Biology subjects

McGregor, L. M.

Publications and source records attributed to McGregor, L. M..

7 recordsLinked to original sources

bioRxiv2025

Expanding the repertoire of chemically induced covalent neoantigens

The ubiquitin-proteasome system generates peptide fragments that are displayed on MHC-I molecules. Recent studies revealed that covalent small molecules can hijack this process, leading to the presentation of hapten-modified peptide fragments, or covalent neoantigens. Here, we report a chemical proteomics platform to systematically investigate this process and harness covalent neoantigens for immune cell recruitment and activation. Using a bioorthogonal cysteine-reactive probe (IA-mTz), we demonstrate that diverse intracellular proteins can be modified, processed, and presented as covalent neoantigens across multiple cell lines. Mass spectrometry confirmed the presence of IA-mTz-modified peptides within the MHC-I immunopeptidome, and structural modeling revealed stable peptide-MHC-I interactions. We further integrated a bioorthogonal strategy that enables immune engagement: IA-mTz-modified cells conjugated with trans-cyclooctene (TCO)-IgG elicited Fc receptor activation in a reporter assay, supporting immune recruitment via antibody-dependent cellular phagocytosis. This approach was extended to additional cysteine-reactive probes and to 2,4-dinitrochlorobenzene (DNCB), a common skin sensitizer, which we show induces covalent neoantigen formation in a cell- and HLA-dependent manner. These findings reveal a previously underappreciated breadth of covalent neoantigen formation and provide a generalizable strategy for immune targeting of covalent neoantigens.

biochemistry↗

bioRxiv2024

Photoaffinity enabled transcriptome-wide identification of splice modulating small molecule-RNA binding events in native cells

Splice modulating small molecules have been developed to promote the U1 snRNP to engage with pre-mRNAs with strong and altered sequence preference. Transcriptomic profiling of bulk RNA from compound treated cells enables detection of RNAs impacted; however, it is difficult to delineate whether transcriptional changes are a consequence of direct compound treatment or trans-acting effects. To identify RNA targets that bind directly with splice modulating compounds, we deployed a photoaffinity labeling (PAL)-based Chem-CLIP approach. Through this workflow, we identify the telomerase lncRNA (TERC) as a previously unknown target of this class of clinically relevant small molecules. Using cellular {Delta} SHAPE-MaP, we orthogonally validate and further define the compound binding site as likely to be the conserved CR4/5 domain. Additionally, a thorough analysis of the PAL-based Chem-CLIP data reveals that considering competed RNAs, irrespective of magnitude of enrichment, adds a rich dimension of hit calling. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/623654v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1ef467eorg.highwire.dtl.DTLVardef@1fd201dorg.highwire.dtl.DTLVardef@1478f83org.highwire.dtl.DTLVardef@11ef59a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Rational Chemical Design of Molecular Glue Degraders

Targeted protein degradation with molecular glue degraders has arisen as a powerful therapeutic modality for eliminating classically undruggable disease-causing proteins through proteasome-mediated degradation. However, we currently lack rational chemical design principles for converting protein-targeting ligands into molecular glue degraders. To overcome this challenge, we sought to identify a transposable chemical handle that would convert protein-targeting ligands into molecular degraders of their corresponding targets. Using the CDK4/6 inhibitor Ribociclib as a prototype, we identified a covalent handle that, when appended to the exit vector of Ribociclib, induced the proteasome-mediated degradation of CDK4 in cancer cells. Covalent chemoproteomic profiling of this CDK4 degrader revealed covalent interactions with cysteine 32 of the RING family E3 ubiquitin ligase RNF126. Structural modification of our initial covalent scaffold led to an improved CDK4 degrader with the development of a but-2-ene, 1,4-dione ("fumarate") handle that showed improved interactions with RNF126. Thereafter, we worked to identify the minimum covalent motif required for interaction with RNF126, which we then transplanted onto chemically related and un-related protein-targeting ligands. This strategy successfully produced molecules which induced the degradation of several proteins across diverse protein classes, including BRD4, BCR-ABL and c-ABL, PDE5, AR and AR-V7, BTK, LRRK2, and SMARCA2. Our study undercovers a design strategy for converting protein-targeting ligands into covalent molecular glue degraders.

biochemistry↗

bioRxiv2021

Deubiquitinase-Targeting Chimeras for Targeted Protein Stabilization

Targeted protein degradation is a powerful therapeutic modality that uses heterobifunctional small-molecules to induce proximity between E3 ubiquitin ligases and target proteins to ubiquitinate and degrade specific proteins of interest. However, many proteins are ubiquitinated and degraded to drive disease pathology; in these cases targeted protein stabilization (TPS), rather than degradation, of the actively degraded target using a small-molecule would be therapeutically beneficial. Here, we present the Deubiquitinase-Targeting Chimera (DUBTAC) platform for TPS of specific proteins. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48 ubiquitin-specific deubiquitinase OTUB1. We then developed a heterobifunctional DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds {Delta}F508-CFTR. We demonstrated proof-of-concept of TPS by showing that this DUBTAC robustly stabilized {Delta}F508-CFTR in human cystic fibrosis bronchial epithelial cells in an OTUB1-dependent manner. Our study underscores the utility of chemoproteomics-enabled covalent ligand discovery approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS. Editorial summaryWe have developed the Deubiquitinase Targeting Chimera (DUBTAC) platform for targeted protein stabilization. We have discovered a covalent recruiter against the deubiquitinase OTUB1 that we have linked to the mutant {Delta}F508-CFTR targeting cystic fibrosis drug Lumacaftor to stabilize mutant CFTR protein in cells.

biochemistry↗

bioRxiv2020

A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes

Targeted protein degradation is a rapidly developing therapeutic modality that promises lower dosing and enhanced selectivity as compared to traditional occupancy-driven inhibitors, and the potential to modulate historically intractable targets. While the well-characterized E3 ligases CRBN and VHL have been successfully redirected to degrade numerous proteins, there are approximately 600 predicted additional E3 family members that may offer improved activity, substrate selectivity, and/or tissue distribution; however, characterizing the potential applications of these many ligases for targeted protein degradation has proven challenging. Here, we report the development of an approach to evaluate the ability of recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method by electroporating recombinant VHL, covalently functionalized with JQ1 or dasatinib, to induce degradation of BRD4 or kinase targets, respectively. Furthermore, by applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase that has not previously been redirected for targeted protein degradation, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligases against neo-substrates.

molecular biology↗

bioRxiv2020

A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific protein targets in a proteasome-dependent manner. However, a major limitation to broader TPD applications is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent ligand for the E3 ligase RNF114. When linked to the BET family inhibitor JQ1, the resulting heterobifunctional PROTAC molecule was capable of selectively degrading BRD4 in cancer cells. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or in some cases inactivity. Further contrasting from cereblon or VHL-recruiting degradation, we show that BT1 treatment not only leads to BCR-ABL degradation, but also stabilizes the endogenous RNF114 substrate and tumor suppressor substrate p21. This leads to additional anti-proliferative effects in leukemia cancer cells beyond those observed with cereblon or VHL-recruiting BCR-ABL PROTACs. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs with unique additional benefits for oncology applications. We also further demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing and unpredictable selectivity for the degradation of neo-substrate proteins.

biochemistry↗

bioRxiv2019

Manumycin Polyketides Act as Molecular Glues Between UBR7 and P53 to Impair Breast Cancer Pathogenicity

Molecular glues are an intriguing therapeutic modality that harness small-molecules to induce interactions between proteins that typically do not interact, thus enabling the creation of novel protein functions not naturally encoded in biology. While molecular glues such as thalidomide and rapamycin have catalyzed drug discovery efforts, such molecules are rare and have often been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention of disease. Historically, natural products have proven to be important sources of molecular glues and we postulated that natural products bearing multiple electrophilic sites may be an unexplored source of such molecules, potentially through multi-covalent attachment. Using activity-based protein profiling (ABPP)-based chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells to impair breast cancer pathogenicity through engaging in molecular glue interactions with the neo-substrate tumor-suppressor TP53, leading to the activation of p53 transcriptional activity and cell death. Our results reveal a previously undiscovered anti-cancer mechanism of this natural product family and highlight the potential for combining chemoproteomics and multi-covalent natural products for the discovery and characterization of new molecular glues.

biochemistry↗