Particle based small molecule-protein complex trap
AssigneeOrionis Biosciences BV
InventorsNikolai KLEY, Samuel LIEVENS, Riccardo SABATINI
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Abstract
The present invention relates to the use of a particle, including a virus-like particle (VLP), for the discovery and analysis of protein-protein interactions that are modulated by small molecules.
Claims27
A method for detecting an interaction between a small molecule/protein complex and another protein, comprising:
- (i) expressing a construct comprising one or more particle-forming (or particle-associated) polypeptides fused to a first interaction polypeptide in a cell;
- (ii) incubating the cell or particle with one or more small molecules;
- (iii) allowing the first interaction polypeptide to interact with the small molecule and form a small molecule/protein complex;
- (iv) allowing a second interaction polypeptide to form a complex with the pre-formed small molecule/protein complex;
- (v) isolating the particle;
- and
- (vi) analyzing the small molecule/protein-protein complex.
A method for detecting a small molecule inhibition or reduction of a protein-protein interaction, comprising:
- (i) expressing a construct comprising one or more particle-forming (or particle-associated) polypeptide fused to a first interaction polypeptide in a cell;
- (ii) allowing the first interaction polypeptide to interact with a second interaction polypeptide present in the cell;
- (iii) incubating the cell or particle with one or more small molecules;
- (iv) allowing the first interaction polypeptide to interact with the small molecule;
- (v) allowing the second interaction polypeptide to dissociate from the first interaction polypeptide;
- (vi) isolating the particle;
- and
- (vii) analyzing the protein-protein complex.
A method for detecting a protein-protein interaction, comprising:
- (i) expressing a construct comprising one or more particle-forming (or particle-associated) polypeptide fused to a second interaction polypeptide in a cell;
- (ii) incubating the cell or particle with one or more small molecules;
- (iii) allowing the small molecule to interact with a first interaction polypeptide and form a complex;
- (iv) allowing the second interaction polypeptide to form a complex with the pre-formed complex comprising the first interaction polypeptide and the small molecule;
- (v) isolating the particle;
- and
- (vi) analyzing the protein-protein complex.
A method for detecting a protein-protein interaction, comprising:
- (i) expressing a construct comprising one or more particle-forming (or particle-associated) polypeptide fused to a second interaction polypeptide in a cell;
- (ii) allowing a first interaction polypeptide to interact with the second interaction polypeptide present in the cell;
- (iii) incubating the cell or particle with one or more small molecules;
- (iv) allowing the first interaction polypeptide to interact with the small molecule;
- (v) allowing the second interaction polypeptide to dissociate from the first interaction polypeptide;
- (vi) isolating the particle;
- and
- (vii) quantifying a protein-protein complex.
The method of any one of the above claims, wherein the particle is a virus-like particle (VLP).
The method of any one of the above claims, wherein the particle forming polypeptide comprises a p55 GAG protein or a variant, derivative, or fragment thereof.
The method of any one of the above claims, wherein the particle further comprises the spike glycoprotein of the vesicular stomatitis virus (VSV-G).
The method of any one of the above claims, wherein the particle lacks the spike glycoprotein of the vesicular stomatitis virus (VSV-G).
The method of any one of the above claims, wherein the construct further comprises one or more linkers.
The method of any of the above claims, wherein the isolating is carried out by affinity chromatography, centrifugation, or any tag-based method.
The method of any of the above claims, wherein the analyzing is carried out by mass spectrometry.
The method of any of the above claims, wherein the first and/or second interaction polypeptide is a protein, cDNA, and/or open reading frame (ORF) library.
The method of any of the above claims, wherein the small molecule is a small molecule library.
The method of any of the above claims, wherein the small molecule is not coupled to another moiety, including another small molecule, a purification handle, a bead, and the like.
The method of any of the above claims, wherein the small molecule does not interact with the second interaction polypeptide.
The method of any of the above claims, wherein the first interaction polypeptide is an E3 ligase substrate binding subunit.
Dependencies are inferred from the claim text.