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.

Illuminating Dark Chemical Matter Using the Cell Painting Assay.

Screening for small-molecule modulators of disease-relevant targets and phenotypes is the first step on the way to new drugs. Large compound libraries have been synthesized by academia and, particularly, pharmaceutical companies to meet the need for novel chemical entities that are as diverse as possible. Screening of these compound libraries revealed a portion of small molecules that is inactive in more than 100 different assays and was therefore termed "dark chemical matter" (DCM). Deorphanization of DCM promises to yield very selective compounds as they are expected to have less off-target effects. We employed morphological profiling using the Cell Painting assay to detect bioactive DCM. Within the DCM collection, we identified bioactive compounds and confirmed several modulators of microtubules, DNA synthesis, and pyrimidine biosynthesis. Profiling approaches are, therefore, powerful tools to probe compound collections for bioactivity in an unbiased manner and are particularly suitable for deorphanization of DCM.

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.

Cooperative Lewis Acid-1,2,3-Triazolium-Aryloxide Catalysis: Pyrazolone Addition to Nitroolefins as Entry to Diaminoamides.

Pyrazolones represent an important structural motif in active pharmaceutical ingredients. Their asymmetric synthesis is thus widely studied. Still, a generally highly enantio- and diastereoselective 1,4-addition to nitroolefins providing products with adjacent stereocenters is elusive. In this article, a new polyfunctional CuII -1,2,3-triazolium-aryloxide catalyst is presented which enables this reaction type with high stereocontrol. DFT studies revealed that the triazolium stabilizes the transition state by hydrogen bonding between C(5)-H and the nitroolefin and verify a cooperative mode of activation. Moreover, they show that the catalyst adopts a rigid chiral cage/pore structure by intramolecular hydrogen bonding, by which stereocontrol is achieved. Control catalyst systems confirm the crucial role of the triazolium, aryloxide and CuII , requiring a sophisticated structural orchestration for high efficiency. The addition products were used to form pyrazolidinones by chemoselective C=N reduction. These heterocycles are shown to be valuable precursors toward ß,γ'-diaminoamides by chemoselective nitro and N-N bond reductions. Morphological profiling using the Cell painting assay identified biological activities for the pyrazolidinones and suggest modulation of DNA synthesis as a potential mode of action. One product showed biological similarity to Camptothecin, a lead structure for cancer therapy.

Unprecedented Combination of Polyketide Natural Product Fragments Identifies the New Hedgehog Signaling Pathway Inhibitor Grismonone.

Pseudo-natural products (pseudo-NPs) are de novo combinations of natural product (NP) fragments that define novel bioactive chemotypes. For their discovery, new design principles are being sought. Previously, pseudo-NPs were synthesized by the combination of fragments originating from biosynthetically unrelated NPs to guarantee structural novelty and novel bioactivity. We report the combination of fragments from biosynthetically related NPs in novel arrangements to yield a novel chemotype with activity not shared by the guiding fragments. We describe the synthesis of the polyketide pseudo-NP grismonone and identify it as a structurally novel and potent inhibitor of Hedgehog signaling. The insight that the de novo combination of fragments derived from biosynthetically related NPs may also yield new biologically relevant compound classes with unexpected bioactivity may be considered a chemical extension or diversion of existing biosynthetic pathways and greatly expands the opportunities for exploration of biologically relevant chemical space by means of the pseudo-NP principle.

Identification of a Novel Pseudo-Natural Product Type IV IDO1 Inhibitor Chemotype.

Natural product (NP)-inspired design principles provide invaluable guidance for bioactive compound discovery. Pseudo-natural products (PNPs) are de novo combinations of NP fragments to target biologically relevant chemical space not covered by NPs. We describe the design and synthesis of apoxidoles, a novel pseudo-NP class, whereby indole- and tetrahydropyridine fragments are linked in monopodal connectivity not found in nature. Apoxidoles are efficiently accessible by an enantioselective [4+2] annulation reaction. Biological evaluation revealed that apoxidoles define a new potent type IV inhibitor chemotype of indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme considered a target for the treatment of neurodegeneration, autoimmunity and cancer. Apoxidoles target apo-IDO1, prevent heme binding and induce unique amino acid positioning as revealed by crystal structure analysis. Novel type IV apo-IDO1 inhibitors are in high demand, and apoxidoles may provide new opportunities for chemical biology and medicinal chemistry research.

Morphological profiling by means of the Cell Painting assay enables identification of tubulin-targeting compounds.

In phenotypic compound discovery, conclusive identification of cellular targets and mode of action are often impaired by off-target binding. In particular, microtubules are frequently targeted in cellular assays. However, in vitro tubulin binding assays do not correctly reflect the cellular context, and conclusive high-throughput phenotypic assays monitoring tubulin binding are scarce, such that tubulin binding is rarely identified. We report that morphological profiling using the Cell Painting assay (CPA) can efficiently detect tubulin modulators in compound collections with a high throughput, including annotated reference compounds and unannotated compound classes with unrelated chemotypes and scaffolds. Small-molecule tubulin binders share similar CPA fingerprints, which enables prediction and experimental validation of microtubule-binding activity. Our findings suggest that CPA or a related morphological profiling approach will be an invaluable addition to small-molecule discovery programs in chemical biology and medicinal chemistry, enabling early identification of one of the most frequently observed off-target activities.

Cell-Based Identification of New IDO1 Modulator Chemotypes.

The immunoregulatory enzyme indoleamine-2,3-dioxygenase (IDO1) strengthens cancer immune escape, and inhibition of IDO1 by means of new chemotypes and mechanisms of action is considered a promising opportunity for IDO1 inhibitor discovery. IDO1 is a cofactor-binding, redox-sensitive protein, which calls for monitoring of IDO1 activity in its native cellular environment. We developed a new, robust fluorescence-based assay amenable to high throughput, which detects kynurenine in cells. Screening of a ca. 150 000-member compound library discovered unprecedented, potent IDO1 modulators with different mechanisms of action, including direct IDO1 inhibitors, regulators of IDO1 expression, and inhibitors of heme synthesis. Three IDO1-modulator chemotypes were identified that bind to apo-IDO1 and compete with the heme cofactor. Our new cell-based technology opens up novel opportunities for medicinal chemistry programs in immuno-oncology.

Discovery of a σ1 receptor antagonist by combination of unbiased cell painting and thermal proteome profiling.

Phenotypic screening for bioactive small molecules is typically combined with affinity-based chemical proteomics to uncover the respective molecular targets. However, such assays and the explored bioactivity are biased toward the monitored phenotype, and target identification often requires chemical derivatization of the hit compound. In contrast, unbiased cellular profiling approaches record hundreds of parameters upon compound perturbation to map bioactivity in a broader biological context and may link a profile to the molecular target or mode of action. Herein we report the discovery of the diaminopyrimidine DP68 as a Sigma 1 (σ1) receptor antagonist by combining morphological profiling using the Cell Painting assay and thermal proteome profiling. Our results highlight that integration of complementary profiling approaches may enable both detection of bioactivity and target identification for small molecules.

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.

Morphological Profiling Identifies a Common Mode of Action for Small Molecules with Different Targets.

Unbiased morphological profiling of bioactivity, for example, in the cell painting assay (CPA), enables the identification of a small molecule's mode of action based on its similarity to the bioactivity of reference compounds, irrespective of the biological target or chemical similarity. This is particularly important for small molecules with nonprotein targets as these are rather difficult to identify with widely employed target-identification methods. We employed morphological profiling using the CPA to identify compounds that are biosimilar to the iron chelator deferoxamine. Structurally different compounds with different annotated cellular targets provoked a shared physiological response, thereby defining a cluster based on their morphological fingerprints. This cluster is based on a shared mode of action and not on a shared target, that is, cell-cycle modulation in the S or G2 phase. Hierarchical clustering of morphological fingerprints revealed subclusters that are based on the mechanism of action and could be used to predict target-related bioactivity.

Discovery of the Hedgehog Pathway Inhibitor Pipinib that Targets PI4KIIIß.

The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIß (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.

Synthesis of Indomorphan Pseudo-Natural Product Inhibitors of Glucose Transporters GLUT-1 and -3.

Bioactive compound design based on natural product (NP) structure may be limited because of partial coverage of NP-like chemical space and biological target space. These limitations can be overcome by combining NP-centered strategies with fragment-based compound design through combination of NP-derived fragments to afford structurally unprecedented "pseudo-natural products" (pseudo-NPs). The design, synthesis, and biological evaluation of a collection of indomorphan pseudo-NPs that combine biosynthetically unrelated indole- and morphan-alkaloid fragments are described. Indomorphane derivative Glupin was identified as a potent inhibitor of glucose uptake by selectively targeting and upregulating glucose transporters GLUT-1 and GLUT-3. Glupin suppresses glycolysis, reduces the levels of glucose-derived metabolites, and attenuates the growth of various cancer cell lines. Our findings underscore the importance of dual GLUT-1 and GLUT-3 inhibition to efficiently suppress tumor cell growth and the cellular rescue mechanism, which counteracts glucose scarcity.

Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth.

Cancer cells sustain growth by altering their metabolism to accelerated aerobic glycolysis accompanied by increased glucose demand and employ glutamine as additional nutrient source. This metabolic adaptation induces upregulation of glucose transporters GLUT-1 and -3, and simultaneous targeting of both transporters and of glutamine metabolism may offer a promising approach to inhibit cancer cell growth. We describe the discovery of the very potent glucose uptake inhibitor Glutor, which targets glucose transporters GLUT-1, -2, and -3, attenuates glycolytic flux and potently and selectively suppresses growth of a variety of cancer cell lines. Co-treatment of colon cancer cells with Glutor and glutaminase inhibitor CB-839 very potently and synergistically inhibits cancer cell growth. Such a dual inhibition promises to be particularly effective because it targets the metabolic plasticity as well as metabolic rescue mechanisms in cancer cells.

Chemical Genetics Reveals a Role of dCTP Pyrophosphatase 1 in Wnt Signaling.

Cell-based screening is a powerful approach to identify novel chemical modulators and biological components of relevant biological processes. The canonical Wnt pathway is essential for normal embryonic development and tissue homeostasis, and its deregulation plays a crucial role in carcinogenesis. Therefore, the identification of new pathway members and regulators is of significant interest. By means of a cell-based assay monitoring Wnt signaling we identified the pyrrolocoumarin Pyrcoumin as inhibitor of canonical Wnt signaling. Target identification and validation revealed that Pyrcoumin is a competitive inhibitor of dCTP pyrophosphatase 1 (dCTPP1). We demonstrate a yet unknown interaction of dCTPP1 with ubiquitin carboxyl-terminal hydrolase (USP7) that is counteracted by dCTPP1 inhibitors. These findings indicate that dCTPP1 plays a role in regulation of Wnt/ß-catenin signaling most likely through a direct interaction with USP7.

The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis.

Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.

Unravelling the Synthesis and Chemistry of Stable, Acyclic, and Double-Deficient 1,3-Butadienes: An endo-Selective Diels-Alder Route to Hedgehog Pathway Inhibitors.

The first synthetic access to stable and acyclic 1,3-butadienes with two electron-withdrawing carbonyl groups and their potential to deliver new molecular scaffolds through intriguing endo-selective Diels-Alder cycloadditions are presented. The bicyclic scaffolds produced through the cycloaddition chemistry of electron-deficient dienes afforded potent Hedgehog signaling pathway inhibitors.

Furo[3,2-b]pyridine: A Privileged Scaffold for Highly Selective Kinase Inhibitors and Effective Modulators of the Hedgehog Pathway.

Reported is the identification of the furo[3,2-b]pyridine core as a novel scaffold for potent and highly selective inhibitors of cdc-like kinases (CLKs) and efficient modulators of the Hedgehog signaling pathway. Initially, a diverse target compound set was prepared by synthetic sequences based on chemoselective metal-mediated couplings, including assembly of the furo[3,2-b]pyridine scaffold by copper-mediated oxidative cyclization. Optimization of the subseries containing 3,5-disubstituted furo[3,2-b]pyridines afforded potent, cell-active, and highly selective inhibitors of CLKs. Profiling of the kinase-inactive subset of 3,5,7-trisubstituted furo[3,2-b]pyridines revealed sub-micromolar modulators of the Hedgehog pathway.

Chromopynones are pseudo natural product glucose uptake inhibitors targeting glucose transporters GLUT-1 and -3.

The principles guiding the design and synthesis of bioactive compounds based on natural product (NP) structure, such as biology-oriented synthesis (BIOS), are limited by their partial coverage of the NP-like chemical space of existing NPs and retainment of bioactivity in the corresponding compound collections. Here we propose and validate a concept to overcome these limitations by de novo combination of NP-derived fragments to structurally unprecedented 'pseudo natural products'. Pseudo NPs inherit characteristic elements of NP structure yet enable the efficient exploration of areas of chemical space not covered by NP-derived chemotypes, and may possess novel bioactivities. We provide a proof of principle by designing, synthesizing and investigating the biological properties of chromopynone pseudo NPs that combine biosynthetically unrelated chromane- and tetrahydropyrimidinone NP fragments. We show that chromopynones define a glucose uptake inhibitor chemotype that selectively targets glucose transporters GLUT-1 and -3, inhibits cancer cell growth and promises to inspire new drug discovery programmes aimed at tumour metabolism.

Combined Proteomic and In Silico Target Identification Reveal a Role for 5-Lipoxygenase in Developmental Signaling Pathways.

Identification and validation of the targets of bioactive small molecules identified in cell-based screening is challenging and often meets with failure, calling for the development of new methodology. We demonstrate that a combination of chemical proteomics with in silico target prediction employing the SPiDER method may provide efficient guidance for target candidate selection and prioritization for experimental in-depth evaluation. We identify 5-lipoxygenase (5-LO) as the target of the Wnt pathway inhibitor Lipoxygenin. Lipoxygenin is a non-redox 5-LO inhibitor, modulates the ß-catenin-5-LO complex and induces reduction of both ß-catenin and 5-LO levels in the nucleus. Lipoxygenin and the structurally unrelated 5-LO inhibitor CJ-13,610 promote cardiac differentiation of human induced pluripotent stem cells and inhibit Hedgehog, TGF-ß, BMP, and Activin A signaling, suggesting an unexpected and yet unknown role of 5-LO in these developmental pathways.