6 research outputs found
PhotoAffinity bits : a photoaffinity-based fragment screening platform for efficient identification of protein ligands
Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space, however, it remains challenging to develop techniques that are both sufficiently high-throughput and sensitive. We present a fragment screening platform, termed PhABits (PhotoAffinity Bits), which utilises a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. We envision that the PhABits will be widely applicable to novel protein targets, identifying starting points in the development of therapeutic
A photoaffinity displacement assay and probes to study the cyclinâdependent kinase family
The CDK family plays a crucial role in the control of the cell cycle. Dysregulation and mutation of the CDKs has been implicated in cancer and the CDKs have been investigated extensively as potential therapeutic targets. Selective inhibition of specific isoforms of the CDKs is crucial to achieve therapeutic effect while minimising toxicity. We present a group of photoaffinity probes designed to bind to the family of CDKs. The site of crosslinking of the optimised probe, as well as its ability to enrich members of the CDK family from cell lysates, was investigated. In a proof of concept study, we subsequently developed a photoaffinity probeâbased competition assay to profile CDK inhibitors. We anticipate that this approach will be widely applicable to the study of small molecule binding to protein families of interest
One-step synthesis of photoaffinity probes for live-cell MS-based proteomics
We present a one-step Ugi reaction protocol for the expedient synthesis of photoaffinity probes for live-cell MS-based proteomics. The reaction couples an amine affinity function with commonly used photoreactive groups, and a variety of handle functionalities. Using this technology, a series of pan-BET (BET: bromodomain and extra-terminal domain) selective bromodomain photoaffinity probes were obtained by parallel synthesis. Studies on the effects of photoreactive group, linker length and irradiation wavelength on photocrosslinking efficiency provide valuable insights into photoaffinity probe design. Optimal probes were progressed to MS-based proteomics to capture the BET family of proteins from live cells and reveal their potential on- and off-target profiles
PhotoAffinity Bits: A Photoaffinity-Based Fragment Screening Platform for Efficient Identification of Protein Ligands
Advances in genomic analyses enable the identification of
new proteins that are associated with disease. To validate these targets, tool
molecules are required to demonstrate that a ligand can have a
disease-modifying effect. Currently, as tools are reported for only a fraction
of the proteome, platforms for ligand discovery are essential to leverage
insights from genomic analyses. Fragment screening offers an efficient approach
to explore chemical space, however, it remains challenging to develop techniques
that are both sufficiently high-throughput and sensitive. We present a fragment
screening platform, termed PhABits (PhotoAffinity Bits), which utilises a
library of photoreactive fragments to covalently capture fragment-protein
interactions. Hits can be profiled to determine potency and site of
crosslinking, and subsequently developed as reporters in a competitive
displacement assay to identify novel hit matter. We envision that the PhABits
will be widely applicable to novel protein targets, identifying starting points
in the development of therapeutics.</p