Detection of a Mitochondrial Fragmentation and Integrated Stress Response Using the Cell Painting Assay.

Mitochondria are cellular powerhouses and are crucial for cell function. However, they are vulnerable to internal and external perturbagens that may impair mitochondrial function and eventually lead to cell death. In particular, small molecules may impact mitochondrial function, and therefore, their influence on mitochondrial homeostasis is at best assessed early on in the characterization of biologically active small molecules and drug discovery. We demonstrate that unbiased morphological profiling by means of the cell painting assay (CPA) can detect mitochondrial stress coupled with the induction of an integrated stress response. This activity is common for compounds addressing different targets, is not shared by direct inhibitors of the electron transport chain, and enables prediction of mitochondrial stress induction for small molecules that are profiled using CPA.

Monovalent Pseudo-Natural Product Degraders Supercharge the Native Degradation of IDO1 by KLHDC3.

Targeted protein degradation (TPD) modulates protein function beyond inhibition of enzyme activity or protein-protein interactions. Most degraders function by proximity induction, and directly bridge an E3 ligase with the target to be degraded. However, many proteins might not be addressable via proximity-based degraders, and other challenges, such as resistance acquisition, exist. Here, we identified pseudo-natural products derived from (-)-myrtanol, termed iDegs, that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs induce a unique conformational change and, thereby, boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2KLHDC3, which we identified to also mediate native IDO1 degradation. Therefore, iDegs supercharge the native proteolytic pathway of IDO1, rendering this mechanism of action distinct from traditional degrader approaches involving proteolysis-targeting chimeras (PROTACs) or molecular-glue degraders (MGDs). In contrast to clinically explored IDO1 inhibitors, iDegs reduce formation of kynurenine by both inhibition and induced degradation of the enzyme and should also modulate non-enzymatic functions of IDO1. This unique mechanism of action may open up new therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1.

The Pseudo-Natural Product Tafbromin Selectively Targets the TAF1 Bromodomain 2.

Phenotypic assays detect small-molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule-target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4-g]quinoline (PQ) pseudo-natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA-box binding protein-associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains.

Discovery of the sEH Inhibitor Epoxykynin as a Potent Kynurenine Pathway Modulator.

Disease-related phenotypic assays enable unbiased discovery of novel bioactive small molecules and may provide novel insights into physiological systems and unprecedented molecular modes of action (MMOA). Herein, we report the identification and characterization of epoxykynin, a potent inhibitor of the soluble epoxide hydrolase (sEH). Epoxykynin was discovered by means of a cellular assay monitoring modulation of kynurenine (Kyn) levels in BxPC-3 cells upon stimulation with the cytokine interferon-γ (IFN-γ) and subsequent target identification employing affinity-based chemical proteomics. Increased Kyn levels are associated with immune suppression in the tumor microenvironment and, thus, the Kyn pathway and its key player indoleamine 2,3-dioxygenase 1 (IDO1) are appealing targets in immuno-oncology. However, targeting IDO1 directly has led to limited success in clinical investigations, demonstrating that alternative approaches to reduce Kyn levels are in high demand. We uncover a cross-talk between sEH and the Kyn pathway that may provide new opportunities to revert cancer-induced immune tolerance.

Yersinia entomophaga Tc toxin is released by T10SS-dependent lysis of specialized cell subpopulations.

Disease-causing bacteria secrete numerous toxins to invade and subjugate their hosts. Unlike many smaller toxins, the secretion machinery of most large toxins remains enigmatic. By combining genomic editing, proteomic profiling and cryo-electron tomography of the insect pathogen Yersinia entomophaga, we demonstrate that a specialized subset of these cells produces a complex toxin cocktail, including the nearly ribosome-sized Tc toxin YenTc, which is subsequently exported by controlled cell lysis using a transcriptionally coupled, pH-dependent type 10 secretion system (T10SS). Our results dissect the Tc toxin export process by a T10SS, identifying that T10SSs operate via a previously unknown lytic mode of action and establishing them as crucial players in the size-insensitive release of cytoplasmically folded toxins. With T10SSs directly embedded in Tc toxin operons of major pathogens, we anticipate that our findings may model an important aspect of pathogenesis in bacteria with substantial impact on agriculture and healthcare.

Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36.

Ubiquitin and ubiquitin-like proteins typically use distinct machineries to facilitate diverse functions. The immunosuppressive ubiquitin-like protein Fubi is synthesized as an N-terminal fusion to a ribosomal protein (Fubi-S30). Its proteolytic maturation by the nucleolar deubiquitinase USP36 is strictly required for translationally competent ribosomes. What endows USP36 with this activity, how Fubi is recognized and whether other Fubi proteases exist are unclear. Here, we report a chemical tool kit that facilitated the discovery of dual ubiquitin/Fubi cleavage activity in USP16 in addition to USP36 by chemoproteomics. Crystal structures of USP36 complexed with Fubi and ubiquitin uncover its substrate recognition mechanism and explain how other deubiquitinases are restricted from Fubi. Furthermore, we introduce Fubi C-terminal hydrolase measurements and reveal a synergistic role of USP16 in Fubi-S30 maturation. Our data highlight how ubiquitin/Fubi specificity is achieved in a subset of human deubiquitinases and open the door to a systematic investigation of the Fubi system.

Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors.

Mutations within Ras proteins represent major drivers in human cancer. In this study, we report the structure-based design, synthesis, as well as biochemical and cellular evaluation of nucleotide-based covalent inhibitors for KRasG13C, an important oncogenic mutant of Ras that has not been successfully addressed in the past. Mass spectrometry experiments and kinetic studies reveal promising molecular properties of these covalent inhibitors, and X-ray crystallographic analysis has yielded the first reported crystal structures of KRasG13C covalently locked with these GDP analogues. Importantly, KRasG13C covalently modified with these inhibitors can no longer undergo SOS-catalysed nucleotide exchange. As a final proof-of-concept, we show that in contrast to KRasG13C, the covalently locked protein is unable to induce oncogenic signalling in cells, further highlighting the possibility of using nucleotide-based inhibitors with covalent warheads in KRasG13C-driven cancer.

Biochemical characterisation of Mer3 helicase interactions and the protection of meiotic recombination intermediates.

Crossing over between homologs is critical for the stable segregation of chromosomes during the first meiotic division. Saccharomyces cerevisiae Mer3 (HFM1 in mammals) is a SF2 helicase and member of the ZMM group of proteins, that facilitates the formation of the majority of crossovers during meiosis. Here, we describe the structural organisation of Mer3 and using AlphaFold modelling and XL-MS we further characterise the previously described interaction with Mlh1-Mlh2. We find that Mer3 also forms a previously undescribed complex with the recombination regulating factors Top3 and Rmi1 and that this interaction is competitive with Sgs1BLM helicase. Using in vitro reconstituted D-loop assays we show that Mer3 inhibits the anti-recombination activity of Sgs1 helicase, but only in the presence of Dmc1. Thus we provide a mechanism whereby Mer3 interacts with a network of proteins to protect Dmc1 derived D-loops from dissolution.

The Highly Potent AhR Agonist Picoberin Modulates Hh-Dependent Osteoblast Differentiation.

Identification and analysis of small molecule bioactivity in target-agnostic cellular assays and monitoring changes in phenotype followed by identification of the biological target are a powerful approach for the identification of novel bioactive chemical matter in particular when the monitored phenotype is disease-related and physiologically relevant. Profiling methods that enable the unbiased analysis of compound-perturbed states can suggest mechanisms of action or even targets for bioactive small molecules and may yield novel insights into biology. Here we report the enantioselective synthesis of natural-product-inspired 8-oxotetrahydroprotoberberines and the identification of Picoberin, a low picomolar inhibitor of Hedgehog (Hh)-induced osteoblast differentiation. Global transcriptome and proteome profiling revealed the aryl hydrocarbon receptor (AhR) as the molecular target of this compound and identified a cross talk between Hh and AhR signaling during osteoblast differentiation.

Structural basis for specific inhibition of the deubiquitinase UCHL1.

Ubiquitination regulates protein homeostasis and is tightly controlled by deubiquitinases (DUBs). Loss of the DUB UCHL1 leads to neurodegeneration, and its dysregulation promotes cancer metastasis and invasiveness. Small molecule probes for UCHL1 and DUBs in general could help investigate their function, yet specific inhibitors and structural information are rare. Here we report the potent and non-toxic chemogenomic pair of activity-based probes GK13S and GK16S for UCHL1. Biochemical characterization of GK13S demonstrates its stereoselective inhibition of cellular UCHL1. The crystal structure of UCHL1 in complex with GK13S shows the enzyme locked in a hybrid conformation of apo and Ubiquitin-bound states, which underlies its UCHL1-specificity within the UCH DUB family. Phenocopying a reported inactivating mutation of UCHL1 in mice, GK13S, but not GK16S, leads to reduced levels of monoubiquitin in a human glioblastoma cell line. Collectively, we introduce a set of structurally characterized, chemogenomic probes suitable for the cellular investigation of UCHL1.

Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin.

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end-directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.

The Pseudo-Natural Product Rhonin Targets RHOGDI.

For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g., natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment-based design. We describe the de novo combination of different 5-membered NP-derived N-heteroatom fragments to structurally unprecedented "pseudo-natural products" in an efficient complexity-generating and enantioselective one-pot synthesis sequence. The pseudo-NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP-derived chemotypes, and may have novel biological targets. Investigation of the pseudo-NPs in unbiased phenotypic assays and target identification led to the discovery of the first small-molecule ligand of the RHO GDP-dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP-bound RHO GTPases and alters the subcellular localization of RHO GTPases.

Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes.

As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities of target proteins and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1-activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CAKs or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a ternary complex consisting of CDK1, Cyclin-B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin-B (CC) and CDK1:Cyclin-B:CKS1 (CCC).

Novel mechanistic insights into the role of Mer2 as the keystone of meiotic DNA break formation.

In meiosis, DNA double-strand break (DSB) formation by Spo11 initiates recombination and enables chromosome segregation. Numerous factors are required for Spo11 activity, and couple the DSB machinery to the development of a meiosis-specific 'axis-tethered loop' chromosome organisation. Through in vitro reconstitution and budding yeast genetics, we here provide architectural insight into the DSB machinery by focussing on a foundational DSB factor, Mer2. We characterise the interaction of Mer2 with the histone reader Spp1, and show that Mer2 directly associates with nucleosomes, likely highlighting a contribution of Mer2 to tethering DSB factors to chromatin. We reveal the biochemical basis of Mer2 association with Hop1, a HORMA domain-containing chromosomal axis factor. Finally, we identify a conserved region within Mer2 crucial for DSB activity, and show that this region of Mer2 interacts with the DSB factor Mre11. In combination with previous work, we establish Mer2 as a keystone of the DSB machinery by bridging key protein complexes involved in the initiation of meiotic recombination.

Organisms are said to be diploid when they carry two copies of each chromosome in their cells, one from each of their biological parents. But in order for each parent to only pass on one copy of their own chromosomes, they need to make haploid cells, which only carry one copy of each chromosome. These cells form by a special kind of cell division called meiosis, in which the two chromosomes from each pair in the parent cells are first linked, and then pulled apart into the daughter cells. Accurate meiosis requires a type of DNA damage called double-stranded DNA breaks. These breaks cut through the chromosomes and can be dangerous to the cell if they are not repaired correctly. During meiosis, a set of proteins gather around the chromosomes to ensure the cuts happen in the right place and to repair the damage. One of these proteins is called Mer2. Previous studies suggest that this protein plays a role in placing the DNA breaks and controlling when they happen. To find out more, Rousova et al. examined Mer2 and the proteins that interact with it in budding yeast cells. This involved taking the proteins out of the cell to get a closer look. The experiments showed that Mer2 sticks directly to the chromosomes and acts as a tether for other proteins. It collaborates with two partners, called Hop1 and Mre11, to make sure that DNA breaks happen safely. These proteins detect the state of the chromosome and repair the damage. Stopping Mer2 from interacting with Mre11 prevented DNA breaks from forming in budding yeast cells. Although Rousova et al. used budding yeast to study the proteins involved in meiosis, similar proteins exist in plant and animal cells too. Understanding how they work could open new avenues of research into cell division. For example, studies on plant proteins could provide tools for creating new crop strains. Studies on human proteins could also provide insights into fertility problems and cancer.

Combined morphological and proteome profiling reveals target-independent impairment of cholesterol homeostasis.

Unbiased profiling approaches are powerful tools for small-molecule target or mode-of-action deconvolution as they generate a holistic view of the bioactivity space. This is particularly important for non-protein targets that are difficult to identify with commonly applied target identification methods. Thereby, unbiased profiling can enable identification of novel bioactivity even for annotated compounds. We report the identification of a large bioactivity cluster comprised of numerous well-characterized drugs with different primary targets using a combination of the morphological Cell Painting Assay and proteome profiling. Cluster members alter cholesterol homeostasis and localization due to their physicochemical properties that lead to protonation and accumulation in lysosomes, an increase in lysosomal pH, and a disturbed cholesterol homeostasis. The identified cluster enables identification of modulators of cholesterol homeostasis and links regulation of genes or proteins involved in cholesterol synthesis or trafficking to physicochemical properties rather than to nominal targets.

Light-Activation of DNA-Methyltransferases.

5-Methylcytosine (5mC), the central epigenetic mark of mammalian DNA, plays fundamental roles in chromatin regulation. 5mC is written onto genomes by DNA methyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis. However, studying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo. We report light-control of DNMT catalysis by genetically encoding a photocaged cysteine as a catalytic residue. This enables translation of inactive DNMTs, their rapid activation by light-decaging, and subsequent monitoring of de novo DNA methylation. We provide insights into how cancer-related DNMT mutations alter de novo methylation in vivo, and demonstrate local and tuneable cytosine methylation by light-controlled DNMTs fused to a programmable transcription activator-like effector domain targeting pericentromeric satellite-3 DNA. We further study early events of transcriptome alterations upon DNMT-catalyzed cytosine methylation. Our study sets a basis to dissect the order and kinetics of diverse chromatin-associated events triggered by normal and aberrant DNA methylation.

Thermal proteome profiling identifies the membrane-bound purinergic receptor P2X4 as a target of the autophagy inhibitor indophagolin.

Signaling pathways are frequently activated through signal-receiving membrane proteins, and the discovery of small molecules targeting these receptors may yield insights into their biology. However, due to their intrinsic properties, membrane protein targets often cannot be identified by means of established approaches, in particular affinity-based proteomics, calling for the exploration of new methods. Here, we report the identification of indophagolin as representative member of an indoline-based class of autophagy inhibitors through a target-agnostic phenotypic assay. Thermal proteome profiling and subsequent biochemical validation identified the purinergic receptor P2X4 as a target of indophagolin, and subsequent investigations suggest that indophagolin targets further purinergic receptors. These results demonstrate that thermal proteome profiling may enable the de novo identification of membrane-bound receptors as cellular targets of bioactive small molecules.

14-3-3 Protein Bmh1 triggers short-range compaction of mitotic chromosomes by recruiting sirtuin deacetylase Hst2.

During mitosis, chromosomes are compacted in length by more than 100-fold into rod-shaped forms. In yeast, this process depends on the presence of a centromere, which promotes condensation in cis by recruiting mitotic kinases such as Aurora B kinase. This licensing mechanism enables the cell to discriminate chromosomal from noncentromeric DNA and to prohibit the propagation of the latter. Aurora B kinase elicits a cascade of events starting with phosphorylation of histone H3 serine 10 (H3S10ph), which signals the recruitment of lysine deacetylase Hst2 and the removal of lysine 16 acetylation in histone 4. The unmasked histone 4 tails interact with the acidic patch of neighboring nucleosomes to drive short-range compaction of chromatin, but the mechanistic details surrounding the Hst2 activity remain unclear. Using in vitro and in vivo assays, we demonstrate that the interaction of Hst2 with H3S10ph is mediated by the yeast 14-3-3 protein Bmh1. As a homodimer, Bmh1 binds simultaneously to H3S10ph and the phosphorylated C-terminus of Hst2. Our pull-down experiments with extracts of synchronized cells show that the Hst2-Bmh1 interaction is cell cycle dependent, peaking in the M phase. Furthermore, we show that phosphorylation of C-terminal residues of Hst2, introduced by genetic code expansion, stimulates its deacetylase activity. Hence, the data presented here identify Bmh1 as a key player in the mechanism of licensing of chromosome compaction in mitosis.

Discovery of small-molecule modulator of heterotrimeric Gi-protein by integrated phenotypic profiling and chemical proteomics.

Discovery of small-molecule inducers of unique phenotypic changes combined with subsequent target identification often provides new insights into cellular functions. Here, we applied integrated profiling based on cellular morphological and proteomic changes to compound screening. We identified an indane derivative, NPD9055, which is mechanistically distinct from reference compounds with known modes of action. Employing a chemical proteomics approach, we then showed that NPD9055 binds subunits of heterotrimeric G-protein Gi. An in vitro [35S]GTPγS-binding assay revealed that NPD9055 inhibited GDP/GTP exchange on a Gαi subunit induced by a G-protein-coupled receptor agonist, but not on another G-protein from the Gαs family. In intact HeLa cells, NPD9055 induced an increase in intracellular Ca2+ levels and ERK/MAPK phosphorylation, both of which are regulated by Gßγ, following its dissociation from Gαi. Our observations suggest that NPD9055 targets Gαi and thus regulates Gßγ-dependent cellular processes, most likely by causing the dissociation of Gßγ from Gαi.

Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets.

Legionella pneumophila governs its interactions with host cells by secreting >300 different "effector" proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection.IMPORTANCELegionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in amoebae and macrophages by employing a "type IV" secretion system, which secretes more than 300 different "effector" proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.