Discovery of novel non-peptide inhibitors of BACE-1 using virtual high-throughput screening

A novel series of isatin-based inhibitors of β-secretase (BACE-1) have been identified using a virtual high-throughput screening approach. Structure–activity relationship studies revealed structural features important for inhibition. Docking studies suggest these inhibitors may bind within the BACE-1 active site through H-bonding interactions involving the catalytic aspartate residues.     

A high-throughput screen for identification of molecular mimics of Smac/DIABLO utilizing a fluorescence polarization assay

Resistance to apoptosis is afforded by inhibitor of apoptosis proteins (IAPs) which bind to and inhibit the caspases responsible for cleavage of substrates leading to apoptotic cell death. Smac (or DIABLO), a proapoptotic protein released from the mitochondrial intermembrane space into the cytosol, promotes apoptosis by binding to IAPs, thus reversing their inhibitory effects on caspases. We have developed a high-throughput fluorescence polarization assay utilizing a fluorescein-labeled peptide similar to the "IAP binding" domain of Smac N terminus complexed with the BIR3 domain of X-linked IAP (XIAP) to identify small-molecule mimics of the action of Smac. The IC(50)s of peptides and a tetrapeptidomimetic homologous to the N terminus of Smac demonstrated the specificity and utility of this assay. We have screened the National Cancer Institute "Training Set" of 230 compounds, with well-defined biological actions, and the "Diversity Set" of 2000 chemically diverse structures for compounds which significantly reduced fluorescence polarization. Highly fluorescing or fluorescence-quenching compounds (false positives) were distinguished from those which interfered with Smac peptide binding to the XIAP-BIR3 in a dose-dependent manner (true positives). This robust assay offers potential for high-throughput screening discovery of novel compounds simulating the action of Smac/DIABLO.     

The identification of novel small-molecule inhibitors targeting WDR5-MLL1 interaction through fluorescence polarization based high-throughput screening

The protein-protein interaction between WDR5 (WD40 repeat protein 5) and MLL1 (mixed-lineage leukemia 1) is important for maintaining optimal H3K4 methyltransferase activity of MLL1. Dysregulation of MLL1 catalytic function is relevant to mixed-lineage leukemia, and targeting WDR5-MLL1 interaction could be a promising therapeutic strategy for leukemia harboring MLL1 fusion proteins. To date, several peptidomimetic and non-peptidomimetic small-molecule inhibitors targeting WDR5-MLL1 interaction have been reported, yet the discovery walk of new drugs inhibiting MLL1 methytransferase activity is still in its infancy. It’s urgent to find other small-molecule WDR5-MLL1 inhibitors with novel scaffolds. In this study, through fluorescence polarization (FP)-based high throughput screening, several small-molecule inhibitors with potent inhibitory activities in vitro against WDR5-MLL1 interaction were discovered. Nuclear Magnetic Resonance (NMR) assays were carried out to confirm the direct binding between hit compounds and WDR5. Subsequent similarity-based analog searching of the 4 hits led to several inhibitors with better activity, among them, DC_M5_2 displayed highest inhibitory activity with IC₅₀ values of 9.63?±?1.46?µM. Furthermore, a molecular docking study was performed and disclosed the binding modes and interaction mechanisms between two most potent inhibitors and WDR5.     

Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT-phosphoprotein interaction

Finding specific small-molecule inhibitors of protein-protein interactions remains a significant challenge. Recently, attention has grown toward "hot spot" interactions where binding is dominated by a limited number of amino acid contacts, theoretically offering an increased opportunity for disruption by small molecules. Inhibitors of the interaction between BRCT (the C-terminal portion of BRCA1, a key tumor suppressor protein with various functions) and phosphorylated proteins (Abraxas/BACH1/CtIP), implicated in DNA damage response and repair pathways, should prove to be useful in studying BRCA1's role in cancer and in potentially sensitizing tumors to chemotherapeutic agents. We developed and miniaturized to a 1536-well format and 3-mul final volume a pair of fluorescence polarization (FP) assays using fluorescein- and rhodamine-labeled pBACH1 fragment. To minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members all were assayed against both the fluorescein- and rhodamine-labeled probe-protein complexes in separate but interleaved reactions. In addition, every library compound was tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm. Analyses of the screening results led to the selection and subsequent confirmation of 16 compounds active in both assays. Faced with a traditionally difficult protein-protein interaction assay, by performing two-fluorophore qHTS, we were able to confidently select a number of actives for further studies.     

Design,synthesis and biological evaluation of new inhibitors of Bax/Bcl-xL interaction in cancer cells

We describe the synthesis of a series of new molecules containing phenol and triazoles moieties, compounds which have been evaluated for their ability to inhibit Bax/Bcl-xL interactions in cancer cells, by using BRET assays, and to induce cell death. Several derivatives exhibit a very promising activity, being more potent than the reference compounds acylpyrogallol A and ABT-737. These preliminary results demonstrate that derivatives of this family can be attractive to develop new molecules with potent anticancer activity.     

Discovery of novel inhibitors targeting the Mycobacterium tuberculosis O-acetylserine sulfhydrylase (CysK1) using virtual high-throughput screening

Cysteine biosynthesis in Mycobacterium tuberculosis (MTB) is crucial for this pathogen to combat oxidative stress and for long term survival in the host. Hence inhibition of this pathway is attractive for developing novel drugs against tuberculosis. In the present study, the crystal structure of the mycobacterial enzyme O-acetylserine sulfhydrylase CysK1 bound to an oligopeptide inhibitor was used as a framework for virtual screening of the BITS-Pilani in-house database to identify new scaffolds as CysK1 inhibitors. Thirty compounds were synthesized and evaluated in vitro for their ability to inhibit CysK1, activity against M. tuberculosis and cytotoxicity as steps towards the derivation of structure–activity relationships (SAR) and lead optimization. Compound 8-nitro-4-(2-(trifluoromethyl)phenyl)-4,4a-dihydro-2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione (4n) emerged as the most promising lead with an IC₅₀ of 17.7 μM for purified CysK1 and MIC of 7.6 μM for M. tuberculosis, with little or no cytotoxicity (>50 μM).     

Discovery of novel HCV polymerase inhibitors using pharmacophore-based virtual screening

We report the use of pharmacophore-based virtual screening as an efficient tool for the discovery of novel HCV polymerase inhibitors. A three-dimensional pharmacophore model for the HCV-796 binding site, NNI site IV inhibitor, to the enzyme was built by means of the structure-based focusing module in Cerius2 program. Using these models as a query for virtual screening, we produced a successful example of using pharmacophore-based virtual screening to identify novel compounds with HCV replicon assay through inhibition of HCV polymerization. Among the hit compounds, compounds 1 and 2 showed 56% and 48% inhibition of NS5B polymerization activity at 20 μM, respectively. In addition, compound 1 also exhibited replicon activity with EC₅₀ value of 2.16 μM. Following up the initial hit, we obtained derivatives of compound 1 and evaluated polymerization inhibition activity and HCV replicon assay. These results provide information necessary for the development of more potent NS5B inhibitors.     

Discovery of furan carboxylate derivatives as novel inhibitors of ATP-citrate lyase via virtual high-throughput screening

The enzyme ATP citrate lyase (ACL) catalyzes the formation of cytosolic acetyl CoA, the starting material for de novo lipid and cholesterol biosynthesis. The dysfunction and upregulation of ACL in numerous cancers makes it an attractive target for developing anticancer therapies. ACL inhibition by shRNA knockdown limits cancer cell proliferation and reduces cancer stemness. We designed and implemented a dual docking protocol to select virtual ACL inhibitors that were scored among the top 10 percentiles by both the Autodock Vina and the Glamdock algorithms. Via this in silico screens of a focused furoic acid library, we discovered four subtypes of furans and benzofurans as novel ACL inhibitors. The hit rate of our in silico protocol was 45.8% with 11 of 24 virtual hits confirmed as active in an in vitro ACL enzymatic assay. The IC₅₀ of the most potent ACL inhibitor A1 is 4.1 μM. Our results demonstrated remarkable hit rate by the dual docking approach and provided novel chemical scaffolds for the development of ACL inhibitors for the treatment of cancer.     

新冠病毒主蛋白酶小分子抑制剂荧光共振能量转移高通量筛选模型的优化与应用北大核心CSCD

基于荧光共振能量转移(fluorescence resonance energy transfer, FRET)原理,以新冠病毒主蛋白酶(main protease, Mpro)为靶标,建立并应用Mpro小分子抑制剂FRET高通量筛选模型,以期快速筛选新型Mpro小分子抑制剂。利用大肠杆菌原核表达与分离纯化高活性的Mpro,再以FRET法进行比活力测定。基于FRET原理,以7-甲氧基香豆素-4-乙酸(7-methoxycoumarin-4-acetic acid, MCA)与2,4-二硝基苯酚(2,4-dinitropheno, Dnp)标记的多肽作为Mpro水解底物,通过优化反应缓冲液、Mpro反应浓度、反应温度与时间及DMSO耐受浓度,建立并应用Mpro小分子抑制剂FRET高通量筛选模型进行苗头化合物的筛选。利用大肠杆菌实现了高活性Mpro的原核表达与分离纯化,且比活力不低于40 000 U/mg。通过一系列优化实验,使用0.4μmol/L Mpro与5μmol/L底物建立了Z′因子值为0.79的Mpro小分子抑制剂FRET高通量筛选模型,且反应体系中含有的二硫苏糖醇(1,4-dithiothreitol,DTT)是影响FRET筛选模型可靠性的重要因素。通过对天然产物化合物库进行高通量筛选,发现白花丹素与银杏酸在体外对Mpro酶活性具有良好的抑制作用。本研究建立了基于FRET原理的Mpro小分子抑制剂高通量筛选模型,初步证实了白花丹素与银杏酸是一类新型苗头化合物,为抗新型冠状病毒药物先导化合物的筛选与发现奠定了基础。     

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.     

An activity-based probe for high-throughput measurements of triacylglycerol lipases

The casitas B-lineage lymphoma (Cbl) proteins play an important role in regulating signal transduction pathways by functioning as E3 ubiquitin ligases. The Cbl proteins contain a conserved tyrosine kinase binding (TKB) domain that binds more than a dozen proteins, including protein tyrosine kinases (PTKs), in a phosphorylation-dependent manner. The cell surface expression levels of the PTKs are regulated by Cbl-mediated ubiquitination, internalization, and degradation. Dysfunction in this signaling cascade has resulted in prolonged activation of the PTKs and, therefore, has been implicated in inflammatory diseases and various cancers. Due to this negative regulatory function, Cbl has been largely ignored as a therapeutic target. However, recent studies, such as the identification of (i) gain of function c-Cbl mutations in subsets of myeloid cancer and (ii) c-Cbl as a prostate basal cell marker that correlates with poor clinical outcome, suggest otherwise. Here we report the development of a competitive high-throughput fluorescence polarization assay in a 384-well format to identify inhibitors of Cbl(TKB). The high-throughput screen readiness of the assay was demonstrated by screening the Prestwick Chemical Library.     

Discovery of antichagasic inhibitors by high-throughput screening with Trypanosoma cruzi glucokinase

A high-throughput screening (HTS) campaign was carried out for Trypanosoma cruzi glucokinase (TcGlcK), a potential drug-target of the pathogenic protozoan parasite. Glycolysis and the pentose phosphate pathway (PPP) are important metabolic pathways for T. cruzi and the inhibition of the glucose kinases (i.e. glucokinase and hexokinase) may be a strategic approach for drug discovery. Glucose kinases phosphorylate d-glucose with co-substrate ATP to yield G6P, and moreover, the produced G6P enters both pathways for catabolism. The TcGlcK – HTS campaign revealed 25 novel enzyme inhibitors that were distributed in nine chemical classes and were discovered from a primary screen of 13,040 compounds. Thirteen of these compounds were found to have low micromolar IC₅₀ enzyme – inhibition values; strikingly, four of those compounds exhibited low toxicity towards NIH-3T3 murine host cells and notable in vitro trypanocidal activity. These compounds were of three chemical classes: (a) the 3-nitro-2-phenyl-2H-chromene scaffold, (b) the N-phenyl-benzenesulfonamide scaffold, and (c) the gossypol scaffold. Two compounds from the 3-nitro-2-phenyl-2H-chromene scaffold were determined to be hit-to-lead candidates that can proceed further down the early-stage drug discovery process.     

Fluorescence polarization assays for high-throughput screening of neuropeptide FF receptors

We have developed the first fluorescence polarization assays of human neuropeptide FF2 receptors in 384-well microtiter plates. Assays are completed in a single well with no transfer, separation, or wash steps. The performance is suitable for high-throughput drug screening applications with regard to speed of analysis, magnitude of displaceable signal, precision, and sensitivity of various reagents. The rank order of potency of agonists and antagonists agrees well relative to the published radiometric filtration assays: DMe NPFF > NPFF > frog PP (Rana temporaria pancreatic polypeptide) > PQRFamide > BIBP 3226. The effect of highly colored compounds is very small on the polarization signal up to micromolar concentrations. The method serves as a simple and fast alternative to radioligand binding assays of antiobesity drug candidates related to NPFF receptors.     

Discovery of small molecule inhibitors of human uridine-cytidine kinase 2 by high-throughput screening

Clinically relevant inhibitors of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in mammalian de novo pyrimidine synthesis, have strong antiviral and anticancer activity in vitro. However, they are ineffective in vivo due to efficient uridine salvage by infected or rapidly dividing cells. The pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2), a ∼29 kDa protein that forms a tetramer in its active state, is necessary for uridine salvage. Notwithstanding the pharmacological potential of this target, no medicinally tractable inhibitors of the human enzyme have been reported to date. We therefore established and miniaturized an in vitro assay for UCK2 activity and undertook a high-throughput screen against a ∼40,000-compound library to generate drug-like leads. The structures, activities, and modes of inhibition of the most promising hits are described. Notably, our screen yielded non-competitive UCK2 inhibitors which were able to suppress nucleoside salvage in cells both in the presence and absence of DHODH inhibitors.     

Discovery of novel 2,4-disubstituted pyrimidines as Aurora kinase inhibitors

In order to explore novel Aurora kinase inhibitors, a series of novel 2,4-disubstituted pyrimidines were designed, synthesized and evaluated their in vitro anti-proliferative activities against a panel of cancerous cell lines (A549, HCT-116 and MCF-7). Among them, compound 12a showed the moderate to high anti-proliferative activities against A549 (IC₅₀ = 12.05 ± 0.45 μM), HCT-116 (IC₅₀ = 1.31 ± 0.41 μM) and MCF-7 (IC₅₀ = 20.53 ± 6.13 μM) cells, as well as the Aurora A and Aurora B inhibitory activities with the IC₅₀ values of 309 nM and 293 nM, respectively. Furthermore, compound 12a induced apoptosis by upregulated the pro-apoptotic proteins Bax and decreased the anti-apoptotic protein Bcl-xl in HCT-116 cells. Moreover, the molecular docking study showed that compound 12a had good binding modes with Aurora A and Aurora B and the bioinformatics prediction discovered that compound 12a exhibited good drug likeness using SwissADME. Taken together, these results indicated that 12a may be a potential anticancer compound that was worthy of further development as Aurora kinase inhibitor.     

Development of a highly sensitive,high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection

Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z′-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators.     

High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1

The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening.     

Discovery of novel pyruvate dehydrogenase kinases inhibitors by screening of an in-house small molecule library for anti-lung cancer therapeutics

Pyruvate dehydrogenase kinases (PDKs) are widely over-expressed in various human solid cancers, making them attractive therapeutic targets for cancer treatment. Herein, we report the identification of structurally novel PDKs inhibitors by screening of an in-house small molecule library. Biochemical assay indicated that the identified compounds 1–4 inhibited PDK1 activity with EC₅₀ values of 0.50, 1.99, 4.64, and 0.42?µM, respectively. The ITC analysis suggested that the identified compounds 1–4 were pan-isoform PDK inhibitors, which bound to and inhibited the four PDK isoforms. Moreover, 1–4 dose-dependently reduced pyruvate dehydrogenase complex phosphorylation in NCI-H1975 cell. Molecular docking suggested that the most potent compound 4 docked well in the ATP binding pocket of the four PDK isoforms, forming direct hydrogen bond interactions with the conserved amino acids Thr and Asp in ATP binding pocket of PDKs. The cell viability assay demonstrated that 4 potently blocked NCI-H1975 cell proliferation (IC₅₀?=?3.32?µM), but had little effect on human normal lung cell MRC-5 even with the tested concentration up to 40?µM. All the data demonstrated that 4 was a promising lead for the development of structurally novel PDKs inhibitor for the cancer treatment.     

Design and characterization of bivalent Smac-based peptides as antagonists of XIAP and development and validation of a fluorescence polarization assay for XIAP containing both BIR2 and BIR3 domains

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis by binding to and inhibition of caspase-3 and caspase-7 through its BIR2 domain and caspase-9 through its BIR3 domain. Smac (second mitochondria-derived activator of caspases) protein is an endogenous antagonist of XIAP. Smac forms a dimer and concurrently binds both the BIR2 and BIR3 domains in XIAP, functioning as a highly efficient and potent cellular inhibitor of XIAP. In this article, we have designed and synthesized a bivalent Smac-based ligand (Smac-1) and its fluorescent labeled analogue (Smac-1F) and characterized their interaction with different constructs of XIAP. Our study demonstrates that bivalent Smac-based ligands bind concurrently to both the BIR2 and BIR3 domains of XIAP and are more than 500 times more potent than the corresponding monovalent Smac-based ligands. Bivalent Smac-based ligands also function as much more potent antagonists of XIAP than do the corresponding monovalent Smac-based ligands in cell-free functional assays. Using Smac-1F and XIAP containing both BIR2 and BIR3 domains, we also developed and validated a new fluorescence polarization-based assay. Hence, our designed bivalent Smac-based peptides mimic the mode of dimeric Smac protein in their interaction with XIAP containing both BIR2 and BIR3 domains and achieve extremely high potency in binding and functional assays. Our study provides new insights into the mode of action of bivalent Smac ligands targeting XIAP and a basis for the design and development of cell-permeable, bivalent Smac mimetics.