Methods and compositions for screening Icrac modulators

Details Methods and compositions for screening Icrac modulators Methods and compositions for screening Icrac modulators

2001-03-30

2004-02-24

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S006120, C435S455000, C435S354000, C435S372300, C435S372000, C435S018000

Reexamination Certificate

active

06696267

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to compositions and methods for screening compounds that modulate calcium entry or calcium-mediated activity within cells. More specifically, this invention discloses compositions and methods, including cell based assays, directed at screening or characterizing compounds that modulate the activity of calcium-release activated channels in cells. The compositions and methods can be used to produce inhibitors or activators of said channel, which represent leads or candidate therapeutic drugs for treating various pathological conditions.
BACKGROUND OF THE INVENTION
Calcium influx and regulation play a critical role in many cellular processes in both excitable and non-excitable mammalian cells, including exocytosis, gene expression, cell differentiation, cell activation, contraction, etc. In non-excitable cells, such as immune cells, calcium concentration and flux are regulated essentially by voltage-independent Ca
++
channels (sometimes referred to as capacitative calcium entry (CCE)), designated store-operated channels (SOC) or receptor-operated channels (ROC). The activity of these channels is essential in the regulation of calcium entry and participates directly in many important cellular processes such as cell activation or maturation for instance. A particularly important type of store-operated calcium channel is the Calcium release-activated channel (Icrac), which is opened in response to depletion of intracellular calcium stores and mediates various intracellular transduction signals.
For instance, the Icrac channel is involved in T cell activation following binding of an antigen to the T Cell Receptor (TCR). The fill activation of T lymphocytes is due to the stimulation of the TCR/CD3 complex and CD2, CD4 or CD28 (see for review (1)). Upon antigenic stimulation of T-cells, (2) nuclear factor of activated T cells (NFAT) is required for the production of immunoregulatory molecules such as interleukins, IFN-&ggr;, or TNF-&agr; (3). NFAT is a complex including a constitutive cytoplasmic component expressed in resting immunomodulatory cells such as T and B-cells, which translocate to the nucleus, and an inducible nuclear component consisting of dimers of fos- and jun-family proteins. Dephosphorylation of the cytoplasmic component of NFAT by Ca
++
/calmodulin-dependent serine/threonine phosphatase, calcineurin, induces its translocation to the nucleus (4). Prolonged elevation of [Ca
++
]
i
level, beyond the initial transient increase resulting from the emptying of calcium intracellular pools, is required to maintain calcineurin phosphatase in activated state. After stimulation of immunomodulatory cells such as T-cell by external components, this calcium mobilization is the outcome of capacitative calcium entry, a process originated by the depletion of Ca
++
store and the inflow of extracellular calcium through the specific Calcium release-activated Ca
++
channel (Icrac) (5, 6). Capacitative Ca
++
entry is triggered by stimulation of various receptors such as T-CR, B-CR, high affinity IgE receptor (Fc&egr;RI) and IgG receptor (Fc&ggr;RI) (via PLC&ggr;, and IP3 receptor activation), CD40 (3) or by calcium ionophore (6, 7). Inhibitors of endoplasmic reticulum Ca
++
/ATPase pump (thapsigargin (TG) (Thastrup et al., PNAS 87 (1990) 2466, ref (9)) or cyclopiazonic acid (CPA) (8)) directly deplete intracellular calcium stores and lead to Icrac activation.
It has also been shown that Icrac is expressed by other blood cells such as T cells. Because of its role in interleukin production, Icrac is seen as a very interesting target for novel anti-inflammatory and immunosuppressing drugs or lead compounds. Furthermore, it has recently become clear that an Icrac like current is also present in endothelial cells (Am. J. Physiol. 269 (1995) C733, ref (10)) and in epithelial cells (J. Biol.Chem. 270 (1995) 169, ref (12)). Since radical damage could be linked to Ca
++
inflow in these cells, it is thought that Icrac blockers would have a positive effect. In addition, since Ca
++
inflow blockade leads to the IL2 synthesis blockade, Icrac blockers are potentially useful in proliferative or progressive diseases such as malignant tumors.
Icrac thus represents a very powerful target for screening of compounds with high therapeutic potential. However, the use of this target has been hampered so far by the fact that this channel has not been isolated or cloned in the art.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods that allow an efficient screening for Icrac modulators. The invention discloses cell based assays allowing selective and efficient determination of the effect of any test compound on Icrac activity. The methods of this invention can be used to screen large amounts of test compounds, including libraries of compounds, on a high throughput basis, by reducing the number of false positives, providing increased selectivity and easy monitoring of the effect of any compound. Icrac modulators represent high potential leads or candidate drugs for the treatment or alleviation of various pathological conditions, including immune-related diseases such as auto-immune diseases, inflammation, allergy, asthma, cancers or other cell proliferative disorders.
In accordance with the present invention, a method is provided for screening a compound that modulates calcium channel activity, preferably calcium release-activated channel (Icrac) activity, comprising:
a. contacting a test compound and a selective calcium channel activator, preferably an Icrac activator with a population of calcium channel expressing cells, preferably Icrac expressing cells, said cells further containing a reporter construct comprising a reporter gene under the control of a NFAT-inducible promoter, and
b. determining the activity of the test compound on the calcium release-activated channel by assessing the expression of the reporter gene in said cells.
In a particular embodiment, the determination of the reporter gene expression comprises:
(i) contacting the cells of a) with a substrate of the reporter gene expression product, and
(ii) determining the activity of the test compound on the calcium release-activated channel by assessing the hydrolysis of the substrate in said cells.
The reporter gene expression (e.g., hydrolysis of the substrate) can be correlated directly to the activity of the test compound: elevated expression levels (e.g. elevated levels of hydrolysis product) or an increase in expression levels (of the hydrolysis product) as compared to a control situation in the absence of the test compound indicates that the compound stimulates Icrac; low expression levels (e.g., low levels of hydrolysis product) or a decrease in the expression levels (of the hydrolysis product) as compared to a control situation in the absence of the test compound indicates that the compound inhibits Icrac.
Within a preferred embodiment, the cells are contacted with a selective Icrac activator, that preferentially does not activate Protein Kinase C (“PKC”). More preferably the selective Icrac activator does not contain phorbol ester myristate acetate (PMA).
According to a preferred embodiment, the reporter gene is a &bgr;-lactamase gene and the substrate is a substrate of &bgr;-lactamase.
According to other preferred embodiments, the substrate is a ratiometric substrate, and/or the population of Icrac-expressing cells comprises a culture of blood cells, particularly lymphocytes or mastocytes.
In a particular variant of the present invention, the method further comprises the screening of the active test compounds detected in step b) above to determine which of these compounds modulate the expression activity of the reporter gene product (e.g., &bgr;-lactamase) in a non-NFAT dependent manner. This secondary screen allows to increase the selectivity of the method, by eliminating various test compounds which would modulate &bgr;-lactamase activity or,

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