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.

Identification of Dihydroorotate Dehydrogenase Inhibitors Using the Cell Painting Assay.

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 complex 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.

Synthesis of Indofulvin Pseudo-Natural Products Yields a New Autophagy Inhibitor Chemotype.

Chemical and biological limitations in bioactive compound design based on natural product (NP) structure can be overcome by the combination of NP-derived fragments in unprecedented arrangements to afford "pseudo-natural products" (pseudo-NPs). A new pseudo-NP design principle is described, i.e., the combination of NP-fragments by transformations that are not part of current biosynthesis pathways. A collection of indofulvin pseudo-NPs is obtained from 2-hydroxyethyl-indoles and ketones derived from the fragment-sized NP griseofulvin by means of an iso-oxa-Pictet-Spengler reaction. Cheminformatic analysis indicates that the indofulvins reside in an area of chemical space sparsely covered by NPs, drugs, and drug-like compounds and they may combine favorable properties of these compound classes. Biological evaluation of the compound collection in different cell-based assays and the unbiased high content cell painting assay reveal that the indofulvins define a new autophagy inhibitor chemotype that targets mitochondrial respiration.

Natural product fragment combination to performance-diverse pseudo-natural products.

Natural product structure and fragment-based compound development inspire pseudo-natural product design through different combinations of a given natural product fragment set to compound classes expected to be chemically and biologically diverse. We describe the synthetic combination of the fragment-sized natural products quinine, quinidine, sinomenine, and griseofulvin with chromanone or indole-containing fragments to provide a 244-member pseudo-natural product collection. Cheminformatic analyses reveal that the resulting eight pseudo-natural product classes are chemically diverse and share both drug- and natural product-like properties. Unbiased biological evaluation by cell painting demonstrates that bioactivity of pseudo-natural products, guiding natural products, and fragments differ and that combination of different fragments dominates establishment of unique bioactivity. Identification of phenotypic fragment dominance enables design of compound classes with correctly predicted bioactivity. The results demonstrate that fusion of natural product fragments in different combinations and arrangements can provide chemically and biologically diverse pseudo-natural product classes for wider exploration of biologically relevant chemical space.

Rescue from Pseudomonas aeruginosa Airway Infection via Stem Cell Transplantation.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to impaired ion transport in epithelial cells. Although lung failure due to chronic infection is the major comorbidity in individuals with cystic fibrosis, the role of CFTR in non-epithelial cells has not been definitively resolved. Given the important role of host defense cells, we evaluated the Cftr deficiency in pulmonary immune cells by hematopoietic stem cell transplantation in cystic fibrosis mice. We transplanted healthy bone marrow stem cells and could reveal a stable chimerism of wild-type cells in peripheral blood. The outcome of stem cell transplantation and the impact of healthy immune cells were evaluated in acute Pseudomonas aeruginosa airway infection. In this study, mice transplanted with wild-type cells displayed better survival, lower lung bacterial numbers, and a milder disease course. This improved physiology of infected mice correlated with successful intrapulmonary engraftment of graft-derived alveolar macrophages, as seen by immunofluorescence microscopy and flow cytometry of graft-specific leucocyte surface marker CD45 and macrophage marker CD68. Given the beneficial effect of hematopoietic stem cell transplantation and stable engraftment of monocyte-derived CD68-positive macrophages, we conclude that replacement of mutant Cftr macrophages attenuates airway infection in cystic fibrosis mice.

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.

Pseudo-natural products and natural product-inspired methods in chemical biology and drug discovery.

Through evolution, nature has provided natural products (NPs) as a rich source of diverse bioactive material. Many drug discovery programs have used nature as an inspiration for the design of NP-like compound classes. These concepts are guided by the prevalidated biological relevance of NPs while going beyond the limitations of nature to produce chemical matter that could have unexpected or novel bioactivities. Herein, we discuss, compare, and highlight recent examples of NP-inspired methods with a focus on the pseudo-NP concept.

Tritiation of azido-labeled diiodo cabazitaxel (Jevtana) and docetaxel (Taxotere) derivatives to generate 3 H-photoaffinity probes.

Radiolabelled azidophenyl analogues can make powerful photoaffinity probes for the identification of molecular targets. We describe our efforts to prepare tritiated azidophenyl analogues of the taxols cabazitaxel and docetaxel. Late-stage tritiation by isotope exchange with diiodo precursors resulted in reduction of the azide moiety, which could only be overcome by addition of high excess of a sacrificial azide. Iodine-deuterium exchange experiments on a model system established that deiodination with concomitant azide reduction is a general problem when performing such isotope-exchange reactions on azide-containing aryl iodides.

Evaluation of a P,N-ligated iridium(I) catalyst in hydrogen isotope exchange reactions of aryl and heteroaryl compounds.

We have developed a novel and efficient iridium-catalyzed hydrogen isotope exchange reaction method with secondary and tertiary sulfonamides at ambient temperatures. Furthermore N-oxides and phosphonamides have been successfully applied in hydrogen isotope exchange reactions with moderate to excellent deuterium introduction.

Burgess iridium(I)-catalyst for selective hydrogen isotope exchange.

We have evaluated the commercially available Burgess catalyst in hydrogen isotope exchange reactions with several substrates bearing different directing group functionalities and have obtained moderate to high (50%-97%D) deuterium incorporations. The broad applicability in hydrogen isotope exchange reactions makes the Burgess catalyst a possible alternative compared to other commercially available iridium(I)-catalysts.