Systematic discovery of mutation-directed neo-protein-protein interactions in cancer.

Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

Discovery of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer's disease.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer's disease (AD) traits and microglia. These proteins are more abundant in Alzheimer's patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) and the cytoplasmic tail of CD44. Inhibiting the MSN-CD44 interaction may help limit AD-associated neuronal damage. Here, we investigated the feasibility of developing inhibitors that target this protein-protein interaction. We have employed structural, mutational, and phage-display studies to examine how CD44 binds to the FERM domain of MSN. Interestingly, we have identified an allosteric site located close to the PIP2 binding pocket that influences CD44 binding. These findings suggest a mechanism in which PIP2 binding to the FERM domain stimulates CD44 binding through an allosteric effect, leading to the formation of a neighboring pocket capable of accommodating a receptor tail. Furthermore, high-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction. One compound series was further optimized for biochemical activity, specificity, and solubility. Our results suggest that the FERM domain holds potential as a drug development target. Small molecule preliminary leads generated from this study could serve as a foundation for additional medicinal chemistry efforts with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

Nicardipine is a putative EED inhibitor and has high selectivity and potency against chemoresistant prostate cancer in preclinical models.

BACKGROUND: It is imperative to develop novel therapeutics to overcome chemoresistance, a significant obstacle in the clinical management of prostate cancer (PCa) and other cancers. METHODS: A phenotypic screen was performed to identify novel inhibitors of chemoresistant PCa cells. The mechanism of action of potential candidate(s) was investigated using in silico docking, and molecular and cellular assays in chemoresistant PCa cells. The in vivo efficacy was evaluated in mouse xenograft models of chemoresistant PCa. RESULTS: Nicardipine exhibited high selectivity and potency against chemoresistant PCa cells via inducing apoptosis and cell cycle arrest. Computational, molecular, and cellular studies identified nicardipine as a putative inhibitor of embryonic ectoderm development (EED) protein, and the results are consistent with a proposed mechanism of action that nicardipine destabilised enhancer of zeste homologue 2 (EZH2) and inhibited key components of noncanonical EZH2 signalling, including transducer and activator of transcription 3, S-phase kinase-associated protein 2, ATP binding cassette B1, and survivin. As a monotherapy, nicardipine effectively inhibited the skeletal growth of chemoresistant C4-2B-TaxR tumours. As a combination regimen, nicardipine synergistically enhanced the in vivo efficacy of docetaxel against C4-2 xenografts. CONCLUSION: Our findings provided the first preclinical evidence supporting nicardipine as a novel EED inhibitor that has the potential to be promptly tested in PCa patients to overcome chemoresistance and improve clinical outcomes.

Tumor microenvironment in a minipig model of spinal cord glioma.

BACKGROUND: Spinal cord glioma (SCG) is considered an orphan disease that lacks effective treatment options with margins that are surgically inaccessible and an overall paucity of literature on the topic. The tumor microenvironment is a critical factor to consider in treatment and modeling design, especially with respect to the unresectable tumor edge. Recently, our group developed a high-grade spinal cord glioma (SCG) model in Göttingen minipigs. METHODS: Immunofluorescence and ELISA were performed to explore the microenvironmental features and inflammation cytokines in this minipig SCG model. Protein carbonyl assay and GSH/GSSG assay were analyzed in the core and edge lesions in the minipig SCG model. The primary core and edge cells proliferation rate were shown in vitro, and the xenograft model in vivo. RESULTS: We identified an elevated Ki-67 proliferative index, vascular and pericyte markers, CD31 and desmin in the tumor edge as compared to the tumor core. In addition, we found that the tumor edge demonstrated increased pro-inflammatory and gliomagenic cytokines including TNF-α, IL-1ß, and IL-6. Furthermore, the mediation of oxidative stress is upregulated in the tumor edge. Hypoxic markers had statistically significant increased staining in the tumor core, but were notably still present in the tumor edge. The edge cells cultures derived from SCG biopsy also demonstrated an increased proliferative rate compared to core cell cultures in a xenotransplantation model. CONCLUSIONS: Our study demonstrates heterogeneity in microenvironmental features in our minipig model of high-grade SCG, with a phenotype at the edge showing increased oxidative stress, proliferation, inflammatory cytokines, neovascularization, and decreased but present staining for hypoxic markers. These findings support the utility of this model as a means for investigating therapeutic approaches targeting the more aggressive and surgically unresectable tumor border.

Constitutively Synergistic Multiagent Drug Formulations Targeting MERTK, FLT3, and BCL-2 for Treatment of AML.

PURPOSE: Although high-dose, multiagent chemotherapy has improved leukemia survival rates, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. The development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. METHODS: The dual MERTK/FLT3 inhibitor MRX-2843 and BCL-2 family protein inhibitors were screened in high-throughput against a panel of AML and MLL-rearranged precursor B-cell ALL (infant ALL) cell lines. A neural network model was built to correlate ratiometric drug synergy and target gene expression. Drugs were loaded into liposomal nanocarriers to assess primary AML cell responses. RESULTS: MRX-2843 synergized with venetoclax to reduce AML cell density in vitro. A neural network classifier based on drug exposure and target gene expression predicted drug synergy and growth inhibition in AML with high accuracy. Combination monovalent liposomal drug formulations delivered defined drug ratios intracellularly and recapitulated synergistic drug activity. The magnitude and frequency of synergistic responses were both maintained and improved following drug formulation in a genotypically diverse set of primary AML bone marrow specimens. CONCLUSIONS: We developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and infant ALL cells. We demonstrate ratiometric drug delivery and synergistic cell killing in AML, a result achieved by a systematic, generalizable approach of combination drug screening and nanoscale formulation that may be extended to other drug pairs or diseases in the future.

Stress induces p38 MAPK-mediated phosphorylation and inhibition of Drosha-dependent cell survival.

MicroRNAs (miRNAs) regulate the translational potential of their mRNA targets and control many cellular processes. The key step in canonical miRNA biogenesis is the cleavage of the primary transcripts by the nuclear RNase III enzyme Drosha. Emerging evidence suggests that the miRNA biogenic cascade is tightly controlled. However, little is known whether Drosha is regulated. Here, we show that Drosha is targeted by stress. Under stress, p38 MAPK directly phosphorylates Drosha at its N terminus. This reduces its interaction with DiGeorge syndrome critical region gene 8 and promotes its nuclear export and degradation by calpain. This regulatory mechanism mediates stress-induced inhibition of Drosha function. Reduction of Drosha sensitizes cells to stress and increases death. In contrast, increase in Drosha attenuates stress-induced death. These findings reveal a critical regulatory mechanism by which stress engages p38 MAPK pathway to destabilize Drosha and inhibit Drosha-mediated cellular survival.

Acquisition of taxane resistance by p53 inactivation in ovarian cancer cells.

Ovarian cancer is one of the most common gynecologic malignancies in women and has a poor prognosis. Taxanes are a class of standard first-line chemotherapeutic agents for the treatment of ovarian cancer. However, tumor-intrinsic and acquired resistance to taxanes poses major challenges to improving clinical outcomes. Hence, there is an urgent clinical need to understand the mechanisms of resistance in order to discover potential biomarkers and therapeutic strategies to increase taxane sensitivity in ovarian cancer. Here, we report the identification of an association between the TP53 status and taxane sensitivity in ovarian cancer cells through complementary experimental and informatics approaches. We found that TP53 inactivation is associated with taxane resistance in ovarian cancer cells, supported by the evidence from (i) drug sensitivity profiling with bioinformatic analysis of large-scale cancer therapeutic response and genomic datasets and (ii) gene signature identification based on experimental isogenic cell line models. Further, our studies revealed TP53-dependent gene expression patterns, such as overexpression of ACSM3, as potential predictive biomarkers of taxane resistance in ovarian cancer. The TP53-dependent hyperactivation of the WNT/ß-catenin pathway discovered herein revealed a potential vulnerability to exploit in developing combination therapeutic strategies. Identification of this genotype-phenotype relationship between the TP53 status and taxane sensitivity sheds light on TP53-directed patient stratification and therapeutic discoveries for ovarian cancer treatment.

Discovery of the first chemical tools to regulate MKK3-mediated MYC activation in cancer.

The transcription master regulator MYC plays an essential role in regulating major cellular programs and is a well-established therapeutic target in cancer. However, MYC targeting for drug discovery is challenging. New therapeutic approaches to control MYC-dependent malignancy are urgently needed. The mitogen-activated protein kinase kinase 3 (MKK3) binds and activates MYC in different cell types, and disruption of MKK3-MYC protein-protein interaction may provide a new strategy to target MYC-driven programs. However, there is no perturbagen available to interrogate and control this signaling arm. In this study, we assessed the drugability of the MKK3-MYC complex and discovered the first chemical tool to regulate MKK3-mediated MYC activation. We have designed a short 44-residue inhibitory peptide and developed a cell lysate-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay to discover the first small molecule MKK3-MYC PPI inhibitor. We have optimized and miniaturized the assay into an ultra-high-throughput screening (uHTS) 1536-well plate format. The pilot screen of ~6,000 compounds of a bioactive chemical library followed by multiple secondary and orthogonal assays revealed a quinoline derivative SGI-1027 as a potent inhibitor of MKK3-MYC PPI. We have shown that SGI-1027 disrupts the MKK3-MYC complex in cells and in vitro and inhibits MYC transcriptional activity in colon and breast cancer cells. In contrast, SGI-1027 does not inhibit MKK3 kinase activity and does not interfere with well-known MKK3-p38 and MYC-MAX complexes. Together, our studies demonstrate the drugability of MKK3-MYC PPI, provide the first chemical tool to interrogate its biological functions, and establish a new uHTS assay to enable future discovery of potent and selective inhibitors to regulate this oncogenic complex.

High expression of MKK3 is associated with worse clinical outcomes in African American breast cancer patients.

BACKGROUND: African American women experience a twofold higher incidence of triple-negative breast cancer (TNBC) and are 40% more likely to die from breast cancer than women of other ethnicities. However, the molecular bases for the survival disparity in breast cancer remain unclear, and no race-specific therapeutic targets have been proposed. To address this knowledge gap, we performed a systematic analysis of the relationship between gene mRNA expression and clinical outcomes determined for The Cancer Genome Atlas (TCGA) breast cancer patient cohort. METHODS: The systematic differential analysis of mRNA expression integrated with the analysis of clinical outcomes was performed for 1055 samples from the breast invasive carcinoma TCGA PanCancer cohorts. A deep learning fully-convolutional model was used to determine the association between gene expression and tumor features based on breast cancer patient histopathological images. RESULTS: We found that more than 30% of all protein-coding genes are differentially expressed in White and African American breast cancer patients. We have determined a set of 32 genes whose overexpression in African American patients strongly correlates with decreased survival of African American but not White breast cancer patients. Among those genes, the overexpression of mitogen-activated protein kinase kinase 3 (MKK3) has one of the most dramatic and race-specific negative impacts on the survival of African American patients, specifically with triple-negative breast cancer. We found that MKK3 can promote the TNBC tumorigenesis in African American patients in part by activating of the epithelial-to-mesenchymal transition induced by master regulator MYC. CONCLUSIONS: The poor clinical outcomes in African American women with breast cancer can be associated with the abnormal elevation of individual gene expression. Such genes, including those identified and prioritized in this study, could represent new targets for therapeutic intervention. A strong correlation between MKK3 overexpression, activation of its binding partner and major oncogene MYC, and worsened clinical outcomes suggests the MKK3-MYC protein-protein interaction as a new promising target to reduce racial disparity in breast cancer survival.

The OncoPPi Portal: an integrative resource to explore and prioritize protein-protein interactions for cancer target discovery.

Motivation: As cancer genomics initiatives move toward comprehensive identification of genetic alterations in cancer, attention is now turning to understanding how interactions among these genes lead to the acquisition of tumor hallmarks. Emerging pharmacological and clinical data suggest a highly promising role of cancer-specific protein-protein interactions (PPIs) as druggable cancer targets. However, large-scale experimental identification of cancer-related PPIs remains challenging, and currently available resources to explore oncogenic PPI networks are limited. Results: Recently, we have developed a PPI high-throughput screening platform to detect PPIs between cancer-associated proteins in the context of cancer cells. Here, we present the OncoPPi Portal, an interactive web resource that allows investigators to access, manipulate and interpret a high-quality cancer-focused network of PPIs experimentally detected in cancer cell lines. To facilitate prioritization of PPIs for further biological studies, this resource combines network connectivity analysis, mutual exclusivity analysis of genomic alterations, cellular co-localization of interacting proteins and domain-domain interactions. Estimates of PPI essentiality allow users to evaluate the functional impact of PPI disruption on cancer cell proliferation. Furthermore, connecting the OncoPPi network with the approved drugs and compounds in clinical trials enables discovery of new tumor dependencies to inform strategies to interrogate undruggable targets like tumor suppressors. The OncoPPi Portal serves as a resource for the cancer research community to facilitate discovery of cancer targets and therapeutic development. Availability and implementation: The OncoPPi Portal is available at http://oncoppi.emory.edu. Contact: andrey.ivanov@emory.edu or hfu@emory.edu.

AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay.

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. In this study, we report the development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells. The NanoPCA system was configured to enable detection of protein-protein interactions (PPI) at the endogenous level, as shown with PRAS40 dimerization, and detection of weak interactions, such as PINCH1-NCK2. Importantly, NanoPCA allows the study of PPI dynamics with reversible interactions. To demonstrate its utility for large-scale PPI detection in mammalian intracellular environment, we have used NanoPCA to examine MYC interaction with 83 cancer-associated proteins in live cancer cell lines. Our new MYC PPI data confirmed known MYC-interacting proteins, such as MAX, GSK3A, and SMARCA4, and revealed a panel of novel MYC interaction partners, such as RAC-α serine/threonine-protein kinase (AKT)1, liver kinase B (LKB)1, and Yes-associated protein (YAP)1. The MYC interactions with AKT1, LKB1, and YAP1 were confirmed by coimmunoprecipitation of endogenous proteins. Importantly, AKT1, LKB1, and YAP1 were able to activate MYC in a transcriptional reporter assay. Thus, these vital growth control proteins may represent promising MYC regulators, suggesting new mechanisms that couple energetic and metabolic pathways and developmental signaling to MYC-regulated cellular programs.

Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis.

TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.

Downregulation of urea transporter UT-A1 activity by 14-3-3 protein.

Urea transporter (UT)-A1 in the kidney inner medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the kidney inner medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione-S-transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.

Chromosome instability in diffuse large B cell lymphomas is suppressed by activation of the noncanonical NF-κB pathway.

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in the United States. DLBCL comprises biologically distinct subtypes including germinal center-like (GCB) and activated-B-cell-like DLBCL (ABC). The most aggressive type, ABC-DLBCL, displays dysregulation of both canonical and noncanonical NF-κB pathway as well as genomic instability. Although, much is known about the tumorigenic roles of the canonical NF-kB pathway, the precise role of the noncanonical NF-kB pathway remains unknown. Here we show that activation of the noncanonical NF-κB pathway regulates chromosome stability, DNA damage response and centrosome duplication in DLBCL. Analysis of 92 DLBCL samples revealed that activation of the noncanonical NF-κB pathway is associated with low levels of DNA damage and centrosome amplification. Inhibiting the noncanonical pathway in lymphoma cells uncovered baseline DNA damage and prevented doxorubicin-induced DNA damage repair. In addition, it triggered centrosome amplification and chromosome instability, indicated by anaphase bridges, multipolar spindles and chromosome missegregation. We determined that the noncanonical NF-κB pathway execute these functions through the regulation of GADD45α and REDD1 in a p53-independent manner, while it collaborates with p53 to regulate cyclin G2 expression. Furthermore, this pathway regulates GADD45α, REDD1 and cyclin G2 through direct binding of NF-κB sites to their promoter region. Overall, these results indicate that the noncanonical NF-κB pathway plays a central role in maintaining genome integrity in DLBCL. Our data suggests that inhibition of the noncanonical NF-kB pathway should be considered as an important component in DLBCL therapeutic approach.

Small molecule antagonist reveals seizure-induced mediation of neuronal injury by prostaglandin E2 receptor subtype EP2.

With interest waning in the use of cyclooxygenase-2 (COX-2) inhibitors for inflammatory disease, prostaglandin receptors provide alternative targets for the treatment of COX-2-mediated pathological conditions in both the periphery and the central nervous system. Activation of prostaglandin E2 receptor (PGE(2)) subtype EP2 promotes inflammation and is just beginning to be explored as a therapeutic target. To better understand physiological and pathological functions of the prostaglandin EP2 receptor, we developed a suite of small molecules with a 3-aryl-acrylamide scaffold as selective EP2 antagonists. The 12 most potent compounds displayed competitive antagonism of the human EP2 receptor with K(B) 2-20 nM in Schild regression analysis and 268- to 4,730-fold selectivity over the prostaglandin EP4 receptor. A brain-permeant compound completely suppressed the up-regulation of COX-2 mRNA in rat cultured microglia by EP2 activation and significantly reduced neuronal injury in hippocampus when administered in mice beginning 1 h after termination of pilocarpine-induced status epilepticus. The salutary actions of this novel group of antagonists raise the possibility that selective block of EP2 signaling via small molecules can be an innovative therapeutic strategy for inflammation-related brain injury.

Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor.

The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kD(a) and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3ζ in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer.

Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F.

Deregulation of cap-dependent translation is associated with cancer initiation and progression. The rate-limiting step of protein synthesis is the loading of ribosomes onto mRNA templates stimulated by the heterotrimeric complex, eukaryotic initiation factor (eIF)4F. This step represents an attractive target for anticancer drug discovery because it resides at the nexus of the TOR signaling pathway. We have undertaken an ultra-high-throughput screen to identify inhibitors that prevent assembly of the eIF4F complex. One of the identified compounds blocks interaction between two subunits of eIF4F. As a consequence, cap-dependent translation is inhibited. This compound can reverse tumor chemoresistance in a genetically engineered lymphoma mouse model by sensitizing cells to the proapoptotic action of DNA damage. Molecular modeling experiments provide insight into the mechanism of action of this small molecule inhibitor. Our experiments validate targeting the eIF4F complex as a strategy for cancer therapy to modulate chemosensitivity.

Composition, characteristics, and treatment technologies of condensable particulate matter present in flue gas emitted by coking plants in China.

To study the total particulate matter (TPM) in flue gas emitted by coking plants, a sampling system that could be used to collect filterable particulate matter (FPM) and condensable particulate matter (CPM) was designed and developed based on Method 202 recommended by the U.S. Environmental Protection Agency in 2017 and HJ 836-2017 issued by China. Using this system, FPM and CPM in flue gas emitted by four coking furnaces named A, B, C, and D were tested in China. Further, 9 water-soluble ions, 20 elements, and organic matter present in the CPM were simultaneously examined to determine their formation mechanisms. Statistical data suggested that the FPM emission level in the coking flue gas was low and the average mass concentration was less than 10 mg/m3. However, with high CPM and TPM emission levels, the TPM mass concentrations of A, B, C, and D were 130 ± 11.1, 84.4 ± 6.36, 35.1 ± 17.0, and 63.8 ± 13.0 mg/m3, respectively. The main component of TPM was CPM, and the average mass concentration of CPM accounted for 98%, 95%, 68%, and 95% of TPM in furnaces A, B, C, and D, respectively. Water-soluble ions were the important components of CPM, and the total concentration of water-soluble ions accounted for 70%, 87%, 42%, and 66% of CPM in furnaces A, B, C, and D, respectively. Toxic and harmful heavy metals, such as Mn, Cr, Ni, Cu, Zn, As, Cd, and Pb, were detected in CPM. The formation mechanism of CPM was analyzed in combination with flue-gas treatment. It was shown that the treatment process "activated carbon- flue-gas countercurrent-integrated purification technology + ammonia spraying" used in furnaces A and B was less effective in removing CPM, water-soluble ions, metals, and compounds than that of "selective catalytic reduction denitrification + limestone-gypsum wet desulfurization (spraying NaOH solution)" in furnaces C and D. Hence, different flue-gas treatment technologies and operation levels played vital roles in the formation, transformation, and removal of CPM from flue-gas. Organic components in CPM discharged from furnace A were determined via gas chromatography-mass spectrometry, and the top 15 organic components in CPM were obtained using the area normalization method. N-alkanes accounted for the highest proportion, followed by esters and phenols, and most of them were toxic and harmful to humans and ecosystems. Therefore, advanced CPM treatment technologies should be developed to reduce atmospheric PM2.5 and its precursors to improve ambient air quality in China.

Development of a Time-Resolved Fluorescence Resonance Energy Transfer ultra-high throughput screening assay targeting SYK and FCER1G interaction.

The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as a TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as an acceptor. We have optimized the assay into a 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z' factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein-protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.

Development of a Time-Resolved Fluorescence Resonance Energy Transfer ultra-high throughput screening assay for targeting SYK and FCER1G interaction.

The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as acceptor. We have optimized the assay into 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z' factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein-protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.