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US 2021/0356467 A1Published application
Systems and method for screening small molecules of interest
AssigneeTechnology Innovation Momentum Fund (Israel) Limited Partnership
InventorGali PRAG
Life of this patent
Estimating the term…
Abstract
Bacterial systems for screening for small molecule agents are disclosed herein. Kits for carrying out the screening are also disclosed.
Claims35
A method of identifying an agent which mediates the binding of a first polypeptide to a second polypeptide comprising:
- (a) contacting a bacterial cell which expresses said first polypeptide and said second polypeptide with the agent, wherein the bacterial cell outputs a detectable or selectable signal which correlates with the non-direct binding of the first polypeptide to said second polypeptide via said agent, wherein said non-direct binding of said first polypeptide to said second polypeptide via said agent brings about the ubiquitination of said first polypeptide or said second polypeptide;
- and
- (b) measuring the level or the rate of accumulation of said detectable or selectable signal, wherein a change in the level as compared to the level in the absence of said agent, is indicative of an agent which mediates the binding of said first polypeptide to said second polypeptide.
9-10. (canceled)
16-20. (canceled)
A method of identifying an agent which mediates the binding of a first polypeptide to a second polypeptide comprising:
- (a) contacting a bacterial cell with the agent, wherein the bacterial cell outputs a detectable or selectable signal which correlates with the non-direct binding of the first polypeptide to said second polypeptide via said agent;
- and
- (b) measuring the level or the rate of accumulation of said detectable or selectable signal, wherein a change in the level as compared to the level in the absence of said agent, is indicative of an agent which mediates the binding of the first polypeptide to said second polypeptide, wherein:
- (i) the first polypeptide is a substrate for ubiquitination;
- (ii) said second polypeptide is attached to ubiquitin;
- (iii) said bacterial cell expresses a ubiquitinating enzyme which attaches said ubiquitin to said first polypeptide if said agent mediates the binding of said first polypeptide to said second polypeptide.
3 more elements
23-24. (canceled)
29-35. (canceled)
A kit comprising:
- (i) a first polynucleotide which encodes a first polypeptide fragment which is operably linked to a bacterial regulatory sequence, and a first cloning site, wherein a position of said first cloning site is selected such that upon insertion of a sequence which encodes a first test polypeptide into said first cloning site, following expression in a bacterial cell, a fusion protein is generated which comprises said first test polypeptide in frame with said first polypeptide fragment;
- and
- (ii) a second polynucleotide comprising a second nucleic acid sequence encoding a second polypeptide fragment which is attached to ubiquitin, the second nucleic acid sequence being operably linked to a bacterial regulatory sequence, the second nucleic acid sequence comprising a second cloning site, wherein a position of said second cloning site is selected such that upon insertion of a sequence which encodes a second test polypeptide into said second cloning site, following expression in a bacterial cell, a fusion protein is generated which comprises said second test polypeptide in frame with said second polypeptide fragment;
- wherein said first polypeptide fragment associates with said second polypeptide fragment to generate a reporter polypeptide dependent on ubiquitination of said first test polypeptide.
1 more element
A method of identifying an agent which mediates the binding of a ubiquitin E3 ligase to a test polypeptide comprising:
- (a) contacting a bacterial cell which expresses said E3 ligase with the agent, wherein the bacterial cell outputs a detectable or selectable signal which correlates with the non-direct binding of the E3 ligase to said test polypeptide via said agent;
- and
- (b) measuring the level or the rate of accumulation of said detectable or selectable signal, wherein a change in the level as compared to the level in the absence of said agent, is indicative of an agent which mediates the binding of the E3 ligase to the test polypeptide.
(canceled)
(canceled)
43-50. (canceled)
A kit comprising:
- (i) a first polynucleotide which encodes a first polypeptide fragment which is operably linked to a bacterial regulatory sequence, and a cloning site, wherein a position of said cloning site is selected such that upon insertion of a sequence which encodes a test polypeptide into said cloning site, following expression in a bacterial cell, a fusion protein is generated which comprises said test polypeptide in frame with said first polypeptide fragment;
- (ii) a second polynucleotide comprising a second nucleic acid sequence encoding a second polypeptide fragment which is attached to ubiquitin, the second nucleic acid sequence being operably linked to a bacterial regulatory sequence, wherein said first polypeptide fragment associates with said second polypeptide fragment via a mediating agent to generate a reporter polypeptide dependent on ubiquitination of said test polypeptide;
- and
- (iii) said mediating agent.
1 more element
A method of identifying an agent which mediates the binding of an E2 conjugating enzyme to an E3 ligase comprising:
- (a) contacting a bacterial cell which expresses said E2 conjugating enzyme and said E3 ligase with the agent, wherein the bacterial cell outputs a detectable or selectable signal which correlates with the non-direct binding of the E2 conjugating enzyme to said E3 ligase via said agent, wherein said non-direct binding of said E2 conjugating enzyme to said E3 ligase via said agent brings about the ubiquitination of a protein of interest;
- and
- (b) measuring the level or the rate of accumulation of said detectable or selectable signal, wherein a change in the level as compared to the level in the absence of said agent, is indicative of an agent which mediates the binding of said E2 conjugating enzyme to said E3 ligase.
Dependencies are inferred from the claim text.