Biology subjects

Ottmann, C.

Publications and source records attributed to Ottmann, C..

9 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

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↗

bioRxiv2023

A simple method for developing lysine targeted covalent protein reagents

There is a growing demand for covalent tool compounds and chemical probes to investigate and perturb protein function and dysregulation. The combination of a covalent electrophile with a peptide or protein-based scaffold with an extended binding footprint enables the targeting of shallow protein surfaces, not typically addressable using small molecules. However, to fully exploit the potential of electrophilic proteins or peptides there is a need for versatile approaches to convert native peptide sequences into covalent binders that can target a broad range of residues. Here we report protein-based thio-methacrylate esters - electrophiles with a diverse reactivity profile that can be installed easily on unprotected peptides and proteins via cysteine side chains, and react efficiently and selectively with cysteine and lysine side chains on the target. Guided by computational modeling, we designed and synthesized methacrylate phosphopeptides derived from 14-3-3-binding proteins and demonstrated these peptides irreversibly label 14-3-3{sigma} via either lysine or cysteine residues, depending on the position of the electrophile. Methacrylate peptides targeting a conserved lysine residue exhibited pan-isoform binding of 14-3-3 proteins, and efficiently labeled 14-3-3 proteins in lysates, as well as secreted 14-3-3 extracellularly. The irreversible binding to the predicted target lysines were confirmed by proteomics and X-ray crystallography of the complexes. Finally, we applied this approach to develop protein-based covalent binders. A methacrylate-modified variant of the colicin E9 immunity protein irreversibly bound to the E9 DNAse, resulting in significantly higher thermal stability relative to the non-covalent complex. Our approach offers a simple and versatile route to convert peptides and proteins into potent covalent binders.

biochemistry↗

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

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

Understanding the interaction of 14-3-3 proteins with hDMX and hDM2: a structural and biophysical study

p53 plays a critical role in regulating diverse biological processes: DNA repair, cell cycle arrest, apoptosis, and senescence. The p53 pathway has therefore served as the focus for drug-discovery efforts. p53 is negatively regulated by hDMX and hDM2; prior studies have identified 14-3-3 proteins as hDMX and hDM2 client proteins. 14-3-3 proteins are adaptor proteins that modulate localisation, degradation and interactions of their targets in response to phosphorylation. Thus 14-3-3 proteins may indirectly modulate the interaction between hDMX or hDM2 and p53 and represent potential targets for modulation of the p53 pathway. In this manuscript we report on the biophysical and structural characterization of peptide/protein interactions that are representative of the interaction between 14-3-3 and hDMX or hDM2. The data establish that proximal phosphosites spaced [~]20-25 residues apart in both hDMX and hDM2 co-operate to facilitate high-affinity 14-3-3 binding and provide structural insight that can be utilized in future stabilizer/inhibitor discovery efforts.

biophysics↗

bioRxiv2021

Covalent Flexible Peptide Docking in Rosetta

Electrophilic peptides that form an irreversible covalent bond with their target have great potential for binding targets that have been previously considered undruggable. However, the discovery of such peptides remains a challenge. Here, we present CovPepDock, a computational pipeline for peptide docking that incorporates covalent binding between the peptide and a receptor cysteine. We applied CovPepDock retrospectively to a dataset of 115 disulfide-bound peptides and a dataset of 54 electrophilic peptides, for which it produced a top-five scoring, near-native model, in 89% and 100% of the cases, respectively. In addition, we developed a protocol for designing electrophilic peptide binders based on known non-covalent binders or protein-protein interfaces. We identified 7,154 peptide candidates in the PDB for application of this protocol. As a proof-of-concept we validated the protocol on the non-covalent complex of 14-3-3{sigma} and YAP1 phosphopeptide. The protocol identified seven highly potent and selective irreversible peptide binders. The predicted binding mode of one of the peptides was validated using X-ray crystallography. This case-study demonstrates the utility and impact of CovPepDock. It suggests that many new electrophilic peptide binders can be rapidly discovered, with significant potential as therapeutic molecules and chemical probes.

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↗