Excited to share my postdoc work in Cravatt lab & Schreiber lab! We described photo-stereoprobes, DOS-inspired photo-reactive probes, for expanding the ligandable proteome. 🧵 https://t.co/Y8TgW2QG4T

#Technology Chemical tools to expand the ligandable proteome: Diversity-oriented synthesis-based photoreactive stereoprobes by @d_ogasa, @brumelillo, @bencravatt at @scrippsresearch @broadinstitute @harvardmed @MassGeneralNews https://t.co/NRD5Co8KEH

16/ This work wouldn’t have been possible without a fantastic contribution by co-authors as well as great mentorship by Ben Cravatt, @brumelillo, Ramnik Xavier, and @SchreiberStuart. Also grateful for the continuous support by @bmsnews for this project.

15/ The screening and subsequent integrated chemoproteomic analysis identified a novel autophagy-modulating compound (DBK-032A) and its biologically relevant molecular target (CLPP).

14/ We also demonstrated that phenotypic screening could provide a ‘function-first’ assay for photo-stereoprobes. We screened photo-stereoprobes for effects on autophagy using an LC3 puncta induction assay as an indicator of autophagosome formation.

13/ We demonstrated that the BRET tracer derived from photo-stereoprobes can monitor the competitive engagement of small molecule competitors to the target protein.

12/ Therefore, we developed a general workflow for converting stereoselective probe-protein interactions into high-throughput screening-compatible NanoBRET assays.

11/ While we succeeded in illuminating many novel ligandable proteins, we acknowledge that the majority of the identified photo-stereoprobe-protein interactions appeared to be low affinity.

10/ Importantly, such binding events would have been missed if the compound screening relied solely on in vitro and/or recombinant forms of protein, underscoring the value of performing small-molecule binding assays with endogenous proteins in their native cellular environments.

9/ For example, we found that the stereoselective engagement of SAE1 by DBK-032A was only recapitulated when this protein was co-expressed with its cognate binding partner SAE2.

8/ Some photo-stereoprobe-protein interactions may depend on an intact protein complex that is not properly assembled when one of the complex members is independently expressed.

7/ For example, we observed a robust stereoselective engagement of CLPP by DBK-032A in situ, but the interaction appears to be completely abolished in vitro for both endogenous and recombinant CLPP.

6/ It is noteworthy that we came across several cases where we observed stark differences in ligandability in situ and in vitro binding profiles.

5/ A subset of those identified interactions was further confirmed by gel-based profiling using recombinantly expressed target proteins. We observed the expected stereoselective probe-protein interactions for the majority of the analyzed proteins.

4/ They represent a wide range of functional classes, including those historically considered challenging to target with small molecules (e.g., transcriptional regulators and adaptors/scaffolding proteins). The majority of the target proteins lack available chemical probes.

3/ We analyzed probe-protein interactions that occur in a probe stereoisomer-specific manner, which is indicative of an authentic liganding event. MS-based profiling identified > 200 proteins that were stereoselectively engaged by the photo-stereoprobes in human cancer cells.

2/ We prepared a focused set of photo-stereoprobes consisting of four stereoisomers per core (4 stereoisomers × 3 cores = 12 photo-stereoprobes in total).

1/ Chemical proteomics offers a versatile strategy for performing small-molecule probe discovery directly in native biological systems. By integrating DOS-inspired scaffolds with photoreactive chemistry, we aimed to expand the scope of ligandable proteome.

Today we report in @nchembio a collaboration led by Haoxin Li in Ben Cravatt's lab to illuminate functional and druggable cysteines in the human proteome by integrated base editing and activity-based protein profiling. https://t.co/rlPei0Vck4 PDF: https://t.co/XxbjjP4LOp

New chemistry and chemical proteomic technology from our group on functional tyrosine profiling in proteomes and live cells. Congrats to Emmanuel, Hahm, Adam, and Jeff. Great job on all of your hard work! @nchembio