Biology subjects

Brunsveld, L.

Publications and source records attributed to Brunsveld, L..

12 recordsLinked to original sources

bioRxiv2025

Induced ubiquitination of the partially disordered Estrogen Receptor alpha protein via a 14-3-3-directed molecular glue-based PROTAC design

Proteins lacking defined ligandable pockets remain challenging drug targets. Here, we develop a molecular glue-based PROTAC (MGPROTACs) approach that chemically conjugates a molecular glue stabilizer to a VHL-recruiting ligand to capture and ubiquitinate the 14-3-3/Estrogen Receptor (ER) complex. Our designed MGPROTACs engage a composite interface between 14-3-3 and the disordered F-domain of ER, promoting cooperative complex formation and target ubiquitination. Biophysical characterization revealed distinct linker-dependent cooperativities across the MGPROTAC series, which influenced both cellular permeability and ubiquitination efficiency. Cryo-EM of the most cooperative MGPROTAC uncovers de novo VHL-14-3-3{zeta} contacts, while molecular dynamics simulations rationalize the stabilizing interactions underlying cooperativity. Strikingly, fine-tuning linker design enables selective ubiquitination of distinct complex subunits. These findings establish a structural and mechanistic framework for integrating molecular glue and PROTAC principles, expanding the scope of drug discovery to previously intractable protein complexes.

biochemistry↗

bioRxiv2025

Modulation of the 14-3-3σ/C-RAF autoinhibited complexby molecular glues

Molecular glues, compounds that bind cooperatively at protein-protein interfaces are revolutionizing chemical biology and drug discovery, allowing the modulation of traditional "undruggable" targets. Here, we focus on the native protein-protein interaction (PPI) of C-RAF, a key component of the MAPK signaling pathway, with the scaffolding protein 14-3-3. Although extensive drug discovery efforts have focused on the MAPK pathway, its central role in oncology and developmental disorders (RASopathies), still requires alternative approaches, moving beyond direct kinase inhibition. Indeed, stabilization of native PPIs is a relatively unexplored territory in this pathway. The function of C-RAF is regulated on multiple levels including dimerization, phosphorylation and complex formation with the hub protein 14-3-3. 14-3-3 prevents C-RAF activation by molecular recognition and binding at the phospho-serine 259. We used a fragment-merging approach to design a molecular glue scaffold that would bind to the composite surface of the 14-3-3/C-RAF "auto"inhibited complex. The synthesized molecular glues stabilized the 14-3-3/C-RAF complex up to 300-fold in biophysical assays; their glue-based mechanism of action was confirmed with several crystal structures of ternary complexes. Selectivity among the other RAF isoforms and other RAF phosphorylation sites was evaluated with biophysical assays. The best compounds showed excellent selectivity among a broad panel of 80 14-3-3 clients. Validation in cell assays showed on-target engagement, enhanced phosphorylation levels of the C-RAF pS259 site, reduced RAF dimerization and reduced ERK phosphorylation. Overall, this approach enables chemical biology studies on a C-RAF site that is intrinsically disordered prior to 14-3-3 binding and has not been targeted previously. These molecular glues will be useful as chemical probes and starting points for further drug discovery efforts to elucidate the effect of native PPI stabilization in the MAPK pathway with applications in oncology and RASopathies.

biochemistry↗

bioRxiv2024

Estrogen Receptor alpha/14-3-3 molecular glues as alternative treatment strategy for endocrine resistant breast cancer

Endocrine resistance in breast cancer treatment is a major clinical hurdle, causing an urgent need for alternative treatment modalities. The suppressive protein-protein interaction (PPI) between Estrogen Receptor alpha (ER) and the adaptor protein 14-3-3 offers such a strategy. Here, we report the biological impact of small-molecule molecular glues of this ER/14-3-3 PPI by using both fusicoccin-derived semi-synthetic natural products and fully synthetic covalent drug-like molecules. We show that the ER/14-3-3 PPI is stabilized by both the natural- and synthetic glues, resulting in a suppression of ER transcriptional activity and a blockade of breast cancer cell proliferation, both in cell lines and in organoids derived from endocrine therapy resistant breast cancer patients. Importantly, the molecular glues effectively blocked ER action even in case of constitutively active clinical ER mutations, providing the foundations for developing alternative classes of ER targeting compounds to improve treatment of patients with endocrine-therapy resistant breast cancer. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/591105v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@173545corg.highwire.dtl.DTLVardef@a78ab7org.highwire.dtl.DTLVardef@183f4b6org.highwire.dtl.DTLVardef@371606_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

14-3-3 binding regulates Tau assembly and microtubule association.

The microtubule (MT) association of protein Tau is decreased upon phosphorylation. Increased levels of phosphorylated Tau in the cytosol pose the risk of pathological aggregation, as observed in neurodegenerative diseases. We show that binding of 14-3-3{zeta} enhances cytosolic Tau solubility by promoting phosphorylated Tau removal from MTs, while simultaneously inhibiting Tau aggregation both directly and indirectly via suppression of condensate formation. These 14-3-3{zeta} activities depend on site-specific binding of 14-3-3 to Tau phosphorylated at S214 and S324. At sub-stoichiometric 14-3-3{zeta} concentrations, or in the presence of other 14-3-3{zeta} binding partners, multivalent electrostatic interactions promote Tau:14-3-3{zeta} co-condensation, offering a phosphorylation-independent mode of Tau-14-3-3{zeta} interactions. Given the high abundance of 14-3-3 proteins in the brain, 14-3-3 binding could provide efficient multi-modal chaperoning activity for Tau in the healthy brain and be important for preventing Tau aggregation in disease.

biochemistry↗

bioRxiv2024

Molecular glues of the regulatory ChREBP/14-3-3 complex protect beta cells from glucolipotoxicity.

The Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-responsive transcription factor (TF) with two major splice isoforms ( and {beta}). In chronic hyperglycemia and glucolipotoxicity, ChREBP-mediated ChREBP{beta} expression surges, leading to insulin-secreting {beta}-cell dedifferentiation and death. 14-3-3 binding to ChREBP results in cytoplasmic retention and suppression of transcriptional activity. Thus, small molecule-mediated stabilization of this protein-protein interaction (PPI) may be of therapeutic value. Here, we show that structure-based optimizations of a molecular glue compound led to potent ChREBP/14-3-3 PPI stabilizers with cellular activity. In primary human {beta}-cells, the most active compound retained ChREBP in the cytoplasm, and efficiently protected {beta}-cells from glucolipotoxicity while maintaining {beta}-cell identity. This study may thus not only provide the basis for the development of a unique class of compounds for the treatment of Type 2 Diabetes but also showcases an alternative molecular glue approach for achieving small molecule control of notoriously difficult to target TFs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/580675v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@18a17c8org.highwire.dtl.DTLVardef@11f6c0forg.highwire.dtl.DTLVardef@1ef4e63org.highwire.dtl.DTLVardef@34799_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Crosstalk interactions between transcription factors ERRalpha and PPARalpha assist PPARalpha-mediated gene expression

The peroxisome proliferator-activated receptor (PPAR) is a crucial transcription factor governing genes associated with fatty acid {beta}-oxidation. How various interacting proteins modulate PPARs transcriptional function remains incompletely understood. Employing an unbiased mammalian protein-protein interaction trap with liganded PPAR as bait, we identified an interaction with the orphan nuclear receptor estrogen-related receptor (ERR). Random mutagenesis scanning of PPARs ligand-binding domain and coregulator profiling experiments implicated bridging coregulators, while in vitro studies suggested a trimeric interaction involving RXR. The PPAR{middle dot}ERR interaction, dependent on three C-terminal residues within ERRs helix 12, was reinforced by PGC1 and serum deprivation. Pharmacological inhibition of ERR reduced its interaction with ligand-activated PPAR, revealing a transcriptome indicative of ERR functioning as a transcriptional repressor on prototypical PPAR target genes. Intriguingly, ERR exhibited opposite behavior on other PPAR targets, including the isolated PDK4 enhancer. Chromatin immunoprecipitation analyses demonstrated PPAR ligand-dependent recruitment of ERR onto specific chromatin regions where PPAR binds in mouse livers. These findings highlight intricate transcriptional crosstalk mechanisms between PPAR and ERR, suggesting a multi-layered regulatory network fine-tuning PPARs activity as a nutrient-sensing transcription factor.

molecular biology↗

bioRxiv2023

A Systematic Approach to the Discovery of Protein-Protein Inter-action Stabilizers

Protein-protein interactions (PPIs) are responsible for the proper function of biological processes and, when dysregulated, commonly lead to disease. PPI stabilization has only recently been systematically explored for drug discovery despite being a powerful approach to selectively target intrinsically disordered proteins and hub proteins, like 14-3-3, with multiple interaction partners. Disulfide tethering is a site-directed fragment-based drug discovery (FBDD) methodology for screening small molecules in a quantitative, high-throughput manner. We explore the scope of the disulfide tethering technology for the discovery of selective fragments as starting points for the development of potent small molecule PPI stabilizers and molecular glues using the hub protein 14-3-3{sigma}. The complexes with 5 biologically and structurally diverse phospho-peptides, derived from the 14-3-3 client proteins ER, FOXO1, C-RAF, USP8, and SOS1, were screened for hit identification. Stabilizing fragments could be found for 4/5 client complexes with a diversified hit-rate and stabilizing efficacy for the different 14-3-3/client phospho-peptides. Extensive structural elucidation revealed the ability and adaptivity of the peptide to make productive interactions with the tethered fragments as key criterion for cooperative complex formation. We validated eight fragment stabilizers, six of which showed selectivity for one phospho-peptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious of these fragments increased 14-3-3{sigma}/C-RAF phospho-peptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3{sigma} provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover molecular glues.

biochemistry↗

bioRxiv2022

Early identification of cooperative fragments for protein-protein interaction stabilization

Modulating protein-protein interactions (PPIs) is an effective approach to drug discovery, with several drugs in the clinic that inhibit PPIs. The orthogonal approach of PPI stabilization has developed slowly, a function of the complicated dynamics of multi-component protein complexes. In contrast to PPI inhibition, where ligand affinity is the driving parameter for efficacy, cooperativity is frequently the directing variable for PPI stabilization. Here we show how STD NMR allows for early-stage detection of cooperativity using the hub protein 14-3-3, a focused library of fragments and several 14-3-3 partner proteins. Further, we validate that the observed enhancement in STD signal is a function of cooperativity of the ternary 14-3-3 complex, using mutagenesis and X-ray crystallography. Additionally, we assess the differential cooperativity of three fragments in a panel of 14-3-3 interaction partners. Finally, we demonstrate how selective 14-3-3 complex formation is a function of cooperativity effects

biochemistry↗

bioRxiv2022

Macrocycle-stabilization of its interaction with 14-3-3 increases plasma membrane localization and activity of CFTR

Impaired activity of the chloride channel CFTR is the cause of cystic fibrosis. 14-3-3 proteins have been shown to stabilize CFTR and increase its biogenesis and activity. Here, we report the identification and mechanism of action of a macrocycle stabilizing the 14-3-3/CFTR complex, a first-in-class molecular glue. This molecule rescues plasma membrane localization and chloride transport of F508del-CFTR and works additively with the CFTR pharmacological chaperone corrector lumacaftor (VX-809).

biochemistry↗

bioRxiv2021

A general framework for straightforward model construction of multi-component thermodynamic equilibrium systems

Mathematical modelling of molecular systems helps elucidating complex phenomena in (bio)chemistry. However, equilibrium conditions in systems consisting of more than two components can typically not be analytically determined without assumptions and resulting (semi-)numerical models are not trivial to derive by the non-expert. Here we present a framework for equilibrium models that utilizes a general derivation method capable of generating custom models for complex molecular systems, based on the simple, reversible reactions describing these systems. Several molecular systems are revisited via the framework and demonstrate the simplicity, the generality and validity of the approach. The ease of use of the framework and the ability to both analyze systems and gain additional insights in the underlying parameters strongly aids the analysis and understanding of molecular equilibrium systems. This conceptual framework severely reduces the time and expertise requirements which currently impede the broad integration of these highly valuable models into chemical research.

biochemistry↗

bioRxiv2020

Molecular basis for inhibition of adhesin-mediated bacterial-host interactions through a novel peptide-binding domain

Modulation of protein-protein interactions (PPIs) with small-molecules is a promising conceptual approach in drug discovery. In the area of bacterial colonization, PPIs contribute to adhesin-mediated biofilm formation that cause most infections. However, the molecular basis underlying these adhesin-ligand interactions is largely unknown. The 1.5-MDa adhesion protein, MpIBP, uses a peptide-binding domain (MpPBD) to help its Antarctic bacterium form symbiotic biofilms on sea ice with microalgae such as diatoms. X-ray crystallography revealed MpPBD uses Camdependent interactions to self-associate with a crystal symmetry mate via the C-terminal threonine-proline-aspartate sequence. Structure-guided optimization derived penta-peptide ligands that bound MpPBD 1,000-fold more tightly, with affinities in the nano-molar range. These ligands act as potent antagonists to block MpPBD from binding to the diatom cells. Since adhesins of some human pathogens contain peptide-binding module homologs of MpPBD, this same conceptual approach could help develop ligand-based PPI modulators to disrupt harmful bacteria-host interactions.

microbiology↗

bioRxiv2019

A DNA-based synthetic apoptosome

Living cells are able to regulate key cellular processes by physically assembling signaling components on dedicated molecular platforms. The spatial organization of proteins in these higher-order signaling complexes facilitates proximity-driven activation and inhibition events, allowing tight regulation of the flow of information. Here, we employ the programmability and modularity of DNA origami as a controllable molecular platform for studying protein-protein interactions involved in intracellular signaling. Specifically, we engineer a synthetic, DNA origami-based version of the apoptosome, a large multi-protein signaling complex that regulates apoptosis by co-localization of multiple caspase-9 monomers. Our in vitro characterization using both wildtype caspase-9 monomers and inactive mutants tethered to a DNA origami platform directly demonstrates that enzymatic activity is induced by proximity-driven dimerization with asymmetric, half-of-sites reactivity. Additionally, experimental results supported by a detailed thermodynamic model reveal a multivalent activity enhancement in tethered caspase-9 oligomers of three and four enzymes, partly originating from a statistical increase in the number of active catalytic units in higher-order enzyme clusters. Our results offer fundamental insights in caspase-9 activity regulation and demonstrate that DNA origami provides a modular platform to construct and characterize higher-order signaling complexes. The engineered DNA-based protein assembly platform has the potential to be broadly applied to inform the function of other important multi-enzyme assemblies involved in inflammation, innate immunity, and necrosis.

synthetic biology↗