Showing papers on "Chemical library published in 2016"

New p32/gC1qR Ligands for Targeted Tumor Drug Delivery.

Abstract: Cell surface p32, the target of LyP-1 homing peptide, is upregulated in tumors and atherosclerotic plaques and has been widely used as a receptor for systemic delivery of payloads. Here, we identified an improved LyP-1-mimicking peptide (TT1, CKRGARSTC). We used this peptide in a fluorescence polarization-based high-throughput screening of a 50 000-compound chemical library and identified a panel of compounds that bind p32 with low micromolar affinity. Among the hits identified in the screen, two compounds were shown to specifically bind to p32 in multiple assays. One of these compounds was chosen for an in vivo study. Nanoparticles surface-functionalized with this compound specifically adhered to surfaces coated with recombinant p32 and, when injected intravenously, homed to p32-expressing breast tumors in mice. This compound provides a lead for the development of p32-targeted affinity ligands that circumvent some of the limitations of peptide-based probes in guided drug delivery.

Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry

Abstract: The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.

Diversity-oriented synthetic strategy for developing a chemical modulator of protein-protein interaction

Abstract: Diversity-oriented synthesis (DOS) can provide a collection of diverse and complex drug-like small molecules, which is critical in the development of new chemical probes for biological research of undruggable targets. However, the design and synthesis of small-molecule libraries with improved biological relevance as well as maximized molecular diversity represent a key challenge. Herein, we employ functional group-pairing strategy for the DOS of a chemical library containing privileged substructures, pyrimidodiazepine or pyrimidine moieties, as chemical navigators towards unexplored bioactive chemical space. To validate the utility of this DOS library, we identify a new small-molecule inhibitor of leucyl-tRNA synthetase-RagD protein-protein interaction, which regulates the amino acid-dependent activation of mechanistic target of rapamycin complex 1 signalling pathway. This work highlights that privileged substructure-based DOS strategy can be a powerful research tool for the construction of drug-like compounds to address challenging biological targets.

Protein–Protein Interaction Inhibition (2P2I)-Oriented Chemical Library Accelerates Hit Discovery

Abstract: Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse "PPI-like" modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using homogeneous time-resolved fluorescence (HTRF) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with nonoriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets.

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening.

Abstract: UNLABELLED Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development. IMPORTANCE This study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale high-throughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for small-molecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism of cooperative resistance to IAV entry blockers.

Target identification in small cell lung cancer via integrated phenotypic screening and activity-based protein profiling

Abstract: To overcome hurdles in identifying key kinases in small cell lung cancer (SCLC), we integrated a target-agnostic phenotypic screen of kinase inhibitors with target identification using activity-based protein profiling (ABPP) in which a desthiobiotin-ATP probe was used. We screened 21 SCLC cell lines with known c-MYC amplification status for alterations in viability using a chemical library of 235 small-molecule kinase inhibitors. One screen hit compound was interrogated with ABPP, and, through this approach, we reidentified Aurora kinase B as a critical kinase in MYC-amplified SCLC cells. We next extended the platform to a second compound that had activity in SCLC cell lines lacking c-MYC amplification and identified TANK-binding kinase 1, a kinase that affects cell viability, polo-like kinase-1 signaling, G2-M arrest, and apoptosis in SCLC cells lacking MYC amplification. These results demonstrate that phenotypic screening combined with ABPP can identify key disease drivers, suggesting that this approach, which combines new chemical probes and disease cell screens, has the potential to identify other important targets in other cancer types. Mol Cancer Ther; 15(2); 334-42. ©2016 AACR.

Identification of Protein Palmitoylation Inhibitors from a Scaffold Ranking Library.

Abstract: The addition of palmitoyl moieties to proteins regulates their membrane targeting, subcellular localization, and stability. Dysregulation of the enzymes which catalyzed the palmitoyl addition and/or the substrates of these enzymes have been linked to cancer, cardiovascular, and neurological disorders, implying these enzymes and substrates are valid targets for pharmaceutical intervention. However, current chemical modulators of zDHHC PAT enzymes lack specificity and affinity, underscoring the need for screening campaigns to identify new specific, high affinity modulators. This report describes a mixture based screening approach to identify inhibitors of Erf2 activity. Erf2 is the Saccharomyces cerevisiae PAT responsible for catalyzing the palmitoylation of Ras2, an ortholog of the human Ras oncogene proteins. A chemical library developed by the Torrey Pines Institute for Molecular Studies consists of more than 30 million compounds designed around 68 molecular scaffolds that are systematically arranged into positional scanning and scaffold ranking formats. We have used this approach to identify and characterize several scaffold backbones and R-groups that reduce or eliminate the activity of Erf2 in vitro. Here, we present the analysis of one of the scaffold backbones, bis-cyclic piperazine. We identified compounds that inhibited Erf2 auto-palmitoylation activity using a fluorescence-based, coupled assay in a high throughput screening (HTS) format and validated the hits utilizing an orthogonal gel-based assay. Finally, we examined the effects of the compounds on cell growth in a yeast cell-based assay. Based on our results, we have identified specific, high affinity palmitoyl transferase inhibitors that will serve as a foundation for future compound design.

An Integrated Approach for Screening and Identification of Positive Allosteric Modulators of N-Methyl-D-Aspartate Receptors

Abstract: N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer's disease and schizophrenia.

Discovery of a novel ROCK2 inhibitor with anti-migration effects via docking and high-content drug screening

Abstract: Rho-associated protein kinase (ROCK) mediated the reorganization of the actin cytoskeleton and has been implicated in the spread and metastatic process of cancer. In this study, structure-based high-throughput virtual screening was used to identify candidate compounds targeting ROCK2 from a chemical library. Moreover, high-content screening based on neurite outgrowth of SH-SY5Y cells (a human neuroblastoma cell line) was used for accelerating the identification of compounds with characteristics of ROCK2 inhibitors. The effects of bioactive ROCK2 inhibitor candidates were further validated using other bioassays including cell migration and wound healing in SH-SY5Y cells. Through the combined virtual and high-content drug screening, the compound 1,3-benzodioxol-5-yl[1-(5-isoquinolinylmethyl)-3-piperidinyl]-methanone (BIPM) was identified as a novel and potent ROCK2 inhibitor. Exposure of SH-SY5Y cells to BIPM led to significant changes in neurite length, cell migration and actin stress fibers. Further experiments demonstrated that BIPM was able to significantly inhibit phosphorylation of cofilin, a regulatory protein of actin cytoskeleton. These results suggest that BIPM could be considered as a promising scaffold for the further development of ROCK2 inhibitors for anti-cancer metastasis.

Novel small molecule modulators of plant growth and development identified by high-content screening with plant pollen

Abstract: Small synthetic molecules provide valuable tools to agricultural biotechnology to circumvent the need for genetic engineering and provide unique benefits to modulate plant growth and development. We developed a method to explore molecular mechanisms of plant growth by high-throughput phenotypic screening of haploid populations of pollen cells. These cells rapidly germinate to develop pollen tubes. Compounds acting as growth inhibitors or stimulators of pollen tube growth are identified in a screen lasting not longer than 8 h high-lighting the potential broad applicability of this assay to prioritize chemicals for future mechanism focused investigations in plants. We identified 65 chemical compounds that influenced pollen development. We demonstrated the usefulness of the identified compounds as promotors or inhibitors of tobacco and Arabidopsis thaliana seed growth. When 7 days old seedlings were grown in the presence of these chemicals twenty two of these compounds caused a reduction in Arabidopsis root length in the range from 4.76 to 49.20 % when compared to controls grown in the absence of the chemicals. Two of the chemicals sharing structural homology with thiazolidines stimulated root growth and increased root length by 129.23 and 119.09 %, respectively. The pollen tube growth stimulating compound (S-02) belongs to benzazepin-type chemicals and increased Arabidopsis root length by 126.24 %. In this study we demonstrate the usefulness of plant pollen tube based assay for screening small chemical compound libraries for new biologically active compounds. The pollen tubes represent an ultra-rapid screening tool with which even large compound libraries can be analyzed in very short time intervals. The broadly applicable high-throughput protocol is suitable for automated phenotypic screening of germinating pollen resulting in combination with seed germination assays in identification of plant growth inhibitors and stimulators.

Discovery of Low Micromolar Dual Inhibitors for Wild Type and L1196M Mutant of Anaplastic Lymphoma Kinase through Structure-Based Virtual Screening.

Abstract: Although anaplastic lymphoma kinase (ALK) is involved in a variety of malignant human cancers, the emergence of constitutively active mutants with drug resistance has rendered it difficult to identify the new medicines for ALK-dependent cancers. To find the common inhibitors of the wild type ALK and the most abundant drug-resistant mutant (L1196M), we performed molecular docking-based virtual screening of a large chemical library in parallel for the two target proteins. As a consequence of augmenting the accuracy of the docking simulation by implementing a sophisticated hydration free energy term in the scoring function, 12 common inhibitors are discovered with the inhibitory activities ranging from submicromolar to low micromolar levels. The results of the binding free energy decomposition indicate that the biochemical potency of ALK inhibitors can be optimized by reducing the dehydration cost for binding to the receptor protein as well as by strengthening the interactions with amino acid residues in the...

Development of a Multiplex Assay for Studying Functional Selectivity of Human Serotonin 5-HT2A Receptors and Identification of Active Compounds by High-Throughput Screening:

Abstract: G protein-coupled receptors (GPCRs) exist as collections of conformations in equilibrium, and the efficacy of drugs has been proposed to be associated with their absolute and relative affinities for these different conformations. The serotonin 2A (5-HT2A) receptor regulates multiple physiological functions, is involved in the pathophysiology of schizophrenia, and serves as an important target of atypical antipsychotic drugs. This receptor was one of the first GPCRs for which the functional selectivity phenomenon was observed, with its various ligands exerting differential effects on the phospholipase A2 (PLA2) and phospholipase C (PLC) signaling pathways. We aimed to develop a multiplex functional assay in 96-well plates for the simultaneous measurement of the PLA2 and PLC pathways coupled to 5-HT2A receptors; this approach enables the detection of either functional selectivity or cooperativity phenomena in early drug screening stages. The suitability of the method for running screening campaigns was tested using the Prestwick Chemical Library, and 22 confirmed hits with activities of more than 90% were identified; 11 of these hits produced statistically significant differences between the two effector pathways. Thus, we have developed a miniaturized multiplex assay in 96-well plates to measure functional selectivity for 5-HT2A receptors in the early stages of the drug discovery process.

Chemical Screens Identify Drugs that Enhance or Mitigate Cellular Responses to Antibody-Toxin Fusion Proteins.

Abstract: The intersection of small molecular weight drugs and antibody-based therapeutics is rarely studied in large scale. Both types of agents are currently part of the cancer armamentarium. However, very little is known about how to combine them in optimal ways. Immunotoxins are antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. For fusion proteins derived from Pseudomonas exotoxin (PE), potency relies on the enzymatic domain of the toxin which catalyzes the ADP-ribosylation of EF2 causing inhibition of protein synthesis leading to cell death. Candidate immunotoxins have demonstrated clear value in clinical trials but generally have not been curative as single agents. Therefore we undertook three screens to discover effective combinations that could act synergistically. From the MIPE-3 library of compounds we identified various enhancers of immunotoxin action and at least one major class of inhibitor. Follow-up experiments confirmed the screening data and suggested that immunotoxins when administered with everolimus or nilotinib exhibit favorable combinatory activity and would be candidates for preclinical development. Mechanistic studies revealed that everolimus-immunotoxin combinations acted synergistically on elements of the protein synthetic machinery, including S61 kinase and 4E-BP1 of the mTORC1 pathway. Conversely, PARP inhibitors antagonized immunotoxins and also blocked the toxicity due to native ADP-ribosylating toxins. Thus, our goal of investigating a chemical library was justified based on the identification of several approved compounds that could be developed preclinically as ‘enhancers’ and at least one class of mitigator to be avoided.

Structure–activity relationships study of mTOR kinase inhibition using QSAR and structure-based drug design approaches

Abstract: The discovery of clinically relevant inhibitors of mammalian target of rapamycin (mTOR) for anticancer therapy has proved to be a challenging task. The quantitative structure-activity relationship (QSAR) approach is a very useful and widespread technique for ligand-based drug design, which can be used to identify novel and potent mTOR inhibitors. In this study, we performed two-dimensional QSAR tests, and molecular docking validation tests of a series of mTOR ATP-competitive inhibitors to elucidate their structural properties associated with their activity. The QSAR tests were performed using partial least square method with a correlation coefficient of r2=0.799 and a cross-validation of q2=0.714. The chemical library screening was done by associating ligand-based to structure-based approach using the three-dimensional structure of mTOR developed by homology modeling. We were able to select 22 compounds from two databases as inhibitors of the mTOR kinase active site. We believe that the method and applications highlighted in this study will help future efforts toward the design of selective ATP-competitive inhibitors.

High-throughput screening against ~6.1 million structurally diverse, lead-like compounds to discover novel ROCK inhibitors for cerebral injury recovery.

Abstract: Rho-associated protein kinase (ROCK) has been recognized as an attractive therapeutic target to promote neurogenesis, neuroregeneration, and neurorecovery after cerebral injury. Here, a high-throughput screening protocol was described to discover novel ROCK inhibitors from a large chemical library containing ~6.1 million structurally diverse, lead-like compounds. The protocol employed empirical rules such as ADMET evaluation and chemical similarity analysis to exclude those of drug-unlike candidates, and then molecular docking and binding affinity predictions were performed to suggest few promising candidates with high scores. Consequently, five compounds were successfully identified to have satisfactory activity profile with IC50 values at nanomolar level. In order to elucidate the molecular mechanism of inhibitor binding to target, the complex structures of ROCK kinase domain with the five identified compounds were modeled and examined in detail. A number of polar chemical forces such as hydrogen bonds and cation-π interactions as well as nonpolar contacts such as π-π stacking and hydrophobic forces were revealed at the complex interface, conferring high affinity and strong specificity to inhibitor binding. In addition, several key residues around the kinase active site, including Val90, Lys105, Asn203, and Phe368, were found to play an important role in binding.

Erratum to: Discovery of new Mycobacterium tuberculosis proteasome inhibitors using a knowledge-based computational screening approach.

Abstract: Mycobacterium tuberculosis bacteria are cause deadly infections in patients. The rise of multidrug resistance associated with tuberculosis further makes the situation worse in treating the disease. M. tuberculosis proteasome is necessary for the pathogenesis of the bacterium validated as an anti-tubercular target, thus making it an attractive enzyme for designing Mtb inhibitors. In this study, a computational screening approach was applied to identify new proteasome inhibitor candidates from a library of 50,000 compounds. This chemical library was procured from the ChemBridge (20,000 compounds) and the ChemDiv (30,000 compounds) databases. After a detailed analysis of the computational screening results, 50 in silico hits were retrieved and tested in vitro finding 15 compounds with \(\hbox {IC}_{50}\) values ranging from 35.32 to 64.15 \(\upmu \)M on lysate. A structural analysis of these hits revealed that 14 of these compounds probably have non-covalent mode of binding to the target and have not reported for anti-tubercular or anti-proteasome activity. The binding interactions of all the 14 protein-inhibitor complexes were analyzed using molecular docking studies. Further, molecular dynamics simulations of the protein in complex with the two most promising hits were carried out so as to identify the key interactions and validate the structural stability.

Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals.

Abstract: Interleukin-7 (IL-7) is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP) gene, drug selection cassette, and modified homologous arms which are considered to enhance the translation of the eGFP reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the IL-7 start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report IL-7 gene transcription based on the eGFP protein signal. Furthermore, we utilized the cells to run a pilot screen campaign for IL-7-upregulating chemicals in a high-throughput format, and identified a chemical that can up-regulate IL-7 gene transcription. Collectively, these results suggest that our IL-7 reporter system can be utilized in large-scale chemical library screening to reveal novel IL-7 regulatory pathways and to identify potential drugs for development of new treatments in immunodeficiency disease.

A simple method for producing multiple copies of controlled release small molecule microarrays for cell-based screening.

Abstract: Traditional drug discovery involves the screening of lead compounds from a chemical library by using cell-based high throughput screening (HTS) procedures. This has created a demand for the development of cell-based microarray chips for HTS of compounds. Although several cell-based microarray devices and procedures for screening of chemical libraries have been reported, each has limitations in terms of simplicity, speed, and cost. Here, we sought to make a simple method for producing multiple copies of microarray chips for the controlled release of small molecules during cell-based screening. Arrays of polytetrafluoroethylene microchannels were set in poly(dimethylsiloxane) and were formed in a metal mold. Subsequently, a biodegradable polymer, PLGAs, with chemical compounds was injected into each channel, and the array was sliced perpendicular to the channels to create multiple copies of the microarray chip. After seeding the cells on the microarray chip, we were able to successfully control the diffusion of small molecules and locally introduce the compounds into cells. The described method enables the production of multiple copies of the chip by using an easy, rapid, and inexpensive array fabrication without any specialized devices. Moreover, screening using the microarray chip minimizes the consumption of cells and chemicals. Both the biodegradable material and compound injected into each channel can be individually tuned for optimized performance. Therefore, we expect that this method will be useful for developing cell-based HTS assays for small chemical compounds to find drug candidates.

In Silico Exploration for Novel Type-I Inhibitors of Tie-2/TEK: The Performance of Different Selection Strategy in Selecting Virtual Screening Candidates.

Abstract: The receptor tyrosine kinase Tie-2 is involved in vessel remodeling and maturation, and has been regarded as a potential target for the treatment of various solid tumors. The absence of novel, potent and selective inhibitors severely hampers the understanding of the therapeutic potential of Tie-2. In the present work, we describe the discovery of novel type-I inhibitors of Tie-2 by structure-based virtual screening. Preliminary SAR was also performed based on one active compound, and several novel inhibitors with low micro-molar affinity were discovered. To directly compare the efficiency between different filtering strategies in selecting VS candidates, two methods were separately carried out to screen the same chemical library, and the selected VS candidates were then experimentally assessed by in vitro enzymatic assays. The results demonstrate that the hit rate is improved when stricter drug-likeness criteria and less number of molecules for clustering analysis are used, and meanwhile, the molecular diversity of the compounds still maintains. As a case study of TIE-2, the information presented in this work underscores the importance of selecting an appropriate selection strategy in VS campaign, and the novel inhibitors identified and the detailed binding modes of action provide a starting point for further hit-to-lead optimization process.

A High-Throughput Screening-Compatible Strategy for the Identification of Inositol Pyrophosphate Kinase Inhibitors

Abstract: Pharmacological tools—‘chemical probes’—that intervene in cell signaling cascades are important for complementing genetically-based experimental approaches. Probe development frequently begins with a high-throughput screen (HTS) of a chemical library. Herein, we describe the design, validation, and implementation of the first HTS-compatible strategy against any inositol phosphate kinase. Our target enzyme, PPIP5K, synthesizes ‘high-energy’ inositol pyrophosphates (PP-InsPs), which regulate cell function at the interface between cellular energy metabolism and signal transduction. We optimized a time-resolved, fluorescence resonance energy transfer ADP-assay to record PPIP5K-catalyzed, ATP-driven phosphorylation of 5-InsP7 to 1,5-InsP8 in 384-well format (Z’ = 0.82 ± 0.06). We screened a library of 4745 compounds, all anticipated to be membrane-permeant, which are known—or conjectured based on their structures—to target the nucleotide binding site of protein kinases. At a screening concentration of 13 μM, fifteen compounds inhibited PPIP5K >50%. The potency of nine of these hits was confirmed by dose-response analyses. Three of these molecules were selected from different structural clusters for analysis of binding to PPIP5K, using isothermal calorimetry. Acceptable thermograms were obtained for two compounds, UNC10112646 (Kd = 7.30 ± 0.03 μM) and UNC10225498 (Kd = 1.37 ± 0.03 μM). These Kd values lie within the 1–10 μM range generally recognized as suitable for further probe development. In silico docking data rationalizes the difference in affinities. HPLC analysis confirmed that UNC10225498 and UNC10112646 directly inhibit PPIP5K-catalyzed phosphorylation of 5-InsP7 to 1,5-InsP8; kinetic experiments showed inhibition to be competitive with ATP. No other biological activity has previously been ascribed to either UNC10225498 or UNC10112646; moreover, at 10 μM, neither compound inhibits IP6K2, a structurally-unrelated PP-InsP kinase. Our screening strategy may be generally applicable to inhibitor discovery campaigns for other inositol phosphate kinases.

Method for screening antithrombotic drug based on magnetic bead separation

Abstract: The invention discloses a method for screening an antithrombotic drug based on magnetic bead separation. According to the method, magnetic separation and chemical information collection (liquid chromatography-mass spectrometry) techniques are adopted; a target protein bonder is analyzed; the method is used for screening and evaluating the antithrombotic action of a drug. According to the method for screening the antithrombotic drug based on the magnetic bead separation, an optimal incubation temperature, an incubation time, a pH (potential of Hydrogen) value of a solution during incubation, ionic strength and a denaturizing cleaning solution are screened out through lots of experiments. A screening and evaluating method provided by the invention can be used for screening a large-scale natural product library or a combinatorial chemical library; in comparison with a conventional ultraviolet screening method, the screening efficiency can be obviously improved; a screening time is shortened; moreover, a synergistic effect between compounds can be discovered; the method for screening the antithrombotic drug based on the magnetic bead separation has the advantages of strong operability, quickness, high efficiency, accuracy, good repeatability and the like.

Chemical Transformation of an Intermediate in the Synthesis of Huperzine A, Leading to a Diverse Array of Molecules.

Abstract: Chemical transformation of an early intermediate in our synthesis of huperzine A provided a diverse array of molecules in which a variety of functional groups could be embedded.

Method for solid-phase synthesis of dna-encoded chemical library

Abstract: Provided is a method for solid-phase synthesis of a DNA-encoded chemical library, the method comprising the steps of: a) reacting a solid-phase carrier G-1 with a linker molecule L-1 to prepare L-G-1; b) reacting DNA with a linker molecule L-0 to prepare L-2; c) reacting L-G-1 with L-2 to prepare L-G-2; d) removing a protecting group from L-G-2 to obtain L-G-2-1; e) reacting with a building block and performing DNA encoding; f) removing the solid-phase carrier to obtain a DNA-encoded chemical library.

Method for fabricating and screening dna-coded library

Abstract: PROBLEM TO BE SOLVED: To provide many methods for identifying one or a plurality of compounds bound to a biological target.SOLUTION: A method for tagging a DNA-coded chemical library includes: (a) a step of binding a first functional group of a bifunctional linker to a 5' end of initiator oligonucleotide, which is a step where the initiator oligonucleotide bound to the bifunctional linker forms a hairpin structure; and (b) a step of binding a second functional group of the bifunctional linker to a component of the chemical library, in which the bifunctional linker or the initiator oligonucleotide is modified under an organic condition so as to increase solubility of a member of the DNA-coded chemical library.SELECTED DRAWING: None