Biology subjects

Wolber, G.

Publications and source records attributed to Wolber, G..

12 recordsLinked to original sources

bioRxiv2026

Assessing State-Specific Accuracy of Cofolding Models for Kinases and GPCRs

AI-driven cofolding models have emerged as powerful tools for predicting protein-ligand complexes, yet whether ligand placement faithfully captures the conformational states of dynamic proteins remains unclear. Here we show that cofolding adaptively remodels binding pockets around bound ligands, but that this local accuracy is frequently decoupled from recovery of the broader conformational state. We benchmark four models, AlphaFold3, RosettaFold3, Boltz-2, and Chai-1, against a set of kinases and class A G protein-coupled receptors (GPCRs), protein families whose pharmacology depends on well-defined structural states. We find that even when ligand root-mean-square deviation (RMSD) is low, critical state markers, including kinase activation-loop geometries and GPCR intracellular arrangements, are frequently mispredicted. Incorporating state-annotated templates and filtered multiple sequence alignments (MSAs) improves conformational recovery in selected cases, yet weakly impacts others. Furthermore, while orthosteric ligand placement is generally reliable, allosteric binders expose a consistent blind spot across all models. These findings establish conformational decoupling as a fundamental limitation of current cofolding approaches, with direct implications for state-selective drug design.

biochemistry↗

bioRxiv2026

The Kifc3 Motor Protein Controls Centrosomal Factor Cep192 in Ontogenic Coordination of Megakaryocyte Development

The distinct features of neonatal megakaryocytes, high proliferation and inefficient platelet production, have clinical repercussions. A diminished capacity for stress thrombopoiesis, the response to acute drops in platelet counts, contributes to the high prevalence of thrombocytopenia in premature infants and to impaired platelet recovery after umbilical cord blood stem cell transplantation. High proliferation also promotes leukemogenesis in babies with Down Syndrome (DS). The transcriptional coactivator Mkl1/MrtfA participates in programming the ontogenic shift from fetal/neonatal to adult-type megakaryopoiesis; in this activity it is opposed by the DS-associated kinase Dyrk1a. In a screen for downstream ontogenic effectors in human progenitors, we identified the kinesin Kifc3 as a factor selectively decreased in adult megakaryocytes and whose knockdown in neonatal megakaryocytes induced adult-type morphogenesis with augmented platelet release. Kifc3 acts as a minus-end directed motor for centrosomal delivery of various cargos. Centrosomal release of Cep192 has recently been found induce cellular process extensions through actin remodeling, reminiscent of megakaryocyte platelet release. In our studies, Cep192 showed striking upregulation and dispersion in adult vs neonatal megakaryocytes, and Kifc3 knockdown recapitulated this effect in neonatal megakaryocytes. A role for Cep192 in promoting megakaryocyte morphogenesis, distinct from its role in centrosome biogenesis, was demonstrated in vitro and in vivo. In silico screening for Kifc3 inhibitors identified a small molecule that affected neonatal megakaryocytes similarly to Kifc3 knockdown, indicating feasibility for therapeutic argeting of the Kifc3-Cep192 pathway in clinical conditions associated with fetal-type megakaryopoiesis. Key PointsO_LIThe motor protein Kifc3 dictates megakaryocyte ontogeny in association with its control of the centrosomal actin-remodeling factor Cep192. C_LIO_LIKnockdown or small molecule targeting of Kifc3 enhances neonatal megakaryocyte morphogenesis and thrombopoiesis. C_LI

developmental biology↗

bioRxiv2025

Dynamic pharmacophores unveil binding mode ensembles for classical partial agonists at the M2 receptor

For the prototypic M2 receptor it has been previously demonstrated that dualsteric partial agonists can stabilize both, active and inactive receptor states, but it remains unclear whether orthosteric partial agonists have a similar mechanism. Here, we apply dynamic pharmacophores to unveil binding mode ensembles for classical M2 partial agonists. We report correlations between the spatial distribution of lipophilic contacts and ligand efficacy and demonstrate the applicability of dynamic pharmacophores to analyze subtle binding mode changes.

bioinformatics↗

bioRxiv2025

Structure-Based Virtual Screening Identifies TREM2-Targeted Small Molecules that Enhance Microglial Phagocytosis

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor whose activation promotes phagocytosis and neuroprotection in Alzheimers disease (AD) and related neurodegenerative disorders. While therapeutic efforts have largely focused on antibodies, small molecule TREM2 modulators remain limited. Here, we applied a structure- based virtual screening workflow targeting a putative allosteric site on TREM2, guided by PyRod-derived pharmacophores from molecular dynamics simulations. Screening of the Enamine Collection yielded 20 candidate compounds, three of which demonstrated binding in TRIC assays. The top hit, EN020, exhibited a KD of 14.2 {micro}M (MST) and 35.9 {micro}M (SPR), and significantly enhanced microglial phagocytosis in BV2 cells outperforming the known TREM2 agonist VG-3927. A preliminary structure-activity relationship (SAR) study, including synthetic and catalog-derived analogs, highlighted a narrow tolerance for scaffold modifications, with only T2V002 retaining partial TREM2 binding affinity. This work identifies EN020 as a novel small molecule TREM2 modulator with functional activity, providing a framework for rational optimization toward potential AD therapeutics.

pharmacology and toxicology↗

bioRxiv2025

Targeting the CAPON-NOS Axis: A Computational Strategy for Small Molecule Modulator Discovery

The carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) serves as a critical regulatory protein controlling nitric oxide (NO) signaling across multiple physiological and pathological processes which encompass neurological, cardiac and metabolic functions. These diverse physiological roles of CAPON marks it as a key therapeutic target for conditions associated with its dysregulation. Despite this therapeutic potential there are no specific CAPON or nNOS/CAPON modulators which have been developed to date, highlighting a significant gap in targeted drug discovery. Herein, we report the first strategy specifically focused on disrupting the nNOS/CAPON protein-protein interface. Through screening of chemical libraries composed of 4.6 million compounds and eight molecular dynamics simulations, two potential hit compounds were identified. Beyond identifying these promising hits, our approach introduces two novel computational tools: a freely available Python-based toolset for NMR structural analysis and visualization and a second toolkit for accelerated ligand preparation. These tools significantly accelerate data preparation timelines while reducing computational costs, providing the research community with accessible resources for structure-based drug discovery efforts. Together, these tools represent a substantial contribution to the computational chemistry toolkit, enabling researchers to conduct high-throughput virtual screening campaigns more efficiently and with greater reproducibility. This work represents a foundational step toward developing targeted therapies for CAPON-mediated disorders and provides a scalable computational framework for future protein-protein interaction drug discovery efforts. HighlightsO_LIA novel structure-based strategy developed to target the CAPON/nNOS protein-protein interaction. C_LIO_LIA Python-based toolset for protein conformation analysis, identification, visualization and separation. C_LIO_LIPython pipeline enables efficient ligand preparation for ultra-large chemical libraries. C_LIO_LIVirtual screening identified 6 promising small-molecule candidates. C_LIO_LIA total of 8*100ns molecular dynamics (MD) simulations performed using DESMOND. C_LIO_LIMM/GBSA and contact-time analysis were conducted to assess binding stability and affinity. C_LI

bioinformatics↗

bioRxiv2025

Virtual Screening-Guided Discovery of Small Molecule CHI3L1 Inhibitors with Functional Activity in Glioblastoma Spheroids

Chitinase-3-like protein 1 (CHI3L1), a glycoprotein implicated in inflammation, fibrosis, and cancer, has emerged as a potential therapeutic target for glioblastoma (GBM). CHI3L1 contributes to tumor progression and immune evasion by promoting STAT3 signaling and mesenchymal transition. To identify small molecule CHI3L1 inhibitors, a structure-based 3D pharmacophore model was developed and applied to virtually screen over 4.4 million compounds from the Enamine collection. Following multi-tiered filtering, 35 candidates were selected for experimental evaluation. Binding validation via microscale thermophoresis (MST) confirmed dose-dependent CHI3L1 interactions for two compounds, 8 and 39, with dissociation constants (Kd) of 6.8 {micro}M and 22 {micro}M, respectively. These affinities were further supported by surface plasmon resonance (SPR), which yielded Kd values of 5.69 {micro}M for compound 8 and 17.09 {micro}M for compound 39. In 3D GBM spheroid models, compound 8 significantly reduced spheroid viability and attenuated phospho-STAT3 levels, consistent with CHI3L1 pathway disruption. These findings identify two promising scaffolds and support the utility of pharmacophore-guided virtual screening for discovering functionally active ligands targeting CHI3L1 in GBM. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/667816v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@291464org.highwire.dtl.DTLVardef@f9d224org.highwire.dtl.DTLVardef@1535eeaorg.highwire.dtl.DTLVardef@7c596e_HPS_FORMAT_FIGEXP M_FIG Table of Contents artwork C_FIG

pharmacology and toxicology↗

bioRxiv2025

Small Molecule-Based Blockade of CD28 Suppresses T Cell Costimulation Across Cellular and Mucosal Co-culture Models

Background and PurposeCD28 is a pivotal costimulatory receptor that governs T cell activation through interaction with B7 ligands (CD80/CD86). While antibody-based inhibitors of CD28 signaling have advanced clinically, the development of small molecule modulators remains limited due to the receptors shallow, flexible surface. We sought to discover small-molecule modulators with favorable pharmacokinetic properties capable of disrupting CD28-B7 interactions in translational models of T cell activation. Experimental ApproachStructure-based virtual screening was conducted using pharmacophore filtering and consensus docking against the human CD28 ectodomain. Hit compounds were validated using temperature-related intensity change (TRIC) and microscale thermophoresis (MST). Functional antagonism was assessed through ELISA, NanoBit luciferase complementation, and a CD28 Blockade Bioassay. In vitro ADME and safety pharmacology profiling were performed, and immunosuppressive activity was evaluated in tumor-PBMC and mucosal-PBMC co-culture assays. Key ResultsLead compound 22VS bound CD28 in biophysical screening, targeting a lipophilic canyon anchored by K24, Q25, and P27. 22VS inhibited CD28-CD80/CD86 interactions in ELISA and cell-based assays with submicromolar potency. 22VS robustly suppressed T cell activation markers in both tumor- PBMC and human mucosal epithelial-PBMC co-culture models, phenocopying the anti-CD28 biologic FR104. It showed no cytotoxicity up to 300 {micro}M and exhibited high solubility, low clearance, strong membrane permeability, and minimal off-target effects in pharmacokinetic screens. Conclusion and ImplicationsThis study identifies a novel druggable site on CD28 and validates 22VS as a selective, non-toxic small molecule inhibitor with translational potential for immune modulation in autoimmunity, transplantation, and cancer. Bullet Point SummaryO_ST_ABSWhat is already knownC_ST_ABSO_LICD28 is a key T cell costimulatory receptor essential for immune activation. C_LIO_LISmall-molecule inhibitors of CD28 are largely unexplored compared to biologics. C_LI What this study addsO_LIIdentifies a novel druggable pocket on CD28 via structure-based virtual screening. C_LIO_LIDiscovers 22VS, a selective small molecule CD28 inhibitor with cellular activity. C_LIO_LIDemonstrates that 22VS suppresses T cell activation in tumor-PBMC and mucosal-PBMC co- culture assays, phenocopying a benchmark biologic (FR104). C_LIO_LIEstablishes 22VS as a drug-like compound with favorable in vitro pharmacokinetic properties, including metabolic stability, permeability, and low off-target toxicity. C_LI Clinical significanceO_LIHighlights the potential of 22VS as a lead for immunomodulatory therapeutic development. C_LIO_LISupports small-molecule targeting of CD28-B7 interactions in T cell-driven diseases. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/665260v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@f1d2forg.highwire.dtl.DTLVardef@451c98org.highwire.dtl.DTLVardef@158446corg.highwire.dtl.DTLVardef@1f42ef1_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

MDPath: Unraveling Allosteric Communication Paths of Drug Targets through Molecular Dynamics Simulations

Understanding allosteric communication in proteins remains a critical challenge for structure-based, rational drug design. We present MDPath, a Python toolkit for analyzing allosteric communication paths in molecular dynamics simulations using NMI-based analysis. We demonstrate MDPaths ability to identify both established and novel GPCR allosteric mechanisms using the {beta}2-adrenoceptor, adenosine A2A receptor, and {micro}-opioid receptor as model systems. The toolkit reveals ligand-specific allosteric effects in {beta}2-adrenoceptor and MOR, illustrating how protein-ligand interactions drive conformational changes. Analysis of ABL1 kinase in complex with allosteric and orthosteric inhibitors demonstrates the broader applicability of the approach. Ultimately, MDPath provides an open-source framework for mapping allosteric communication within proteins, advancing structure-based drug design (https://github.com/wolberlab/mdpath).

bioinformatics↗

bioRxiv2024

From Virtual Screens to Cellular Target Engagement: New Small Molecule Ligands for the Immune Checkpoint LAG-3

Herein, we performed a virtual screening study to discover new scaffolds for small molecule-based ligands of the immune checkpoint lymphocyte-activation gene 3 (LAG-3). Molecular dynamics (MD) simulations using the LAG-3 structure revealed two putative binding sites for small molecules: the antibody interface and a lipophilic canyon. A 3D pharmacophore screening resulted in the identification of potential ligands for these binding sites and afforded a library of 25 compounds. We then evaluated the screening hits for LAG-3 binding via microscale thermophoresis (MST) and surface plasmon resonance (SPR). Our biophysical screening identified two binders with KD values in the low micromolar range, compounds 3 (antibody interface) and 25 (lipophilic canyon). Furthermore, we investigated the ability of LAG-3 hits to en-gage LAG-3 on a cellular level using a cellular thermal shift assay (CETSA), where compound 3 emerged as a promising candidate for future development.

pharmacology and toxicology↗

bioRxiv2022

Crystal structures of glycoprotein D of equine alphaherpesviruses reveal potential binding sites to the entry receptor MHC-I

Cell entry of most alphaherpesviruses is mediated by the binding of glycoprotein D (gD) to different cell surface receptors. Equine herpesvirus type 1 (EHV-1) and EHV-4 gDs interact with equine major histocompatibility complex I (MHC-I) to initiate entry into equine cells. We have characterized the gD-MHC-I interaction by solving the crystal structures of EHV-1 and EHV-4 gDs (gD1, gD4), performing protein-protein docking simulations, surface plasmon resonance (SPR) analysis, and biological assays. The structures of gD1 and gD4 revealed the existence of a common V-set immunoglobulin-like (IgV-like) core comparable to those of other gD homologs. Molecular modeling yielded plausible binding hypotheses and identified key residues (F213 and D261) that are important for virus binding. Altering the key residues resulted in impaired virus growth in cells, which highlights the important role of these residues in the gD-MHC-I interaction. Taken together, our results add to our understanding of the initial herpesvirus-cell interactions and will contribute to the targeted design of antiviral drugs and vaccine development. Author summaryEquine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) are endemic in horses and cause great suffering as well as substantial economic losses to the equine industry. Current vaccines do not prevent infections and treatment is difficult. A prerequisite for vaccine and drug development is an in-depth understanding of the virus replication cycle, especially the virus entry process in order to block the infection at early stages. Entry of alphaherpesviruses into the host cell is mediated by a set of virus envelope glycoproteins including glycoprotein D (gD) that triggers the internalization of the virus particle. The structure of gD and the interaction with the entry receptor equine major histocompatibility complex class I (MHC-I) remains elusive. Here, we solved the crystal structures of gD1 and gD4 that allowed us to model virus-receptor interaction and to determine the key residues for virus entry. Alterations of these key residues impaired virus growth in cell culture. The overall structure of gD1 and gD4 shows classical features of other alphaherpesvirus gDs making it possible to gain further insights into human pathogens as well.

molecular biology↗

bioRxiv2020

ACE2-Variants Indicate Potential SARS-CoV-2-Susceptibility in Animals: An Extensive Molecular Dynamics Study

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late 2019 and since evolved into a global threat with nearly 4.4 million infected people and over 290,000 confirmed deaths worldwide.1 SARS-CoV-2 is an enveloped virus presenting spike (S) glycoproteins on its outer surface. Binding of S to host cell angiotensin converting enzyme 2 (ACE2) is thought to be critical for cellular entry. The host range of the virus extends far beyond humans and non-human primates. Natural and experimental infections have confirmed high susceptibility of cats, ferrets, and hamsters, whereas dogs, mice, rats, pigs, and chickens seem refractory to SARS-CoV-2 infection. To investigate the reason for the variable susceptibility observed in different species, we have developed molecular descriptors to efficiently analyze our dynamic simulation models of complexes between SARS-CoV-2 S and ACE2. Based on our analyses we predict that: (i) the red squirrel is likely susceptible to SARS-CoV-2 infection and (ii) specific mutations in ACE2 of dogs, rats, and mice render them susceptible to SARS-CoV-2 infection.

pharmacology and toxicology↗

bioRxiv2019

BiasDB: A Comprehensive Database for Biased GPCR Ligands

G protein-coupled receptors transmit signals across membranes via interaction with intracellular binding partners. While there is an imprinted signaling profile for each receptor, biased ligands are able to shift intracellular pathways resulting in different recruitment profiles. We present the first comprehensive database of all literature-known biased ligands as a resource for medicinal chemistry and pharmacology. In addition to careful manual curation, we provide an analysis of the data. BiasDB is available at https://biasdb.drug-design.de/.

pharmacology and toxicology↗