Biology subjects

McLoughlin, S. M.

Publications and source records attributed to McLoughlin, S. M..

3 recordsLinked to original sources

bioRxiv2026

Native Mass Spectrometry Reveals Distinct Effects of Linker Length and Targeting-Ligand Identity on PROTAC Ternary Complexes

Proteolysis-targeting chimeras (PROTACs) promote targeted protein degradation by inducing a transient ternary complex between a protein of interest and an E3 ubiquitin ligase. Despite the clinical promise of these event-driven therapeutics, defining the conformational landscape and interfacial organization of these complexes remains challenging. Here, we use an integrated native mass spectrometry (nMS) approach combining ion mobility, surface-induced dissociation (SID), and variable-temperature electrospray ionization (vT-ESI) to determine how small-molecule architecture shapes the structural and thermodynamic landscapes of bromodomain-containing protein 4 (BRD4) with the VCB E3 ligase complex. Using a set of PROTACs that differ in targeting ligand identity (JQ1 versus I-BET726) and PEG linker length, ion mobility measurements show that shorter linkers constrain the ternary complex into more extended topologies, whereas longer, more flexible linkers allow dynamic sampling of relaxed, native-like conformations. SID directly probes noncovalent interfaces and shows that JQ1 complexes favor prompt PROTAC ejection, whereas tighter-binding I-BET726 complexes are more likely to retain the PROTAC at the interface. Complementing these kinetically-controlled gas-phase measurements, vT-ESI reveals a monophasic melting transition for a JQ1-mediated complex and a distinct biphasic melting profile for an I-BET726-mediated complex. The gas-phase activation measurements revealed targeting-ligand-driven differences in PROTAC ejection and retention, whereas vT-ESI uncovered distinct temperature-dependent disassembly pathways for the matched-linker-length JQ10L and IBET10L complexes. Together, these complementary techniques show how PROTAC architecture influences ternary-complex conformation, interfacial connectivity, and disassembly.

biochemistry↗

bioRxiv2025

Proteome-Wide Discovery of Degradable Proteins Using Bifunctional Molecules

Targeted protein degradation (TPD) is an emergent therapeutic strategy with the potential to circumvent challenges associated with targets unamenable to conventional pharmacological inhibition. Among TPD approaches, Proteolysis Targeting Chimeras (PROTACs) have shown marked advancement with numerous candidates in clinical development. Despite their potential, most PROTACs utilize advanced small molecule inhibitors, inherently limiting the scope of this approach. More generally, the fraction of the proteome tractable to PROTAC-type strategies is unknown. Here, we describe a chemical proteomic strategy for the agnostic discovery of degradable human proteins in cells using a new class of bifunctional degrader molecules called "AgnoTACs". Proteome-wide screening of 72 AgnoTACs in human cells uncovered downregulation events spanning >50 functionally and structurally diverse proteins, most of which lack chemical probes. Our findings highlight the potential of function-biased chemical libraries coupled with proteomic profiling to discover degrader starting points as well as furnish a blueprint for expanding our understanding of the chemically degradable proteome.

biochemistry↗

bioRxiv2023

Metabolite Fingerprinting for Phenotypic Screening by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry

The adoption of mass spectrometry for high-throughput screening in drug discovery has become increasingly prevalent and has enabled label-free screening against diverse targets. Cellular assays for phenotypic screening, however, are primarily conducted by microscopy as there remain many challenges associated with conducting phenotypic screens via ultra-high throughput mass spectrometry. Following a simple on-plate extraction, infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) was employed to directly sample the cell lysate at a speed of one second per sample. A549 cells were treated with compounds identified as hits in literature including a recently reported glutaminase cellular screen. Among the test compounds, there were confirmed glutaminase inhibitors, proposed nuisance compounds, and cell-active but enzyme-inactive compounds. Filtered data were further processed in R for dimensionality reduction and unsupervised clustering. Though it was observed that all compounds affected the intracellular conversion of glutamine to glutamate, there were clear metabolic differences between the biochemically active compounds and the off-target hits. Moreover, two nuisance compounds were observed to cluster separately from the confirmed glutaminase inhibitors in the observed metabolite fingerprints. This proof-of-concept work establishes a complete workflow that enables highthroughput mass spectrometry-based phenotypic screening. The methods proposed herein, at the throughput enabled by IR-MALDESI, could offer a new avenue for discovery of novel drugs.

cell biology↗