Biology subjects

Young, L. C.

Publications and source records attributed to Young, L. C..

4 recordsLinked to original sources

bioRxiv2025

Nutrient-limited conditions reveal the activity of a minor groove binder, MGB-BP-3, against Escherichia coli

BackgroundAntimicrobial resistance demands new antibiotics, but conventional screening in nutrient-rich media often fails to predict in vivo efficacy, especially against Gram-negative bacteria. Minor groove binders (MGB), such as MGB-BP-3, are a promising new antibacterial with potent activity against Gram-positive bacteria but thought to have limited potency against Gram-negative bacteria. ObjectivesThis study evaluated the antibacterial activity of MGB-BP-3 under nutrient-limited, physiologically relevant conditions to better mimic in vivo environments. MethodsMinimum inhibitory concentrations (MICs) were determined against a panel of ESKAPEE pathogens in both rich (Cation Adjusted Mueller-Hinton Broth, CA-MHB) and nutrient-limited media (Roswell Park Memorial Institute, RPMI-1640 + 10% Luria-Bertani, LB). Synergy with efflux pump inhibitors and membrane permeabilizers was evaluated via checkerboard assays. Permeability was assessed using a fluorescent DNA-binding cell permeant (Hoechst 33342) accumulation assay. MICs were determined in the presence of different concentrations of ions, using both wild-type and efflux-compromised strains. In vivo efficacy was assessed using the Galleria mellonella infection model. ResultsThe activity of MGB-BP-3 against Gram-negative bacteria was improved by shifting the medium from rich to nutrient-limited conditions. Specifically, it showed potent activity against E. coli in nutrient-limited media (MIC 3.1 {micro}M) but was inactive in rich media (MIC 700 {micro}M). Synergy with efflux inhibitors occurred mainly in rich media, indicating barriers to intracellular accumulation were not present in nutrient-limited conditions. Increased permeability correlated with enhanced susceptibility and efflux-deficient strains were more susceptible, confirming the role of efflux pumps in resistance. MGB-BP-3 provided dose-dependent protection in G. mellonella larvae infected with E. coli or S. aureus. For example, at concentrations of 12.5 {micro}M and 100 {micro}M, MGB-BP-3 was able to protect the larvae by more than 70% over 5 days of treatment, respectively. ConclusionsNutrient-limited media better reveal MGB-BP-3s activity against Gram-negative bacteria and align with in vivo efficacy, highlighting the need for host-mimicking conditions in antibiotic screening.

microbiology↗

bioRxiv2025

Modulation of the 14-3-3σ/C-RAF autoinhibited complexby molecular glues

Molecular glues, compounds that bind cooperatively at protein-protein interfaces are revolutionizing chemical biology and drug discovery, allowing the modulation of traditional "undruggable" targets. Here, we focus on the native protein-protein interaction (PPI) of C-RAF, a key component of the MAPK signaling pathway, with the scaffolding protein 14-3-3. Although extensive drug discovery efforts have focused on the MAPK pathway, its central role in oncology and developmental disorders (RASopathies), still requires alternative approaches, moving beyond direct kinase inhibition. Indeed, stabilization of native PPIs is a relatively unexplored territory in this pathway. The function of C-RAF is regulated on multiple levels including dimerization, phosphorylation and complex formation with the hub protein 14-3-3. 14-3-3 prevents C-RAF activation by molecular recognition and binding at the phospho-serine 259. We used a fragment-merging approach to design a molecular glue scaffold that would bind to the composite surface of the 14-3-3/C-RAF "auto"inhibited complex. The synthesized molecular glues stabilized the 14-3-3/C-RAF complex up to 300-fold in biophysical assays; their glue-based mechanism of action was confirmed with several crystal structures of ternary complexes. Selectivity among the other RAF isoforms and other RAF phosphorylation sites was evaluated with biophysical assays. The best compounds showed excellent selectivity among a broad panel of 80 14-3-3 clients. Validation in cell assays showed on-target engagement, enhanced phosphorylation levels of the C-RAF pS259 site, reduced RAF dimerization and reduced ERK phosphorylation. Overall, this approach enables chemical biology studies on a C-RAF site that is intrinsically disordered prior to 14-3-3 binding and has not been targeted previously. These molecular glues will be useful as chemical probes and starting points for further drug discovery efforts to elucidate the effect of native PPI stabilization in the MAPK pathway with applications in oncology and RASopathies.

biochemistry↗

bioRxiv2020

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

A vital first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we solved crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures revealed that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutation at the KRAS-CRD interface resulted in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

biophysics↗

bioRxiv2019

Role for Merlin/NF2 in transcription elongation through interaction with the PAF complex

The PAF complex (PAFC) coordinates transcription elongation and mRNA processing events and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions at the cell cortex and nucleus and is a key mediator of contact inhibition. Here we provide a direct link between nuclear Merlin and transcription elongation controlled by cell-cell adhesion. Merlin interacts with the PAFC in a cell density-dependent manner and tumour-derived inactivating mutations in both Merlin and CDC73 mutually disrupt their interaction. Growth suppression by Merlin requires CDC73 and Merlin regulates PAFC association with chromatin in a subset of genes. We also identify by CDC73 affinity-proteomics a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, nuclear Merlin is part of a tumour suppressor network which coordinates postinitiation steps of the transcription cycle of genes mediating in contact inhibition.

cancer biology↗