BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation.

The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization (MOMP). The BCL-2 family effectors BAX and BAK are thought to be absolutely required for this process. Here, we report that BCL-2 ovarian killer (BOK) is a bona fide yet unconventional effector of MOMP that can trigger apoptosis in the absence of both BAX and BAK. However, unlike the canonical effectors, BOK appears to be constitutively active and unresponsive to antagonistic effects of the antiapoptotic BCL-2 proteins. Rather, BOK is controlled at the level of protein stability by components of the endoplasmic reticulum (ER)-associated degradation pathway. BOK is ubiquitylated by the AMFR/gp78 E3 ubiquitin ligase complex and targeted for proteasomal degradation in a VCP/p97-dependent manner, which allows survival of the cell. When proteasome function, VCP, or gp78 activity is compromised, BOK is stabilized to induce MOMP and apoptosis independently of other BCL-2 proteins.

E3 ligase autoinhibition by C-degron mimicry maintains C-degron substrate fidelity.

E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.

NSD1 mediates antagonism between SWI/SNF and polycomb complexes and is required for transcriptional activation upon EZH2 inhibition.

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.

Acute myeloid leukemias with UBTF tandem duplications are sensitive to menin inhibitors.

ABSTRACT: UBTF tandem duplications (UBTF-TDs) have recently emerged as a recurrent alteration in pediatric and adult acute myeloid leukemia (AML). UBTF-TD leukemias are characterized by a poor response to conventional chemotherapy and a transcriptional signature that mirrors NUP98-rearranged and NPM1-mutant AMLs, including HOX-gene dysregulation. However, the mechanism by which UBTF-TD drives leukemogenesis remains unknown. In this study, we investigated the genomic occupancy of UBTF-TD in transformed cord blood CD34+ cells and patient-derived xenograft models. We found that UBTF-TD protein maintained genomic occupancy at ribosomal DNA loci while also occupying genomic targets commonly dysregulated in UBTF-TD myeloid malignancies, such as the HOXA/HOXB gene clusters and MEIS1. These data suggest that UBTF-TD is a gain-of-function alteration that results in mislocalization to genomic loci dysregulated in UBTF-TD leukemias. UBTF-TD also co-occupies key genomic loci with KMT2A and menin, which are known to be key partners involved in HOX-dysregulated leukemias. Using a protein degradation system, we showed that stemness, proliferation, and transcriptional signatures are dependent on sustained UBTF-TD localization to chromatin. Finally, we demonstrate that primary cells from UBTF-TD leukemias are sensitive to the menin inhibitor SNDX-5613, resulting in markedly reduced in vitro and in vivo tumor growth, myeloid differentiation, and abrogation of the UBTF-TD leukemic expression signature. These findings provide a viable therapeutic strategy for patients with this high-risk AML subtype.

A bromodomain-independent mechanism of gene regulation by the BET inhibitor JQ1: direct activation of nuclear receptor PXR.

Bromodomain and extraterminal (BET) proteins are extensively studied in multiple pathologies, including cancer. BET proteins modulate transcription of various genes, including those synonymous with cancer, such as MYC. Thus, BET inhibitors are a major area of drug development efforts. (+)-JQ1 (JQ1) is the prototype inhibitor and is a common tool to probe BET functions. While showing therapeutic promise, JQ1 is not clinically usable, partly due to metabolic instability. Here, we show that JQ1 and the BET-inactive (-)-JQ1 are agonists of pregnane X receptor (PXR), a nuclear receptor that transcriptionally regulates genes encoding drug-metabolizing enzymes such as CYP3A4, which was previously shown to oxidize JQ1. A PXR-JQ1 co-crystal structure identified JQ1's tert-butyl moiety as a PXR anchor and explains binding by (-)-JQ1. Analogs differing at the tert-butyl lost PXR binding, validating our structural findings. Evaluation in liver cell models revealed both PXR-dependent and PXR-independent modulation of CYP3A4 expression by BET inhibitors. We have characterized a non-BET JQ1 target, a mechanism of physiological JQ1 instability, a biological function of (-)-JQ1, and BET-dependent transcriptional regulation of drug metabolism genes.

Structure-guided approach to modulate small molecule binding to a promiscuous ligand-activated protein.

Ligand-binding promiscuity in detoxification systems protects the body from toxicological harm but is a roadblock to drug development due to the difficulty in optimizing small molecules to both retain target potency and avoid metabolic events. Immense effort is invested in evaluating metabolism of molecules to develop safer, more effective treatments, but engineering specificity into or out of promiscuous proteins and their ligands is a challenging task. To better understand the promiscuous nature of detoxification networks, we have used X-ray crystallography to characterize a structural feature of pregnane X receptor (PXR), a nuclear receptor that is activated by diverse molecules (with different structures and sizes) to up-regulate transcription of drug metabolism genes. We found that large ligands expand PXR's ligand-binding pocket, and the ligand-induced expansion occurs through a specific unfavorable compound-protein clash that likely contributes to reduced binding affinity. Removing the clash by compound modification resulted in more favorable binding modes with significantly enhanced binding affinity. We then engineered the unfavorable ligand-protein clash into a potent, small PXR ligand, resulting in marked reduction in PXR binding and activation. Structural analysis showed that PXR is remodeled, and the modified ligands reposition in the binding pocket to avoid clashes, but the conformational changes result in less favorable binding modes. Thus, ligand-induced binding pocket expansion increases ligand-binding potential of PXR but is an unfavorable event; therefore, drug candidates can be engineered to expand PXR's ligand-binding pocket and reduce their safety liability due to PXR binding.

Molecular basis of crosstalk in nuclear receptors: heterodimerization between PXR and CAR and the implication in gene regulation.

The 48 human nuclear receptors (NRs) form a superfamily of transcription factors that regulate major physiological and pathological processes. Emerging evidence suggests that NR crosstalk can fundamentally change our understanding of NR biology, but detailed molecular mechanisms of crosstalk are lacking. Here, we report the molecular basis of crosstalk between the pregnane X receptor (PXR) and constitutive androstane receptor (CAR), where they form a novel heterodimer, resulting in their mutual inhibition. PXR and CAR regulate drug metabolism and energy metabolism. Although they have been broadly perceived as functionally redundant, a growing number of reports suggests a mutual inhibitory relation, but their precise mode of coordinated action remains unknown. Using methods including RNA sequencing, small-angle X-ray scattering and crosslinking mass spectrometry we demonstrate that the mutual inhibition altered gene expression globally and is attributed to the novel PXR-CAR heterodimerization via the same interface used by each receptor to heterodimerize with its functional partner, retinoid X receptor (RXR). These findings establish an unexpected functional relation between PXR, CAR and RXR, change the perceived functional relation between PXR and CAR, open new perspectives on elucidating their role and designing approaches to regulate them, and highlight the importance to comprehensively investigate nuclear receptor crosstalk.

Targeting OCT3 attenuates doxorubicin-induced cardiac injury.

Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell-derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.

Evaluation of a Pooling Chemoproteomics Strategy with an FDA-Approved Drug Library.

Chemoproteomics is a key platform for characterizing the mode of action for compounds, especially for targeted protein degraders such as proteolysis targeting chimeras (PROTACs) and molecular glues. With deep proteome coverage, multiplexed tandem mass tag-mass spectrometry (TMT-MS) can tackle up to 18 samples in a single experiment. Here, we present a pooling strategy for further enhancing the throughput and apply the strategy to an FDA-approved drug library (95 best-in-class compounds). The TMT-MS-based pooling strategy was evaluated in the following steps. First, we demonstrated the capability of TMT-MS by analyzing more than 15 000 unique proteins (> 12 000 gene products) in HEK293 cells treated with five PROTACs (two BRD/BET degraders and three degraders for FAK, ALK, and BTK kinases). We then introduced a rationalized pooling strategy to separate structurally similar compounds in different pools and identified the proteomic response to 14 pools from the drug library. Finally, we validated the proteomic response from one pool by reprofiling the cells via treatment with individual drugs with sufficient replicates. Interestingly, numerous proteins were found to change upon drug treatment, including AMD1, ODC1, PRKX, PRKY, EXO1, AEN, and LRRC58 with 7-hydroxystaurosporine; C6orf64, HMGCR, and RRM2 with Sorafenib; SYS1 and ALAS1 with Venetoclax; and ATF3, CLK1, and CLK4 with Palbocilib. Thus, pooling chemoproteomics screening provides an efficient method for dissecting the molecular targets of compound libraries.

Regulation of Nuclear Receptors PXR and CAR by Small Molecules and Signal Crosstalk: Roles in Drug Metabolism and Beyond.

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are ligand-activated transcription factors that regulate the expression of drug metabolizing enzymes and drug transporters. Since their discoveries, they have been studied as important factors for regulating processes related to drug efficacy, drug toxicity, and drug-drug interactions. However, their vast ligand-binding profiles extend into additional spaces, such as endogenously produced chemicals, microbiome metabolites, dietary compounds, and environmental pollutants. Therefore, PXR and CAR can respond to an enormous abundance of stimuli, resulting in significant shifts in metabolic programs and physiologic homeostasis. Naturally, PXR and CAR have been implicated in various diseases related to homeostatic perturbations, such as inflammatory bowel disorders, diabetes, and certain cancers. Recent findings have injected the field with new signaling mechanisms and tools to dissect the complex PXR and CAR biology and have strengthened the potential for future PXR and CAR modulators in the clinic. Here, we describe the historical and ongoing importance of PXR and CAR in drug metabolism pathways and how this history has evolved into new mechanisms that regulate and are regulated by these xenobiotic receptors, with a specific focus on small molecule ligands. To effectively convey the impact of newly emerging research, we have arranged five diverse and representative key recent advances, four specific challenges, and four perspectives on future directions. SIGNIFICANCE STATEMENT: PXR and CAR are key transcription factors that regulate homeostatic detoxification of the liver and intestines. Diverse chemicals bind to these nuclear receptors, triggering their transcriptional tuning of the cellular metabolic response. This minireview revisits the importance of PXR and CAR in pharmaceutical drug responses and highlights recent results with implications beyond drug metabolism.

Mutation of a single amino acid of pregnane X receptor switches an antagonist to agonist by altering AF-2 helix positioning.

Pregnane X receptor (PXR) is activated by chemicals to transcriptionally regulate drug disposition and possibly decrease drug efficacy and increase resistance, suggesting therapeutic value for PXR antagonists. We previously reported the antagonist SPA70 and its analog SJB7, which unexpectedly is an agonist. Here, we describe another unexpected observation: mutating a single residue (W299A) within the PXR ligand-binding domain converts SPA70 to an agonist. After characterizing wild-type and W299A PXR activity profiles, we used molecular dynamics simulations to reveal that in wild-type PXR, agonists stabilize the activation function 2 (AF-2) helix in an "inward" position, but SPA70 displaces the AF-2. In W299A, however, SPA70 stabilizes the AF-2 "inward", like agonists. We validated our model by predicting the antagonist SJC2 to be a W299A agonist, which was confirmed experimentally. Our work correlates previously unobserved ligand-induced conformational changes to PXR cellular activity and, for the first time, reveals how PXR antagonists work.

Unraveling the Structural Basis of Selective Inhibition of Human Cytochrome P450 3A5.

The human cytochrome P450 (CYP) CYP3A4 and CYP3A5 enzymes metabolize more than one-half of marketed drugs. They share high structural and substrate similarity and are often studied together as CYP3A4/5. However, CYP3A5 preferentially metabolizes several clinically prescribed drugs, such as tacrolimus. Genetic polymorphism in CYP3A5 makes race-based dosing adjustment of tacrolimus necessary to minimize acute rejection after organ transplantation. Moreover, the differential tissue distribution and expression levels of CYP3A4 and CYP3A5 can aggravate toxicity during treatment. Therefore, selective inhibitors of CYP3A5 are needed to distinguish the role of CYP3A5 from that of CYP3A4 and serve as starting points for potential therapeutic development. To this end, we report the crystal structure of CYP3A5 in complex with a previously reported selective inhibitor, clobetasol propionate (CBZ). This is the first CYP3A5 structure with a type I inhibitor, which along with the previously reported substrate-free and type II inhibitor-bound structures, constitute the main CYP3A5 structural modalities. Supported by structure-guided mutagenesis analyses, the CYP3A5-CBZ structure showed that a unique conformation of the F-F' loop in CYP3A5 enables selective binding of CBZ to CYP3A5. Several polar interactions, including hydrogen bonds, stabilize the position of CBZ to interact with this unique F-F' loop conformation. In addition, functional and biophysical assays using CBZ analogs highlight the importance of heme-adjacent moieties for selective CYP3A5 inhibition. Our findings can be used to guide further development of more potent and selective CYP3A5 inhibitors.

ABCG2 requires a single aromatic amino acid to "clamp" substrates and inhibitors into the binding pocket.

ATP-binding cassette sub-family G member 2 (ABCG2) is a homodimeric ATP-binding cassette (ABC) transporter that not only has a key role in helping cancer cells to evade the cytotoxic effects of chemotherapy, but also in protecting organisms from multiple xeno- and endobiotics. Structural studies indicate that substrate and inhibitor (ligands) binding to ABCG2 can be differentiated quantitatively by the number of amino acid contacts, with inhibitors displaying more contacts. Although binding is the obligate initial step in the transport cycle, there is no empirical evidence for one amino acid being primarily responsible for ligand binding. By mutagenesis and biochemical studies, we demonstrated that the phylogenetically conserved amino acid residue, F439, was critical for both transport and the binding of multiple substrates and inhibitors. Structural modeling implied that the π-π interactions from each F439 monomer mediated the binding of a surprisingly diverse array of structurally unrelated substrates and inhibitors and that this symmetrical π-π interaction "clamps" the ligand into the binding pocket. Key molecular features of diverse ABCG2 ligands using the π-π clamp along with structural studies created a pharmacophore model. These novel findings have important therapeutic implications because key properties of ligands interacting with ABCG2 have been disovered. Furthermore, mechanistic insights have been revealed by demonstrating that for ABCG2 a single amino acid is essential for engaging and initiating transport of multiple drugs and xenobiotics.

Biological evaluation and synthesis of calcitroic acid.

We describe the synthesis and broad profiling of calcitroic acid (CTA) as vitamin D receptor (VDR) ligand. The x-ray co-crystal structure of the Danio Rerio VDR ligand binding domain in complex with CTA and peptide MED1 confirmed an agonistic conformation of the receptor. CTA adopted a similar conformation as 1,25(OH)2D3 in the binding pocket. A hydrogen bond with His333 and a water molecule were observed in the binding pocket, which was accommodated due to the shorter CTA side chain. In contrast, 1,25(OH)2D3 interacted with His423 and His333 due to its longer side chain. In vitro, the EC50 values of CTA and CTA-ME for VDR-mediated transcription were 2.89 µM and 0.66 µM, respectively, confirming both compounds as VDR agonists. CTA was further evaluated for interaction with fourteen nuclear receptors demonstrating selective activation of VDR. VDR mediated gene regulation by CTA in intestinal cells was observed for the VDR target gene CYP24A1. CTA at 10 µM upregulated CYP24A1 with similar efficacy as 1,25(OH)2D3 at 20 nM and 100-fold stronger compared to lithocholic acid at 10 µM. CTA reduced the transcription of iNOS and IL-1ß in interferon γ and lipopolysaccharide stimulated mouse macrophages resulting in a reduction of nitric oxide production and secretion of IL-1ß. These observed anti-inflammatory properties of 20 µM CTA were similar to 20 nM 1,25(OH)2D3.

Metabolic activation of CaMKII by coenzyme A.

Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis by directly activating CaMKII. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca(2+) to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes but also in Murine oocytes. These findings uncover a direct mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

Strategies for developing pregnane X receptor antagonists: Implications from metabolism to cancer.

Pregnane X receptor (PXR) is a ligand-activated nuclear receptor (NR) that was originally identified as a master regulator of xenobiotic detoxification. It regulates the expression of drug-metabolizing enzymes and transporters to control the degradation and excretion of endobiotics and xenobiotics, including therapeutic agents. The metabolism and disposition of drugs might compromise their efficacy and possibly cause drug toxicity and/or drug resistance. Because many drugs can promiscuously bind and activate PXR, PXR antagonists might have therapeutic value in preventing and overcoming drug-induced PXR-mediated drug toxicity and drug resistance. Furthermore, PXR is now known to have broader cellular functions, including the regulation of cell proliferation, and glucose and lipid metabolism. Thus, PXR might be involved in human diseases such as cancer and metabolic diseases. The importance of PXR antagonists is discussed in the context of the role of PXR in xenobiotic sensing and other disease-related pathways. This review focuses on the development of PXR antagonists, which has been hampered by the promiscuity of PXR ligand binding. However, substantial progress has been made in recent years, suggesting that it is feasible to develop selective PXR antagonists. We discuss the current status, challenges, and strategies in developing selective PXR antagonists. The strategies are based on the molecular mechanisms of antagonism in related NRs that can be applied to the design of PXR antagonists, primarily driven by structural information.

CITCO Directly Binds to and Activates Human Pregnane X Receptor.

The xenobiotic receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are activated by structurally diverse chemicals to regulate the expression of target genes, and they have overlapping regulation in terms of ligands and target genes. Receptor-selective agonists are, therefore, critical for studying the overlapping function of PXR and CAR. An early effort identified 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) as a selective human CAR (hCAR) agonist, and this has since been widely used to distinguish the function of hCAR from that of human PXR (hPXR). The selectivity was demonstrated in a green monkey kidney cell line, CV-1, in which CITCO displayed >100-fold selectivity for hCAR over hPXR. However, whether the selectivity observed in CV-1 cells also represented CITCO activity in liver cell models was not hitherto investigated. In this study, we showed that CITCO: 1) binds directly to hPXR; 2) activates hPXR in HepG2 cells, with activation being blocked by an hPXR-specific antagonist, SPA70; 3) does not activate mouse PXR; 4) depends on tryptophan-299 to activate hPXR; 5) recruits steroid receptor coactivator 1 to hPXR; 6) activates hPXR in HepaRG cell lines even when hCAR is knocked out; and 7) activates hPXR in primary human hepatocytes. Together, these data indicate that CITCO binds directly to the hPXR ligand-binding domain to activate hPXR. As CITCO has been widely used, its confirmation as a dual agonist for hCAR and hPXR is important for appropriately interpreting existing data and designing future experiments to understand the regulation of hPXR and hCAR. SIGNIFICANCE STATEMENT: The results of this study demonstrate that 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is a dual agonist for human constitutive androstane receptor (hCAR) and human pregnane X receptor (hPXR). As CITCO has been widely used to activate hCAR, and hPXR and hCAR have distinct and overlapping biological functions, these results highlight the value of receptor-selective agonists and the importance of appropriately interpreting data in the context of receptor selectivity of such agonists.

Development of BODIPY FL Thalidomide As a High-Affinity Fluorescent Probe for Cereblon in a Time-Resolved Fluorescence Resonance Energy Transfer Assay.

Ligands for cereblon, a component of a functional E3 ligase complex that targets proteins for proteolysis, are critical for developing molecular glues and proteolysis-targeting chimeras (PROTACs), which have therapeutic implications for various diseases. However, the lack of sensitivity of previously reported assays limits characterization of cereblon ligands. To address this shortcoming, we developed BODIPY FL thalidomide (10) as a high-affinity fluorescent probe for the human cereblon protein, with a Kd value of 3.6 nM. We then used BODIPY FL thalidomide (10) to develop a cereblon time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay. The IC50 values of the cereblon ligand pomalidomide (8) were 6.4 nM in our cereblon TR-FRET binding assay, 264.8 nM in a previously reported Cy5-conjugated thalidomide (7)-mediated fluorescence polarization (FP) assay, and 1.2 µM in a previously reported Cy5-conjugated cereblon modulator (compound 7) (9)-mediated TR-FRET assay, indicating that our cereblon TR-FRET binding assay is 41- and 187-fold more sensitive than these two previously published assays. With our cereblon TR-FRET binding assay, we detected binding of cereblon ligands but not binding of bromodomain-containing protein 4 or von Hippel-Lindau ligands, thereby demonstrating its selectivity. Our cereblon TR-FRET binding assay was very stable and detected changes in phthalimide activity due to thalidomide isomerization. Therefore, the BODIPY FL thalidomide (10)-mediated cereblon TR-FRET binding assay we designed is highly sensitive, selective, and stable and will aid the development and characterization of novel cereblon ligands.

Genome-wide CRISPR screen reveals PSMA6 to be an essential gene in pancreatic cancer cells.

BACKGROUND: Despite its relatively low incidence, pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths because of the aggressive growth/metastasis of the tumor, the lack of early symptoms, and the poor treatment options. Basic research to identify potential therapeutic targets for PDAC is greatly needed. METHODS: We used a negative-selection genome-wide CRISPR screen to identify essential genes in the PANC-1 human pancreatic carcinoma cell line. We validated the top hits with follow-up siRNA screens, using the HPNE, HPAF-II, AsPC-1, and Mia PaCa-2 cell lines. RESULTS: The PSMA6 gene was an identified candidate hit after the CRISPR screen, siRNA validation screen, and siRNA deconvolution screen. Spheroid formation assays and flow cytometry analysis showed that PSMA6 is critical for survival in many pancreatic ductal carcinoma cell models. Lastly, as PSMA6 protein is a proteosomal subunit of the 20S core complex, we showed that bortezomib, a proteasome inhibitor, was especially toxic in PANC-1 cells. CONCLUSIONS: Further study of PSMA6 and the proteasome subunit that it encodes, along with other hits identified in our CRISPR screens, may provide valuable insights into potential therapeutic targets for PDAC.