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Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by N-myristoyltransferases (NMT). Myristoylation is emerging as a promising cancer therapeutic target; however, the molecular determinants of sensitivity to NMT inhibition or the mechanism by which it induces cancer cell death are not completely understood. We report that NMTs are a novel therapeutic target in lung carcinoma cells with LKB1 and/or KEAP1 mutations in a KRAS-mutant background. Inhibition of myristoylation decreases cell viability in vitro and tumor growth in vivo. Inhibition of myristoylation causes mitochondrial ferrous iron overload, oxidative stress, elevated protein poly (ADP)-ribosylation, and death by parthanatos. Furthermore, NMT inhibitors sensitized lung carcinoma cells to platinum-based chemotherapy. Unexpectedly, the mitochondrial transporter translocase of inner mitochondrial membrane 17 homolog A (TIM17A) is a critical target of myristoylation inhibitors in these cells. TIM17A silencing recapitulated the effects of NMT inhibition at inducing mitochondrial ferrous iron overload and parthanatos. Furthermore, sensitivity of lung carcinoma cells to myristoylation inhibition correlated with their dependency on TIM17A. This study reveals the unexpected connection between protein myristoylation, the mitochondrial import machinery, and iron homeostasis. It also uncovers myristoylation inhibitors as novel inducers of parthanatos in cancer, and the novel axis NMT-TIM17A as a potential therapeutic target in highly aggressive lung carcinomas.SignificanceKRAS-mutant lung carcinomas with LKB1 and/or KEAP1 co-mutations have intrinsic therapeutic resistance. We show that these tumors are sensitive to NMT inhibitors, which slow tumor growth in vivo and sensitize cells to platinum-based chemotherapy in vitro. Inhibition of myristoylation causes death by parthanatos and thus has the potential to kill apoptosis and ferroptosis-resistant cancer cells. Our findings warrant investigation of NMT as a therapeutic target in highly aggressive lung carcinomas.

PurposeSmall molecule inhibitors that target oncogenic driver kinases are an important class of therapies for non-small cell lung cancer (NSCLC) and other malignancies. However, these therapies are not without their challenges. Each inhibitor works on only a subset of patients, the pharmacokinetics of these inhibitors is variable, and these inhibitors are associated with significant side effects. Many of these inhibitors lack non-invasive biomarkers to confirm pharmacodynamic efficacy, and our understanding of how these inhibitors block cancer cell growth remains incomplete. Limited clinical studies suggest that early (

Squamous cell lung cancer maintains its growth through elevated glucose consumption, but selective glucose consumption inhibitors are lacking. Here, we discovered using a high-throughput screen new compounds that block glucose consumption in three squamous cell lung cancer cell lines and identified 79 compounds that block glucose consumption in one or more of these cell lines. Based on its ability to block glucose consumption in all three cell lines, pacritinib, an inhibitor of FMS Related Receptor Tyrosine Kinase 3 (FLT3) and Janus Kinase 2 (JAK2), was further studied. Pacritinib decreased glucose consumption in squamous cell lung cancer cells in cell culture and in vivo without affecting glucose consumption in healthy tissues. Pacritinib blocked hexokinase activity, and Hexokinase 1 and 2 mRNA and protein expression. Overexpression of Hexokinase 1 blocked the ability of pacritinib to inhibit glucose consumption in squamous cell lung cancer cells. Overexpression of FLT3 but not JAK2 significantly increased glucose consumption and blocked the ability of pacritinib to inhibit glucose consumption in squamous cell lung cancer cells. Additional FLT3 inhibitors blocked glucose consumption in squamous cell lung cancer cells. Our study identifies FLT3 inhibitors as a new class of inhibitors that can block glucose consumption in squamous cell lung cancer.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg.

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.

We previously reported several vignettes on types and classes of drugs able to mitigate acute and, in at least one case, late radiation syndromes in mice. Most of these had emerged from high throughput screening (HTS) of bioactive and chemical drug libraries using ionizing radiation-induced lymphocytic apoptosis as a readout. Here we report the full analysis of the HTS screen of libraries with 85,000 small molecule chemicals that identified 220 "hits." Most of these hits could be allocated by maximal common substructure analysis to one of 11 clusters each containing at least three active compounds. Further screening validated 23 compounds as being most active; 15 of these were cherry-picked based on drug availability and tested for their ability to mitigate acute hematopoietic radiation syndrome (H-ARS) in mice. Of these, five bore a 4-nitrophenylsulfonamide motif while 4 had a quinoline scaffold. All but two of the 15 significantly (p < 0.05) mitigated H-ARS in mice. We had previously reported that the lead 4-(nitrophenylsulfonyl)-4-phenylpiperazine compound (NPSP512), was active in mitigating multiple acute and late radiation syndromes in mice of more than one sex and strain. Unfortunately, the formulation of this drug had to be changed for regulatory reasons and we report here on the synthesis and testing of active analogs of NPSP512 (QS1 and 52A1) that have increased solubility in water and in vivo bioavailability while retaining mitigator activity against H-ARS (p < 0.0001) and other radiation syndromes. The lead quinoline 057 was also active in multiple murine models of radiation damage. Taken together, HTS of a total of 150,000 bioactive or chemical substances, combined with maximal common substructure analysis has resulted in the discovery of diverse groups of compounds that can mitigate H-ARS and at least some of which can mitigate multiple radiation syndromes when given starting 24 h after exposure. We discuss what is known about how these agents might work, and the importance of formulation and bioavailability.

We previously reported several vignettes on types and classes of drugs able to mitigate acute and, in at least one case, late radiation syndromes in mice. Most of these had emerged from high throughput screening (HTS) of bioactive and chemical drug libraries using ionizing radiation-induced lymphocytic apoptosis as a readout. Here we report the full analysis of the HTS screen of libraries with 85,000 small molecule chemicals that identified 220 "hits." Most of these hits could be allocated by maximal common substructure analysis to one of 11 clusters each containing at least three active compounds. Further screening validated 23 compounds as being most active; 15 of these were cherry-picked based on drug availability and tested for their ability to mitigate acute hematopoietic radiation syndrome (H-ARS) in mice. Of these, five bore a 4-nitrophenylsulfonamide motif while 4 had a quinoline scaffold. All but two of the 15 significantly (p < 0.05) mitigated H-ARS in mice. We had previously reported that the lead 4-(nitrophenylsulfonyl)-4-phenylpiperazine compound (NPSP512), was active in mitigating multiple acute and late radiation syndromes in mice of more than one sex and strain. Unfortunately, the formulation of this drug had to be changed for regulatory reasons and we report here on the synthesis and testing of active analogs of NPSP512 (QS1 and 52A1) that have increased solubility in water and in vivo bioavailability while retaining mitigator activity against H-ARS (p < 0.0001) and other radiation syndromes. The lead quinoline 057 was also active in multiple murine models of radiation damage. Taken together, HTS of a total of 150,000 bioactive or chemical substances, combined with maximal common substructure analysis has resulted in the discovery of diverse groups of compounds that can mitigate H-ARS and at least some of which can mitigate multiple radiation syndromes when given starting 24 h after exposure. We discuss what is known about how these agents might work, and the importance of formulation and bioavailability.

BackgroundDuchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by mutations in the dystrophin gene. Loss of dystrophin prevents the formation of a critical connection between the muscle cell membrane and the extracellular matrix. Overexpression of sarcospan (SSPN) in the mouse model of DMD restores the membrane connection and reduces disease severity, making SSPN a promising therapeutic target for pharmacological upregulation.MethodsUsing a previously described cell-based promoter reporter assay of SSPN gene expression (hSSPN-EGFP), we conducted high-throughput screening on libraries of over 200,000 curated small molecules to identify SSPN modulators. The hits were validated in both hSSPN-EGFP and hSSPN-luciferase reporter cells. Hit selection was conducted on dystrophin-deficient mouse and human myotubes with assessments of (1) SSPN gene expression using quantitative PCR and (2) SSPN protein expression using immunoblotting and an ELISA. A membrane stability assay using osmotic shock was used to validate the functional effects of treatment followed by cell surface biotinylation to label cell surface proteins. Dystrophin-deficient mdx mice were treated with compound, and muscle was subjected to quantitative PCR to assess SSPN gene expression.ResultsWe identified and validated lead compounds that increased SSPN gene and protein expression in dystrophin-deficient mouse and human muscle cells. The lead compound OT-9 increased cell membrane localization of compensatory laminin-binding adhesion complexes and improved membrane stability in DMD myotubes. We demonstrated that the membrane stabilizing benefit is dependent on SSPN. Intramuscular injection of OT-9 in the mouse model of DMD increased SSPN gene expression.ConclusionsThis study identifies a pharmacological approach to treat DMD and sets the path for the development of SSPN-based therapies.

Patients with pancreatic adenocarcinoma (PDAC) have a 5-year survival rate of 8%, the lowest of any cancer in the United States. Traditional chemotherapeutic regimens, such as gemcitabine- and fluorouracil-based regimens, often only prolong survival by months. Effective precision targeted therapy is therefore urgently needed to substantially improve survival. In an effort to expedite approval and delivery of targeted therapy to patients, we utilized a platform to develop a novel combination of FDA approved drugs that would target pancreaticoduodenal homeobox1 (PDX1) and baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) utilizing super-promoters of the target genes to interrogate an FDA approved drug library. We identified and selected metformin, simvastatin and digoxin (C3) as a novel combination of FDA approved drugs, which were shown to effectively target PDX1 and BIRC5 in human PDAC tumors in mice with no toxicity.

Mutations in CAPN3 cause limb girdle muscular dystrophy R1 (LGMDR1, formerly LGMD2A) and lead to progressive and debilitating muscle wasting. Calpain 3 deficiency is associated with impaired CaMKIIβ signaling and blunted transcriptional programs that encode the slow-oxidative muscle phenotype. We conducted a high-throughput screen on a target of CaMKII (Myl2) to identify compounds to override this signaling defect; 4 were tested in vivo in the Capn3 knockout (C3KO) model of LGMDR1. The leading compound, AMBMP, showed good exposure and was able to reverse the LGMDR1 phenotype in vivo, including improved oxidative properties, increased slow fiber size, and enhanced exercise performance. AMBMP also activated CaMKIIβ signaling, but it did not alter other pathways known to be associated with muscle growth. Thus, AMBMP treatment activates CaMKII and metabolically reprograms skeletal muscle toward a slow muscle phenotype. These proof-of-concept studies lend support for an approach to the development of therapeutics for LGMDR1.

Hu11B6 is a monoclonal antibody that internalizes in cells expressing androgen receptor (AR)-regulated prostate-specific enzyme human kallikrein-related peptidase 2 (hK2; KLK2). In multiple rodent models, Actinium-225-labeled hu11B6-IgG1 ([225Ac]hu11B6-IgG1) has shown promising treatment efficacy. In the present study, we investigated options to enhance and optimize [225Ac]hu11B6 treatment. First, we evaluated the possibility of exploiting IgG3, the IgG subclass with superior activation of complement and ability to mediate FC-γ-receptor binding, for immunotherapeutically enhanced hK2 targeted α-radioimmunotherapy. Second, we compared the therapeutic efficacy of a single high activity vs. fractionated activity. Finally, we used RNA sequencing to analyze the genomic signatures of prostate cancer that progressed after targeted α-therapy. [225Ac]hu11B6-IgG3 was a functionally enhanced alternative to [225Ac]hu11B6-IgG1 but offered no improvement of therapeutic efficacy. Progression-free survival was slightly increased with a single high activity compared to fractionated activity. Tumor-free animals succumbing after treatment revealed no evidence of treatment-associated toxicity. In addition to up-regulation of canonical aggressive prostate cancer genes, such as MMP7, ETV1, NTS, and SCHLAP1, we also noted a significant decrease in both KLK3 (prostate-specific antigen ) and FOLH1 (prostate-specific membrane antigen) but not in AR and KLK2, demonstrating efficacy of sequential [225Ac]hu11B6 in a mouse model.

Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris may be a useful platform for discovery efforts targeting sortase.

A potent class of isoquinoline-based α-N-heterocyclic carboxaldehyde thiosemicarbazone (HCT) compounds has been rediscovered; based upon this scaffold, three series of antiproliferative agents were synthesized through iterative rounds of methylation and fluorination modifications, with anticancer activities being potentiated by physiologically relevant levels of copper. The lead compound, HCT-13, was highly potent against a panel of pancreatic, small cell lung carcinoma, prostate cancer, and leukemia models, with IC50 values in the low-to-mid nanomolar range. Density functional theory (DFT) calculations showed that fluorination at the 6-position of HCT-13 was beneficial for ligand-copper complex formation, stability, and ease of metal-center reduction. Through a chemical genomics screen, we identify DNA damage response/replication stress response (DDR/RSR) pathways, specifically those mediated by ataxia-telangiectasia and Rad3-related protein kinase (ATR), as potential compensatory mechanism(s) of action following HCT-13 treatment. We further show that the cytotoxicity of HCT-13 is copper-dependent, that it promotes mitochondrial electron transport chain (mtETC) dysfunction, induces production of reactive oxygen species (ROS), and selectively depletes guanosine nucleotide pools. Lastly, we identify metabolic hallmarks for therapeutic target stratification and demonstrate the in vivo efficacy of HCT-13 against aggressive models of acute leukemias in mice.

Mechanisms underpinning airway epithelial homeostatic maintenance and ways to prevent its dysregulation remain elusive. Herein, we identify that β-catenin phosphorylated at Y489 (p-β-cateninY489) emerges during human squamous lung cancer progression. This led us to develop a model of airway basal stem cell (ABSC) hyperproliferation by driving Wnt/β-catenin signaling, resulting in a morphology that resembles premalignant lesions and loss of ciliated cell differentiation. To identify small molecules that could reverse this process, we performed a high-throughput drug screen for inhibitors of Wnt/β-catenin signaling. Our studies unveil Wnt inhibitor compound 1 (WIC1), which suppresses T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) activity, reduces ABSC proliferation, induces ciliated cell differentiation, and decreases nuclear p-β-cateninY489. Collectively, our work elucidates a dysregulated Wnt/p-β-cateninY489 axis in lung premalignancy that can be modeled in vitro and identifies a Wnt/β-catenin inhibitor that promotes airway homeostasis. WIC1 may therefore serve as a tool compound in regenerative medicine studies with implications for restoring normal airway homeostasis after injury.

Progressive organ fibrosis accounts for one-third of all deaths worldwide, yet preclinical models that mimic the complex, progressive nature of the disease are lacking, and hence, there are no curative therapies. Progressive fibrosis across organs shares common cellular and molecular pathways involving chronic injury, inflammation, and aberrant repair resulting in deposition of extracellular matrix, organ remodeling, and ultimately organ failure. We describe the generation and characterization of an in vitro progressive fibrosis model that uses cell types derived from induced pluripotent stem cells. Our model produces endogenous activated transforming growth factor β (TGF-β) and contains activated fibroblastic aggregates that progressively increase in size and stiffness with activation of known fibrotic molecular and cellular changes. We used this model as a phenotypic drug discovery platform for modulators of fibrosis. We validated this platform by identifying a compound that promotes resolution of fibrosis in in vivo and ex vivo models of ocular and lung fibrosis.

BackgroundDuchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion.MethodsStandard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting.ResultsWe investigated the gene expression profiles of SSPN and its associated proteins during myoblast differentiation into myotubes, revealing an increase in expression after 3 days of differentiation. We created C2C12 muscle cells expressing an EGFP reporter for SSPN promoter activity and observed a comparable increase in reporter levels during differentiation. Assay conditions for high-throughput screening were optimized for a 384-well microplate format and a high-content imager for the visualization of reporter levels. We conducted a screen of 3200 compounds and identified seven hits, which include an overrepresentation of L-type calcium channel antagonists, suggesting that SSPN gene activity is sensitive to calcium. Further validation of a select hit revealed that the calcium channel inhibitor felodipine increased SSPN transcript and protein levels in both wild-type and dystrophin-deficient myotubes, without increasing differentiation.ConclusionsWe developed a stable muscle cell line containing the promoter region of the human SSPN protein fused to a fluorescent reporter. Using the reporter cells, we created and validated a scalable, cell-based assay that is able to identify compounds that increase SSPN promoter reporter, transcript, and protein levels in wild-type and dystrophin-deficient muscle cells.

Targeting the leukemia proliferation cycle has been a successful approach to developing antileukemic therapies. However, drug screening efforts to identify novel antileukemic agents have been hampered by the lack of a suitable high-throughput screening platform for suspension cells that does not rely on flow-cytometry analyses. We report the development of a novel leukemia cell-based high-throughput chemical screening platform for the discovery of cell cycle phase specific inhibitors that utilizes chemical cell cycle profiling. We have used this approach to analyze the cell cycle response of acute lymphoblastic leukemia CCRF-CEM cells to each of 181420 druglike compounds. This approach yielded cell cycle phase specific inhibitors of leukemia cell proliferation. Further analyses of the top G2-phase and M-phase inhibitors identified the leukemia specific inhibitor 1 (Leusin-1). Leusin-1 arrests cells in G2 phase and triggers an apoptotic cell death. Most importantly, Leusin-1 was more active in acute lymphoblastic leukemia cells than other types of leukemias, non-blood cancers, or normal cells and represents a lead molecule for developing antileukemic drugs.

The transcription factor nuclear factor of activated T cells (NFAT) has a key role in both T cell activation and tolerance and has emerged as an important target of immune modulation. NFAT directs the effector arm of the immune response in the presence of activator protein-1 (AP-1), and T cell anergy/exhaustion in the absence of AP-1. Envisioning a strategy for selective modulation of the immune response, we designed a FRET-based high-throughput screen to identify compounds that disrupt the NFAT:AP-1:DNA complex. We screened ∼202,000 small organic compounds and identified 337 candidate inhibitors. We focus here on one compound, N-(3-acetamidophenyl)-2-[5-(1H-benzimidazol-2-yl)pyridin-2-yl]sulfanylacetamide (Compound 10), which disrupts the NFAT:AP-1 interaction at the composite antigen-receptor response element-2 site without affecting the binding of NFAT or AP-1 alone to DNA. Compound 10 binds to DNA in a sequence-selective manner and inhibits the transcription of the Il2 gene and several other cyclosporin A-sensitive cytokine genes important for the effector immune response. This study provides proof-of-concept that small molecules can inhibit the assembly of specific DNA-protein complexes, and opens a potential new approach to treat human diseases where known transcription factors are deregulated.

ObjectiveExposure to lethal doses of radiation has severe effects on normal tissues. Exposed individuals experience a plethora of symptoms in different organ systems including the gastrointestinal (GI) tract, summarized as Acute Radiation Syndrome (ARS). There are currently no approved drugs for mitigating GI-ARS. A recent high-throughput screen performed at the UCLA Center for Medical Countermeasures against Radiation identified compounds containing sulfonylpiperazine groups with radiation mitigation properties to the hematopoietic system and the gut. Among these 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine (Compound #5) efficiently mitigated gastrointestinal ARS. However, the mechanism of action and target cells of this drug is still unknown. In this study we examined if Compound #5 affects gut-associated lymphoid tissue (GALT) with its subepithelial domes called Peyer's patches.MethodsC3H mice were irradiated with 0 or 12 Gy total body irradiation (TBI). A single dose of Compound #5 or solvent was administered subcutaneously 24 h later. 48 h after irradiation the mice were sacrificed, and the guts examined for changes in the number of visible Peyer's patches. In some experiments the mice received 4 daily injections of treatment and were sacrificed 96 h after TBI. For immune histochemistry gut tissues were fixed in formalin and embedded in paraffin blocks. Sections were stained with H&E, anti-Ki67 or a TUNEL assay to assess the number of regenerating crypts, mitotic and apoptotic indices. Cells isolated from Peyer's patches were subjected to immune profiling using flow cytometry.ResultsCompound #5 significantly increased the number of visible Peyer's patches when compared to its control in non-irradiated and irradiated mice. Additionally, assessment of total cells per Peyer's patch isolated from these mice demonstrated an overall increase in the total number of Peyer's patch cells per mouse in Compound #5-treated mice. In non-irradiated animals the number of CD11bhigh in Peyer's patches increased significantly. These Compound #5-driven increases did not coincide with a decrease in apoptosis or an increase in proliferation in the germinal centers inside Peyer's patches 24 h after drug treatment. A single dose of Compound #5 significantly increased the number of CD45+ cells after 12 Gy TBI. Importantly, 96 h after 12 Gy TBI Compound #5 induced a significant rise in the number of visible Peyer's patches and the number of Peyer's patch-associated regenerating crypts.ConclusionIn summary, our study provides evidence that Compound #5 leads to an influx of immune cells into GALT, thereby supporting crypt regeneration preferentially in the proximity of Peyer's patches.

Tumor organoids maintain cell-cell interactions, heterogeneity, microenvironment, and drug response of the sample they originate from. Thus, there is increasing interest in developing tumor organoid models for drug development and personalized medicine applications. Although organoids are in principle amenable to high-throughput screenings, progress has been hampered by technical constraints and extensive manipulations required by current methods. Here we introduce a miniaturized method that uses a simplified geometry by seeding cells around the rim of the wells (mini-rings). This allows high-throughput screenings in a format compatible with automation as shown using four patient-derived tumor organoids established from two ovarian and one peritoneal high-grade serous carcinomas and one carcinosarcoma of the ovary. Using our automated screening platform, we identified personalized responses by measuring viability, number, and size of organoids after exposure to 240 kinase inhibitors. Results are available within a week from surgery, a timeline compatible with therapeutic decision-making.

Tumor organoids maintain cell-cell interactions, heterogeneity, microenvironment, and drug response of the sample they originate from. Thus, there is increasing interest in developing tumor organoid models for drug development and personalized medicine applications. Although organoids are in principle amenable to high-throughput screenings, progress has been hampered by technical constraints and extensive manipulations required by current methods. Here we introduce a miniaturized method that uses a simplified geometry by seeding cells around the rim of the wells (mini-rings). This allows high-throughput screenings in a format compatible with automation as shown using four patient-derived tumor organoids established from two ovarian and one peritoneal high-grade serous carcinomas and one carcinosarcoma of the ovary. Using our automated screening platform, we identified personalized responses by measuring viability, number, and size of organoids after exposure to 240 kinase inhibitors. Results are available within a week from surgery, a timeline compatible with therapeutic decision-making.

Elevated glucose consumption is fundamental to cancer, but selectively targeting this pathway is challenging. We develop a high-throughput assay for measuring glucose consumption and use it to screen non-small-cell lung cancer cell lines against bioactive small molecules. We identify Milciclib that blocks glucose consumption in H460 and H1975, but not in HCC827 or A549 cells, by decreasing SLC2A1 (GLUT1) mRNA and protein levels and by inhibiting glucose transport. Milciclib blocks glucose consumption by targeting cyclin-dependent kinase 7 (CDK7) similar to other CDK7 inhibitors including THZ1 and LDC4297. Enhanced PIK3CA signaling leads to CDK7 phosphorylation, which promotes RNA Polymerase II phosphorylation and transcription. Milciclib, THZ1, and LDC4297 lead to a reduction in RNA Polymerase II phosphorylation on the SLC2A1 promoter. These data indicate that our high-throughput assay can identify compounds that regulate glucose consumption and that CDK7 is a key regulator of glucose consumption in cells with an activated PI3K pathway.

Receptor type protein tyrosine phosphatase-sigma (PTPσ) is primarily expressed by adult neurons and regulates neural regeneration. We recently discovered that PTPσ is also expressed by hematopoietic stem cells (HSCs). Here, we describe small molecule inhibitors of PTPσ that promote HSC regeneration in vivo. Systemic administration of the PTPσ inhibitor, DJ001, or its analog, to irradiated mice promotes HSC regeneration, accelerates hematologic recovery, and improves survival. Similarly, DJ001 administration accelerates hematologic recovery in mice treated with 5-fluorouracil chemotherapy. DJ001 displays high specificity for PTPσ and antagonizes PTPσ via unique non-competitive, allosteric binding. Mechanistically, DJ001 suppresses radiation-induced HSC apoptosis via activation of the RhoGTPase, RAC1, and induction of BCL-XL. Furthermore, treatment of irradiated human HSCs with DJ001 promotes the regeneration of human HSCs capable of multilineage in vivo repopulation. These studies demonstrate the therapeutic potential of selective, small-molecule PTPσ inhibitors for human hematopoietic regeneration.

BackgroundPrecision medicine therapies require identification of unique molecular cancer characteristics. Hexokinase (HK) activity has been proposed as a therapeutic target; however, different hexokinase isoforms have not been well characterized as alternative targets. While HK2 is highly expressed in the majority of cancers, cancer subtypes with differential HK1 and HK2 expression have not been characterized for their sensitivities to HK2 silencing.MethodsHK1 and HK2 expression in the Cancer Cell Line Encyclopedia dataset was analyzed. A doxycycline-inducible shRNA silencing system was used to examine the effect of HK2 knockdown in cultured cells and in xenograft models of HK1-HK2+ and HK1+HK2+ cancers. Glucose consumption and lactate production rates were measured to monitor HK activity in cell culture, and 18F-FDG PET/CT was used to monitor HK activity in xenograft tumors. A high-throughput screen was performed to search for synthetically lethal compounds in combination with HK2 inhibition in HK1-HK2+ liver cancer cells, and a combination therapy for liver cancers with this phenotype was developed. A metabolomic analysis was performed to examine changes in cellular energy levels and key metabolites in HK1-HK2+ cells treated with this combination therapy. The CRISPR Cas9 method was used to establish isogenic HK1+HK2+ and HK1-HK2+ cell lines to evaluate HK1-HK2+ cancer cell sensitivity to the combination therapy.ResultsMost tumors express both HK1 and HK2, and subsets of cancers from a wide variety of tissues of origin express only HK2. Unlike HK1+HK2+ cancers, HK1-HK2+ cancers are sensitive to HK2 silencing-induced cytostasis. Synthetic lethality was achieved in HK1-HK2+ liver cancer cells, by the combination of DPI, a mitochondrial complex I inhibitor, and HK2 inhibition, in HK1-HK2+ liver cancer cells. Perhexiline, a fatty acid oxidation inhibitor, further sensitizes HK1-HK2+ liver cancer cells to the complex I/HK2-targeted therapeutic combination. Although HK1+HK2+ lung cancer H460 cells are resistant to this therapeutic combination, isogenic HK1KOHK2+ cells are sensitive to this therapy.ConclusionsThe HK1-HK2+ cancer subsets exist among a wide variety of cancer types. Selective inhibition of the HK1-HK2+ cancer cell-specific energy production pathways (HK2-driven glycolysis, oxidative phosphorylation and fatty acid oxidation), due to the unique presence of only the HK2 isoform, appears promising to treat HK1-HK2+ cancers. This therapeutic strategy will likely be tolerated by most normal tissues, where only HK1 is expressed.

BackgroundClinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy.MethodsTo determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures.ResultsAn unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization.ConclusionsThese data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.

Background:Clinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy. Methods:To determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures. Results:An unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization. Conclusions:These data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.

Interactions and emergent processes are essential for research on complex systems involving many components. Most studies focus solely on pairwise interactions and ignore higher-order interactions among three or more components. To gain deeper insights into higher-order interactions and complex environments, we study antibiotic combinations applied to pathogenic Escherichia coli and obtain unprecedented amounts of detailed data (251 two-drug combinations, 1512 three-drug combinations, 5670 four-drug combinations, and 13608 five-drug combinations). Directly opposite to previous assumptions and reports, we find higher-order interactions increase in frequency with the number of drugs in the bacteria's environment. Specifically, as more drugs are added, we observe an elevated frequency of net synergy (effect greater than expected based on independent individual effects) and also increased instances of emergent antagonism (effect less than expected based on lower-order interaction effects). These findings have implications for the potential efficacy of drug combinations and are crucial for better navigating problems associated with the combinatorial complexity of multi-component systems.

Melanoma resistant to MAPK inhibitors (MAPKi) displays loss of fitness upon experimental MAPKi withdrawal and, clinically, may be resensitized to MAPKi therapy after a drug holiday. Here, we uncovered and therapeutically exploited the mechanisms of MAPKi addiction in MAPKi-resistant BRAFMUT or NRASMUT melanoma. MAPKi-addiction phenotypes evident upon drug withdrawal spanned transient cell-cycle slowdown to cell-death responses, the latter of which required a robust phosphorylated ERK (pERK) rebound. Generally, drug withdrawal-induced pERK rebound upregulated p38-FRA1-JUNB-CDKN1A and downregulated proliferation, but only a robust pERK rebound resulted in DNA damage and parthanatos-related cell death. Importantly, pharmacologically impairing DNA damage repair during MAPKi withdrawal augmented MAPKi addiction across the board by converting a cell-cycle deceleration to a caspase-dependent cell-death response or by furthering parthanatos-related cell death. Specifically in MEKi-resistant NRASMUT or atypical BRAFMUT melanoma, treatment with a type I RAF inhibitor intensified pERK rebound elicited by MEKi withdrawal, thereby promoting a cell death-predominant MAPKi-addiction phenotype. Thus, MAPKi discontinuation upon disease progression should be coupled with specific strategies that augment MAPKi addiction.Significance: Discontinuing targeted therapy may select against drug-resistant tumor clones, but drug-addiction mechanisms are ill-defined. Using melanoma resistant to but withdrawn from MAPKi, we defined a synthetic lethality between supraphysiologic levels of pERK and DNA damage. Actively promoting this synthetic lethality could rationalize sequential/rotational regimens that address evolving vulnerabilities. Cancer Discov; 8(1); 74-93. ©2017 AACR.See related commentary by Stern, p. 20This article is highlighted in the In This Issue feature, p. 1.

Melanoma resistant to MAPK inhibitors (MAPKi) displays loss of fitness upon experimental MAPKi withdrawal and, clinically, may be resensitized to MAPKi therapy after a drug holiday. Here, we uncovered and therapeutically exploited the mechanisms of MAPKi addiction in MAPKi-resistant BRAFMUT or NRASMUT melanoma. MAPKi-addiction phenotypes evident upon drug withdrawal spanned transient cell-cycle slowdown to cell-death responses, the latter of which required a robust phosphorylated ERK (pERK) rebound. Generally, drug withdrawal-induced pERK rebound upregulated p38-FRA1-JUNB-CDKN1A and downregulated proliferation, but only a robust pERK rebound resulted in DNA damage and parthanatos-related cell death. Importantly, pharmacologically impairing DNA damage repair during MAPKi withdrawal augmented MAPKi addiction across the board by converting a cell-cycle deceleration to a caspase-dependent cell-death response or by furthering parthanatos-related cell death. Specifically in MEKi-resistant NRASMUT or atypical BRAFMUT melanoma, treatment with a type I RAF inhibitor intensified pERK rebound elicited by MEKi withdrawal, thereby promoting a cell death-predominant MAPKi-addiction phenotype. Thus, MAPKi discontinuation upon disease progression should be coupled with specific strategies that augment MAPKi addiction.Significance: Discontinuing targeted therapy may select against drug-resistant tumor clones, but drug-addiction mechanisms are ill-defined. Using melanoma resistant to but withdrawn from MAPKi, we defined a synthetic lethality between supraphysiologic levels of pERK and DNA damage. Actively promoting this synthetic lethality could rationalize sequential/rotational regimens that address evolving vulnerabilities. Cancer Discov; 8(1); 74-93. ©2017 AACR.See related commentary by Stern, p. 20This article is highlighted in the In This Issue feature, p. 1.

BackgroundPrecision medicine therapies require identification of unique molecular cancer characteristics. Hexokinase (HK) activity has been proposed as a therapeutic target; however, different hexokinase isoforms have not been well characterized as alternative targets. While HK2 is highly expressed in the majority of cancers, cancer subtypes with differential HK1 and HK2 expression have not been characterized for their sensitivities to HK2 silencing.MethodsHK1 and HK2 expression in the Cancer Cell Line Encyclopedia dataset was analyzed. A doxycycline-inducible shRNA silencing system was used to examine the effect of HK2 knockdown in cultured cells and in xenograft models of HK1-HK2+ and HK1+HK2+ cancers. Glucose consumption and lactate production rates were measured to monitor HK activity in cell culture, and 18F-FDG PET/CT was used to monitor HK activity in xenograft tumors. A high-throughput screen was performed to search for synthetically lethal compounds in combination with HK2 inhibition in HK1-HK2+ liver cancer cells, and a combination therapy for liver cancers with this phenotype was developed. A metabolomic analysis was performed to examine changes in cellular energy levels and key metabolites in HK1-HK2+ cells treated with this combination therapy. The CRISPR Cas9 method was used to establish isogenic HK1+HK2+ and HK1-HK2+ cell lines to evaluate HK1-HK2+ cancer cell sensitivity to the combination therapy.ResultsMost tumors express both HK1 and HK2, and subsets of cancers from a wide variety of tissues of origin express only HK2. Unlike HK1+HK2+ cancers, HK1-HK2+ cancers are sensitive to HK2 silencing-induced cytostasis. Synthetic lethality was achieved in HK1-HK2+ liver cancer cells, by the combination of DPI, a mitochondrial complex I inhibitor, and HK2 inhibition, in HK1-HK2+ liver cancer cells. Perhexiline, a fatty acid oxidation inhibitor, further sensitizes HK1-HK2+ liver cancer cells to the complex I/HK2-targeted therapeutic combination. Although HK1+HK2+ lung cancer H460 cells are resistant to this therapeutic combination, isogenic HK1KOHK2+ cells are sensitive to this therapy.ConclusionsThe HK1-HK2+ cancer subsets exist among a wide variety of cancer types. Selective inhibition of the HK1-HK2+ cancer cell-specific energy production pathways (HK2-driven glycolysis, oxidative phosphorylation and fatty acid oxidation), due to the unique presence of only the HK2 isoform, appears promising to treat HK1-HK2+ cancers. This therapeutic strategy will likely be tolerated by most normal tissues, where only HK1 is expressed.

BackgroundIn drug-drug interactions, there are surprising cases in which the growth inhibition of bacteria by a single antibiotic decreases when a second antibiotic is added. These interactions are termed suppressive and have been argued to have the potential to limit the evolution of resistance. Nevertheless, little attention has been given to suppressive interactions because clinical studies typically search for increases in killing efficiency and because suppressive interactions are believed to be rare based on pairwise studies.ResultsHere, we quantify the effects of single-, double-, and triple-drug combinations from a set of 14 antibiotics and 3 bacteria strains, totaling 364 unique three-drug combinations per bacteria strain. We find that increasing the number of drugs can increase the prevalence of suppressive interactions: 17% of three-drug combinations are suppressive compared to 5% of two-drug combinations in this study. Most cases of suppression we find (97%) are "hidden" cases for which the triple-drug bacterial growth is less than the single-drug treatments but exceeds that of a pairwise combination.ConclusionsWe find a surprising number of suppressive interactions in higher-order drug combinations. Without examining lower-order (pairwise) bacterial growth, emergent suppressive effects would be missed, potentially affecting our understanding of evolution of resistance and treatment strategies for resistant pathogens. These findings suggest that careful examination of the full factorial of drug combinations is needed to uncover suppressive interactions in higher-order combinations.

Microtubule targeting drugs like taxanes, vinca alkaloids, and epothilones are widely-used and effective chemotherapeutic agents that target the dynamic instability of microtubules and inhibit spindle functioning. However, these drugs have limitations associated with their production, solubility, efficacy and unwanted toxicities, thus driving the need to identify novel antimitotic drugs that can be used as anticancer agents. We have discovered and characterized the Microtubins (Microtubule inhibitors), a novel class of small synthetic compounds, which target tubulin to inhibit microtubule polymerization, arrest cancer cells predominantly in mitosis, activate the spindle assembly checkpoint and trigger an apoptotic cell death. Importantly, the Microtubins do not compete for the known vinca or colchicine binding sites. Additionally, through chemical synthesis and structure-activity relationship studies, we have determined that specific modifications to the Microtubin phenyl ring can activate or inhibit its bioactivity. Combined, these data define the Microtubins as a novel class of compounds that inhibit cancer cell proliferation by perturbing microtubule polymerization and they could be used to develop novel cancer therapeutics.

Microtubule targeting drugs like taxanes, vinca alkaloids, and epothilones are widely-used and effective chemotherapeutic agents that target the dynamic instability of microtubules and inhibit spindle functioning. However, these drugs have limitations associated with their production, solubility, efficacy and unwanted toxicities, thus driving the need to identify novel antimitotic drugs that can be used as anticancer agents. We have discovered and characterized the Microtubins (Microtubule inhibitors), a novel class of small synthetic compounds, which target tubulin to inhibit microtubule polymerization, arrest cancer cells predominantly in mitosis, activate the spindle assembly checkpoint and trigger an apoptotic cell death. Importantly, the Microtubins do not compete for the known vinca or colchicine binding sites. Additionally, through chemical synthesis and structure-activity relationship studies, we have determined that specific modifications to the Microtubin phenyl ring can activate or inhibit its bioactivity. Combined, these data define the Microtubins as a novel class of compounds that inhibit cancer cell proliferation by perturbing microtubule polymerization and they could be used to develop novel cancer therapeutics.

Discovery of first-in-class medicines for treating cancer is limited by concerns with their toxicity and safety profiles, while repurposing known drugs for new anticancer indications has become a viable alternative. Here, we have developed a new approach that utilizes cell cycle arresting patterns as unique molecular signatures for prioritizing FDA-approved drugs with repurposing potential. As proof-of-principle, we conducted large-scale cell cycle profiling of 884 FDA-approved drugs. Using cell cycle indexes that measure changes in cell cycle profile patterns upon chemical perturbation, we identified 36 compounds that inhibited cancer cell viability including 6 compounds that were previously undescribed. Further cell cycle fingerprint analysis and 3D chemical structural similarity clustering identified unexpected FDA-approved drugs that induced DNA damage, including clinically relevant microtubule destabilizers, which was confirmed experimentally via cell-based assays. Our study shows that computational cell cycle profiling can be used as an approach for prioritizing FDA-approved drugs with repurposing potential, which could aid the development of cancer therapeutics.

BackgroundIn drug-drug interactions, there are surprising cases in which the growth inhibition of bacteria by a single antibiotic decreases when a second antibiotic is added. These interactions are termed suppressive and have been argued to have the potential to limit the evolution of resistance. Nevertheless, little attention has been given to suppressive interactions because clinical studies typically search for increases in killing efficiency and because suppressive interactions are believed to be rare based on pairwise studies.ResultsHere, we quantify the effects of single-, double-, and triple-drug combinations from a set of 14 antibiotics and 3 bacteria strains, totaling 364 unique three-drug combinations per bacteria strain. We find that increasing the number of drugs can increase the prevalence of suppressive interactions: 17% of three-drug combinations are suppressive compared to 5% of two-drug combinations in this study. Most cases of suppression we find (97%) are "hidden" cases for which the triple-drug bacterial growth is less than the single-drug treatments but exceeds that of a pairwise combination.ConclusionsWe find a surprising number of suppressive interactions in higher-order drug combinations. Without examining lower-order (pairwise) bacterial growth, emergent suppressive effects would be missed, potentially affecting our understanding of evolution of resistance and treatment strategies for resistant pathogens. These findings suggest that careful examination of the full factorial of drug combinations is needed to uncover suppressive interactions in higher-order combinations.

Our ability to use ionizing radiation as an energy source, as a therapeutic agent, and, unfortunately, as a weapon, has evolved tremendously over the past 120 years, yet our tool box to handle the consequences of accidental and unwanted radiation exposure remains very limited. We have identified a novel group of small molecule compounds with a 4-nitrophenylsulfonamide (NPS) backbone in common that dramatically decrease mortality from the hematopoietic acute radiation syndrome (hARS). The group emerged from an in vitro high throughput screen (HTS) for inhibitors of radiation-induced apoptosis. The lead compound also mitigates against death after local abdominal irradiation and after local thoracic irradiation (LTI) in models of subacute radiation pneumonitis and late radiation fibrosis. Mitigation of hARS is through activation of radiation-induced CD11b+Ly6G+Ly6C+ immature myeloid cells. This is consistent with the notion that myeloerythroid-restricted progenitors protect against WBI-induced lethality and extends the possible involvement of the myeloid lineage in radiation effects. The lead compound was active if given to mice before or after WBI and had some anti-tumor action, suggesting that these compounds may find broader applications to cancer radiation therapy.

Our ability to use ionizing radiation as an energy source, as a therapeutic agent, and, unfortunately, as a weapon, has evolved tremendously over the past 120 years, yet our tool box to handle the consequences of accidental and unwanted radiation exposure remains very limited. We have identified a novel group of small molecule compounds with a 4-nitrophenylsulfonamide (NPS) backbone in common that dramatically decrease mortality from the hematopoietic acute radiation syndrome (hARS). The group emerged from an in vitro high throughput screen (HTS) for inhibitors of radiation-induced apoptosis. The lead compound also mitigates against death after local abdominal irradiation and after local thoracic irradiation (LTI) in models of subacute radiation pneumonitis and late radiation fibrosis. Mitigation of hARS is through activation of radiation-induced CD11b+Ly6G+Ly6C+ immature myeloid cells. This is consistent with the notion that myeloerythroid-restricted progenitors protect against WBI-induced lethality and extends the possible involvement of the myeloid lineage in radiation effects. The lead compound was active if given to mice before or after WBI and had some anti-tumor action, suggesting that these compounds may find broader applications to cancer radiation therapy.

Target identification remains a major challenge for modern drug discovery programs aimed at understanding the molecular mechanisms of drugs. Computational target prediction approaches like 2D chemical similarity searches have been widely used but are limited to structures sharing high chemical similarity. Here, we present a new computational approach called chemical similarity network analysis pull-down 3D (CSNAP3D) that combines 3D chemical similarity metrics and network algorithms for structure-based drug target profiling, ligand deorphanization, and automated identification of scaffold hopping compounds. In conjunction with 2D chemical similarity fingerprints, CSNAP3D achieved a >95% success rate in correctly predicting the drug targets of 206 known drugs. Significant improvement in target prediction was observed for HIV reverse transcriptase (HIVRT) compounds, which consist of diverse scaffold hopping compounds targeting the nucleotidyltransferase binding site. CSNAP3D was further applied to a set of antimitotic compounds identified in a cell-based chemical screen and identified novel small molecules that share a pharmacophore with Taxol and display a Taxol-like mechanism of action, which were validated experimentally using in vitro microtubule polymerization assays and cell-based assays.

Antiviral drugs do not currently exist for the treatment of enterovirus infections, which are often severe and potentially life-threatening. We conducted high-throughput molecular screening and identified a structurally diverse set of compounds that inhibit the replication of coxsackievirus B3, a commonly encountered enterovirus. These compounds did not interfere with the function of the viral internal ribosome entry site or with the activity of the viral proteases, but they did drastically reduce the synthesis of viral RNA and viral proteins in infected cells. Sequence analysis of compound-resistant mutants suggests that the viral 2C protein is targeted by most of these compounds. These compounds demonstrated antiviral activity against a panel of the most commonly encountered enteroviruses and thus represent potential leads for the development of broad-spectrum anti-enteroviral drugs.

Antiviral drugs do not currently exist for the treatment of enterovirus infections, which are often severe and potentially life-threatening. We conducted high-throughput molecular screening and identified a structurally diverse set of compounds that inhibit the replication of coxsackievirus B3, a commonly encountered enterovirus. These compounds did not interfere with the function of the viral internal ribosome entry site or with the activity of the viral proteases, but they did drastically reduce the synthesis of viral RNA and viral proteins in infected cells. Sequence analysis of compound-resistant mutants suggests that the viral 2C protein is targeted by most of these compounds. These compounds demonstrated antiviral activity against a panel of the most commonly encountered enteroviruses and thus represent potential leads for the development of broad-spectrum anti-enteroviral drugs.

Glucocorticoids are commonly used as palliative or chemotherapeutic clinical agents for treatment of a variety of cancers. Although steroid treatment is beneficial, the mechanisms by which steroids improve outcome in cancer patients are not well understood. Na,K-ATPase beta-subunit isoform 1 (NaK-β1) is a cell-cell adhesion molecule, and its expression is down-regulated in cancer cells undergoing epithelial-to mesenchymal-transition (EMT), a key event associated with cancer progression to metastatic disease. In this study, we performed high-throughput screening to identify small molecules that could up-regulate NaK-β1 expression in cancer cells. Compounds related to the glucocorticoids were identified as drug candidates enhancing NaK-β1 expression. Of these compounds, triamcinolone, dexamethasone, and fluorometholone were validated to increase NaK-β1 expression at the cell surface, enhance cell-cell adhesion, attenuate motility and invasiveness and induce mesenchymal to epithelial like transition of renal cell carcinoma (RCC) cells in vitro. Treatment of NaK-β1 knockdown cells with these drug candidates confirmed that these compounds mediate their effects through up-regulating NaK-β1. Furthermore, we demonstrated that these compounds attenuate tumor growth in subcutaneous RCC xenografts and reduce local invasiveness in orthotopically-implanted tumors. Our results strongly indicate that the addition of glucocorticoids in the treatment of RCC may improve outcome for RCC patients by augmenting NaK-β1 cell-cell adhesion function.

Constitutively active BCR-ABL kinase fusions are causative mutations in the pathogenesis of hematopoietic neoplasias including chronic myelogenous leukemia (CML). Although these fusions have been successfully targeted with kinase inhibitors, drug-resistance and relapse continue to limit long-term survival, highlighting the need for continued innovative drug discovery. We developed a time-resolved Förster resonance energy transfer (TR-FRET) -based assay to identify compounds that disrupt stimulation of the ABL kinase by blocking its ability to bind the positive regulator RIN1. This assay was used in a high throughput screen (HTS) of two small molecule libraries totaling 444,743 compounds. 708 confirmed hits were counter-screened to eliminate off-target inhibitors and reanalyzed to prioritize compounds with IC50 values below 10 μM. The CML cell line K562 was then used to identify five compounds that decrease MAPK1/3 phosphorylation, which we determined to be an indicator of RIN1-dependent ABL signaling. One of these compounds is a thiadiazole, and the other four are structurally related acyl piperidine amides. Notably, these five compounds lower cellular BCR-ABL1 kinase activity by blocking a positive regulatory interaction rather than directly inhibiting ABL catalytic function.

Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60-70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/).

Constitutively active BCR-ABL kinase fusions are causative mutations in the pathogenesis of hematopoietic neoplasias including chronic myelogenous leukemia (CML). Although these fusions have been successfully targeted with kinase inhibitors, drug-resistance and relapse continue to limit long-term survival, highlighting the need for continued innovative drug discovery. We developed a time-resolved Förster resonance energy transfer (TR-FRET) -based assay to identify compounds that disrupt stimulation of the ABL kinase by blocking its ability to bind the positive regulator RIN1. This assay was used in a high throughput screen (HTS) of two small molecule libraries totaling 444,743 compounds. 708 confirmed hits were counter-screened to eliminate off-target inhibitors and reanalyzed to prioritize compounds with IC50 values below 10 μM. The CML cell line K562 was then used to identify five compounds that decrease MAPK1/3 phosphorylation, which we determined to be an indicator of RIN1-dependent ABL signaling. One of these compounds is a thiadiazole, and the other four are structurally related acyl piperidine amides. Notably, these five compounds lower cellular BCR-ABL1 kinase activity by blocking a positive regulatory interaction rather than directly inhibiting ABL catalytic function.

Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.

Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.