US20090221691A1 - Compositions and methods for inhibiting g protein signaling - Google Patents

Compositions and methods for inhibiting g protein signaling Download PDF

Info

Publication number
US20090221691A1
US20090221691A1 US11/885,981 US88598106A US2009221691A1 US 20090221691 A1 US20090221691 A1 US 20090221691A1 US 88598106 A US88598106 A US 88598106A US 2009221691 A1 US2009221691 A1 US 2009221691A1
Authority
US
United States
Prior art keywords
protein
sigk
binding
subunit
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/885,981
Other languages
English (en)
Inventor
Alan V. Smrcka
Jose Font
Tabetha Bonacci
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Rochester
Original Assignee
University of Rochester
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Rochester filed Critical University of Rochester
Priority to US11/885,981 priority Critical patent/US20090221691A1/en
Assigned to ROCHESTER, UNIVERSITY OF reassignment ROCHESTER, UNIVERSITY OF ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BONACCI, TABETHA, FONT, JOSE, SMRCKA, ALAN V.
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF ROCHESTER
Publication of US20090221691A1 publication Critical patent/US20090221691A1/en
Assigned to UNIVERSITY OF ROCHESTER reassignment UNIVERSITY OF ROCHESTER ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BONACCI, TABETHA, FONT, JOSE, SMRCKA, ALAN V
Assigned to NATIONAL INSTITUTES OF HEALTH - DIRECTOR DEITR reassignment NATIONAL INSTITUTES OF HEALTH - DIRECTOR DEITR CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF ROCHESTER
Abandoned legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/36—Opioid-abuse
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • 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
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4719—G-proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • G protein ⁇ subunits Five mammalian isoforms of the G protein ⁇ subunit (37 kDa) and twelve isoforms of G protein ⁇ (7.8 kDa) have been identified (Offermanns (2003) Prog. Biophys. Mol. Biol 83:101-30). Obligate heterodimers composed of G protein ⁇ and ⁇ subunits (G ⁇ ) function as regulatory molecules in various pathways in eukaryotic cells (Neves, et al. (2002) Science 296:1636-9; Clapham and Neer (1997) Annu. Rev. Pharmacol. Toxicol. 37:167-203).
  • G ⁇ associates tightly with GDP-bound G protein ⁇ subunits (G ⁇ ) and thereby constitutes the basal form of the G protein heterotrimer in which neither G ⁇ nor G ⁇ are active in signaling.
  • GPCRs Agonist-stimulated G protein coupled receptors (GPCRs) catalyze the exchange of GDP for GTP upon G ⁇ and release of G ⁇ from the heterotrimer complex, liberating two active signaling species: G ⁇ •GTP and G ⁇ .
  • GPCRs G protein-regulated inward-rectifying potassium channel (GIRK) (Krapivinsky, et al. (1993) J. Biol. Chem.
  • G ⁇ is a cone-shaped toroidal structure composed of seven four-stranded ⁇ -sheets arranged radially about a central axis (Wall, et al. (1995) Cell 83:1047-58; Lambright, et al. (1996) Nature 379:311-9). Each ⁇ -sheet is formed from elements of two consecutive WD-40 repeats, named for a conserved C-terminal Trp-Asp sequence in each repeat (Gettemans, et al. (2003) Sci STKE 2003:PE27). The G ⁇ subunit, an extended helical molecule, is nested in a hydrophobic channel that runs across the base of the cone.
  • the slightly narrower, “top” surface of the G ⁇ cone is the main binding site of G ⁇ (through its switch II region) (Wall, et al. (1995) supra; Lambright, et al. (1996) supra), phosducin (Loew, et al. (1998) Structure 6:1007-19; Gaudet, et al. (1996) Cell 87:577-88), and GRK2 (Lodowski, et al. (2003) Science 300:1256-62), as shown by the crystal structures of these complexes. Mutational analysis indicates that many interaction partners of G ⁇ , including PLC ⁇ 2 and adenylyl cyclase, bind to the same surface (Li, et al.
  • Phage display of randomized peptide libraries has been used to identify sequence requirements for binding and screen for peptide that bind to G ⁇ 1 ⁇ 2 dimers (Scott, et al. (2001) EMBO J. 20:767-76). Although billions of individual clones were screened, most of the peptides that bound G ⁇ 1 ⁇ 2 could be classified into four, unrelated groups based on amino acid sequence. One of these groups included a linear peptide (the “SIRK” peptide) with the sequence Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp (SEQ ID NO:1).
  • the SIRK peptide inhibited PLC ⁇ 2 activation by G ⁇ 1 ⁇ 2 subunits with an IC 50 of 5 ⁇ M and blocked activation of PI3K. In contrast, the SIRK peptide had little or no effect on G ⁇ 1 ⁇ 2 regulation of type I adenylyl cyclase or voltage-gated N-type Ca ++ channel activity (Scott, et al. (2001) supra). This demonstrated that selective inhibition of G ⁇ binding partners could be achieved.
  • G ⁇ binding peptides such as QEHA, derived from adenylyl cyclase II (Weng, et al. (1996) J. Biol. Chem. 271:26445-26448; Chen, et al. (1997) Proc. Natl. Acad. Sci. USA 94:2711-2714) and amino acids 643-670 from the C-terminal region of PARK (GRK2) (Koch, et al. (1993) supra) could not promote dissociation of the heterotrimer, despite competing for G ⁇ subunit binding (Ghosh, et al. (2003) supra). This indicates that competition for G ⁇ -G ⁇ subunit binding is not sufficient for these peptides to accelerate subunit dissociation.
  • a peptide similar to the SIRK peptide was derived that had higher affinity for G ⁇ 1 ⁇ 2 .
  • the sequence of this peptide is Ser-Ile-Gly-Lys-Ala-Phe-Lys-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp (SEQ ID NO:2) (SIGK).
  • SIGK Ser-Ile-Gly-Lys-Ala-Phe-Lys-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp
  • the present invention relates to a method for identifying an agent that modulates at least one activity of a G protein. This method involves contacting a G protein ⁇ subunit with a test agent and determining whether the agent interacts with at least one amino acid residue of the protein interaction site of the ⁇ subunit thereby identifying an agent that modulates at least one activity of the G protein.
  • the present invention also relates to a method for identifying an agent that binds at least one amino acid residue of the protein interaction site of the ⁇ subunit.
  • the method involves the steps of contacting a G protein ⁇ subunit with a test agent in the presence of a peptide that binds at least one amino acid residue of the protein interaction site of ⁇ subunit, and determining whether the agent inhibits the binding of the peptide to the at least one amino acid residue of the protein interaction site of the ⁇ subunit thereby identifying an agent that binds at least one amino acid residue of the protein interaction site of the ⁇ subunit.
  • the present invention further relates to a method for modulating at least one activity of a G protein.
  • This method involves contacting a G protein with an effective amount of an agent that interacts with at least one amino acid residue of the protein interaction site of the G protein ⁇ subunit so that at least one activity of the G protein is modulated.
  • the present invention is also a method for preventing or treating a disease or condition involving at least one G protein ⁇ subunit activity.
  • the method involves administering to a patient having or at risk of having a disease or condition involving at least one G protein ⁇ subunit activity an effective amount of an agent that interacts with at least one amino acid residue of the protein interaction site of the G protein ⁇ subunit so that the at least one activity of the G protein is modulated thereby preventing or treating the disease or condition involving the at least one G protein ⁇ subunit activity.
  • G protein ⁇ subunit activities include heart failure, addiction, inflammation, and opioid tolerance.
  • kits for identifying an agent that binds at least one amino acid residue of the protein interaction site of the ⁇ subunit is also provided.
  • the kit of the invention contains a SIGK peptide or SIGK peptide derivative.
  • Agents identified in accordance with the screening methods of the present invention are further provided, wherein said agents have a structure of Formula I, II, or III.
  • FIG. 1 shows that small molecules predicted to bind to the G ⁇ protein interaction site can interfere with peptide interactions at the protein interaction site.
  • FIG. 2 illustrates that NSC119910 binds to G ⁇ and interferes with physiologically relevant protein interactions such as with the G ⁇ subunit.
  • FIG. 3 demonstrates the inhibition of phospholipase C-G ⁇ interactions by NSC119910. Phospholipase enzymatic activity was determined using well-established methods (Ghosh and Smrcka (2003) Meth. Mol. Biol. 237:67-75).
  • FIG. 4 depicts the peak cytosolic Ca 2+ concentrations for neutrophils activated with fMLP or ATP agonists in the presence or absence of 10 ⁇ M NSC119910.
  • FIG. 5 shows representative experiments demonstrating peak cytosolic Ca 2+ concentrations, as well as the time taken for fluorescence intensity to decline to half-peak (t 1/2 ) values, for neutrophils activated with fMLP or ATP in the absence and absence of 10 ⁇ M NSC119910.
  • FIG. 6 shows inhibition of PLC- ⁇ 2 and PLC- ⁇ 3 activation in the presence of exemplary compounds of the instant invention.
  • the protein interaction site for G proteins has now been appreciated.
  • the structure of G ⁇ bound to SIGK was elucidated and indicates that SIGK binds to G ⁇ as an a helix across the G ⁇ interaction surface, in a position occupied by an ⁇ helical region of the switch II domain of G ⁇ in the heterotrimer.
  • the conformations of G ⁇ in the presence and absence of SIGK are very similar.
  • the crystal structure reveals how the peptide blocks G ⁇ -G ⁇ interactions.
  • the structure further indicates that G ⁇ has evolved a highly reactive and specialized surface for interaction with diverse protein partners. This specialized surface is referred to herein as the “protein interaction site” or “protein interaction site of G ⁇ ”.
  • the present invention relates to a method for identifying an agent that modulates (i.e., blocks or inhibits, or activates or potentiates) at least one activity of a G protein by contacting a G protein ⁇ subunit with a test agent (e.g., in a high-throughput screen) and determining whether the test agent interacts with at least one amino acid residue of the protein interaction site of the G protein ⁇ subunit.
  • a test agent e.g., in a high-throughput screen
  • a G protein ⁇ subunit is intended to include any one of the five known mammalian G protein ⁇ subunit isoforms (Offermanns (2003) supra).
  • G protein-regulated inward-rectifying potassium channel GIRK
  • type I, type II, and type IV isoforms of adenylyl cyclase mitogen-activated protein kinase (MAPK); phosphotidylinositol-3-kinase (PI3K); G protein receptor kinase (GRK) family members; and other plextrinhomology (PH) domain-containing proteins including the dynamins and the ⁇ 1, ⁇ 2, and ⁇ 3 isoforms of phospholipase C ⁇ (PLC ⁇ ).
  • Modulation of G protein activity occurs via binding of the agent to at least one amino acid residue of the protein interaction site thereby blocking interactions between the G ⁇ subunits and G ⁇ subunit or the G ⁇ subunits and the downstream proteins described herein.
  • G ⁇ 1 bound to SIGK revealed that the SIGK peptide interacts with residues of G ⁇ 1 subunit that are utilized by several G ⁇ binding proteins (e.g., downstream proteins).
  • G ⁇ binding proteins e.g., downstream proteins.
  • Lys57, Tyr59, Trp99, Met101, Leu117, Tyr145, Met188, Asp246, and Trp332 of G ⁇ 1 are involved in contacts with the GRK2 PH domain in the crystal structure of the G ⁇ 1 ⁇ 2 •GRK2 complex, and all of these residues of G ⁇ 1 are involved in SIGK contacts as well (Table 1).
  • switch II of G ⁇ i1 has no sequence similarity to the SIGK peptide, although it contains a lysine (Lys210) which is oriented in almost the same position as Lys4 of SIGK (Goubaeva, et al. (2003) supra).
  • SIGK SIGK binding residues that are most sensitive to mutational perturbation are also the most frequently involved in interactions with other G ⁇ binding partners.
  • SIGK was identified from a random peptide phage display where multiple peptides, unrelated by sequence, appeared to bind to a common protein interaction site on G ⁇ 1 .
  • SIGK is a competitive inhibitor of multiple G ⁇ binding reactions.
  • the closely related SIRK peptide has effects on several G ⁇ -dependent pathways; it blocks G ⁇ -mediated activation of PLC ⁇ 2, PLC ⁇ 3 and PI3K in enzyme assays, and induces ERK I/II activation in a cell-based assay (Scott, et al. (2001) supra; Goubaeva, et al. (2003) supra).
  • SIRK does not block inhibition of adenylyl cyclase type I or N-type Ca 2+ channel regulation, even though their footprints are quite similar to those of G ⁇ and PLC ⁇ 2 (Scott, et al. (2001) supra). Conversely, mutations in GP that abrogate SIGK binding do not equally affect interaction with other G ⁇ binding partners.
  • mutation of Leu117 to alanine decreases the ability of G ⁇ 1 ⁇ 2 to activate adenylyl cyclase type II and PLC ⁇ 3 and to bind GRK2 and SIGK, but has no effect on GIRK1/GIRK4 potassium channel activation, CC ⁇ 1B calcium channel activation, or PLC ⁇ 2 activation (Table 1) (Li, et al. (1998) supra; Ford, et al. (1998) supra).
  • Trp332 of G ⁇ 1 ⁇ 2 reduces affinity of G ⁇ 1 ⁇ 2 for SIGK and impairs stimulatory activity towards adenylyl cyclase type II, CC ⁇ 1B and both PLC ⁇ 2 and PLC ⁇ 3, but does not affect interaction with GRK2, activation of GIRK1/GIRK4, or inhibition of adenylyl cyclase type I (Li, et al. (1998) supra; Ford, et al. (1998) supra). Both Leu117 and Trp332 of G ⁇ 1 ⁇ 2 form part of the G ⁇ t and G ⁇ i1 binding sites of G ⁇ 1 (Wall, et al.
  • SIGK promotes nucleotide exchange-independent dissociation of G ⁇ 1 ⁇ 2 from G ⁇ i1 (Ghosh, et al. (2003) supra; Goubaeva, et al. (2003) supra).
  • a peptide derived from the C-terminus of GRK2 blocks heterotrimer formation (Ghosh, et al.
  • SIGK could promote heterotrimer dissociation by either of two mechanisms. SIGK may induce conformational changes on G ⁇ 1 that propagate beyond the SIGK binding site and disrupt other interactions between G ⁇ 1 and G ⁇ i1 . However, the G ⁇ 1 ⁇ 2 •SIGK structure shows that SIGK does not induce substantial conformational change in G ⁇ 1 outside of the SIGK binding site itself.
  • the second mechanism relies on the assumption that G ⁇ i1 can dynamically detach from and rebind to either of two surfaces on G ⁇ : the switch II interaction site on the top face of G ⁇ 1 , where SIGK binds in a similar orientation, and the N-terminal interaction surface on blade one of G ⁇ 1 .
  • Transient release from G ⁇ i1 at the switch II interface would allow SIGK access to G ⁇ 1 .
  • Complete release of G ⁇ i1 from G ⁇ could then occur if the off-rate for SIGK is slower than that for dissociation of the N-terminus of G ⁇ i1 .
  • the GRK2 peptide which binds the top surface of G ⁇ , may dissociate too quickly to promote dissociation of G ⁇ .
  • This dynamic model of G ⁇ interactions is biologically relevant, since many G ⁇ binding targets exhibit binding outside of the top surface of G ⁇ and may also transiently sample alternate surfaces on G ⁇ .
  • Preferential binding surfaces are characterized as having high solvent accessibility, low polarity, and a large degree of conformational flexibility (Scott, et al. (2001) supra; Ma, et al. (2001) Curr. Opin. Struct. Biol. 11:364-9; Bogan and Thorn (1998) J. Mol. Biol. 280:1-9; Clackson and Wells (1995) Science 267:383-6; DeLano (2002) Curr.
  • preferential binding sites are likely to contain an unusually high concentration of so-called “hot spots”, i.e., residues that, if mutated to alanine, reduce binding energy at least ten-fold (DeLano (2002) supra).
  • Hot spots have been described for both protein-protein and protein-small molecule interfaces; often point mutations to any hot spot on a surface completely abrogate complex formation, even when the binding interfaces bury several hundred ⁇ 2 of total surface area (Bogan and Thorn (1998) supra; Clackson and Wells (1995) supra; Thanos, et al. (2003) J. Am. Chem. Soc. 125:15280-1; Zhang, et al.
  • the SIGK binding surface of G ⁇ 1 contains several residues that have been shown to be enriched in hot spots (Bogan and Thorn (1998) supra). These include tyrosine, tryptophan and arginine; bulky residues that are capable of forming both polar and non-polar interactions.
  • the protein interaction site of G ⁇ is significantly more populated with aromatic residues than the rest of the G ⁇ surface. 38% of the SIGK binding surface versus 8.5% of the total non-glycine surface accessible G ⁇ residues is composed of Phe, Tyr, His, or Trp. Therefore, the protein interaction site of G ⁇ is more nonpolar; in total, 62% of the protein interaction site of G ⁇ is nonpolar compared to 29% of G ⁇ surface accessible residues.
  • asparagine and aspartic acid which have a moderately favorable distribution among hot spot surfaces, account for four of the thirteen residues in the protein interaction site of G ⁇ .
  • This combination of aromatic and charged residues allows for accommodation of binding partners with diverse chemical properties at the G ⁇ protein interaction site.
  • Preferential binding surfaces are expected to have high surface accessibility (DeLano, et al. (2000) supra).
  • the total surface accessible area was calculated for the G ⁇ molecule on a residue, main chain, and side chain basis.
  • Most amino acids in the protein interaction site of G ⁇ were not significantly more accessible than others of their type in G ⁇ .
  • five residues showed significant deviation from the mean: Tyr59, Trp99, Met101, Leu117, and Trp332.
  • side chain surface accessibility was significantly greater than the type average; the main chain of Tyr59, Trp99, and Met101 were more accessible than the mean.
  • Leu117 had significantly higher main chain and side chain accessibility than the mean.
  • Conformational flexibility or adaptability has been cited as an important determinant of a preferential binding surface, since such surfaces are better able to bind to structurally unrelated protein targets (DeLano, et al. (2000) supra). Residue flexibility can be quantified in terms of relative positional variation in the context of several protein complexes.
  • Histogram analysis of the RMSD relative to uncomplexed G ⁇ 1 ⁇ 1 of all G ⁇ residues in four crystal structures shows that the protein interaction site residues of GP exhibit only slightly greater than average side chain positional dispersity (1.42 ⁇ compared to 1.35 ⁇ ), with the side chains of Trp99, Asp228, and Trp332 having the largest positive deviation from the average (each greater than 2 ⁇ ).
  • Arg314 and Trp332 in blade seven move more than 10 ⁇ towards the outside of the G ⁇ 1 torus to interact with phosducin.
  • Atomic B factors also provide a measure of conformational flexibility.
  • the B factors for Trp99, Val100, and Met101 exceed the mean value by least one standard deviation (Trp99 is greater than two standard deviations from the mean).
  • Trp99 is greater than two standard deviations from the mean.
  • these binding site residues become more well-ordered with B values close to the mean and in some cases up to one standard deviation below the mean.
  • G ⁇ to recognize structurally diverse binding partners does not require a high degree of conformational flexibility for most residues in the protein interaction site of G ⁇ .
  • Small structural adaptations in G ⁇ 1 are sufficient to accommodate a range of co-evolving binding partners.
  • Structural and mutagenic analysis demonstrates that the protein interaction site on GP can be regarded as a hot surface, co-evolved to promote tight binding with multiple protein targets.
  • the mechanism by which G ⁇ acts as a hot surface is complex. Trp332 is the only residue which meets all four of the criteria for a hot spot, although Tyr59 and Trp99 have three of the four characteristics of hot spot residues that were tested.
  • an amino acid residue of the protein interaction site of a G ⁇ is intended to include Lys57, Tyr59, Trp99, Val100, Met101, Leu117, Tyr145, Asp186, Met188, Asp228, Asn230, Asp246, and Trp332.
  • the location of these residues is provided in the rat G ⁇ amino acid sequence of:
  • MSELEQLRQE AEQLRNQIRD ARKACGDSTL TQITAGLDPV GRIQMRTRRT LRGHLA K I Y A MHWGTDSRLL VSASQDGKLI IWDSYTTNKV HAIPLRSS WV M TCAYAXSGN FVACGG L DNI CSIYSLKTRE GNVRVSRELP GHTG Y LSCCR FLDDNQIITS SGDTTCALWD IETGQQTVGF AGHSG D V M SL SLAPNGRTFV SGACDASIKL WDVRDSMCRQ TFIGHES D I N AVAFFP N GYA FTTGSDDATC RLFDLRADQE LLMYSHDNII CGITSVAFSR SGRLLLAGYD DFNCNIWDAM KGDRAGVLAG HDNRVSCLGV TDDGMAVATG S W DSFLKIWN (GENBANK Accession No. AAA35922; SEQ ID NO:4), wherein the protein interaction site residues are underlined
  • An agent which interacts with at least one of these amino acid residues of the protein interaction site of G ⁇ can bind via various heterogeneous non-bonded interactions including, but not limited to van der Waals contacts (e.g., with methionine or leucine), polar contacts (e.g., with aspartate or asparagine), or both (e.g., with lysine, tryptophan, or tyrosine) to contribute to the energy of binding.
  • van der Waals contacts e.g., with methionine or leucine
  • polar contacts e.g., with aspartate or asparagine
  • both e.g., with lysine, tryptophan, or tyrosine
  • the agent interacts with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the amino acid residues of the protein interaction site of G ⁇ to enhance the specificity of the agent for one or more G protein interacting proteins and therefore one or more G protein-mediated signaling pathways.
  • Determining whether the agent interacts with at least one amino acid residue of the protein interaction site of the ⁇ subunit can be accomplished using various in vitro or in vivo assays based on detecting protein-protein interactions between the G ⁇ subunits and other peptides or proteins known to interact with G ⁇ subunits (e.g., SIGK peptide, G ⁇ subunit, or downstream proteins).
  • An exemplary in vitro assay has been disclosed herein.
  • This assay consists of obtaining an isolated G ⁇ complex; contacting the G ⁇ complex with a test agent in the presence of a peptide that binds at least one amino acid residue of the protein interaction site of ⁇ subunit, (e.g., a SIGK peptide or SIGK peptide derivative of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13); and detecting the ability of the agent to inhibit the binding of the peptide to the protein interaction site of the ⁇ subunit using, for example, an ELISA assay.
  • a peptide that binds at least one amino acid residue of the protein interaction site of ⁇ subunit e.g., a SIGK peptide or SIGK peptide derivative of SEQ ID NO:1, SEQ ID NO:2, S
  • an in vivo assay can be used to determine whether a test agent interacts with at least one amino acid residue of the protein interaction site of the ⁇ subunit.
  • a two-hybrid assay is contemplated where the test agent is contacted with a cell expressing G ⁇ subunits and a peptide such as SIGK, wherein the ⁇ subunit is fused to, e.g., a DNA-binding domain and the SIGK peptide is fused to an activation domain.
  • SIGK peptide is bound to the protein interaction site of G ⁇
  • reporter protein expression is induced. If the test agent disrupts the binding of the SIGK peptide to the protein interaction site of G ⁇ , reporter protein expression is blocked.
  • Additional screens such as well-established computational screens or screens that detect the activity of G protein-dependent downstream proteins (e.g., PLC ⁇ enzymatic activity) are also contemplated for use in conjunction with the assays disclosed herein.
  • G protein-dependent downstream proteins e.g., PLC ⁇ enzymatic activity
  • Test agents also referred to herein as compounds, which can be screened in accordance with the methods of the present invention are generally derived from libraries of agents or compounds. Such libraries can contain either collections of pure agents or collections of agent mixtures. Examples of pure agents include, but are not limited to, proteins, polypeptides, peptides, nucleic acids, oligonucleotides, carbohydrates, lipids, synthetic or semi-synthetic chemicals, and purified natural products. Examples of agent mixtures include, but are not limited to, extracts of prokaryotic or eukaryotic cells and tissues, as well as fermentation broths and cell or tissue culture supernates.
  • the methods of this invention are not only used to identify those crude mixtures that possess the desired activity, but also provide the means to monitor purification of the active agent from the mixture for characterization and development as a therapeutic drug.
  • the mixture so identified can be sequentially fractionated by methods commonly known to those skilled in the art which can include, but are not limited to, precipitation, centrifugation, filtration, ultrafiltration, selective digestion, extraction, chromatography, electrophoresis or complex formation. Each resulting subfraction can be assayed for the desired activity using the original assay until a pure, biologically active agent is obtained.
  • Library screening can be performed as exemplified herein or can be performed in any format that allows rapid preparation and processing of multiple reactions.
  • Stock solutions of the test agents as well as assay components are prepared manually and all subsequent pipetting, diluting, mixing, washing, incubating, sample readout and data collecting is done using commercially available robotic pipetting equipment, automated work stations, and analytical instruments for detecting the signal generated by the assay.
  • detectors include, but are not limited to, luminometers, spectrophotometers, and fluorimeters, and devices that measure the decay of radioisotopes.
  • a model system of any particular disease or disorder involving G protein signaling can be utilized to evaluate the adsorption, distribution, metabolism and excretion of a compound as well as its potential toxicity in acute, sub-chronic and chronic studies.
  • overexpression of ⁇ inhibitors in NG108-15/D2 cells and rat primary hippocampal neurons has been shown to block ⁇ -opioid and cannabinoid receptor-induced PKA C ⁇ translocation and gene expression by preventing ⁇ activation of adenylyl cyclase (Yao, et al. (2003) Proc. Natl. Acad. Sci. USA 100:14379-84).
  • NG108-15/D2 cells and/or rat primary hippocampal neurons are contacted with said compound and the effect on PKA C ⁇ translocation is determined.
  • Compounds which block ⁇ -opioid and cannabinoid receptor-induced PKA C ⁇ translocation will be useful in treating addiction.
  • Efficacy of compounds of the instant invention for preventing or treating heart failure can be analyzed in a genetic model of murine-dilated cardiomyopathy which involves the ablation of a muscle-restricted gene that encodes the muscle LIM protein (MLP ⁇ / ⁇ ) (Arber, et al. 1997) Cell 88:393-403).
  • MLP ⁇ / ⁇ muscle LIM protein
  • BARK-ct beta-adrenergic receptor kinase 1 inhibitor
  • BARK-ct which binds to ⁇ and blocks ⁇ -dependent activation of beta-adrenergic receptor kinase 1 activity, can enhance cardiac contractility in vivo with or without isoproterenol (Koch, et al.
  • mice are injected intracerebroventricularly with a compound of the instant invention and tolerance to a select opioid (e.g., morphine) is determined.
  • a select opioid e.g., morphine
  • PLC- ⁇ 2 and - ⁇ 3 and PI3K ⁇ have been shown to be involved in the chemoattractant-mediated signal transduction pathway.
  • Mice deficient in PI3K ⁇ lack neutrophil production of PtdIns(3,4,5)P 3 , neutrophil migration, and production of antibodies containing the chain when immunized with T cell-independent antigen hydroxylnitrophenyl-FICOLLTM (Li, et al. (2000) Science 287:1046-1049).
  • Mice lacking PLC- ⁇ 2 and - ⁇ 3 are deficient in Ca 2+ release, superoxide production, and MAC-1 upregulation in neutrophils (Li, et al. (2000) supra).
  • mice exhibit enhanced chemotaxis of different leukocyte populations and are sensitized to bacteria, viruses, and immune complexes (Jiang, et al. (1997) Proc. Natl. Acad. Sci. USA 94(15):7971-5). Accordingly, to analyze the efficacy of a compound of the instant invention for modulating an inflammatory response, mice can be administered said compound and the effect on neutrophil production of PtdIns(3,4,5)P 3 , neutrophil migration, Ca 2+ efflux, superoxide production, production of antibodies containing the ⁇ chain when immunized with T cell-independent antigen hydroxylnitrophenyl-FICOLLTM is determined.
  • one embodiment of the present invention is a compound having a structure of Formula I:
  • Exemplary compounds having the structure of Formula I which depict various substituent R groups include, but are not limited to, the following:
  • Exemplary compounds having the structure of Formula II which depict various substituent R groups include, but are not limited to, the following:
  • Additional exemplary compounds which bind to the protein interaction site of G ⁇ include, but are not limited to, the following:
  • Exemplary compounds disclosed herein are intended to include all enantiomers, isomers or tautomers, as well as any derivatives of such compounds that retain the same biological activity as the original compound.
  • Exemplary compounds of the present invention were initially selected from a computational screen to identify ligands that bind to the novel protein interaction site of G ⁇ .
  • the computational screen involved using SYBYL molecular modeling software to model the protein interaction site of G ⁇ as determined in the X-ray structure disclosed herein.
  • the computational docking screen was performed with the National Cancer Institute 1900 compound library wherein the compounds were tested for docking to the protein interaction site of G ⁇ using FLEXXTM (Tripos, Inc., St. Louis, Mo.) for docking and CSCORETM (Tripos, Inc.) to evaluate the energetics and fitness of the docked structure.
  • FLEXXTM Tripos, Inc., St. Louis, Mo.
  • CSCORETM Tripos, Inc.
  • Compounds NSC201400 and NSC119916 had IC 50 values of 100 nM and 5 ⁇ M, respectively, and the remaining compounds were found to bind in the ELISA-based assay to G ⁇ with an affinity of at least 50 ⁇ M and interfere with peptide interactions at the protein interaction site ( FIG. 1 ). These compounds were further analyzed in the phage ELISA assay and found to have high affinities for the protein interaction site of G ⁇ and interacted with similar amino acid residues as SIGK.
  • NSC119910 blocked interactions of G ⁇ subunit with G ⁇ subunits ( FIG. 2 ) and inhibited the ability of G ⁇ subunits to inhibit interactions with a physiological target such as PLC ⁇ in vitro ( FIG. 3 ) based on a decrease in the enzymatic activity of PLC ⁇ .
  • PI3K ⁇ is involved in the production of TI-Ig ⁇ L and mice deficient in PI3K ⁇ , lack neutrophil migration (Li, et al. (2000) Science 287:1046-9).
  • the PLC pathway is involved in down-modulation of chemotaxis and in hyperinflammatory conditions (Li, et al. (2000) supra). Therefore, it was determined whether NSC119910 could inhibit the G ⁇ /PLC interaction and block PLC activation.
  • Opioid receptors ⁇ , ⁇ , and ⁇ , couple to G i and G o proteins through ⁇ and ⁇ subunits, and regulate a number of signaling pathways.
  • the efficacy of opioid signal transduction in PLC- ⁇ 3-deficient mice has been shown to increase, indicating that PLC suppresses opioid signaling by modification of opioid-dependent signaling components (Xie, et al. (1999) Proc. Natl. Acad. Sci. USA 96:10385-10390).
  • PLC- ⁇ 3 plays a significant role as a negative regulator of opioid responses, it was determined whether NSC119910 could inhibit PLC- ⁇ 3 activation thereby enhancing morphine-induced analgesia.
  • mice were intracerebroventricularly injected in accordance with standard protocols (Xu, et al. (1998) J. Pharmacol. Exp. Therapeut. 284:196-201) with 100 nmol of NSC119910 in combination with varying doses (0.1, 0.3, 1, and 3 nmol) of morphine. Mice were tested 20 minutes after the injection for an analgesic response in a 55° C. warm-water tail-flick test (Wells, et al. (2001) J. Pharmacol. Exp. Therapeut. 297:597-605). The ED 50 value for morphine alone was 0.74 nmol, while the ED 50 value for NSC119910 plus morphine was 0.065 nmol.
  • NSC119910 effectively modulates G-protein interactions
  • R 1 can be a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl; and R 2 and R 3 are independently hydrogen or a hydroxyl group. In particular embodiments, R 2 and R 3 are both hydroxyl.
  • alkyl refers to a straight or branched hydrocarbon chain consisting solely of carbon and hydrogen atoms, containing no saturation, having from one to eight carbon atoms.
  • Alkenyl is intended to mean an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be a straight or branched chain having from 2 to about 10 carbon atoms.
  • Cycloalkyl denotes a non-aromatic mono or multicyclic ring system of about 3 to 12 carbon atoms.
  • cycloalkenyl refers to a mono or multicyclic ring system containing in the range of about 3 to 12 carbon atoms with at least one carbon-carbon double bond.
  • Substituents in the substituted alkyl, cycloalkyl, alkenyl or cycloalkenyl groups include, but are not limited to, hydroxy, carboxyl, halogen (e.g., fluorine, chlorine, bromine, or iodine), or substituted or unsubstituted alkyl.
  • halogen e.g., fluorine, chlorine, bromine, or iodine
  • analogs of NSC119910 generally contained hydroxyl groups in the R 2 and R 3 positions of Formula III, which appeared to facilitate binding; and a carboxyl-substituted alkyl, cycloalkyl, alkenyl or cycloalkenyl substituent at R 1 of Formula III, which appeared to modulate activity, but was not required for binding.
  • a further embodiment of the present invention is a compound having a structure of Formula III and pharmaceutically acceptable salts and complexes thereof.
  • NSC119910 analogs to selectively modulate activation PLC- ⁇ 2 and - ⁇ 3 was analyzed.
  • PLC- ⁇ 2 and PLC- ⁇ 3 were purified and PLC enzymatic activity was measured in the presence or absence of purified ⁇ and in the presence or absence of analog.
  • the results of this analysis indicated that NSC119911 appeared to block PLC- ⁇ 2 activation more effectively than PLC- ⁇ 3 activation and NSC201400 selectively potentiated PLC- ⁇ 3 activation despite blocking peptide binding to ⁇ ( FIG. 6 ).
  • NSC119910, NSC and analog NSC119893 block Ca 2+ mobilization, they do so without interfering with fMLP-dependent ERK activation.
  • NSC119911, NSC158110, and NSC201400 also do not interfere with fMLP-dependent ERK activation.
  • the compounds disclosed herein as well as those found to bind to the protein interaction site of G ⁇ and interfere with protein interactions at this surface can be used in a method for modulating (i.e., blocking or inhibiting, or enhancing or potentiating) at least one activity of a G protein.
  • a method for modulating i.e., blocking or inhibiting, or enhancing or potentiating
  • Such a method involves contacting a G protein either in vitro or in vivo with an effective amount of an agent that interacts with at least one amino acid residue of the protein interaction site of the G protein ⁇ subunit so that at least one activity of the G protein is modulated.
  • An effective amount of an agent is an amount which reduces or increases the activity of the G protein by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
  • Such activity can be monitored based on protein-protein interactions or enzymatic assays detecting activity of downstream proteins.
  • modulating one or more G protein activities can be useful in selectively analyzing G protein signaling events in model systems as well as in preventing or treating diseases and disorders involving G protein ⁇ subunit signaling.
  • the selection of the compound for use in preventing or treating a particular disease or disorder will be dependent upon the particular G protein-dependent downstream protein involved in the disease or disorder.
  • a compound which interacts with Lys57, Trp99, Met101, Leu117, Asp186, Asp228, Asp246 and/or Trp332 of GP would be useful in preventing or treating adenylyl cyclase-associated diseases or disorders, whereas a compound which interacts with Lys57, Tyr59, Trp99, Met101, Leu117, Tyr146, Met188, Asp246, and/or Trp332 may be more suitable for GRK2-associated diseases or disorders. It is contemplated that this selectivity for specific downstream proteins may reduce side effects associated with antagonists which inhibit all activities associated the G protein ⁇ subunit signaling.
  • Prevention or treatment typically involves the steps of first identifying a patient at risk of having or having a disease or disorder involving at least one G protein ⁇ subunit activity (e.g., congestive heart failure, addiction, hyper- or hypo-inflammation, or opioid tolerance). Once such an individual is identified using, for example, standard clinical practices, said individual is administered a pharmaceutical composition containing an effective of a selective compound disclosed herein or identified in the screening methods of the invention. In most cases this will be a human being, but treatment of agricultural animals, e.g., livestock and poultry, and companion animals, e.g., dogs, cats and horses, is expressly covered herein.
  • agricultural animals e.g., livestock and poultry
  • companion animals e.g., dogs, cats and horses
  • the selection of the dosage or effective amount of a compound is that which has the desired outcome of reducing or reversing at least one sign or symptom of a disease or disorder involving G protein ⁇ subunit signaling in a patient.
  • some of the general signs or symptoms associated with congestive heart failure include increased heart rate, increased respiratory rate, breathing faster and deeper than normal, breathlessness, irritability, restlessness, an unexplained fussiness, swelling, puffiness, edema, sudden weight gain or poor weight gain, decrease in appetite, diaphoresis, cough, congestion or wheezing, a decrease in activity level, fatigue, listlessness, decrease in urine output, or pale, mottled or grayish appearance in skin color.
  • General signs or symptoms associated with addiction include, but are not limited to, changes in attitude, appearance, and relationships with others, whether at home, school or work and other behavioral changes.
  • the selective modulation of either the PLC pathway or PI3K ⁇ will be useful in treating different inflammatory conditions.
  • a compound which selectively inhibits the activation of PI3K ⁇ thereby reducing the injury to tissues that contribute to the pathophysiology of the inflammatory diseases.
  • a compound which selectively inhibits the activation of the PLC pathway it is desirable to administer a compound which selectively inhibits the activation of the PLC pathway.
  • compositions can be in the form of pharmaceutically acceptable salts and complexes and can be provided in a pharmaceutically acceptable carrier and at an appropriate dose.
  • Such pharmaceutical compositions can be prepared by methods and contain carriers which are well known in the art. A generally recognized compendium of such methods and ingredients is Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, Pa., 2000.
  • a pharmaceutically-acceptable carrier, composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, is involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • Examples of materials which can serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • compositions of the present invention can be administered parenterally (for example, by intravenous, intraperitoneal, subcutaneous or intramuscular injection), topically (including buccal and sublingual), orally, intranasally, intravaginally, or rectally according to standard medical practices.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of a compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. This is considered to be within the skill of the artisan and one can review the existing literature on a specific compound or similar compounds to determine optimal dosing.
  • Additional compounds to those exemplified herein can be identified routinely in accordance with the screening methods taught herein. Additional compounds for screening can be selected randomly by one skilled in the art, based upon computational prediction, and/or based upon their containing a structure of Formula I, II or III or a structure similar to that of the exemplary compounds disclosed herein.
  • Peptides were purchased from Alpha Diagnostic International (San Antonio, Tex.) or SIGMA®-Genosys (St. Louis, Mo.), HPLC purified to greater than 90% and masses confirmed by mass spectroscopy.
  • Ni-NTA agarose was from QIAGEN® (Valencia, Calif.).
  • Streptavidin-coated poly-styrene beads were from Spherotec (Libertyville, Ill.).
  • HRP-conjugated anti-M13 antibody was from Amersham Biosciences (Piscataway, N.J.).
  • HRP-conjugated Neutravidin was from Pierce (Rockford, Ill.). All molecular biology reagents were from INVITROGENTM (Carlsbad, Calif.) unless otherwise indicated.
  • G ⁇ 1 ⁇ 2 Baculoviruses harboring cDNA for wild-type bovine G ⁇ 1 and N-terminally (His) 6 -tagged bovine G ⁇ 2 were used to produce proteins of the same. High 5 cells (INVITROGENTM, Carlsbad, Calif.; 2 ⁇ 10 6 cells/mL) were infected with high titer G ⁇ 1 and G ⁇ 2 baculoviruses. G ⁇ 1 ⁇ 2 was purified according to standard methods (Kozaza and Gilman (1995) J. Biol. Chem. 270:1734-41), with modifications. All steps were carried out at 4° C.
  • Cells were harvested 60 hours post-infection by centrifugation at 2600 g, then resuspended in 50 mL of lysis buffer (20 mM HEPES, pH 8, 150 mM NaCl, 5 mM ⁇ -ME, 1 mM EDTA, 1 mL SIGMA® protease inhibitor cocktail P-2714) per liter of cell culture. Cells were lysed by sonication and centrifuged at 2600 g to pellet the membranes. Resuspension and homogenization of membranes was accomplished by douncing in 100 mL lysis buffer. The membranes were solubilized by adding 1% Lubrol (Cl 2 E10, SIGMA®, St.
  • Lubrol Cl 2 E10, SIGMA®, St.
  • G ⁇ 1 ⁇ 2 eluted in Ni-C (20 mM HEPES pH 8, 0.01 M NaCl, 5 mM ⁇ -ME, 1% cholate, 200 mM imidazole).
  • the eluate was loaded onto a HITRAPTM Q (Amersham Biosciences, Piscataway, N.J.) column pre-equilibrated with QA (20 mM HEPES, pH 8, 5 mM ⁇ -ME, 0.7% CHAPS, 1 mM EDTA).
  • G ⁇ 1 ⁇ 2 eluted in a gradient using QB (QA+1.0 M NaCl). Fractions containing G ⁇ 1 ⁇ 2 were analyzed by SDS-PAGE and pooled.
  • SIGK peptide (Ser-Ile-Gly-Lys-Ala-Phe-Lys-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp; SEQ ID NO:2) was synthesized using well-established methods. No modifications were made to the peptide termini; purification was by reverse phase-HPLC chromatography on a VYDAC® C4 semi-preparative column.
  • SIGK peptide was added to G ⁇ 1 ⁇ 2 in 1.5 molar excess, and the G ⁇ 1 ⁇ 2 •SIGK complex was used at 7 mg/mL for crystallization.
  • Crystals were grown by vapor diffusion using equal volumes (2 ⁇ L) of protein and reservoir solution (15-17% PEG 4000, 100 mM HEPES, pH 7.5, 0.01-0.05 M Na-Acetate, 10% glycerol) at 20° C. Crystals attained dimensions of 150 ⁇ m ⁇ 50 ⁇ m ⁇ 20 ⁇ m within one week. Crystals were cryoprotected in 15% glycerol and frozen in liquid nitrogen.
  • the final model contains residues 2-340 of G ⁇ 1 (of 340), 7-52 of G ⁇ 2 (of 68), and 1-13 of SIGK (of 15).
  • R work ⁇ h
  • An I/ ⁇ cutoff was not used in the final calculations of R-factors.
  • 5 R free is the R-factor obtained for a test set of reflections consisting of a randomly selected 8% of the data. 6 B-factors at the N-termini, including G ⁇ 1 residues 2-41 and G ⁇ 2 residues 7-13, are greater than 80 ⁇ 2 .
  • the structure of the G ⁇ 1 ⁇ 2 •SIGK complex was solved by the molecular replacement method using the program PHASER (Storoni, et al. (2004) Acta Crystallogr. D Biol. Crystallogr. 60:432-8; Read (2001) Acta Crystallogr. D Biol. Crystallogr. 57:1373-82).
  • the coordinates of G ⁇ 1 ⁇ 2 in the G ⁇ 1 ⁇ 2 •GRK2 complex (10 MW, 100% sequence identity) were used as the search model.
  • rigid body refinement using the maximum likelihood minimization target in CNS version 1.1 Adams, et al. (1997) Proc. Natl. Acad. Sci. USA 94:5018-23; Brunger, et al.
  • Crystallography 26:283-291 analysis indicates that all residues exhibit main chain conformations in most favored or additional allowed regions of ⁇ , ⁇ space (Table 4). Calculations of surface accessibility, G ⁇ 1 ⁇ 2•SIGK contacts and RMSD between structures were carried out using programs in the CNS suite.
  • Wild-type G ⁇ 1 and G ⁇ 1 mutants were made in the baculovirus vector PDW464 which encodes a biotinylation site at a lysine upstream of the amino terminus of G ⁇ 1 (Goubaeva, et al. (2003) supra). Mutants were generated by overlap extension PCR using standard protocols.
  • the wild-type and mutant G ⁇ 1 constructs consisted of a 20 amino acid biotin acceptor peptide (BAP) sequence fused in-frame with the amino-terminus of rat G ⁇ 1 subunit.
  • the G ⁇ 1 subunit becomes covalently biotinylated in vivo at the specific lysine acceptor residue in the BAP.
  • 1-2 mg protein of purified protein can be obtained per liter of Sf9 insect cells.
  • 45 ng of protein is used in the phage ELISA assay, a single purification is sufficient for 10,000 to 30,000 binding assays.
  • Baculoviruses were generated via the BAC-TO-BAC® system following the manufacturer's instructions (GIBCO/BRL, Gaithersburg, Md.). Sf9 cells (200 mL) were triply infected with 0.5 mL baculovirus encoding (H is) 6 -G ⁇ i1 , 4 mL of G ⁇ 2 virus, and 4 mL of either wild-type or mutated G ⁇ 1 virus. G ⁇ 1 ⁇ 2 dimers were purified 60 hours post-infection using a well-established method with modifications as indicated (Kozasa and Gilman (1995) supra).
  • Cell pellets were lysed in 4 mL lysis buffer (50 mM HEPES, pH 8.0, 3 mM MgCl 2 , 10 mM ⁇ -mercaptoethanol, 1 mM EDTA, 100 mM NaCl, 10 ⁇ M GDP, and protease inhibitors) by four freeze-thaw cycles in liquid nitrogen.
  • lysis buffer 50 mM HEPES, pH 8.0, 3 mM MgCl 2 , 10 mM ⁇ -mercaptoethanol, 1 mM EDTA, 100 mM NaCl, 10 ⁇ M GDP, and protease inhibitors
  • G ⁇ 1 ⁇ 2 subunits were eluted from bound G ⁇ i1 by mixing beads with buffer containing 50 mM MgCl 2 , 10 mM NaF, 10 ⁇ M AlCl 3 , 1% cholate, and 5 mM imidazole at room temperature for one hour.
  • concentrations of b- ⁇ and b- ⁇ mutants were analyzed by comparative immunoblotting and chemiluminescence. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with HRP-neutravidin (Pierce, Rockford, Ill.). The chemiluminescent signal was measured using an EPI-CHEM IITM darkroom system (UVP Bioimaging Systems, Upland, Calif.). Concentrations of eluted b- ⁇ dimers were determined by comparing to a standard curve of fully purified 100% biotinylated G ⁇ 1 ⁇ 2 from at least two separate gels.
  • Phage ELISA assays used to assess peptide binding to wild-type and mutant b- ⁇ were performed according to standard methods (Smrcka and Scott (2002) Methods Enzymol. 344:557-76). Briefly, 1 ⁇ g streptavidin was immobilized in the well of a 96-six well plate overnight at 4° C. The wells were blocked with 100 ⁇ L of 2% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 hour at 4° C. followed by three washes of 1 ⁇ TBS/0.5% TWEEN®.
  • BSA bovine serum albumin
  • TBS Tris-buffered saline
  • amino acid residues having the prefix “s” are indicative of SIGK residues.
  • G ⁇ 1 is a ⁇ -propeller composed of seven four-stranded ⁇ -sheets (“blades”) and an N-terminal extended helix that interacts extensively with G ⁇ 2 . Each sheet is composed of WD-40 repeats connected by loops of variable length. Residues 2-340 of G ⁇ 1 are included in the model. B factors throughout the core of G ⁇ 1 are less than 40 ⁇ 2 . Residues with B factors>60 ⁇ 2 are found in three loop regions: Lys127-Ser136 in blade two, Arg214-Met217 in blade four, and Ser265-Ile269 in the loop connecting blades six and seven.
  • G ⁇ 2 forms a helix with a kink made by residues Asn24-Lys29 and a coil region beginning at residue His44.
  • the average B factor within the G ⁇ 2 molecule is 44 ⁇ 2 .
  • No electron density was observed for the N-terminal seven residues and the C-terminal sixteen residues of G ⁇ 2 or the prenyl lipid modification at the C-terminus of G ⁇ 2 .
  • SIGK forms an ⁇ -helical structure broken by a glycine at position 10.
  • the C-terminal three residues form an extended structure that stretches away from the G ⁇ 1 molecule and is supported by crystal contacts between sPro12 and sAsp13 with Thr47 and Lys337 from a symmetry-related G ⁇ 1 molecule.
  • the B factors for the N-(sSer1, sIle2) and C-terminal (sGly10-sAsp13) residues of SIGK are greater than 50 ⁇ 2 ; those for all other residues are between 30-50 ⁇ 2 .
  • the electron density for the main chain atoms in residues 1-13 is well-defined; three of the SIGK side chains that do not contact G ⁇ 1 (sIle2, sLys7, and sAsp13) are disordered.
  • the peptide binds across the “top” face of G ⁇ 1 and is buried 970 ⁇ 2 total solvent-accessible surface area. The peptide makes no contact with the G ⁇ 2 subunit, which is bound to the “bottom” surface of the G ⁇ 1 torus.
  • the SIGK contact surface on G ⁇ 1 was separated into two regions: an acidic region on G ⁇ 1 that interacts with the N-terminus of the peptide, and a largely nonpolar region that interacts with the C-terminus of the peptide. In total, thirteen G ⁇ 1 residues directly contact SIGK, contributed by six of the seven blades of the ⁇ -propeller (Table 5).
  • the N-terminal binding surface is centered on an electrostatic interaction in which sLys4 projects into a negatively charged binding pocket on G ⁇ 1 ⁇ 2 where it forms hydrogen-bonded or charge interactions with Asp228, Asn230, and Asp246.
  • Met188 participates in van der Waals interactions with the alkyl chain of sLys4, and Asp186 forms a polar contact with the carbonyl oxygen of sSer1 and also makes a hydrogen bond to the amide of Cys204.
  • Tyr145 forms van der Waals interactions with the main chain oxygen of sSer1, the sLys4 side chain, and the C ⁇ atom of sAla5, and forms a hydrogen bond with the nearby amide of Gly162.
  • the side chain of Leu117 is within van der Waals contact distances of the side chains of sIle2 and sAla5. Together, these nine G ⁇ 1 residues form a surface that tethers SIGK to G ⁇ 1 using charged and nonpolar interactions.
  • SIRK and SIGK peptides can now be interpreted in the context of the SIGK•G ⁇ 1 ⁇ 2 structure (Scott, et al. (2001) supra; Goubaeva, et al. (2003) supra).
  • Wild-type SIRK peptide inhibits the activation of PLC ⁇ 2 by G ⁇ 1 ⁇ 2 with an IC 50 of 5 ⁇ M.
  • Substitution of sLys4 with alanine in the SIRK peptide lowers the IC 50 of the peptide 12-fold, and mutation of sAla5 to glycine lowers the IC 50 by 13-fold.
  • Immobilized b- ⁇ was incubated with phage displaying SIGK peptide. Phage binding was detected using an ⁇ -phage antibody; the raw data was absorbance at 405 nm. Data shown are the mean ⁇ SD of triplicate determinations from three independent experiments.
  • SIGK and FITC- ⁇ i1 were simultaneously added to streptavidin beads coated with wild-type or mutant b- ⁇ protein and the amount of FITC- ⁇ i1 bound to the beads was assayed by flow cytometry. Data are shown as the mean of triplicate determinants +/ ⁇ standard deviation of a representative experiment. The experiment was repeated two (Met188A) or three (wild-type, Arg314A, Trp332A) times with similar results.
  • the second area of binding involves most of the C-terminal residues of SIGK (sAla5-sGly11), which pack against a largely hydrophobic pocket on G ⁇ 1 .
  • This pocket extends 11 ⁇ from Trp332 on blade seven to Met188 in blade two.
  • Eight G ⁇ 1 residues are in direct contact with the C-terminal surface of SIGK, and two more G ⁇ 1 residues support the residues directly involved in the SIGK interaction.
  • Met188 which interacts with sLys4 in the N-terminal interface, is also within contact distance of the side chain of sLeu8.
  • SIGK residues sAla5, sLeu8 and sLeu9 are complimented by van der Waals interactions with Leu117, Met101, Trp99, Tyr59 and the alkyl chain of Lys57.
  • the main chain oxygen of Val100 interacts with the side chain of sLeu9.
  • the indole imine of Trp99 forms a hydrogen bond with the hydroxyl group of sTyr11 and the side chain of Trp332 makes contact with the main chain oxygen of sIle8 and the C ⁇ of sGly10.
  • the side chains of Lys57 and Arg314 are positioned on either side of Trp332 and support its orientation in the binding site.
  • Arg314 also forms a hydrogen bond with Trp332, and Lys57 with the nitrogen of Gln75, further stabilizing this interaction surface on G ⁇ 1 .
  • Data from alanine scanning of the peptide (Scott, et al. (2001) supra; Goubaeva, et al. (2003) supra) validate these structural observations.
  • Mutation of sIle8, sLeu9 or sGly10 to alanine increases the IC 50 for inhibition of PLC activation by 40-fold (5 ⁇ M to 200 ⁇ M), 60-fold and 12-fold, respectively (Scott, et al. (2001) supra).
  • the same mutation of sLeu9 also blocks the ability of SIRK to enhance ERK1/2 phosphorylation in RASM cells (Goubaeva, et al. (2003) supra).
  • the binding surface of G ⁇ 1 in the G ⁇ 1 ⁇ 2 •SIGK complex is not significantly changed upon SIGK binding.
  • the RMSD between the core residues of G ⁇ 1 in the G ⁇ 3 ⁇ 2 •SIGK complex and that in the uncomplexed G ⁇ 1 ⁇ 1 heterodimer (1TBG (Sondek, et al. (1996) Nature 379:369-74); Val40-Asn340, C ⁇ only) is 0.88 ⁇ .
  • Trp99, Tyr59, Asp228, Leu117 and Met101 rotate to accommodate SIGK such that atoms within these residues undergo maximum displacements of 4.0 ⁇ , 3.6 ⁇ , 2.9 ⁇ , 2.8 ⁇ and 2.3 ⁇ , respectively, relative to their positions in uncomplexed G ⁇ 1 .
  • the B factors for residues in the SIGK binding surface are close to the overall average for the complex.
  • the B factor for Trp99 is reduced two-fold upon binding to SIGK, as indicated by comparison of normalized B factors of the respective structures. In this analysis, there are no large conformational changes or disorder to order transitions in G ⁇ upon SIGK binding.
  • the SIGK•G ⁇ 1 ⁇ 2 complex may be compared to those of five G ⁇ 1 ⁇ 2 complexes with protein targets: the G ⁇ 1 ⁇ 2 •G ⁇ i1 heterotrimer (1GG2) (Wall, et al. (1995) supra; Wall, et al. (1998) supra) and the G ⁇ 1 ⁇ 1 •G ⁇ t/i heterotrimer (1GOT) (Lambright, et al. (1996) supra), the G ⁇ 1 ⁇ 1 •phosducin complex (1AOR and 2TRC) (Loew, et al. (1998) supra; Gaudet, et al. (1996) supra), and the G ⁇ 1 ⁇ 2 •GRK2 complex (1OMW) (Lodowski, et al.
  • Fluorescein-labeled G ⁇ i1 (F ⁇ i1 ) was prepared in accordance with standard methods (Sarvazyan, et al. (1998) supra). Assays were used to determine peptide effects on G ⁇ -G ⁇ interactions included competition and dissociation assays (Ghosh, et al. (2003) supra). Briefly, for competition-based assays, 100 pM F ⁇ i1 and indicated concentrations of peptides were added to 50 pM b-G ⁇ 1 ⁇ 2 immobilized on 10 5 beads per mL and incubated at room temperature for 30 minutes to reach equilibrium. The bead-associated fluorescence was then recorded in a BD Biosciences FACSCALIBURTM flow cytometer.
  • the nine peptides were selected to represent the different consensus groups of peptides previously identified (See Scott et al. (2001) supra; Table 9) and to compare binding characteristics within and between consensus groups. Binding of phage displaying these peptides to wild-type G ⁇ 1 ⁇ 2 gave ELISA signals that were different, but fell within a similar range (25 to 100% binding relative to phage 3.14). As disclosed herein, the binding signal obtained in the ELISA assay was correlated to a loss in affinity by comparing the results to behavior of the peptide in a solution based assay. For example, a mutant displaying an 80% loss of binding in an ELISA had a corresponding 10-fold shift in peptide affinity in solution. For the purposes of present disclosure, any substitution that decreased the binding to less than 20% of the wild-type binding was considered to be a critical binding contact for that peptide. Data obtained from this analysis is presented in Table 10.
  • each of the peptides utilized unique combinations of amino acids within the SIGK binding surface for its particular interaction.
  • a dominant feature amongst the peptides was a strong requirement for Trp332, within the C-terminal interface. Lys57, Tyr59, Leu117, also within this interface, generally contributed significantly to binding the peptides, though there were cases where their effects were not absolutely required. The remainder of the amino acids had more variable effects on binding of each peptide.
  • SIGK has a minimal requirement for Trp99 while Ser-Cys-Lys-Arg-Thr-Lys-Ala-Gln-Ile-Leu-Leu-Ala-Pro-Cys-Thr (C1; SEQ ID NO:7) absolutely requires Trp99 for binding.
  • SIGK binding has an absolute requirement for Tyr145 and Ser-Cys-Lys-Arg-Thr-Lys-Ala-Gln-Ile-Leu-Leu-Ala-Pro-Cys-Thr (C1; SEQ ID NO:7) binding is not affected by this mutation.
  • the N-terminus of SIGK interacts with the GP subunit through two main contacts: sSer1 interactions with ⁇ Asp186 and ⁇ Tyr145 residues, and sLys4 interactions with ⁇ Met188 through a Van der Waals interaction and ⁇ Asn230, ⁇ Asp246 and ⁇ Asp228 through hydrogen bonded or charged interactions.
  • Asp228Ala and Asp246Ala did not dimerize with gamma and could not be purified; however, Asp246Ser was expressed and purified.
  • peptides in groups I, II and IV have a substantial requirement for binding to the N-terminal region, reflected by an almost complete loss of binding to the Met188Ala and Asp246Ser (except SIGK) mutants and various requirements for Asn230.
  • Peptides in groups I, II and IV have a conserved motif where a lysine is spaced three amino acids away from a hydrophobic core motif (see Table 9).
  • This motif in SIGK provides the appropriate spacing in a single alpha-helical turn between the lysine that interacts with the N-terminal binding surface and the Ile-Leu-Gly motif that interacts with the C-terminus. It is believed that some of the other peptides adopt a similar ⁇ -helical structure that may make this spacing critical.
  • the peptides in group III bind the C-terminal interaction region, but lack a requirement for Met188 and have minimal requirements for Asn230 and Asp246, indicating they do not use the N-terminal binding surface for their interaction with ⁇ .
  • Arg314 Two amino acids that do not apparently bind directly to SIGK were also analyzed, Arg314 and His311. Replacement of Arg314 results in a modest decrease in SIGK binding; however, for other peptides, Arg314 is absolutely required indicating that they may directly interact with this amino acid. His311 lies well outside the SIGK peptide binding site but was mutated because of its potential involvement in a conformation change in ⁇ subunits (Gaudet, et al. (1996) supra; Loew, et al. (1998) supra). The imidazole side chain of His311 is 13 ⁇ from the guanido nitrogen of Arg314, the closest amino acid that apparently interacts with any of the peptides.
  • His311 could directly interact with amino acids from the phage display-derived peptides. Nevertheless, mutation of His311 to alanine affected binding of various peptides to varying extents. Peptides whose binding was affected by His311A also required Arg314 for binding, an effect possibly due to an alteration in the position of Arg314.
  • amino acids Trp99, Trp332, and Try59 within the hydrophobic pocket are common interaction sites in all three structures.
  • the SIGK peptide and ⁇ switch II have a lysine residue occupying nearly identical positions on G ⁇ .
  • the ⁇ ARK-ct peptide has a lysine residue in a similar position, the geometry and nature of the interaction is different. ⁇ ARK interacts only with Asp228 whereas SIGK and G ⁇ interact with Asp228, Asp246, Asn230 and Met188. Based on this difference, it was determined whether the specific interactions of SIGK at this interface were critical for promoting dissociation.
  • SCAR peptide another peptide derived from the phage display screen.
  • SCAR lacks a lysine residue with the correct positioning relative to the hydrophobic core motif to reach the lysine-binding N-terminal surface (Table 9). Therefore, SCAR would not be able to promote subunit dissociation.
  • SIGK and SCAR can compete with G ⁇ i for binding to G ⁇ 1 ⁇ 2 , with IC 50 's of 0.5 and 1.7 ⁇ M, respectively.
  • SIRK(Lys4Ala) is low affinity blocker, and is not effective at preventing rebinding of F ⁇ i1 .
  • a peptide with comparable affinity to SIRK(Lys4Ala), SIRK(Gly10Ala) (IC 50 ⁇ 80 ⁇ M) was tested. This peptide has Lys4 but Ala is substituted for Gly at position 10, thus SIRK(Gly10Ala) retains binding to the N-terminal interface but has a reduced affinity due to decreased interactions with the C-terminal region.
  • SIRK(Gly10Ala) blocked heterotrimer formation at high peptide concentrations and despite having a low affinity for G ⁇ , could still accelerate heterotrimer dissociation.
  • SIGK binds to G ⁇ 1 at a region occupied by the switch II domain of G ⁇ subunits in the heterotrimer.
  • the crystal structure of the heterotrimer reveals the switch interface (composed of switch I and switch II) of G ⁇ buries approximately 1,800 ⁇ of G ⁇ through numerous contacts (Lambright, et al. (1996) supra; Wall, et al. (1995) supra); however, the effects of mutations of ⁇ subunit amino acids at this interface on ⁇ subunit binding have not been measured in direct binding assays near the K d for G ⁇ -G ⁇ interactions.
  • Switch I and switch II undergo large conformational changes upon GTP binding and it is thought these changes mediate heterotrimer dissociation.
  • G ⁇ 1 subunit mutants disclosed herein were isolated from insect cells as a complex with G ⁇ 2 and hexa-histidine-tagged G ⁇ i1 indicating that many of these contacts between the subunits predicted from the crystal structures were not individually critical for G ⁇ subunit binding.
  • the dissociation rate constant (k off ) for F ⁇ i1 from each of the individually substituted b- ⁇ 1 ⁇ 2 mutants was measured.
  • the intrinsic off-rate for wild-type was 0.123 s ⁇ 1, corresponding well with previous measurements (Sarvazyan, et al. (1998) J. Biol. Chem. 273:7934-7940). Data from all of these mutants are shown in Table 11.
  • a phage ELISA assay was used to determine whether small molecules identified in the computational screen could interact with the G ⁇ protein interaction surface.
  • Phage displaying the SIGK peptide were used in accordance with established methods (Scott, et al. (2001) supra; Smrcka and Scott (2002) supra). The screen was based on a reduction in the optical density (OD) of wells containing G ⁇ subunits and phage. In each plate, three wells contained positive controls for binding that included b- ⁇ subunits, SIGK-phage, and the appropriate amount of vehicle. Three background wells contained no ⁇ subunits.
  • biotinylated G ⁇ subunits were immobilized on the surface of a 96-well plate coated with streptavidin, phage displaying G ⁇ -binding peptides were subsequently added and binding in the presence and absence of test compounds detected with an anti-phage antibody.
  • Ca 2+ fluxes were measured using two 35 mL cultures of differentiated HL-60 neutrophil cultures (0.2 ⁇ 10 6 cells/mL). Cells were cultured for three days with in DMSO (1.2%), washed in HSS and resuspended in 2 mL HBSS at a concentration of 7 ⁇ 10 6 cells/mL. Addition of DMSO to the growth medium induces differentiation of these cells into morphologically and functionally mature neutrophils (Collins, et al. (1978) Proc. Natl. Acad. Sci. USA 75:2458; Collins, et al. (1979) J. Exp. Med. 149:969).
  • Neutrophils were preloaded with fura-2 (1 ⁇ M), a fluorescent Ca 2+ -sensitive indicator (Suh, et al. (1996) J. Biol. Chem. 271:32753), for 45 minutes, washed with HBSS and resuspended in 2 mL of indicator-free HBSS. An 140 ⁇ L aliquote of cells was added to a total of 2 mL HBSS. Fluorescence ratios were taken by dual excitation at 340 and 380 nm and emission at 510 nm. After a stable baseline was established, either DMSO or NSC119910 was added and incubated for 5 minutes. Subsequently, either fMLP or ATP agonists were added to activate release of Ca 2+ from intracellular stores.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Addiction (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Cardiology (AREA)
  • Epidemiology (AREA)
  • Pain & Pain Management (AREA)
  • Neurology (AREA)
  • Psychiatry (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Rheumatology (AREA)
  • Hospice & Palliative Care (AREA)
  • Neurosurgery (AREA)
US11/885,981 2005-03-07 2006-03-07 Compositions and methods for inhibiting g protein signaling Abandoned US20090221691A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US11/885,981 US20090221691A1 (en) 2005-03-07 2006-03-07 Compositions and methods for inhibiting g protein signaling

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US65926705P 2005-03-07 2005-03-07
PCT/US2006/008031 WO2006096690A2 (en) 2005-03-07 2006-03-07 Compositions and methods for inhibiting g protein signaling
US11/885,981 US20090221691A1 (en) 2005-03-07 2006-03-07 Compositions and methods for inhibiting g protein signaling

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/008031 A-371-Of-International WO2006096690A2 (en) 2005-03-07 2006-03-07 Compositions and methods for inhibiting g protein signaling

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/630,995 Continuation US8748480B2 (en) 2005-03-07 2012-09-28 Methods for treating opioid tolerance

Publications (1)

Publication Number Publication Date
US20090221691A1 true US20090221691A1 (en) 2009-09-03

Family

ID=36953955

Family Applications (2)

Application Number Title Priority Date Filing Date
US11/885,981 Abandoned US20090221691A1 (en) 2005-03-07 2006-03-07 Compositions and methods for inhibiting g protein signaling
US13/630,995 Expired - Fee Related US8748480B2 (en) 2005-03-07 2012-09-28 Methods for treating opioid tolerance

Family Applications After (1)

Application Number Title Priority Date Filing Date
US13/630,995 Expired - Fee Related US8748480B2 (en) 2005-03-07 2012-09-28 Methods for treating opioid tolerance

Country Status (7)

Country Link
US (2) US20090221691A1 (ja)
EP (1) EP1868634A4 (ja)
JP (1) JP2008537481A (ja)
CN (1) CN101171022A (ja)
AU (1) AU2006220652A1 (ja)
CA (1) CA2600946A1 (ja)
WO (1) WO2006096690A2 (ja)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10436795B2 (en) * 2016-09-16 2019-10-08 Institute For Cancer Research Carbon-detected NMR for mapping binding sites in intrinsically disordered regions of a protein
CN115368386A (zh) * 2022-08-09 2022-11-22 浙江省中医院、浙江中医药大学附属第一医院（浙江省东方医院） 一种防治骨性关节炎的小分子化合物及其用途

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100523092C (zh) * 2005-05-16 2009-08-05 徐州开达精细化工有限公司 蒽醌型还原染料16,17-二溴紫蒽酮和16,17-二(1’-亚胺基蒽醌基)紫蒽酮及其制备方法
US8975259B2 (en) 2007-04-27 2015-03-10 University Of Rochester Compositions and methods for inhibiting G protein signaling
WO2011040564A1 (ja) * 2009-09-30 2011-04-07 千寿製薬株式会社 特定のペプチド化合物を有効成分として含む眼科用剤
CN105802970A (zh) * 2016-05-30 2016-07-27 东北师范大学 靶向沉默Gβ1的shRNA
CN106908610B (zh) * 2017-03-13 2018-11-02 绍兴康知生物科技有限公司 一种基于受体配体结合技术筛查吸食吗啡的试剂、制备方法及应用
CN108802366B (zh) * 2017-05-04 2020-01-21 中国科学院微生物研究所 一种检测待测样本中目标蛋白的含量的方法

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU652576B2 (en) * 1990-09-13 1994-09-01 Duke University Expression of G protein coupled receptors in yeast
CA2219958A1 (en) * 1998-01-07 1999-07-07 Ekkehard Leberer The g-protein .beta.subunit interaction domain of ste20p/pak family of protein kinases and uses thereof in bioassays
US7189519B2 (en) * 1998-08-10 2007-03-13 Stc.Unm Bead-based detection of ligand-GPCR-G protein complexes
ATE445335T1 (de) * 1998-12-23 2009-10-15 Sinai School Medicine Hemmstoffe zur unterdrückung des bitteren geschmacks
AU2976000A (en) * 1999-01-27 2000-08-18 Regents Of The University Of California, The Assays for sensory modulators using a sensory cell specific g-pr otein beta subunit
WO2001021797A1 (en) * 1999-09-21 2001-03-29 Forsyth Dental Infirmary For Children Rgs10b, a g-protein regulator expressed in osteoclasts
WO2003062393A2 (en) * 2002-01-22 2003-07-31 Bristol-Myers Squibb Company G protein-coupled receptor variants
US20040121330A1 (en) * 2002-12-18 2004-06-24 Feder John N. Novel human G-protein coupled receptor, HGPRBMY4, and methods of use thereof
US7097993B2 (en) 2003-06-25 2006-08-29 Wisconsin Alumni Research Foundation Method for identifying an agent that modulates type 1 phosphatidylinositol phosphate kinase isoform β661 activity

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10436795B2 (en) * 2016-09-16 2019-10-08 Institute For Cancer Research Carbon-detected NMR for mapping binding sites in intrinsically disordered regions of a protein
CN115368386A (zh) * 2022-08-09 2022-11-22 浙江省中医院、浙江中医药大学附属第一医院（浙江省东方医院） 一种防治骨性关节炎的小分子化合物及其用途

Also Published As

Publication number Publication date
JP2008537481A (ja) 2008-09-18
US20130116313A1 (en) 2013-05-09
EP1868634A4 (en) 2010-10-20
CN101171022A (zh) 2008-04-30
CA2600946A1 (en) 2006-09-14
AU2006220652A1 (en) 2006-09-14
WO2006096690A2 (en) 2006-09-14
EP1868634A2 (en) 2007-12-26
US8748480B2 (en) 2014-06-10
WO2006096690A3 (en) 2007-11-08

Similar Documents

Publication Publication Date Title
US8748480B2 (en) Methods for treating opioid tolerance
Campbell et al. Activation of JAK2 tyrosine kinase by prolactin receptors in Nb2 cells and mouse mammary gland explants.
Dietrich et al. Cation channels of the transient receptor potential superfamily: their role in physiological and pathophysiological processes of smooth muscle cells
Huang et al. Defining the specificity space of the human SRC homology 2 domain
Wang et al. Ezrin-anchored protein kinase A coordinates phosphorylation-dependent disassembly of a NHERF1 ternary complex to regulate hormone-sensitive phosphate transport
Martinu et al. Endocytosis of epidermal growth factor receptor regulated by Grb2-mediated recruitment of the Rab5 GTPase-activating protein RN-tre
JP2002098702A (ja) プロスタグランジンｄ２により媒介される疾病状態の治療に有用な化合物の同定方法
JP2002531384A (ja) ＢクラスｅｐｈｒｉｎとＰＤＺ領域との相互作用を変調するペプチド
Ishii et al. Phosphatidylinositol 3, 4, 5-trisphosphate and Ca2+/calmodulin competitively bind to the regulators of G-protein-signalling (RGS) domain of RGS4 and reciprocally regulate its action
Li et al. Cdc2/cyclin B1 interacts with and modulates inositol 1, 4, 5-trisphosphate receptor (type 1) functions
JP2002508961A (ja) 内皮細胞の形態および細胞骨格編成に影響を与える、ヒトリンパ節由来のｇｔｐアーゼ
US6812335B1 (en) Human polypeptide receptors for lysophospholipids and sphingolipids and nucleic acids encoding the same
Wang et al. A novel peptide exerts potent immunosuppression by blocking the two-site interaction of NFAT with calcineurin
Faitar et al. EVI5 is a novel centrosomal protein that binds to α-and γ-tubulin
Kerov et al. Interaction of transducin-α with LGN, a G-protein modulator expressed in photoreceptor cells
US6498139B1 (en) Remedies for diseases caused by insulin resistance
JP2007537711A (ja) Ｉｇｆ結合タンパク質
CA2405311A1 (en) Multiprotein-complexes comprising a nmda receptor and uses thereof
JP2007533309A (ja) プロテインキナーゼｃシータの構造
US7122655B2 (en) Compositions involving M-RIP, and related methods for screening for anti-hypertensive agents, and uses thereof
JP2008504224A (ja) Ｅｃｔ２のプレクストリン相同ドメインを含むポリペプチド、及びホスファチジルイノシトールに対するその結合特異性
Dorsam et al. Development and use of ELISA to quantify type II phospholipase A2 in normal and uremic serum
WO2005009469A1 (ja) 新規血糖調節薬及びそのスクリーニング方法
Li et al. Kiss1/Gpr54 Prevents Bone Loss through Src Dephosphorylation by Dusp18 in Osteoclasts
WO2022076947A1 (en) Peptide inhibitors of glioma-associated oncogene derived from the coiled-coil dimerization domain of kif7

Legal Events

Date Code Title Description
AS Assignment

Owner name: ROCHESTER, UNIVERSITY OF, NEW YORK

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SMRCKA, ALAN V.;FONT, JOSE;BONACCI, TABETHA;REEL/FRAME:017742/0001

Effective date: 20060322

AS Assignment

Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF ROCHESTER;REEL/FRAME:022276/0905

Effective date: 20080929

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: UNIVERSITY OF ROCHESTER, NEW YORK

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SMRCKA, ALAN V;FONT, JOSE;BONACCI, TABETHA;SIGNING DATES FROM 20130320 TO 20140131;REEL/FRAME:032793/0413

AS Assignment

Owner name: NATIONAL INSTITUTES OF HEALTH - DIRECTOR DEITR, MA

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF ROCHESTER;REEL/FRAME:048963/0577

Effective date: 20190311