Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting OSBP.

Metabolic alterations in cancer precipitate in associated dependencies that can be therapeutically exploited. To meet this goal, natural product-inspired small molecules can provide a resource of invaluable chemotypes. Here, we identify orpinolide, a synthetic withanolide analog with pronounced antileukemic properties, via orthogonal chemical screening. Through multiomics profiling and genome-scale CRISPR-Cas9 screens, we identify that orpinolide disrupts Golgi homeostasis via a mechanism that requires active phosphatidylinositol 4-phosphate signaling at the endoplasmic reticulum-Golgi membrane interface. Thermal proteome profiling and genetic validation studies reveal the oxysterol-binding protein OSBP as the direct and phenotypically relevant target of orpinolide. Collectively, these data reaffirm sterol transport as a therapeutically actionable dependency in leukemia and motivate ensuing translational investigation via the probe-like compound orpinolide.

Inhibition of OSBP blocks retrograde trafficking by inducing partial Golgi degradation.

Sterol-binding proteins are important regulators of lipid homeostasis and membrane integrity; however, the discovery of selective modulators can be challenging due to structural similarities in the sterol-binding domains. We report the discovery of potent and selective inhibitors of oxysterol-binding protein (OSBP), which we term oxybipins. Sterol-containing chemical chimeras aimed at identifying new sterol-binding proteins by targeted degradation, led to a significant reduction in levels of Golgi-associated proteins. The degradation occurred in lysosomes, concomitant with changes in protein glycosylation, indicating that the degradation of Golgi proteins was a downstream effect. By establishing a sterol transport protein biophysical assay panel, we discovered that the oxybipins potently inhibited OSBP, resulting in blockage of retrograde trafficking and attenuating Shiga toxin toxicity. As the oxybipins do not target other sterol transporters and only stabilized OSBP in intact cells, we advocate their use as tools to study OSBP function and therapeutic relevance.

Fluorescent probes and degraders of the sterol transport protein Aster-A.

Our understanding of sterol transport proteins (STPs) has increased exponentially in the last decades with advances in the cellular and structural biology of these important proteins. However, small molecule probes have only recently been developed for a few selected STPs. Here we describe the synthesis and evaluation of potential proteolysis-targeting chimeras (PROTACs) based on inhibitors of the STP Aster-A. Based on the reported Aster-A inhibitor autogramin-2, ten PROTACs were synthesized. Pomalidomide-based PROTACs functioned as fluorescent probes due to the intrinsic fluorescent properties of the aminophthalimide core, which in some cases was significantly enhanced upon Aster-A binding. Most PROTACs maintained excellent binary affinity to Aster-A, and one compound, NGF3, showed promising Aster-A degradation in cells. The tools developed here lay the foundation for optimizing Aster-A fluorescent probes and degraders and studying its activity and function in vitro and in cells.

Identification of Biologically Diverse Tetrahydronaphthalen-2-ols through the Synthesis and Phenotypic Profiling of Chemically Diverse, Estradiol-Inspired Compounds.

Combining natural product fragments to design new scaffolds with unprecedented bioactivity is a powerful strategy for the discovery of tool compounds and potential therapeutics. However, the choice of fragments to couple and the biological screens to employ remain open questions in the field. By choosing a primary fragment containing the A/B ring system of estradiol and fusing it to nine different secondary fragments, we were able to identify compounds that modulated four different phenotypes: inhibition of autophagy and osteoblast differentiation, as well as potassium channel and tubulin modulation. The latter two were uncovered by using unbiased morphological profiling with a cell-painting assay. The number of hits and variety in bioactivity discovered validates the use of recombining natural product fragments coupled to phenotypic screening for the rapid identification of biologically diverse compounds.

PSL Chemical Biology Symposia Third Edition: A Branch of Science in its Explosive Phase.

This symposium is the third PSL (Paris Sciences & Lettres) Chemical Biology meeting (2016, 2019, 2023) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette (2013, 2014), under the directorship of Professor Max Malacria, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition was postponed twice for the reasons that we know. This has given us the opportunity to invite additional speakers of great standing. This year, Institut Curie hosted around 300 participants, including 220 on site and over 80 online. The pandemic has had, at least, the virtue of promoting online meetings, which we came to realize is not perfect but has its own merits. In particular, it enables those with restricted time and resources to take part in events and meetings, which can now accommodate unlimited participants. We apologize to all those who could not attend in person this time due to space limitation at Institut Curie.

Small molecule probes for targeting autophagy.

Autophagy is implicated in a wide range of (patho)physiological processes including maintenance of cellular homeostasis, neurodegenerative disorders, aging and cancer. As such, small molecule autophagy modulators are in great demand, both for their ability to act as tools to better understand this essential process and as potential therapeutics. Despite substantial advances in the field, major challenges remain in the development and comprehensive characterization of probes that are specific to autophagy. In this Review, we discuss recent developments in autophagy-modulating small molecules, including the specific challenges faced in the development of activators and inhibitors, and recommend guidelines for their use. Finally, we discuss the potential to hijack the process for targeted protein degradation, an area of great importance in chemical biology and drug discovery.

Structure, function and small molecule modulation of intracellular sterol transport proteins.

Intracellular sterol transport proteins (STPs) are crucial for maintaining cellular lipid homeostasis by regulating local sterol pools. Despite structural similarities in their sterol binding domains, STPs have different substrate specificities, intracellular localisation and biological functions. In this review, we highlight recent advances in the determination of STP structures and how this regulates their lipid specificities. Furthermore, we cover the important discoveries relating to the intracellular localisation of STPs, and the organelles between which lipid transport is carried out, giving rise to specific functions in health and disease. Finally, serendipitous and targeted efforts to identify small molecule modulators of STPs, as well as their ability to act as tool compounds and potential therapeutics, will be discussed.

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.

Identification of Inhibitors of Cholesterol Transport Proteins Through the Synthesis of a Diverse, Sterol-Inspired Compound Collection.

Cholesterol transport proteins regulate a vast array of cellular processes including lipid metabolism, vesicular and non-vesicular trafficking, organelle contact sites, and autophagy. Despite their undoubted importance, the identification of selective modulators of this class of proteins has been challenging due to the structural similarities in the cholesterol-binding site. Herein we report a general strategy for the identification of selective inhibitors of cholesterol transport proteins via the synthesis of a diverse sterol-inspired compound collection. Fusion of a primary sterol fragment to an array of secondary privileged scaffolds led to the identification of potent and selective inhibitors of the cholesterol transport protein Aster-C, which displayed a surprising preference for the unnatural-sterol AB-ring stereochemistry and new inhibitors of Aster-A. We propose that this strategy can and should be applied to any therapeutically relevant sterol-binding protein.

Pseudo Natural Products-Chemical Evolution of Natural Product Structure.

Pseudo-natural products (PNPs) combine natural product (NP) fragments in novel arrangements not accessible by current biosynthesis pathways. As such they can be regarded as non-biogenic fusions of NP-derived fragments. They inherit key biological characteristics of the guiding natural product, such as chemical and physiological properties, yet define small molecule chemotypes with unprecedented or unexpected bioactivity. We iterate the design principles underpinning PNP scaffolds and highlight their syntheses and biological investigations. We provide a cheminformatic analysis of PNP collections assessing their molecular properties and shape diversity. We propose and discuss how the iterative analysis of NP structure, design, synthesis, and biological evaluation of PNPs can be regarded as a human-driven branch of the evolution of natural products, that is, a chemical evolution of natural product structure.

Phenotyping Reveals Targets of a Pseudo-Natural-Product Autophagy Inhibitor.

Pseudo-natural-product (NP) design combines natural product fragments to provide unprecedented NP-inspired compounds not accessible by biosynthesis, but endowed with biological relevance. Since the bioactivity of pseudo-NPs may be unprecedented or unexpected, they are best evaluated in target agnostic cell-based assays monitoring entire cellular programs or complex phenotypes. Here, the Cinchona alkaloid scaffold was merged with the indole ring system to synthesize indocinchona alkaloids by Pd-catalyzed annulation. Exploration of indocinchona alkaloid bioactivities in phenotypic assays revealed a novel class of azaindole-containing autophagy inhibitors, the azaquindoles. Subsequent characterization of the most potent compound, azaquindole-1, in the morphological cell painting assay, guided target identification efforts. In contrast to the parent Cinchona alkaloids, azaquindoles selectively inhibit starvation- and rapamycin-induced autophagy by targeting the lipid kinase VPS34.

Image-Based Morphological Profiling Identifies a Lysosomotropic, Iron-Sequestering Autophagy Inhibitor.

Chemical proteomics is widely applied in small-molecule target identification. However, in general it does not identify non-protein small-molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image-based morphological profiling in the cell painting assay. A compound-induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe2+ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.

Modulation of autophagy by the novel mitochondrial complex I inhibitor Authipyrin.

Autophagy ensures cellular homeostasis by the degradation of long-lived proteins, damaged organelles and pathogens. This catabolic process provides essential cellular building blocks upon nutrient deprivation. Cellular metabolism, especially mitochondrial respiration, has a significant influence on autophagic flux, and complex I function is required for maximal autophagy. In Parkinson's disease mitochondrial function is frequently impaired and autophagic flux is altered. Thus, dysfunctional organelles and protein aggregates accumulate and cause cellular damage. In order to investigate the interdependency between mitochondrial function and autophagy, novel tool compounds are required. Herein, we report the discovery of a structurally novel autophagy inhibitor (Authipyrin) using a high content screening approach. Target identification and validation led to the discovery that Authipyrin targets mitochondrial complex I directly, leading to the potent inhibition of mitochondrial respiration as well as autophagy.

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.

Natural product inspired compound collections: evolutionary principle, chemical synthesis, phenotypic screening, and target identification.

Natural products have been an excellent and abundant source of therapeutics for many decades. To expand on their success, and explore areas of chemical space not covered by biosynthesis, the synthesis of natural product-inspired compound collections has emerged as a viable strategy. Herein we describe the principles behind biology-oriented synthesis and related approaches, the requirements for development of novel chemistry and how phenotypic screens are a very fruitful way to explore the bioactivity of compounds made using these approaches. Finally, we summarize state-of-the-art techniques to identify the biological targets of any hits identified.

Highly Enantioselective Catalytic Vinylogous Propargylation of Coumarins Yields a Class of Autophagy Inhibitors.

A highly enantioselective copper-catalyzed vinylogous propargylic substitution has been developed. Aromatic and aliphatic propargylic esters react smoothly with substituted coumarins under mild reaction conditions to give the desired products with excellent yields and enantioselectivities. Subsequent single-step transformations enable the synthesis of a wide range of multifunctional and diverse compounds, and allow the efficient combination of different natural product fragments. Investigation of the obtained compound collection in cell-based assays monitoring changes in phenotype led to the discovery of a novel class of autophagy inhibitors.

Discovery of Novel Cinchona-Alkaloid-Inspired Oxazatwistane Autophagy Inhibitors.

The cinchona alkaloids are a privileged class of natural products and are endowed with diverse bioactivities. However, for compounds with the closely-related oxazatricyclo[4.4.0.0]decane ("oxazatwistane") scaffold, which are accessible from cinchonidine and quinidine by means of ring distortion and modification, biological activity has not been identified. We report the synthesis of an oxazatwistane compound collection through employing state-of-the-art C-H functionalization, and metal-catalyzed cross-coupling reactions as key late diversity-generating steps. Exploration of oxazatwistane bioactivity in phenotypic assays monitoring different cellular processes revealed a novel class of autophagy inhibitors termed oxautins, which, in contrast to the guiding natural products, selectively inhibit autophagy by inhibiting both autophagosome biogenesis and autophagosome maturation.

Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS34.

Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small-molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy-induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.

Making bubbles: Targeting VPS41 induces vacuolization and methuosis.

In this issue of Cell Chemical Biology, Liu et al. describe the natural product DMBP as the first tool compound for VPS41. Treatment with DMBP induced vacuolization and methuosis and inhibited autophagic flux in lung and pancreatic cancer cell lines, validating VPS41 as a potential therapeutic target.

Redshifted and thermally bistable one-way quantitative hemithioindigo-derived photoswitches enabled by isomer-specific excited state intramolecular proton transfer.

We report bistable indole-containing hemithioindigos (HTIs) with one-way quantitative photoswitching properties. Supported by state-averaged CASPT2/CASSCF calculations, we propose a mechanism for the observed one-way photoswitching that involves an isomer-specific excited state intramolecular proton transfer (ESIPT). Additionally, we developed a thermally bistable oligomer-inspired bipyrrole-containing HTI, which displays large band separation and bidirectional near-quantitative photoisomerization in the near-infrared, bio-optical window.