METHODS FOR IDENTIFYING MODULATORS OF RAS USING NONLINEAR TECHNIQUES

• US 20150051110A1
• Filed: 11/02/2012
• Published: 02/19/2015
• Est. Priority Date: 02/05/2012
• Status: Active Grant

• Current Assignee: Quanta Therapeutics, Inc.
• Original Assignee: Biodesy, Inc.
• Inventors: Salafsky, Joshua S., McGuinness, Ryan P.
• Granted Patent: US 9,938,560 B2
• Time in Patent Office: Days
• US Class Current: 506/11
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: G01N 2333/82 Translation products from oncogenes
• CPC Class Codes: G01N 2333/916 acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• CPC Class Codes: G01N 2440/00 Post-translational modifications [PTMs] in chemical analysis of biological material
• CPC Class Codes: G01N 2458/00 Labels used in chemical analysis of biological material

Abstract

Provided herein are compositions and methods for identifying and detecting modulators of Ras protein conformational states through the use of second harmonic generation (SHG) technology. Also provided herein are methods for detecting a conformational changes in the three dimensional structure of a protein bound to a supported lipid bilayer.

Claims

1-126. -126. (canceled)

127. A method for identifying an agent that induces a conformational change of a target protein, the method comprising:

a. contacting the target protein with the agent, wherein the target protein is bound to a binding partner, and wherein the binding partner is labeled with a nonlinear active moiety that has a net orientation at an interface; and

b. detecting a conformational change in the structure of the target protein induced when the agent binds to the target protein by analyzing a detectable signal generated by the nonlinear active moiety using a surface selective technique.

128. The method of claim 127, wherein the nonlinear active moiety is a second harmonic-active label.

129. The method of claim 127, wherein the binding partner is GDP, GDP analog, GTP or GTP analog.

130. The method of claim 127, wherein the binding partner is a second harmonic generation (SHG)-active GTP analog.

131. The method of claim 127, wherein the target protein is a G protein-coupled receptor (GPCR).

132. The method of claim 131, wherein the binding partner is a known ligand that binds the GPCR.

133. The method of claim 127, wherein the target protein is a wild type or mutant Ras protein.

134. The method of claim 127, wherein the interface is selected from the group consisting of a glass surface, a polyethylene glycol surface, a supported lipid bilayer surface, a lipid analog bilayer surface, a plastic surface, a metal surface, a latex surface, a rubber surface, a ceramic surface, a polymeric surface, a polypropylene surface, a polyvinylidene difluoride surface, and a polyethylene surface.

135. The method of claim 134, wherein the interface is derivatized with oligo-PEG molecules or lipids.

136. The method of claim 135, wherein the oligo-PEG molecules or lipids are Ni-NTA-bearing oligo-PEG molecules or Ni-NTA-bearing lipids.

137. The method of claim 135, wherein the interface is a supported lipid bilayer or a lipid analog bilayer.

138. The method of claim 127, wherein the target protein comprises an affinity tag.

139. The method of claim 127, wherein the agent is a small molecule chemical compound, an antibody, a non-antibody polypeptide, a carbohydrate, an inhibitory nucleic acid, or any combination thereof.

140. A method for identifying an agent comprising:

a. contacting a wild type target protein with a binding partner that binds to a site of the wild type target protein, wherein the wild type target protein is labeled with a second harmonic-active label having a net orientation at an interface;

b. contacting a mutant target protein with the agent, wherein the mutant target protein is labeled with a second harmonic-active label having a net orientation at an interface, and

c. comparing a detectable signal generated by the second harmonic-active label of the wild type target protein due to contacting in step (a) with a detectable signal generated by the second harmonic-active label of the mutant target protein due to contacting in step (b).

141. The method of claim 140, wherein the mutant target protein has an activity different from the wild type target protein bound to the binding partner.

142. The method of claim 140, wherein the binding partner stimulates the activity of the wild type target protein.

143. The method of claim 140, wherein the agent modulates an activity of the mutant target protein.

144. The method of claim 140, wherein the agent modulates a conformational structure of the mutant target protein.

145. The method of claim 140, wherein the binding partner stabilizes the conformation of the wild type target protein.

146. The method of claim 140, wherein the detectable signal generated in step (a) indicates a conformational change in the structure of the wild type target protein.

147. The method of claim 140, wherein the detectable signal generated in step (b) indicates a conformational change in the structure of the mutant target protein.

148. The method of claim 140, wherein the binding partner is GDP, GDP analog, GTP or GTP analog.

149. The method of claim 140, wherein the second harmonic-active label is PyMPO-MALEIMIDE™

(1-(2-Maleimidylethyl)-4-(5-(4-Methoxyphenyl)Oxazol-2-yl) Pyridinium Methanesulfonate).

150. The method of claim 140, wherein the second harmonic-active label is PyMPO-succinimidyl ester.

151. The method of claim 140, wherein the second harmonic-active label is bound to the wild type or mutant target protein by one or more amine groups on the surface of the target protein.

152. The method of claim 151, wherein said one or more amine groups are native amine groups.

153. The method of claim 151, wherein said one or more amine groups are engineered amine groups.

154. The method of claim 140, wherein the second harmonic-active label is an unnatural amino acid.

155. The method of claim 154, wherein the unnatural amino acid is Aladan.

156. The method of claim 140, wherein the second harmonic-active label is bound to the wild type or mutant target protein by one or more sulfhydryl groups on the surface of the target protein.

157. The method of claim 156, wherein said one or more sulfhydryl groups are native sulfhydryl groups.

158. The method of claim 156, wherein said one or more sulfhydryl groups are engineered sulfhydryl groups.

159. The method of claim 140, wherein the agent stabilizes the structure of the mutant target protein into an active conformation.

160. The method of claim 140, wherein the agent stabilizes the structure of the mutant target protein into an inactive conformation.

161. The method of claim 140, wherein the agent is a small molecule chemical compound, an antibody, a non-antibody polypeptide, a carbohydrate, an inhibitory nucleic acid, or any combination thereof.

162. The method of claim 140, wherein the interface is a supported lipid bilayer or a lipid analog bilayer.

163. A method for determining a structure change of a protein, the method comprising:

a) inducing a conformational change upon contacting of a binding partner to the protein labeled with a second harmonic-active label;

b) determining the induced conformational change,

wherein the structure change is within a root mean square standard deviation (RMSD) of about 0.5-2 Å

.

164. The method of claim 163, wherein the conformational change is in a three dimensional structure of the protein.

165. The method of claim 163, wherein the conformational change is in a specific domain of the protein.

166. The method of claim 163, wherein the conformational change is in an α

-helical or β

-sheet secondary structure of the protein.

167. The method of claim 163, wherein the protein is in a lipid bilayer.

168. The method of claim 167, wherein the lipid bilayer comprises phospholipids.

169. The method of claim 167, wherein the lipid bilayer further comprises a membrane protein.

170. The method of claim 163, wherein the protein is in a biological cell or liposome.

171. The method of claim 163, wherein the second harmonic-active label has a net orientation at an interface.

172. The method of claim 163, wherein b) further comprises detecting light emitted from the interface using a surface selective technique.