their activity in pathological contexts is a crucial, unmet need in drug development. The regulation of low molecular weight GTPases is highly complex, as GTP-binding GTPases link the GDP / GTP switch and the cytosolic / membrane cycle when attached to the membrane. In addition, the output of GTPase signaling involves multiple activators (guanine nucleotide exchange factors or GEFs that stimulate GDP / GTP exchange), inhibitors (GTPase-activating proteins or GAPs that accelerate GTP hydrolysis and GDIs that encapsulate their lipid anchors to solubilize them), and effectors that assemble the signaling platform as a whole. GEFs, GAPs, and effectors highly modulate themselves through structural rearrangement and protein-membrane interactions. To date, several strategies have inhibited the membrane / cytosolic cycle by targeting either the enzymes or GDIs involved in anchoring their lipid post-translational modifications. However, inhibitors that directly target regulatory protein-membrane interactions have never been described.

[0004] Superficial membrane proteins modulate cellular responses to signals originating from their environment, and therefore are involved in numerous diseases. Despite their significance, they remain unresolved targets for conventional antagonistic inhibitors, and alternative approaches are urgently needed. [Overview of the Initiative]

[0005] Objective of the present invention The present invention aims to solve the technical problem of providing BRAG2 inhibitors and their applications. The present invention aims to solve the technical problem of providing a novel pathway for the treatment of cancer or angiogenesis.

[0006] More particularly, the present invention aims to solve the technical problem of providing a novel pathway for the treatment of cancer or angiogenesis by BRAG2 inhibitors. The present invention also aims to solve the technical problem of providing a molecule that binds to the protein-membrane interface.

[0007] Detailed Description of the Invention Detailed Description The present invention solves at least one, and preferably all, of the technical problems defined in the present invention.

[0008] In particular, the present invention relates to the following chemical structure (I) for use in a method of therapeutic treatment: [Chemical formula] {Wherein, R1 is a fluoroalkyl, preferably CF3; R3 is a chemical group containing at least one oxygen and / or nitrogen; R2, R4, R5 and R6 are independently atoms or atomic groups}, or a pharmaceutically acceptable salt thereof or a prodrug thereof.

[0009] The present invention also relates to the following chemical structure (I): [Chemical formula] {Wherein R1 is a fluoroalkyl, preferably CF3; R3 is a chemical group containing at least one oxygen and / or nitrogen; R6 is an atom or atomic group different from hydrogen; R2, R4 and R5 are independently atoms or atomic groups} Regarding molecules that possess the following properties.

[0010] In particular, the present invention relates to a BRAG2 inhibitor having the molecular structure defined in the present invention. BRAG2 refers to the protein Brefeldin-resistant Arf-GEF2 protein (SEQ ID NO: 1) (IQ motif and SEC7 domain-containing protein 1, i.e., see UniProtKB-Q6DN90(IQEC1_HUMAN)-SEQ ID NO: 1). This protein is also called ADP-ribosylation factor guanine nucleotide exchange protein 100 or ADP-ribosylation factor guanine nucleotide exchange protein 2. Another isoform of BRAG2 exists, called UniProtKB-A0A087WWK8 (SEQ ID NO: 10). This isoform shares a high degree of sequence identity (same PH domain) but differs mainly in its longer N-terminus. The BRAG2 inhibitors of the present invention that inhibit BRAG2 (Q6DN90) are thought to also inhibit this isoform (A0A087WWK8) because they bind to the PH domain.

[0011] In particular, the present invention relates to non-competitive inhibition of protein-membrane interactions having potent and selective inhibition of membrane-binding regulators of low molecular weight GTPases. The molecule according to the present invention is called Bragsin and inhibits the activation of Arf GTPases. Such inhibition is carried out, for example, in vitro by their guanine nucleotide exchanger BRAG2, and this effect is specific and manifest only in the presence of a membrane. Advantageously, in cells, the molecule according to the present invention affects the trans-Golgi network, and this effect is rescued by ectopic expression of BRAG2 or constitutive activity of Arf, and phenotypicly mimicked by silencing of the BRAG2 gene. [Brief explanation of the drawing]

[0012] Regarding the drawings: [Figure 1]Bragsin2 affects the Arf pathway in cells. a. Chemical structures of Bragsin1, Bragsin2 and derivatives used in this study. Chemical synthesis and structural characterization of the compounds are described in Example 6. b. Bragsin2 disperses the TGN46 and GM130 markers. HeLa cells were treated with either DMSO (0.25%) or Bragsin2 (50 μM) for 30 minutes, immunostained for TGN46, GM130, or EEA1 (green channel), and analyzed by confocal microscopy. The effect of Bragsin1 treatment is shown in Figure 11. c. Dispersion of GM130 and TGN46 by Bragsin2 is reversible. HeLa cells were treated as shown in Figure 1a, incubated in fresh medium for a further 30 minutes (wash panel), and then immunostained and analyzed by confocal microscopy. The reversibility of the effect of Bragsin1 is shown in Figure 12. d. Expression of an Arf-mCherry construct carrying an activating mutation restores the effect of Bragsin2. HeLa cells were transfected with a constitutively active Q / L mutant of Arf-mCherry. Note the difference between untransfected cells (white asterisks) and transfected cells. The restoration of the Bragsin1 phenotype by the constitutively active Arf mutant is shown in Figure 13. Scale bar, 10 μm. [Figure 2]Bragsin is a specific inhibitor of ArfGEF BRAG2. a. Bragsin1 has no effect on the Sec7 domain of human ArfGEF in solution. Nucleotide exchange kinetics were measured by fluorescence kinetics in the presence of Bragsin1 (50 μM) or DMSO, using a purified Sec7 domain and a shortened version of Arf1 (D17Arf1) (which can be activated in solution). A representative kinetic profile is shown in Figure 14. b. Bragsin1 specifically inhibits BRAG2 in the presence of liposomes. Nucleotide exchange kinetics were measured in the presence of Bragsin1 (50 μM) or DMSO, using an ArfGEF construct carrying a membrane-bound domain and myristoylated Arf1. Rac1 was artificially ligated to liposomes by a C-terminal hexahistidine tag as described in (Peurois, F. et al. Biochem J 474, 1259-1272 (2017)). A typical kinetic profile is shown in Figure 15. c. Bragsin1 inhibits the activation of myristoylated Arf6 by BRAG2 on liposomes. The experiment was performed as shown in Figure 2b. A typical kinetic profile is shown in Figure 16. d. Dose-response of Bragsin1 and Bragsin2 directed toward myristoylated Arf1 and BRAG2 on liposomes. The experiment was performed as shown in Figure 2b. e. Silencing of BRAG2 phenotypic mimicry, effect of Bragsin2 on the TGN46 compartment. HeLa cells were treated with DMSO or Bragsin2 (50 μM) or transfected with siRNA targeting BRAG2, ARNO, or GBF1 or control siRNA (siCTRL). Immunofluorescence staining of TGN46 is green (gray in black and white figures). Cell boundaries were highlighted by actin staining (magenta (gray in black and white figures)). SiORNA silencing efficiency is shown in Figure 17. Scale bar, 10 μm. Statistical analysis of TGN46 staining dispersion is shown in Figure 19. f. Overexpression of BRAG2 restores Bragsin-induced dispersion of the TGN46 compartment.HeLa cells were transfected with BRAG2-mCherry (magenta channel) and treated with Bragsin2 (50 μM). Immunofluorescence staining of TGN46 is green (gray in the black and white figure). [Figure 3] Bragsin binds to the PH domain of BRAG2. a. Crystal structure of Bragsin1 binding to the PH domain of BRAG2. The inset shows an electron density omit map of the inhibitor. The Sec7 domain is pink, the linker is yellow, and the PH domain is blue. b. Interaction between residues in the PH domain and Bragsin1. Hydrogen bonds are shown by dotted lines. c. Analysis of inhibition of BRAG2 mutants by Bragsin2. GEF efficiency was measured by fluorescence kinetics using myristoylated Arf1 as shown in Figure 2b. d. Structure-activity relationship analysis of Bragsin analogs. All compounds were used at 20 mM. GEF activity was measured by fluorescence kinetics using myristoylated Arf1 as shown in Figure 2b. e. Effect of Bragsin analogs on the TGN46 compartment. HeLa cells treated with the compound (50 mM) were immunostained as shown in Figure 1b and analyzed by confocal microscopy. [Figure 4]Bragsin is a non-competitive inhibitor of protein-membrane interactions. a. Bragsin1 (violet) overlaps with the standard phosphoinositide binding site of the PH domain of BRAG2. IP3 (red) is derived from the GRP1-IP3 complex (DiNitto, JP et al. Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors. Mol Cell 28, 569-83 (2007)). Membrane locations are indicated by arrows. b. Bragsin2 does not impair the binding of BRAG2 to liposomes. Binding was measured by liposome suspension. U: Bottom fraction containing unbound proteins. B: Top fraction containing liposome-bound proteins. Proteins are revealed by Instant Blue staining after SDS-PAGE. Quantification is shown below. c. Inhibition of BRAG2 by Bragsin2 is more potent in the presence of liposomes containing PIP2 lipids. The dynamics were measured as shown in Figure 2b. [Figure 5] Bragsin affects breast cancer stem cells. a. Development of the percentage of ALDHbr cells after treatment with Bragsin2 using the Aldefluor assay. Results are expressed as mean ± SD. b. Tumor mass formation efficiency (SFE) was calculated using the extreme dilution analysis (ELDA) algorithm. Results are expressed as the estimated number of tumor masses obtained for 100 cells in a plate culture. [Figure 6] A model of protein-membrane boundary inhibition by Bragsin. [Figure 7] Figures 7-9. Analysis of the stability of Bragsin1 and Bragsin2. Figure 7. Bragsin1, kept in its original state in DMSO, loses its activity toward BRAG2 over several months (not shown). 1H-NMR analysis showed that it was completely degraded into a hydrated compound (13). [Figure 8]Figures 8-9 show that mass spectrometry results indicate that Bragsin1 is less stable than Bragsin2. Both Bragsin1 and Bragsin2 were dissolved in PBS. While Bragsin2 remained stable (Figure c), Bragsin1 slowly degraded into hydrolyzable compounds within 72 hours (Figure 8). [Figure 9] Figures 8-9 show that mass spectrometry results indicate that Bragsin1 is less stable than Bragsin2. Both Bragsin1 and Bragsin2 were dissolved in PBS. While Bragsin2 remained stable (Figure c), Bragsin1 slowly degraded into hydrolyzable compounds within 72 hours (Figure 8). [Figure 10] Figures 10-13. Bragsin1 has the same cellular effect as Bragsin2. Figure 10: Bragsin2 has no effect on the tubulin and actin networks in HeLa cells. HeLa cells were treated with either DMSO (0.25%) or Bragsin2 (50 μM) for 30 minutes, immunostained for α-tubulin (green channels) or F-actin (magenta channels), and analyzed by confocal microscopy. [Figure 11] Figure 11: Bragsin1 disperses TGN46 and GM130 markers. HeLa cells were treated with 50 μM Bragsin1 as shown in Figure 10, immunostained for TGN46 or GM130 (green channel), and then subjected to confocal microscopy analysis. [Figure 12] Figure 12: Dispersion of GM130 and TGN46 by Bragsin1 is reversible. After 30 minutes of treatment with either DMSO (0.25%) or Bragsin1 (50 μM), HeLa cells were washed and incubated in fresh medium for another 30 minutes (wash), then immunostained with TGN46 or GM130 (green channel), and confocal microscopy analysis was performed. [Figure 13]Figure 13: Expression of constitutively active Arf1, Arf5, and Arf6 constructs restores the effect of Bragsin1. HeLa cells were transiently transfected with plasmids encoding Q / L mutants of the indicated Arf-mCherry isoforms; untransfected cells are marked with white asterisks. Scale bar, 10 μm. [Figure 14] Figures 14-19. Bragsin is a specific inhibitor of BRAG2. 14: Tracking of representative fluorescence dynamics from the experiment shown in Figure 2a. GDP / GTP nucleotide exchange of N-terminal cleaved Arf-GDP was tracked in solution by tryptophan fluorescence in the presence of 50 μM Bragsin1 (light gray) or 0.25% DMSO (dark gray) after the addition of a catalytic amount of the indicated ArfGEF Sec7 domain and an excess amount of GTP (see Materials and Methods for details). The natural curve (black) corresponds to Arf activation in the absence of GEF. [Figure 15] 15: Tracking of typical fluorescence dynamics in the experiment shown in Figure 2b. MyrArf1 or Rac1 activation by catalytically controlled GEF was performed as in Figure 14, except that the experiment contained 100 μM liposomes (see Materials and Methods for details; Example 7). [Figure 16] 16: Tracking of typical fluorescence dynamics in the experiment shown in Figure 2c. Activation of myrArf6 was performed in the presence of liposomes, as described in Figure 15. [Figure 17]17-18: RT-qPCR analysis of ARNO, BRAG2, and GBF1 mRNA levels. HeLa cells were transfected with siRNA-targeted BRAG2 (siBRAG2, light gray bar graph), ARNO (siARNO, medium gray bar graph), GBF1 (siGBF1, dark gray bar graph), or untargeted siRNA (siCTRL, white bar graph) as a control (Figure 17), or treated with Bragsin2 (dark gray bar graph) or DMSO as a control (white bar graph) (Figure 18). Total RNA was extracted, reverse transcribed, and subjected to real-time PCR using primer pairs specific to mRNA encoding ARNO, BRAG2, or GBF1 (as shown in the graph below). The graph shows the amount of mRNA relative to a control value set to 100%. [Figure 18] 17-18: RT-qPCR analysis of ARNO, BRAG2, and GBF1 mRNA levels. HeLa cells were transfected with siRNA-targeted BRAG2 (siBRAG2, light gray bar graph), ARNO (siARNO, medium gray bar graph), GBF1 (siGBF1, dark gray bar graph), or untargeted siRNA (siCTRL, white bar graph) as a control (Figure 17), or treated with Bragsin2 (dark gray bar graph) or DMSO as a control (white bar graph) (Figure 18). Total RNA was extracted, reverse transcribed, and subjected to real-time PCR using primer pairs specific to mRNA encoding ARNO, BRAG2, or GBF1 (as shown in the graph below). The graph shows the amount of mRNA relative to a control value set to 100%. [Figure 19] 19: Statistical analysis of the variance of TGN46 staining in the experiment shown in Figure 2e. The areas of cells (polygons) and TGN (minimum ellipse) were manually determined after total Z projection of the deposit and measured using Fiji / ImageJ. Their ratios represent the relative area (%) occupied by TGN46 staining in the cells. Statistical analysis was performed using ANOVA with Tukeys' post-hoc test. Counted cells > 200, *p < 0.05, ****p < 0.0001. [Figure 20] 20: This study demonstrates that Bragsin3 (compound 14) potently inhibits BRAG2 in the presence of liposomes. Nucleotide exchange dynamics were measured in the presence of Bragsin3 (50 μM) or DMSO with the BRAG2Sec7PH construct and myristoylated Arf1 (see Materials and Methods for details). [Figure 21] 21: Bragsin3 disperses the TGN46 marker. HeLa cells were treated with 50 mM Bragsin3 as shown in Figure 1b, then immunostained for TGN46, and subsequently subjected to confocal microscopy analysis. [Figure 22] 22: The viability and cytotoxicity of Bragsine2, compound 14, and compound 3 against the cell line MDA MB231 are shown. [Figure 23] 23: The viability and cytotoxicity of Bragsine2, compound 14, and compound 3 against the cell line MCF7 are shown. [Figure 24] 24: Shows the survival rates of Bragsine2, compound 14, and compound 3 in cell line SUM149. [Figure 25] 25: The viability and cytotoxicity of Bragsine2, compound 14, and compound 3 against the cell line SUM159 are shown. [Figure 26] 26: The viability and cytotoxicity of Bragsine2, compound 14, and compound 3 against cell line A549 are shown. [Figure 27] 27: The viability and cytotoxicity of Bragsine2, compound 14, and compound 3 against the cell line U87-MG are shown. [Figure 28] 28: The viability and cytotoxicity of Bragsine2, compound 14, and compound 3 against the cell line PANC-1 are shown. [Figure 29]29: Shows the viability and cytotoxicity of Bragsin2, compound 14, and compound 3 against cell lines. Viability tests (measurement of ATP levels after cell lysis, CelltiterGlo, Promega) and cytotoxicity (measurement of LDH levels after cell lysis, CytoTox-One, Promega). The results in Figures 22-29 were analyzed by one-way ANOVA and subsequent Tukey multiple comparisons (vs. DMSO), with *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Bragsin2: 6-Methoxy-5-nitro-2-(trifluoromethyl)-4H-chromen-4-one. Compound 14(2MetO): 6,8-Dimethoxy-5-nitro-2-(trifluoromethyl)-4H-chromen-4-one. Compound 3(-NO2):6-methoxy-2-(trifluoromethyl)-4H-chromen-4-one (negative control). [Modes for carrying out the invention]

[0013] In one embodiment, the molecule is as follows: [ka] {In the formula, R' is a chemical group of an atom, for example, an optionally substituted alkyl, or COOR' forms an ester salt, for example, a sodium ester} Selected from the group consisting of

[0014] In one embodiment, R4 is selected from the group consisting of hydrogen, hydroxyl, alkyl, preferably methyl (Me) or ethyl (Et), O-alkyl (or alkoxy), preferably OMe or OEt, alkene, O-alkylene, alkyne, preferably -CCH, or O-alkyne, preferably -OCH2-CCH.

[0015] In one embodiment, R6 is selected from the group consisting of hydrogen, hydroxyl, alkyl, preferably methyl (Me) or ethyl (Et), O-alkyl, preferably OMe or OEt, alkene, O-alkylene, alkyne, preferably -CCH, or O-alkyne, preferably -OCH2-CCH.

[0016] In one embodiment, R5 is hydrogen, hydroxy, alkyl, preferably methyl (Me) Alternatively, it is selected from the group consisting of ethyl (Et), O-alkyl, preferably OMe or OEt, alkene, O-alkylene, alkyne, preferably -CCH, or O-alkyne, preferably -OCH2-CCH.

[0017] In one embodiment, R2 is H. In one embodiment, R4 is H. In one embodiment, R5 is H. In one embodiment, R6 is H.

[0018] In one embodiment, R6 is selected from the group consisting of hydroxy, alkyl, preferably methyl (Me) or ethyl (Et), O-alkyl (or alkoxy), preferably OMe or OEt, alkene, O-alkylene, alkyne, preferably -CCH, or O-alkyne, preferably -OCH2-CCH.

[0019] In one embodiment, R6 is OMe. In one embodiment, R2, R5, and R6 are hydrogen atoms.

[0020] In one embodiment, the molecule is as follows: [ka] Selected from the group consisting of

[0021] In one embodiment, the molecule is as follows: [ka] Selected from the group consisting of

[0022] In one embodiment, R1 is CF3. In one embodiment, R3 is NO2. In one embodiment, R3 is NO2 and R1 is CF3.

[0023] As used herein, the term “alkyl group” means a saturated linear or branched acyclic hydrocarbon having 1 to 10 carbon atoms. Representative saturated linear alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl; on the other hand, saturated branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, and 2,4-dimethylhexyl. Examples include xyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3-dimethylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and 3,3-diethylhexyl. The alkyl group contained in the compounds of the present invention may be optionally substituted with one or more substituents.

[0024] As used herein, the terms “alkoxy” or “O-alkyl” refer to an alkyl group linked to another part by an oxygen atom. Examples of alkoxy groups include methoxy, isopropoxyethoxy, and tert-butoxy. The alkoxy group may be optionally substituted with one or more substituents.

[0025] As used herein, the term "heterocycloalkyl" means a monocyclic or polycyclic group having at least one heteroatom selected from O, N, or S, having 2 to 11 carbon atoms, and being saturated or unsaturated but not aromatic. Examples of heterocycloalkyl groups include (but are not limited to) piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 4-piperidonyl, pyrrolidinyl, hydantoinyl, valerolactamyl, oxylanil, oxetanyl, tetrahydropyranyl, tetrahydrothiopyranil, tetrahydropyrindinyl, tetrahydropyrimidinyl, tetrahydrothiopyranylsulfone, tetrahydrothiopyranylsulfoxide, morpholinyl, thiomorpholinyl, thiomorpholinylsulfoxide, thiomorpholinylsulfone, 1,3-dioxolane, tetrahydrofuranyl, dihydrofuranyl-2-one, tetrahydrothienyl, and tetrahydro-1,1-dioxothienyl. Generally, monocyclic heterocycloalkyl groups have 3 to 7 members. Preferred 3 to 7-membered monocyclic heterocycloalkyl groups have 5 or 6 ring atoms. Heteroatoms may be substituted with protecting groups known to those skilled in the art; for example, a hydrogen atom on the nitrogen may be substituted with a tert-butoxycarbonyl group. Furthermore, heterocycloalkyl groups may be optionally substituted with one or more substituents. In addition, the linkage point to another group of the heterocycle may be either a carbon atom or a heteroatom of the heterocycle. Only stable isomers of such substituted heterocyclic groups are considered in this definition.

[0026] As used herein, the terms “substituent” or “substituted” mean that a hydrogen group on a compound or group is substituted with a desired atom or group of atoms. Examples of substituents are found in the representative compounds and embodiments disclosed herein, where the molecule still exhibits BRAG2 inhibitory activity. Examples of substituents include halogens; alkyls; alkenyls; alkynyls; hydroxyls; alkoxys; nitros; thiols; thioethers; imines; cyanos; amides; phosphonates; phosphines; carboxyls; thiocarbonyls; sulfonyls; sulfonamides; ketones; aldehydes; esters; oxygen (-O); haloalkyls (e.g., trifluoromethyl); cycloalkyls which may be monocyclic or condensed or uncondensed polycyclic; heterocycloalkyls which may be monocyclic or condensed or uncondensed polycyclic; monocyclic or condensed or uncondensed polycyclic aryls or heteroaryls; aminos (primary, secondary or tertiary); CO2CH3; CONH2; OCH2CONH2; NH2; SO2NH2; OCHF2; CF3; OCF3; and such parts may also be optionally substituted by condensed ring structures or crosslinks, e.g., -OCH2O-. These substituents may be optionally further substituted with substituents selected from such groups.In one embodiment, the term "substituent" or the adjective "substituted" may refer to alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, haloalkyl, -C(O)NR11R12, -NR13C(O)R14, halo, -OR13, cyano, nitro, haloalkoxy, -C(O)R13, ​​-NR11R12, -SR13, -C(O)OR13, -OC(O)R13 -NR13C(O)NR11R12, -OC(O)NR11R12, -NR13C(O)OR14, -S(O)rR13, -NR13S(O)rR14, -OS(O)rR14, S(O)rNR11R12, -O, -S and -N-R13 {wherein r is 1 or 2; R11 and R12 are independently of each occurrence: H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally R11 and R12, together with the nitrogen to which they are linked, become an optionally substituted heterocycloalkyl or an optionally substituted heteroaryl; R13 and R14, independently for each occurrence, are H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl or an optionally substituted heteroaralkyl. This refers to substituents selected from the group consisting of [the specified elements].

[0027] The term "halogen" means -F, -Cl, -Br, or -I. In one embodiment, the halogen is Br. In one embodiment, the halogen is -Cl.

[0028] The crystal structure of the complex between BRAG2 and the molecule according to the present invention, and structure-activity analysis using BRAG2 variants and analogues of the molecule according to the present invention, revealed this. Preferably, the molecule according to the present invention binds to the interface between the PH domain of BRAG2 and the lipid bilayer in such a manner that BRAG2 is unable to activate lipid-modified Arf. Preferably, the molecule according to the present invention drastically reduces the cancer stem cell population of breast cancer cell lines. Preferably, the molecules according to the present invention are intended for use in the treatment of breast cancer, particularly breast cancer with BRAG2 overexpression. This opens up a new class of cellular activity inhibitors that weaken protein-membrane interactions without disrupting them. The present invention represents a new class of drugs that target superficial membrane proteins.

[0029] The present invention also relates to inhibitors that have one or more protein-membrane interactions and inhibit mammalian BRAG2, and preferably human BRAG2, for use in the treatment of cancer. The present invention also relates to an inhibitor of mammalian BRAG2, and preferably human BRAG2, the inhibitor having the structure described in any one of claims 1 to 6. The present invention also relates to a method for inhibiting mammalian BRAG2, and preferably human BRAG2, particularly in vitro or in cellulose, the method comprising contacting BRAG2 with a BRAG2 inhibitor as defined in the present invention. The present invention also relates to an inhibitor of mammalian BRAG2, and preferably human BRAG2, wherein the inhibitor has the structure defined in the present invention. The present invention also relates to a method for inhibiting mammalian BRAG2, and preferably human BRAG2, particularly in vitro or in cellulose, wherein the method comprises contacting BRAG2 with the BRAG2 inhibitor defined in the present invention.

[0030] This invention focuses particularly on Arf GTPases and their GEF BRAG2 (which modulate various regulatory functions of lipid and membrane transport). BRAG2 belongs to the BRAG family, and its members activate Arf GTPases to regulate signaling and / or endocytosis of integrins and other receptors, as well as cell adhesion (see D'Souza, RS, & Casanova, JE (2016) - below).

[0031] In one embodiment, the molecule according to the present invention is an inhibitor of the Arf pathway. In one embodiment, the molecule according to the present invention is an inhibitor of the activation of myristoylated Arf1 and myristoylated Arf6 by BRAG2. In one embodiment, the molecule according to the present invention does not inhibit the nucleotide exchange activity of BIG1, Golgi ArfGEF, ARNO, and EFA6a. In one embodiment, the molecule according to the present invention binds to the PH domain of BRAG2. In one embodiment, the molecule according to the present invention is an inhibitor of the Sec7 domain of BRAG2.

[0032] Surprisingly, in one preferred embodiment, the molecule according to the present invention is more specific to BRAG2 than to BIG1, Golgi ArfGEF, and ARNO, EFA6a. Surprisingly, in one preferred embodiment, the molecule according to the present invention is a BRAG2 inhibitor in the presence of an artificial membrane, and even more surprisingly, in the presence of a membrane containing PIP2. In one embodiment, the molecule according to the present invention binds at the interface between the PH domain and the membrane. In one embodiment, the molecule according to the present invention disperses TGN46 immunostaining in cells. The present invention also relates to molecules according to the present invention, and BRAG2, in particular human BRAG2, and more particularly BRAG2 Sec7-PH This also relates to the complex with.

[0033] BRAG2 is involved in serious medical conditions including breast cancer (Morishige, M. et al. (2008)), uveal melanoma (Yoo, JH et al. (2016)), diabetic retinopathy (Zhu, W. et al. (2017)), and intellectual disability (Shoubridge, C. et al. (2010)). Members of the BRAG family contain a Sec7 domain that is involved in stimulating GDP / GTP exchange and subsequently stimulating the plextrin homology (PH) domain (which binds to the PIP2-containing membrane with high affinity). Previous studies have shown that PIP2-containing membranes enhance BRAG2 GEF activity by more than three orders of magnitude (Aizel, K. et al. Integrated conformational and lipid-sensing regulation of endosomal ArfGEF BRAG2. PLoS Biol 11, e1001652 (2013) and Jian, X., Gruschus, JM, Sztul, E. & Randazzo, PA). The plextrin homology (PH) domain of the Arf exchange factor Brag2 is an allosteric binding site (J Biol Chem 287, 24273-83 (2012)). This significant increase in activity is measured by the interaction of multiple lipids with BRAG2, resulting in its precisely directed juxtaposition to the membrane (Karandur, D., Nawrotek, A., Kuriyan, J. & Cherfils, J.). Multiple interactions between the Arf / GEF complex and charged lipids determine the activation rate on the membrane (Proc Natl Acad Sci USA 114, 11416-11421 (2017)).

[0034] The present invention also relates to a pharmaceutical composition comprising at least one molecule as defined herein, wherein the composition comprises one or more excipients and optionally one other pharmaceutically active ingredient.

[0035] As is well known to those skilled in the art, various forms of excipients can be used in accordance with the administration method, some of which can enhance the efficacy of the active molecule, for example, by promoting a release profile that makes the active molecule more effective overall for the desired treatment.

[0036] Accordingly, the pharmaceutical compositions of the present invention can be administered in various forms, more specifically, in injectable, micronized, or ingestible forms, via routes such as intramuscular, intravenous, subcutaneous, intradermal, oral, topical, rectal, vaginal, ocular, nasal, transdermal, or parenteral. The preferred route is oral administration. The present invention, in particular, encompasses the use of the compounds according to the present invention for the manufacture of pharmaceutical compositions.

[0037] Such drugs may take the form of orally administered pharmaceutical compositions that can be formulated using pharmaceutically acceptable carriers known in the art at appropriate dosages. Such carriers allow the pharmaceutical compositions to be formulated for patient ingestion as tablets, pills, coated tablets, capsules, liquids, gels, syrups, slurries, turbidities, etc. In addition to the active ingredient, these pharmaceutical compositions may contain appropriate pharmaceutically acceptable carriers, including excipients and adjuvants that facilitate the processing of the active compound into a pharmaceutically usable formulation. Further technical details regarding formulation and administration can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

[0038] The present invention also relates to molecules or pharmaceutical compositions as defined herein for use in methods of therapeutic treatment for diseases exhibiting deregulated expression, preferably overexpression, of BRAG2. The present invention also relates to molecules or pharmaceutical compositions as defined herein, as drugs having protein-membrane interactions for use in methods of therapeutic treatment of diseases requiring protein-membrane interactions. In one embodiment, the disease is selected from the group consisting of cancer, particularly invasive cancer, metastatic cancer, cancer resistant to EGFR and / or ErbB2 modulators, angiogenesis, diabetic retinopathy, and non-symptomatic intellectual disability. The present invention also relates to molecules or pharmaceutical compositions as defined herein for use in methods of therapeutic treatment of diseases selected from the group consisting of cancer, particularly invasive cancer, metastatic cancer, cancer resistant to EGFR and / or ErbB2 modulators, angiogenesis, diabetic retinopathy, and non-symptomatic intellectual disability.

[0039] The present invention also relates to a method of therapeutic treatment, the method comprising administering an effective amount of at least one molecule as defined in the present invention to a mammal in need thereof. Preferably, the mammal is a human patient. In one embodiment, the treatment method is for treating breast cancer, particularly breast cancer that requires targeting of the bCSC population.

[0040] For example, the cancer or angiogenesis treatment method according to the present invention is supported by the following: Brag2 and cancer D'Souza, RS, & Casanova, JE (2016). The BRAG / IQSec family of Arf GEF. Small GTPases, 7(4), 257-264. http: / / doi.org / 10.1080 / 21541248.2016.1219442 Matsumoto, Y., Sakurai, H., Kogashiwa, Y., Kimura, T., Matsumoto, Y., Shionome, T., et al. (2017). Inhibition of epithelial-mesenchymal transition by cetuximab via the EGFR-GEP100-Arf6-AMAP1 pathway in head and neck cancer. Head & Neck, 39(3), 476-485. http: / / doi.org / 10.1002 / hed.24626 Xie, C.-G., Wei, S.-M., Chen, J.-M., Xu, X.-F., Cai, J.-T., Chen, Q.-Y., & Jia, L.-T. (2012). Down-Regulation of GEP100 Causes Increase in E-Cadherin Levels and Inhibits Pancreatic Cancer Cell Invasion. PLoS ONE, 7(5), e37854. http: / / doi.org / 10.1371 / journal.pone.0037854 Hu, Z., Du, J., Yang, L., Zhu, Y., Yang, Y., Zheng, D., et al. (2012). GEP100 / Arf6 is required for epidermal growth factor-induced ERK / Rac1 signaling and cell migration in human hepatoma HepG2 cells. PLoS ONE, 7(6), e38777. http: / / doi.org / 10.1371 / journal.pone.0038777 Menju, T., Hashimoto, S., Hashimoto, A., Otsuka, Y., Handa, H., Ogawa, E., et al. (2011). Engagement of overexpressed Her2 with GEP100 induces autonomous invasive activities and provides a biomarker for metastases of lung adenocarcinoma. PLoS ONE, 6(9), e25301. http: / / doi.org / 10.1371 / journal.pone.0025301 Yoo, J.H. et al. ARF6 Is an Actionable Node that Orchestrates Oncogenic GNAQ Signaling in Uveal Melanoma. Cancer Cell 29, 889-904 (2016)

[0041] Breast cancer Hu, Z., Xu, R., Liu, J., Zhang, Y., Du, J., Li, W., et al. (2013). GEP100 regulates epidermal growth factor-induced MDA-MB-231 breast cancer cell invasion through the activation of Arf6 / ERK / uPAR signaling pathway. Experimental Cell Research, 319(13), 1932-1941. http: / / doi.org / 10.1016 / j.yexcr.2013.05.028 Sabe, H., Hashimoto, S., Morishige, M., Ogawa, E., Hashimoto, A., Nam, J.-M., et al. (2009). The EGFR-GEP100-Arf6-AMAP1 signaling pathway specific to breast cancer invasion and metastasis. Traffic (Copenhagen, Denmark), 10(8), 982-993. http: / / doi.org / 10.1111 / j.1600-0854.2009.00917.x Sabe, H., Hashimoto, S., Morishige, M., Ogawa, E., Hashimoto, A., Nam, J.-M., et al. (2009). The EGFR-GEP100-Arf6-AMAP1 signaling pathway specific to breast cancer invasion and metastasis. Traffic (Copenhagen, Denmark), 10(8), 982-993. http: / / doi.org / 10.1111 / j.1600-0854.2009.00917.x Morishige, M. et al. GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion. Nat Cell Biol 10, 85-92 (2008)

[0042] Angiogenesis Manavski, Y., Carmona, G., Bennewitz, K., Tang, Z., Zhang, F., Sakurai, A., et al. (2014). Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis. Basic Research in Cardiology, 109(2), 404. http: / / doi.org / 10.1007 / s00395-014-0404-2 Hashimoto, A., Hashimoto, S., Ando, R., Noda, K., Ogawa, E., Kotani, H., et al. (2011). GEP100-Arf6-AMAP1-cortactin pathway frequently used in cancer invasion is activated by VEGFR2 to promote angiogenesis. PLoS ONE, 6(8), e23359. http: / / doi.org / 10.1371 / journal.pone.0023359 Zhu, W., Shi, D. S., Winter, J. M., Rich, B. E., Tong, Z., Sorensen, L. K., et al. (2017). Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy. The Journal of Clinical Investigation, 127(12). http: / / doi.org / 10.1172 / JCI91770 Sakurai, A., Jian, 286(39), 34335-34345. http: / / doi.org / 10.1074 / jbc. M111.259499

[0043] And again: Zhu, W. et al. Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy. J Clin Invest (2017) Shoubridge, C. et al. Mutations in the guanine nucleotide exchange factor gene IQSEC2 cause nonsyndromic intellectual disability. Nat Genet 42, 486-8 (2010) [Examples]

[0044] The present invention is further illustrated by the following embodiments. The data presented in these examples and in portions of the patent description are obtained to some extent from preliminary analyses that are very close to the final, validated dataset. However, this fully supports the present invention.

[0045] Other objects, features, and advantages of the present invention will likely become clear to those skilled in the art by reading the descriptive description which refers to examples given merely as illustrations and which do not limit the scope of the invention. The examples constitute an essential part of the present invention, and any features from the entire description, including the examples, that appear novel compared to any prior art constitute an essential part of the present invention in terms of their functionality and generality. Therefore, all examples have a general scope. Furthermore, in the examples, all percentages are by mass unless otherwise indicated, temperatures are expressed in degrees Celsius unless otherwise indicated, and pressures are atmospheric pressure unless otherwise indicated.

[0046] Examples The small molecule Bragsin1 was originally discovered in a yeast chemogemics screen; Bragsin disrupts the function of yeast ArfGEF Sec7p, and related analogues, such as Bragsin2 or any of the structures described in the claims, as well as the molecules according to the present invention, are collectively called Bragsin (Figure 1a). Bragsin is a specific inhibitor of ArfGEF BRAG2 in vitro and intracellularly. Cell-based assays and in vitro reconstitution of GEF activity in artificial membranes using pure protein were performed. Bragsin is a non-antagonistic inhibitor, and it binds at the GEF / membrane interface and acts to position GEF correctly on the membrane in a manner that neutralizes BRAG2's nucleotide exchange activity, according to crystallography linked to structure-activity relationship (SAR) analysis. Consistent with the role of BRAG2 in breast cancer, Bragsin drastically reduces the cancer stem cell population in breast cancer cell lines. Bragsin exists as a new type of inhibitor that targets protein-membrane interactions, which have potential in breast cancer treatment.

[0047] Example 1 - Bragsin inhibits the activation of Arf GTPases in cells. Bragsin1 affects the cellular Arf pathway because it disrupts the function of yeast ArfGEF Sec7p. Bragsin1 was found to become chemically unstable after several days in aqueous solution, resulting in a biologically inactive hydrated derivative. A related analogue (Bragsin2) carrying a methoxy group instead of a methyl group was stable because it is resistant to hydration (Figures 7-9). In initial experiments, Bragsin2 was used to evaluate its expected effects on the structure of the cis-Golgi and trans-Golgi network (TGN) compartments, which are modulated by the Arf-dependent pathway. In HeLa cells, the cis-Golgi marker GM130 and the TGN marker TGN46 were dispersed into heterogeneously sized punctate structures by Bragsin2 (Figure 1b), and this effect was reversible (Figure 1c). In contrast, Bragsin2 had no effect on the early endosomal marker EEA1 (Figure 1b), and it did not affect the tubulin and actin networks (Figure 10). To assess whether this effect was mediated by interference with the Arf GTPase pathway, cells were transfected with an Arf-mCherry construct carrying a QL mutation that constitutively activates Arf. Arf1, Arf5, and Arf6 expression restored Bragsin2-induced dispersion in the TGN46 compartment, consistent with the compound's effect on the Arf pathway (Figure 1d). The phenotype of cells treated with newly prepared Bragsin1 was identical and reversible to that of cells treated with Bragsin2 (Figure 11) (Figure 12), and was restored by the expression of constitutively active Arf GTPases (Figure 13). Furthermore, these data suggest that Bragsin1 and Bragsin2 inhibit the Arf pathway in cells, consistent with the intrinsic activity observed in chemogenomics screening of Bragsin1 toward yeast ArfGEF Sec7p.

[0048] Example 2 - Bragsin is a specific inhibitor of BRAG2 that requires the presence of a membrane for inhibition. Arf GTPases are activated in human cells by several Arf GEF subfamilies, all of which contain a conserved Sec7 domain decorated with a variable add-on domain (Nastou, KC, Tsaousis, GN, Kremizas, KE, Litou, ZI & Hamodrakas, SJ The human plasma membrane peripherome: visualization and analysis of interactions. Biomed Res Int 2014, 397145 (2014); DiNitto, JP et al. Mol Cell 28, 569-83 (2007)).

[0049] The effect of Bragsin1 on the GEF efficiency of representative human ArfGEFs was evaluated by fluorescence kinetics using highly purified recombinant Arf GTPases and ArfGEFs. In the first series of assays, the Sec7 domains of BIG1, Golgi ArfGEF, and ARNO (which function in the plasma membrane), as well as EFA6a and BRAG2, were used. When tested in solution, Bragsin1 had no effect on Arf1 activation by any of these Sec7 domains (Figures 2a and 14). Separately, we reconstituted the activation of myristoylated Arf1 on liposomes by the same ArfGEFs using ArfGEF constructs containing membrane-bound domains (DCB-HUS domain for BIG1 and PH domain for other GEFs). In particular, Bragsin1 potently inhibited BRAG2 on liposomes, while it had no effect on other ArfGEFs (Figures 2b and 15) or Rac1 / Trio, or unrelated low molecular weight GTPase / GEF systems (Figures 2b and 15). BRAG2 activates several Arf isoforms, including the Arf6 isoform located in the plasma membrane. Bragsin1 also inhibited BRAG2-mediated activation of myristoylated Arf6, and this inhibition was shown to be independent of the Arf isoform used (Figures 2c and 16). According to dose-response analysis, Bragsin1 and Bragsin2 had the same IC50 for BRAG2 (3 mM). 50Since Bragsin1 and Bragsin2 possess the characteristic of being interchangeable, we confirmed that they can be used interchangeably (Figure 2d). Specific inhibition of BRAG2 by Bragsin in vitro predicts that siRNA-mediated BRAG2 silencing should yield the same phenotype as treatment with small molecules. Silencing of BRAG2 induced the redistribution of TGN46 into tubular or punctate structures, which phenotypicly mimicked the effect induced by Bragsin (Figures 2e, 17, and 18). In contrast, silencing of Golgi ArfGEF GBF1 resulted in a significantly different phenotype (either TGN46 becoming concentrated in punctate structures or diffusing into the cytosol). Silencing of ARNO located in the plasma membrane had no effect on TGN46 dispersion. Overexpression of BRAG2-mCherry restored the dispersion of TGN46 structures induced by Bragsin, which was consistent with the silencing experiment (Figure 2f). According to these experiments, Bragsin is a specific inhibitor of ArfGEF BRAG2 in vitro and in cells. These experiments identify an inhibitory mechanism that requires a membrane to manifest itself.

[0050] Example 3 - Bragsin binds to the PH domain of BRAG2. In vitro and cell assays were performed using BRAG2 in vitro. Sec7-PH The construct supports the idea that it summarizes the inhibitory effect observed in cells using full-length BRAG2. Sec7-PHThe crystal structure of the Bragsin1 complex was obtained using the method described in Table 1. A distinct electron density was observed near the PH domain, which allowed us to model the inhibitor (Figure 3a), but its location was not related to crystal contact. Bragsin1 interacts with the PH domain through a combination of polar and hydrophobic contacts, including Leu651, His652, and Arg654 on chain b1, Lys667 on chain b3, and Arg681 on chain b4 (Figure 3b). The carbonyl groups of CF3 and Bragsin2 interact with the PH domain but not with the methyl group, which explains why this latter group is as potent as Bragsin1 when substituted with a methoxy group. Conversely, hydration of Bragsin1, which leads to the sp3 shape and various orientations of the carbon supporting the CF3 substituent, was consistent with the loss of Bragsin1 activity over time (see above). R654E or R681E mutations were introduced into the PH domain of BRAG2, and the location of the binding site was confirmed. Inhibition was affected by both mutants (Figure 3c). The R681E mutant provided BRAG2 that was insensitive to Bragsin, and interestingly, the R654E mutant was more inhibited by Bragsin than wild-type BRAG2. Next, Bragsin analogs were synthesized, and the NO2 or CF3 functional group (establishing interaction with BRAG2 in crystal) was removed or modified in the formula (compounds (3) to (6), Figure 1a and Example 6). All analogs showed reduced inhibition of BRAG2 in vitro (Figure 3d), and either failed to disperse TGN46 (compounds 3, 4, and 6) or had less effect in cells (compound 5) (Figure 3e). Along with this, crystallography, mutagenesis, and structure-activity relationship analysis consistently and accurately indicated the specific binding site of Bragsin within the PH domain of BRAG2.

[0051] Example 4 - Bragsin is a non-competitive inhibitor of BRAG2 / membrane interaction. Bragsin may partially overlap with the lipid bound at this site, as predicted by comparing the phosphoinositide head group bound to the standard lipid binding site with the PH domain of cytohesin ArfGEF (DiNitto, JP et al. Mol Cell 28, 569-83 (2007)) (Figure 4a). Bragsin inhibition may also act by interfering with the interaction between the membrane and BRAG2. Surprisingly, Bragsin2 does not replace BRAG2 from PIP2-containing liposomes, indicating that it does not inhibit BRAG2 by interfering with the membrane and its relationship (Figure 4b). Alternatively, if inhibition is influenced by the lipid composition of the membrane, Bragsin may bind at the interface between the PH domain and the membrane. Using liposomes that enhance potent BRAG2 activity, the inventors found that Bragsin2 inhibits BRAG2 more efficiently against PIP2-containing liposomes compared to liposomes containing PS as the sole negatively charged lipid (Figure 4c). These experiments support the idea that Bragsin inhibits BRAG2 by binding to the BRAG2-lipid bilayer interface in a non-competitive manner, thereby inducing a binding mode in which BRAG2 is ineffective in promoting nucleotide exchange with membrane-bound myristoylated Arf.

[0052] Example 5 - Bragsin affects breast cancer stem cells. Cancer stem cells represent a population of cells that maintain tumor growth, metastasis, resistance to chemotherapy and radiotherapy, and recurrence after treatment. Targeting stem cell populations has become a key step in designing effective anti-cancer strategies, as their relative amounts in tumors correlate with poor patient prognosis. Since BRAG2 had been reported to be involved in breast cancer cell invasion, we tested whether Bragsin affects breast cancer stem cells (bCSCs) (Morishige, M. et al. GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion. Nat Cell Biol 10, 85-92 (2008)). It was initially used as a marker for malignant bCSC populations, specifically targeting aldehyde dehydrogenase (ALDH brThe effect of Bragsin on the bCSC population in three different breast cancer cell lines (SUM159, SUM149, S68) was evaluated using its activity (Charafe-Jauffret, E. et al. ALDH1-positive cancer stem cells predict engraftment of primary breast tumors and are governed by a common stem cell program. Cancer Res 73, 7290-300 (2013); Ginestier, C. et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555-67 (2007)). Bragsin treatment (50 μM) drastically reduced the bCSC population in two of the three cell lines tested (SUM149 and S68) (Figure 5a). A single-cell tumor-like aggregate assay was performed to confirm the functional validity of these observations. A significant decrease was observed in the tumor-like mass formation efficiency (SFE) of SUM149 and S68 cell lines after treatment with Bragsin, and bCSC ALDH br This was consistent with the decrease in the population (Figure 5b). In contrast, the bCSC ALDH br SUM159 cell lines whose activity was not reduced by Bragsin showed similar SFE regardless of whether they were treated with Bragsin or not. Together, these results suggest that Bragsin may be able to target several bCSC populations of breast cancer.

[0053] Example 6 - Synthesis of Bragsin1, Bragsin2 and analogs used in this test (Figure 1a) 6.1 General Unless otherwise stated, all glassware was dried by heating or in an oven before use, and all reactions were carried out under an argon atmosphere. Dichloromethane, acetonitrile, toluene, methanol and DMSO were purchased as anhydrous grades from Sigma-Aldrich and used as received; all other solvents were distilled before use. All reagents were used as received from commercial suppliers unless otherwise stated. The progress of the reaction was monitored by thin layer chromatography (TLC) on aluminum plates coated with silica gel F 254 and visualized by fluorescence quenching using UV light at 254 nm or by staining with potassium permanganate, phosphomolybdic acid solution, p-anisaldehyde solution or vanillin solution followed by heating. Flash column chromatography was performed using silica gel 60 (230 - 400 mesh, Merck and co.). 1 1H NMR and 13 13C NMR spectra were recorded at 300 K using Bruker AV-300, AV-400 and AV-500 spectrometers. Chemical shifts were reported in parts per million (ppm, δ) relative to the solvent peak of CDCl3 defined as δ = 7.26 ppm ( 1 1H NMR) and δ = 77.16 ( 13 13C NMR). Coupling constants were reported in Hz (J). 1 1H NMR splitting patterns were designated as singlet (s), doublet (d), doublet of doublet (dd), triplet (t), quartet (q), pentet (p). Splitting patterns that could not be interpreted or readily distinguished were designated as multiplet (m) or broad (br).

[0054] 6.2. Experimental Procedures Synthesis of Bragsin1, Bragsin2 and Compound (3)

Chem.

[0055] 6.2.1. General Procedure Steps 1 and 2 Dry THF (1.5 ml) and finely dispersed LiH (3.4 equivalents) were placed in a double-necked round-bottom flask under argon, and the mixture was brought to reflux temperature. A solution of the desired ketone (1.0 equivalent) and ethyl 2,2,2-trifluoroacetate (1.5 equivalents) in the dry THF was added over 10-15 minutes with stirring. The mixture was refluxed for 2 hours and then quenched with a 1N aqueous solution of HCl. The reaction mixture was extracted three times with HCl, the organic layers were combined, washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The product was used in the next step without further purification.

[0056] A droplet of 37% aqueous HCl was added, while stirring, to a solution containing the reaction product (1.0 equivalent) obtained after step 1 in acetic acid (5 ml), and the solution was refluxed for 1 hour. After this time, the reactants were diluted with water, and the solvent was removed under reduced pressure. The unpurified mixture was obtained and purified by column chromatography (cyclohexane:ethyl acetate, gradient from 0% to 60% ethyl acetate) to obtain the designed chromenone derivative.

[0057] Step 3 A mixture of concentrated H2SO4 (0.6 ml) and concentrated HNO3 (0.6 ml) was added to the solution (1 equivalent) of the purified product from Step 2 in concentrated H2SO4 (2 ml). The reaction mixture was stirred at 75°C for 35 minutes and then diluted with ice water. The reaction mixture was extracted three times using ethyl acetate, washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The unpurified mixture was purified by chromatography (reverse phase / forward phase) to obtain the designed nitro-chromenone product.

[0058] 6-methyl-5-nitro-2-(trifluoromethyl)-4H-chromen-4-one (Bragsin1, Figure 1a): [ka]

[0059] The title compound was prepared from commercially available 2'-hydroxy-5'-methylacetophenone following a general procedure. Bragsin 1 was obtained in 11% yield (in 3 steps). The data is consistent with the literature. 1 H-NMR (500MHz, CDCl3): δ 7.71 (d, J = 8.9 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H), 6.74 (s, 1H), 2.39 (s, 3H).

[0060] 6-Methoxy-5-nitro-2-(trifluoromethyl)-4H-chromen-4-one (Bragsin2, Figure 1a): [ka]

[0061] The title compound was prepared from commercially available 2'-hydroxy-5'-methoxyacetophenone following a general procedure. Bragsin 2 was obtained in 78% yield (in 3 steps). The data is consistent with the literature. 1 H-NMR (500MHz, CDCl3): δ 7.71 (d, J = 9.5 Hz, 1H), 7.54 (d, J = 9.5 Hz, 1H), 6.70 (s, 1H), 3.98 (s, 3H).

[0062] 6-Methoxy-2-(trifluoromethyl)-4H-chromen-4-one (3) (Compound 3, Figure 1a): [ka]

[0063] The title compound was prepared from commercially available 2'-hydroxy-5'-methoxyacetophenone according to a general procedure (steps 1 and 2). Compound 3 was obtained in 90% yield (in 2 steps). The data is consistent with the literature. 1H-NMR (300MHz, CDCl3): δ 7.54 (d, J = 3.1 Hz, 1H), 7.48 (d, J = 9.2 Hz, 1H), 7.34 (dd, J = 9.2, 3.1 Hz, 1H), 6.70 (s, 1H), 3.91 (s, 3H).

[0064] Quality analysis of compound (4) (Compound 4, Figure 1a) 6-methoxy-5-nitro-2-(trifluoromethyl)-4H-chromen-4-one (4): [ka]

[0065] The compound in question is a commercially available compound (CAS: 354128-13-5). 1 H-NMR (500MHz, CDCl3): δ 7.88 (d, J = 6.0 Hz, 1H), 7.00 (s, 1H), 6.37 (d, J = 6.0 Hz, 1H), 4.03 (s, 3H), 2.37 (s, 3H).

[0066] Synthesis of compound (5) (Compound 5, Figure 1a) [ka]

[0067] 6-Hydroxy-2-(trifluoromethyl)-4H-chromen-4-one(7): [ka]

[0068] Clomenone 3 (0.1 g, 0.41 mmol, 1.0 equivalent) was dissolved in DCM. To the reaction mixture, 1 mL of BBr3 (1 M, 0.82 mmol, 2.0 equivalents) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 2 hours. Next, it was quenched with water, extracted with DCM, washed with NaHCO3, and dried over MgSO4. Volatile substances were removed under reduced pressure. The collected residue was purified by column chromatography (hexane:siRNA = 1:2). Compound 7 was obtained in 69% yield (65 mg, 0.28 mmol). 1 H-NMR (300MHz, CDCl3): δ 7.84 (d, J = 3.0 Hz, 1H), 7.50 (d, J = 9.2 Hz, 1H), 7.38 (dd, J = 9.2, 3.0 Hz, 1H), 6.75 (s, 1H). 13 C-NMR (75MHz, CDCl3): δ 178.2, 155.3, 152.9 (q, J = 39.2 Hz), 150.4, 125.2, 124.7, 120.1, 118.7 (q, J = 274.2 Hz), 109. 4 (q, J = 2.8 Hz), 109.2. HRMS(ESI):C 10 H6F3O3[M+H] + Calculated value for this: 231.0259, measured value: 231.0264.

[0069] 6-Hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromene-5-carbaldehyde(8): [ka]

[0070] A mixture of clomenone 7 (100 mg, 0.44 mmol, 1.0 equivalent) and hexamethylenetetramine (121.9 mg, 0.87 mmol, 2.0 equivalents) was dissolved in TFA (2 ml). The mixture was heated in a microwave apparatus to 120°C for 30 minutes. The reaction product was then cooled to room temperature, quenched with ice water, extracted with Et2O, and dried over MgSO4. After removing the solvent, the collected residue was purified by column chromatography (hexane:SiO=3:2). Compound 8 was obtained in 48% yield (55 mg, 0.21 mmol). 1 H-NMR (300MHz, CDCl3): δ 13.09 (s, 1H), 11.43 (s, 1H), 7.75 (d, J = 9.4 Hz, 1H), 7.43 (d, J = 9.4 Hz, 1H), 6.78 (s, 1H). 13 C-NMR (75MHz, CDCl3): δ 198.6, 178.1, 162.5, 151.3 (q, J = 39.7 Hz), 150.3, 128.0, 127.2, 122.4, 118.5 (q, J = 273.9 Hz), 115.3, 111.8 (q, J = 2.7 Hz). HRMS(ESI):C 11 H6F3O4[M+H] + Calculated value for this: 259.0218, measured value: 259.0212.

[0071] 6-Methoxy-4-oxo-2-(trifluoromethyl)-4H-chromene-5-carbaldehyde(5) (Compound 5, Figure 1a) [ka]

[0072] Under argon, compound 8 (50 mg, 0.19 mmol, 1.0 equivalent) was dissolved in dry DMF at 0°C, followed by the addition of K2CO3 (80 mg, 0.582 mmol, 3.0 equivalents). Next, methyl iodide (15 μL, 0.23 mmol, 1.2 equivalents) was added dropwise to the mixture at 0°C. The reaction mixture was stirred at room temperature for 12 hours. The mixture was quenched with NH4Cl, extracted with ethyl acetate, washed with brine, and dried over MgSO4. The organic layers were combined, and volatiles were removed under reduced pressure. The unpurified product was purified by column chromatography (hexane:siRNA, 3:2) to obtain the title product in 86% yield (55 mg, 0.21 mmol). 1 H-NMR (300MHz, CDCl3): δ 10.57 (s, 1H), 7.67 (d, J = 9.3 Hz, 1H), 7.46 (d, J = 9.3 Hz, 1H), 6.67 (s, 1H), 3.91 (s, 3H). 13 C-NMR (75MHz, CDCl3): δ 191.9, 177.5, 154.7, 152.5 (q, J = 39.4 Hz), 149.6, 126.0, 123.6, 121.9, 120.0, 118. 6 (q, J = 274.2 Hz), 110.0 (q, J = 2.8 Hz), 57.1. HRMS(ESI):C 12 H8F3O4[M+H] + Calculated value for this: 273.0374, measured value: 273.0369.

[0073] Synthesis of compound (6) (Compound 6, Figure 1a) [ka]

[0074] Methyl 2-acetyl-3-hydroxy-6-methoxybenzoate (11): [ka]

[0075] To a stirred solution of furan 9 (389 mg, 3.96 mmol, 1.0 equivalent) in toluene (1.5 mL) at 0°C, ketoester 10 (500 mg, 3.96 mmol, 1.0 equivalent) was added all at once. Upon completion of the addition, the amber-colored solution was warmed to 90°C. After 1 hour, the reaction mixture was concentrated in vacuum to obtain a viscous reddish-purple oil, a bicyclic mixture with 1:3 regiospecificity (observed by NMR). To a stirred solution of this bicyclic mixture (875 mg, 3.97 mmol, 1.0 equivalent) in THF (4 mL), a solution of dry hydrochloric acid in ether (1.0 M, 0.8 mL, 0.78 mmol, 0.2 equivalent) was slowly added over 5 minutes at 0°C. Upon completion of the addition, the amber-colored solution was warmed to room temperature. After 2 hours, the reaction mixture was concentrated in vacuum to obtain an amber-colored oil. The unpurified substance (412 mg, 43% over two steps) was purified by silica gel column chromatography (hexane:Â, 10:1) to obtain product 11 (89 mg) as a clear, pale yellow oil. 1 H-NMR (300MHz, CDCl3): δ 11.91 (s, 1H), 7.17 (d, J = 9.2 Hz, 1H), 7.04 (d, J = 9.2 Hz, 1H), 3.95 (s, 3H), 3.81 (s, 3H), 2.52 (s, 3H). 13 C-NMR (75MHz, CDCl3): δ 203.8, 168.6, 156.5, 149.1, 124.0, 121.3, 121.0, 117.5, 57. 7 53.2, 29.3. HRMS(ESI):C 11 H 12 Calculated value for O5Na[M+Na]: 247.0582, measured value: 247.0580.

[0076] Methyl 6-methoxy-4-oxo-2-(trifluoromethyl)-4H-chromene-5-carboxylate(12): [ka]

[0077] The title compound was prepared from benzoquinone 11 according to a general procedure. Compound 12 was obtained in 85% yield. 1 H-NMR (300MHz, CDCl3): δ 7.63 (d, J = 9.4 Hz, 1H), 7.42 (d, J = 9.4 Hz, 1H), 6.65 (s, 1H), 4.01 (s, 3H), 3.93 (s, 3H). 13 C-NMR (75MHz, CDCl3): δ 176.0, 166.9, 154.2, 152.0 (q, J = 39.4 Hz), 149.6, 121.9, 120.7, 120.4, 119.0, 118. 6 (q, J = 274.5 Hz), 109.9 (q, J = 2.7 Hz), 57.0, 53.3. HRMS(ESI):C 13 H 10 F3O5[M+H] + Calculated value for this: 303.0430, measured value: 303.0475.

[0078] 6-Methoxy-4-oxo-2-(trifluoromethyl)-4H-chromene-5-carboxylic acid (6) (Compound 6, Figure 1a) [ka]

[0079] Clomenone 12 (50 mg, 0.16 mmol, 1.0 equivalent) was dissolved in DCM. To the reaction mixture, 0.3 mL of BBr3 (1 M, 0.33 mmol, 2.0 equivalents) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 1 hour. Next, it was quenched with water, extracted with DCM, washed with NaHCO3, and dried over MgSO4. Volatile substances were removed under reduced pressure. The collected residue was purified by column chromatography (hexane:siRNA = 2:3). After chromatographic separation, compound 6 was obtained as a white solid in an amount of 22 mg (46% yield). 1H-NMR (300MHz, CDCl3): δ 7.56 (d, J = 9.3 Hz, 1H), 7.40 (d, J = 9.3 Hz, 1H), 6.69 (s, 1H), 3.96 (s, 3H). 13 C-NMR (75MHz, CDCl3): δ 175.6, 168.8, 154.8, 151.7 (q, J = 39.3 Hz), 150.2, 125.3, 122.9, 122.8, 118. 6 (q, J = 274.3 Hz), 113.3, 110.7 (q, J = 2.7 Hz), 53.3. HRMS(ESI):C 12 H8F3O5[M+H] + Calculated value for this: 289.0324, measured value: 289.0317.

[0080] 2-Hydroxy-6-methyl-5-nitro-2-(trifluoromethyl)chroman-4-one(13): [ka]

[0081] The data is consistent with the literature. 1 H-NMR (300MHz, CDCl3): δ 7.49 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 5.71 (s (br), 1H, OH), 3.10 (q, J = 9.6 Hz, 2H), 2.24 (s, 3H).

[0082] 6,8-Dimethoxy-5-nitro-2-(trifluoromethyl)-4H-chromen-4-one(14) Step 1 A 4 mL intillation vial was packed with Pd(OAc)2 (4.4 mg, 0.02 mmol) and K2S2O8 (216 mg, 0.8 mmol), followed by 2 mL of TFA and ketone substrate (0.4 mmol). The reaction mixture was sealed with a Teflon®-lined cap. The reaction mixture was heated on a pipe block at 50°C for 1.5 hours. The reaction was observed by TLC. The solvent was removed under vacuum. The residue was subjected to flash chromatography (silica gel) using Hex / DCM to obtain hydroxyl-3E 1 , 5E 1 -Dimethoxyacetophenone was obtained. [ka]

[0083] 1 H-NMR (400MHz, CDCl3): δ 12.21 (s, 1H), 6.69 (d, J = 0.7 Hz, 2H), 3.86 (s, 3H), 3.79 (s, 3H), 2.61 (s, 3H).

[0084] General procedure Step 2 Dry THF (1.5 ml) and finely dispersed LiH (3.4 equivalents) were placed in a double-necked round-bottom flask under argon, and the mixture was brought to reflux temperature. A solution containing the corresponding ketone in the dry THF, a commercially available starting material (1.0 equivalent), and ethyl 2,2,2-trifluoroacetate (1.5 equivalents) was added over 10-15 minutes with stirring. The mixture was refluxed for 2 hours and then quenched with a 1N aqueous solution of HCl. The reaction mixture was extracted three times with HCl, the organic layers were combined, washed with brine, dried over MgSO4, filtered, and concentrated under vacuum. The product was used in the next step without further purification.

[0085] Step 3 A drop of concentrated HCl was added to the unpurified solution from Step 1 in acetic acid while stirring, and the solution was refluxed for 1 hour. After this time, the reactants were diluted with water, and the solvent was removed under reduced pressure. The unpurified mixture was obtained and purified by chromatography to obtain the designed chromen derivative. [ka]

[0086] Step 4 (Nitration) A mixture of concentrated H2SO4 (0.6 ml) and concentrated HNO3 (0.6 ml) was added to the solution (1 equivalent) of the purified product from Step 2 in concentrated H2SO4 (2.3 ml). The reaction mixture was stirred at 75°C for 35 minutes, and then diluted with ice-cold water. The precipitate was filtered, washed with water, dried, and recrystallized from BuOH. Compound 14 (Bragsin3) was obtained as a white crystalline solid. [ka]

[0087] Example 7 - Materials and Methods 7.1. Chemicals Nucleotides were purchased from Jena Bioscience. BFA was purchased from Sigma. Bragsin1 (6-methyl-5-nitro-2-(trifluoromethyl)-4H-chromen-4-one) and Bragsin2 (6-methoxy-5-nitro-2-(trifluoromethyl)-4H-chromen-4-one) were purchased from Vitas-M laboratory, and further purified and analyzed as described in Example 6. The stability analysis of Bragsin1 and Bragsin2 is shown in Figures 7-9. Since Bragsin1 became commercially unavailable during the course of this study, some experiments were performed using only Bragsin2 (effect on BRAG2 mutants - Figure 3c, effect on BRAG2 membrane binding - Figure 4b, and effect of membrane composition on Bragsin efficiency - Figure 4c). Bragsin1 and Bragsin2 can be used without distinction. The synthesis of the Bragsin analog shown in Figure 1a is described in Example 6.

[0088] 7.2. Antibodies and cDNA Mouse monoclonal anti-GM130 (130 kDa cis-Golgi substrate protein) and anti-EEA1 (early endosomal antigen 1) were derived from Transduction Laboratories. Mouse monoclonal anti-α-tubulin was purchased from Sigma. Sheep antibody against TGN46 (46 kDa trans-Golgi network protein) was purchased from AbD Serotec. Alexa647-labeled phalloidin was derived from Invitrogen. For secondary antibodies, Alexa488 conjugated with goat anti-mouse antibody or donkey anti-sheep IgGs (Invitrogen) were used for immunofluorescence, and horseradish peroxidase-conjugated chicken anti-mouse IgG (Santa Cruz Biotechnology) was used for Western blotting. Plasmids encoding full-length Arf1 Q71L, Arf5 Q71L, and Arf6 Q67L mutants were kindly provided by Julie Menetrey (LEBS, CNRS, Gif-sur-Yvette, France) and used as templates for subcloning the pmCherry-N1 vector (Clontech) for the expression of the C-terminal fusion mutant protein of mCherry in mammalian cells. The full-length sequence of human BRAG2b(1-963) was synthesized by ProteoGenix and subcloned into pmCherry-N1 (Clontech) for transient expression of BRAG2-mCherry in mammalian cells. Sec7PH The mutants were created by site-directed mutagenesis using a Quickchange kit (Stratagene) according to the manufacturer's protocol.

[0089] 7.3. Cell culture, transfection, and treatment with inhibitors HeLa cells were cultured in Dulbecco's modified Eagle medium supplemented with Glutamax® and 10% fetal bovine serum (Invitrogen). For immunofluorescence studies, cells were cultured on Labtek glass slides (Nunc) and transfected with Lipofectamine 2000 (Invitrogen) for 18–24 hours according to the supplier's instructions. In detail, cells were treated with small molecules or the corresponding volume of vehicle (DMSO) at 37°C for 30 minutes at the indicated concentration in the culture medium.

[0090] 7.4. Immunofluorescence and Confocal Microscopy Immunostaining was performed as described in (Viaud, J. et al. Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes. Proc Natl Acad Sci USA 104, 10370-5 (2007)), except that secondary antibody incubation was performed for 1 hour using Alexa488-conjugated goat anti-mouse (1:600) or donkey anti-sheep (1:500) IgG and Alexa647-conjugated phalloidin (1:100). Anti-GM130 was used at a dilution of 1:200. Images were recorded bidirectionally using an inverted Leica TCS SP8 laser scanning confocal microscope equipped with a 100× (NA 1.40) oil immersion objective lens (HCX APO, Leica). Fluorescent dyes were continuously detected using excitation lasers at 488 nm (Alexa488), 594 nm (mCherry), and 633 nm (Alexa647). Stacks were created using a 0.5 μm z-step and processed using Fiji / ImageJ (Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-82 (2012)). Images are representative of at least two independent experiments.

[0091] 7.5. siRNA knockdown Gene silencing was achieved using siRNAs targeting all known transcript variants of each GEF gene: siBRAG2, Hs_IQSEC1_5 (Qiagen, SI03019408); siARNO, Hs_PSCD2_3 (Qiagen, SI00061299); siGBF1, Hs_GBF1_3 (Qiagen, SI00425418). Untargeted control siRNA: siCTRL, AllStars Negative Control siRNA (Qiagen, 1027280). HeLa cells were subjected to double reverse transfection using Lipofectamine® RNAiMAX (Invitrogen, 12323563) according to the manufacturer's instructions. Simply put, trypsin-treated cells are transfected into 12-well plates, with 15,000 cells / cm³ per well, each containing 10 pmol of siRNA and 3 μl of Lipofectamine® RNAiMAX in 200 μl of Opti-MEMR per 1 ml of transfection mix. 2Cells were seeded in [specimen name]. After 48 hours, the cells were again cultured on plates and transfected again according to the same protocol into 6-well plates (final volume 2.5 ml / well) for RNA extraction and 8-well Lab-Tek® glass slides (final volume 200 μl / well) for immunofluorescence staining. Analysis was performed 48 hours after the second transfection. As determined by RT-qPCR, BRAG2, ARNO, and GBF1 mRNA levels were reduced by approximately 50%, 83%, and 88% in the presence of their respective siRNAs (Figure 17), and these levels were not modified by treatment with Bragsin (Figure 18). Total RNA was extracted using the RNeasy® Mini Kit (Qiagen, 74104) according to the manufacturer's instructions. Each sample RNA (1 μg) was reverse transcribed using iScript® Reverse Transcription Supermix (Bio-Rad, 170-8840) for RT-qPCR. cDNA (100 ng) was quantified by real-time PCR using a CFX Connect instrument (Bio-Rad) with SsoAdvanced® Universal SYBR® Green Supermix (Bio-Rad, 172-5270). Amplification was performed according to Bio-Rad's standard Prime-PCR protocol. Primers were selected using the NCBI Primer-BLAST algorithm as follows: BRAG2, 5'-CGTGGCATTTCTTTGGTGTC-3' (SEQ ID NO: 2) and 5'-ACCCGAGAATTGATGAGTCG-3' (SEQ ID NO: 3); ARNO, 5'-TTGTGTCAAGGATAGGGCTG-3' (SEQ ID NO: 4) and 5'-ACTTCACCTTCATAGAGGCG-3' (SEQ ID NO: 5); GBF1, 5'-TGTCACTCTCTACCTTTGCG-3' (SEQ ID NO: 6) and 5'-AAATCTCCGCTGTGTCCATC-3' (SEQ ID NO: 7); GAPDH, 5'-ACAAGAGGAAGAGAGAGACCC-3' (SEQ ID NO: 8) and 5'-TACATGACAAGGTGCGGCTC-3' (SEQ ID NO: 9).

[0092] 7.6. Protein Bovine Δ17Arf1 and human Δ13Arf6, full-length myristoylated Arf1 and Arf6, human BRAG2 Sec7 (390~594) and BRAG2 Sec7PH (390~811), Human EFA6 Sec7 (527~727) and EFA6 Sec7PHCt (527~1024), ARNO Sec7 (50~256), BIG1 Sec7 (691~889), BIG1 DcbHusSec7 (2~888) and ARNO Sec7PH (50-399) was expressed and purified as described in (Benabdi, S. et al. Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles. Biochemistry 56, 5125-5133 (2017)) (incorporated herein). BRAG2 Sec7PH The mutant was purified as the wild-type protein. Human full-length Rac1 and with a 6xHis tag at the C-terminus. Human Trio DH1PH1 The purification of (1232~1550) is described in (Peurois, F. et al. Characterization of the activation of small GTPases by their GEFs on membranes using artificial membrane tethering. Biochem J 474, 1259-1272 (2017)).

[0093] 7.7. Liposomes and Suspension Assays The (naturally occurring) lipids were from Avanti Polar Lipids, with the exception of NBD-PE from Sigma. Liposomes were prepared as described in (Aizel, K. et al. Integrated conformational and lipid-sensing regulation of endosomal ArfGEF BRAG2. PLoS Biol 11, e1001652 (2013)) and extruded to 0.2 μm. For the study of ArfGEF specificity, the liposomes contained 48% phosphatidylcholine (PC), 20% phosphatidylethanolamine (PE), 30% phosphatidylserine (PS), and 2% phosphatidylinositol-4,5-bisphosphate (PIP2). For the Rac activation assay, liposomes contained 43% PC, 20% PE, 10% PS, 20% cholesterol, 2% PIP2, 5% NiNTA lipid, and 0.2% NBD-PE. To eliminate liposome aggregation or breakage, their size distribution was controlled before and after the experiment by dynamic light scattering as described in (Benabdi, S. et al. Biochemistry 56, 5125-5133 (2017)). Dose-response and suspension assays were performed using liposomes containing 37.9% PC, 20% PE, 20% PS, 2% PIP2, 20% cholesterol, and 0.1% NBD-PE. 18 It was implemented as follows.

[0094] 7.8. Nucleotide exchange assay The nucleotide exchange dynamics, 50、51As described, the samples were observed at 37°C under continuous stirring using a Cary Eclipse fluorimeter (Varian) with excitation / emission wavelengths of 292 / 340 nm by tryptophan fluorescence. For specificity assays, 50 μM Bragsin1 or 0.25% DMSO were added in the presence of 100 μM liposomes and 2-100 nM ArfGEF, as described in (Benabdi, S. et al. Biochemistry 56, 5125-5133 (2017) and Peurois, F. et al. Biochem J 474, 1259-1272 (2017)), along with 1 μM N-terminal shortened Arf-GDP and 100 nM indicated Sec7 domain (in solution) or 0.4 μM myr The nucleotides were incubated at 37°C for 2 minutes in an HKM solution containing one of the Arf1 nucleotides (50 mM HEPES pH 7.4, 120 mM potassium acetate, 1 mM MgCl2, and 1 mM DTT). The nucleotide exchange rate (K) was measured. obs The activity was determined from single-exponential fitting, and the mean is shown as a percentage ± SD relative to the control activity. All experiments were performed in triplicate.

[0095] 7.9. Crystallization and Structure Determination BRAG2 was concentrated to 5 mg / ml for crystallization, and crystals were obtained at 293 K by vapor diffusion in 18% PEG20000 and 0.1 M Tris HCl pH 8.5. The crystals were transferred to a reservoir solution supplemented with 10% glycerol, immersed in 20 μM Bragsin1 in 100 μl of final volume, and incubated at room temperature for 24 hours. Diffraction data was collected from single crystals at the PROXIMA2 beamline (SOLEIL Synchrotron, Gif-sur-Yvette, France) and processed using autoPROC (Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr D Biol Crystallogr 67, 293-302 (2011)). The structure was resolved by molecular substitution using Phaser (McCoy, AJ et al. Phaser crystallographic software. J Appl Crystallogr 40, 658-674 (2007)) with unbound BRAG2 (PDB5NLY) (Karandur, D. et al., J. Proc Natl Acad Sci USA 114, 11416-11421 (2017)) as a model. Ligand matching was performed using ligand matching options in RHOFIT (Global Phasing Ltd.) or Phenix, and both showed the same ligand orientation.The structures were refined using Phenix (Adams, PD et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica. Section D, Biological crystallography 60, 2126-32 (2004)) and Buster (Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr D Biol Crystallogr 66, 213-21 (2010)) instead of the Coot model (Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta crystallographica. Section D, Biological crystallography 60, 2126-32 (2004)). Statistical data for data processing and refinement are reported in Table 1. The coordinates and structural elements were deposited in the Protein Data Bank with entry code 6FNE.

[0096] 7.10. Cancer stem cell assay Three breast cancer cell lines (BCLs) derived from three different molecular subtypes (SUM149 / basal, SUM159 / mesenchymal, and S68 / luminal) were used in this study. All BCLs were cultured in standard media as previously described (Charafe-Jauffret, E. et al. Cancer Res 73, 7290-300 (2013)). The ALDEFLUOR Kit (Stem Cell Technologies) was used to isolate populations using high aldehyde dehydrogenase enzyme activity with an LSR2 hemocytometer (Becton-Dickinson Biosciences) as previously described (Ginestier, C. et al. Cell Stem Cell 1, 555-67 (2007)). For the tumor-like mass assay, BCL cells were cultured for 72 hours under Bragsin 2 treatment (50 mM) or under vehicle-based adhesion conditions, and then seeded as single cells in ultra-low adhesion plates (Corning) according to limiting dilutions. The tumor-like masses were cultured in serum-free mammary epithelial basal medium. The ability of cells to form tumor-like masses was quantified under a microscope. Statistical analysis of tumor-like mass formation efficiency was performed using Extreme LDA software (http: / / bioinf.wehi.edu.au / software / elda / ).

[0097] Conclusion of the Examples In this invention, Bragsin was confirmed, in conjunction with cell-based assays, to be a potent and selective inhibitor of BRAG2, which affects breast cancer stem cells in terms of the reconstitution of lipid-modified Arf GTPases and GEFs on artificial membranes. The inhibitory mechanism was supported by crystallography, mutagenesis, SAR, and membrane binding assays. The inventors support this by the implications of these experiments on the cellular physiology of BRAG2 and its role in breast cancer. Boundary inhibition of protein-membrane interactions as a novel concept in drug development was also established.

[0098] Characterization of Bragsin reveals previously overlooked aspects of BRAG2 function in regulating the integrity of the TGN compartment. Previous studies have reported general roles of BRAG2 in regulating signaling of plasma membrane receptors such as AMPA, EGF, VEGF, and GNAQ receptors, and in the trafficking of adhesion proteins including β1-integrin and N-cadherin, but the underlying functional pathways remained unclear. Interestingly, recent studies have shown that β1-integrin is used in a retrograde pathway so that TGN is secreted in a polarized manner, leading to cell adhesion or persistent migration, and that β1-integrin regeneration transiently localizes to TGN46-positive post-Golgi carriers. The distinctive effects of Bragsin on TGN structure suggest a role for BRAG2 in regulating the main aspects of trafficking of β1-integrin and other receptors to and from TGN.

[0099] Bragsin is involved in a unique mechanism of action at the protein-membrane interface. Bragsin binds to the edge of the standard lipid-binding site of the PH domain without interfering with the interaction between liposomes and BRAG2. This allows Bragsin to contact both BRAG2 and the membrane simultaneously.

[0100] PIP2-containing membranes enhance the GEF activity of BRAG2, as highlighted by previous studies (Aizel, K. et al. Integrated conformational and lipid-sensing regulation of endosomal ArfGEF BRAG2. PLoS Biol 11, e1001652 (2013); Jian, X., Gruschus, JM, Sztul, E. & Randazzo, PA The pleckstrin homology (PH) domain of the Arf exchange factor Brag2 is an allosteric binding site. J Biol Chem 287, 24273-83 (2012)). This involves contact with multiple lipids, leading to the clear juxtaposition of the Arf-BRAG2 complex on the membrane (Karandur, D., Nawrotek, A., Kuriyan, J. & Cherfils, J. Multiple interactions between an Arf / GEF complex and charged lipids determine activation kinetics on the membrane. Proc Natl Acad Sci USA 114, 11416-11421 (2017)). It is predicted that mispositioning of BRAG2 on the membrane would affect its efficiency. The biophysical and structural data reported in this invention, summarized in the model shown in Figure 6, strongly suggest that this is the mechanism by which Bragsin inhibits BRAG2.If Bragsin targets PH domains (widely present in signaling superficial membrane proteins) (Lemmon, MA Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Biol 9, 99-111 (2008)), it is noteworthy and surprising that it did not inhibit the exchange activity of ArfGEF, ARNO, and EFA6, as well as the RacGEF Trio (all of which also carry plasma membrane-bound PH domains). Such selectivity is supported by a mountain of evidence that lipid cooperativeness and / or the recognition of certain types of phosphoinositides are the general principles of the specific membrane mobilization of many PH domains and other superficial membrane-binding domains. As a result, the collective physicochemical properties of the lipid bilayer are recognized by superficial membrane proteins, in addition to, or instead of, specific interactions with individual lipids, and encode specificity-determining sites that lead to unique protein-membrane interfaces. Therefore, the specificity of Bragsin may arise from its boundary mechanism, thereby allowing it to recognize BRAG2 in relation to its unique interface with the lipid bilayer.

[0101] Membrane-surfaced proteins constitute a large class of signaling proteins that regulate life-sustaining cellular processes; however, "peripheromes" are still considered to have limited drug potential according to conventional techniques. Targeting superficial membrane proteins and membrane interactions represents an emerging paradigm shift in drug development, where the focus has traditionally been rather on inhibiting catalytic activity or protein-protein interactions, particularly in cancer. Bragsin represents an entirely new class of inhibitors, and it utilizes the protein-membrane interface for effective inhibition. This mode of inhibition is reminiscent of some protein-protein interaction boundary inhibitors, such as the natural compound Brefeldin A (capturing a non-productive complex with the small GTPase Arf1, and another subfamily of ArfGEFs in the growing list of protein-protein stabilizers that operate on the same principle). This invention demonstrates that the protein-membrane interface is a new weak point for superficial membrane proteins due to the specificity determinants and energy properties available to small molecules for inhibition. As illustrated by the inventor's observations, Bragsin affects the stemness of breast cancer cells.

[0102] This invention features novel inhibitors that utilize protein-membrane interactions. It presents a best-in-class tool for analyzing the BRAG2-mediated pathway involved in receptor signaling and cancer. The invention is also applicable to a wide range of superficial membrane signaling proteins and defines a new concept that paves the way for drug development based on the mechanisms described in this study.

[0103] [Table 1]

[0104] Human BRAG2 (protein 1 containing the IQ motif and SEC7 domain) has the following sequence: (https: / / www.uniprot.org / uniprot / Q6DN90.fasta) >sp|Q6DN90|IQEC1_HUMAN Protein containing IQ motif and SEC7 domain 1 OS=Homo sapiens OX=9606 GN=IQSEC1 PE=1 SV=1 [ka]

[0105] Human IQ motif and SEC7 domain-containing protein 1 is also known by the following sequence: (https: / / www.uniprot.org / uniprot / A0A087WWK8.fasta): >tr|A0A087WWK8|A0A087WWK8_HUMAN Protein containing IQ motif and SEC7 domain 1 OS=Homo sapiens OX=9606 GN=IQSEC1 PE=1 SV=1 [ka]

Claims

1. A pharmaceutical composition for use in therapeutic treatment methods, wherein the pharmaceutical composition has the following chemical structure (I): 【Chemistry 1】 A molecule having, or a pharmaceutically acceptable salt thereof, wherein, The aforementioned molecules are as follows: 【Chemistry 2】 {During the ceremony, R1 is an alkyl fluoride; R2 is hydrogen; R4 is alkyl or O-alkyl; R5 is hydrogen; R6 is either hydrogen or an O-alkyl group. A pharmaceutical composition selected from the group consisting of the following.

2. The aforementioned molecule is as follows: 【Transformation 3】 A pharmaceutical composition for use according to claim 1, selected from the group consisting of the following.

3. An inhibitor for use in the treatment of cancer, wherein the inhibitor comprises the PH domain of BRAG2 and It binds to the interface with the membrane and inhibits mammalian BRAG2, and has the structure described in claim 1 or 2. Inhibitors.

4. The following chemical structure (I): 【Chemistry 4】 A molecule that has, The aforementioned molecule is as follows: 【Transformation 5】 {During the ceremony, R1 is an alkyl fluoride; R2 is hydrogen; R4 is alkyl or O-alkyl; R5 is hydrogen; R6 is O-alkyl. A molecule selected from the group consisting of the following.

5. The aforementioned molecule is as follows: 【Transformation 6】 A molecule according to claim 4, selected from the group consisting of the following.

6. An inhibitor of mammalian BRAG2, having the structure described in any one of claims 1 to 5.

7. A method for inhibiting mammalian BRAG2 in vitro or in cellulose, comprising contacting BRAG2 with the BRAG2 inhibitor described in claim 3 or 6.

8. A pharmaceutical composition comprising at least one molecule according to claim 4 or 5, comprising one or more excipients and optionally one other pharmaceutically active ingredient.

9. A pharmaceutical composition according to any one of claims 1, 2, and 8, for use in a method of therapeutic treatment for a disease exhibiting BRAG2 overexpression, The aforementioned disease is cancer, particularly invasive cancer, metastatic cancer, EGFR and / or ErbB2 modulator A pharmaceutical composition selected from the group consisting of cancer, angiogenesis, and diabetic retinopathy, which have resistance to the following.

10. Any one of claims 1, 2, and 8, as a drug that binds to the interface between the PH domain of BRAG2 and the membrane, for use in a method of therapeutic treatment for diseases requiring protein-membrane interactions. A pharmaceutical composition described above, The aforementioned disease is cancer, particularly invasive cancer, metastatic cancer, EGFR and / or ErbB2 modulator A pharmaceutical composition selected from the group consisting of cancer, angiogenesis, and diabetic retinopathy, which have resistance to the following.

11. Cancer, especially invasive cancer, metastatic cancer, and resistance to EGFR and / or ErbB2 modulators. A pharmaceutical composition according to any one of claims 1, 2, and 8, for use in a method of therapeutic treatment for a disease selected from the group consisting of cancer, angiogenesis, and diabetic retinopathy.

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