Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2002-11-19
2003-11-04
Stockton, Laura L. (Department: 1626)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C548S172000
Reexamination Certificate
active
06642263
ABSTRACT:
FIELD OF THE INVENTION
The claimed invention relates generally to the fields of medicine and enzyme biochemistry. More particularly, the invention relates to compounds and methods for modulating the activity of Rho C.
BACKGROUND OF THE INVENTION
The small GTPase family of proteins are central regulators of cell physiology. Five homologous subfamilies are found in the genomes of all eukaryotes; the
S. cerevisiae
genome includes 29 proteins in all five families, and the human genome encodes approximately 100 proteins. These five subfamilies have five overlapping but partially distinct functional roles. Ras family members regulate cell growth and division (A. Hall,
Curr Opin Cell Biol
(1993) 5(2):265-68; A. B. Vojtek et al.,
J Biol Chem
(1998) 273(32):19925-28). Rho family members regulate cell motility, and shape through the actin skeleton (A. Hall,
Science
(1998) 279:509-14; D. J. Mackay et al.,
J Biol Chem
(1998) 273:20685-88). ARF family members regulate cell adhesion and vesicle trafficking to and from the plasma membrane (A. L. Boman et al.,
Trends Biochem Sci
(1995) 20(4):147-50; P. Chavrier et al.,
Curr Opin Cell Biol
(1999) 1(4):466-75). Rab family members regulate intra-vesicular organelle trafficking (0. Martinez et al.,
Biochim Biophys Acta
(1998) 1404(1-2):101-12; P. Chavrier et al., supra; F. Schimmoller et al.,
J Biol Chem
(1998) 273(35):22161-14) and Ran family members regulate nuclear translocation and chromosomal segregation through regulation of microtubule assembly at the spindle pole (M. S. Moore,
J Biol Chem
(1998) 273(36):22857-60; M. G. Rush et al., Bioessays (1996) 18(2):103-12). These proteins stimulate other proteins in their GTP-bound state via physical interactions, and lose these associations and activities in the post-hydrolytic GDP-bound state. The hydrolysis reaction thus serves as molecular timer for the events triggered by the GTP-bound small G-protein. In addition, these GTPases also serve as signal integrators since the GTPases are regulated by other signaling pathway proteins; these signaling proteins are themselves regulated and promote or inhibit exchange of GDP for GTP or accelerate the GTP hydrolysis reaction. Ras was the first human small-GTPase to be appreciated in detail due to its identification as a human oncogene mutated in greater than 20% of human cancers (J. L. Bos,
Cancer Res
(1989) 49(17):4682-89). The ras mutants found in human cancers create a GTPase deficient form of ras which thus exists predominantly in the GTP bound-activated state.
The Rho family of small GTPase comprises more than 10 members in humans and 6 members in yeast. In both organisms, control of the actin skeleton organization and localization is a major Rho function. The human Rho family is composed of three sub-families: Rho, Rac and CDC42 (K. Kaibuchi et al.,
Ann. Rev. Biochem
. (1999) 68:459-86). These sub-families are all involved in control of the actin skeleton and cell adhesion. RhoA is the best-studied of RhoA-G group (collectively Rho) and has been closely associated with actin stress fiber formation in fibroblasts, and through its interaction with ROCK (Rho activated kinase) actin-myosin contraction leading to smooth muscle contraction. Yeast Rho1 is most homologous to human RhoA, and is found at the main site of organized actin in yeast (the bud), where it appears to regulate actin organization associated with budding. In addition, Rho1p controls cell wall bio-synthetic enzyme activity of 1,3-beta-D-glucan synthase (FKS 1) during its physical association with the GTP-bound Rho1p. CDC42 and Rac-1 have also been well studied. CDC42 is closely associated with filopodia or microspike formation in fibroblasts and integrin activation. Rac-1 is a downstream component of the Ras signaling pathway from growth factor receptors and is closely associated with actin rearrangements leading to lamellipodia formation in fibroblasts (A. Hall,
Science
(1998) 279:509-14).
Rho proteins interact with several upstream and downstream components in signaling pathways that originate at the cell membrane with either G-protein coupled receptors, CDC42 and RhoA, or growth factor receptors, such as Rac-1.
The upstream pathways from membrane receptors to the Rho protein involves P13-Kinase, PIP3, and a Db1-homology protein that is a PIP3 receptor and catalyzes guanine nucleotide exchange of Rho; it is thus termed a GEF (“guanine nucleotide exchange factor”). The GEFs for RhoA, and its close homologue RhoC, include Db1, Net1, Ost and Vav. These proteins all have Db1 homology domains (also known as RhoGEF domains) and pleckstrin homology domains, and all activate guanine nucleotide exchange by interaction with Rho proteins through their Db1-homology domain (R. A. Cerione et al.,
Curr Opin Cell Biol
(1996) 8(2):216-22; I. P. Whitehead et al.,
Biochim Biophys Acta
(1997) 1332(1):F1-23). The yeast upstream pathways from the cell membrane to Rho and beyond are highly related to those found in mammalian cells and include Tor2 (yeast PI3-kinase), and Rom1/Rom2 yeast Db1-homology and pleckstrin containing GEFs (K. Tanaka et al.,
Curr Opin Cell Biol
(1998) 10(1):112-16). In both yeast and humans, Rho proteins are prenylated and associate, in their GDP bound states, with a guanine nucleotide-dissociation inhibitor (“GDI”). The GDI, known as RhoGDI in humans, and Rdi1p in yeast, solublizes the Rho protein and prevents its membrane association until activation by a GEF exchanges its GDP for GTP and allows its association with the membrane (T. K. Nomanbhoy et al.,
Biochemistry
(1999) 38(6):1744-50; P. W. Read et al.,
Protein Sci
(2000) 9(2):376-86).
The downstream pathways from Rho family members include many functionally and structurally homologous proteins. RhoA interacts with formin family members Dia1/Dia2, and yeast Rho1p interacts with Bni1 (another formin family member), while CDC42 interacts with WASP and WASP-N, a pair of proteins organized and regulated similarly to formin members. The formin family members have binding sites for the GTP-bound forms of Rho and also actin-nucleating domains whose exposure is controlled by binding of the GTP-Rho (N. Watanabe et al.,
Nat Cell Biol
(1999) 1(3):136-43). In addition to formin interactions, Rho proteins interact with serine/threonine kinases. RhoA interacts with ROCK kinase, which then phosphorylates proteins that control actin polymerization; it also phosphorylates myosin regulators which control contraction in smooth muscles. Yeast Rho1p interacts with PKC1 which launches a MAP kinase cascade leading to control of transcription and the actin skeleton (S. B. Helliwell et al.,
Curr Biol
(1998) 8(22):1211-14; K. Tanaka et al.,
Curr Opin Cell Biol
(1998) 10(1):112-16).
The 3-dimensional structure of RhoA in its GTP, GDP and Mg
2+
depleted states are known (K. Ihara et al.,
J Biol Chem
(1998) 273(16):9656-66; R. Maesaki et al.,
Mol Cell
(1999) 4(5):793-803; T. Shimizu et al.,
J Biol Chem
(2000) 275(24):18311-17) as is the structure RhoA-GTP in complex with an interaction domain of the downstream effector PKN (R. Maesaki et al., supra; R. Maesaki et al.,
J Struct Biol
(1999) 126:166-70), and the structure of the complex of RhoA-GDP with Rho-GDI (K. Longenecker et al.,
Acta Crystallogr D Biol Crystallogr
(1999) 55(Pt 9):1503-15). The structure of RhoA in complex with rhoGAP is also known (K. Rittinger et al.,
Nature
(1997) 388:693-97). The structure of these molecules combined with similarly detailed data regarding Ras and the Rho family member CDC42 yield consensus molecular mechanism for the GTPase function, GEF's promotion of GDP exchange, GAP's acceleration of GTPase activity and effector stimulation by RhoA-GTP. These studies show the guanine nucleotide bound in a pocket surrounded by three protein loops, known as switch region-I, switch region-II and the P-loop (the phosphate-binding loop). Switch region-I and -II interact extensively with GDI, GEF and effector domains in regions that occlude each other's binding site. Switch region I and II are dramatically rearra
Lee Ving J.
Perkins Edward L.
Sun Dongxu
Tugendreich Stuart
Cooley & Godward LLP
Iconix Pharmaceuticals Inc.
Stockton Laura L.
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