Methods and compositions for identifying receptor effectors

• US 20050059135A1
• Filed: 01/05/2004
• Published: 03/17/2005
• Est. Priority Date: 03/31/1993
• Status: Active Grant

• Current Assignee: Cadus Pharmaceutical Corporation
• Original Assignee: Cadus Pharmaceutical Corporation
• Inventors: Murphy, Andrew J.M., Trueheart, Joshua, Paul, Jeremy, Klein, Christine A., Fowlkes, Dana M., Broach, James, Manfredi, John
• Granted Patent: US 7,250,263 B2
• Time in Patent Office: Days
• US Class Current: 435/254.200
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/81 for yeasts

Abstract

The present invention makes available a rapid, effective assay for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular receptor or ion channel. The subject assay enables rapid screening of large numbers of polypeptides in a library to identifying those polypeptides which induce or antagonize receptor bioactivity. The subject assay is particularly amenable for identifying surrogate ligands for orphan receptors.

Claims

1-76. -76. (Canceled)

77. A mixture of recombinant yeast cells, each cell of which has a cell membrane and each cell of which comprises:

(a) a heterologous receptor selected from the group consisting of;

an FPRL-1 receptor, a C5a receptor and a thrombin receptor, which receptor is expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal; and

(b) a heterologous polypeptide, wherein the heterologous polypeptide is transported to a location allowing interaction with the extracellular region of the heterologous receptor expressed in the cell membrane;

wherein collectively the mixture of cells expresses a library of said heterologous polypeptides, said library being expressible at a sufficient level such that modulation of the signal transduction activity of the heterologous receptor by a heterologous polypeptide within the library provides a detectable signal.

78. The yeast cells of claim 77, wherein each yeast cell further comprises a chimeric G protein subunit.

79. The yeast cells of claim 77, which are a mutant strain of yeast cells having a pheromone system pathway that is desensitized at slower rate than a wild type strain under the same conditions of continuous stimulation of the pheromone system pathway.

80. The yeast cells of claim 77, wherein the yeast cells have a mutation in a gene selected from the group consisting of:

STE50, bar1, pik1, msg5, sig1, and aft1.

81. The yeast cells of claim 77, wherein the yeast cells have a ste2 or ste3 mutation.

82. The yeast cells of claim 77, wherein an endogenous pheromone gene is not functionally expressed in the yeast cells.

83. The yeast cells of claim 77, wherein an endogenous FAR1 gene is not functionally expressed in the yeast cells.

84. The yeast cells of claim 77, wherein an endogenous SST2 gene is not functionally expressed in the yeast cells.

85. The yeast cells of claim 78, which comprise a chimeric Gα

i2 subunit consisting of the first 41 amino acids of GPA1 fused to a region of human Gα

i2.

86. The yeast cells of claim 77, which further comprise a selectable marker that is activated by a pheromone system pathway of the yeast cells, thereby providing the detectable signal.

87. The yeast cells of claim 86, wherein the selectable marker comprises a pheromone-responsive promoter operably linked to a selectable gene.

88. The yeast cells of claim 87, wherein the pheromone-responsive promoter is the FUS1 promoter.

89. The yeast cells of claim 87, wherein the selectable gene is a HIS 3 gene.

90. The yeast cells of claim 77, wherein the heterologous polypeptide includes a signal sequence that facilitates transport of the polypeptide to a location allowing interaction with the extracellular region of the receptor.

91. The yeast cells of claim 80, wherein the signal sequence corresponds to a leader peptide of the Saccharomyces cerevisiae a factor or a-factor.

92. The yeast cells of claim 77, which belong to the species Saccharomyces cerevisiae.

93. The yeast cells of claim 77, wherein the library of heterologous polypeptides includes more than 1000 different polypeptide sequences.

109. The mixture of claim 77, wherein said receptor is a C5a receptor.

110. The mixture of claim 77, wherein said heterologous receptor acts as a surrogate for an endogenous yeast pheromone receptor in a pheromone system pathway of said cell.

111. The yeast cells of claim 77, which further comprise a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity of the receptor protein which thereby provides the detectable signal.

112. The yeast cells of claim 111, wherein said reporter gene encodes a gene product which confers a growth signal.

113. The yeast cells of claim 77, wherein said detectable signal is selected from the group consisting of:

color, fluorescence, luminescence, cell viability, relief of a cell nutritional requirement, cell growth, and drug resistance.

94. A mixture of recombinant yeast cells, each cell of which has a cell membrane and each cell of which comprises:

(a) a heterologous receptor selected from the group consisting of an FPRL-1 receptor, a C 5 a receptor, and a thrombin receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal, said heterologous receptor acting as a surrogate for an endogenous yeast pheromone receptor in a pheromone system pathway of the yeast cell;

(b) a heterologous polypeptide, wherein the heterologous polypeptide is transported to a location allowing interaction with the extracellular region of the heterologous receptor expressed in the cell membrane; and

(c) a selectable marker that is activated by the pheromone system pathway;

wherein collectively the mixture of cells expresses a library of said heterologous polypeptides, said library being expressible at a sufficient level such that modulation of the signal transduction activity of the heterologous receptor by a heterologous polypeptide within the library provides a detectable signal mediated by the selectable marker; and

wherein said yeast cells have a mutation in at least one gene selected from the group consisting of FAR1, SST2, BAR1, SVG1, STE2, STE3, MFa1, MFa2, MFα

1 and MFα

2.

95. The yeast cells of claim 94, wherein the selectable marker comprises a pheromone-responsive promoter operably linked to a selectable gene.

96. The yeast cells of claim 94, wherein the heterologous polypeptide includes a signal sequence that facilitates transport of the polypeptide to a location allowing interaction with the extracellular region of the receptor.

97. A recombinant yeast cell comprising a heterologous FPRL-1 receptor, which receptor is expressed in a cell membrane of said yeast cell such that signal transduction activity via said heterologous receptor is modulated by interaction of an extracellular region of the heterologous receptor with an extracellular signal, said heterologous receptor acting as a surrogate for an endogenous yeast pheromone receptor in a pheromone system pathway of the yeast cell, wherein modulation of the signal transduction activity of the heterologous receptor by an extracellular signal provides a detectable signal.

98. The yeast cell of claim 97, which further comprises a heterologous polypeptide, wherein the heterologous polypeptide is transported to a location allowing interaction with the extracellular region of the FPRL-1 receptor expressed in the cell membrane and wherein modulation of the signal transduction activity of the FPRL-1 receptor by the heterologous polypeptide provides a detectable signal.

99. The yeast cell of claim 98, wherein the heterologous polypeptide is an agonist of the FPRL-1 receptor.

100. The yeast cell of claim 99, which further comprises a second heterologous polypeptide, said second heterologous polypeptide also being transported to a location allowing interaction with the extracellular region of the FPRL-1 receptor expressed in the cell membrane.

101. A recombinant yeast cell comprising a heterologous C5a receptor, which receptor is expressed in a cell membrane of said yeast cell such that signal transduction activity via said heterologous receptor is modulated by interaction of an extracellular region of the heterologous receptor with an extracellular signal, said heterologous receptor acting as a surrogate for an endogenous yeast pheromone receptor in a pheromone system pathway of the yeast cell, wherein modulation of the signal transduction activity of the heterologous receptor by an extracellular signal provides a detectable signal.

102. The yeast cell of claim 101, which further comprises a heterologous polypeptide, wherein the heterologous polypeptide is transported to a location allowing interaction with the extracellular region of the C5a receptor expressed in the cell membrane and wherein modulation of the signal transduction activity of the C5a receptor by the heterologous polypeptide provides a detectable signal.

103. The yeast cell of claim 102, wherein the heterologous polypeptide is an agonist of the C5a receptor.

104. The yeast cell of claim 103, which further comprises a second heterologous polypeptide, said second heterologous polypeptide also being transported to a location allowing interaction with the extracellular region of the C5a receptor expressed in the cell membrane.

105. A recombinant yeast cell comprising a heterologous thrombin receptor, which receptor is expressed in a cell membrane of said yeast cell such that signal transduction activity via said heterologous receptor is modulated by interaction of an extracellular region of the heterologous receptor with an extracellular signal, said heterologous receptor acting as a surrogate for an endogenous yeast pheromone receptor in a pheromone system pathway of the yeast cell, wherein modulation of the signal transduction activity of the heterologous receptor by an extracellular signal provides a detectable signal.

106. The yeast cell of claim 105, which further comprises a heterologous polypeptide, wherein the heterologous polypeptide is transported to a location allowing interaction with the extracellular region of the thrombin receptor expressed in the cell membrane and wherein modulation of the signal transduction activity of the thrombin receptor by the heterologous polypeptide provides a detectable signal.

107. The yeast cell of claim 106, wherein the heterologous polypeptide is an agonist of the thrombin receptor.

108. The yeast cell of claim 107, which further comprises a second heterologous polypeptide, said second heterologous polypeptide also being transported to a location allowing interaction with the extracellular region of the thrombin receptor expressed in the cell membrane.