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Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Here, we describe a set of stereo- and regiochemically defined spirocycle acrylamides and the analysis of these electrophilic “stereoprobes” in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamide stereoprobes, the spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound termed ZL-12A promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL-12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation but instead causes collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another’s protein degradation profiles. Finally, we provide evidence that the antihypertension drug spironolactone─previously found to promote ERCC3 degradation through an enigmatic mechanism─also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.

Covalent chemistry is a versatile approach for expanding the ligandability of the human proteome. Activity-based protein profiling (ABPP) can infer the specific residues modified by electrophilic compounds through competition with broadly reactive probes. However, the extent to which such residue-directed platforms fully assess the protein targets of electrophilic compounds in cells remains unclear. Here we evaluate a complementary protein-directed ABPP method that identifies proteins showing stereoselective reactivity with alkynylated, chiral electrophilic compounds—termed stereoprobes. Integration of protein- and cysteine-directed data from cancer cells treated with tryptoline acrylamide stereoprobes revealed generally well-correlated ligandability maps and highlighted features, such as protein size and the proteotypicity of cysteine-containing peptides, that explain gaps in each ABPP platform. In total, we identified stereoprobe binding events for >300 structurally and functionally diverse proteins, including compounds that stereoselectively and site-specifically disrupt MAD2L1BP interactions with the spindle assembly checkpoint complex leading to delayed mitotic exit in cancer cells.

The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better understand and potentially manipulate protein localization for therapeutic purposes, we engineered bifunctional compounds that bind to proteins in separate cellular compartments. We show these compounds induce nuclear import of cytosolic cargoes, using nuclear-localized BRD4 as a “carrier” for co-import and nuclear trapping of cytosolic proteins. We use this system to calculate kinetic constants for passive diffusion across the nuclear pore and demonstrate single-cell heterogeneity in response to these bifunctional molecules with cells requiring high carrier to cargo expression for complete import. We also observe incorporation of cargo into BRD4-containing condensates. Proteins shown to be substrates for nuclear transport include oncogenic mutant nucleophosmin (NPM1c) and mutant PI3K catalytic subunit alpha (PIK3CAE545K), suggesting potential applications to cancer treatment. In addition, we demonstrate that chemically induced localization of BRD4 to cytosolic-localized DNA-binding proteins, namely, IRF1 with a nuclear export signal, induces target gene expression. These results suggest that induced localization of proteins with bifunctional molecules enables the rewiring of cell circuitry, with significant implications for disease therapy.

Small molecules that induce protein–protein associations represent powerful tools to modulate cell circuitry. We sought to develop a platform for the direct discovery of compounds able to induce association of any two preselected proteins, using the E3 ligase von Hippel–Lindau (VHL) and bromodomains as test systems. Leveraging the screening power of DNA-encoded libraries (DELs), we synthesized ~1 million DNA-encoded compounds that possess a VHL-targeting ligand, a variety of connectors and a diversity element generated by split-and-pool combinatorial chemistry. By screening our DEL against bromodomains in the presence and absence of VHL, we could identify VHL-bound molecules that simultaneously bind bromodomains. For highly barcode-enriched library members, ternary complex formation leading to bromodomain degradation was confirmed in cells. Furthermore, a ternary complex crystal structure was obtained for our most enriched library member with BRD4BD1and a VHL complex. Our work provides a foundation for adapting DEL screening to the discovery of proximity-inducing small molecules.

Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

Targeted protein degradation induced by heterobifunctional compounds and molecular glues presents an exciting avenue for chemical probe and drug discovery. To date, small-molecule ligands have been discovered for only a limited number of E3 ligases, which is an important limiting factor for realizing the full potential of targeted protein degradation. We report herein the discovery by chemical proteomics of azetidine acrylamides that stereoselectively and site-specifically react with a cysteine (C1113) in the E3 ligase substrate receptor DCAF1. We demonstrate that the azetidine acrylamide ligands for DCAF1 can be developed into electrophilic proteolysis-targeting chimeras (PROTACs) that mediated targeted protein degradation in human cells. We show that this process is stereoselective and does not occur in cells expressing a C1113A mutant of DCAF1. Mechanistic studies indicate that only low fractional engagement of DCAF1 is required to support protein degradation by electrophilic PROTACs. These findings, taken together, demonstrate how the chemical proteomic analysis of stereochemically defined electrophilic compound sets can uncover ligandable sites on E3 ligases that support targeted protein degradation.

This lengthy article shows how use of small molecules are helping to change the field of organic chemical research.

The development of an entirely crystallization-based synthetic route to the antimalarial BRD5018 is described, which assembles a structurally complex bicyclic azetidine scaffold adorned with five stereogenic centers without the need for any chromatographic separations. A diastereoselective glycine ester Claisen rearrangement, diastereomeric salt resolution, and diastereoselective iodo-lactonization are utilized to provide an efficient access to three contiguous stereogenic centers on an acyclic template with the desired relative and absolute configurations. A tandem aziridine ring-opening/azetidine ring-closure on the derived 2-amino-1,4-diol template was developed to efficiently establish the all-cistrisubstituted azetidine scaffold with the proper ancillary functionality for end-game maneuvers. d-Ribose-2,3-acetonide provided a conveniently differentiated vicinal syn-diol suitable for the planned reductive amination/periodate cleavage/Staudinger-aza-Wittig sequence to form the eight-membered diazocene ring. An early quantitative installation of the diaryl acetylene moiety via a Sonogashira coupling on an electronically matched methyl 4-bromocinnamate circumvented a low-yielding, late-stage reaction in the first-generation synthesis. Multiple crystalline intermediates enabled the complete removal of chromatography from the synthesis resulting in a substantially reduced cost and waste generation with enhanced throughput and quality control.

Small-molecule discovery typically involves large-scale screening campaigns, spanning multiple compound collections. However, such activities can be cost- or time-prohibitive, especially when using complex assay systems, limiting the number of compounds tested. Further, low hit rates can make the process inefficient. Sparse coverage of chemical structure or biological activity space can lead to limited success in a primary screen and represents a missed opportunity by virtue of selecting the “wrong” compounds to test. Thus, the choice of screening collections becomes of paramount importance. In this perspective, we discuss the utility of generating “informer sets” for small-molecule discovery, and how this strategy can be leveraged to prioritize probe candidates. While many researchers may assume that informer sets are focused on particular targets (e.g., kinases) or processes (e.g., autophagy), efforts to assemble informer sets based on historical bioactivity or successful human exposure (e.g., repurposing collections) have shown promise as well. Another method for generating informer sets is based on chemical structure, particularly when the compounds have unknown activities and targets. We describe our efforts to screen an informer set representing a collection of 100,000 small molecules synthesized through diversity-oriented synthesis (DOS). This process enables researchers to identify activity early and more extensively screen only a few chemical scaffolds, rather than the entire collection. This elegant and economic outcome is a goal of the informer set approach. Here, we aim not only to shed light on this process, but also to promote the use of informer sets more widely in small-molecule discovery projects.

Ferroptosis—an iron-dependent, non-apoptotic cell death process—is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2–5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR–Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome–ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.

Small molecules have been classically developed to inhibit enzyme activity; however, new classes of small molecules that endow new functions to enzymes via proximity-mediated effect are emerging. Phosphorylation (native or neo) of any given protein-of-interest can alter its structure and function, and we hypothesized that such modifications can be accomplished by small molecules that bring a kinase in proximity to the protein-of-interest. Herein, we describe phosphorylation-inducing chimeric small molecules (PHICS), which enable two example kinases—AMPK and PKC—to phosphorylate target proteins that are not otherwise substrates for these kinases. PHICS are formed by linking small-molecule binders of the kinase and the target protein, and exhibit several features of a bifunctional molecule, including the hook-effect, turnover, isoform specificity, dose and temporal control of phosphorylation, and activity dependent on proximity (i.e., linker length). Using PHICS, we were able to induce native and neo-phosphorylations of BRD4 by AMPK or PKC. Furthermore, PHICS induced a signaling-relevant phosphorylation of the target protein Bruton’s tyrosine kinase in cells. We envision that PHICS-mediated native or neo-phosphorylations will find utility in basic research and medicine.

Naturally occurring human genetic variants that are predicted to inactivate protein-coding genes provide an in vivo model of human gene inactivation that complements knockout studies in cells and model organisms. Here we report three key findings regarding the assessment of candidate drug targets using human loss-of-function variants. First, even essential genes, in which loss-of-function variants are not tolerated, can be highly successful as targets of inhibitory drugs. Second, in most genes, loss-of-function variants are sufficiently rare that genotype-based ascertainment of homozygous or compound heterozygous ‘knockout’ humans will await sample sizes that are approximately 1,000 times those presently available, unless recruitment focuses on consanguineous individuals. Third, automated variant annotation and filtering are powerful, but manual curation remains crucial for removing artefacts, and is a prerequisite for recall-by-genotype efforts. Our results provide a roadmap for human knockout studies and should guide the interpretation of loss-of-function variants in drug development.

We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.

DNA-encoded libraries of small molecules are being explored extensively for the identification of binders in early drug-discovery efforts. Combinatorial syntheses of such libraries require water- and DNA-compatible reactions, and the paucity of these reactions currently limit the chemical features of resulting barcoded products. The present work introduces strain-promoted cycloadditions of cyclic allenes under mild conditions to DNA-encoded library synthesis. Owing to distinct cycloaddition modes of these reactive intermediates with activated olefins, 1,3-dipoles, and dienes, the process generates diverse molecular architectures from a single precursor. The resulting DNA-barcoded compounds exhibit unprecedented ring and topographic features, related to elements found to be powerful in phenotypic screening.

Nature uses a variety of tools to mediate the flow of information in cells, many of which control distances between key biomacromolecules. Researchers have thus generated compounds whose activities stem from interactions with two (or more) proteins simultaneously. In this Perspective, we describe how these ‘bifunctional’ small molecules facilitate the study of an increasingly wide range of complex biological phenomena and enable the drugging of otherwise challenging therapeutic targets and processes. Despite their structural and functional differences, all bifunctional molecules employ Nature’s strategy of altering interactomes and inducing proximity to modulate biology. They therefore exhibit a shared set of chemical and biophysical principles that have not yet been appreciated fully. By highlighting these commonalities—and their wide-ranging consequences—we hope to chip away at the artificial barriers that threaten to constrain this interdisciplinary field. Doing so promises to yield remarkable benefits for biological research and therapeutics discovery.

Ferroptosis is widely involved in degenerative diseases in various tissues including kidney, liver and brain, and is a targetable vulnerability in multiple primary and therapy-resistant cancers. Accumulation of phospholipid hydroperoxides in cellular membranes is the hallmark and rate-limiting step of ferroptosis; however, the enzymes contributing to lipid peroxidation remain poorly characterized. Using genome-wide, CRISPR–Cas9-mediated suppressor screens, we identify cytochrome P450 oxidoreductase (POR) as necessary for ferroptotic cell death in cancer cells exhibiting inherent and induced susceptibility to ferroptosis. By genetic depletion of POR in cancer cells, we reveal that POR contributes to ferroptosis across a wide range of lineages and cell states, and in response to distinct mechanisms of ferroptosis induction. Using systematic lipidomic profiling, we further map POR’s activity to the lipid peroxidation step in ferroptosis. Hence, our work suggests that POR is a key mediator of ferroptosis and potential druggable target for developing antiferroptosis therapeutics.

GPX4 represents a promising yet difficult-to-drug therapeutic target for the treatment of, among others, drug-resistant cancers. Although most GPX4 inhibitors rely on a chloroacetamide moiety to modify covalently the protein’s catalytic selenocysteine residue, the discovery and mechanistic elucidation of structurally diverse GPX4-inhibiting molecules have uncovered novel electrophilic warheads that bind and inhibit GPX4. Here, we report our discovery that diacylfuroxans can act as masked nitrile oxide prodrugs that inhibit GPX4 covalently with unique cellular and biochemical reactivity compared to existing classes of GPX4 inhibitors. These observations illuminate a novel molecular mechanism of action for biologically active furoxans and also expand the collection of reactive groups capable of targeting GPX4.

PrP in cerebrospinal fluid is measured by targeted mass spectrometry using peptides across protein domains in humans and preclinical species of interest. Peptides are uniformly reduced in patients with prion disease, suggesting that dose-finding studies of PrP-lowering drugs may be most informative in presymptomatic individuals.

The development of new antimalarial therapeutics is necessary to address the increasing resistance to current drugs. Bicyclic azetidines targeting Plasmodium falciparumphenylalanyl-tRNA synthetase comprise one promising new class of antimalarials, especially due to their activities against three stages of the parasite’s life cycle, but a lengthy synthetic route to these compounds may affect the feasibility of delivering new therapeutic agents within the cost constraints of antimalarial drugs. Here, we report an efficient synthesis of antimalarial compound BRD3914 (EC50= 15 nM) that hinges on a Pd-catalyzed, directed C(sp3)–H arylation of azetidines at the C3 position. This newly developed protocol exhibits a broad substrate scope and provides access to valuable, stereochemically defined building blocks. BRD3914 was evaluated in P. falciparum-infected mice, providing a cure after four oral doses.

It is challenging to incorporate stereochemical diversity and topographic complexity into DNA-encoded libraries (DELs) because DEL syntheses cannot fully exploit the capabilities of modern synthetic organic chemistry. Here, we describe the design, construction, and validation of DOS-DEL-1, a library of 107 616 DNA-barcoded chiral 2,3-disubsituted azetidines and pyrrolidines. We used stereospecific C–H arylation chemistry to furnish complex scaffolds primed for DEL synthesis, and we developed an improved on-DNA Suzuki reaction to maximize library quality. We then studied both the structural diversity of the library and the physicochemical properties of individual compounds using Tanimoto multifusion similarity analysis, among other techniques. These analyses revealed not only that most DOS-DEL-1 members have “drug-like” properties, but also that the library more closely resembles compound collections derived from diversity synthesis than those from other sources (e.g., commercial vendors). Finally, we performed validation screens against horseradish peroxidase and carbonic anhydrase IX, and we developed a novel, Poisson-based statistical framework to analyze the results. A set of assay positives were successfully translated into potent carbonic anhydrase inhibitors (IC50= 20.1–68.7 nM), which confirmed the success of the synthesis and screening procedures. These results establish a strategy to synthesize DELs with scaffold-based stereochemical diversity and complexity that does not require the development of novel DNA-compatible chemistry.

The limited scope of DNA-compatible chemistry restricts the types of chemical features that can be incorporated into DNA-encoded libraries (DELs). Here, a method to synthesize DNA-conjugated polycyclic isoxazolidines via a [3 + 2] nitrone–olefin cycloaddition is described. The reaction is compatible with many olefin-containing substrates and diverse N-alkylhydroxylamines. The ability to perform subsequent DNA ligation and PCR amplification was also confirmed. This methodology facilitates the synthesis of DELs containing topographically complex compounds with underexplored chemical features.

Chordoma is a primary bone cancer with no approved therapy1. The identification of therapeutic targets in this disease has been challenging due to the infrequent occurrence of clinically actionable somatic mutations in chordoma tumors2,3. Here we describe the discovery of therapeutically targetable chordoma dependencies via genome-scale CRISPR-Cas9 screening and focused small-molecule sensitivity profiling. These systematic approaches reveal that the developmental transcription factor T(brachyury; TBXT) is the top selectively essential gene in chordoma, and that transcriptional cyclin-dependent kinase (CDK) inhibitors targeting CDK7/12/13 and CDK9 potently suppress chordoma cell proliferation. In other cancer types, transcriptional CDK inhibitors have been observed to downregulate highly expressed, enhancer-associated oncogenic transcription factors4,5. In chordoma, we find that Tis associated with a 1.5-Mb region containing ‘super-enhancers’ and is the most highly expressed super-enhancer-associated transcription factor. Notably, transcriptional CDK inhibition leads to preferential and concentration-dependent downregulation of cellular brachyury protein levels in all models tested. In vivo, CDK7/12/13-inhibitor treatment substantially reduces tumor growth. Together, these data demonstrate small-molecule targeting of brachyury transcription factor addiction in chordoma, identify a mechanism of Tgene regulation that underlies this therapeutic strategy, and provide a blueprint for applying systematic genetic and chemical screening approaches to discover vulnerabilities in genomically quiet cancers.

Target- and phenotype-agnostic assessments of biological activity have emerged as viable strategies for prioritizing scaffolds, structural features, and synthetic pathways in screening sets, with the goal of increasing performance diversity. Here, we describe the synthesis of a small library of functionalized stereoisomeric azetidines and its biological annotation by “cell painting,” a multiplexed, high-content imaging assay capable of measuring many hundreds of compound-induced changes in cell morphology in a quantitative and unbiased fashion. Using this approach, we systematically compare the degrees to which a core scaffold’s biological activity, inferred from its effects on cell morphology, is affected by variations in stereochemistry and appendages. We show that stereoisomerism and appendage diversification can produce effects of similar magnitude, and that the concurrent use of these strategies results in a broader sampling of biological activity.

Strategies such as diversity-oriented synthesis aim to explore novel areas of chemical space efficiently by populating small-molecule screening libraries with compounds containing structural features that are typically under-represented in commercially available screening collections. This article highlights how the design and synthesis of such libraries have been enabled by modern synthetic chemistry and illustrates the impact of the resultant chemical probes and drug leads in a wide range of diseases.

Cancer persister cells, which survive cytotoxic treatments, are shown to be sensitive to inhibition of the lipid hydroperoxidase GPX4.

Enhancing production of the anti-inflammatory cytokine interleukin-10 (IL-10) is a promising strategy to suppress pathogenic inflammation. To identify new mechanisms regulating IL-10 production, we conducted a phenotypic screen for small molecules that enhance IL-10 secretion from activated dendritic cells. Mechanism-of-action studies using a prioritized hit from the screen, BRD6989, identified the Mediator-associated kinase CDK8, and its paralog CDK19, as negative regulators of IL-10 production during innate immune activation. The ability of BRD6989 to upregulate IL-10 is recapitulated by multiple, structurally differentiated CDK8 and CDK19 inhibitors and requires an intact cyclin C–CDK8 complex. Using a highly parallel pathway reporter assay, we identified a role for enhanced AP-1 activity in IL-10 potentiation following CDK8 and CDK19 inhibition, an effect associated with reduced phosphorylation of a negative regulatory site on c-Jun. These findings identify a function for CDK8 and CDK19 in regulating innate immune activation and suggest that these kinases may warrant consideration as therapeutic targets for inflammatory disorders.

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α–β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial–mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

The mechanism proposed for the transformation of cyclopentanone to the dienoic acid 1, as published in this journal, is revealed to be in error. We show that carbon 11 derives not from dimethyl sulfoxide as proposed but from the dichloromethane present in the “quenching” solution. The intermediacy of an α-chloromethyl ketone and its subsequent fragmentation in the presence of a hydroxide ion is supported by experiments described herein and by extensive literature precedent.

The hallmark of a molecular glue is its ability to induce cooperative protein–protein interactions, leading to the formation of a ternary complex, despite weaker binding toward one or both individual proteins. Notably, the extent of cooperativity distinguishes molecular glues from bifunctional compounds, which constitute a second class of inducers of protein–protein interactions. However, apart from serendipitous discovery, there have been limited rational screening strategies for the high cooperativity exhibited by molecular glues. Here, we propose a binding-based screen of DNA-barcoded compounds on a target protein in the presence or absence of a presenter protein, using the “presenter ratio”, the ratio of ternary enrichment to binary enrichment, as a predictive measure of cooperativity. Through this approach, we identified a range of cooperative, noncooperative, and uncooperative compounds in a single DNA-encoded library screen with bromodomain containing protein (BRD)9 and the VHL–elongin C–elongin B (VCB) complex. Our most cooperative hit compound, 13-7, exhibits micromolar binding affinity to BRD9 but nanomolar affinity for the ternary complex with BRD9 and VCB, with cooperativity comparable to classical molecular glues. This approach may enable the rational discovery of molecular glues for preselected proteins and thus facilitate the transition to a new paradigm of small-molecule therapeutics.

Antimalarial drugs have thus far been chiefly derived from two sources—natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

Salt-inducible kinases (SIKs) are promising therapeutic targets for modulating cytokine responses during innate immune activation. The study of SIK inhibition in animal models of disease has been limited by the lack of selective small-molecule probes suitable for modulating SIK function in vivo. We used the pan-SIK inhibitor HG-9-91-01 as a starting point to develop improved analogs, yielding a novel probe 5(YKL-05-099) that displays increased selectivity for SIKs versus other kinases and enhanced pharmacokinetic properties. Well-tolerated doses of YKL-05-099 achieve free serum concentrations above its IC50for SIK2 inhibition for >16 h and reduce phosphorylation of a known SIK substrate in vivo. While in vivoactive doses of YKL-05-099 recapitulate the effects of SIK inhibition on inflammatory cytokine responses, they did not induce metabolic abnormalities observed in Sik2knockout mice. These results identify YKL-05-099 as a useful probe to investigate SIK function in vivoand further support the development of SIK inhibitors for treatment of inflammatory disorders.

The mood stabilizer lithium, the first-line treatment for bipolar disorder, is hypothesized to exert its effects through direct inhibition of glycogen synthase kinase 3 (GSK3) and indirectly by increasing GSK3’s inhibitory serine phosphorylation. GSK3 comprises two highly similar paralogs, GSK3α and GSK3β, which are key regulatory kinases in the canonical Wnt pathway. GSK3 stands as a nodal target within this pathway and is an attractive therapeutic target for multiple indications. Despite being an active field of research for the past 20 years, many GSK3 inhibitors demonstrate either poor to moderate selectivity versus the broader human kinome or physicochemical properties unsuitable for use in in vitrosystems or in vivomodels. A nonconventional analysis of data from a GSK3β inhibitor high-throughput screening campaign, which excluded known GSK3 inhibitor chemotypes, led to the discovery of a novel pyrazolo-tetrahydroquinolinone scaffold with unparalleled kinome-wide selectivity for the GSK3 kinases. Taking advantage of an uncommon tridentate interaction with the hinge region of GSK3, we developed highly selective and potent GSK3 inhibitors, BRD1652and BRD0209, which demonstrated in vivoefficacy in a dopaminergic signaling paradigm modeling mood-related disorders. These new chemical probes open the way for exclusive analyses of the function of GSK3 kinases in multiple signaling pathways involved in many prevalent disorders.

Unbiased binding assays involving small-molecule microarrays were used to identify compounds that display unique patterns of selectivity among members of the zinc-dependent histone deacetylase family of enzymes. A novel, hydroxyquinoline-containing compound, BRD4354, was shown to preferentially inhibit activity of HDAC5 and HDAC9 in vitro. Inhibition of deacetylase activity appears to be time-dependent and reversible. Mechanistic studies suggest that the compound undergoes zinc-catalyzed decomposition to an ortho-quinone methide, which covalently modifies nucleophilic cysteines within the proteins. The covalent nature of the compound–enzyme interaction has been demonstrated in experiments with biotinylated probe compound and with electrospray ionization–mass spectrometry.

Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with ∼19,000 basal transcript levels across 823 different human cancer cell lines and identified selective outlier transcripts. This process yielded many novel mechanistic insights, including the identification of activation mechanisms, cellular transporters and direct protein targets. We found that ML239, originally identified in a phenotypic screen for selective cytotoxicity in breast cancer stem-like cells, most likely acts through activation of fatty acid desaturase 2 (FADS2). These data and analytical tools are available to the research community through the Cancer Therapeutics Response Portal.

High cancer death rates indicate the need for new anticancer therapeutic agents. Approaches to discovering new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds through phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the gene PDE3A, encoding phosphodiesterase 3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells, whereas others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggestive of a neomorphic activity. Coexpression of SLFN12 with PDE3A correlates with DNMDP sensitivity, whereas depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.

We examined the effects of isoform-specific histone deacetylase (HDAC) inhibitors on β-catenin posttranslational modifications in neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs). β-catenin is a multifunctional protein with important roles in the developing and adult central nervous system. Activation of the Wnt pathway results in stabilization and nuclear translocation of β-catenin, resulting in activation of multiple target genes. In addition, β-catenin forms a complex with cadherins at the plasma membrane as part of the adherens junctions. The N-terminus of β-catenin has phosphorylation, ubiquitination, and acetylation sites that regulate its stability and signaling. In the absence of a Wnt signal, Ser33, Ser37, and Thr41 are constitutively phosphorylated by glycogen synthase kinase 3β (GSK3β). β-Catenin phosphorylated at these sites is recognized by β-transducin repeat-containing protein (βTrCP), which results in ubiquitination and degradation by the ubiquitin-proteasome pathway. The N-terminal regulatory domain of β-catenin also includes Ser45, a phosphorylation site for Casein Kinase 1α (CK1α) and Lys49, which is acetylated by the acetyltransferase p300/CBP-associated factor (PCAF). The relevance of Lys49 acetylation and Ser45 phosphorylation to the function of β-catenin is an active area of investigation. We find that HDAC6 inhibitors increase Lys49 acetylation and Ser45 phosphorylation but do not affect Ser33, Ser37, and Thr41 phosphorylation. Lys49 acetylation results in decreased ubiquitination of β-catenin in the presence of proteasome inhibition. While increased Lys49 acetylation does not affect total levels of β-catenin, it results in increased membrane localization of β-catenin.

Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876’s mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.

The small-molecule probes STF-31 and its analogue compound 146 were discovered while searching for compounds that kill VHL-deficient renal cell carcinoma cell lines selectively and have been reported to act via direct inhibition of the glucose transporter GLUT1. We profiled the sensitivity of 679 cancer cell lines to STF-31 and found that the pattern of response is tightly correlated with sensitivity to three different inhibitors of nicotinamide phosphoribosyltransferase (NAMPT). We also performed whole-exome next-generation sequencing of compound 146-resistant HCT116 clones and identified a recurrent NAMPT-H191R mutation. Ectopic expression of NAMPT-H191R conferred resistance to both STF-31 and compound 146 in cell lines. We further demonstrated that both STF-31 and compound 146 inhibit the enzymatic activity of NAMPT in a biochemical assay in vitro. Together, our cancer-cell profiling and genomic approaches identify NAMPT inhibition as a critical mechanism by which STF-31-like compounds inhibit cancer cells.

Pseudomonas aeruginosaproduces the peptide siderophore pyoverdine, which is used to acquire essential Fe3+ions from the environment. PvdQ, an Ntn hydrolase, is required for the biosynthesis of pyoverdine. PvdQknockout strains are not infectious in model systems, suggesting that disruption of siderophore production via PvdQ inhibition could be exploited as a target for novel antibacterial agents, by preventing cells from acquiring iron in the low iron environments of most biological settings. We have previously described a high-throughput screen to identify inhibitors of PvdQ that identified inhibitors with IC50values of ∼100 μM. Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase. ML318 inhibits PvdQ in vitro(IC50= 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318. Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosaunder iron-limiting conditions.

Understanding the structure–activity relationships (SARs) of small molecules is important for developing probes and novel therapeutic agents in chemical biology and drug discovery. Increasingly, multiplexed small-molecule profiling assays allow simultaneous measurement of many biological response parameters for the same compound (e.g., expression levels for many genes or binding constants against many proteins). Although such methods promise to capture SARs with high granularity, few computational methods are available to support SAR analyses of high-dimensional compound activity profiles. Many of these methods are not generally applicable or reduce the activity space to scalar summary statistics before establishing SARs. In this article, we present a versatile computational method that automatically extracts interpretable SAR rules from high-dimensional profiling data. The rules connect chemical structural features of compounds to patterns in their biological activity profiles. We applied our method to data from novel cell-based gene-expression and imaging assays collected on more than 30,000 small molecules. Based on the rules identified for this data set, we prioritized groups of compounds for further study, including a novel set of putative histone deacetylase inhibitors.

Autophagy is an evolutionarily conserved catabolic process that directs cytoplasmic proteins, organelles and microbes to lysosomes for degradation. Autophagy acts at the intersection of pathways involved in cellular stress, host defense, and modulation of inflammatory and immune responses; however, the details of how the autophagy network intersects with these processes remain largely undefined. Given the role of autophagy in several human diseases, it is important to determine the extent to which modulators of autophagy also modify inflammatory or immune pathways and whether it is possible to modulate a subset of these pathways selectively. Here, we identify small-molecule inducers of basal autophagy (including several FDA-approved drugs) and characterize their effects on IL-1β production, autophagic engulfment and killing of intracellular bacteria, and development of Treg, TH17, and TH1 subsets from naı̈ve T cells. Autophagy inducers with distinct, selective activity profiles were identified that reveal the functional architecture of connections between autophagy, and innate and adaptive immunity. In macrophages from mice bearing a conditional deletion of the essential autophagy gene Atg16L1, the small molecules inhibit IL-1β production to varying degrees suggesting that individual compounds may possess both autophagy-dependent and autophagy-independent activity on immune pathways. The small molecule autophagy inducers constitute useful probes to test the contributions of autophagy-related pathways in diseases marked by impaired autophagy or elevated IL-1β and to test novel therapeutic hypotheses.

Elevation of reactive oxygen species (ROS) levels has been observed in many cancer cells relative to nontransformed cells, and recent reports have suggested that small-molecule enhancers of ROS may selectively kill cancer cells in various in vitroand in vivomodels. We used a high-throughput screening approach to identify several hundred small-molecule enhancers of ROS in a human osteosarcoma cell line. A minority of these compounds diminished the viability of cancer cell lines, indicating that ROS elevation by small molecules is insufficient to induce death of cancer cell lines. Three chemical probes (BRD5459, BRD56491, BRD9092) are highlighted that most strongly elevate markers of oxidative stress without causing cell death and may be of use in a variety of cellular settings. For example, combining nontoxic ROS-enhancing probes with nontoxic doses of l-buthionine sulfoximine, an inhibitor of glutathione synthesis previously studied in cancer patients, led to potent cell death in more than 20 cases, suggesting that even nontoxic ROS-enhancing treatments may warrant exploration in combination strategies. Additionally, a few ROS-enhancing compounds that contain sites of electrophilicity, including piperlongumine, show selective toxicity for transformed cells over nontransformed cells in an engineered cell-line model of tumorigenesis. These studies suggest that cancer cell lines are more resilient to chemically induced increases in ROS levels than previously thought and highlight electrophilicity as a property that may be more closely associated with cancer-selective cell death than ROS elevation.

Post-translational modifications of histones alter chromatin structure and play key roles in gene expression and specification of cell states. Small molecules that target chromatin-modifying enzymes selectively are useful as probes and have promise as therapeutics, although very few are currently available. G9a (also named euchromatin histone methyltransferase 2 (EHMT2)) catalyzes methylation of lysine 9 on histone H3 (H3K9), a modification linked to aberrant silencing of tumor-suppressor genes, among others. Here, we report the discovery of a novel histone methyltransferase inhibitor, BRD4770. This compound reduced cellular levels of di- and trimethylated H3K9 without inducing apoptosis, induced senescence, and inhibited both anchorage-dependent and -independent proliferation in the pancreatic cancer cell line PANC-1. ATM-pathway activation, caused by either genetic or small-molecule inhibition of G9a, may mediate BRD4770-induced cell senescence. BRD4770 may be a useful tool to study G9a and its role in senescence and cancer cell biology.

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D1, known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.

High grade epithelial ovarian cancers are relatively sensitive to DNA damaging platinum-based chemotherapy, suggesting that the dependencies of ovarian tumors on DNA damage response pathways can be harnessed for therapeutic purposes. Our goal was to determine if the DNA damage mark gamma-H2AX phosphorylation (pH2AX) could be used to identify suitable cytotoxic histone deacetylase inhibitors (HDACi) for ovarian cancer treatment. Nineteen chemically diverse HDACi compounds were tested in 7 ovarian cancer cell lines. Fluorescent, biochemical and cell-based assays were performed to assess DNA damage by induction of pH2AX and to measure cell viability and apoptosis. The relationships between pH2AX and the cellular effects of cell viability and apoptosis were calculated. Selected HDACi were tested in combination with cisplatin and other DNA damaging agents to determine if the HDACi improved upon the effects of the DNA damaging agents. The HDACi compounds induced differing levels of pH2AX expression. High levels of pH2AX in HDACi-treated ovarian cancer cells were tightly associated with decreased cell viability and increased apoptosis. Consequently, a ketone-based HDACi was chosen and found to enhance the effects of cisplatin, even in ovarian cancer cells with extreme resistance to DNA damaging drugs. In conclusion, a fluorescent-based assay for pH2AX can be used to determine cellular responses to HDACi in vitro and may be a useful tool to identify potentially more effective HDACi for the treatment of ovarian cancer. In addition, these results lend support to the inclusion of ketone-derived HDACi compounds for future development.

Echter Mehrwert: Die erste diastereoselektive Petasis‐Reaktion in Gegenwart chiraler Biphenolkatalysatoren ermöglicht die Synthese von syn‐ und anti‐β‐Aminoalkoholen in reiner Form. In dieser Mehrkomponentenreaktion kommen Boronate, α‐Hydroxyaldehyde und Amine zur Anwendung (siehe Schema).

Pancreatic β-cell apoptosis is a critical event during the development of type-1 diabetes. The identification of small molecules capable of preventing cytokine-induced apoptosis could lead to avenues for therapeutic intervention. We developed a set of phenotypic cell-based assays designed to identify such small-molecule suppressors. Rat INS-1E cells were simultaneously treated with a cocktail of inflammatory cytokines and a collection of 2,240 diverse small molecules and screened using an assay for cellular ATP levels. Forty-nine top-scoring compounds included glucocorticoids, several pyrazole derivatives, and known inhibitors of glycogen synthase kinase-3β. Two compounds were able to increase cellular ATP levels, reduce caspase-3 activity and nitrite production, and increase glucose-stimulated insulin secretion in the presence of cytokines. These results indicate that small molecules identified by this screening approach may protect β cells from autoimmune attack and may be good candidates for therapeutic intervention in early stages of type-1 diabetes.