C646 degrades Exportin-1 to modulate p300 chromatin occupancy and function.

Molecular glues can induce proximity between a target protein and ubiquitin ligases to induce target degradation, but strategies for their discovery remain limited. We screened 3,200 bioactive small molecules and identified that C646 requires neddylation-dependent protein degradation to induce cytotoxicity. Although the histone acetyltransferase p300 is the canonical target of C646, we provide extensive evidence that C646 directly targets and degrades Exportin-1 (XPO1). Multiple cellular phenotypes induced by C646 were abrogated in cells expressing the known XPO1C528S drug-resistance allele. While XPO1 catalyzes nuclear-to-cytoplasmic transport of many cargo proteins, it also directly binds chromatin. We demonstrate that p300 and XPO1 co-occupy hundreds of chromatin loci. Degrading XPO1 using C646 or the known XPO1 modulator S109 diminishes the chromatin occupancy of both XPO1 and p300, enabling direct targeting of XPO1 to phenocopy p300 inhibition. This work highlights the utility of drug-resistant alleles and further validates XPO1 as a targetable regulator of chromatin state.

HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity.

Proteasomes degrade intracellular proteins to generate antigenic peptides that are recognized by the adaptive immune system and promote anticancer immunity. However, tumors subvert the antigen presentation machinery to escape immunosurveillance. We hypothesized that proteasome activation could concomitantly increase antigen abundance and diversity in multiple myeloma cells. High-throughput screens revealed that histone deacetylase 6 (HDAC6) inhibitors activated proteasomes to unmask neoantigens and amplify the tumor-specific antigenic landscape. Treatment of patient CD138+ cells with HDAC6 inhibitors significantly promoted the antimyeloma activity of autologous CD8+ T cells. Pharmacologic blockade and genetic ablation of the HDAC6 ubiquitin-binding domain released HR23B, which shuttles ubiquitinylated cargo to proteasomes, while silencing HDAC6 or HR23B in multiple myeloma cells abolished the effect of HDAC6 inhibitors on proteasomes, antigen presentation, and T-cell cytotoxicity. Taken together, our results demonstrate the paradigm-shifting translational impact of proteasome activators to expand the myeloma immunopeptidome and have revealed novel, actionable antigenic targets for T cell-directed immunotherapy. SIGNIFICANCE: The elimination of therapy-resistant tumor cells remains a major challenge in the treatment of multiple myeloma. Our study identifies and functionally validates agents that amplify MHC class I-presented antigens and pave the way for the development of proteasome activators as immune adjuvants to enhance immunotherapeutic responses in patients with multiple myeloma.

Lipid-Restricted Culture Media Reveal Unexpected Cancer Cell Sensitivities.

Cancer cell culture models frequently rely on fetal bovine serum as a source of protein and lipid factors that support cell survival and proliferation; however, serum-containing media imperfectly mimic the in vivo cancer environment. Recent studies suggest that typical serum-containing cell culture conditions can mask cancer dependencies, for example, on cholesterol biosynthesis enzymes, that exist in vivo and emerge when cells are cultured in media that provide more realistic levels of lipids. Here, we describe a high-throughput screen that identified fenretinide and ivermectin as small molecules whose cytotoxicity is greatly enhanced in lipid-restricted media formulations. The mechanism of action studies indicates that ivermectin-induced cell death involves oxidative stress, while fenretinide likely targets delta 4-desaturase, sphingolipid 1, a lipid desaturase necessary for ceramide synthesis, to induce cell death. Notably, both fenretinide and ivermectin have previously demonstrated in vivo anticancer efficacy despite their low cytotoxicity under typical cell culture conditions. These studies suggest ceramide synthesis as a targetable vulnerability of cancer cells cultured under lipid-restricted conditions and reveal a general screening strategy for identifying additional cancer dependencies masked by the superabundance of medium lipids.

Targeting the chromatin binding of exportin-1 disrupts NFAT and T cell activation.

Exportin-1 (XPO1/CRM1) plays a central role in the nuclear-to-cytoplasmic transport of hundreds of proteins and contributes to other cellular processes, such as centrosome duplication. Small molecules targeting XPO1 induce cytotoxicity, and selinexor was approved by the Food and Drug Administration in 2019 as a cancer chemotherapy for relapsed multiple myeloma. Here, we describe a cell-type-dependent chromatin-binding function for XPO1 that is essential for the chromatin occupancy of NFAT transcription factors and thus the appropriate activation of T cells. Additionally, we establish a class of XPO1-targeting small molecules capable of disrupting the chromatin binding of XPO1 without perturbing nuclear export or inducing cytotoxicity. This work defines a broad transcription regulatory role for XPO1 that is essential for T cell activation as well as a new class of XPO1 modulators to enable therapeutic targeting of XPO1 beyond oncology including in T cell-driven autoimmune disorders.

Discovery and Optimization of Selective Brain-Penetrant EBP Inhibitors that Enhance Oligodendrocyte Formation.

The inhibition of emopamil binding protein (EBP), a sterol isomerase within the cholesterol biosynthesis pathway, promotes oligodendrocyte formation, which has been proposed as a potential therapeutic approach for treating multiple sclerosis. Herein, we describe the discovery and optimization of brain-penetrant, orally bioavailable inhibitors of EBP. A structure-based drug design approach from literature compound 1 led to the discovery of a hydantoin-based scaffold, which provided balanced physicochemical properties and potency and an improved in vitro safety profile. The long half-lives of early hydantoin-based EBP inhibitors in rodents prompted an unconventional optimization strategy, focused on increasing metabolic turnover while maintaining potency and a brain-penetrant profile. The resulting EBP inhibitor 11 demonstrated strong in vivo target engagement in the brain, as illustrated by the accumulation of EBP substrate zymostenol after repeated dosing. Furthermore, compound 11 enhanced the formation of oligodendrocytes in human cortical organoids, providing additional support for our therapeutic hypothesis.

Development of novel cytoprotective small compounds inhibiting mitochondria-dependent cell death.

We identified cytoprotective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in ocular disease mouse models. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only mouse embryonic fibroblasts. M109S also inhibited apoptosis induced by staurosporine, etoposide, and obatoclax. M109S decreased maximal mitochondrial oxygen consumption rate and reactive oxygen species production, whereas it increased glycolysis. These effects on cellular metabolism may contribute to the cytoprotective activity of M109S. M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both in vitro and in vivo. M109S has the potential to become a research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway.

Discovery of Nonretinoid Inhibitors of CRBP1: Structural and Dynamic Insights for Ligand-Binding Mechanisms.

The dysregulation of retinoid metabolism has been linked to prevalent ocular diseases including age-related macular degeneration and Stargardt disease. Modulating retinoid metabolism through pharmacological approaches holds promise for the treatment of these eye diseases. Cellular retinol-binding protein 1 (CRBP1) is the primary transporter of all-trans-retinol (atROL) in the eye, and its inhibition has recently been shown to protect mouse retinas from light-induced retinal damage. In this report, we employed high-throughput screening to identify new chemical scaffolds for competitive, nonretinoid inhibitors of CRBP1. To understand the mechanisms of interaction between CRBP1 and these inhibitors, we solved high-resolution X-ray crystal structures of the protein in complex with six selected compounds. By combining protein crystallography with hydrogen/deuterium exchange mass spectrometry, we quantified the conformational changes in CRBP1 caused by different inhibitors and correlated their magnitude with apparent binding affinities. Furthermore, using molecular dynamic simulations, we provided evidence for the functional significance of the "closed" conformation of CRBP1 in retaining ligands within the binding pocket. Collectively, our study outlines the molecular foundations for understanding the mechanism of high-affinity interactions between small molecules and CRBPs, offering a framework for the rational design of improved inhibitors for this class of lipid-binding proteins.

Definition of fatty acid transport protein-2 (FATP2) structure facilitates identification of small molecule inhibitors for the treatment of diabetic complications.

Diabetes is a major public health problem due to morbidity and mortality associated with end organ complications. Uptake of fatty acids by Fatty Acid Transport Protein-2 (FATP2) contributes to hyperglycemia, diabetic kidney and liver disease pathogenesis. Because FATP2 structure is unknown, a homology model was constructed, validated by AlphaFold2 prediction and site-directed mutagenesis, and then used to conduct a virtual drug discovery screen. In silico similarity searches to two low-micromolar IC50 FATP2 inhibitors, followed by docking and pharmacokinetics predictions, narrowed a diverse 800,000 compound library to 23 hits. These candidates were further evaluated for inhibition of FATP2-dependent fatty acid uptake and apoptosis in cells. Two compounds demonstrated nanomolar IC50, and were further characterized by molecular dynamic simulations. The results highlight the feasibility of combining a homology model with in silico and in vitro screening, to economically identify high affinity inhibitors of FATP2, as potential treatment for diabetes and its complications.

The landscape of targets and lead molecules for remyelination.

Remyelination, or the restoration of myelin sheaths around axons in the central nervous system, is a multi-stage repair process that remains a major need for millions of patients with multiple sclerosis and other diseases of myelin. Even into adulthood, rodents and humans can generate new myelin-producing oligodendrocytes, leading to the therapeutic hypothesis that enhancing remyelination could lessen disease burden in multiple sclerosis. Multiple labs have used phenotypic screening to identify dozens of drugs that enhance oligodendrocyte formation, and several hit molecules have now advanced to clinical evaluation. Target identification studies have revealed that a large majority of these hits share the ability to inhibit a narrow range of cholesterol pathway enzymes and thereby induce cellular accumulation of specific sterol precursors to cholesterol. This Perspective surveys the recent fruitful intersection of chemical biology and remyelination and suggests multiple approaches toward new targets and lead molecules to promote remyelination.

Enhancers of Human and Rodent Oligodendrocyte Formation Predominantly Induce Cholesterol Precursor Accumulation.

Regeneration of myelin in the central nervous system is being pursued as a potential therapeutic approach for multiple sclerosis. Several labs have reported small molecules that promote oligodendrocyte formation and remyelination in vivo. Recently, we reported that many such molecules function by inhibiting a narrow window of enzymes in the cholesterol biosynthesis pathway. Here we describe a new high-throughput screen of 1,836 bioactive molecules and a thorough re-analysis of more than 60 molecules previously identified as promoting oligodendrocyte formation from human, rat, or mouse oligodendrocyte progenitor cells. These studies highlight that an overwhelming fraction of validated screening hits, including several molecules being evaluated clinically for remyelination, inhibit cholesterol pathway enzymes like emopamil-binding protein (EBP). To rationalize these findings, we suggest a model that relies on the high druggability of sterol-metabolizing enzymes and the ability of cationic amphiphiles to mimic the transition state of EBP. These studies further establish cholesterol pathway inhibition as a dominant mechanism among screening hits that enhance human, rat, or mouse oligodendrocyte formation.

ATAD3A oligomerization promotes neuropathology and cognitive deficits in Alzheimer's disease models.

Predisposition to Alzheimer's disease (AD) may arise from lipid metabolism perturbation, however, the underlying mechanism remains elusive. Here, we identify ATPase family AAA-domain containing protein 3A (ATAD3A), a mitochondrial AAA-ATPase, as a molecular switch that links cholesterol metabolism impairment to AD phenotypes. In neuronal models of AD, the 5XFAD mouse model and post-mortem AD brains, ATAD3A is oligomerized and accumulated at the mitochondria-associated ER membranes (MAMs), where it induces cholesterol accumulation by inhibiting gene expression of CYP46A1, an enzyme governing brain cholesterol clearance. ATAD3A and CYP46A1 cooperate to promote APP processing and synaptic loss. Suppressing ATAD3A oligomerization by heterozygous ATAD3A knockout or pharmacological inhibition with DA1 restores neuronal CYP46A1 levels, normalizes brain cholesterol turnover and MAM integrity, suppresses APP processing and synaptic loss, and consequently reduces AD neuropathology and cognitive deficits in AD transgenic mice. These findings reveal a role for ATAD3A oligomerization in AD pathogenesis and suggest ATAD3A as a potential therapeutic target for AD.

Inhibition of SC4MOL and HSD17B7 shifts cellular sterol composition and promotes oligodendrocyte formation.

While the cholesterol biosynthesis pathway has been extensively studied, recent work has forged new links between inhibition of specific sterol pathway enzymes, accumulation of their unique sterol substrates, and biological areas as diverse as cancer, immunology, and neurodegenerative disease. We recently reported that dozens of small molecules enhance formation of oligodendrocytes, a glial cell type lost in multiple sclerosis, by inhibiting CYP51, Sterol 14-reductase, or EBP and inducing cellular accumulation of their 8,9-unsaturated sterol substrates. Several adjacent pathway enzymes also have 8,9-unsaturated sterol substrates but have not yet been evaluated as potential targets for oligodendrocyte formation or in many other biological contexts, in part due to a lack of available small-molecule probes. Here, we show that genetic suppression of SC4MOL or HSD17B7 increases the formation of oligodendrocytes. Additionally, we have identified and optimized multiple potent new series of SC4MOL and HSD17B7 inhibitors and shown that these small molecules enhance oligodendrocyte formation. SC4MOL inhibitor CW4142 induced accumulation of SC4MOL's sterol substrates in mouse brain and represents an in vivo probe of SC4MOL activity. Mechanistically, the cellular accumulation of these 8,9-unsaturated sterols represents a central driver of enhanced oligodendrocyte formation, as exogenous addition of purified SC4MOL and HSD17B7 substrates but not their 8,9-saturated analogs promotes OPC differentiation. Our work validates SC4MOL and HSD17B7 as novel targets for promoting oligodendrocyte formation, underlines a broad role for 8,9-unsaturated sterols as enhancers of oligodendrocyte formation, and establishes the first high-quality small molecules targeting SC4MOL and HSD17B7 as novel tools for probing diverse areas of biology.

Small-molecule suppression of calpastatin degradation reduces neuropathology in models of Huntington's disease.

Mitochondrial dysfunction is a common hallmark of neurological disorders, and reducing mitochondrial damage is considered a promising neuroprotective therapeutic strategy. Here, we used high-throughput small molecule screening to identify CHIR99021 as a potent enhancer of mitochondrial function. CHIR99021 improved mitochondrial phenotypes and enhanced cell viability in several models of Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Notably, CHIR99201 treatment reduced HD-associated neuropathology and behavioral defects in HD mice and improved mitochondrial function and cell survival in HD patient-derived neurons. Independent of its known inhibitory activity against glycogen synthase kinase 3 (GSK3), CHIR99021 treatment in HD models suppressed the proteasomal degradation of calpastatin (CAST), and subsequently inhibited calpain activation, a well-established effector of neural death, and Drp1, a driver of mitochondrial fragmentation. Our results established CAST-Drp1 as a druggable signaling axis in HD pathogenesis and highlighted CHIR99021 as a mitochondrial function enhancer and a potential lead for developing HD therapies.

Screening Reveals Sterol Derivatives with Pro-Differentiation, Pro-Survival, or Potent Cytotoxic Effects on Oligodendrocyte Progenitor Cells.

Inducing the formation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs) represents a potential approach to repairing the loss of myelin observed in multiple sclerosis and other diseases. Recently, we demonstrated that accumulation of specific cholesterol precursors, 8,9-unsaturated sterols, is a dominant mechanism by which dozens of small molecules enhance oligodendrocyte formation. Here, we evaluated a library of 56 sterols and steroids to evaluate whether other classes of bioactive sterol derivatives may also influence mouse oligodendrocyte precursor cell (OPC) differentiation or survival. From this library, we identified U-73343 as a potent enhancer of oligodendrocyte formation that induces 8,9-unsaturated sterol accumulation by inhibition of the cholesterol biosynthesis enzyme sterol 14-reductase. In contrast, we found that mouse OPCs are remarkably vulnerable to treatment with the glycosterol OSW-1, an oxysterol-binding protein (OSBP) modulator that induces Golgi stress and OPC death in the low picomolar range. A subsequent small-molecule suppressor screen identified mTOR signaling as a key effector pathway mediating OSW-1's cytotoxic effects in mouse OPCs. Finally, evaluation of a panel of ER and Golgi stress-inducing small molecules revealed that mouse OPCs are highly sensitive to these perturbations, more so than closely related neural progenitor cells. Together, these studies highlight the wide-ranging influence of sterols and steroids on OPC cell fate, with 8,9-unsaturated sterols positively enhancing differentiation to oligodendrocytes and OSW-1 able to induce lethal Golgi stress with remarkable potency.

Modulation of lanosterol synthase drives 24,25-epoxysterol synthesis and oligodendrocyte formation.

Small molecules that promote the formation of new myelinating oligodendrocytes from oligodendrocyte progenitor cells (OPCs) are potential therapeutics for demyelinating diseases. We recently established inhibition of specific cholesterol biosynthesis enzymes and resulting accumulation of 8,9-unsaturated sterols as a unifying mechanism through which many such molecules act. To identify more potent sterol enhancers of oligodendrocyte formation, we synthesized a collection of 8,9-unsaturated sterol derivatives and found that 24,25-epoxylanosterol potently promoted oligodendrocyte formation. In OPCs, 24,25-epoxylanosterol was metabolized to 24,25-epoxycholesterol via the epoxycholesterol shunt pathway. Increasing flux through the epoxycholesterol shunt using genetic manipulation or small-molecule inhibition of lanosterol synthase (LSS) increased endogenous 24,25-epoxycholesterol levels and OPC differentiation. Notably, exogenously supplied 24,25-epoxycholesterol promoted oligodendrocyte formation despite lacking an 8,9-unsaturation. This work highlights epoxycholesterol shunt usage, controlled by inhibitors of LSS, as a target to promote oligodendrocyte formation. Additionally, sterols beyond the 8,9-unsaturated sterols, including 24,25-epoxycholesterol, drive oligodendrocyte formation.

Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS.

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.

Clonal selection confers distinct evolutionary trajectories in BRAF-driven cancers.

Molecular determinants governing the evolution of tumor subclones toward phylogenetic branches or fixation remain unknown. Using sequencing data, we model the propagation and selection of clones expressing distinct categories of BRAF mutations to estimate their evolutionary trajectories. We show that strongly activating BRAF mutations demonstrate hard sweep dynamics, whereas mutations with less pronounced activation of the BRAF signaling pathway confer soft sweeps or are subclonal. We use clonal reconstructions to estimate the strength of "driver" selection in individual tumors. Using tumors cells and human-derived murine xenografts, we show that tumor sweep dynamics can significantly affect responses to targeted inhibitors of BRAF/MEK or DNA damaging agents. Our study uncovers patterns of distinct BRAF clonal evolutionary dynamics and nominates therapeutic strategies based on the identity of the BRAF mutation and its clonal composition.

Vesiculopolins, a New Class of Anti-Vesiculoviral Compounds, Inhibit Transcription Initiation of Vesiculoviruses.

Vesicular stomatitis virus (VSV) represents a promising platform for developing oncolytic viruses, as well as vaccines against significant human pathogens. To safely control VSV infection in humans, small-molecule drugs that selectively inhibit VSV infection may be needed. Here, using a cell-based high-throughput screening assay followed by an in vitro transcription assay, compounds with a 7-hydroxy-6-methyl-3,4-dihydroquinolin-2(1H)-one structure and an aromatic group at position 4 (named vesiculopolins, VPIs) were identified as VSV RNA polymerase inhibitors. The most effective compound, VPI A, inhibited VSV-induced cytopathic effects and in vitro mRNA synthesis with micromolar to submicromolar 50% inhibitory concentrations. VPI A was found to inhibit terminal de novo initiation rather than elongation for leader RNA synthesis, but not mRNA capping, with the VSV L protein, suggesting that VPI A is targeted to the polymerase domain in the L protein. VPI A inhibited transcription of Chandipura virus, but not of human parainfluenza virus 3, suggesting that it specifically acts on vesiculoviral L proteins. These results suggest that VPIs may serve not only as molecular probes to elucidate the mechanisms of transcription of vesiculoviruses, but also as lead compounds to develop antiviral drugs against vesiculoviruses and other related rhabdoviruses.

Ex vivo screen identifies CDK12 as a metastatic vulnerability in osteosarcoma.

Despite progress in intensification of therapy, outcomes for patients with metastatic osteosarcoma (OS) have not improved in thirty years. We developed a system that enabled preclinical screening of compounds against metastatic OS cells in the context of the native lung microenvironment. Using this strategy to screen a library of epigenetically targeted compounds, we identified inhibitors of CDK12 to be most effective, reducing OS cell outgrowth in the lung by more than 90% at submicromolar doses. We found that knockout of CDK12 in an in vivo model of lung metastasis significantly decreased the ability of OS to colonize the lung. CDK12 inhibition led to defects in transcription elongation in a gene length- and expression-dependent manner. These effects were accompanied by defects in RNA processing and altered the expression of genes involved in transcription regulation and the DNA damage response. We further identified OS models that differ in their sensitivity to CDK12 inhibition in the lung and provided evidence that upregulated MYC levels may mediate these differences. Our studies provided a framework for rapid preclinical testing of compounds with antimetastatic activity and highlighted CDK12 as a potential therapeutic target in OS.

Diverse Chemical Scaffolds Enhance Oligodendrocyte Formation by Inhibiting CYP51, TM7SF2, or EBP.

Small molecules that promote oligodendrocyte formation have been identified in "drug repurposing" screens to nominate candidate therapeutics for diseases in which myelin is lost, including multiple sclerosis. We recently reported that many such molecules enhance oligodendrocyte formation not by their canonical targets but by inhibiting a narrow range of enzymes in cholesterol biosynthesis. Here we identify enhancers of oligodendrocyte formation obtained by screening a structurally diverse library of 10,000 small molecules. Identification of the cellular targets of these validated hits revealed a majority inhibited the cholesterol biosynthesis enzymes CYP51, TM7SF2, or EBP. In addition, evaluation of analogs led to identification of CW3388, a potent EBP-inhibiting enhancer of oligodendrocyte formation poised for further optimization.