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US 2011/0212476 A1Published application
Genetically engineered g-alpha proteins and uses thereof
AssigneeBellbrook Labs, LLC
InventorsRobert G. Lowery, Thomas K. Zielinski
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AssigneeBellbrook Labs, LLC
InventorsRobert G. Lowery, Thomas K. Zielinski
Estimating the term…
The present invention relates to novel engineered Ga proteins and assay methods of using such proteins to advance drug discovery. Engineered Ga proteins described by the invention contain alterations of at least one and preferably two or more amino acid residues that are highly conserved among all four subfamilies of Ga proteins. A preferred engineered protein disclosed here is a double mutant, Gαπ R178M A326S. This specific combination of mutations yields an unexpectedly amplified effect on Ga function both in terms of GTPase activity (GTP hydrolysis) and GDP dissociation. This synergistic effect may have a profound influence on the way GPCR signaling pathways are examined for the development of new pharmacotherapies, particularly in the field of central nervous system disorders such as Parkinson's disease.
An engineered protein comprising a Gα protein differing in amino acid sequence from a reference native Gα protein, wherein the difference comprises at least two amino acid substitutions, wherein the substitutions have a net effect of an increase in the GDP dissociation rate and a decrease in the GTP hydrolysis rate, so that the rate of GDP dissociation is no longer rate limiting for catalysis relative to a Gα protein without said amino acid substitutions.
An engineered Gα protein differing in amino acid sequence from a reference native Gα protein, wherein the difference consists of two amino acid substitutions, wherein the first substitution is at (i) the conserved Arginine located in the native Gα protein at any one of amino acid positions ranging from about 178 to about 205, and the second substitution is at (ii) the conserved Alanine located in the native Gα protein at any one of amino acid positions ranging from about 326 and about 367, wherein both substitution result in a net effect of an increase in the GDP dissociation rate and a decrease in the GTP hydrolysis rate, so that the rate of GDP dissociation is no longer rate limiting for catalysis relative to a Gα protein without said amino acid substitutions.
A method of using an engineered Gα protein, wherein the method comprises incubating the engineered Gα protein in the presence or absence of a protein containing an RGS domain, wherein the engineered Gα protein differs in amino acid sequence from a reference native Gα protein in at least two amino acid substitutions, wherein the substitutions have a net effect of an increase in the GDP dissociation rate and a decrease in the GTP hydrolysis rate, so that the rate of GDP dissociation is no longer rate limiting for catalysis relative to a Gα protein without said amino acid substitutions.
Dependencies are inferred from the claim text.