Biology subjects

Lokey, S.

Publications and source records attributed to Lokey, S..

5 recordsLinked to original sources

bioRxiv2026

Minimal N-methylated and stapled peptide ligands for the autophagy protein GABARAP

The LC3/GABARAP protein family is a promising target for selective inhibition of autophagy. Further, LC3/GABARAP ligands have been used as targeted degraders of soluble proteins, protein aggregates, mitochondria, lipid droplets, and RNA. However, the small molecules used for such applications have poor binding affinity and known off-target effects. LC3/GABARAP proteins are challenging targets for small-molecule drug development due to their long, shallow binding grooves. In this work, we evaluate multiple approaches to stabilizing the extended structure of the native binding motif, producing N-methylated peptides and stapled peptides with low nanomolar affinity. A crystal structure and molecular dynamics simulations support a model where the N-methylation pre-organizes the motif into an extended, strand-like structure. N-methylation allowed minimization of the binding motif to a tetrapeptide that retained sub-micromolar affinity while minimizing charge and overall molecular weight. The truncated, N-methylated tetrapeptide showed passive permeability in artificial membrane and cell-based transwell assays. These results highlight new drug-like space for LC3/GABARAP ligands with high affinity and subfamily selectivity. Table of Contents GraphicDrawn and developed by Mollie McGibbon and Joshua Kritzer O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/710535v2_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@1b74a1org.highwire.dtl.DTLVardef@861c09org.highwire.dtl.DTLVardef@9abc2corg.highwire.dtl.DTLVardef@b7f745_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

MOAST: Mechanism of Action Similarity Tool

Determining the mechanism of action (MOA) for natural products remains a significant bottleneck in drug discovery, particularly for researchers with limited computational resources or small compound libraries. Traditional approaches require screening large numbers of annotated compounds alongside unknowns, which is cost-prohibitive, or depend on complex machine learning models that need substantial computational resources and large datasets. Here, we present a dissertation chapter excerpt: MOAST (Mechanism of Action Similarity Tool), a BLAST-inspired computational workflow that addresses these limitations by providing rapid MOA hypotheses for newly screened compounds. This chapter investigates two complementary approaches: a kernel density estimation (KDE) method providing statistical significance measures and E-values for MOA class membership, and a CatBoost machine learning classifier for multi-class prediction with ranked outputs. Using cytological profiling data from HeLa and A549 cell lines, MOAST achieved 22% accuracy for the top 5 predictions among [~] 300 MOA classes, with the CatBoost classifier reaching 10% balanced accuracy--significantly better than the [~] 3% reported in literature. The tool suggests a 0.8 prediction probability threshold for trustworthy results and demonstrates robust performance across multiple feature reduction strategies. MOAST provides a practical, accessible solution that bridges traditional phenotypic screening and modern computational approaches, making MOA determination feasible for researchers with limited resources while maintaining statistical rigor and interpretability.

bioinformatics↗

bioRxiv2025

Cell Painting in activated cells illuminates phenotypic dark space and uncovers novel drug mechanisms of action.

As drug and natural product libraries expand, assays for assessing mechanisms of action (MoA) are increasingly critical. Performing cytological profiling using the Cell Painting (CP) assay enables image-based profiling of cellular states upon treatment, yet many bioactive compounds remain uncharacterized due to undetectable cellular effects under standard conditions. To address this, we combined drug dosing with cell activation using the protein kinase C (PKC) agonist phorbol myristate acetate (PMA). Profiling A549 lung cancer cells treated with 8,387 compounds at two concentrations (1 and 10 {micro}M) in both resting and PMA-activated states allowed us to detect phenotypic effects for up to 40% of all screened compounds, effectively illuminating new phenotypic "dark space". Over 1,000 compounds exhibited phenotypes exclusively under PMA activation, establishing its advantage for MoA studies. We introduce novel quality control measures for CP screens and demonstrate that integrating phenotypic signatures enhances MoA discovery. Notably, 2-methoxycinnamaldehyde clustered with glucocorticoid receptor modulators and induced nuclear translocation, emphasizing the power of this approach in uncovering novel drug mechanisms and, therefore, aiding in improving therapeutic strategies.

systems biology↗

bioRxiv2022

Inability to make facial expressions dampens emotion perception.

Humans rely heavily on facial expressions for social communication to convey their thoughts and emotions and to understand them in others. One prominent but controversial view is that humans learn to recognize the significance of facial expressions by mimicking the expressions of others. This view predicts that an inability to make facial expressions (e.g., facial paralysis) would result in reduced perceptual sensitivity to others facial expressions. To test this hypothesis, we developed a diverse battery of sensitive emotion recognition tasks to characterize emotion perception in individuals with Moebius Syndrome (MBS), a congenital neurological disorder that causes facial palsy. Using computer-based emotion detection tasks we systematically assessed emotion perception thresholds for static and dynamic face and body expressions. We found that while MBS individuals were able to perform challenging perceptual control tasks, they were less efficient at extracting emotion from facial expressions, compared to matched controls. Exploratory analyses of fMRI data from a small group of MBS participants suggested potentially reduced engagement of the amygdala in MBS participants during expression processing relative to matched controls. Collectively, these results support the role of facial mimicry and consequent facial feedback and motor experience in the perception of others expressions.

neuroscience↗

bioRxiv2020

Developing Cyclic Peptomers as Broad-Spectrum Gram negative Bacterial Type III Secretion System Inhibitors

Antibiotic resistant bacteria are an emerging global health threat. New antimicrobials are urgently needed. The injectisome type III secretion system (T3SS), required by dozens of Gram-negative bacteria for virulence but largely absent from non-pathogenic bacteria, is an attractive antimicrobial target. We previously identified synthetic cyclic peptomers, inspired by the natural product phepropeptin D, that inhibit protein secretion through the Yersinia Ysc and Pseudomonas aeruginosa Psc T3SSs, but do not inhibit bacterial growth. Here we describe identification of an isomer, 4EpDN, that is two-fold more potent (IC50 4 M) than its parental compound. Furthermore, 4EpDN inhibited the Yersinia Ysa and the Salmonella SPI-1 T3SSs, suggesting that this cyclic peptomer has broad efficacy against evolutionarily distant injectisome T3SSs. Indeed, 4EpDN strongly inhibited intracellular growth of Chlamydia trachomatis in HeLa cells, which requires the T3SS. 4EpDN did not inhibit the unrelated Twin arginine translocation (Tat) system, nor did it impact T3SS gene transcription. Moreover, although the injectisome and flagellar T3SSs are evolutionarily and structurally related, the 4EpDN cyclic peptomer did not inhibit secretion of substrates through the Salmonella flagellar T3SS, indicating that cyclic peptomers broadly but specifically target the injestisome T3SS. 4EpDN reduced the number of T3SS basal bodies detected on the surface of Y. enterocolitica, as visualized using a fluorescent derivative of YscD, an inner membrane ring with low homology to flagellar protein FliG. Collectively, these data suggest that cyclic peptomers specifically inhibit the injectisome T3SS from a variety of Gram-negative bacteria, possibly by preventing complete T3SS assembly. IMPORTANCETraditional antibiotics target both pathogenic and commensal bacteria, resulting in a disruption of the microbiota, which in turn is tied to a number of acute and chronic diseases. The bacterial type III secretion system (T3SS) is an appendage used by many bacterial pathogens to establish infection, but is largely absent from commensal members of the microbiota. In this study, we identify a new derivative of the cyclic peptomer class of T3SS inhibitors. These compounds inhibit the T3SS of the nosocomial ESKAPE pathogen Pseudomonas aeruginosa and enteropathogenic Yersinia and Salmonella. The impact of cyclic peptomers is specific to the T3SS, as other bacterial secretory systems are unaffected. Importantly, cyclic peptomers completely block replication of Chlamydia trachomatis, the causative agent of genital, eye, and lung infections, in human cells, a process that requires the T3SS. Therefore, cyclic peptomers represent promising virulence blockers that can specifically disarm a broad spectrum of Gram-negative pathogens.

microbiology↗