2026-09-20T19:46:17Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/1501782023-04-01T03:31:48Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022
Koehler, Angela N.
Henry, Catherine Campbell
Massachusetts Institute of Technology. Department of Biological Engineering
2023-03-31T14:37:49Z
2023-03-31T14:37:49Z
2023-02
2023-03-22T16:44:13.836Z
https://hdl.handle.net/1721.1/150178
Target identification is a major challenge in probe and drug discovery. Current binding assays are unable to detect interactions between unoptimized probes and difficult targets, such as transcription factors. Here, we developed generalizable, high-throughput platforms that can rapidly identify the mechanism(s) of action of small molecules emerging from high-throughput screening (HTS) campaigns. Specifically, this project established a solid phase method to rapidly modify small molecules with moieties of interest using isocyanate-based chemistries. This chemical method can be used to quickly generate photoaffinity labeling analogs of small molecules that can be used in a covalent ELISA and mass spectrometry workflow to determine whether small molecules bind to a target of interest and identify off-target binders. Additionally, we created a synergistic critical path for assessing the mechanism of action and on-target activity of small molecules through the generation of an on-target transcriptional profile and application of the L1000 gene-expression platform. Together, these workflows and chemical tools will enable high-throughput studies of small molecule-protein interactions with a wide range of affinities and abundances and facilitate prioritization of small molecules that bind and modulate the function of difficult targets.
Ph.D.
Massachusetts Institute of Technology
In Copyright - Educational Use Permitted
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http://rightsstatements.org/page/InC-EDU/1.0/
Toward High-throughput, Quantitative Platforms to Identify the Targets of Small Molecules
Thesis
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Doctoral
Doctor of Philosophy
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<Abstract>Target identification is a major challenge in probe and drug discovery. Current binding assays are unable to detect interactions between unoptimized probes and difficult targets, such as transcription factors. Here, we developed generalizable, high-throughput platforms that can rapidly identify the mechanism(s) of action of small molecules emerging from high-throughput screening (HTS) campaigns. Specifically, this project established a solid phase method to rapidly modify small molecules with moieties of interest using isocyanate-based chemistries. This chemical method can be used to quickly generate photoaffinity labeling analogs of small molecules that can be used in a covalent ELISA and mass spectrometry workflow to determine whether small molecules bind to a target of interest and identify off-target binders. Additionally, we created a synergistic critical path for assessing the mechanism of action and on-target activity of small molecules through the generation of an on-target transcriptional profile and application of the L1000 gene-expression platform. Together, these workflows and chemical tools will enable high-throughput studies of small molecule-protein interactions with a wide range of affinities and abundances and facilitate prioritization of small molecules that bind and modulate the function of difficult targets.</Abstract>
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