Leibniz-ForschungsInstitut für Molekulare Pharmakologie

The delivery of functional proteins remains a major challenge in advancing biological and pharmaceutical sciences. Herein, we describe a powerful, simple, and highly effective strategy for the intracellular delivery of functional cargoes. Previously, we demonstrated that cell-penetrating peptide (CPP) additives equipped with electrophilic thiol-reactive moieties temporarily attach to the cellular membrane, thereby facilitating the cellular uptake of protein- and antibody-CPP cargoes through direct membrane transduction at low concentrations. Now, we hypothesize that CPP-additives with an increased retention on the cellular membrane will further enhance intracellular uptake. We discovered that adding a small hydrophobic peptide sequence to an arginine-rich electrophilic CPP-additive further improved the uptake of protein-CPP conjugates, whereas larger hydrophobic anchors showed increased cytotoxicity. Cell viability and membrane integrity measurements, structure–activity relationship studies, and quantitative evaluation of protein-CPP uptake revealed important design principles for cell-surface-retained CPP-additives. These investigations allowed us to identify a nontoxic, thiol-reactive CPP-additive containing the hydrophobic ILFF sequence, which can deliver fluorescent model proteins at low micromolar concentrations. This hydrophobic CPP-additive allowed the addition of protein cargoes for intracellular delivery after initial additive incubation. Time-lapse fluorescence microscopy and membrane tension analysis of cells treated with fluorescent ILFF-CPP-additives supported the claim of increased cell surface retention and suggested that the protein-CPP cargoes enter the cell through a mechanism involving lowered cell membrane tension. Finally, we demonstrated that our newly engineered hydrophobic CPP-additive enabled the uptake of a functional macrocyclic peptidic MDM2-inhibitor and a recombinant genome editing protein. This indicates that the developed hydrophobic CPP-additive holds promise as a tool to enhance the intracellular delivery of peptide and protein cargoes.

CB₂R ligands to treat inflammatory diabetes Researchers discuss how scientific innovations might influence the discovery of future tailor-made CB2R-based anti-inflammatory treatments. The G-protein-coupled receptor (GPCR) type-2 cannabinoid receptor (CB2R) is a 2 highly promising pharmacological target for novel anti-inflammatory medicines. (1) CB2R is an essential component of a vital lipid signaling system that tremendously influences multiple human health and disease states ranging from metabolic disorders to neurodegenerative diseases and is termed endocannabinoid system (ECS). Few CB2R-activation-based drugs have been launched. (2) Several candidates are under active clinical development, aiming at treating diseases in which inflammatory processes play a significant role or are the underlying reason. Recent scientific innovations in ECS research and drug discovery in general (Figure 1) are now being incorporated into the design, synthesis and evaluation of novel ligands targeting CB2R. They will impact the generation of further improved tailor-made medicines for inflammatory diseases.

Light perception for orientation in zoospore-forming fungi is linked to homo- or heterodimeric rhodopsin-guanylyl cyclases (RGCs). Heterodimeric RGCs, first identified in the chytrid Rhizoclosmatium globosum , consist of an unusual near-infrared absorbing highly fluorescent sensitizer neorhodopsin (NeoR) that is paired with a visual light-absorbing rhodopsin responsible for enzyme activation. Here, we present a comprehensive analysis of the distribution of RGC genes in early-branching fungi using currently available genetic data. Among the characterized RGCs, we identified red-sensitive homodimeric RGC variants with maximal light activation close to 600 nm, which allow for red-light control of GTP to cGMP conversion in mammalian cells. Heterodimeric RGC complexes have evolved due to a single gene duplication within the branching of Chytridiales and show a spectral range for maximal light activation between 480 to 600 nm. In contrast, the spectral sensitivity of NeoRs is reaching into the near-infrared range with maximal absorption between 641 and 721 nm, setting the low energy spectral edge of rhodopsins so far. Based on natural NeoR variants and mutational studies, we reevaluated the role of the counterion-triad proposed to cause the extreme redshift. With the help of chimera constructs, we disclose that the cyclase domain is crucial for functioning as homo- or heterodimers, which enables the adaptation of the spectral sensitivity by modular exchange of the photosensor. The extreme spectral plasticity of retinal chromophores in native photoreceptors provides broad perspectives on the achievable spectral adaptation for rhodopsin-based molecular tools ranging from UVB into the near-infrared.

Metastasis is directly linked to poor prognosis of cancer patients and warrants search for effective anti-metastatic drugs. MACC1 is a causal key molecule for metastasis. High MACC1 expression is prognostic for metastasis and poor survival. Here, we developed novel small molecule inhibitors targeting MACC1 expression to impede metastasis formation. We performed a human MACC1 promoter-driven luciferase reporter-based high-throughput screen (HTS; 118.500 compound library) to identify MACC1 transcriptional inhibitors. HTS revealed 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds as efficient transcriptional inhibitors of MACC1 expression, able to decrease MACC1-induced cancer cell motility in vitro. Structure-activity relationships identified the essential inhibitory core structure. Best candidates were evaluated for metastasis inhibition in xenografted mouse models demonstrating metastasis restriction. ADMET showed high drug-likeness of these new candidates for cancer therapy. The NFκB pathway was identified as one mode of action targeted by these compounds. Taken together, 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds are effective MACC1 inhibitors and pose promising candidates for anti-metastatic therapies particularly for patients with MACC1-overexpressing cancers, that are at high risk to develop metastases. Although further preclinical and clinical development is necessary, these compounds represent important building blocks for an individualized anti-metastatic therapy for solid cancers.

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the α C helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases.

Neurons relay information via specialized presynaptic compartments for neurotransmission. Unlike conventional organelles, the specialized apparatus characterizing the neuronal presynapse must form de novo. How the components for presynaptic neurotransmission are transported and assembled is poorly understood. Our results show that the rare late endosomal signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ] directs the axonal cotransport of synaptic vesicle and active zone proteins in precursor vesicles in human neurons. Precursor vesicles are distinct from conventional secretory organelles, endosomes, and degradative lysosomes and are transported by coincident detection of PI(3,5)P 2 and active ARL8 via kinesin KIF1A to the presynaptic compartment. Our findings identify a crucial mechanism that mediates the delivery of synaptic vesicle and active zone proteins to developing synapses.

ClC-6 is a late endosomal voltage-gated chloride-proton exchanger that is predominantly expressed in the nervous system. Mutated forms of ClC-6 are associated with severe neurological disease. However, the mechanistic role of ClC-6 in normal and pathological states remains largely unknown. Here, we present cryo-EM structures of ClC-6 that guided subsequent functional studies. Previously unrecognized ATP binding to cytosolic ClC-6 domains enhanced ion transport activity. Guided by a disease-causing mutation (p.Y553C), we identified an interaction network formed by Y553/F317/T520 as potential hotspot for disease-causing mutations. This was validated by the identification of a patient with a de novo pathogenic variant p.T520A. Extending these findings, we found contacts between intramembrane helices and connecting loops that modulate the voltage dependence of ClC-6 gating and constitute additional candidate regions for disease-associated gain-of-function mutations. Besides providing insights into the structure, function, and regulation of ClC-6, our work correctly predicts hotspots for CLCN6 mutations in neurodegenerative disorders.

Topoisomerase I (TOP1) Inhibitors constitute an emerging payload class to engineer Antibody-Drug-Conjugates (ADCs) as next-generation biopharmaceutical for cancer treatment. Existing ADCs are utilizing camptothecin payloads with lower potency and suffer from limited stability in circulation. With this study, we introduce a novel camptothecin-based linker-payload platform based on the highly potent camptothecin derivative exatecan. First, we describe general challenges that arise from the hydrophobic combination of exatecan and established dipeptidyl p-aminobenzyl-carbamate (PAB) cleavage sites such as reduced antibody conjugation yields and ADC aggregation. After evaluating several linker-payload structures we identified ethynyl-phosphonamidates in combination with a discrete PEG24 chain to compensate for the hydrophobic PAB-exatecan moiety. Furthermore, we demonstrate that the identified linker-payload structure enables the construction of highly loaded DAR8 ADCs with excellent solubility properties. Head-to-head comparison to Enhertu, an approved camptothecin-based ADC, revealed improved target-mediated killing of tumor cells, excellent bystander killing, drastically improved linker stability in vitro and in vivo and superior in vivo efficacy over four tested dose levels in a xenograft model. Moreover, we show that ADCs based on the novel exatecan linker-payload platform exhibit antibody-like pharmacokinetic properties, even when the ADCs are highly loaded with eight drug molecules per antibody. This ADC platform constitutes a new and general solution to deliver TOP1-inhibitors with highest efficiency to the site of the tumor, independent of the antibody and its target, and is thereby broadly applicable to various cancer indications.

Herein, we introduce a dual-labelling technique for covalent modification of cell surfaces with viscosity-sensitive and viscosity-insensitive fluorescent dyes. For this purpose, we combined an improved method for chemical reactions at sialic acid residues with fluorescent molecular rotor (FMR) dyes and a dye commonly used to stain nucleic acids. Fluorescence of FMR dyes increases with increasing viscosity providing options for detecting changes of cell surface viscosity during disease and therapeutic intervention. The method involves mild periodate oxidation of cell surface glycans followed by organocatalysed dye conjugation. Both steps are performed within a total of 15 min at 25 °C, avoiding cold stress of cells. We show that FMR dyes such as 9-(2-Carboxy-2-cyanovinyl)julolidine (CCVJ) or Cyanine 3 (Cy3) and the nucleic acid stain thiazole orange (TO) emit sufficiently strong signals to allow fluorescence microscopy imaging of surfaces of A549, HeLa and CEM cells. We demonstrate that the fluorescence of CCVJ and TO on cell surface glycans and isolated mucin is influenced by the action of the disulfide-cleaving agent TCEP. Co-labelling of cells with CCVJ and an environmentally insensitive AF568 dye revealed a TCEP-induced decrease in the intensity CCVJ fluorescence, which did not occur when the dye was studied in glycerol solution, consistent with a decrease of the fluorescence lifetime determined by fluorescence lifetime imaging microscopy (FLIM). This indicates that glycan-bound FMR dyes respond to changes in friction that hinder dye intramolecular rotation, potentially enabling the detection of viscosity variations in cell surface layers.

Disclosure: A. Gulbins: Grant Recipient; Self; Sling therapeutics, Inc. M. Horstmann: Grant Recipient; Self; Sling therapeutics, Inc. A. Daser: None. U. Flögel: None. M. Oeverhaus: Grant Recipient; Self; Sling therapeutics, Inc. B.E. Nikolaos: None. J.P. Banga: None. G. Krause: None. G.D. Hammer: Research Investigator; Self; Sling therapeutics, Inc. A.G. Spencer: Research Investigator; Self; Sling therapeutics, Inc. R. Zeidan: Research Investigator; Self; Sling therapeutics, Inc. A. Eckstein: Grant Recipient; Self; Sling therapeutics, Inc. S. Philipp: Grant Recipient; Self; Sling therapeutics, Inc. G. Görtz: Grant Recipient; Self; Sling therapeutics, Inc. Study objective: We investigated the effect of linsitinib, a small molecule inhibitor of the Insulin like growth factor 1 receptor (IGF-1R), on Graves’ disease and thyroid eye disease. Graves‘ disease (GD), also known as “Basedow's disease“, is the most common cause for hyperthyroidism, typically presenting in patients between 40-60 years. GD is an autoimmune condition of the thyroid which is caused by autoantibodies against the thyroid stimulating hormone receptor (TSHR). Thyroid eye disease (TED) is the most common extra thyroidal manifestation of GD and occurs in about 50% of the clinical cases. Methods: To induce Graves’ Disease in mice we immunized mice 3-times with a plasmid encoding for the A-subunit of the TSHR. During each active (early) and chronic (late) states of the autoimmune disease, linsitinib was administered orally for four weeks. Endocrine orbitopathy and inflammation were determined by histology and MRI. Results: As seen in the histology, linsitinib prevented autoimmune hyperthyroidism, morphological changes, formation of brown adipose tissue in the orbita and orbital T-cell and macrophage infiltration into the orbit in the active state as well as the chronic phase. To evaluate the effect of linsitinib during the course of therapy, living mice were examined via MRI. A distinctive migration of immune cells in the orbit with consecutive inflammation can be seen in the TSHR-immunized group, which is completely blocked by treatment with linsitinib. In addition, the orbital inflammation was partnered with the onset of muscle edema and formation of brown adipose tissue in TSHR-immunized mice, effects that were abrogated upon application of linsitinib. Conclusion: In summary, we demonstrate the development of GD and TED in a mouse model upon immunization against the TSHR. The IGF-1R antagonist linsitinib blocks the development of the local pathologies of GD and TED in an early and late phase of the autoimmune disorder and also prevents development of the autoimmune response. We show that treatment of immunized mice with linsitinib after disease onset significantly limited the severity of the disease, indicating the clinical significance of the findings and providing a path to therapeutic intervention of Graves’ Disease. Our data support the use of linsitinib as a novel first line treatment of thyroid eye disease. Presentation: Saturday, June 17, 2023

Using unnatural amino acid mutagenesis, we made a mutant of CaMKII that forms a covalent linkage to Calmodulin upon illumination by UV light. Like wild‐type CaMKII, the L308BzF mutant stoichiometrically binds to Calmodulin, in a calcium‐dependent manner. Using this construct, we demonstrate that Calmodulin binding to CaMKII, even under these stochiometric conditions, does not perturb the CaMKII oligomeric state. Further, we were able to achieve activation of CaMKII L308BzF by UV‐induced binding of Calmodulin, which, once established, is further insensitive to calcium depletion. In addition to the canonical auto‐inhibitory role of the regulatory segment, inter‐subunit crosslinking in the absence of CaM indicates that kinase domains and regulatory segments are substantially mobile in basal conditions. Characterization of CaMKII L308BzF in vitro , and its expression in mammalian cells, suggests it could be a promising candidate for control of CaMKII activity in mammalian cells with light. This article is protected by copyright. All rights reserved.

Inositol pyrophosphates (PP‐IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP‐IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo‐inositol ring. 5PP‐IP5 is the most abundant PP‐IP in human cells. To investigate the function and regulation by PP‐IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP‐IP5. Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co‐crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations.

We still face fundamental gaps in understanding how molecular plastic changes of synapses intersect with circuit operation to define behavioral states. Here we show that an antagonism between two conserved regulatory proteins, Spinophilin (Spn) and Syd-1, controls presynaptic long-term plasticity and the maintenance of olfactory memories in Drosophila . While Spn mutants could not trigger nanoscopic active zone remodeling under homeostatic challenge and failed to stably potentiate neurotransmitter release, concomitant reduction of Syd-1 rescued all these deficits. The Spn/Syd-1 antagonism converged on active zone close F-actin, and genetic or acute pharmacological depolymerization of F-actin rescued the Spn deficits by allowing access to synaptic vesicle release sites. Within the intrinsic mushroom body neurons, the Spn/Syd-1 antagonism specifically controlled olfactory memory stabilization but not initial learning. Thus, this evolutionarily conserved protein complex controls behaviorally relevant presynaptic long-term plasticity, also observed in the mammalian brain but still enigmatic concerning its molecular mechanisms and behavioral relevance.

Uridine diphosphate N‐acetylglucosamine 2‐epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high‐throughput screening campaign of 68,640 drug‐like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real‐time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems

Viroporins are small ion channels in membranes of enveloped viruses that play key roles during viral life cycles. To use viroporins as drug targets against viral infection requires in‐depth mechanistic understanding and, with that, methods that enable investigations under in situ conditions. Here, we apply surface‐enhanced infrared absorption (SEIRA) spectroscopy to Influenza A M2 reconstituted within a solid‐supported membrane, to shed light on the mechanics of its viroporin function. M2 is a paradigm of pH‐activated proton channels and controls the proton flux into the viral interior during viral infection. We use SEIRA to track the large‐scale reorientation of M2’s transmembrane α‐helices in situ during pH‐activated channel opening. We quantify this event as a helical tilt from 26° to 40° by correlating the experimental results with solid‐state nuclear magnetic resonance‐informed computational spectroscopy. This mechanical motion is impeded upon addition of the inhibitor rimantadine, giving a direct spectroscopic marker to test antiviral activity. The presented approach provides a spectroscopic tool to quantify large‐scale structural changes and to track the function and inhibition of the growing number of viroporins from pathogenic viruses in future studies.

Viroporins are small ion channels in membranes of enveloped viruses that play key roles during viral life cycles. To use viroporins as drug targets against viral infection requires in‐depth mechanistic understanding and, with that, methods that enable investigations under in situ conditions. Here, we apply surface‐enhanced infrared absorption (SEIRA) spectroscopy to Influenza A M2 reconstituted within a solid‐supported membrane, to shed light on the mechanics of its viroporin function. M2 is a paradigm of pH‐activated proton channels and controls the proton flux into the viral interior during viral infection. We use SEIRA to track the large‐scale reorientation of M2’s transmembrane α‐helices in situ during pH‐activated channel opening. We quantify this event as a helical tilt from 26° to 40° by correlating the experimental results with solid‐state nuclear magnetic resonance‐informed computational spectroscopy. This mechanical motion is impeded upon addition of the inhibitor rimantadine, giving a direct spectroscopic marker to test antiviral activity. The presented approach provides a spectroscopic tool to quantify large‐scale structural changes and to track the function and inhibition of the growing number of viroporins from pathogenic viruses in future studies.