Leibniz-ForschungsInstitut für Molekulare Pharmakologie
Recent publications
Tryptophan hydroxylases catalyze the first and rate-limiting step in the synthesis of serotonin. Serotonin is a key neurotransmitter in the central nervous system and, in the periphery, functions as a local hormone with multiple physiological functions. Studies in genetically altered mouse models have shown that dysregulation of peripheral serotonin levels leads to metabolic, inflammatory, and fibrotic diseases. Overproduction of serotonin by tumor cells causes severe symptoms typical for the carcinoid syndrome, and tryptophan hydroxylase inhibitors are already in clinical use for patients suffering from this disease. Here, we describe a novel class of potent tryptophan hydroxylase inhibitors, characterized by spanning all active binding sites important for catalysis, specifically those of the cosubstrate pterin, the substrate tryptophan as well as directly chelating the catalytic iron ion. The inhibitors were designed to efficiently reduce serotonin in the periphery while not passing the blood-brain barrier, thus preserving serotonin levels in the brain.
Blinding diseases that are caused by degeneration of rod and cone photoreceptor cells often spare the rest of the retinal circuit, from bipolar cells, which are directly innervated by photoreceptor cells, to the output ganglion cells that project axons to the brain. A strategy for restoring vision is to introduce light sensitivity to the surviving cells of the retina. One approach is optogenetics, in which surviving cells are virally transfected with a gene encoding a signaling protein that becomes sensitive to light by binding to the biologically available chromophore retinal, the same chromophore that is used by the opsin photo-detectors of rods and cones. A second approach uses photopharmacology, in which a synthetic photoswitch associates with a native or engineered ion channel or receptor. We review these approaches and look ahead to the next generation of advances that could reconstitute core aspects of natural vision.
Introduction: The G-protein coupled receptor LPAR5 plays a prominent role in LPA-mediated pain and itch signaling. In this study we focus on the LPAR5-antagonist compound 3 (cpd3) and its ability to affect pain and itch signaling, both in vitro and in vivo. Methods: Nociceptive behavior in wild type mice was induced by formalin, carrageenan or prostaglandin E2 (PGE2) injection in the hind paw, and the effect of oral cpd3 administration was measured. Scratch activity was measured after oral administration of cpd3, in mice overexpressing phospholipase A2 (sPLAtg2), in wild type mice (WT) and in TRPA1-deficient mice (Trpa1 KO). In vitro effects of cpd3 were assessed by measuring intracellular calcium release in HMC-1 and HEK-TRPA1 cells. Results: As expected, nociceptive behavior (induced by formalin, carrageenan or PGE2) was reduced after treatment with cpd3. Unexpectedly, cpd3 induced scratch activity in mice. In vitro addition of cpd3 to HEK-TRPA1 cells induced an intracellular calcium wave that could be inhibited by the TRPA1-antagonist A-967079. In Trpa1 KO mice, however, the increase in scratch activity after cpd3 administration was not reduced. Conclusions: Cpd3 has in vivo antinociceptive effects but induces scratch activity in mice, probably by activation of multiple pruriceptors, including TRPA1. These results urge screening of antinociceptive candidate drugs for activity with pruriceptors.
Ion channels mediate information from the outside to the inside of cells, and are particularly important in electrical excitability. Several tissues rely on this transmission, such as the brain, the heart, and the pancreas, and accordingly, ion channels are not only in the focus of basic research but also for drug development programs. While traditional pharmacology has its limitations in terms of selectivity and speed, approaches to use light as a trigger for channel function have been developed in the last 15 years, coining the term “photopharmacology.” Herein, we highlight how remote control of channel activity with light can achieved and how it shapes our understanding of channel function in different organs.
Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is the actin ADP-ribosyltransferase TccC3, whose activity leads to the clustering of the cellular cytoskeleton and ultimately cell death. Here, we show in atomic detail how TccC3 modifies actin. We find that the ADP-ribosyltransferase does not bind to G-actin but interacts with two consecutive actin subunits of F-actin. The binding of TccC3 to F-actin occurs via an induced-fit mechanism that facilitates access of NAD+ to the nucleotide binding pocket. The following nucleophilic substitution reaction results in the transfer of ADP-ribose to threonine-148 of F-actin. We demonstrate that this site-specific modification of F-actin prevents its interaction with depolymerization factors, such as cofilin, which impairs actin network turnover and leads to steady actin polymerization. Our findings reveal in atomic detail a mechanism of action of a bacterial toxin through specific targeting and modification of F-actin. Entomopathogenic bacteria used for pest control secrete potent Tc toxins. Here, the authors combine biochemistry, solution and solid-state NMR spectroscopy and cryo-EM to show in atomic detail how the toxin disrupts the host cell cytoskeleton and kills the target cell.
We report the density functional theory (DFT) guided discovery of ethynyl‐triazolyl‐phosphinates (ETP) as a new class of electrophilic warheads for cysteine selective bioconjugation. By using Cu I ‐catalysed azide alkyne cycloaddition (CuAAC) in aqueous buffer we were able to access a variety of functional electrophilic building blocks, including proteins, from diethynyl‐phosphinate. ETP‐reagents were used to obtain fluorescent peptide‐conjugates for receptor labelling on live cells and a stable and a biologically active antibody‐drug‐conjugate. Moreover, we were able to incorporate ETP‐electrophiles into an azide‐containing ubiquitin under native conditions and demonstrate their potential in protein‐protein conjugation. Finally, we showcase the excellent cysteine‐selectivity of this new class of electrophile in mass spectrometry based, proteome‐wide cysteine profiling, underscoring the applicability in homogeneous bioconjugation strategies to connect two complex biomolecules.
We report the density functional theory (DFT) guided discovery of ethynyl‐triazolyl‐phosphinates (ETP) as a new class of electrophilic warheads for cysteine selective bioconjugation. By using Cu I ‐catalysed azide alkyne cycloaddition (CuAAC) in aqueous buffer we were able to access a variety of functional electrophilic building blocks, including proteins, from diethynyl‐phosphinate. ETP‐reagents were used to obtain fluorescent peptide‐conjugates for receptor labelling on live cells and a stable and a biologically active antibody‐drug‐conjugate. Moreover, we were able to incorporate ETP‐electrophiles into an azide‐containing ubiquitin under native conditions and demonstrate their potential in protein‐protein conjugation. Finally, we showcase the excellent cysteine‐selectivity of this new class of electrophile in mass spectrometry based, proteome‐wide cysteine profiling, underscoring the applicability in homogeneous bioconjugation strategies to connect two complex biomolecules.
Natural product dimers have intriguing structural features and often have remarkable pharmacological activities. We report here two uncommon marine gorgonian-derived symmetric dimers, weizhouochrones A (1) and B (2), with indenone-derived monomers, that were isolated from the coral Anthogorgia ochracea collected from the South China Sea. These dimers are difficult targets for structure elucidation that solely relies upon conventional NMR data such as NOEs and J-couplings. Here, to explore the application of emerging methods on the structure elucidation of challenging molecules, we explored a number of different anisotropic and computational NMR approaches. The measurements of anisotropic NMR parameters of weizhouochrone A, including residual dipolar couplings (RDCs) and residual chemical shift anisotropy (RCSA), allowed us to successfully determine the planar structure and its relative configuration. This result was corroborated by a computational NMR analysis based on DP4+ probability and computer-assisted 3D structure elucidation (CASE-3D).
We report an efficient method to install electrophilic cysteine-selective ethynyl-phosphonamidates on peptides during Fmoc-based solid phase peptide synthesis (SPPS). By performing Staudinger-phosphonite reactions between different solid supported azido-peptides and varying ethynylphosphonites, we obtained ethynyl-phosphonamidate containing peptidic compounds after acidic deprotection, including an electrophilic cell-penetrating peptide that showed high efficiency as an additive for cellular delivery of proteins.
Epilepsy is one of the most frequent neurological diseases, with focal epilepsy accounting for the largest number of cases. The genetic alterations involved in focal epilepsy are far from being fully elucidated. Here, we show that defective lipid signalling caused by heterozygous ultra-rare variants in PIK3C2B, encoding for the class II phosphatidylinositol 3-kinase PI3K-C2β, underlie focal epilepsy in humans. We demonstrate that patients' variants act as loss-of-function alleles, leading to impaired synthesis of the rare signalling lipid phosphatidylinositol 3,4-bisphosphate, resulting in mTORC1 hyperactivation. In vivo, mutant Pik3c2b alleles caused dose-dependent neuronal hyperexcitability and increased seizure susceptibility, indicating haploinsufficiency as a key driver of disease. Moreover, acute mTORC1 inhibition in mutant mice prevented experimentally induced seizures, providing a potential therapeutic option for a selective group of patients with focal epilepsy. Our findings reveal an unexpected role for class II PI3K-mediated lipid signalling in regulating mTORC1-dependent neuronal excitability in mice and humans.
Discovery of protein‐binding fragments for precisely defined binding sites is an unmet challenge so far. Here, we investigate formylglycine as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site. Formylglycine peptide 3 was derived from a phosphotyrosine‐containing peptide substrate of protein tyrosine phosphatase PTP1B by replacing the phosphorylated amino acid with the reactive electrophile. Fragment ligation with formylglycine occurred in‐situ in aqueous physiological buffer. Structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non‐fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low‐affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4‐amino‐phenyl‐acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.
During viral cell entry, the spike protein of SARS‐CoV‐2 binds to the α1‐helix motif of human angiotensin‐converting enzyme 2 (ACE2). Thus, alpha‐helical peptides mimicking this motif may serve as inhibitors of viral cell entry. For this purpose, we employed the rigidified diproline‐derived module ProM‐5 to induce α‐helicity in short peptide sequences inspired by the ACE2 α1‐helix. Starting with Ac‐QAKTFLDKFNHEAEDLFYQ‐NH 2 as a relevant section of α1, a series of peptides, N‐ capped with either Ac‐βHAsp‐[ ProM‐5 ] or Ac‐βHAsp‐PP, were prepared and their α‐helicities were investiga­ted. While ProM‐5 clearly showed a pro­nounced effect, an even increased degree of helicity (up to 63%) was observed in sequences in which non‐binding amino acids were replaced by alanine. The binding affinities of the peptides towards the spike protein, as determined by means of microscale thermo­phoresis (MST), revealed only a subtle influence of the α‐helical content and, noteworthy, led to the identification of an Ac‐βHAsp‐PP‐capped peptide displaying a very strong binding affinity (K D = 62 nM).
Focal adhesions are multifunctional organelles that couple cell-matrix adhesion to cytoskeletal force transmission and signaling and to steer cell migration and collective cell behavior. Whereas proteomic changes at focal adhesions are well understood, little is known about signaling lipids in focal adhesion dynamics. Through the characterization of cells from mice with a kinase-inactivating point mutation in the class II PI3K-C2β, we find that generation of the phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) membrane lipid promotes focal adhesion disassembly in response to changing environmental conditions. We show that reduced growth factor signaling sensed by protein kinase N, an mTORC2 target and effector of RhoA, synergizes with the adhesion disassembly factor DEPDC1B to induce local synthesis of PtdIns(3,4)P2 by PI3K-C2β. PtdIns(3,4)P2 then promotes turnover of RhoA-dependent stress fibers by recruiting the PtdIns(3,4)P2-dependent RhoA-GTPase-activating protein ARAP3. Our findings uncover a pathway by which cessation of growth factor signaling facilitates cell-matrix adhesion disassembly via a phosphoinositide lipid switch.
Identifying the protein targets of drugs is an important but tedious process. Existing proteomic approaches enable unbiased target identification but lack the throughput needed to screen larger compound libraries. Here, we present a compound interaction screen on a photoactivatable cellulose membrane (CISCM) that enables target identification of several drugs in parallel. To this end, we use diazirine‐based undirected photoaffinity labeling (PAL) to immobilize compounds on cellulose membranes. Functionalized membranes are then incubated with protein extract and specific targets are identified via quantitative affinity purification and mass spectrometry. CISCM reliably identifies known targets of natural products in less than three hours of analysis time per compound. In summary, we show that combining undirected photoimmobilization of compounds on cellulose with quantitative interaction proteomics provides an efficient means to identify the targets of natural products.
Inositol pyrophosphates (PP-InsPs) constitute a group of highly charged messengers, which regulate central biological processes in health and disease, such as cellular phosphate and general energy homeostasis. Deciphering the molecular mechanisms underlying PP-InsP-mediated signaling remains a challenge due to the unique properties of these molecules, the different modes of action they can access, and a somewhat limited chemical and analytical toolset. Herein, we summarize the most recent mechanistic insights into PP-InsP signaling, which illustrate our progress in connecting mechanism and function of PP-InsPs.
Multiple conjugation of virus-binding ligands to multivalent carriers is a prominent strategy to construct highly affine virus binders for the inhibition of viral entry into host cells. In a previous study, we introduced rationally designed sialic acid conjugates of bacteriophages (Qβ) that match the triangular binding site geometry on hemagglutinin spike proteins of influenza A virions, resulting in effective infection inhibition in vitro and in vivo. In this work, we demonstrate that even partially sialylated Qβ conjugates retain the inhibitory effect despite reduced activity. These observations not only support the importance of trivalent binding events in preserving high affinity, as supported by computational modeling, but also allow us to construct heterobifunctional modalities. Capsids carrying two different sialic acid ligand-linker structures showed higher viral inhibition than their monofunctional counterparts. Furthermore, capsids carrying a fluorescent dye in addition to sialic acid ligands were used to track their interaction with cells. These findings support exploring broader applications as multivalent inhibitors in the future.
Synapses maintain their molecular composition, plasticity and function through the concerted action of protein synthesis and removal. The complex and polarized neuronal architecture poses specific challenges to the logistics of protein and organelle turnover since protein synthesis and degradation mainly happen in the cell soma. In addition, post-mitotic neurons accumulate damage over a lifetime, challenging neuronal degradative pathways and making them particularly susceptible to the effects of aging. This review will summarize the current knowledge on neuronal protein turnover mechanisms with a particular focus on the presynapse, including the proteasome, autophagy and the endolysosomal route and their roles in regulating presynaptic proteostasis and function. In addition, I will discuss how physiological brain aging, which entails a progressive decline in cognitive functions, affects synapses and the degradative machinery.
Intracellular organelles change their size during trafficking and maturation. This requires the transport of ions and water across their membranes. Macropinocytosis, a ubiquitous form of endocytosis of particular importance for immune and cancer cells, generates large vacuoles that can be followed optically. Shrinkage of macrophage macropinosomes depends on TPC-mediated Na⁺ efflux and Cl⁻ exit through unknown channels. Relieving osmotic pressure facilitates vesicle budding, positioning osmotic shrinkage upstream of vesicular sorting and trafficking. Here we identify the missing macrophage Cl⁻ channel as the proton-activated Cl⁻ channel ASOR/TMEM206. ASOR activation requires Na⁺-mediated depolarization and luminal acidification by redundant transporters including H⁺-ATPases and CLC 2Cl⁻/H⁺ exchangers. As corroborated by mathematical modelling, feedback loops requiring the steep voltage and pH dependencies of ASOR and CLCs render vacuole resolution resilient towards transporter copy numbers. TMEM206 disruption increased albumin-dependent survival of cancer cells. Our work suggests a function for the voltage and pH dependence of ASOR and CLCs, provides a comprehensive model for ion-transport-dependent vacuole maturation and reveals biological roles of ASOR.
Bacterial toxins gain growing attention as potential cancer treatment due to their potent cytotoxic effects. Among the very different toxins with diverse modes of action, the Clostridium perfringens enterotoxin (CPE) is in focus to treat solid cancers. This toxin targets the tight junction proteins claudin-3 and -4 (Cldn-3/4), which are frequently overexpressed in solid cancers. Binding to these claudins induces pore formation in the host cell plasma membrane leading to rapid oncoleaking cell death of tumor cells. Based on this, extending the targeting of CPE beyond Cldn-3/4 is of interest, since other claudins, such as claudin-1 or -5 are often overexpressed in various cancer entities such as non–small-cell lung cancer (NSCLC) or papillary thyroid carcinoma. In this chapter we describe the modification of a CPE-encoding vector by structure-directed mutagenesis to either preferentially target Cldn-1 and -5 or to expand targeting to Cldn1–9 for improved broadened cytotoxic targeting of claudin-overexpressing tumors such as but not limited to lung cancer via CPE gene transfer.
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163 members
Jan Schmoranzer
  • Super-Resolution Microscopy
Barth-Jan Van Rossum
  • NMR-Supported Structural Biology
Volker Haucke
  • Molecular Physiology and Cell Biology
Jens Furkert
  • Protein Trafficking
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