Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog pathway inhibitor-1.

Target deconvolution of small molecule hits from phenotypic screens presents a major challenge. Many screens have been conducted to find inhibitors for the Hedgehog signaling pathway - a developmental pathway with many implications in health and disease - yielding many hits but only few identified cellular targets. We here present a strategy for target identification based on Proteolysis-Targeting Chimeras (PROTACs), combined with label-free quantitative proteomics. We develop a PROTAC based on Hedgehog Pathway Inhibitor-1 (HPI-1), a phenotypic screen hit with unknown cellular target. Using this Hedgehog Pathway PROTAC (HPP) we identify and validate BET bromodomains as the cellular targets of HPI-1. Furthermore, we find that HPP-9 is a long-acting Hedgehog pathway inhibitor through prolonged BET bromodomain degradation. Collectively, we provide a powerful PROTAC-based approach for target deconvolution, that answers the longstanding question of the cellular target of HPI-1 and yields a PROTAC that acts on the Hedgehog pathway.

Nitrogen Fixation and Hydrogen Evolution by Sterically Encumbered Mo-Nitrogenase.

The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as α2ß2(γ2) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases in vivo, previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.4 kDa peptide was covalently introduced in the proximity of the P cluster, corresponding to the Fe protein docking position. The Strep-tag carried by the added peptide simultaneously sterically inhibits electron delivery to the MoFe protein and allows the isolation of partially inhibited MoFe proteins (where the half-inhibited MoFe protein was targeted). We confirm that the partially functional MoFe protein retains its ability to reduce N2 to NH3, with no significant difference in selectivity over obligatory/parasitic H2 formation. Our experiment concludes that wild-type nitrogenase exhibits negative cooperativity during the steady state regarding H2 and NH3 formation (under Ar or N2), with one-half of the MoFe protein inhibiting turnover in the second half. This emphasizes the presence and importance of long-range (>95 Å) protein-protein communication in biological N2 fixation in Azotobacter vinelandii.

Out With a Bang: Celebrating Global Chemical Biology.

On November 8-10, 2022, 163 participants from all over the world gathered at the Campus Biotech in Geneva, Switzerland to share in the latest research in chemical biology. The fourth international symposium of the Swiss National Centres of Competence in Research (NCCR) Chemical Biology coincided with the end of this successful research consortium, and as such this event marked a celebration of the past 12 years of chemical biology research in Switzerland. The inspiring talks delivered by the 15 well-known scientists, balanced in gender, expertise, and geographic location, as well as the numerous poster presentations by junior scientists showcased the breadth of global chemical biology and the bright future ahead.

Photocleavable Fluorescent Membrane Tension Probes: Fast Release with Spatiotemporal Control in Inner Leaflets of Plasma Membrane, Nuclear Envelope, and Secretory Pathway.

Mechanosensitive flipper probes are attracting interest as fluorescent reporters of membrane order and tension in biological systems. We introduce PhotoFlippers, which contain a photocleavable linker and an ultralong tether between mechanophore and various targeting motifs. Upon irradiation, the original probe is released and labels the most ordered membrane that is accessible by intermembrane transfer. Spatiotemporal control from photocleavable flippers is essential to access open, dynamic or elusive membrane motifs without chemical or physical interference. For instance, fast release with light is shown to place the original small-molecule probes into the innermost leaflet of the nuclear envelope to image changes in membrane tension, at specific points in time of membrane trafficking along the secretory pathway, or in the inner leaflet of the plasma membrane to explore membrane asymmetry. These results identify PhotoFlippers as useful chemistry tools to enable research in biology.

Flipper Probes for the Community.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

From Phenotypic Hit to Chemical Probe: Chemical Biology Approaches to Elucidate Small Molecule Action in Complex Biological Systems.

Biologically active small molecules have a central role in drug development, and as chemical probes and tool compounds to perturb and elucidate biological processes. Small molecules can be rationally designed for a given target, or a library of molecules can be screened against a target or phenotype of interest. Especially in the case of phenotypic screening approaches, a major challenge is to translate the compound-induced phenotype into a well-defined cellular target and mode of action of the hit compound. There is no "one size fits all" approach, and recent years have seen an increase in available target deconvolution strategies, rooted in organic chemistry, proteomics, and genetics. This review provides an overview of advances in target identification and mechanism of action studies, describes the strengths and weaknesses of the different approaches, and illustrates the need for chemical biologists to integrate and expand the existing tools to increase the probability of evolving screen hits to robust chemical probes.

Small Molecules Targeting the Hedgehog Pathway: From Phenotype to Mechanistic Understanding.

Since the beginning of 2019, the Hoogendoorn lab is active at the University of Geneva. We are a Chemical Biology lab and our research focuses on the Hedgehog (Hh) signalling pathway and the primary cilium, a small cellular organelle which corrects structure and function, is required to conduct the Hh signal. Ciliary Hh signalling plays an important role in embryonic development, and its dysregulation consequently results in developmental disorders as well as a variety of cancers. We use an interdisciplinary approach, ranging from organic chemistry to cell biology and genetics, to develop chemical tools to study and perturb ciliary signalling. In this account, I will highlight existing small molecules that target the Hh pathway, our efforts to discover new compounds, and the methodologies that we employ for target deconvolution and mechanism of action studies.

Empowering Global Chemical Biology at the Dawn of the New Decade.

On January 22-24, 2020, scientific luminaries across the far-flung corners of chemical biology gathered in Geneva, Switzerland, to deliver their latest and greatest discoveries in the field. Generously supported by the Swiss National Science Foundation (SNSF), our academic partners, and industrial and journal sponsors, this chemical biology symposium in our opinion will remain memorable for several years to come, not only because of the diversity in scientific topics delivered by our invited eminent speakers as detailed herein, but it is also one-of-a-kind conference which reflected multidimensional balance-balance in age and gender, across these speakers. Such a remarkable speaker line-up doubtless attracted >200 attendees from academia and industry in and around Switzerland and beyond, representing a huge swathe of subfields of science interfacing chemistry and biology. Poster presentations from students and postdocs further spotlighted the exciting diversity in the field: spanning biosynthesis, optochemical genetics, genetic code expansion, lipid chemical biology, redox perturbation, microfluidics screening, membrane signaling, immune modulation, DNA circuits, and synthetic and computational biology. This notable heterogeneity in scientific topics also went hand-in-hand with the diverse representations of student/postdoc trainees from 56 institutions covering 14 countries worldwide, allowing us to witness science as a truly global enterprise.

Fluorescent small-molecule agonists as follicle-stimulating hormone receptor imaging tools.

Fluorescent cell surface receptor agonists allow visualization of processes that are set in motion by receptor activation. This study describes the synthesis of two fluorescent, low molecular weight ligands for the follicle-stimulating hormone receptor (FSHR), based on a dihydropyridine (DHP) agonist. We show that both BODIPY- and Cy5-conjugated DHP (m-DHP-BDP and m-DHP-Cy5) are potent FSHR agonists, able to activate receptor signalling with nanomolar potencies and to effect receptor internalisation at higher concentrations. FSHR-dependent uptake of m-DHP-Cy5 is in stark contrast to the cellular uptake of m-DHP-BDP which was efficiently internalised also in the absence of FSHR. Our results comprise a first-in-class fluorescent low molecular weight ligand for in situ FSHR imaging and pertain the potential means for targeted delivery of drugs into the endolysosomal pathway of FSHR-expressing cells.

A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies.

Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

Investigations on therapeutic glucocerebrosidases through paired detection with fluorescent activity-based probes.

Deficiency of glucocerebrosidase (GBA) causes Gaucher disease (GD). In the common non-neuronopathic GD type I variant, glucosylceramide accumulates primarily in the lysosomes of visceral macrophages. Supplementing storage cells with lacking enzyme is accomplished via chronic intravenous administration of recombinant GBA containing mannose-terminated N-linked glycans, mediating the selective uptake by macrophages expressing mannose-binding lectin(s). Two recombinant GBA preparations with distinct N-linked glycans are registered in Europe for treatment of type I GD: imiglucerase (Genzyme), contains predominantly Man(3) glycans, and velaglucerase (Shire PLC) Man(9) glycans. Activity-based probes (ABPs) enable fluorescent labeling of recombinant GBA preparations through their covalent attachment to the catalytic nucleophile E340 of GBA. We comparatively studied binding and uptake of ABP-labeled imiglucerase and velaglucerase in isolated dendritic cells, cultured human macrophages and living mice, through simultaneous detection of different GBAs by paired measurements. Uptake of ABP-labeled rGBAs by dendritic cells was comparable, as well as the bio-distribution following equimolar intravenous administration to mice. ABP-labeled rGBAs were recovered largely in liver, white-blood cells, bone marrow and spleen. Lungs, brain and skin, affected tissues in severe GD types II and III, were only poorly supplemented. Small, but significant differences were noted in binding and uptake of rGBAs in cultured human macrophages, in the absence and presence of mannan. Mannan-competed binding and uptake were largest for velaglucerase, when determined with single enzymes or as equimolar mixtures of both enzymes. Vice versa, imiglucerase showed more prominent binding and uptake not competed by mannan. Uptake of recombinant GBAs by cultured macrophages seems to involve multiple receptors, including several mannose-binding lectins. Differences among cells from different donors (n = 12) were noted, but the same trends were always observed. Our study suggests that further insight in targeting and efficacy of enzyme therapy of individual Gaucher patients could be obtained by the use of recombinant GBA, trace-labeled with an ABP, preferably equipped with an infrared fluorophore or other reporter tag suitable for in vivo imaging.

Enantioselective synthesis of adamantylalanine and carboranylalanine and their incorporation into the proteasome inhibitor bortezomib.

The incorporation of adamantylalanine and carboranylalanine at the P2 site of bortezomib is well tolerated and provided potent cell permeable proteasome inhibitors with increased off-rates compared to bortezomib. Adamantylalanine and carboranylalanine were synthesized enantioselectively by an asymmetric Strecker reaction on Ellmans tert-butyl sulfinimines.

Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity.

Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.

Synthesis and Evaluation of Hybrid Structures Composed of Two Glucosylceramide Synthase Inhibitors.

Glucosylceramide metabolism and the enzymes involved have attracted significant interest in medicinal chemistry, because aberrations in the levels of glycolipids that are derived from glucosylceramide are causative in a range of human diseases including lysosomal storage disorders, type 2 diabetes, and neurodegenerative diseases. Selective modulation of one of the glycoprocessing enzymes involved in glucosylceramide metabolism-glucosylceramide synthase (GCS), acid glucosylceramidase (GBA1), or neutral glucosylceramidase (GBA2)-is therefore an attractive research objective. In this study we took two established GCS inhibitors, one based on deoxynojirimycin and the other a ceramide analogue, and merged characteristic features to obtain hybrid compounds. The resulting 39-compound library does not contain new GCS inhibitors; however, a potent (200 nm) GBA1 inhibitor was identified that has little activity toward GBA2 and might therefore serve as a lead for further biomedical development as a selective GBA1 modulator.

Direct and two-step bioorthogonal probes for Bruton's tyrosine kinase based on ibrutinib: a comparative study.

Ibrutinib is a covalent and irreversible inhibitor of Bruton's tyrosine kinase (BTK) and has been approved for the treatment of haematological malignancies, such as chronic lymphocytic leukaemia, mantle cell lymphoma and Waldenström's macroglobulinemia. The covalent and irreversible nature of its molecular mode of action allows identification and monitoring of its target in an activity-based protein profiling (ABPP) setting. Fluorescent and biotinylated ibrutinib derivatives have appeared in the literature in recent years to monitor BTK in vitro and in situ. The work described here complements this existing methodology and pertains a comparative study on the efficacy of direct and two-step bioorthogonal ABPP of BTK.

Targeted Delivery of Fluorescent High-Mannose-Type Oligosaccharide Cathepsin Inhibitor Conjugates.

Three fluorescent cathepsin inhibitor glycoconjugates have been designed, synthesized, and evaluated in terms of their cell internalization and cathepsin inhibitory properties. The conjugates are composed of a peptide epoxysuccinate, capable of covalent and irreversible binding to cysteine proteases, coupled to a fluorescent BODIPY dye and functionalized with a mono-, tri-, or heptamannoside. Mannose-receptor-dependent uptake of the probes in live dendritic cells is shown to depend on the type of carbohydrate attached. Where uptake of the monomannoside is poor and mannose-receptor-independent, the intracellular labeling of cathepsins by the probes equipped with a tri- or heptamannoside conjugate appeared concentration- and mannose-receptor-dependent.

A multivalent ligand for the mannose-6-phosphate receptor for endolysosomal targeting of an activity-based probe.

The ubiquitously expressed mannose-6-phosphate receptors (MPRs) are a promising class of receptors for targeted compound delivery into the endolysosomal compartments of a variety of cell types. The development of a synthetic, multivalent, mannose-6-phosphate (M6P) glycopeptide-based MPR ligand is described. The conjugation of this ligand to fluorescent DCG-04, an activity-based probe for cysteine cathepsins, enabled fluorescent readout of its receptor-targeting properties. The resulting M6P-cluster-BODIPY-DCG-04 probe was shown to efficiently label cathepsins in cell lysates as well as in live cells. Furthermore, the introduction of the 6-O-phosphates leads to a completely altered uptake profile in COS and dendritic cells compared to a mannose-containing ligand. Competition with mannose-6-phosphate abolished all uptake of the probe in COS cells, and we conclude that the mannose-6-phosphate cluster targets the MPR and ensures the targeted delivery of cargo bound to the cluster into the endolysosomal pathway.

Two-step bioorthogonal activity-based proteasome profiling using copper-free click reagents: a comparative study.

The development and application of bioorthogonal two-step labeling techniques receives much attention. Employing bifunctional proteasome probe 2 the efficiency of two-step labeling of recently published biotinylated cyclooctynes 3-5 is compared to Staudinger-Bertozzi ligation in cell extracts and living cells. While cyclooctynes 3-5 react faster and at a much lower concentration then the Staudinger-Bertozzi benchmark, background labeling is considerable with these reagents.

Synthesis of pH-activatable red fluorescent BODIPY dyes with distinct functionalities.

A series of tunable pH-dependent BODIPY dyes were synthesized and further functionalized in a Knoevenagel condensation reaction with various aldehydes. In this fashion, monofunctional dyes containing an alkyne, azide, or carboxylic acid (masked as its methyl ester) as ligation sites as well as asymmetrical bifunctional dyes were obtained, without compromising their pH-dependency. In addition, fluorescence excitation and emission maxima for these dyes were shown to be significantly red-shifted in comparison to their tetramethyl precursors.