Orthogonal Screening Research Articles

Identification of ATP-Competitive Human CMG Helicase Inhibitors for Cancer Intervention that Disrupt CMG-Replisome Function.

The human CMG helicase (Cdc45-MCM-GINS) is a novel target for anti-cancer therapy. Tumor-specific weaknesses in the CMG are caused by oncogene-driven changes that adversely affect CMG function, and a requirement for CMG activity during recovery from replicative stresses such as chemotherapy. Here, we developed an orthogonal biochemical screening approach and identified CMG inhibitors (CMGi) that inhibit ATPase and helicase activities in an ATP-competitive manner at low micromolar concentrations. Structure-activity information, in silico docking, and testing with synthetic chemical compounds indicate that CMGi require specific chemical elements and occupy ATP binding sites and channels within MCM subunits leading to the ATP clefts, which are likely used for ATP/ADP ingress or egress. CMGi are therefore also MCM complex inhibitors (MCMi). Biological testing shows that CMGi/MCMi inhibit cell growth and DNA replication using multiple molecular mechanisms distinct from other chemotherapy agents. CMGi/MCMi block helicase assembly steps that require ATP binding/hydrolysis by the MCM complex, specifically MCM ring assembly on DNA and GINS recruitment to DNA-loaded MCM hexamers. During S-phase, inhibition of MCM ATP binding/hydrolysis by CMGi/MCMi causes a 'reverse allosteric' dissociation of Cdc45/GINS from the CMG that destabilizes replisome components Ctf4, Mcm10, and DNA polymerase-a, -d, -e, resulting in DNA damage. CMGi/MCMi display selective toxicity toward multiple solid tumor cell types with K-Ras mutations, targeting the CMG and inducing DNA damage, Parp cleavage, and loss of viability. This new class of CMGi/MCMi provides a basis for small chemical development of CMG helicase-targeted anti-cancer compounds with distinct mechanisms of action.

Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting OSBP.

Metabolic alterations in cancer precipitate in associated dependencies that can be therapeutically exploited. To meet this goal, natural product-inspired small molecules can provide a resource of invaluable chemotypes. Here, we identify orpinolide, a synthetic withanolide analog with pronounced antileukemic properties, via orthogonal chemical screening. Through multiomics profiling and genome-scale CRISPR-Cas9 screens, we identify that orpinolide disrupts Golgi homeostasis via a mechanism that requires active phosphatidylinositol 4-phosphate signaling at the endoplasmic reticulum-Golgi membrane interface. Thermal proteome profiling and genetic validation studies reveal the oxysterol-binding protein OSBP as the direct and phenotypically relevant target of orpinolide. Collectively, these data reaffirm sterol transport as a therapeutically actionable dependency in leukemia and motivate ensuing translational investigation via the probe-like compound orpinolide.

A machine learning algorithm based on multi-omics biomarkers for the detection of tumor microsatellite instability.

1554 Background: Microsatellite instability (MSI) occurs via defects in mismatch repair (MMR) and is characterized by high tumor mutational burden (TMB) and a robust inflammatory response. Accurate MSI-high classification of immune-evasive tumors is vital due to the effectiveness of immune checkpoint inhibitor (ICI) therapy, such as pembrolizumab, which is FDA approved for all MSI-high tumors irrespective of anatomic origin. Detection methods of MSI-status vary widely, including immunohistochemistry (IHC), polymerase chain reaction (PCR), and next-generation sequencing (NGS). We developed a machine learning (ML) model to predict MSI status in patients with solid tumors using comprehensive genomic and immune profiling (CGIP), independent of direct sequencing data from microsatellite sites. Methods: We analyzed samples from 1,838 patients with colorectal cancer (CRC) by CGIP, which included DNA panel testing (523 genes) for pathogenic single nucleotide variants (pSNV) and determination of TMB (mut/Mb), and RNA sequencing (397 genes) for gene expression (GEX). The Boruta algorithm was used to select key genomic and GEX changes associated with MSI status. A distributed gradient boosting algorithm created a predictive model that was trained on 70% of the cohort. The model was tested on the remaining CRC cohort (Test), and in the PanCancer Atlas CRCs (TCGA), endometrial adenocarcinomas (EMCA), and CRC with indeterminate MSI testing by CGIP. Performance of the trained model was assessed using sensitivity, specificity, and positive (PPV) and negative (NPV) predictive value. Results: Feature selection identified 79 genes with pSNVs, 63 GEX changes, and TMB as informative for MSI prediction. The model showed high predictive accuracy in differentiating MSI-H tumors. Of the 39 cases that failed MSI component of CGIP (Indeterminate), 17 had MMR IHC results available, of which 1 demonstrated loss of MLH1/PMS2 and the other 16 showed intact (normal) expression. Testing of this cohort identified 3 cases as MSI-high, at least one of which was confirmed by IHC. No cases with intact MMR protein IHC were identified as MSI-high by the algorithm. Conclusions: Our ML-driven approach accurately assessed MSI status of CRC and EMCA using CGIP data. This approach also identified potential cases with MSI-high status where direct sequencing of microsatellites failed. This study highlights a method to identify patients with potential MSI-high status for orthogonal screening with the MSI component of a test fails. [Table: see text]

Targeted protein degradation via intramolecular bivalent glues

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)—bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1–4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change ‘glues’ BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target–ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4–IBG1–DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

Lean-and-Green Datacentric Engineering in Laser Cutting: Non-Linear Orthogonal Multivariate Screening Using Gibbs Sampling and Pareto Frontier

Metal processing may benefit from innovative lean-and-green datacentric engineering techniques. Broad process improvement opportunities in the efficient usage of materials and energy are anticipated (United Nations Sustainable Development Goals #9, 12). A CO2 laser cutting method is investigated in this study in terms of product characteristics (surface roughness (SR)) and process characteristics (energy (EC) and gas consumption (GC) as well as cutting time (CT)). The examined laser cutter controlling factors were as follows: (1) the laser power (LP), (2) the cutting speed (CS), (3) the gas pressure (GP) and, (4) the laser focus length (F). The selected 10mm-thick carbon steel (EN10025 St37-2) workpiece was arranged to have various geometric configurations so as to simulate a variety of real industrial milling demands. Non-linear saturated screening/optimization trials were planned using the Taguchi-type L9(34) orthogonal array. The resulting multivariate dataset was treated using a combination of the Gibbs sampler and the Pareto frontier method in order to approximate the strength of the studied effects and to find a solution that comprises the minimization of all the tested process/product characteristics. The Pareto frontier optimal solution was (EC, GC, CT, SR) = (4.67 kWh, 20.35 Nm3, 21 s, 5.992 μm) for the synchronous screening/optimization of the four characteristics. The respective factorial settings were optimally adjusted at the four inputs (LP, CS, GP, F) located at (4 kW, 1.9 mm/min, 0.75 bar, +2.25 mm). The linear regression analysis was aided by the Gibbs sampler and promoted the laser power and the cutting speed on energy consumption to be stronger effects. Similarly, a strong effect was identified of the cutting speed and the gas pressure on gas consumption as well as a reciprocal effect of the cutting speed on the cutting time. Further industrial explorations may involve more intricate workpiece geometries, burr formation phenomena, and process economics.

5-adenosine monophosphate promotes signal adaptation in PKA signaling.

Protein Kinase A (PKA) plays a central role in several cellular processes and its activity is tightly regulated by 2nd messenger 3,5-cyclic adenosine monophosphate (cAMP)-signaling. While the role of cAMP in PKA activation is well established, the significance of its hydrolysis end-product, 5-adenosine monophosphate (AMP) is poorly understood. AMP is generated upon hydrolysis of cAMP bound to the PKA regulatory subunits (RIα) catalyzed by phosphodiesterases. All components of the cAMP-PKA signaling pathway that include PDEs are compartmentalized into signalosomal units. Our research has shown that PDEs in proximity with PKA RIα in signalosomes lead to formation of a signal termination complex of PDEs and RIα, that facilitates cAMP substrate channeling from the PKA binding site to the PDE catalytic site. In contrast to the long held view that AMP diffused away from the PKA signalosome following cAMP hydrolysis, AMP is a regulatory end-product of each round of PKA activation and termination. Here we report high resolution structures of the AMP bound conformations of RIα and PDE8A catalytic domain (PDE8c) by cryo-EM. We have further captured AMP-bound end-state signal termination complex of PDE8c with RIα, by orthogonal electron microscopy screening, amide hydrogen/deuterium exchange mass spectrometry. Our results describe an undiscovered active role of AMP in signal termination and adaptation in PKA signaling.

Identification of cell type specific ACE2 modifiers by CRISPR screening.

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. In liver-derived HuH7 cells, we identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual HuH7 cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Orthogonal screening of lung-derived Calu-3 cells revealed a distinct set of ACE2 modifiers comprised of ACE2, KDM6A, MOGS, GPAA1, and UGP2. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry, highlight the cell type specificity of ACE2 regulatory networks, and suggest potential targets for therapeutic development.

Synthesis and Evaluation of CQC-1—An Oil Field Scale Inhibitor

Most of the oil wells enter the middle and late stages of extraction. After one oil extraction, water injection is needed for secondary extraction. With a large amount of calcium and magnesium ions in the water, which is easy to form a large amount of insoluble scale, its formed barium sulfate and strontium sulfate scale are more difficult to handle than carbonate scale. In order to reduce the difficulty of oil production work, this paper mainly targeted the sulfate scale for experiments, and prepared the scale retardant agent CQC-1. Through orthogonal experimental screening, the mass ratio of itaconate acid:methacrylic acid:AMPS:sodium acrylic sulfonate was 2:1:1:1, increase the amount of evocating agent was 8% and the reaction time was 4h, the sulfate scale inhibitor synthesized at the temperature of 80°C had the best performance. Using scale resistance rate, degradation and salt-resistance, the experimental results showed that the scale resistance rate of CQC-1 reached 90.46%. In the four-week degradation experiment, the degradation rate reached over 71% and was maintained by 80.65% at 150°C, while the scale resistance rate still reached 80.35% in simulated saline. It has excellent scale resistance, temperature resistance, salt-resistance and degradation, and has good development prospects.

OTME-20. Chitinase-3-like-1(CHI3L1) Protein Complexes Regulate the immunosuppressive Microenvironment in Glioblastoma

Glioblastoma (GBM) is the most common and highly malignant brain tumor in adults. Despite advances in multimodal treatment, GBM remains largely incurable. While immunotherapies have been highly effective in some types of cancer, the disappointing results from clinical trials for GBM immunotherapy represent continued challenges. GBM is highly immunosuppressive and resistant to immunotherapy because of glioma cells escaping from immune surveillance by reprograming the tumor microenvironment (TME). However, understanding the mechanisms of immune evasion by GBM remains elusive. Based on unbiased approaches, we found that Chitinase-3-like-1 (CHI3L1), also known as human homolog YKL-40, is highly expressed in GBM, which is regulated by the CHI3L1-PI3K/AKT/mTOR signaling in a positive feedback loop. Gain- and loss-function studies reveal that CHI3L1 plays a predominant role in regulating an immunosuppressive microenvironment by reprogramming tumor-associated macrophages (TAMs). Using the liquid chromatography-mass spectrometry and orthogonal structure-based screening, we found that Galectin-3 binding protein (Gal3BP) and its binding partner, Galectin-3 (Gal3), can interact competitively with the same binding motif on CHI3L1, leading to selective migration of M2-like versus M1-like bone marrow-derived macrophages (BMDMs) and resident microglia (MG). Mechanistically, the CHI3L1-Gal3 protein complex governs a transcriptional program of NFκB/CEBPβ to control the protumor phenotype of BMDMs, leading to inhibition of T cell infiltration and activation in the GBM TME. However, Gal3BP can reverse CHI3L1-Gal3 induced signaling pathway activation and subsequent protumor phenotype in TAMs. Based on protein binding motifs, a newly developed Gal3BP mimetic peptide can attenuate immune suppression and tumor progression in the syngeneic GBM mouse models, including decreasing M2-like TAMs and increasing M1-like TAMs and T cell infiltration. Together, these results shed light on the role of CHI3L1 protein complexes in immune evasion by glioblastoma and as a potential immunotherapeutic target for this devastating disease.

Double-Layer Simulated Moving Bed Chromatography for Ternary Separations: Serialized Layer Configurations

Double-layer simulated moving bed chromatography introduced for the complete ternary separation problems utilizes parallel column configurations. The inlet and outlet streams of paired columns need to be properly split and merged to realize parallel operations with the packed bed columns. To make a pair of split streams, at least one flow control unit is additionally required. Therefore, the double-layer simulated moving bed chromatography that consists of six zones with parallelized two columns required at least 10 pumps to control five internal flow splits and five external inlet and outlet flows. In this work, two alternative layer configurations of the double-layer simulated moving bed were introduced to simplify the internal flow patterns. Applying an orthogonal parameter screening method, the design parameters of the double-layer simulated moving bed processes were optimized. The simplified layer configurations were compared with the standard parallel double-layer configuration in terms of process performances and system pressure decrease.

Two-level orthogonal screening designs with 80, 96, and 112 runs, and up to 29 factors

Due to recent advances in the development of laboratory equipment, large screening experiments can now be conducted to study the joint impact of up to a few dozen factors. While much is known about orthogonal designs involving 64 and 128 runs, there is a lack of literature on screening designs with intermediate run sizes. In this article, we therefore construct screening designs with 80, 96 and 112 runs which allow the main effects to be estimated independently from the two-factor interactions and limit the aliasing among the interactions. We motivate our work using a 14-factor tuberculosis inhibition experiment and compare our new designs with alternatives from the literature using simulations.

Generating Selective Leads for Mer Kinase Inhibitors-Example of a Comprehensive Lead-Generation Strategy.

Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.

Bifunctional Doscadenamides Activate Quorum Sensing in Gram-Negative Bacteria and Synergize with TRAIL to Induce Apoptosis in Cancer Cells.

New cyanobacteria-derived bifunctional analogues of doscadenamide A, a LasR-dependent quorum sensing (QS) activator in Pseudomonas aeruginosa, characterized by dual acylation of the pyrrolinone core structure and the pendant side chain primary amine to form an imide/amide hybrid are reported. The identities of doscadenamides B-J were confirmed through total synthesis and a strategic focused library with different acylation and unsaturation patterns was created. Key molecular interactions for binding with LasR and a functional response through mutation studies coupled with molecular docking were identified. The structure-activity relationships (SARs) were probed in various Gram-negative bacteria, including P. aeruginosa and Vibrio harveyi, indicating that the pyrrolinone-N acyl chain is critical for full agonist activity, while the other acyl chain is dispensable or can result in antagonist activity, depending on the bacterial system. Since homoserine lactone (HSL) quorum sensing activators have been shown to act in synergy with TRAIL to induce apoptosis in cancer cells, selected doscadenamides were tested in orthogonal eukaryotic screening systems. The most potent QS agonists, doscadenamides S10-S12, along with doscadenamides F and S4 with partial or complete saturation of the acyl side chains, exhibited the most pronounced synergistic effects with TRAIL in triple negative MDA-MB-231 breast cancer cells. The overall correlation of the SAR with respect to prokaryotic and eukaryotic targets may hint at coevolutionary processes and intriguing host-bacteria relationships. The doscadenamide scaffold represents a non-HSL template for combination therapy with TRAIL pathway stimulators.

Rapid orthogonal screening of forensic drugs using a compact UHPLC-PDA/SQD; a complement to GC-MS

With the recent development of hand-held and miniature mass spectrometers, low cost and compact mass detectors have become more available and accessible for on-site and routine laboratory use. Here we present a rapid ultra-high performance liquid chromatography-photodiode array/mass spectrometry (UHPLC-PDA/MS) method for the routine screening of a wide variety of illicit drugs using a small and cost effective single quadrupole MS (SQD) with electron spray ionization (ESI). Two libraries for common drugs of forensic interest, a PDA library including retention time (RT) and PDA spectra and a MS library including RT and [M+H]+/[M−H]−, were developed. Using a BEH phenyl column and gradient elution of acetonitrile and 0.1% formic acid, drugs of forensic interest were detected within 8.5min and identified based on their RT/RRT to an internal standard (Codeine-d6), UV spectrum, and/or MS spectrum/(de)protonated molecular mass. The method proved to be efficient, reproducible, and sensitive with a limit of detection (LOD) ≤0.5μg/mL for untargeted screening using PDA and MS in full scan mode and ≤0.1μg/mL for targeted screening by MS in select ion recording (SIR) mode. Improved isomer discrimination was achieved, when a second injection and separation by a charged column (CSH C18) was incorporated into the same acquisition sequence. The method was applied to 20 seized drugs with similar detection limits and identifications by GC-MS. Using an orthogonal separation technique and dual detection systems, the UHPLC-PDA/SQD method serves as an attractive technique for a direct and fast screening of illicit drugs with excellent discrimination power and detection capacity. The developed method can be an alternative screening method to GC-FID and complementary technique to GC-MS for routine forensic drug screening.

Cooling load dynamics and simplified calculation method for radiant ceiling panel and dedicated outdoor air system

Compared with all-air system, the cooling load dynamics for integration operation of radiant ceiling panel (RCP) and dedicated outdoor air system (DOAS) is more complex, and there is a lack of practical cooling load calculation method in current standards and guidebooks. This study aims to explore their cooling load dynamics and develop a simplified calculation method for such integrated system. A heat balance based model to calculate the cooling load for the integrated system was established. The results showed that RCP with DOAS removed more room heat gain than all-air system at the same indoor air temperature set-point, and the peak cooling load of RCP with DOAS is 16% larger. It was also found that ignoring DOAS in cooling load calculation could make the results 6% larger than DOAS integrated. In addition, there is a condensation risk if RCP alone was used to maintain air set-point temperature. To help the application of the integrated system at design stage, we proposed cooling load correction coefficient rrad as the cooling load difference between radiant and all-air system, and the cooling load split fraction rDOAS for DOAS. Through orthogonal screening, 6 significant factors on rrad and rDOAS were selected. Simplified models for rrad and rDOAS calculation in both continuous and intermittent operation modes were established using simulation data, and a revised design process for the integrated system was proposed.

A Whole-Cell Screen for Adjunctive and Direct Antimicrobials Active against Carbapenem-Resistant Enterobacteriaceae

Carbapenem-resistant Enterobacteriaceae (CRE) are an emerging antimicrobial resistance threat for which few if any therapeutic options remain. Identification of new agents that either inhibit CRE or restore activity of existing antimicrobials is highly desirable. Therefore, a high-throughput screen of 182,427 commercially available compounds was used to identify small molecules which either enhanced activity of meropenem against a carbapenem-resistant Klebsiella pneumoniae ST258 screening strain and/or directly inhibited its growth. The primary screening methodology was a whole-cell screen/counterscreen combination assay that tested for reduction of microbial growth in the presence or absence of meropenem, respectively. Screening hits demonstrating eukaryotic cell toxicity based on an orthogonal screening effort or identified as pan-assay interference compounds (PAINS) by computational methods were triaged. Primary screening hits were then clustered and ranked according to favorable physicochemical properties. Among remaining hits, we found 10 compounds that enhanced activity of carbapenems against a subset of CRE. Direct antimicrobials that passed toxicity and PAINS filters were not, however, identified in this relatively large screening effort. It was previously shown that the same screening strategy was productive for identifying candidates for further development when screening known bioactive libraries inclusive of natural products. Our findings therefore further highlight liabilities of commercially available small-molecule screening libraries in the Gram-negative antimicrobial space. In particular, there was especially low yield in identifying compelling activity against a representative, highly multidrug-resistant, carbapenemase-producing K. pneumoniae strain.

Determination of aflatoxins in milk sample with ionic liquid modified magnetic zeolitic imidazolate frameworks

The ionic liquid (IL) was introduced to the synthesis system of magnetic zeolite imidazolate framework-8 (M/ZIF-8), which was benefit to the formation of binary imidazole and the co-modification of M/ZIF-8. The morphology and textural properties of ILM/ZIF-8 were characterized by SEM, TEM, BET and BJH. The crystal structural shape and size of MZIF-8 was unvaried with the interventional of IL. The ILM/ZIF-8 was applied to the concentration and determination of aflaoxins (AFB1, AFB2, AFG1 and AFG2) in milk samples based on magnetic solid phase extraction (MSPE) coupled with ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The experimental parameters of the MSPE, including amount of ILM/ZIF-8, pH, type and amount of desorption solvent, extraction time and sample volume were investigated by a univariate method and orthogonal screening. The four AFs were concentrated from the 20 mL milk when 90 mg ILM/ZIF-8 was used as magnetic adsorbent. The extraction efficiency of AFs was higher than 80.0% within 15 min. The limits of quantitative and detection were 7.5–26.7 and 2.3–8.1 ng/L, respectively. The proposed method was applied to the determination of milk samples containing trace amounts of AFs and the recoveries ranged from 79.0% to 102.5%, with RSD below 7.7%.

A Functional Landscape of Resistance to MEK1/2 and CDK4/6 Inhibition in NRAS-Mutant Melanoma.

Combinatorial inhibition of MEK1/2 and CDK4/6 is currently undergoing clinical investigation in NRAS-mutant melanoma. To prospectively map the landscape of resistance to this investigational regimen, we utilized a series of gain- and loss-of-function forward genetic screens to identify modulators of resistance to clinical inhibitors of MEK1/2 and CDK4/6 alone and in combination. First, we identified NRAS-mutant melanoma cell lines that were dependent on NRAS for proliferation and sensitive to MEK1/2 and CDK4/6 combination treatment. We then used a genome-scale ORF overexpression screen and a CRISPR knockout screen to identify modulators of resistance to each inhibitor alone or in combination. These orthogonal screening approaches revealed concordant means of achieving resistance to this therapeutic modality, including tyrosine kinases, RAF, RAS, AKT, and PI3K signaling. Activated KRAS was sufficient to cause resistance to combined MEK/CDK inhibition and to replace genetic depletion of oncogenic NRAS. In summary, our comprehensive functional genetic screening approach revealed modulation of resistance to the inhibition of MEK1/2, CDK4/6, or their combination in NRAS-mutant melanoma. SIGNIFICANCE: These findings reveal that NRAS-mutant melanomas can acquire resistance to genetic ablation of NRAS or combination MEK1/2 and CDK4/6 inhibition by upregulating activity of the RTK-RAS-RAF and RTK-PI3K-AKT signaling cascade.

Determination of microcystins in environmental water samples with ionic liquid magnetic graphene

Microcystins is a class of monocyclic of heptapeptides with many different isomerides. It has become potential hazardous material in water environment for its toxic, distribution and stability. This project worked on a method for determination of trace microcystin (MC-LR and MC-RR) in environmental waters. The ionic liquid magnetic graphene (IL@MG) was prepared and applied to the concentration and determination of microcystins, based on magnetic solid phase extraction (MSPE), and coupled with ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The ionic liquid magnetic graphene was prepared by coprecipitatial synthesis and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), specific surface area (BET), pore size distribution (BJH) and magnetic hysteresis loop. The experimental parameters of magnetic solid phase extraction, including amount of IL@MG, pH, extraction time and elution solvent were investigated by a univariate method and orthogonal screening. The method showed good linearity in the range of 0.01–10.0 g/L and 0.005–10.0 μg/L for MC-LR and MC-RR, when the pH of water samples was 4.00 and 10.0 mg adsorbents were used to extract targets for 18 min. The lowest detection limit was 0.414 ng/L and 0.216 ng/L for MC-LR and MC-RR respectively. The recoveries of the microcystins were in the range of 83.6–100.9%, and the relative standard deviation was less than 7.59%. The trace amount of MC-LR (0.020 μg/L) and MC-RR (0.003 μg/L and 0.021 μg/L) was detected in actural water samples. Attributed to its simple operator, low detection limit and high sensitivity, this method could be used for the detection of trace microcystins in water samples.

Discovery and Characterization of Two Classes of Selective Inhibitors of the Suppressor of the TCR Signaling Family of Proteins.

The suppressor of T-cell receptor signaling (Sts) proteins, Sts-1, has recently emerged as a potential immunostimulatory target for drug development. Genetic inactivation of the Sts proteins dramatically increases host survival of systemic infection and leads to improved pathogen clearance. The protein tyrosine phosphatase (PTP) activity of these proteins arises from a C-terminal 2-histidine phosphatase (HP) domain. To identify new inhibitors of the HP activity of Sts-1, we miniaturized a phosphatase assay to a 1536-well format and conducted a 20 580 compound screen. Among the hits were two classes of structurally related compounds, tetracycline variants and sulfonated azo dyes. These hits had low micromolar to nanomolar IC50 values. Orthogonal screening confirmed the validity of these inhibitors and demonstrated that both act competitively on Sts-1 phosphatase activity. When tested on other PTPs, PTP1B and SHP1, it was found that the tetracycline PTP1B, SHP1, the tetracycline variant (doxycycline), and the sulfonated azo dye (Congo red) are selective inhibitors of Sts-1HP, with selectivity indices ranging from 19 to as high as 200. The planar polyaromatic moieties present in both classes of compounds suggested a common binding mode. The mutation of either tryptophan 494 or tyrosine 596, located near the active site of the protein, reduced the Ki of the inhibitors from 3- to 18-fold, indicating that these residues may help to promote the binding of substrates with aromatic groups. This work provides new insights into substrate selectivity mechanisms and describes two classes of compounds that can serve as probes of function or as a basis for future drug discovery.