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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. [ABSTRACT FROM AUTHOR]

Abstract: Complexity‐generating chemical transformations that afford novel molecular scaffolds enriched in sp3 character are highly desired. Here, we present a highly stereoselective scaffold diversity synthesis approach that utilizes cascade double‐annulation reactions of diverse pairs of zwitterionic and non‐zwitterionic partners with 3‐formylchromones to generate highly complex tetracyclic benzopyrones. Each pair of annulation partners adds to the common chroman‐4‐one scaffold to build two new rings, supporting up to four contiguous chiral centers that include an all‐carbon quaternary center. Differently ring‐fused benzopyrones display different biological activities, thus demonstrating their immense potential in medicinal chemistry and chemical biology research. [ABSTRACT FROM AUTHOR]

N-Terminal myristoylation facilitates membrane binding and activity of proteins, in particular of Src family kinases, but the underlying mechanisms are only beginning to be understood. The chaperones UNC119A/B regulate the cellular distribution and signaling of N-myristoylated proteins. Selective small-molecule modulators of the UNC119-cargo interaction would be invaluable tools, but have not been reported yet. We herein report the development of the first UNC119-cargo interaction inhibitor, squarunkin A. Squarunkin A selectively inhibits the binding of a myristoylated peptide representing the N-terminus of Src kinase to UNC119A with an IC50 value of 10 n m. It binds to UNC119 proteins in cell lysate and interferes with the activation of Src kinase. Our results demonstrate that small-molecule inhibition of the UNC119-cargo interaction might provide new opportunities for modulating the activity of Src kinases that are independent of direct inhibition of the enzymatic kinase activity. [ABSTRACT FROM AUTHOR]

Biology-oriented synthesis employs the structural information encoded in complex natural products to guide the synthesis of compound collections enriched in bioactivity. The trans-hydrindane dehydro-δ-lactone motif defines the characteristic scaffold of the steroid-like withanolides, a plant-derived natural product class with a diverse pattern of bioactivity. A withanolide-inspired compound collection was synthesized by making use of three key intermediates that contain this characteristic framework derivatized with different reactive functional groups. Biological evaluation of the compound collection in cell-based assays that monitored biological signaltransduction processes revealed a novel class of Hedgehog signaling inhibitors that target the protein Smoothened. [ABSTRACT FROM AUTHOR]

In biology-oriented synthesis, the scaffolds of biologically relevant compound classes inspire the synthesis of focused compound collections enriched in bioactivity. This criterion is, in particular, met by the scaffolds of natural products selected in evolution. The synthesis of natural product-inspired compound collections calls for efficient reaction sequences that preferably combine multiple individual transformations in one operation. Here we report the development of a one-pot, twelve-step cascade reaction sequence that includes nine different reactions and two opposing kinds of organocatalysis. The cascade sequence proceeds within 10-30 min and transforms readily available substrates into complex indoloquinolizines that resemble the core tetracyclic scaffold of numerous polycyclic indole alkaloids. Biological investigation of a corresponding focused compound collection revealed modulators of centrosome integrity, termed centrocountins, which caused fragmented and supernumerary centrosomes, chromosome congression defects, multipolar mitotic spindles, acentrosomal spindle poles and multipolar cell division by targeting the centrosome-associated proteins nucleophosmin and Crm1. [ABSTRACT FROM AUTHOR]

The quest for small molecule perturbators of protein function or a given cellular process lies at the heart of chemical biology and pharmaceutical research. Bioactive compounds need to be extensively characterized in the context of the modulated protein(s) or process(es) in living systems to unravel and confirm their mode of action. A crucial step in this workflow is the identification of the molecular targets for these small molecules, for which a generic methodology is lacking. Herein we summarize recently developed approaches for target identification spurred by advances in omics techniques and chemo- and bioinformatics analysis. [ABSTRACT FROM AUTHOR]

Fast prediction of the mode of action (MoA) for bioactive compounds would immensely foster bioactivity annotation in compound collections and may early on reveal off-targets in chemical biology research and drug discovery. Morphological profiling, e.g., using the Cell Painting assay, offers a fast, unbiased assessment of compound activity on various targets in one experiment. However, due to incomplete bioactivity annotation and unknown activities of reference compounds, prediction of bioactivity is not straightforward. Here we introduce the concept of subprofile analysis to map the MoA for both, reference and unexplored compounds. We defined MoA clusters and extracted cluster subprofiles that contain only a subset of morphological features. Subprofile analysis allows for the assignment of compounds to, currently, twelve targets or MoA. This approach enables rapid bioactivity annotation of compounds and will be extended to further clusters in the future. [Display omitted] • Morphological profiles of compound-perturbed cells are grouped into clusters • Characteristic features define subprofiles that describe each cluster • Subprofile analysis allows fast detection of twelve different modes of action • Non-cluster features can be used for further bioactivity analysis Pahl et al. define twelve bioactivity clusters that can be mapped using the Cell Painting assay. From the full morphological profiles, they extracted features that constitute subprofiles and are characteristic of the given cluster. Subprofile analysis enables fast assignment of compounds to the twelve clusters. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Morphological profiling detects small-molecule modulators of microtubules • Identification of tubulin-targeting compounds using the fingerprint of nocodazole • Confirmation of unanticipated tubulin-targeting activity for reference compounds • Detection of microtubule modulation for several unexplored small molecules Akbarzadeh et al. employ the Cell Painting assay to identify small-molecule modulators of microtubules in compound collections. They detect unanticipated tubulin-targeting activity for several reference compounds and for various unexplored small molecules. Morphological profiling enables early identification of microtubule modulation, which is one of the most frequently observed off-target activities. [ABSTRACT FROM AUTHOR]

Small-molecule chemotypes with unexpected bioactivity may be identified by combining strategies built on the biological relevance of, e.g., natural products (NPs), such as biology-oriented synthesis, with principles that enable efficient coverage of chemical space, such as fragment-based compound design. Evaluation in target-agnostic phenotypic assays and target identification may link biologically relevant chemotypes to unexpected and unknown targets. We describe the phenotypic identification of an unprecedented kinase inhibitor chemotype obtained by synthetic combination of two biosynthetically unrelated NP fragment types. Target identification and biological characterization revealed that the inhibitor, termed Myokinasib, impairs cytokinesis, induces formation of multinucleated cells, and reduces phosphorylated myosin II light chain abundance on stress fibers by selective inhibition of myosin light chain kinase 1. • De novo combination of natural product fragments yields bioactive small molecules • Target-agnostic phenotypic assays identified the MLCK inhibitor Myokinasib • Indotropanes induce cytokinesis failure and multinucleated cells • Myokinasib defines an unprecedented chemotype for kinase inhibition Schneidewind et al. present Myokinasib, a truly unique chiral kinase inhibitor with unprecedented chemotype. It promises to be a tool for the analysis of cellular processes mediated by its target MLCK1. [ABSTRACT FROM AUTHOR]