Compound screening methods

Details Compound screening methods Compound screening methods

2000-04-14

2003-04-01

Fredman, Jeffrey (Department: 1635)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C800S003000, C800S008000, C800S013000, C435S004000

Reexamination Certificate

active

06540996

ABSTRACT:

The invention is concerned with methods for use in the identification of compounds which affect the activity of a physiologically important calcium pump, the sarco/endoplasmic reticulum Ca
2+
ATPase (SERCA).
In most animal cells and plant cells, the normal concentration of free cytosolic Ca
2+
is 50 to 100 nM. Since Ca
2+
acts as a major intracellular messenger, elevating these levels affects a wide range of cellular processes including contraction, secretion and cell cycling (Dawson, 1990, Essays Biochem. 25:1-37; Evans et al., 1991, J. Exp. Botany 42:285-303). Intracellular Ca
2+
stores hold a key position in the intracellular signalling. They allow the rapid establishment of Ca
2+
gradients, and accumulate and release Ca
2+
in order to control cytosolic Ca
2+
levels. Moreover, lumenal Ca
2+
intervenes in the regulation of the synthesis, folding and sorting of proteins in the endoplasmic reticulum (Brostrom and Brostrom, 1990, Ann. Rev. Physiol. 52:577-590; Suzuki et al., 1991, J. Cell. Biol. 114:189-205; Wileman et al., 1991, J. Biol. Chem. 266:4500-4507). Furthermore it controls signal-mediated and passive diffusion through the nuclear pore (Greber and Gerace, 1995, J. Cell. Biol. 128:5-14).
Three genes that code for five different isoforms of the sarco/endoplasmic reticulum Ca
2+
ATPase (SERCA) are known in vertebrates, SERCA1a/b, SERCA2a/b and SERCA3. The SERCA isoforms are usually tagged to the endoplasmic reticulum (ER) or ER subdomains like the sarcoplasmic reticulum, although the precise subcellular location is often not known. The SERCA proteins belong to the group of ATP-driven ion-motive ATPases, which also includes, amongst others, the plasma membrane Ca
2+
-transport ATPases (PMCA), the Na+-K+-ATPases, and the gastric H+-K+-ATPases. The SERCA Ca
2+
-transport ATPases can be distinguished from their plasma membrane counterparts like PMCA by the specific SERCA inhibitors: thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)-1,4-benzohydroquinone (Thastrup et al., 1990, PNAS 87:2466-2477; Seidler et al., 1989, J. Biol. Chem. 264:17816-17823; Oldershaw and Taylor, 1990, FEBS Lett. 274:214-216). In view of the diverse role of Ca
2+
in the cell and the fact that Ca
2+
is stored in diverse organelles, the diversity in Ca
2+
-accumulation pump isoforms is not surprising.
SERCA1 is only expressed in fast-twitch skeletal muscle fibres. The gene encodes two different isoforms; SERCA1b which is the neonatal isoform and SERCA1a the adult isoform (Brandl et al., 1986, Cell 44:597-607; Brandl et al., 1987, J. Biol. Chem. 262:3768-3774). The difference between the two isoforms is the result of an alternative splice. As a consequence, the neonatal isoform contains a highly charged carboxyl-terminal extension (Korczak et al., 1988, J. Biol. Chem. 263:4813-4819). The reason for this alternative splicing is as yet unknown; the functional significance of this extension is not yet clear. When expressed in COS cells, SERCA1a and SERCA1b exhibit nearly identical maximal Ca
2+
-turnover rate, Ca
2
+-affinity and ATP-dependency of Ca
2+
transport (Maruyama and MacLennan, 1988, PNAS 85:3314-3318). The human SERCA1 gene is mapped on chromosome 16P12.1 and is about 26 kb long (MacLennan et al., 1987, Somatic Cell Mol. Genet. 13:341-346; Callen et al., 1991, Am. J. Hum. Genet. 49:1372-1377).
SERCA2 is expressed in muscle and non-muscle cells. The human SERCA2 gene maps to chromosome 12q23-q24.1 (Otsu et al., 1993, Genomics 17:507-509). Partial sequence analysis suggests that the same exon/intron layout is conserved between SERCA1 and SERCA2. mRNA of SERCA2 can be divided in 4 different classes; class 1 encodes SERCA2a and is mainly expressed in muscle, the other classes encode SERCA2b and are mainly expressed in non-muscle tissues. SERCA2b harbors a 49 amino acid extension, which contains a highly hydrophobic stretch. As with SERCA1, no functional difference can be measured between the two SERCA2 isoforms when expressed in COS cells (Campbell, 1991, J. Biol. Chem. 266:16050-16055). However, differences in Ca
2+
affinity and turnover rate of the phosphoprotein intermediate have been observed (Lytton et al., 1992, 267:14483-14489; Verboomen et al., 1992, J. Biochem. 286:591-596). Both isoforms are expressed in a tissue-dependent pattern, both qualitatively and quantitatively (Eggermont et al., 1990, J. Biochem. 271:649-653). Cardiac muscle expresses 5- to 20-fold higher levels of SERCA2 than smooth muscle. Slow-twitch skeletal and cardiac muscle only express SERCA2a, while SERCA2b (referred to as the “housekeeping” isoform) is expressed in all non-muscle tissue, and represents about 75% of the Ca
2+
-transporting ATPase activity in smooth-muscle tissue. Different protein-to-message ratios for SERCA2a and SERCA2b have been observed. Cardiac muscle expresses 70 times more protein and only 7 times more SERCA2a mRNA compared to stomach smooth muscle which expresses SERCA2b (Khan et al., 1990, J. Biochem. 268:415-419).
SERCA3 is considered to be the non-muscle SERCA isoform. SERCA3 lacks the putative interacting domain for phospholamban, and hence, does not respond to this modulator (Toyofuku et al., 1993, J. Biol. Chem. 268:2809-2815). When expressed in COS cells, SERCA3 shows approximately 5-fold lower activity for Ca
2+
and a slightly higher pH optimum (Toyofuku et al., 1992, J. Biol. Chem. 267:14490-14496). In platelets, mast cells and lymphoid cells SERCA3 is co-expressed with SERCA2b (Wuytack et al., 1994, J. Biol. Chem. 269:1410-1416; Wuytack et al., 1995, Bioscience Rep. 15:299-306). Expression has also been observed in some arterial endothelial cells, in early developing rat heart, in some secretory epithelial cells of endodermal origin and in cerebellar Purkinje neurons.
In slow-twitch skeletal muscle, cardiac muscle and smooth-muscle tissues, SERCA2 activity is modulated by phosphorylation of the regulatory protein phospholamban (PLB) (see Fuji et al., 1991, FEBS Lett. 273:232-234). In cardiac muscle, in vivo phosphorylation of PLB by cAMP- or Ca
2+
/Calmodulin-dependent protein kinase has a positive effect on the Ca
2+
transport (Le Peuch et al., 1997, Biochemistry 18:5150-5157; Tada et al., 1979, J. Biol. Chem. 254: 319-326; Davis et al., 1983, J. Biol. Chem. 258:13587-13591; Wegener et al., 1989, J. Biol. Chem. 264:11468-11474). In order to determine the exact in vivo role of phospholamban, PLB-deficient mice have been generated (Luo et al., 1994, Circ. Res. 75:401-409). A marked effect is observed on Ca
2+
uptake, whereas no effect is measured in Vmax. The ablation of the PLB gene in mice is associated with increased myocardial contractility, and a loss of the positive inotropic response to adrenergic stimulation. The precise molecular mechanism underlying the modulation of SERCA by PLB is not apparent. An electrostatic mechanism has been proposed, as a direct interaction between PLB and SERCA, in which the unphosphorylated PLB inhibits the SERCA pump (Kirchberger et al., 1986, Biochemistry 25:5484-5492; Chiesi and Schwaller, 1989, FEBS Lett. 244:241-244; Xu and Kirchberger, 1989, J. Biol. Chem. 264:16644-16651). Alternatively, PLB and the SERCA Ca
2+
pump are able to interact and phosphorylation of PLB alters its properties, as confirmed by cross-linking experiments (James et al., 1989, Nature 342:90-92). In some experiments, inhibitory effects of PLB have been observed on co-transfection of PLB and SERCA2a in COS-1 cells (Fuji et al. 1990, FEBS Lett. 273:232-234). Several models have been proposed to explain the regulatory effect of PLB on Ca
2+
ATPases. These include the aggregation of SERCA2 around a pentameric form of PLB (Voss et al., 1994, Biophys J. 67:190-196). Another explanation starts from the electrostatic inhibition of Ca
2+
binding due to the SERCA-PLB interaction (Toyoftiku et al., 1994, J. Biol. Chem. 269:3088-3094). The interaction between PLB and SERCA2a has been studied in more detail, reveali

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