Granted Patent
B2
US 12,269,900 · App. 16/988,287 · Granted Apr 8, 2025
Inhibitors of β integrin-G protein α subunit binding interactions
View Patent ↗
Loading inventors, assignments & file history…
Abstract
Provided herein are compounds that inhibit a binding interaction between a β integrin and a G protein subunit, as well as compositions, e.g., pharmaceutical compositions, comprising the same, and related kits. In some embodiments, the compound is an antibody or antibody analog, and, in other embodiments, the compound is a peptide or peptide analog. Also provided are methods of using the compounds, including methods of treating or preventing a medical condition, such as stroke, heart attack, cancer, or inflammation.
Claims (31)
1. A six-mer peptide consisting of the amino acid sequence
Phe Xaa 1 Xaa 2 Glu Xaa 3 Xaa 4
wherein Xaa 1 is Glu and Xaa 2 is Glu or Lys,
wherein Xaa 3 is Lys or Arg and Xaa 4 is Met or Leu,
and
wherein the peptide inhibits a binding interaction between a β integrin and a G protein α subunit.
2. A pharmaceutical composition comprising the peptide of claim 1 , or a micelle comprising the peptide and a pharmaceutically acceptable carrier, diluent, or excipient.
3. The peptide of claim 1 , wherein Xaa 3 is Arg.
4. The peptide of claim 3 , wherein Xaa 4 is Met.
5. The peptide of claim 3 , wherein Xaa 4 is Leu.
6. The peptide of claim 1 , wherein Xaa 3 is Lys.
7. The peptide of claim 6 , wherein Xaa 4 is Met.
8. The peptide of claim 6 , wherein Xaa 4 is Leu.
9. A peptide comprising the amino acid sequence Phe Xaa 1 Xaa 2 Glu Xaa 3 Xaa 4
wherein Xaa 1 is Glu and Xaa 2 is Glu or Lys,
wherein Xaa 3 is Lys or Arg and Xaa 4 is Met or Leu,
wherein the peptide is a 6-mer, 7-mer or 8-mer and the peptide is cyclized.
10. The peptide of claim 9 , further comprising two Cys residues, the sulfur atoms of which form a disulfide bridge.
11. A pharmaceutical composition comprising the peptide of claim 9 , or a micelle comprising the peptide and a pharmaceutically acceptable carrier, diluent, or excipient.
12. The peptide of claim 9 , wherein Xaa3 is Arg.
13. The peptide of claim 12 , wherein Xaa4 is Met.
14. The peptide of claim 12 , wherein Xaa4 is Leu.
15. The peptide of claim 9 , wherein Xaa3 is Lys.
16. The peptide of claim 15 , wherein Xaa4 is Met.
17. The peptide of claim 15 , wherein Xaa4 is Leu.
18. A peptide comprising the amino acid sequence of FEEERL (SEQ ID NO: 419), FEEEKM (SEQ ID NO: 420), FEKERM (SEQ ID NO: 421), FEEEKL (SEQ ID NO: 422, or FEKERL (SEQ ID NO: 423), wherein the peptide is a 6-mer, 7-mer or 8-mer, and wherein the peptide inhibits a binding interaction between a β integrin and a G protein α subunit.
19. A pharmaceutical composition comprising the peptide of claim 18 , or a micelle comprising the peptide and a pharmaceutically acceptable carrier, diluent, or excipient.
20. The peptide of claim 18 , further comprising two Cys residues, the sulfur atoms of which form a disulfide bridge.
21. The peptide of claim 18 , wherein the peptide is cyclized.
22. A method of inhibiting thrombosis in a subject in need thereof comprising administering to the subject the pharmaceutical composition of claim 2 in an amount effective to inhibit thrombosis.
23. A method of treating stroke or heart attack in a subject in need thereof comprising administering to the subject the pharmaceutical composition of claim 2 in an amount effective to treat stroke or heart attack.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Oct 1, 2021
From: DU, XIAOPING
To: THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ILLINOIS
Reel/Frame 057684/0626 →
Continuity (8)
Continuation In Part
PCTUS2011028567
· Mar 15, 2011
Provisional Application
61433037
· Jan 14, 2011
Provisional Application
61314027
· Mar 15, 2010
References Cited (130)
US 8685921B2
· Du
· 2014
[cited by applicant]
US 9156884B2
· Du
· 2015
[cited by applicant]
WO WO9209302A1
· 1992
[cited by applicant]
WO WO0063236A2
· 2000
[cited by applicant]
WO WO02072759A2
· 2002
[cited by applicant]
WO WO2004098627A1
· 2004
[cited by applicant]
WO WO2006028393A1
· 2006
[cited by applicant]
WO WO2008011513A2
· 2008
[cited by applicant]
WO WO2008087216A1
· 2008
[cited by applicant]
WO WO2008102557A1
· 2008
[cited by applicant]
WO WO2011116026A2
· 2011
[cited by applicant]
Aapptec (aapptec Practical Guide to Solid Phase Peptide Synthesis, pp. 1-76, published on 2009).
[cited by examiner]
Richard Clark, Engineering stable peptide toxins by means of backbone cyclization; PNAS Sep. 27, 2005 102 (39) 13767-13772).
[cited by examiner]
UniProtKB—A0A618TEU0, Integrin beta, Uniprot Protein Database, accessed on Apr. 4, 2022.
[cited by examiner]
Integrin beta, UniProtKB—S4R4Y5, Uniprot Protein Database, accessed on Apr. 4, 2022.
[cited by examiner]
UniProtKB—P07228 (ITB1_CHICK), Integrin beta-1, Uniprot Protein Database, accessed on Apr. 4, 2022.
[cited by examiner]
UniProtKB—Q65NK5 (Q65NK5_BACLD), Lichenysin synthetase A, Uniprot Protein Database, accessed on Apr. 4, 2022.
[cited by examiner]
Anthis et al., Beta integrin tyrosine phosphorylation is a conserved mechanism for regulating talin-induced integrin activation, J. Biol. Chem., 284(52):36700-10 (2009).
[cited by applicant]
Arias-Salgado et al., Src kinase activation by direct interaction with the integrin beta cytoplasmic domain, Proc. Natl. Acad. Sci. USA, 100(23): 13298-302 (2003).
[cited by applicant]
Arthur et al., Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism, Curr. Biol., 10(12):719-22 (2000).
[cited by applicant]
Berendsen, A glimpse of the holy grail?, Science, 282:642-3 (1998).
[cited by applicant]
Bradley et al., Limits of cooperativity in a structurally modular protein: Response of the notch ankyrin domain to analogous alanine substitutions in each repeat, J. Mol. Biol., 324:373-86 (2002).
[cited by applicant]
Brass et al., GTP-binding proteins and platelet activation, Prog. Hemost. Thromb., 10:127-74 (1991).
[cited by applicant]
Calderwood et al., The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation, J. Biol. Chem., 274(40):28071-4 (1999).
[cited by applicant]
Cho et al., A critical role for extracellular protein disulfide isomerase during thrombus formation in mice, J. Clin. Invest., 118(3):1123-31 (2008).
[cited by applicant]
Coller, Anti-GPIIb/IIIa drugs: current strategies and future directions, Thromb. Haemost., 86(1):427-43 (2001).
[cited by applicant]
Coller, Interaction of normal, thrombasthenic, and Bernard-Soulier platelets with immobilized fibrinogen: defective platelet-fibrinogen interaction in thrombasthenia, Blood, 55(2):169-78 (1980).
[cited by applicant]
Critchley et al., Talin at a glance, J. Cell Sci., 121(9):1345-7 (2008).
[cited by applicant]
Dai et al., A critical role for 14-3-3zeta protein in regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic implications, Blood, 106(6):1975-81 (2005).
[cited by applicant]
Day et al., Murine thrombosis models, Thromb. Haemost., 92(3):486-94 (2004).
[cited by applicant]
Designing Custom Peptides Technical Bulleting, Sigma-Genosys, 2 pages (2004).
[cited by applicant]
Dominguez et al., Antigenic homology of HIV-1 GP41 and human platelet glycoprotein GPIIIa (integrin beta3),
[cited by applicant]
Du et al., Calpain cleavage of the cytoplasmic domain of the integrin B2 subunit, J. Biol. Chem., 270:26146-51 (1995).
[cited by applicant]
Du et al., Identification of a binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein Ib alpha, J. Biol. Chem., 271(13):7362-7 (1996).
[cited by applicant]
Du et al., Ligands “activate” integrin alpha IIb beta 3 (platelet GPIIb-IIIa), Cell, 65(3):409-16 (1991).
[cited by applicant]
Dyke, Safety of glycoprotein IIb-IIIa inhibitors: A heart surgeon's perspective, Am. Heart J., 138(Pt. 2):307-16 (1999).
[cited by applicant]
Edelstein et al., Computer control of microscopes using μManager, Unit 14.20.1-14.20.17 Current Protocols in Molecular Biology (Oct. 2010).
[cited by applicant]
Falati et al., Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse, Nat. Med., 8(10):1175-81 (2002).
[cited by applicant]
Flevaris et al., A molecular switch that controls cell spreading and retraction, J. Cell Biol., 179(3):553-65 (2007).
[cited by applicant]
Flevaris et al., Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signaling pathway, Blood, 113(4):893-901 (2009).
[cited by applicant]
Giannone et al., Talin1 is critical for force-dependent reinforcement of initial integrin-cytoskeleton bonds but not tyrosine kinase activation, J. Cell Biol., 163(2):409-19 (2003).
[cited by applicant]
Ginsberg et al., Integrin regulation, Curr. Opin. Cell Biol., 17(5):509-16 (2005).
[cited by applicant]
Goksoy et al., Structural basis for the autoinhibition of talin in regulating integrin activation, Mol. Cell, 31(1):124-33 (2008).
[cited by applicant]
Gong et al., G protein subunit Galpha13 binds to integrin alphallbbeta3 and mediates integrin “outside-in” signaling, Science, 327(5963):340-3 (2010).
[cited by applicant]
Gu et al., Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model, J. Cell Biol., 147(5):1085-96 (1999).
[cited by applicant]
Guanine nucleotide-binding protein subunit alpha-12 [
[cited by applicant]
Guanine nucleotide-binding protein subunit alpha-13 [
[cited by applicant]
Haling et al., Talin-dependent integrin activation is required for fibrin clot retraction by platelets, Blood, 117(5):1719-22 (2011).
[cited by applicant]
Hart et al., Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13, Science, 280(5372):2112-4 (1998).
[cited by applicant]
Hayward et al., Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function, J. Thromb. Haemost., 4(2):312-9 (2006).
[cited by applicant]
Hu et al., Lsc activity is controlled by oligomerization and regulates integrin adhesion, Mol. Immunol., 45(7):1825-36 (2008).
[cited by applicant]
Huang et al., Signaling through G(alpha) 13 switch region I is essential for protease-activated receptor 1-mediated human platelet shape change, aggregation, and secretion, J. Biol. Chem., 282(14):10210-22 (2007).
[cited by applicant]
Humphries et al., Vinculin controls focal adhesion formation by direct interactions with talin and actin, J. Cell Biol., 179(5):1043-57 (2007).
[cited by applicant]
Hynes, Integrins: bidirectional, allosteric signaling machines, Cell, 110(6):673-87 (2002).
[cited by applicant]
Integrin beta-1 isoform 1A precursor [
[cited by applicant]
Integrin beta-1 isoform 1D precursor [
[cited by applicant]
Integrin beta-2 precursor [
[cited by applicant]
Integrin beta-3 precursor [
[cited by applicant]
Integrin beta-6 precursor [
[cited by applicant]
International Search Report and Written Opinion from corresponding international application No. PCT/US11/28567, mailing date Oct. 18, 2011.
[cited by applicant]
International Search Report and Written Opinion from corresponding international application No. PCT/US2013/059833, dated Feb. 25, 2014.
[cited by applicant]
Kataoka et al., Block copolymer micelles for drug delivery: design, characterization and biological significance, Adv. Drug Deliv. Rev., 47(1):113-31 (2001).
[cited by applicant]
Katz, Inhibition of inflammatory responses by leukcyte Ig-like receptors, Adv. Immunol., 91:251-72 (2006).
[cited by applicant]
Klages et al., Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets, J. Cell Biol., 144(4):745-54 (1999).
[cited by applicant]
Kong et al., Regulation of integrin expression by G?12: An additional potential mechanism modulating cell attachment, Cell Adh. Migr., 4(3):372-6 (2010).
[cited by applicant]
Kozasa et al., p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13, Science, 280(5372):2109-11 (1998).
[cited by applicant]
Krishnadas et al., Sterically stabilized phospholipid mixed micelles: in vitro evaluation as a novel carrier for water-insoluble drugs, Pharm. Res., 20(2):297-302 (2003).
[cited by applicant]
Kuchroo et al., TIM family of genes in immunity and tolerance, Adv. Immunol., 91:227-49 (2006).
[cited by applicant]
Law et al., Integrin cytoplasmic tyrosine motif is required for outside-in alphallbbeta3 signalling and platelet function, Nature, 401(6755):808-11 (1999).
[cited by applicant]
Li et al., A platelet secretion pathway mediated by cGMP-dependent protein kinase, J. Biol. Chem., 279(41):42469-75 (2004).
[cited by applicant]
Li et al., A stimulatory role for cGMP-dependent protein kinase in platelet activation, Cell, 112(1):77-86 (2003).
[cited by applicant]
Li et al., Sequential activation of p38 and ERK pathways by cGMP-dependent protein kinase leading to activation of the platelet integrin alphallb beta3, Blood, 107(3):965-72 (2006).
[cited by applicant]
Li et al., Signaling during platelet adhesion and activation, Arterioscler. Thromb. Vasc. Biol., 30(12):2341-9 (2010).
[cited by applicant]
Ma et al., Kindlin-2 (Mig-2): a co-activator of beta3 integrins, J. Cell Biol., 181(3):439-46 (2008).
[cited by applicant]
Ma et al., Platelet integrin alpha(IIb)beta(3): activation mechanisms, J. Thromb. Haemost., 5(7):1345-52 (2007).
[cited by applicant]
Marjanovic et al., Stimulatory roles of nitric-oxide synthase 3 and guanylyl cyclase in platelet activation, J. Biol. Chem., 280(45):37430-8 (2005).
[cited by applicant]
Maynor, Supramolecular Nanomimetics: Replication of Micelles, Viruses, and Other Naturally Occurring Nanoscale Objects, Small: 3(5):845-9 (2007).
[cited by applicant]
Moers et al., G13 is an essential mediator of platelet activation in hemostasis and thrombosis, Nat. Med., 9(11):1418-22 (2003).
[cited by applicant]
Moissoglu et al., Integrin signalling in directed cell migration, Biol. Cell, 98(9):547-55 (2006).
[cited by applicant]
Montanez et al., Kindlin-2 controls bidirectional signaling of integrins, Genes Dev., 22(10):1325-30 (2008).
[cited by applicant]
Moser et al., Kindlin-3 is essential for integrin activation and platelet aggregation, Nat. Med., 14(3):325-30 (2008).
[cited by applicant]
Nayal et al., Talin: an emerging focal point of adhesion dynamics, Curr. Opin. Cell Biol., 16(1):94-8 (2004).
[cited by applicant]
NCBI Reference Sequence: NP_000770.2, cytochrome P450 4B1 isoform b [
[cited by applicant]
NCBI Reference Sequence: NP_002204.2, Integrin beta-5 precursor [
[cited by applicant]
Nelson et al., Myristoyl-based transport of peptides into living cells, Biochem., 46(51):14771-81 (2007).
[cited by applicant]
Ngo et al., Computational Complexity, Protein Structure Prediction, and the Levinthal Paradox, in: Merz et al. (eds.), The Protein Folding Problem and Tertiary Structure Prediction, Birchauser (1994).
[cited by applicant]
Nieves et al., The NPIY motif in the integrin beta1 tail dictates the requirement for talin-1 in outside-in signaling, J. Cell Sci., 123(Pt. 8):1216-26 (2010).
[cited by applicant]
Niu et al., G Protein betagamma subunits stimulate p114RhoGEF, a guanine nucleotide exchange factor for RhoA and Rac1: regulation of cell shape and reactive oxygen species production, Circ. Res., 93(9):848-56 (2003).
[cited by applicant]
O'Brien et al., An important role for Akt3 in platelet activation and thrombosis, Blood, 118(15):4215-23 (2011).
[cited by applicant]
Obergfell et al., Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton, J. Cell Biol., 157(2):265-75 (2002).
[cited by applicant]
Patil et al., Identification of a talin-binding site in the integrin beta(3) subunit distinct from the NPLY regulatory motif of post-ligand binding functions. The talin n-terminal head domain interacts with the membrane…
[cited by applicant]
Petrich et al., Talin is required for integrin-mediated platelet function in hemostasis and thrombosis, J. Exp. Med., 204(13):3103-11 (2007).
[cited by applicant]
Petrich et al., The antithrombotic potential of selective blockade of talin-dependent integrin alpha IIb beta 3 (platelet GPIIb-IIIa) activation, J. Clin. Invest., 117(8):2250-9 (2007).
[cited by applicant]
Ren et al., Determination of GTP loading on Rho, Methods Enzymol., 325:264-72 (2000).
[cited by applicant]
Ren et al., The platelet release reaction: just when you thought platelet secretion was simple, Curr. Opin. Hematol., 15(5):537-41 (2008).
[cited by applicant]
Riobo et al., Receptors coupled to heterotrimeric G proteins of the G12 family, Trends Pharmacol. Sci., 26(3):146-54 (2005).
[cited by applicant]
Roger et al., Executive summary: heart disease and stroke statistics—2012 update: a report from the American Heart Association, Circulation, 125(1):188-97 (2012).
[cited by applicant]
Rudinger, Characteristics of the amino acids as components of a peptide hormone sequence, pp. 1-7 in: Parsons (ed.), Peptide Hormones, University Park Press (1976).
[cited by applicant]
Ruggeri, Platelets in atherothrombosis, Nat. Med., 8(11):1227-34 (2002).
[cited by applicant]
Saab et al., Bleeding risk and safety profile related to the use of eptifibatide: a current review, Expert Opin. Drug Saf., 11(2):315-24 (2012).
[cited by applicant]
Salsmann et al., A new functional role of the fibrinogen RGD motif as the molecular switch that selectively triggers integrin alphaIIbbeta3-dependent RhoA activation during cell spreading, J. Biol. Chem., 280(39):33610-…
[cited by applicant]
Senyuk et al., Consistent up-regulation of Stat3 Independently of Jak2 mutations in a new murine model of essential thrombocythemia, Cancer Res., 69(1):262-71 (2009).
[cited by applicant]
Shattil et al., Integrin signaling: the platelet paradigm, Blood, 91(8):2645-57 (1998).
[cited by applicant]
Shattil et al., Integrins: dynamic scaffolds for adhesion and signaling in platelets, Blood, 104(6):1606-15 (2004).
[cited by applicant]
Shattil, Integrins and Src: dynamic duo of adhesion signaling, Trends Cell Biol., 15(8):399-403 (2005).
[cited by applicant]
Shen et al. A directional switch of integrin signalizing and a new anti-thrombotic strategy, Nature, 503(7474): 131-5 (2013).
[cited by applicant]
Staunton et al., Targeting integrin structure and function in disease. Adv. Immunol., 91:111-57 (2006).
[cited by applicant]
Su et al., RGT, a synthetic peptide corresponding to the integrin beta 3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin alpha IIb …
[cited by applicant]
Tadokoro et al., Talin binding to integrin beta tails: a final common step in integrin activation, Science, 302(5642):103-6 (2003).
[cited by applicant]
Tanabe et al., Regulation of RGS-RhoGEFs by Galpha12 and Galpha13 proteins, Methods Enzymol., 390:285-94 (2004).
[cited by applicant]
The Online Medical Dictionary, Definition of Analogue, accessed on Jul. 7, 2005 (pp. 1-5).
[cited by applicant]
Thrombosis entry, p. 561, in: Lackie, “A Dictionary of Biomedicine”, Oxford University Press (2010).
[cited by applicant]
Ugarova et al., Conformational changes in fibrinogen elicited by its interaction with platelet membrane glycoprotein GPIIb-IIIa, J. Biol. Chem., 268(28):21080-7 (1993).
[cited by applicant]
UniProt Protein Database, G protein-coupled receptor kinase 4, protein Accession G9K3F8, accessed on Aug. 26, 2014.
[cited by applicant]
Uniprot Protein Database, protein Accession G5BLN3, Ninein protein, accessed on Oct. 1, 2016.
[cited by applicant]
Uniprot Protein Database, Protein Accession Q2HR82, accessed on Oct. 31, 2019.
[cited by applicant]
Vagner, Peptidomimetic, a synthetic tool of drug discovery, Curr. Opin. Chem. Biol., 12(3):292-6 (2008).
[cited by applicant]
Vidal et al., Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin, Blood, 100(13):4462-9 (2002).
[cited by applicant]
Voet et al., Abnormal Hemoglobins, in: Voet et al., Biochemistry, second edition, John Wiley & Sons, Inc., pp. 235-241 (1995).
[cited by applicant]
Wang et al., Truncation of the cytoplasmic domain of beta3 in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin alpha(IIb)beta3 complex, J. Clin. Invest., 100(9):2393-403 (1997).
[cited by applicant]
Wegener et al., Structural basis of integrin activation by talin, Cell, 128(1):171-82 (2007).
[cited by applicant]
Xi et al., Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling, J. Cell Biol., 162(2):329-39 (2003).
[cited by applicant]
Ye et al., Molecular mechanism of inside-out integrin regulation, J. Thromb. Haemost., 9 Suppl 1:20-5 (2011).
[cited by applicant]
Yin et al., Src family tyrosine kinase Lyn mediates VWF/GPIb-IX-induced platelet activation via the cGMP signaling pathway, Blood, 112(4):1139-46 (2008).
[cited by applicant]
Yin et al., The role of Akt in the signaling pathway of the glycoprotein Ib-IX induced platelet activation, Blood, 111(2):658-65 (2007).
[cited by applicant]
Zhang et al., Talin depletion reveals independence of initial cell spreading from integrin activation and traction, Nat. Cell Biol., 10(9):1062-8 (2008).
[cited by applicant]
Zuckermann et al., Efficient method for the preparation of peptoids {oligo(N-substituted glycines)} by sunmonomer solid-phase synthesis, J. Am. Chem. Soc., 114(26):10646-7 (1992).
[cited by applicant]
Cited By (1)