Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

Chaetocin-mediated SUV39H1 inhibition targets stemness and oncogenic networks of diffuse midline gliomas and synergizes with ONC201.

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPG/DMG) are devastating pediatric brain tumors with extraordinarily limited treatment options and uniformly fatal prognosis. Histone H3K27M mutation is a common recurrent alteration in DIPG and disrupts epigenetic regulation. We hypothesize that genome-wide H3K27M-induced epigenetic dysregulation makes tumors vulnerable to epigenetic targeting. METHODS: We performed a screen of compounds targeting epigenetic enzymes to identify potential inhibitors for the growth of patient-derived DIPG cells. We further carried out transcriptomic and genomic landscape profiling including RNA-seq and CUT&RUN-seq as well as shRNA-mediated knockdown to assess the effects of chaetocin and SUV39H1, a target of chaetocin, on DIPG growth. RESULTS: High-throughput small-molecule screening identified an epigenetic compound chaetocin as a potent blocker of DIPG cell growth. Chaetocin treatment selectively decreased proliferation and increased apoptosis of DIPG cells and significantly extended survival in DIPG xenograft models, while restoring H3K27me3 levels. Moreover, the loss of H3K9 methyltransferase SUV39H1 inhibited DIPG cell growth. Transcriptomic and epigenomic profiling indicated that SUV39H1 loss or inhibition led to the downregulation of stemness and oncogenic networks including growth factor receptor signaling and stemness-related programs; however, D2 dopamine receptor (DRD2) signaling adaptively underwent compensatory upregulation conferring resistance. Consistently, a combination of chaetocin treatment with a DRD2 antagonist ONC201 synergistically increased the antitumor efficacy. CONCLUSIONS: Our studies reveal a therapeutic vulnerability of DIPG cells through targeting the SUV39H1-H3K9me3 pathway and compensatory signaling loops for treating this devastating disease. Combining SUV39H1-targeting chaetocin with other agents such as ONC201 may offer a new strategy for effective DIPG treatment.

Structure-Activity Relationship Analysis of Rhosin, a RhoA GTPase Inhibitor, Reveals a New Class of Antiplatelet Agents.

Current antiplatelet therapies have several clinical complications and are mostly irreversible in terms of suppressing platelet activity; hence, there is a need to develop improved therapeutic agents. Previous studies have implicated RhoA in platelet activation. Here, we further characterized the lead RhoA inhibitor, Rhosin/G04, in platelet function and present structure-activity relationship (SAR) analysis. A screening for Rhosin/G04 analogs in our chemical library by similarity and substructure searches revealed compounds that showed enhanced antiplatelet activity and suppressed RhoA activity and signaling. A screening for Rhosin/G04 analogs in our chemical library using similarity and substructure searches revealed compounds that showed enhanced antiplatelet activity and suppressed RhoA activity and signaling. SAR analysis revealed that the active compounds have a quinoline group optimally attached to the hydrazine at the 4-position and halogen substituents at the 7- or 8-position. Having indole, methylphenyl, or dichloro-phenyl substituents led to better potency. Rhosin/G04 contains a pair of enantiomers, and S-G04 is significantly more potent than R-G04 in inhibiting RhoA activation and platelet aggregation. Furthermore, the inhibitory effect is reversible, and S-G04 is capable of inhibiting diverse-agonist-stimulated platelet activation. This study identified a new generation of small-molecule RhoA inhibitors, including an enantiomer capable of broadly and reversibly modulating platelet activity.

Rational polypharmacological targeting of FLT3, JAK2, ABL, and ERK1 suppresses the adaptive resistance to FLT3 inhibitors in AML.

Despite significant advancements in developing selective FMS-like tyrosine kinase 3 (FLT3) inhibitors, resistance to treatment is common even on continued therapy. Acquisition of on-target mutations or adaptation to MAPK, JAK2, and ABL signaling pathways drive treatment failure and disease relapse. Although combinatorial targeting of all escape routes in preclinical models demonstrated its efficacy, the clinical application is challenging owing to drug-drug interaction and differing pharmacokinetics of the inhibitors. We reasoned that selective polypharmacological targeting could lead to a durable response with reduced toxicity. A cell-based screening was carried out to identify inhibitors targeting FLT3, RAS-MAPK, BCR-ABL, and JAK2 to target the adaptive resistance observed with FLT3 inhibitors. Here, we show that pluripotin is an equipotent inhibitor of FLT3, BCR-ABL, and JAK2 in addition to inhibiting Ras-GAP and extracellular signal-regulated kinase 1 (ERK1). Structural modeling studies revealed that pluripotin is a type II kinase inhibitor that selectively binds with inactive conformations of FLT3, ABL, and JAK2. Pluripotin showed potent inhibitory activity on both mouse and human cells expressing FLT3ITD, including clinically challenging resistant mutations of the gatekeeper residue, F691L. Likewise, pluripotin suppressed the adaptive resistance conferred by the activation of RAS-MAPK pathways, BCR-ABL, and JAK2 signaling. Treatment with pluripotin curbed the progression of acute myeloid leukemia (AML) in multiple in vivo models including patient-derived primary AML cells in mouse xenotransplants. As a proof of concept, we demonstrate that targeted polypharmacological inhibition of key signaling nodes driving adaptive resistance can provide a durable response.

Brain pharmacokinetics and metabolism of the AMP-activated protein kinase selective inhibitor SBI-0206965, an investigational agent for the treatment of glioblastoma.

PURPOSE: Emerging evidence suggests that 5' Adenosine Monophosphate-Activated Protein Kinase (AMPK), a key regulator of cellular bioenergetics, is a novel target for the treatment of glioblastoma (GBM), a lethal brain tumor. SBI-0206965, an aminopyrimidine derivative, is a potent AMPK inhibitor being investigated for the treatment of GBM. Here we characterized the systemic and brain pharmacokinetics (PK) and hepatic metabolism of SBI-0206965. METHODS: We performed intracerebral microdialysis to determine brain partitioning of SBI-0206965 in jugular vein cannulated rats. We assessed systemic PK of SBI-0206965 in rats and C57BL/6 mice following oral administration. Employing human, mouse, and rat liver microsomes we characterized the metabolism of SBI-0206965. RESULTS: SBI-0206965 is quickly absorbed, achieving plasma and brain extracellular fluid (ECF) peak levels within 0.25 - 0.65 h. Based on the ratio of Cmax and AUC in brain ECF to plasma (corrected for protein binding), brain partitioning is ~ 0.6-0.9 in rats. However, the compound has a short elimination half-life (1-2 h) and low relative oral bioavailability (~ 0.15). The estimated in-vitro hepatic intrinsic clearance of SBI-0206965 in mouse, rat and human was 325, 76 and 68 mL/min/kg, respectively. SBI-0206965 metabolites included desmethylated products, and the metabolism was strongly inhibited by ketoconazole, a CYP3A inhibitor. CONCLUSION: SBI-0206965 has adequate brain permeability but low relative oral bioavailability which may be due to rapid hepatic metabolism, likely catalyzed by CYP3A enzymes. Our observations will facilitate further development of SBI-0206965, and/or other structurally related molecules, for the treatment of GBM and other brain tumors.

Blocking UBE2N abrogates oncogenic immune signaling in acute myeloid leukemia.

Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.

Inhibition of the RacGEF VAV3 by the small molecule IODVA1 impedes RAC signaling and overcomes resistance to tyrosine kinase inhibition in acute lymphoblastic leukemia.

Aberrant RHO guanine nucleotide exchange factor (RhoGEF) activation is chief mechanism driving abnormal activation of their GTPase targets in transformation and tumorigenesis. Consequently, a small-molecule inhibitor of RhoGEF can make an anti-cancer drug. We used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like acute lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation-dependent RacGEF, as the target of the small molecule IODVA1. We show that through binding to VAV3, IODVA1 inhibits RAC activation and signaling and increases pro-apoptotic activity in BCR-ABL1-transformed cells. Consistent with this mechanism of action, cellular and animal models of BCR-ABL1-induced leukemia in Vav3-null background do not respond to IODVA1. By durably decreasing in vivo RAC signaling, IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal. Importantly, IODVA1 suppresses the leukemic burden in the treatment refractory pediatric Ph+ and TKI-resistant Ph+ B-ALL patient-derived xenograft models better than standard-of-care dasatinib or ponatinib and provides a more durable response after treatment withdrawal. Pediatric leukemia samples with diverse genetic lesions show high sensitivity to IODVA1 ex vivo and this sensitivity is VAV3 dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapy.

Pharmacological clearance of misfolded rhodopsin for the treatment of RHO-associated retinitis pigmentosa.

Rhodopsin mutation and misfolding is a common cause of autosomal dominant retinitis pigmentosa (RP). Using a luciferase reporter assay, we undertook a small-molecule high-throughput screening (HTS) of 68, 979 compounds and identified nine compounds that selectively reduced the misfolded P23H rhodopsin without an effect on the wild type (WT) rhodopsin protein. Further, we found five of these compounds, including methotrexate (MTX), promoted P23H rhodopsin degradation that also cleared out other misfolded rhodopsin mutant proteins. We showed MTX increased P23H rhodopsin degradation via the lysosomal but not the proteasomal pathway. Importantly, one intravitreal injection (IVI) of 25 pmol MTX increased electroretinogram (ERG) response and rhodopsin level in the retinae of RhoP23H/+ knock-in mice at 1 month of age. Additionally, four weekly IVIs increased the photoreceptor cell number in the retinae of RhoP23H/+ mice compared to vehicle control. Our study indicates a therapeutic potential of repurposing MTX for the treatment of rhodopsin-associated RP.

IODVA1, a guanidinobenzimidazole derivative, targets Rac activity and Ras-driven cancer models.

We report the synthesis and preliminary characterization of IODVA1, a potent small molecule that is active in xenograft mouse models of Ras-driven lung and breast cancers. In an effort to inhibit oncogenic Ras signaling, we combined in silico screening with inhibition of proliferation and colony formation of Ras-driven cells. NSC124205 fulfilled all criteria. HPLC analysis revealed that NSC124205 was a mixture of at least three compounds, from which IODVA1 was determined to be the active component. IODVA1 decreased 2D and 3D cell proliferation, cell spreading and ruffle and lamellipodia formation through downregulation of Rac activity. IODVA1 significantly impaired xenograft tumor growth of Ras-driven cancer cells with no observable toxicity. Immuno-histochemistry analysis of tumor sections suggests that cell death occurs by increased apoptosis. Our data suggest that IODVA1 targets Rac signaling to induce death of Ras-transformed cells. Therefore, IODVA1 holds promise as an anti-tumor therapeutic agent.

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.

A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration.

Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC50 similar to 9-cis-retinal. The chaperone activity of YC-001 is evidenced by its ability to rescue the transport of multiple rod opsin mutants in mammalian cells. YC-001 is also an inverse agonist that non-competitively antagonizes rod opsin signaling. Significantly, a single dose of YC-001 protects Abca4 -/- Rdh8 -/- mice from bright light-induced retinal degeneration, suggesting its broad therapeutic potential.

Exploiting mitochondrial and metabolic homeostasis as a vulnerability in NF1 deficient cells.

Neurofibromatosis type 1 is a disease caused by mutation of neurofibromin 1 (NF1), loss of which results in hyperactive Ras signaling and a concomitant increase in cell proliferation and survival. Patients with neurofibromatosis type 1 frequently develop tumors such as plexiform neurofibromas and malignant peripheral nerve sheath tumors. Mutation of NF1 or loss of the NF1 protein is also observed in glioblastoma, lung adenocarcinoma, and ovarian cancer among other sporadic cancers. A therapy that selectively targets NF1 deficient tumors would substantially advance our ability to treat these malignancies. To address the need for these therapeutics, we developed and conducted a synthetic lethality screen to discover molecules that target yeast lacking the homolog of NF1, IRA2. One of the lead candidates that was observed to be synthetic lethal with ira2Δ yeast is Y100. Here, we describe the mechanisms by which Y100 targets ira2Δ yeast and NF1-deficient tumor cells. Y100 treatment disrupted proteostasis, metabolic homeostasis, and induced the formation of mitochondrial superoxide in NF1-deficient cancer cells. Previous studies also indicate that NF1/Ras-dysregulated tumors may be sensitive to modulators of oxidative and ER stress. We hypothesize that the use of Y100 and molecules with related mechanisms of action represent a feasible therapeutic strategy for targeting NF1 deficient cells.

Compound C/Dorsomorphin: Its Use and Misuse as an AMPK Inhibitor.

The evolutionary conserved energy sensor AMPK plays crucial roles in many biological processes-both during normal development and pathology. Loss-of-function genetic studies in mice as well as in lower organisms underscore its importance in embryonic development, stress physiology in the adult, and in key metabolic disorders including cardiovascular disease, diabetes, cancer, and metabolic syndrome. In contrast to several other kinases important in human health and medicine where specific/selective inhibitors are available, no AMPK-specific inhibitors are available. The only reagent called dorsomorphin or compound C that is occasionally used as an AMPK inhibitor unfortunately inhibits several other kinases much more potently than AMPK and is therefore highly non-specific. In this chapter, we discuss the pros and cons of using this reagent to study AMPK functions.

Author Correction: Targeted inhibition of STAT/TET1 axis as a therapeutic strategy for acute myeloid leukemia.

The original version of this Article contained an error in the spelling of the author James C. Mulloy, which was incorrectly given as James Mulloy. This has now been corrected in both the PDF and HTML versions of the Article.

Targeted inhibition of STAT/TET1 axis as a therapeutic strategy for acute myeloid leukemia.

Effective therapy of acute myeloid leukemia (AML) remains an unmet need. DNA methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is a critical oncoprotein in AML. Through a series of data analysis and drug screening, we identified two compounds (i.e., NSC-311068 and NSC-370284) that selectively suppress TET1 transcription and 5-hydroxymethylcytosine (5hmC) modification, and effectively inhibit cell viability in AML with high expression of TET1 (i.e., TET1-high AML), including AML carrying t(11q23)/MLL-rearrangements and t(8;21) AML. NSC-311068 and especially NSC-370284 significantly repressed TET1-high AML progression in vivo. UC-514321, a structural analog of NSC-370284, exhibited a more potent therapeutic effect and prolonged the median survival of TET1-high AML mice over three fold. NSC-370284 and UC-514321 both directly target STAT3/5, transcriptional activators of TET1, and thus repress TET1 expression. They also exhibit strong synergistic effects with standard chemotherapy. Our results highlight the therapeutic potential of targeting the STAT/TET1 axis by selective inhibitors in AML treatment.

Identification of Novel Bisbenzimidazole Derivatives as Anticancer Vacuolar (H⁺)-ATPase Inhibitors.

The vacuolar (H⁺)-ATPases (V-ATPases) are a family of ATP-driven proton pumps and they have been associated with cancer invasion, metastasis, and drug resistance. Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V) as an initial hit from a similarity search using four known V-ATPase inhibitors (I-IV). Based on the initial hit (V), we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a-e). All newly prepared compounds have been screened for selected human breast cancer (MDA-MB-468, MDA-MB-231, and MCF7) and ovarian cancer (A2780, Cis-A2780, and PA-1) cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e) is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 µM). Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.

Combined rational design and a high throughput screening platform for identifying chemical inhibitors of a Ras-activating enzyme.

The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions.

Target validation and structure-activity analysis of a series of novel PCNA inhibitors.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair. Tumor cells express high levels of PCNA, identifying it as a potentially ideal target for cancer therapy. Previously, we identified nine compounds termed PCNA inhibitors (PCNA-Is) that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, and selectively inhibit tumor cell growth. Of these compounds, PCNA-I1 was most potent. The purpose of this study is to further establish targeting of PCNA by PCNA-I1 and to identify PCNA-I1 analogs with superior potencies. We found that PCNA-I1 does not affect the level of chromatin-associated PCNA harboring point mutations at the predicted binding site of PCNA-I1. Forty-six PCNA-I1 analogs with structures of 1-hydrazonomethyl-2-hydroxy (scaffold A), 2-hydrazonomethyl-1-hydroxy (scaffold B), 2-hydrazonomethyl-3-hydroxy (scaffold C), and 4-pyridyl hydrazine (scaffold D) were analyzed for their effects on cell growth in four tumor cell lines and PCNA trimer stabilization. Compounds in scaffold group A and group B showed the highest trimer stabilization and the most potent cell growth inhibitory activities with a significant potency advantage observed in the Z isomers of scaffold A. The absence of trimer stabilization and growth inhibitory effects in compounds of scaffold group D confirms the essentiality of the hydroxynaphthyl substructure. Compounds structure-activity relationship (SAR)-6 and SAR-24 were analyzed for their effects on and found to reduce chromatin-associated PCNA in tumor cells. This study led to the identification of SAR-24, a compound with superior potencies and potentially improved solubility, which will be used for future development of PCNA-targeting cancer therapies.

A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation.

PURPOSE: Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding. METHODS: Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses. RESULTS: Two stabilization assays of P23H mutant opsin were developed and validated, one based on ß-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identified changes in the aggregation and glycosylation of P23H mutant opsin. CONCLUSIONS: Our findings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for difficult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.

A novel, soluble compound, C25, sensitizes to TRAIL-induced apoptosis through upregulation of DR5 expression.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential therapeutic agent that induces apoptosis selectively in tumor cells. However, numerous solid tumor types are resistant to TRAIL. Sensitization to TRAIL has been an area of great research interest, but has met significant challenges because of poor bioavailability, half-life, and solubility of sensitizing compounds such as curcumin. Soluble, TRAIL-sensitizing compounds were screened on the basis of similarity to the redox-active substructure of curcumin and sensitization to TRAIL-induced apoptosis. We determined the effect of the lead compound, C25, in combination with TRAIL in human cancer cell lines using MTS proliferation assays, apoptosis assays, and western blotting. Short hairpin RNA knockdown of death receptor 5 (DR5) was performed to determine whether DR5 upregulation was required for TRAIL-mediated apoptosis. In-vivo efficacy was determined using human lung tumor xenograft models. C25 helped overcome TRAIL resistance by upregulating the expression of the TRAIL receptor DR5 and apoptosis in several tumor cell lines. Blockade of DR5 expression abrogated C25 sensitization to TRAIL, demonstrating the requirement for DR5 upregulation for C25-mediated potentiation of TRAIL-mediated apoptosis. The combination of C25 and TRAIL effectively inhibited tumorigenesis in vivo. This study demonstrates the synergistic efficacy of C25 in sensitization to TRAIL-induced apoptosis in multiple tumor cell types, including highly resistant lung and ovarian tumor cell lines. Furthermore, C25 was efficacious against tumor growth in vivo. Thus, C25 may be a potential therapeutic for cancer in combination with TRAIL or DR5 agonist therapy.