US20140194451A1 - Cdc42 Inhibitor and Uses Thereof - Google Patents

Cdc42 Inhibitor and Uses Thereof Download PDF

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US20140194451A1
US20140194451A1 US14/087,662 US201314087662A US2014194451A1 US 20140194451 A1 US20140194451 A1 US 20140194451A1 US 201314087662 A US201314087662 A US 201314087662A US 2014194451 A1 US2014194451 A1 US 2014194451A1
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cdc42
compound
treatment
zcl278
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Qun Lu
Huchen Zhou
Yanhua Chen
Amy Friesland
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    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32—One oxygen, sulfur or nitrogen atom
    • C07D239/42—One nitrogen atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/06—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to acyclic carbon atoms
    • C07C335/10—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C07C335/12—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to acyclic carbon atoms of an unsaturated carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/69—Benzenesulfonamido-pyrimidines

Definitions

  • the present invention relates to compounds which are inhibitors of the small G protein Rho GTPase Cdc42 and uses of the described compounds.
  • Cell division cycle protein Cdc42 is a sub-class of the small G protein Rho GTPase family and is an important regulatory protein of many cell biological functions. First identified in Saccharomyces cerevisiae for its involvement in cell polarization, Cdc42 was then recognized to play important roles in cytoskeletal reorganization, cellular endocytosis, regulation of cell cycle and cell transcription. Activation of Cdc42, like that of most GTPases, is achieved through the exchange of guanosine-5′-diphosphate (GDP) for guanosine-5′-triphsopahte (GTP) binding.
  • GDP guanosine-5′-diphosphate
  • GTP guanosine-5′-triphsopahte
  • GEF guanine nucleotide exchange factor
  • GAP GTPase activating protein
  • GDI Guanosine nucleotide dissociation inhibitors
  • Cdc42 chronic myelogenous leukemia
  • EMT epithelial to mesenchymal cell transition
  • Rho GTPase family members studies of Cdc42 lag far behind the RhoA and Rac1. This is partly due to fast activation/inactivation cycles of Cdc42, but also to the lack of selective small molecule research tools to help understand this process directly.
  • Rho GTPase family proteins are involved in the signaling pathways that regulate a variety of biochemical and cellular functions, e. g. cell membrane and material transport, cell cycle regulation and cytoskeletal organization which is related to the control of cell morphology, cell motility and cell fate.
  • cell membrane and material transport e. g. cell membrane and material transport
  • cell cycle regulation e. g. cell cycle regulation
  • cytoskeletal organization e. g. cell cellular function of cells
  • deregulated Rho GTPase signaling is involved in the pathogenesis of many diseases, and therefore it has become an important target for drug development.
  • Rho GTPase family proteins relate to the study of the functions of Rho GTPase family proteins.
  • RhoA downstream effector signaling molecules target RhoA downstream effector signaling molecules and are recognized potent Rho/p160 ROCK inhibitors.
  • RhoA downstream effector signaling molecules As a Rac1-selective inhibitor, NSC23786 in the recent study of the small molecule compounds targeting Rac1-GEF also greatly facilitate the understanding of Rac1 protein function.
  • Cdc42 selective inhibitors Secramine analogues of natural products galantamine recently showed to behave like a RhoGDI and inhibited Cdc42-dependent Golgi-mediated protein transport through cell membranes.
  • Cdc42 alteration is closely involved in tumorigenesis in many ways, including tumor transformation and metastasis: in addition, the development and maintenance of neurons is also heavily dependent on the normal Cdc42 activity.
  • NSCG23766 was identified through a computer simulation of the structure of the compounds screened, and it fits the Rac1 molecular surface structure and the known Rac1 essential binding GEF (Gao, Y., J B Dickerson et al (2004) Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci USA. 101 (20): 7618-7623).
  • NSC23766 can inhibit serum or growth factor-induced Rac1 activation and Rac1 mediated lamellipodia formation.
  • NSC23766 inhibits cell proliferation in human prostate cancer cell lines and tumor growth, and reduces cell invasion phenotype of the tumor cells which are dependent on the activity of endogenous Rac1.
  • NSC23766 treatment can improve Rac1-mediated spinal cord injury (SCI)-induced neuropathic pain (Tan, A M, S. Stamboulian, et al (2008). Neuropathic pain memory is maintained by Rac1-regulated dendritic spine remodeling after spinal cord injury. J Neurosci. 28 (49): 13173-13183).
  • SCI spinal cord injury
  • FIGS. 1A-1C show characterization of described compounds according to one embodiment, and referred to as ZCL compounds, in targeting Cdc42-intersectin (ITSN) interaction:
  • FIG. 1A shows docked pose of ZCL278 in the Cdc42 binding pocket with protein shown as gray surface (Arrow 1 ) and ligand is shown as sticks (Arrow 2 );
  • FIG. 1B shows proposed interactions between ZCL278 and Cdc42 residues, with ZCL278 shown as sticks (Arrow 3 ), Cdc42 is shown as gray cartoon, residues of Cdc42 are shown as sticks (Arrow 4 ), and hydrogen bonds are represented as dashed lines (Arrow 5 ); and
  • FIG. 1A shows docked pose of ZCL278 in the Cdc42 binding pocket with protein shown as gray surface (Arrow 1 ) and ligand is shown as sticks (Arrow 2 );
  • FIG. 1B shows proposed interactions between ZCL278 and Cdc42 residues, with ZCL278 shown as sticks
  • FIG. 1C shows superposition of GMP-PCP (Protein DataBank ID code 2QRZ) and the docked Cdc42-ZCL278 complex, with Cdc42 indicated by gray cartoon, ZCL278 by sticks, and GMP-PCP (GMP-PCP is GTP analogs, beta, gamma-methylene diphosphate guanylate) by sticks, as indicated.
  • GMP-PCP Protein DataBank ID code 2QRZ
  • GMP-PCP GTP analogs, beta, gamma-methylene diphosphate guanylate
  • FIGS. 2 A.- 2 C show properties of ZCL278: FIG. 2A shows the inhibition of Cdc42-mediated cell microspike formation; FIG. 2B shows ZCL278 inhibits the activation of endogenous Rac/Cdc42; and FIG. 2C shows ZCL278 inhibits the stimulated Cdc42 activation.
  • FIGS. 3A-3C show immunofluorescence staining of active Cdc42 and the phosphorylated RhoA.
  • FIG. 4 shows ZCL278 disrupts the GM130-docked Golgi organization.
  • FIGS. 5A-5C show ZCL278 suppresses cell migration without affecting cell viability.
  • FIGS. 6A-6C show ZCL278 inhibits neuronal branching and growth cone dynamics.
  • compounds which inhibit Cdc42 are provided.
  • the compound has the following structure A:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 are each independently selected from the group consisting of fluoro, chloro, bromo, nitro, cyano, amino, methyl, hydroxylmethyl, trifluoromethyl, methoxy, trifluoromethyoxy, and ethyl.
  • the compound has the following structure B:
  • A, D, E, G, J, M, W, X, Y, Z are each selected from the group consisting of nitrogen, carbon, and substituted carbon, and any of A, D, E, G, J, M, W, X, Y, Z may be missing, such that the ring structure is less than a six-membered ring.
  • the compound is a combination of structures A and B.
  • Y and Z are N and R 1 , R 2 and R 3 , as described for structure A, are attached to X, W, and M of structure B, respectively.
  • the compound has the following structure C:
  • R 1 and R 2 are independently selected from the group consisting of alkyl, cyclic alkyl, aryl, and substituted aryl. In one embodiment, R 1 is selected from the group consisting of
  • R2 is selected from the group consisting of
  • the compound has the structure of formula I:
  • the compound may be a variant of the compound of Formula I selected from the group consisting of Cl-2, Cl-3, Cl-4, and Cl-5, as shown below:
  • the present compounds are used to inhibit Cdc42 and Cdc42 mediated cellular processes.
  • Cdc42 has important roles in cell cycle regulation, cell movement, adhesion, apoptosis and intracellular transport, and in the same time it plays important roles in the development of cancer and invasion, cardiovascular system and respiratory diseases, neurological diseases, and many other diseases
  • the presently described compounds, including ZCL278, can be prepared for the treatment of malignant tumors.
  • the described compounds may be prepared for the prevention and treatment of malignant tumor development and invasion.
  • the described compounds can be prepared for the treatment of cardiovascular diseases.
  • the described compounds can be prepared for the treatment of pulmonary and respiratory diseases.
  • the described compounds can be prepared for the treatment of diseases of the nervous system.
  • the described compounds function through inhibition of Cdc42.
  • malignant tumor is meant to encompass any malignant proliferative cell disorder such as carcinoma, sarcoma, lymphoma and blastoma.
  • cancers that may be treated using the present method include, but are not limited to, colorectal, prostate, testes, lung, stomach, pancreas, uterine, cervix, bone, spleen, head and neck, brain such as glioblastoma multiforme, breast, ovary, stem cell tumors, non-Hodgkin's lymphoma, Kaposi's sarcoma and leukemia.
  • the terms “treat”, “treating” or “treatment” means alleviating, inhibiting the progression of, or preventing the cancer, or one or more symptoms thereof.
  • the present compound is provided as a pharmaceutical composition.
  • the pharmaceutical composition may also include one or more pharmaceutically acceptable excipients such as, but not limited to, carriers, diluents, adjuvants and vehicles.
  • Excipients that may be included in the present formulation include preserving or antioxidant agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, lubricants such as sodium lauryl sulfate, stabilizers, solvents, dispersion media, tableting agents, colouring and flavouring agents, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents.
  • Supplementary active agents or ingredients may also be included in the present pharmaceutical composition.
  • ZCL278 effectively suppressed GTP-binding activity of Cdc42.
  • ZCL278 affects Cdc42 regulation of subcellular structures of two of the most important Cdc42 functions: the elimination of the formation of microspikes or filopodia and disruption of GM130-docked Golgi structures.
  • Rac-selective inhibitor NSC23766 ZCL278 reduced the peri-nuclear accumulation of active Cdc42.
  • ZCL278 inhibits Cdc42-mediated neuronal branching and growth cone dynamics, and inhibits metastatic prostate cancer cells PC-3 cell actin-based movement and migration without disrupting the cell survival.
  • the compound can play an important role in modulating the development and invasion of cancer, cardiovascular system and respiratory diseases, and nervous system diseases.
  • the putative binding pocket on Cdc42 was created within 7 ⁇ of the center of the above ITSN residues that interact with Cdc42.
  • the binding pocket consists of 16 Cdc42 residues, including Thr35, Val36, Asn39, Phe56, and Asp57 ( FIGS. 1A-1C ).
  • Cdc42-ITSN The structure pose of Cdc42-ITSN complex was from the protein databank (PDB: 1 Kl1).
  • the ITSN residues that occupy the Cdc42 binding interface are Leu1376, Met1379, Gin1380, Thr1383, and Arg1384.
  • the binding pocket on Cdc42 was created with residues of Cdc42 within 7.0 ⁇ of the center of the above five ITSN residues.
  • the docking grid was generated in the Receptor Grid Generation module. The 197,000 compounds from SPECS were screened using HTVS (high-throughput virtual screening) and SP (standard precision) docking sequentially.
  • top ranked 50,000- molecules From the top ranked 50,000- molecules, more stringent SP (standard precision) docking resulted in the top ranked 100 molecules.
  • the top ranked 100 molecules were subjected to manual inspection according to the following criteria. ITSN-like binding posture and occupation for the Leu1376, Gln1380, Arg1384, Met1379, and Thr1383 residue space of ITSN should be observed; at least three hydrogen bonds should be formed, a conserved hydrogen bond with Asn39 or Phe37 of Cdc42 should exist; and diversity of scaffolds should be considered. A selection of 30 compounds was eventually tested on their ability to disrupt Cdc42 activity and/or functions.
  • Computed binding mode of ZCL278 in Cdc42 as shown in FIG. 1A , one small molecule, termed ZCL278, bound to a well-formed Cdc42 pocket lined by residues Tbr35, Val36, Asp38, Asn38, Phe56, Tyr64, Leu67, and Leu70. Extensive favorable interactions were found between ZCL278 and Cdc42 residues. Five hydrogen bonds involving residues Thr35, Asn39, and Asp57, as well as hydrophobic interactions associated with residues Val36 and Phe58 were observed ( FIG. 1B ). The bromophenyl ring was inserted into the adjacent GTP/GDP binding pocket. The computed binding mode suggests that ZCL278 should be able to disrupt the Cdc42-ITSN interaction as well as GTP/GDP binding ( FIG. 1C ).
  • ZCL278 was synthesized by the following synthetic method. This route of synthesis method is used for illustration, rather than limiting the present invention. Those skilled in the art can understand and expect other synthetic methods to synthesize ZCL278, which also belong to the scope of protection of the present invention.
  • Reaction Reagents and Conditions (a) K 2 CO 3 , DMF (N, N-dimethyl formamide), 70° C.; (b) NaOH, dioxane (dioxane. Embankments B)/H 2 0; (c) SOCl 2 (thionyl chloride), DMF, reflux (reflux); (d) NaSCN (sodium thiocyanate), acetone (acetone), 0 C-room temperature; (e) 4-amino-N-( 4,6-dimethylpyrimidin-2-yl) benzene-sulfonamide (4-amino-N-(4,6-dimethyl-2-pyrimidinyl) benzenesulfonamide), 0° C.-room temperature.
  • Step 1 2-(4-bromo-2-chlorophenoxy) acetate (i.e. Compound 2) Synthesis.
  • Anhydrous K 2 CO 3 (3.45 g, 25.0 mM) was added to a solution of 4-bromo-2-chlorophenol (1) (5.2 g, 25.0 mM) and ethyl 2-bromoacetate (4.3 g, 25.7 mM) in 50 mL DMF. After stirring overnight at 70° C., the mixture was poured into 150 mL water and extracted with ethyl acetate (70 mL ⁇ 4). The organic layer was combined, washed with brine (100 ml ⁇ 3), and dried over anhydrous Na 2 SO 4 .
  • Step 3 Preparation of 4-(3-(2-(4-bromo-2-chlorophenoxy) acetyl) thioureido)-N-(4,6-dimethyl-2-pyrimidinyl:) benzene—Synthesis of the sulfonamide (i.e., compound 5).
  • Compound 3 (539 mg, 2.0 mM) in 25 mL SOCl 2 and a drop of DMF were heated to reflux. After 3 hours, SOCl 2 was removed by distillation and the residue was dried in vacuo for 5 minutes to give crude acyl chloride 4.
  • the binding affinity of ZCL278 and Cdc42 was assessed by using two independent biophysical methods.
  • fluorescence titration of purified Cdc42 by ZCL278 was carried out by monitoring the change of fluorescence intensity of a tryptophan residue on Cdc42 upon ZCL278 binding.
  • ZCL278 has a weak absorption peak at 310 nm, to avoid any experimental error that might result from potential fluorescence quenching by ZCL278, the fluorescence emission of Cdc42 was monitored at 350 nm, at which ZCL278 has a negligible absorption.
  • a Kd value of 6.4 ⁇ M was obtained.
  • a surface plasmon resonance (SPR) experiment was performed by covalently immobilizing purified Cdc42 onto CM5 chips and varying ZCL278 concentration.
  • the SPR response was observed to increase along with elevated ZCL278 concentrations, and eventually gave a Kd of 11.4 ⁇ M.
  • the solubility of ZCL278 was determined to be 181 ⁇ M and was greater than the concentrations used in all examples.
  • the experimental pKa values of ZCL278 were determined to be 3.48 ⁇ 0.04, 6.61 ⁇ 0.02, and 7.45 ⁇ 0.01.
  • the pKa value of 3.48 is associated to pyrimidine nitrogen that should stay in a neutral form at pH 7.4
  • the NH groups corresponding to pKa values of 6.61 and 7.45 should be partially deprotonated and give a population of charged species in solution. These species in solution may have modifying effects on membrane transport and binding to Cdc42.
  • ZCL278 inhibits Cdc42-mediated microspike formation.
  • Compound ZCL278 was applied at 50 ⁇ M for 1 hour and then stimulated with the Cdc42 activator for 2 minute.
  • the cell periphery of ZCL278-treated cells resembles control cells with few microspikes ( FIG. 2A , ZCL278).
  • Cdc42 activation was investigated in human metastatic prostate cancer PC-3 cells that were treated with the Cdc42 activator or 50 ⁇ M ZCL278 for 5, 10, and 15 minutes.
  • Serine 71 phosphorylation is known to negatively regulate Rac/Cdc42 activity, thus an increase in phospho-Rac/Cdc42 expression is indicative of a decrease in active (GTP-bound) Rac/Cdc42.
  • activation of Cdc42 shows an expected decrease in phospho-Rac/Cdc42.
  • the application of ZCL278 resulted in a time-dependent increase in Rac/CDc42 phosphorylation.
  • Wiskott-Aldrich syndrome Protein is a downstream effector of Cdc42 activation. Tyrosine phosphorylation of WASP is linked to rapid Cdc42 degradation following its activation. As shown in FIG. 2B , the Cdc42 activator leads to a decreased expression of phospho-WASP by 15 minutes while ZCL278 does not suppress phospho-WASP activity. Thus, ZCL278 inhibits Rac/Cdc42 phosphorylation in a time-dependent manner and maintains tyrosine phosphorylation of WASP.
  • Serine 71 phosphorylation can occur on both Rac and Cdc42.
  • a G-LISA an ELISA-based assay that allows a quantitative determination of the levels of GTP-bound (active) Cdc42 in cellular lysates was utilized.
  • Serum-starved Swiss 3T3 fibroblasts were incubated for 1 hour with 50 ⁇ M ZCL278 or 10 ⁇ M NSC23766 (Rac inhibitor), followed by 2 minutes of stimulation with 1 unit/mL Cdc42 activator.
  • This analysis revealed a significant increase (70%) in GTP-bound Cdc42 in cells treated with the activator as compared to control (untreated) cells ( FIG. 2C ).
  • Cells treated with ZCL278 showed a dramatic (nearly 80%) decrease in GTP-Cdc42 content as compared with cells treated solely with the activator.
  • NSC23766 was developed in a similar manner as ZCL278; however, it is specific to Rac and should therefore act as an additional negative control in this assay. As expected, NSC23766 does not reduce GTP-Cdc42 content ( FIG. 2C ). These data establish that ZCL278 inhibits Cdc42 in two different cell types.
  • Immunofluorescent staining Swiss3T3 cells were grown on coverslips to 30% confluence. In serum-deprived culture, cells were treated with 10 uM NSC23766 or 50 uM ZCL278 for 1 hour followed by 1 unit/mL Cdc42 agonists (Cytoskeleton Products) treatment for 1 minute. Added agonist alone was used as a positive control while DMSO was used as a negative control. Cells on the coverslips were fixed in 4% paraformaldehyde for 15 minutes, and in 0.2% Triton X-100 for permeabilization for 15 minutes and then blocked in 10% BSA for 30 minutes.
  • Rhodamine Phalloidin was incubated for 1 hour at room temperature to observe filamentous actin under the Zeiss Axiovert fluorescent light microscopy of cells staining. Image processing software MetaMorph-5 was used to randomly select cells in randomly selected five-point pixels to obtain the average number of each group for pixel intensity values.
  • the Western blot PC3 cells cultured to approximately 70% of the density, then removed serum and cultured for 16 hours, with addition of drugs; 1 unit/mL Cdc42 agonist (cytoskeleton) 5 uM, 50 uM ZCL278 to treat cells for 5, 10, and 15 minutes respectively.
  • Cell lysis buffer (Formulation: 50 mM Tris buffer solution pH 7.5, 10 mM magnesium chloride, 0.5 M sodium chloride. 1% Triton X-100 and protease inhibitors) cell lysis and centrifuged at 14000 rpm and 4° C to obtain extracted protein samples.
  • G-LISA kit Swiss 3T3 cells were cultured to 40% of the growth density, then removed serum and cultured for 48 hours, treated with Cdc42 agonist for 1 minute, and treated with the 50 uM ZCL278 and 10 uM NSC23766. Cell lysis proteins were extracted according to kit instructions, and analysis of the quantification of total protein concentration to be 0.15 mg/ml. Untreated cells or cells treated with buffer were used as negative control while the agonist treated cells and active Cdc42 protein were used as a positive control. Enzyme linked immunosorbent assay measured the absorbance value of each sample 490 nm light wave.
  • FIG. 3B shows the number of cells of each group with Golgi-like distribution, randomly selected 6 cells were independently counted for Golgi-endoplasmic reticulum network (*; p ⁇ 0.05).
  • GM130 a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and helps to maintain cis-Golgi structures, was examined.
  • Filopodia are dynamic structures that aid cells in pathfinding and migration, and are largely controlled by Cdc42 activity.
  • a wound healing assay was used in order to elucidate the effects of ZCL278 on cellular migration.
  • Cells were photographed at 0 and 24 hours following drug treatments and MetaMorph software was used to determine the distances cells had migrated. Black line indicates the boundary of wounded area. Each experiment was tested with p value (p ⁇ 0.05).
  • PC3 cells were plated at 75000 /ml for 48 hours, serum was removed and 50 uM ZCL278 or 10 uM NSC23766 was added, and then continue to culture for 24 hours before staining with trypan blue to determine cell survival rate.
  • Cdc42 activation resulted in a significant increase (58%) in wound healing ability in comparison to controls (41%).
  • Bar graph shows the wound area compared to that before drug treatment. The experiment was repeated three times and the means were obtained: **; p ⁇ 0.01, *:p ⁇ 0.05. Wound closure was less pronounced at 50 ⁇ M (8%) than 5 ⁇ M (30%) concentrations. Cellular migration was also significantly reduced with NSC23766 treatment. This result is to be expected since Rac regulates the formation of lamellipodia, which are well-described motile structures.
  • FIG. 5C demonstrates that there was no difference in viability between treated and non-treated (control) cells. Therefore, the differences seen in migratory ability is due to ZCL278-mediated Cdc42 inhibition or NSC23766-mediated Rac inhibition and not cell death.
  • ZCL278 inhibits neuronal branching and growth cone dynamics
  • Cdc42 plays a crucial role in the establishment of neuronal morphogenesis. Cdc42's absence in neurons results in a significantly reduced number of neurites and severely disrupted filopodia function. Therefore, the ability of ZCL278 to inhibit neuronal branching in primary neonatal cortical neurons was tested.
  • Cdc42 is also widely known to control filopodia and microspikes at the leading edge of migrating growth cones.
  • Time-lapse video light microscopy shows a control cortical neuron with multiple microspikes or filopodia extended from the growth cone ( FIG. 6C ).
  • ZCL278 treatments resulted in rapid retraction of filopodia within 4 minutes ( FIG. 6C ).
  • these studies further support ZCL278 as an effective small-molecule inhibitor of Cdc42-mediated neuronal branching and growth cone motility.
  • the present description provided computer simulation methods which were applied for high-throughput in silico screening of compounds that target chimeric Cdc42-GEF structures. Based on the structure characteristics of Cdc42 and its specific GEF intersectin (ITSN), the three-dimensional structure of the described compounds can fit exactly into the pocket that intersectin interacts with Cdc42. Additional research successfully screened and identified a compound ZCL278 as a cell permeable Cdc42 specific inhibitor.
  • ZCL278 As the first small molecule inhibitor of Cdc42, selectively targeting Cdc42 and its GEF intersectin.
  • Cell wound healing experiments showed that activated Cdc42 promotes the wound closure and tumor cell metastasis.
  • ZCL278 significantly inhibits the migration of PC3 cells in a concentration-dependent manner.
  • ZCL278 inhibits cell migration but it is not cytotoxic and does not cause cell death.
  • Cdc42 plays an important role in the development of neurons.
  • Garalov et al. J. Neurosci. 27(48): 13117-13129
  • Cdc42-deficient mice exhibited brain and neuronal development which was severely disrupted.
  • These mice showed a series of brain malformations, including the reduction of the axon bundles, as well as neurons filamentous pseudopodia dynamics and reduced growth cone, and suppression of axonal extension.
  • the movement of axons and dendrites is mainly actin-based, the process also regulated by Cdc42.
  • ZCL278 can reduce the number of branches of the newborn central neurons, and inhibition of the growth cone dynamics.
  • ZCL278, which targets Cdc42-ITSN is the first small molecule inhibitor that can be effectively used in the studies of molecular functions of Cdc42 in cancer and neurological disorders.

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US20190091184A1 (en) * 2017-09-25 2019-03-28 East Carolina University Roles of modulators of intersectin-cdc42 signaling in alzheimer's disease
WO2021011800A1 (fr) * 2019-07-17 2021-01-21 Children's Hospital Medical Center Méthode de traitement de maladies néoplasiques à l'aide d'un inhibiteur spécifique de cdc42
WO2021034616A1 (fr) * 2019-08-16 2021-02-25 Children' S Hospital Medical Center Méthodes de traitement d'un sujet au moyen d'un inhibiteur spécifique de cdc42

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EP2716292B1 (fr) 2011-05-23 2017-09-06 ONCO Biomedical Technology (Suzhou) Co., Ltd Inhibiteur de cdc42 et ses utilisations
CN115068480B (zh) * 2022-08-09 2023-10-20 郑州大学第一附属医院 细胞分裂周期蛋白42小分子抑制剂在制备治疗慢性肾脏病药物中的应用

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US20190091184A1 (en) * 2017-09-25 2019-03-28 East Carolina University Roles of modulators of intersectin-cdc42 signaling in alzheimer's disease
US11439608B2 (en) * 2017-09-25 2022-09-13 Qun Lu Roles of modulators of intersectin-CDC42 signaling in Alzheimer's disease
WO2021011800A1 (fr) * 2019-07-17 2021-01-21 Children's Hospital Medical Center Méthode de traitement de maladies néoplasiques à l'aide d'un inhibiteur spécifique de cdc42
WO2021034616A1 (fr) * 2019-08-16 2021-02-25 Children' S Hospital Medical Center Méthodes de traitement d'un sujet au moyen d'un inhibiteur spécifique de cdc42

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CN102796050A (zh) 2012-11-28
KR20140028078A (ko) 2014-03-07
US9725417B2 (en) 2017-08-08
EP2716292A4 (fr) 2014-12-03
JP2014515357A (ja) 2014-06-30
US20150329496A1 (en) 2015-11-19
CN102796050B (zh) 2014-07-23
WO2012159456A1 (fr) 2012-11-29
EP2716292B1 (fr) 2017-09-06
EP2716292A1 (fr) 2014-04-09
JP6001650B2 (ja) 2016-10-05
US20170334863A1 (en) 2017-11-23

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