Discovery of novel aromatase inhibitors using a homogeneous time-resolved fluorescence assay

Aim:

Aromatase is an important target for drugs to treat hormone-dependent diseases, including breast cancer. The aim of this study was to develop a homogeneous time-resolved fluorescence (HTRF) aromatase assay suitable for high-throughput screening (HTS).

Methods:

A 384-well aromatase HTRF assay was established, and used to screen about 7000 compounds from a compound library. Anti-proliferation activity of the hit was evaluated using alamarBlue(R) assay in a hormone-dependent breast cancer cell line T47D. Molecular docking was conducted to elucidate the binding mode of the hit using the Discovery Studio program.

Results:

The Z′ value and signal to background (S/B) ratio were 0.74 and 5.4, respectively. Among the 7000 compounds, 4 hits (XHN22, XHN26, XHN27 and triptoquinone A) were found to inhibit aromatase with IC₅₀ values of 1.60±0.07, 2.76±0.24, 0.81±0.08 and 45.8±11.3 μmol /L, respectively. The hits XHN22, XHN26 and XHN27 shared the same chemical scaffold of 4-imidazolyl quinoline. Moreover, the most potent hit XHN27 at 10 and 50 μmol/L inhibited the proliferation of T47D cells by 45.3% and 35.2%, respectively. The docking study revealed that XHN27 docked within the active site of aromatase and might form a hydrogen bond and had a π-cation interaction with amino acid residues of the protein.

Conclusion:

XHN27, an imidazolyl quinoline derivative of flavonoid, is a potent aromatase inhibitor with anti-proliferation activity against breast cancer in vitro. The established assay can be used in HTS for discovering novel aromatase inhibitor.     

A simple and rapid assay for heparanase activity using homogeneous time-resolved fluorescence

Degradation of heparan sulfate proteoglycan by heparanase is an important process in tissue invasion by malignant tumor cells and inflammatory cells. Several heparanase assays have been reported previously, but all of them are cumbersome and time-consuming for separating degradative products from uncleaved substrates and detecting these products. This paper describes the development of a simple and rapid assay for heparanase activity using homogeneous time-resolved fluorescence based on non-radiative energy transfer. In this assay, heparan sulfate proteoglycan labeled with biotin and europium cryptate (fluorescence donor) was used as a substrate for heparanase. Degradation of the substrate by incubation with murine melanoma cell extract was detected by measuring the time-resolved fluorescence after addition of XL665 (cross-linked allophycocyanin, fluorescence acceptor)-labeled streptavidin. As heparanase activity can be simply measured by successive addition of substrate, enzyme and detection reagent onto one assay plate, our heparanase assay allows rapid processing of large numbers of samples.     

Development of BACE1 inhibitors for Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia. The production and accumulation of beta-amyloid peptides (Abeta) from the beta-amyloid precursor protein (APP) are believed to play a key role in the onset and progression of AD. BACE1 (beta-site APP cleaving enzyme 1) is the protease responsible for the N-terminal cleavage of APP leading to the production of Abeta peptides and the development of BACE1 inhibitors as potential therapeutic agents for AD has generated tremendous interests from both academia and the pharmaceutical industry. A wide variety of BACE1 inhibitors have been reported, several of which have demonstrated highly promising efficacy in animal models of AD. This review focuses on recent disclosures of BACE1 inhibitors in the patent and scientific literature, covering the period from approximately May 2004 to November 2005.     

Development and implementation of a miniaturized high-throughput time-resolved fluorescence energy transfer assay to identify small molecule inhibitors of polo-like kinase 1

Polo-like kinase (Plk) 1 is a key enzyme involved in regulating the mammalian cell cycle that is also a validated anticancer drug target. Nonetheless, there are relatively few readily available potent and selective small molecule inhibitors of Plk1. To increase the availability of pharmacologically valuable Plk1 inhibitors, we describe herein the development, variability assessment, validation, and implementation of a 384-well automated, miniaturized high-throughput time-resolved fluorescence energy transfer screening assay designed to identify Plk1 kinase inhibitors. Using a small molecule library of pharmaceutically active compounds to gauge high-throughput assay robustness and reproducibility, we found nine general kinase inhibitors, including H-89, which was selected as the minimum control. We then interrogated a 97,101 compound library from the National Institutes of Health repository for small molecule inhibitors of Plk1 kinase activity. The initial primary hit rate in a single 10 microM concentration format was 0.21%. Hit compounds were subjected to concentration-response confirmation and interference assays. Identified in the screen were seven compounds with 50% inhibitory concentration (IC50) values below 1 microM, 20 compounds with IC50 values between 1 microM and 5 microM, and eight compounds with IC50 values between 5 and 10 microM, which could be assigned to seven distinct chemotype classes. Hit compounds were also examined for their ability to inhibit other kinases such as protein kinase D, focal adhesion kinase, rho-associated coiled coil protein kinase 2, c-jun NH2-terminal kinase 3, and protein kinase A via experimentation or data-mining. These compounds should be useful as probes for the biological activity of Plk1 and as leads for the development of new selective inhibitors of Plk1.     

Conformationally controlled mechanistic aspects of BACE 1 inhibitors

This study highlights conformationally controlled mechanistic aspects of peptide inhibitors for BACE 1. Peptide inhibitors with reduced molecular weight tend to have cyclic conformation leading to reduced interactions with catalytic motif. Conformation plays a major role in determining potency of peptide inhibitors. An attempt has been made at designing lead compound with reduced molecular weight along with proper conformation suitable for active site and retention of specificity analogous to natural substrate. Reduced molecular weight should hopefully lead to enhanced bioavailability.     

BACE1抑制剂分子水平高通量筛选体系的建立

目的建立体外分子水平β-分泌酶（BACE1）抑制剂高通量筛选体系。方法采用均相时间分辨荧光法（HTRF）,通过优化反应时间、酶浓度、检测仪器等实验条件,建立BACE1抑制剂高通量筛选体系,根据待测样品对BACE1活性抑制程度筛选其抑制剂。结果采用HTRF法建立了BACE1抑制剂高通量筛选体系,其中反应时间确定为6h、BACE1浓度为670U/L,采用VICTOR3酶标仪测得信噪比为649.6,信背比为44.6,Z′因子为0.91,变异系数小于10%,并筛选到一系列IC50小于10-6mol/L的化合物。结论采用HTRF法建立的BACE1抑制剂筛选体系灵敏度高、特异性和稳定性好,可用于BACE1抑制剂的高通量筛选。     

β-分泌酶抑制剂的研究进展

β-淀粉样多肽(Aβ)聚积被认为是导致阿尔茨海默病(AD)病理变化的重要因素,而β-分泌酶(BACE1)是产生Aβ的限速酶.因此,以BACE1为靶标,抑制BACE1减少或阻断Aβ的生成已成为防治AD药物研究的热点,该文对近几年BACE1抑制剂的设计与进展进行简要的综述.     

The potential for BACE1 inhibitors in the treatment of Alzheimer's disease

Alzheimer's disease (AD) is a debilitating neurodegenerative disease of the elderly characterized by the loss of memory and other cognitive functions. Currently marketed drugs for the treatment of this disease only offer symptomatic relief, and, consequently, there is a large unmet clinical need for disease-modifying therapies for the treatment of AD. Substantial research efforts are focused on inhibiting the proteases involved in the generation of the amyloidogenic beta-amyloid (A beta) peptide. One of these key proteases, beta-secretase, was identified as a novel transmembrane aspartic protease and named BACE1. Due to its pivotal role in A beta production, many pharmaceutical companies are actively pursuing the challenging task of developing BACE1 inhibitors for evaluation in the clinic.     

A time-resolved fluorescence immunoassay for insulin in rodent plasma

We describe a time-resolved fluoroimmunoassay (TR-FIA) for quantification of insulin in rodent serum and plasma in the picomolar levels typical of these samples. The method is a solid-phase, sequential saturation assay based on competition of unlabeled insulin and biotinamidocaproyl-labeled insulin for anti-insulin antibody. Europium-labeled streptavidin allows the DELFIA system (Wallac) to be used for detection. The assay is sensitive (0.1 fmol detection limit, EC50 = 58 +/- 3 pM), accurate ( > 95% recovery of 88-880 pM insulin added to the samples), and simple enough to be automated in a 96-well microtiter plate format. Blood samples of 5 microl can be quickly processed and analyzed within a working concentration range of 40-200 pM, allowing direct measurement of insulin levels in rodents from a tail bleed. We used the TR-FIA to assess insulin levels in mouse and rat samples. In studies of streptozotocin-induced diabetes, as well as glucose load experiments, the assay gave results consistent with known literature. The measured insulin levels correlated significantly with values obtained by radioimmunoassay (R2 = 0.996). The intra-assay and inter-assay coefficients of variation were 2.3% and 15%, respectively. We compared results of this assay with an enzyme-linked immunosorbent assay (ELISA) method. The TR-FIA method was comparable to the ELISA but had higher sensitivity and required only one-tenth as much sample. The assay can be performed using commercially available reagents that allow for high sensitivity and practicability.     

A homogeneous G protein-coupled receptor ligand binding assay based on time-resolved fluorescence resonance energy transfer

Abstract: Fluorescence resonance energy transfer (FRET) has emerged as a powerful tool to the study of protein-protein interactions, such as receptor-ligand binding. However, the application of FRET to the study of G protein-coupled receptors (GPCRs) has been limited by the method of labeling receptor with fluorescence probes. Here we described a novel time-resolved (TR)-FRET method to study GPCR-ligand binding by using human complement 5a (C5a) receptor (C5aR) as a model system. Human C5aR was expressed in human embryonic kidney 293 cells with a hemagglutinin (HA) epitope at the N-terminus. Purified human C5a was labeled with terbium chelate and used as the fluorescence donor. Monoclonal anti-HA antibody conjugated with Alexa Fluor 488 was used as the fluorescence acceptor. Robust FRET signal was observed when the labeled ligand and C5aR membrane were mixed in the presence of the conjugated anti-HA antibody. This FRET signal was specific and saturable. C5a binding affinity to C5aR measured by the FRET assay was consistent with the data as determined by competition binding analysis using radiolabeled C5a. The FRET assay was also used to determine affinity of C5aR antagonists by competition binding analysis, and the data are similar to those from radioligand binding studies. Compared to the commonly used radioligand binding assay, this TR-FRET-based assay provides a nonradioactive, faster, and sensitive homogeneous assay format that could be easily adapted to high-throughput screening. The principle of this assay should also be applicable to other GPCRs, especially to those receptors with peptide or protein ligands.     

苯甲脒类BACE1抑制剂的设计和合成(英文)

本文通过对已有小分子库进行虚拟筛选, 得到了可与BACE1活性中心Asp228、Asp32形成氢键作用的苯甲脒片段, 并在其基础上, 设计出了3-苯乙基苯甲脒类BACE1抑制剂。虽然活性测试结果显示这些化合物对BACE1的抑制活性较弱, 但是可以通过进一步的结构优化3-苯乙基苯甲脒类化合物来提高对BACE1的抑制活性。     

Serotonin derivatives as inhibitors of beta-secretase (BACE 1)

All serotonin derivatives described here (1-9) inhibited BACE 1 in a dose dependent manner. The 50% Inhibition Concentration (IC50) of N-cinnamoyl serotonin (1) was 86.7 +/- 4.0 microM. The peptide conjugation of serotonin derivatives influenced the BACE 1 inhibitory activity. Among serotonin derivatives (1-8), introduction of substituents, such as hydroxyl and methoxy groups at the 4'-position decreased the inhibitory activity (N-p-coumaroyl serotonin (2), N-p-methoxy cinnamoyl serotonin (3)). With a hydroxylgroup at the 4'-position, and the meta-hydroxy function being substituted by a hydroxyl group or methoxy group (N-caffeoyl serotonin (4), N-feruloyl serotonin (5)), inhibitory activity was weakened, (IC50 >400 microM). BACE 1 inhibitory activity was effected by the substituents of the cinnamic acid moiety. This is the first report on Structure-Activity-Relationships (SAR) for the BACE 1-inhibiting activity of serotonin derivatives. These serotonin derivatives, which have anti-oxidative effects as well are expected to be useful in the study of the mechanisms of Alzheimer's disease.     

Development of filtration-based time-resolved fluorescence assay for the high-throughput screening of urotensin II receptor antagonist

The time-resolved fluorescence (TRF) receptor binding assay has many advantages over the traditional radioligand binding assay in terms of sensitivity and reproducibility for the screening of receptor ligands. The TRF-based urotensin receptor (UT) binding assay with an automatic vacuum filtration system was developed and evaluated for the high-throughput screening of UT receptor antagonists. For this assay development, the human recombinant urotensin II (UII) was modified by labeling europium at its N-terminal position (Eu-UII) and used as a fluorescent tracer. The microsomal membrane fraction of UT receptor was prepared from HEK293 cells stably expressing the human UT receptor. The 50% inhibitory concentration (IC(50)) values of UII from competition binding assays with Eu-UII were 2.76 nM, which is very similar to that of fluorescence polarization (FP)-based UT receptor binding experiment (2.18 nM). Comparing with the FP-based receptor binding assay for UII (Z' factor, 0.36), the current TRF assay presented improved Z' factor (0.76) with a relatively higher signal-to-background ratio (1.5 and 2.1, respectively). The known high-affinity UT receptor antagonists, palosuran and SB657510, exhibited IC(50) values of 23.6 and 73.4 nM, respectively, which were consistent with the IC(50) values from FP-based receptor binding assay (30.6 and 78.7 nM, respectively). These results suggest that our filtration-based TRF UT receptor binding assay can achieve the desired sensitivity with higher reproducibility to adapt for the high-throughput screening of compound libraries.     

L-酒石酸衍生物的合成及其抑制-分泌酶活性

摘 要: 目的 设计并合成&;#61538;-分泌酶(BACE1)的小分子抑制剂。方法 基于BACE1的晶体结构及其配体的关键结构特征，设计并合成新结构的BACE1配体。结果与结论 合成了26个酒石酸衍生物。筛选结果表明，部分化合物对BACE1有一定的抑制作用。     

Recent progress in the drug discovery of non-peptidic BACE1 inhibitors

Background: β-Secretase, also called BACE1, is a promising molecular target for developing anti-Alzheimer's disease drugs. Several approaches of drug discovery for this therapy have been attempted, for example, substrate-based and structure-based designs, high-throughput screening, fragment-based lead generation and in silico screening. Method: In this review, we describe the design of non-peptidic BACE1 inhibitors from a historical perspective and not by the inhibitor's category. Conclusion: The respective methods have both merits and demerits and should be used in a mutually complementary manner.     

Design and synthesis of 2-aminobenzimidazoles as potential BACE1 inhibitors

Fragment-based drug discovery (FBDD) has been widely applied in the research of aspartyl protease inhibitors. In the present study, we reported our work on 2-aminobenzimidazole as the original fragment, which was predicted to bind with the catalytic aspartyl dyad (Asp228 and Asp32) of β-site amyloid precursor protein cleaving enzyme 1 (BACE1). A series of novel 2-aminobenzimidazole derivatives were designed and synthesized. The results from FRET assay revealed that three out of the 12 designed 2-aminobenzimidazoles could inhibit more than 50% of the enzymatic potency of BACE1 at 10 μM. Docking study showed that 2-aminobenzimidazole could form multiple hydrogen bonds and occupy S1/S2’ pockets well.     

Cell-based assay strategy for identification of motif-specific RANK signaling pathway inhibitors

Osteoclasts, the principal bone-resorbing cells, not only play a pivotal role in skeletal development and maintenance but are also implicated in the pathogenesis of various bone disorders such as postmenopausal osteoporosis, bone erosion in inflammatory conditions, and tumor-induced osteolysis. As a result, several antiresorptive drugs (agents capable of inhibiting osteoclast formation and/or function) have been developed and are widely used to prevent and treat these bone diseases. However, current antiresorptive agents either lack satisfactory efficacy or cause serious side effects in clinical management of these bone disorders. Almost a decade ago, the receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) was identified as an essential factor required for osteoclast formation. RANKL exerts the effect by binding to its receptor RANK on osteoclast precursors. RANKL also has a decoy receptor, osteoprotegerin (OPG), which inhibits RANKL function by competing with RANK for RANKL. The unraveling of the critical role for the RANKL/RANK/OPG system in osteoclast biology provides an unprecedented opportunity to develop more effective antiresorptive drugs. Unfortunately, the agents currently under development, such as OPG, RANK-Fc, and anti-RANKL antibodies, all inherit a serious drawback--lack of specificity, due to the involvement of the RANKL/RANK/OPG system in other biological processes such as immune response and mammary gland development. Thus, future efforts may need to shift to explore RANK signaling pathways as more effective therapeutic targets. Here, we review our current understanding of RANK signaling in osteoclasts and then discuss the potential of RANK signaling pathways as therapeutic pathways. Moreover, we further describe a strategy for constructing novel cell-based systems for identifying compounds inhibiting signaling from two recently identified RANK motifs through high throughput screening. We hope that this review will not only provide readers with an update on progress in this area of research but, more importantly, will also serve as a starting point for further discussion and eventual development of new strategies for harnessing the ultimate potential of the RANKL/RANK/OPG system as antiresorptive therapeutic targets.     

Identification of a series of potent telomerase inhibitors using a time-resolved fluorescence-based assay

A high-throughput, nonradioactive telomerase assay using a europium-labeled oligonucleotide probe and time-resolved fluorescence has been developed to detect PCR-amplified telomerase products. The use of a thermally activated TAQ polymerase, rather than a wax barrier, to implement a PCR hot-start facilitated the use of laboratory automation equipment. Results obtained with this high-throughput protocol correlate well with those obtained with the TRAP protocol. Screening a set of 125,000 compounds from the Berlex library, we identified a set of isothiazolone-containing telomerase inhibitors. The most potent of these inhibitors have submicromolar IC₅₀ values and may be reacting with a telomerase thiol. These compounds may be useful tools to evaluate the effects of telomerase in cancer cells. Drug Dev. Res. 43:109–116, 1998. © 1998 Wiley-Liss, Inc.     

Iminoheterocycle as a druggable motif: BACE1 inhibitors and beyond

Beta-secretase 1 (BACE1) is a central nervous system (CNS) aspartyl protease required for production of amyloid beta (Aβ) peptides. Brain-penetrant BACE1 inhibitors are central to test the 'amyloid hypothesis', which suggests that a reduction of Aβ species in the CNS would halt or even reverse Alzheimer's disease. Discovery of the iminoheterocycle class of BACE1 inhibitors (which show robust efficacy in reduction of CNS Aβ species in animal models) marked an important milestone. These discoveries arose independently from multiple research laboratories that took different paths to end at the same scaffolds. These druggable motifs should be applicable to other aspartyl proteases of therapeutic importance and also to other protein targets involving crucial interactions with carboxylic acid side chains. Here, a comparison of these approaches to BACE1 discovery will showcase how it is important to pay attention to the intrinsic physicochemical properties of the lead series, even at the very early stage of drug discovery.     

A high throughput colorimetric cell proliferation assay for the identification of human cytomegalovirus inhibitors.

A colorimetric assay based on the cleavage of the tetrazolium salt WST-1 has been developed for human cytomegalovirus (HCMV) antiviral susceptibility testing and adapted to a microtiter plate format. Optimal conditions were determined and the standard routine assay was calibrated with a viral input of 0.05-0.10 plaque forming unit (p.f.u.)/cell with a density of 2000 cells/well in a 96-well microtiter plate for an incubation period of 7 days. Ganciclovir (9-(2-hydroxy-1(hydroxymethyl) ethyoxymethyl) guanine; DHPG), and cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine; HPMPC) were used as positive control test compounds to validate the assay. The effective EC50 concentration values obtained with the two antiviral compounds in the present assay were in good agreement with plaque reduction assay results performed in parallel experiments. This method presents the advantage of being easy and rapid to perform, reliable, reproducible, and convenient for use in a high throughput screening capacity.