Biology subjects

Boyer, N.

Publications and source records attributed to Boyer, N..

6 recordsLinked to original sources

bioRxiv2026

Mirror-image mRNA display uncovers isoform-selective D-peptide macrocycles targeting a cryptic KRAS pocket

Activating KRAS mutations drive millions of cancers diagnosed worldwide,1 yet for decades this oncoprotein was deemed "undruggable", reflecting the challenge of discovering molecules capable of perturbing its complex biological functions, and of translating these discoveries into effective cancer therapeutics.2 Recent advances propelled by innovative screening have identified diverse modalities that bind at or near the switch-II pocket (SII-P) of RAS proteins, including molecular glues,3 macrocyclic peptides,4 fragment-derived small molecules,5 and approved therapies that covalently target KRASG12C.6,7 Unfortunately, resistance to approved therapies has emerged,8,9 highlighting the need for molecules that engage new or underexploited binding sites on RAS oncoproteins with mechanisms complementary to established SII-P inhibitors.10,11 Here we show that mirror-image mRNA display12 enabled the discovery of all-D macrocyclic peptide ligands targeting a cryptic RAS back pocket (CRB-P).13 These ligands engage KRAS(OFF) and KRAS(ON) with equal affinity, exploit a single-residue difference among isoforms to bind KRAS selectively, and successfully inhibit oncogenic signaling in KRAS-mutant cells through a mechanism distinct from SII-P binders. Mirror-image screening directly afforded nanomolar peptide ligands stable toward cellular proteolysis and delivered probes targeting distinct epitopes not accessible by homochiral peptide-display methods. Together, these findings establish the CRB-P as a specifically druggable and mechanistically differentiated site on KRAS with potential for combination with emerging RAS-targeting therapies and substantiate mirror-image mRNA display as a strategy for discovering stable all-D macrocyclic peptides targeting previously inaccessible epitopes on challenging targets.

cancer biology↗

bioRxiv2026

NEUROMUSCULAR ELECTRICAL STIMULATION LIMITS MUSCLE WEAKNESS, ATROPHY, MODULATES SATELLITE CELL FUNCTION AND REDUCES INFLAMMATION IN CANCER CACHEXIA

BackgroundCancer cachexia (CC) is characterized by skeletal muscle atrophy and reduced strength, partly linked to dysfunction of muscle stem cells (MuSCs) and alterations in their niche. Although exercise may mitigate muscle loss, its effects in CC remain debated and its feasibility is often limited in advanced patients. Neuromuscular electrical stimulation (NMES) offers a promising alternative, by promoting MuSC proliferation and fusion, increasing muscle size and macrophage content in healthy muscle. This study investigated whether NMES, initiated at tumor onset, could improve MuSC regulation and its niche while limiting muscle atrophy and weakness in a tumor-bearing mouse model. MethodsTen-week-old male BALB/c mice were subcutaneously injected with C26 tumor cells or PBS. Tumor-bearing mice were divided into NMES-treated (C26 NMES) and non-stimulated controls (C26). NMES consisted of six sessions (two series of three consecutive daily sessions separated by one rest day), starting seven days post-inoculation when tumors became visible. Each session was delivered at a submaximal intensity corresponding to 15% of maximal strength. Muscle mass, myofiber size, strength and cellular composition were assessed. ResultsMuscle mass was decreased by 13% in C26 mice as compared to PBS controls, while C26 NMES mice showed a [~]7% improvement over C26 mice. Mean myofiber size decreased similarly in both tumor-bearing groups as compared to PBS controls (-12-14%). However, NMES reduced the proportion of small myofibers (400-600 {micro}m{superscript 2}) as compared to C26 mice. Maximal torque loss was less severe in C26 NMES mice (-28%) than in C26 mice (-34%). As compared with PBS mice, C26 mice exhibited increased MuSC proliferation (+97%) but reduced differentiation (-61%), as indicated by fewer myogenin-positive cells. NMES normalized MuSC proliferation, restored myogenin-positive cell number, and enhanced MuSC fusion, reflected by an increased number of PCM1-positive myonuclei (+8-11%). NMES also modulated inflammation, reducing neutrophils (-42%) and increasing macrophages (+35%), through the proliferation of CD169-positive resident macrophages (+106%). In vitro, macrophages exposed to C26 muscle extracts showed elevated pro-inflammatory markers (COX2 and TNF-; +21% and +16%) as compared to PBS controls. This effect was abolished with extracts from C26 NMES muscles. Additionally, C26 extracts reduced the expression of anti-inflammatory markers by macrophages (CD206 and IL-10; -23%), whereas NMES restored their levels to those of controls. ConclusionNMES-induced mild contractile activity is an effective stimulus for preserving muscle strength and mass, improving MuSC regulation, and modulating muscle inflammation in a mouse model of CC.

cell biology↗

bioRxiv2025

Application of Large Language Models for Annotating Genes into Reactome Pathways

Reactome is the most comprehensive, open source, open access biological pathway knowledgebase, widely used in the research community. To ensure the highest quality of its content, human pathway data in Reactome is manually curated. However, manual curation is labor-intensive, time-consuming, and increasingly difficult to keep up with the ever-growing biomedical literature. Large language model (LLM)-driven artificial intelligence (AI) technologies are transforming many fields, including bioinformatics resource development. Applying LLM/AI technologies in Reactome may offer a powerful way to scale curation and consolidate pathway-related data into a single resource. This manuscript describes the first stage of our attempt to adopt LLM/AI technologies for Reactome manual curation. We developed an LLM workflow that can assist curators in adding new genes to existing pathways and refining the functional annotations of existing ones. The workflow predicts pathways in which genes are likely to function, identifies PubMed-indexed literature that may support these predictions, generates text summaries describing potential molecular mechanisms, and extracts functional relationships among biological entities from full-text PDF papers. To validate the workflow output, we used a computational approach based on semantic similarity between LLM workflow-generated summaries and Reactome manual annotations. The results show significant enrichment of high-similarity matches. Manual evaluation of 19 genes indicated that more than half of the outputs are useful for supporting curation. Based on these results, we developed an enhanced workflow that incorporates protein-protein interaction data, facilitating Reactomes reaction-based annotation. In summary, our initial adoption of LLM/AI technologies produced encouraging results and provides a practical framework for integrating AI-assisted methods into Reactomes curation pipeline. The strategies described here may be broadly applicable to community knowledgebases in general.

bioinformatics↗

bioRxiv2023

Oncostatin M is a Master Regulator of an Inflammatory Network in Dnmt3a-Mutant Hematopoietic Stem Cells

Age-associated clonal hematopoiesis (CH) occurs due to somatic mutations accrued in hematopoietic stem cells (HSCs) that confer a selective advantage in the context of aging. The mechanisms by which CH-mutant HSCs gain this advantage with aging are not comprehensively understood. Using unbiased transcriptomic approaches, we identify Oncostatin M (OSM) signaling as a candidate contributor to aging-driven Dnmt3a-mutant CH. We find that Dnmt3a-mutant HSCs from young mice do not functionally respond to acute OSM stimulation with respect to proliferation, apoptosis, hematopoietic engraftment, or myeloid differentiation. However, young Dnmt3a-mutant HSCs transcriptionally upregulate an inflammatory cytokine network in response to acute OSM stimulation including genes encoding IL-6, IL-1{beta} and TNF. In addition, OSM-stimulated Dnmt3a-mutant HSCs upregulate the anti-inflammatory genes Socs3, Atf3 and Nr4a1, creating a negative feedback loop limiting sustained activation of the inflammatory network. In the context of an aged bone marrow (BM) microenvironment with chronically elevated levels of OSM, Dnmt3a-mutant HSCs upregulate pro-inflammatory genes but do not upregulate Socs3, Atf3 and Nr4a1. Together, our work suggests that chronic inflammation with aging exhausts the regulatory mechanisms in young CH-mutant HSCs that resolve inflammatory states, and that OSM is a master regulator of an inflammatory network that contributes to age-associated CH.

cell biology↗

bioRxiv2022

Development of a high-throughput barrier function assay using primary human gut organoids

Disruptions in the gut epithelial barrier can lead to the development of chronic indications such as inflammatory bowel disease (IBD). Historically, barrier function has been assessed in cancer cell lines, which do not contain all human intestinal cell types, leading to poor translatability. To bridge this gap, we adapted human primary gut organoids grown as monolayers to assess barrier function. In this work we describe and characterize a novel 96-well human gut organoid-derived monolayer system that enables medium/high-throughput quantitative assessment of candidate therapeutics. Normal human intestine differentiation patterns and barrier function were characterized and confirmed to recapitulate key aspects of in vivo biology. Next, cellular response to TNF- (a central driver of IBD) was determined using a diverse cadre of readouts.These outputs included transcription factor phosphorylation, target gene expression, cytokine/chemokine production, and barrier function. Additionally, we showed that TNF- pathway antagonists rescued damage caused by TNF- in a dose-dependent manner, indicating that this system is suitable for quantitative assessment of barrier modulating factors. Taken together, we have established a robust primary cell-based 96-well system capable of interrogating questions around mucosal response. This system is well suited to provide pivotal functional data to support translational target and drug discovery efforts.

immunology↗

bioRxiv2021

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

RAS is the most commonly mutated oncogene in human cancers and RAS-driven tumors are amongst the most difficult to treat. Historically, discovery of therapeutics targeting this protein has proven challenging due to a lack of deep hydrophobic pockets to which a small molecule might bind. The single such pocket available is normally occupied by GDP or GTP which have millimolar cellular concentrations and picomolar affinities for KRAS and hence is challenging to target. The recent FDA approval of sotorasib, a covalent modifier of the KRASG12C mutant protein, has clinically validated KRAS as an oncology target. However, traditional challenges remain for targeting the more common KRAS mutations such as G12D and G12V. As an alternative approach, we investigated the superior binding capacity of macrocyclic peptides to identify KRAS inhibitory molecules. We focused on the recently reported disulfide-cyclized arginine-rich peptide KRpep-2d, discovered through phage display and previously independently confirmed by us as a bona fide KRAS binder. To mitigate intracellular disulfide reduction and loss of binding, we identified an alternate cyclization motif by inverting the configuration of Cys5 and linking it to Cys15 through a thioacetal bridge. The corresponding peptide bound KRAS through cis isomerization of the peptide bond between D-Cys5 and Pro6 as observed through x-ray crystallography. Prototypic molecules displayed measurable cellular inhibition of RAS signaling without membrane lysis and counter-screen off-target activity. An analogue containing azido-lysine confirmed the cell penetrant nature of this molecule using our recently reported NanoClick assay. To improve cellular activity, we sought to improve proteolytic stability. Structure-activity relationship studies identified key scaffold residues critical for binding and revealed that replacement of N- and C-terminal arginine residues with D-arginines and introduction of an -methyl moiety at Ser10 resulted in a molecule with improved proteolytic stability as indicated by its persistence in whole cell homogenate. The resulting peptide MP-3995 had improved and sustained cell-based efficacy. On-target activity was validated through confirmation of target engagement, lack of signaling in irrelevant pathways, and use of non-binding control peptides. Mechanism of action studies suggested that peptide binding to both GDP- and GTP-states of KRAS may contribute to cellular activity. Although validated as bona fide and on-target inhibitors of cell based KRAS signaling, this series is unlikely to advance to the clinic in its current form due to its arginine-dependent cell entry mechanism. Indeed, we showed a strong correlation between net positive charge and histamine release in an ex vivo assay making this series challenging to study in vivo. Nonetheless, these molecules provide valuable templates for further medicinal chemistry efforts aimed at leveraging this unique inhibitory binding site on KRAS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/459913v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@17f4764org.highwire.dtl.DTLVardef@12818daorg.highwire.dtl.DTLVardef@177bdf0org.highwire.dtl.DTLVardef@57046c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗