Biology subjects

Han, S. B.

Publications and source records attributed to Han, S. B..

9 recordsLinked to original sources

bioRxiv2025

Outward-facing P-glycoprotein bound to drug displays nucleotide-dependent drug egress mechanism

ABCB1/P-glycoprotein requires coordination between ligand movement in the trans-membrane cavity and ATP binding and hydrolysis at the nucleotide binding domains, yet the sequence of these events remains incompletely resolved. Here, atomistic simulations with paclitaxel and verapamil across outward facing P-glycoprotein ensembles in various nucleotide states were integrated with analysis of transmembrane domain contacts, geometric solvent/ligand pathways, extracellular gating distances, and nucleotide coordi-nation characterization. Collectively, our simulations delineate a mechanistic framework for P-glycoprotein in which ATP binding organizes an outward-facing ensemble and sequential hydrolysis at the two nucleotide-binding sites drives substrate progression toward egress. Asymmetry in the nucleotide state is correlated to distinct motions within the transmembrane domains, which suggests a possible transient extracellular gating mechanism that enables staged release and limits re-entry of the ligand. The resulting model connects nucleotide chemistry to transmembrane domain motions that regulate gate dynamics and may guide experiments and development of modulators for ABC exporter activity in physiological and pharmacological contexts.

molecular biology↗

bioRxiv2025

Drug-bound P-glycoprotein reveals nucleotide-dependent ligand pockets and ingress channels

P-glycoprotein, an ATP-binding cassette exporter, coordinates drug recognition in a central cavity with nucleotide handling at nucleotide binding domains, yet the sequence and coupling of these events remain incompletely defined. Here, we report an integrated computational analysis of inward facing P-glycoprotein ensembles across varying drug locations, nucleotide states, and starting conformations. The sampled inward-facing ensembles of P-glycoprotein display continuous ligand cavity, reshaped by nucleotide occupancy, which enables polyspecific ligand recognition while transmitting allosteric signals to the nucleotide binding domains through intracellular coupling loops. Asymmetry between the two nucleotide binding sites follows from this communication, which alters the nucleotide coordination to potentially affect the catalytic progression. Our simulations reveal dynamic cavity access routes from cytosol and inner membrane, together with transient secondary structure within the interdomain flexible linker. Overall, our study gives insights into how ligand redistribution, asymmetric nucleotide coordination, and linker flexibility may correlate to form a unified view of early transport steps, yielding hypotheses for mutational and kinetic evaluation and informing strategies for small-molecule modulation of transporter function in cellular contexts where multidrug transport affects therapeutic response.

molecular biology↗

bioRxiv2025

A high-throughput cell-based platform for Rhinovirus C research and antiviral drug discovery.

Rhinoviruses (RVs) are the most common cause of upper respiratory infections, yet RV-C is particularly associated with severe respiratory disease, including exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Despite its clinical significance, RV-C research and antiviral development have been hindered by the lack of scalable and biologically relevant models for high-throughput screening (HTS). Here, we present the development, optimization, and validation of a scalable high-content imaging-based platform for RV-C research. Our approach combines engineered HeLa cells expressing CDHR3C529Y, a genetically stable mGreenLantern-reporter RV-C15 virus, and a phenotypic selection strategy to identify monoclonal cell lines with maximal and sustained permissiveness. This approach enabled assay optimization in both 96- and 384-well formats, achieving robust performance (Z' > 0.7) and reproducibility across more than 100 runs with reference antiviral compounds. The platform was readily adapted to RV-A16, RV-B14, and additional RV-C types (C11 and C41), demonstrating broad applicability across rhinovirus species. A pilot screen of 10 240 compounds identified confirmed RV-C inhibitors, highlighting the readiness of the platform for integration into drug discovery pipelines. This platform provides a robust and scalable tool for systematic antiviral discovery and a foundation for mechanistic studies of RV-C replication. Significance statementRhinovirus C (RV-C) is strongly associated with severe respiratory disease such as asthma and COPD exacerbations, yet its study has been limited by the absence of scalable cell-based systems. We developed a high-content imaging-based high-throughput screening platform that supports efficient RV-C replication in engineered CDHR3-expressing cells and enables systematic antiviral discovery. The platform integrates a genetically stable fluorescent reporter virus with phenotypically selected permissive monoclonal cell lines, achieving high reproducibility and scalability in both 96- and 384-well formats. Its adaptability across multiple rhinovirus species establishes a unified framework for antiviral testing and mechanistic investigation. A pilot screen of 10K compounds confirmed the applicability of the platform to drug discovery campaigns.

microbiology↗

bioRxiv2025

Nucleotide Asymmetry and Flexible Linker Dynamics Modulate Drug Efflux Cycle of P-glycoprotein, a Computational Study

Despite advancements in oncology, multidrug resistance (MDR) mediated by P-glycoprotein (P-gp/ABCB1) remains a major barrier to chemotherapy. P-gp is an ATP-binding cassette transporter that undergoes nucleotide-driven structural rearrangements to efflux chemotherapeutics, but the mechanistic details of the substrate transport remain poorly resolved. Here, we performed high-throughput molecular dynamics to simulate P-gp in a lipid bilayer (totaling [~]110 {micro}s) to dissect nucleotide-dependent conformational changes across the transport cycle. Our adaptive sampling strategy reveals asymmetric nucleotide coordination at nucleotide-binding sites (NBS), which correlates with transmembrane domain (TMD) restructuring for substrate efflux. The experimentally unresolved flexible linker transiently forms up to five turn -helix that affects nucleotide binding domain (NBD) dimerization process. We identified conformation-dependent substrate/allocrite pathways including nucleotide-specific access routes, while TMD-linker interaction facilitates substrate access tunnel formation. Together, these pathways reveal that the concerted interplay of nucleotide occupancy, linker dynamics, and overall protein conformation governs the structural plasticity and broad substrate promiscuity of the substrate binding cavity in P-gp. By integrating these findings, this work bridges static structural data with dynamic functional insights to further our understanding of the P-gp substrate translocation cycle.

biochemistry↗

bioRxiv2024

Multiresolution molecular dynamics simulations reveal the interplay between conformational variability and functional interactions in membrane-bound cytochrome 2B4

Cytochrome P450 2B4 (CYP 2B4) is one of the best characterized CYPs and serves as a key model system for understanding the mechanisms of microsomal class II CYPs, which metabolize most known drugs. The highly flexible nature of CYP 2B4 is apparent from crystal structures that show the active site with either a wide open or a closed heme binding cavity. Here, we investigated the conformational ensemble of the full-length CYP 2B4 in a phospholipid bilayer, using multiresolution molecular dynamics (MD) simulations. Coarse-grained MD simulations revealed two predominant orientations of CYP 2B4s globular domain with respect to the bilayer. Their refinement by atomistic resolution MD showed adaptation of the enzymes interaction with the lipid bilayer, leading to open configurations that facilitate ligand access to the heme binding cavity. CAVER analysis of enzyme tunnels, AquaDuct analysis of water routes, and Random Acceleration Molecular Dynamics simulations of ligand dissociation support the conformation-dependent passage of molecules between the active site and the protein surroundings. Furthermore, simulation of the re-entry of the inhibitor bifonazole into the open conformation of CYP 2B4 resulted in binding at a transient hydrophobic pocket within the active site cavity that may play a role in substrate binding or allosteric regulation. Together, these results show how the open conformation of CYP 2B4 facilitates binding of substrates from and release of products to the membrane, whereas the closed conformation prolongs the residence time of substrates or inhibitors and selectively allows the passage of smaller reactants via the solvent and water channels. ImpactOur findings from multiresolution molecular dynamics simulations elucidate the structural dynamics of the membrane-bound CYP 2B4 enzyme and how these affect substrate recognition and processing, as well as inhibitor binding. As CYP 2B4 is a representative of the class of cytochrome P450 enzymes that metabolize most known drugs, the insights from this study are pertinent to the prediction of drug metabolism and the design of CYP inhibitors as therapeutics.

biochemistry↗

bioRxiv2022

Synapsing with oligodendrocyte precursor cells stop sensory axons regenerating into the spinal cord

Primary sensory axons stop regenerating as they re-enter the spinal cord, resulting in incurable sensory loss. What arrests them has remained unclear. We previously showed that axons stop by forming synaptic contacts with unknown non-neuronal cells. Here, we identified these cells in adult mice as oligodendrocyte precursor cells (OPCs). We also found that only a few axons stop regenerating by forming dystrophic endings, exclusively at the CNS:PNS borderline where OPCs are absent. Most axons stop in contacts with a dense network of OPC processes. Live imaging, immuno-EM and OPC-DRG co-culture additionally suggest that axons are rapidly immobilized by forming synapses with OPCs. Genetic OPC ablation enables many axons to continue regenerating deep into the spinal cord. We propose that sensory axons stop regenerating by encountering OPCs that induce presynaptic differentiation. Our findings identify OPCs as a major regenerative barrier that prevents intraspinal restoration of sensory circuits following spinal root injury.

neuroscience↗

bioRxiv2020

Co-targeting myelin inhibitors and CSPGs enhances sensory axon regeneration within, but not into, the spinal cord

A major barrier to intraspinal regeneration after dorsal root (DR) injury is the DR entry zone (DREZ), the CNS/PNS interface. DR axons stop regenerating at the DREZ, even if regenerative capacity is increased by a nerve conditioning lesion. This potent blockade has long been attributed to myelin-associated inhibitors and CSPGs, but incomplete lesions and conflicting reports have prevented conclusive agreement. Here we evaluated DR regeneration in mice, using novel strategies to facilitate complete lesions and analyses, selective tracing of proprioceptive and mechanoreceptive axons, and the first simultaneous targeting of Nogo/Reticulon-4, MAG, OMgp, CSPGs and GDNF. Co-eliminating myelin inhibitors and CSPGs elicited regeneration of only a few conditioning-lesioned DR axons across the DREZ. Their absence, however, markedly and synergistically enhanced regeneration of GDNF-stimulated axons, highlighting the importance of sufficiently elevating intrinsic growth capacity. We also conclude that myelin inhibitors and CSPGs are not the primary mechanism stopping axons at the DREZ.

neuroscience↗

bioRxiv2019

Druggability Assessment in TRAPP using Machine Learning Approaches

Accurate protein druggability predictions are important for the selection of drug targets in the early stages of drug discovery. Due to the flexible nature of proteins, the druggability of a binding pocket may vary due to conformational changes. We have therefore developed two statistical models, a logistic regression model (TRAPP-LR) and a convolutional neural network model (TRAPP-CNN), for predicting druggability and how it varies with changes in the spatial and physicochemical properties of a binding pocket. These models are integrated into TRAPP (TRAnsient Pockets in Proteins), a tool for the analysis of binding pocket variations along a protein motion trajectory. The models, which were trained on publicly available and self-augmented data sets, show equivalent or superior performance to existing methods on test sets of protein crystal structures, and have sufficient sensitivity to identify potentially druggable protein conformations in trajectories from molecular dynamics simulations. Visualization of the evidence for the decisions of the models in TRAPP facilitates identification of the factors affecting the druggability of protein binding pockets.

bioinformatics↗

bioRxiv2016

Parkin knockout inhibits neuronal development via regulation of proteasomal degradation of p21

PARK2 encodes for the E3 ubiquitin ligase parkin and iimplicates in the development of Parkinsons disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of parkins function in neural stem differentiation is not clear. Co-expressions network analysis showed that SNAP25 and BDNF were positively correlated with parkin, but negatively correlated with p21 in human patient brain. Therefore, we investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin resulting in the loss of cell differentiation ability. Tranfection of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.\n\nSummary statementThe present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.

cell biology↗