Biology subjects

Loehr, F.

Publications and source records attributed to Loehr, F..

5 recordsLinked to original sources

bioRxiv2025

Overcoming Ligand Discovery Challenges: Developing Peptide-Based Tracers for SPSB2

Developing new E3 ligase ligands for the design of heterobivalent molecules, such as PROteolysis TArgeting Chimeras (PROTACs), requires careful evaluation of target engagement (TE). Characterizing protein-protein interactions (PPIs) is therefore essential in drug discovery, as it enables the assessment of ligand binding to sites that are often difficult to target. Degrons, peptide motifs recognized by E3 ligases, may serve as valuable starting points for designing E3 ligands. However, many degrons are highly polar and lack intrinsic membrane permeability, requiring alternative strategies for efficient cellular delivery. In this study, we used the SPRY domain-containing SOCS box protein 2 (SPSB2) E3 ligase as a model system to develop TE strategies for in vitro and in cellulo using polar degron-based peptides. By conjugating various polycationic cell-penetrating peptides (CPPs) to the degron sequence, we present a study demonstrating efficient cellular delivery. We obtained a high-resolution crystal structure and used various biophysical techniques to assess the influence of each modification, while confocal microscopy and BRET-based assays confirmed successful cellular delivery as well as potent target engagement. O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/673904v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@191848borg.highwire.dtl.DTLVardef@94fae6org.highwire.dtl.DTLVardef@e43016org.highwire.dtl.DTLVardef@110f9f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Targeting LC3/GABARAP for degrader development and autophagy modulation

Recent successes in developing small-molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the target recruitment to LC3/GABARAP, a family of membrane-bound proteins that tether autophagy receptors to the autophagosome. In order to validate the existing ligands, we rigorously tested target engagement of reported ATTEC ligands and handles. Surprisingly, using various biophysical methods, most available ligands did not interact with their designated target LC3. Intrigued by the idea of developing ATTECs, we evaluated the druggability of LC3/GABARAP by in silico docking and large scale crystallographic fragment screening. The data revealed that most fragments bound to the HP2, but not the HP1 pocket of the LC3-interacting region (LIR) docking site, suggesting favorable druggability of this binding pocket. Here, we present diverse comprehensively validated ligands for future ATTEC development.

biochemistry↗

bioRxiv2021

Design, Synthesis and Evaluation of WD-repeat containing protein 5 (WDR5) degraders

Histone H3K4 methylation serves as post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferases (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat containing protein 5 (WDR5) that has also been associated with controlling long non-coding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMTs complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic function. This study presents the design, synthesis and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.

cancer biology↗

bioRxiv2019

p63 sets the threshold for induction of apoptosis using a kinetically encoded ‘doorbell-like’ mechanism

Cell fate decisions such as apoptosis require cells to translate signaling input into a binary yes/no response. A tight control of the process is required to avoid loss of cells by accidental activation of cell death pathways. One particularly critical situation exists in primary oocytes because their finite number determines the reproductive capacity of females. On the one hand a stringent genetic quality control is necessary to maintain the genetic integrity of the entire species; on the other hand an overly stringent mechanism that kills oocytes with even minor DNA damage can deplete the whole primary oocyte pool leading to infertility. The p53 homolog TAp63 is the key regulator of genome integrity in oocytes. After DNA damage TAp63 is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer. By measuring activation kinetics in ovaries and single site phosphorylation kinetics in vitro with peptides and full length protein we show that TAp63 phosphorylation follows a biphasic behavior. While the first two CK1 phosphorylation events are fast, the third one that constitutes the decisive step to form the active conformation is slow. We reveal the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63 substrate interaction and demonstrate by quantitative simulation that the slow phosphorylation phase determines the threshold of DNA damage required for induction of apoptosis.

biochemistry↗

bioRxiv2016

Structural and Functional analysis of the GABARAP Interaction Motif (GIM).

Through the canonical LC3 interaction motif (LIR), [W/F/Y]-X1-X2-[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence (W/F-V-X2-V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1-LIR is indeed eleven-fold more specific for GABARAP than LC3B. Selective mutation of the X1 and X2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1-GIM selectivity 20-fold towards LC3B. Finally, we show that conversion of the canonical p62/SQSTM1-LIR into our newly defined GIM, by introducing two valine residues, enhances p62/SQSTM1 interaction with endogenous GABARAP over LC3B. The identification of a GABARAP-specific interaction motif will aid the identification and characterization of the continually expanding array of autophagy receptor and adaptor proteins and their in vivo functions.

biochemistry↗