RESUMO
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.
Assuntos
Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/química , Fator de Transcrição TFIID/metabolismo , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/antagonistas & inibidores , Humanos , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/antagonistas & inibidores , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/citologia , Diferenciação Celular/efeitos dos fármacos , Quinolinas/química , Quinolinas/farmacologia , Estrutura MolecularRESUMO
Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death.
Assuntos
Autofagia , Lisossomos , Transporte Biológico , Galectina 3 , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismoRESUMO
Oxindoles and iso-oxindoles are natural product-derived scaffolds that provide inspiration for the design and synthesis of novel biologically relevant compound classes. Notably, the spirocyclic connection of oxindoles with iso-oxindoles has not been explored by nature but promises to provide structurally related compounds endowed with novel bioactivity. Therefore, methods for their efficient synthesis and the conclusive discovery of their cellular targets are highly desirable. We describe a selective RhIII -catalyzed scaffold-divergent synthesis of spirooxindole-isooxindoles and spirooxindole-oxindoles from differently protected diazooxindoles and N-pivaloyloxy aryl amides which includes a functional group-controlled Lossen rearrangement as key step. Unbiased morphological profiling of a corresponding compound collection in the Cell Painting assay efficiently identified the mitotic kinesin Eg5 as the cellular target of the spirooxindoles, defining a unique Eg5 inhibitor chemotype.
Assuntos
Cinesinas , OxindóisRESUMO
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.
RESUMO
Monoterpene indole alkaloids (MIAs) are endowed with high structural and spatial complexity and characterized by diverse biological activities. Given this complexity-activity combination in MIAs, rapid and efficient access to chemical matter related to and with complexity similar to these alkaloids would be highly desirable, since such compound classes might display novel bioactivity. We describe the design and synthesis of a pseudo-natural product (pseudo-NP) collection obtained by the unprecedented combination of MIA fragments through complexity-generating transformations, resulting in arrangements not currently accessible by biosynthetic pathways. Cheminformatic analyses revealed that both the pseudo-NPs and the MIAs reside in a unique and common area of chemical space with high spatial complexity-density that is only sparsely populated by other natural products and drugs. Investigation of bioactivity guided by morphological profiling identified pseudo-NPs that inhibit DNA synthesis and modulate tubulin. These results demonstrate that the pseudo-NP collection occupies similar biologically relevant chemical space that Nature has endowed MIAs with.
Assuntos
Alcaloides , Monoterpenos , Alcaloides IndólicosRESUMO
Profiling approaches have been increasingly employed for the characterization of disease-relevant phenotypes or compound perturbation as they provide a broad, unbiased view on impaired cellular states. We report that morphological profiling using the cell painting assay (CPA) can detect modulators of de novo pyrimidine biosynthesis and of dihydroorotate dehydrogenase (DHODH) in particular. The CPA can differentiate between impairment of pyrimidine and folate metabolism, which both affect cellular nucleotide pools. The identified morphological signature is shared by inhibitors of DHODH and the functionally tightly coupled complexâ III of the mitochondrial respiratory chain as well as by UMP synthase, which is downstream of DHODH. The CPA appears to be particularly suited for the detection of DHODH inhibitors at the site of their action in cells. As DHODH is a validated therapeutic target, the CPA will enable unbiased identification of DHODH inhibitors and inhibitors of de novo pyrimidine biosynthesis for biological research and drug discovery.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/farmacologia , Pirimidinas/farmacologia , Descoberta de DrogasRESUMO
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.
Assuntos
Antineoplásicos , Produtos Biológicos , Policetídeos , Produtos Biológicos/química , Proteínas Hedgehog/metabolismo , Vias BiossintéticasRESUMO
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.
Assuntos
Produtos Biológicos , Aminoácidos , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Inibidores Enzimáticos/química , Heme , Indolamina-Pirrol 2,3,-Dioxigenase , Indóis , Pirrolidinas , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Produtos Biológicos , Produtos Biológicos/química , Ligantes , Proteínas rho de Ligação ao GTP , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
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.
Assuntos
Autofagossomos/metabolismo , Proteínas de Membrana/metabolismo , Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Humanos , Proteínas de Membrana/antagonistas & inibidores , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Linhagem Celular Tumoral , Cumarínicos/química , Inibidores Enzimáticos/química , Corantes Fluorescentes/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/análise , Estrutura MolecularRESUMO
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.
Assuntos
Quelantes de Ferro/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Quelantes de Ferro/química , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/químicaRESUMO
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.
Assuntos
Descoberta de Drogas , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Fenótipo , Proteômica , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , HumanosRESUMO
The prenyl-protein chaperone PDEδ modulates the localization of lipidated proteins in the cell, but current knowledge about its biological function is limited. Small-molecule inhibitors that target the PDEδ prenyl-binding site have proven invaluable in the analysis of biological processes mediated by PDEδ, like KRas cellular trafficking. However, allosteric inhibitor release from PDEδ by the Arl2/3 GTPases limits their application. We describe the development of new proteolysis-targeting chimeras (PROTACs) that efficiently and selectively reduce PDEδ levels in cells through induced proteasomal degradation. Application of the PDEδ PROTACs increased sterol regulatory element binding protein (SREBP)-mediated gene expression of enzymes involved in lipid metabolism, which was accompanied by elevated levels of cholesterol precursors. This finding for the first time demonstrates that PDEδ function plays a role in the regulation of enzymes of the mevalonate pathway.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Metabolismo dos Lipídeos , Sondas Moleculares/química , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidores , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Sondas Moleculares/metabolismo , Sondas Moleculares/farmacologia , Proteólise , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismoRESUMO
A "branching-folding" synthetic strategy that affords a range of diverse cyclic benzo-sulfonamide scaffolds is presented. Whereas different annulation reactions on common ketimine substrates build the branching phase of the scaffold synthesis, a common hydrogenative ring-expansion method, facilitated by an increase of the ring-strain during the branching phase, led to sulfonamides bearing medium-sized rings in a folding pathway. Cell painting assay was successfully employed to identify tubulin targeting sulfonamides as novel mitotic inhibitors.
Assuntos
Sulfonamidas/síntese química , Ciclização , Sulfonamidas/químicaRESUMO
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.
RESUMO
Herein, we report the first enantioselective annulation of α-arylidene pyrazolones through a formal C(sp3 )-H activation under mild conditions enabled by highly variable RhIII -Cpx catalysts. The method has a wide substrate scope and proceeds with good to excellent yields and enantioselectivities. Its synthetic utility was demonstrated by the late-stage functionalization of drugs and natural products as well as the preparation of enantioenriched [3]dendralenes. Preliminary biological investigations also identified the spiropyrazolones as a novel class of Hedgehog pathway inhibitors.
Assuntos
Compostos de Espiro/química , Catálise , Estrutura Molecular , EstereoisomerismoRESUMO
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.
Assuntos
Pirofosfatases/metabolismo , Via de Sinalização Wnt , Inibidores Enzimáticos/farmacologia , Células HCT116 , Células HEK293 , Humanos , Mapas de Interação de Proteínas/efeitos dos fármacos , Pirofosfatases/antagonistas & inibidores , Peptidase 7 Específica de Ubiquitina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
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.
Assuntos
Produtos Biológicos/farmacologia , Proliferação de Células , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 3/antagonistas & inibidores , Glucose/metabolismo , Morfinanos/síntese química , Neoplasias/tratamento farmacológico , Transporte Biológico , Ciclo Celular , Glicólise , Humanos , Células Tumorais CultivadasRESUMO
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.