Biology subjects

Kosloff, M.

Publications and source records attributed to Kosloff, M..

8 recordsLinked to original sources

bioRxiv2026

An engineered nanobody inhibitor for molecular-to-circuit control of opioid receptor function

Opioid receptors (ORs) orchestrate pain relief, reward, and dependence, yet their signaling arises from diverse cell types and subcellular compartments that cannot be selectively interrogated with existing pharmacological or genetic approaches. Single-domain antibodies, or nanobodies (Nbs), can probe receptor states, but their potential as tools for controlling native receptor signaling at the system level has remained unexplored. Here, we engineer a suite of high-affinity intracellular Nbs that bind active ORs through structure-guided evolution and in silico design. Iterative optimization yields Nb64, a potent inhibitor that rapidly suppresses transducer engagement, receptor internalization, and downstream signaling, including endogenous pathways in neuronal cells. Organelle targeting highlights Nb64s capacity to control OR activity with subcellular precision, while bio-reversible cell-penetrating peptide (CPP) conjugation enables non-genetic cytosolic delivery. Cell-type-specific expression of Nb64 in VTA interneurons attenuates fentanyl-evoked dopamine release and behavioral responses in mice, demonstrating targeted control of opioid actions in vivo. Nb64 provides a versatile strategy for dissecting OR biology and establishes a generalizable framework for precision inhibition of native GPCR signaling in vivo.

neuroscience↗

bioRxiv2025

Deciphering the mechanistic basis for the pathological effect of the Gαo E246K mutation in neurodevelopmental disorder

Mutations in the GNAO1 gene, which encodes for Go, a major neuronal G protein, are associated with neurodevelopmental disorders, epilepsy, and movement disorders. We identified and characterized a spontaneous heterozygous GNAO1 E246K mutation in an Israeli female infant with complex developmental delays and substantial motor difficulties. This mutation has been reported in other cases as a prevalent pathogenic mutation in patients with motor dysfunction and a broad range of neurological outcomes. To investigate the molecular and functional consequences of the Go E246K mutation, we employed structural modeling and analysis, biochemical assays, mass spectrometry-based proteomics, and cellular functional assays. We show that this mutation does not affect nucleotide binding, nor basal or RGS- accelerated GTP hydrolysis. Despite the E246 position located within a predicted effector binding region, proteomics analysis did not identify any new cellular partners. Instead, we demonstrate that the E246K mutation disrupts the Go regulatory GTPase cycle by directly impairing G{beta}{gamma} dissociation. This impairment overrides the presence of wild-type Go, explaining the dominant effect of the severe neurogenetic phenotype in the heterozygous background. These findings establish a new molecular mechanism for a GNAO1 mutation with dominant-negative effects on the GTPase regulatory cycle. The insights gained from studying this mechanism of action provide a basis for developing specific and personalized therapeutic strategies based on the outcome of a missense mutation in GNAO1.

neuroscience↗

bioRxiv2025

Developing inhibitors of the guanosine triphosphate hydrolysis accelerating activity of Regulator of G protein Signaling-14

Regulator of G protein Signaling-14 (RGS14), an intracellular inactivator of G protein-coupled receptor (GPCR) signaling, is considered an undruggable protein given its shallow and relatively featureless protein-protein interaction interface combined with a distal allosteric site prone to nonspecific inhibition by thiol-reactive compounds. Here, we identify and validate a tractable chemotype that selectively and non-covalently inhibits RGS14 GTPase-accelerating protein (GAP) activity. Combining structure-guided virtual screening, ligand docking across multiple receptor conformers, and enrichment validation, we progressed from a first-generation active, Z90276197, to over 40 second-generation analogs with improved potency. These inhibitors are predicted to engage the solvent-exposed "canyon" in the RGS14 RGS-box that interacts with the G switch I region. Binding pose predictions underscored the importance of non-polar interactions and shape complementarity over polar interactions in engaging this G-binding canyon and revealed an "ambidextrous" pattern of R1- and R2-group orientations. GAP inhibition was confirmed in fluorescence-based and gold-standard radioactive GTP hydrolysis assays. Two second-generation analogs, Z55660043 and Z55627844, inhibited RGS14 GAP activity in both assays and without measurable cytotoxicity. Deep learning-based scoring of predicted docking poses further supported observed affinity gains from R3-group additions. One analog demonstrated favorable in vivo pharmacokinetics and CNS penetration. Collectively, our findings establish tractable, non-covalent, small molecule inhibition of a G protein regulatory interface and illustrate how machine learning-enhanced docking can guide ligand optimization for shallow protein surfaces. This work opens the door to future development of RGS14 inhibitors as potential therapeutics for central nervous system and metabolic disorders.

pharmacology and toxicology↗

bioRxiv2025

Structural conservation and divergence across the Receptor Tyrosine Kinase superfamily

Members of the Receptor Tyrosine Kinase (RTK) superfamily are regulators of cellular signaling, playing essential roles in cellular growth, differentiation, and survival. Dysregulation of RTKs leads to diseases such as cancer, diabetes, and inflammatory disorders, making them important therapeutic targets. Despite extensive research on RTKs, the structural diversity and evolutionary relationships across the superfamily are not fully understood. Here, we systematically compared structural conservation and divergence among 245 extracellular domains from 54 RTKs, across 18 RTK families. Using experimentally-resolved structures and AlphaFold2 models, we conducted an all-versus-all structural alignment to explore domain architecture and quantify significant structural similarities within the RTK superfamily. We curated a comprehensive database encompassing PDB structures, 3D folds, ligand-binding properties, and sequence information of all RTK domains analyzed (https://fasslero.github.io/RTK-domains). Our analysis revealed numerous inter-family similarities and remote evolutionary connections, in particular among ligand-binding domains (LBDs), and distinct structural domain types and unexpected dissimilarities among domains previously classified as related. Our work highlights the intricate balance between structural conservation and divergence in RTKs and sheds light on the evolutionary mechanisms shaping this critical superfamily.

bioinformatics↗

bioRxiv2024

Molecular determinants underlying differential recruitment of p115RhoGEF and PDZRhoGEF to activated Gα13

Heterotrimeric G proteins, particularly G12 and G13, are pivotal regulators of cellular signaling pathways. Their direct downstream effectors, which include p115RhoGEF and PDZRhoGEF, engage downstream signaling via Rho activation. Yet the molecular determinants that dictate their differential recruitment by G12/13 are not fully understood. Here, we combined quantitative computational residue-level analysis with site-directed mutagenesis and bioluminescence resonance energy transfer (BRET)-based assays to dissect G13 interactions with these RhoGEFs. We mapped the contributions of individual residues to binding and identified specific G13 residues in its helical domain, switch regions, and effector-binding site as key yet differential contributors to p115RhoGEF and PDZRhoGEF recruitment. Experimental validation with BRET confirmed that changes in many G13 residues impact p115RhoGEF more substantially than PDZRhoGEF, underscoring the specificity of G13 interactions with p115RhoGEF. Investigation of the p115RhoGEFs identified critical residues that contribute to interactions with G13 and G12. Our findings highlight residue-level differences in the molecular interactions of G13 with p115RhoGEF and PDZRhoGEF, providing insights into the specificity and regulation of G13-mediated signaling pathways. The resulting residue-level maps lay the groundwork for development of selective therapeutic strategies targeting G13-RhoGEF interactions.

biochemistry↗

bioRxiv2024

Structure-based mechanistic principles for the paradoxical effects of pathological RET mutations

The RET receptor is a transmembrane protein that belongs to the receptor tyrosine kinase family. RET activates signaling pathways regulating cell growth, differentiation, and survival in diverse tissues that include the thyroid and enteric nervous system. Mutations that reduce RET levels or disrupt function can cause Hirschsprungs disease (HSCR), characterized by abnormal distal colon innervation in the developing embryo. In contrast, mutations that constitutively activate RET can lead to tumorigenesis, most notably in multiple endocrine neoplasia type 2 (MEN2) syndromes usually involving the thyroid. Paradoxically, some RET mutations can both reduce RET activity in enteric ganglia, and increase signaling in other tissues, resulting in patients suffering from both HSCR and MEN2A. Although extensive research has been conducted on RET mutations, the structural and mechanistic bases underlying these paradoxical effects remain unclear. Here, we curated data on 70 positions in RET extracellular domains where point-mutations were associated with HSCR, MEN2A, or both. Taking a structure-based approach, we predict the potential effects of mutations in these positions on RET structure. Our analysis suggests that approximately 90% of positions associated with HSCR can, upon mutation, disrupt intramolecular interactions that stabilize RET tertiary structure: residues buried in the protein core, calcium-binding sites, or residues participating in stabilizing intramolecular electrostatic/covalent bonds. A smaller subset of mutations involves substitutions to/from glycines or prolines in key positions. Only a small minority of HSCR-associated positions affect protein-protein interactions needed for signal activation. On the other hand, our analysis showed that [~]75% of mutations in positions that cause MEN2A lead to an unpaired cysteine that can form an intermolecular disulfide bond between two RET monomers. Other mutations that cause MEN2A are also predicted to enhance RET homodimerization via extracellular domains that are proximal to the membrane. Importantly, substitutions that concurrently destabilizes RET tertiary structure and lead to an unpaired cysteine are predicted to cause the paradoxical co-occurrence of HSCR and MEN2A. Our findings suggest a mechanistic basis for almost all identified pathological mutations in RET and imply that therapeutic strategies for targeting RET activity in HSCR and MEN2A may need to be orthogonal.

bioinformatics↗

bioRxiv2024

Gαs-specific structural elements attenuate interactions with RGS proteins

Heterotrimeric ({beta}{gamma}) G proteins are molecular switches that are activated by G protein-coupled receptors (GPCRs) and regulate numerous intracellular signaling cascades. Most active G subunits are inactivated by Regulators of G protein Signaling (RGS) proteins, which determine the duration of G protein-mediated signaling by accelerating the catalytic turn-off of the G subunit. However, the G protein Gs does not interact with known RGS proteins. To understand the molecular basis for this divergent phenomenon, we combined a comparative structural analysis of experimental and modelled structures with functional biochemical assays. This analysis showed that Gs contains unique structural elements in both the helical and the GTPase domains. Modeling identified helical domain insertions, missing in experimental structures, that project towards the interface with RGS proteins, and residues in the GTPase domain that might interfere with RGS binding. Mutagenesis of Gs and measurements of RGS GAP activity showed that three residues in the Gs GTPase domain are both necessary and sufficient to prevent Gs inactivation by RGSs. Indeed, substitution of all three Gs residues with the corresponding residues from Gi1 enabled efficient inactivation by RGS proteins. These results shed new light on the mechanistic bases for G protein specificity towards RGS proteins.

biochemistry↗

bioRxiv2019

Signaling via GABAB receptors regulates early development and neurogenesis in the basal metazoan Nematostella vectensis

The metabotropic gamma-amino-butyric acid B receptor (GABABR) is a G protein-coupled receptor that mediates neuronal inhibition by the neurotransmitter GABA. Here, we identified putative GABAB receptors and signaling modulators in the basal sea anemone Nematostella vectensis. Activation of GABABR signaling reversibly arrests planula-to-polyp transformation during early development and affects the neurogenic program. We identified four Nematostella GABABR homologs that have the conserved 3D extracellular domains and residues needed for binding of GABA and the GABABR agonist baclofen. Transcriptomic analysis, combined with spatial analysis of baclofen-treated planulae, revealed that baclofen down-regulated pro-neural factors such as NvSoxB(2), NvNeuroD1 and NvElav1. Baclofen also inhibited neuron development and extended neurites, resulting in an under-developed and less organized nervous system. Our results shed light on cnidarian development and suggest an evolutionarily conserved function for GABABR in regulation of neurogenesis, highlighting Nematostella as a new model system to study GABABR signaling.

molecular biology↗