Androgen receptor transactivation assay using green fluorescent protein as a reporter

For screening of a large number of samples for androgenic activity, a robust system with minimal handling is required. The coding sequence for human androgen receptor (AR) was inserted into expression plasmid YEpBUbi-FLAG1, resulting in the plasmid YEpBUbiFLAG-AR, and the estrogen response element (ERE) on the reporter vector YRpE2 was replaced by an androgen response element (ARE), resulting in the plasmid YRpE2-ARE. Thus, a fully functional transactivation assay system with beta-galactosidase as a reporter gene could be created. Furthermore, green fluorescent protein (GFP) was introduced as an alternative reporter gene that resulted in a simplification of the whole assay procedure. For evaluation of both reporter systems, seven steroidal compounds with known AR agonistic properties (5 alpha-dihydrotestosterone, testosterone, androstenedione, 17 alpha-methyltestosterone, progesterone, epitestosterone, and d-norgestrel) were tested, and their potencies obtained in the different assays were compared. Furthermore, potencies from the transactivation assays were compared with IC(50) values obtained in radioligand binding assays. The newly developed androgen receptor transactivation assay is a useful tool for characterizing compounds with androgenic activity.     

Synthesis, conformational analysis and CB1 binding affinity of hairpin-like anandamide pseudopeptide mimetics.

We have designed, synthesized and evaluated the CB(1) binding affinity of a number of new conformationally restricted lipopeptides (1-17). All of them present some of the AEA key structural elements incorporated in a hairpinlike peptide framework. Among them, compounds 1-3 and 8 showed CB(1) affinities in competitive binding assays with K(i) values in the micromolar range (K(i) of AEA = 0.8 microM in the same assay). The remaining pseudopeptides showed little binding to the CB(1) receptor (with K(i) values >or= 50 microM). Conformational analysis on two representative compounds, performed by a combination of NMR studies, restrained molecular dynamics and QM calculations, allowed us to shed light on the structure-activity relationships (SAR), pointing to a correlation between the predominance of the hairpin-like structural motif and the CB(1) binding affinity. In a more general context, the present study may also prove useful in gaining additional insight into the biological relevance of the various AEA conformations.     

Arginine-aminoglycoside conjugates that bind to HIV transactivation responsive element RNA in vitro

HIV gene expression is crucially dependent on binding of the viral Tat protein to the transactivation RNA response element. A number of synthetic Tat-transactivation responsive element interaction inhibitors of peptide/peptoid nature were described as potential antiviral drug prototypes. We present a new class of peptidomimetic inhibitors, conjugates of L-arginine with aminoglycosides. Using a gel-shift assay and affinity chromatography on an L-arginine column we found that these compounds bind specifically to the transactivation responsive element RNA in vitro with Kd values in the range of 20-400 nM, which is comparable to the Kd of native Tat bound to the transactivation responsive element (10-12 nM). Confocal microscopy studies demonstrated that fluorescein-labelled conjugate penetrates into live cells. High affinity to the transactivation responsive element, low toxicity, and relative simplicity of synthesis make these compounds attractive candidates for antiviral drug design.     

Development and validation of competition binding assays for affinity to the extracellular matrix receptors, α(v)β(3) and α(IIb)β(3) integrin

The RGD (Arg-Gly-Asp) binding integrins α(v)β(3) and α(IIb)β(3) are integral components of various pathological and physiological processes, including tumor angiogenesis, osteoclast function, and thrombus formation. Because of this, there is interest in identifying novel compounds and proteins binding to these receptors as well as investigating the mechanism of these interactions. In this article, we describe the development and validation of competition binding assays for determining the affinity of test compounds to α(v)β(3) and α(IIb)β(3) integrin. Assays were successfully developed for each receptor, and the affinity of known compounds was comparable to published results. However, the inability of binding between α(IIb)β(3) integrin and the labeled echistatin protein ligand to reach equilibrium resulted in an assay that did not meet the assumptions of the competition binding model. Nevertheless, there was good agreement between this assay and known literature values, and intra- and interassay variability was acceptable. Binding by conformation-specific antibodies provided evidence that solid-phase bound α(IIb)β(3) receptor was in an activated conformation. This study also demonstrated that current models and methods for determining receptor affinity are simplistic and fail to account for common receptor-ligand interactions such as nondissociable interactions and varying receptor activation states.     

Investigation of the lipophilic behaviour of some thiazolidinediones. Relationships with PPAR-gamma activity

Various lipophilicity aspects of five well-known PPAR-gamma ligands, belonging to the thiazolidinedione (TZD) class, ciglitazone (CSZ), troglitazone (TGZ), netoglitazone (NGZ) and the ampholytic pioglitazone (PGZ) and rosiglitazone (RGZ), have been explored. The compounds were found to be highly lipophilic as assessed by direct octanol-water partitioning experiments and further confirmed by reversed phase HPLC measurements under different conditions. Immobilised artificial membrane (IAM) chromatographic indices were also determined as an alternative expression of lipophilicity. They were found to show less diversity forming two clusters. Experimental logD/logP values were compared to those predicted by three widely used calculation systems. For the two ampholytic TZDs, the lipophilicity and retention/pH profiles were established over a broad pH range and compared to the corresponding calculated profiles. Lipophilicity indices derived under the different conditions were further compared to biological activity, concerning in vitro transactivation (pEC(50)) and binding affinity (pK(i)) data, taken from literature. The most active TZD (RGZ) in both transactivation and binding assay proved to be the less lipophilic analogue. An equation relating pEC(50) data to experimental logD(7.4) or reversed-phase logk(w) values could be established, while pK(i) data did not lead to satisfactory correlation.     

Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.     

Kinetic studies of small molecule interactions with protein kinases using biosensor technology

Protein kinases are among the most commonly targeted groups of molecules in drug discovery today. Despite this, there are few examples of using surface plasmon resonance (SPR) for kinase inhibitor interaction studies, probably reflecting the need for better developed assays for these proteins. In this article, we present a general methodology that uses biosensor technology to study small molecule binding to eight different serine/threonine and tyrosine kinases. Mild immobilization conditions and a carefully composed assay buffer were identified as key success factors. The methodology package consists of direct binding studies of compounds to immobilized kinases, kinase activity assays to confirm inhibitory effects, detailed kinetic analyses of inhibitor binding, and competition assays with ATP for identification of competitive inhibitors. The kinetic assays resolve affinity into the rates of inhibitor binding and dissociation. Therefore, more detailed information on the relation between inhibitor structure and function is obtained. This might be of key importance for the development of effective kinase inhibitors.     

Synthesis and opioid activity of 2-substituted dynorphin A-(1-13) amide analogues.

A series of 2-substituted dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues was prepared by solid phase peptide synthesis and evaluated for opioid receptor affinities in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI) assay. Amino acid substitution at the 2 position produced marked differences in both opioid receptor affinities and potency in the GPI assay; Ki values for the analogues in the radioligand binding assays and IC50 values in the GPI assay varied over three to four orders of magnitude. The parent peptide, Dyn A-(1-13)NH2, exhibited the greatest affinity and selectivity for kappa receptors and was the most potent peptide examined in the GPI assay. The most important determinant of opioid receptor selectivity and opioid potency for the synthetic analogues was the stereochemistry of the amino acid at the 2 position. Except for [D-Lys2]Dyn A-(1-13)NH2 in the kappa receptor binding assay, the analogues containing a D-amino acid at position 2 were much more potent in all of the assays than their corresponding isomers containing an L-amino acid at this position. The L-amino acid-substituted analogues generally retained some selectivity for kappa opioid receptors. The more potent derivatives with a D-amino acid in position 2, however, preferentially interacted with mu opioid receptors. Introduction of a positively charged amino acid into the 2 position generally decreased opioid receptor affinities and potency in the GPI assay.     

4-(3-Aryloxyaryl)quinoline sulfones are potent liver X receptor agonists

A series of 4-(3-aryloxyaryl)quinolines with sulfone substituents on the terminal aryl ring (7) was prepared as LXR agonists. High affinity LXR ligands with excellent agonist potency and efficacy in functional assays of LXR activity were identified. In general, these sulfone agonists were equal to or superior to previously described alcohol and amide analogs in terms of affinity, functional potency, and microsomal stability. Many of the sulfones had LXRβ binding IC₅₀ values <10 nM while the most potent compounds in an ABCA1 mRNA induction assay in J774 mouse cells had EC₅₀ values <10 nM and were as efficacious as T0901317.     

Synthesis of 4-(3-biaryl)quinoline sulfones as potent liver X receptor agonists

A series of 4-(3-biaryl)quinolines with sulfone substituents on the terminal aryl ring (8) was prepared as potential LXR agonists. High affinity LXRβ ligands with generally modest binding selectivity over LXRα and excellent agonist potency in LXR functional assays were identified. Many compounds had LXRβ binding IC₅₀ values <10 nM while the most potent had EC₅₀ values <1.0 nM in an ABCA1 mRNA induction assay in J774 mouse cells with efficacy comparable to T0901317. Sulfone 8a was further evaluated in LDL (?/?) mice and shown to reduce atherosclerotic lesion progression.     

Characterization of a new class of selective nonsteroidal progesterone receptor agonists

The identification of a new series of selective nonsteroidal progesterone receptor (PR) agonists is reported. Using a high-throughput screening assay based on the measurement of transactivation of a mouse mammary tumor virus promoter-driven luciferase reporter (MMTV-Luc) in human breast cancer T47D cells, a benzimidazole-2-thione analog was identified. Compound 1 showed an apparent EC50 of 53 nM and efficacy of 93% with respect to progesterone. It binds to PR with high affinity (Ki nM), but had no or very low affinity for other steroid hormone receptors. Structure-activity relationship studies of a series of benzimidazole-2-thione analogs revealed critical positions for high PR binding affinity and transactivation potency as well as receptor selectivity, as exemplified by 25. Compound 25 binds to human PR with high affinity (Ki nM) and had at least > 1000-fold selectivity for PR versus other steroid receptors. Molecular modeling studies suggested that these agonists overlap favorably with progesterone in the ligand-binding domain of PR. In T47D cells, compound 25 acted as a full agonist in the MMTV-Luc reporter assay, as well as in the induction of endogenous alkaline phosphatase activity with apparent EC50 values of 4 and 9 nM, respectively. In the immature rat model, compound 25 provided a significant suppression of estrogen-induced endometrium hypertrophy as measured by luminal epithelial height. In contrast, compound 25 was inactive in the luteinizing hormone release assay in young ovariectomized rats. These benzimidazole-2-thione analogs constitute a new series of nonsteroidal PR agonists with an excellent steroid receptor selectivity profile. The differential activities observed in the in vivo progestogenic assays in rat models suggest that these analogs can act as selective PR modulators.     

Influence of the substitution of 11-methylene, delta(15), and/or 18-methyl groups in norethisterone on receptor binding, transactivation assays and biological activities in animals

The profile of norethisterone and newly developed derivatives thereof were assessed by in vitro binding and transactivation assays on progesterone (PR) as well as on androgen (AR) receptors and by subcutaneous treatment in in vivo models. The following in vivo models were performed: A McPhail test for progestational activity in immature rabbits, an ovulation inhibition test in cycling rats and a Hershberger test for androgenic activity in immature orchidectomised rats. The compounds tested were: norethisterone (NET), 11-methylene-NET (11-NET), Delta(15)-NET (15-NET), 18-methyl-NET (18-NET, Levonorgestrel, LNG), 11-methylene-Delta(15)-NET (11, 15-NET), 11-methylene-18-methyl-NET (11,18-NET, 3-keto-desogestrel, Etonogestrel, ETG), (Delta(15)-18-methyl-NET (15,18-NET, Gestodene, GSD) and 11-methylene-Delta(15)-18-methyl-NET (11,15,18-NET). Compared to the non-substituted compound NET, the binding to and agonistic activity via PR was increased for all the three mono-substituted compounds, although the stimulatory effect of 15-NET was only twofold. Compounds with 18-methyl in combination with Delta(15) (GSD), with 11-methylene (ETG) or with both combined showed clear synergistic effects, leading to equipotent compounds. If the 18-methyl group was lacking as in 11,15-NET, potency was lower than for ETG or GSD, but higher than for 18-NET (LNG). A correlation coefficient of 0.9 was found between binding affinity and agonistic potency. With respect to the AR binding and transactivation activities, the 18-methyl group potentiated androgenic in vitro activity (LNG). The 11-methylene group increased relative binding affinity in NET, but reduced androgenic activity clearly when also other substituents were present (11,15-NET, ETG and 11,15,18-NET). The Delta(15) bond alone did not change the binding in NET, but decreased androgen binding, induced by the 18-methyl substituent, in GSD and 11,15,18-NET. Transactivation activity was also diminished in the compounds having a Delta(15) bond. In the McPhail test mono-substitution of NET increased the progestagenic in vivo activity three to five times. Bi- and tri-substitution enhanced the activity further. With respect to ovulation inhibition mono-substitution of NET resulted in three to nine times more potent compounds, whereas bi- and tri-substitution increased potency further, except for 11,15-NET, which was as active as 11-NET. The relative progestagenic potencies in the McPhail and ovulation inhibition tests, correlated significantly with those of the relative binding affinity values (correlation coefficient of 0. 91 and 0.93, respectively) and relative transactivation activity values (0.88 and 0.81) for the PR. In the Hershberger test, all the compounds increased androgenic activity with respect to growth of ventral prostate weight compared to NET, with the exception of 11, 15-NET and 11,15,18-NET. The androgenic activity was negligible for these latter compounds. The androgenicity of both 18-NET (LNG) and 15,18-NET (GSD), on the other hand, was significantly higher than that of 11,18-NET (ETG). The results of this in vivo test are in line with the AR binding and transactivation activity values (correlation coefficients of 0.86 and 0.88). In addition, selectivity indices were calculated by dividing the progestational potencies by androgenic potencies for both in vitro and in vivo assays. ETG and GSD had clearly higher in vitro and in vivo indices than the other compounds with NET and LNG having the lowest indices. Because the androgenicity of 11,15-NET and 11,15,18-NET was very low, no exact selectivity ratios could be calculated for these compounds. From these experiments we may conclude that small structural modifications exert enhancement of progestational activity and a clear reduction in androgenicity leading to very selective progestagenic compounds. The influence of bi-substitution is additive over mono-substitution, whereas tri-substition is not additive. (ABSTRACT TRUNCATED)     

Comparison of assay technologies for a tyrosine kinase assay generates different results in high throughput screening

In today's high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC(50) values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC(50) values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.     

Cytotoxicity and topoisomerase I/II inhibition of glycosylated 2-phenyl-indoles, 2-phenyl-benzo[b]thiophenes and 2-phenyl-benzo[b]furans

A panel of glycosylated DNA binding agents (1-12) designed as functional anthracycline mimics was screened against three solid-tumor cell lines (MCF-7, HT 29 and HepG2/C3A) and three non-tumor cell lines by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) cell viability assay. Several compounds showed better in vitro cytotoxicity and selectivity against MCF-7 cells than daunomycin and doxorubicin, two known DNA binding agents that are clinically-used anti-cancer agents. Although the selectivity for HT 29 and HepG2/C3A cells is generally lower, the IC₅₀ values of some analogs against these two cancer cell lines were of the same magnitude as doxorubicin. Because there was no correlation between DNA binding affinity and cytotoxicity, and because topoisomerase (Topo) inhibition is another biological mechanism of action of most anthracycline drugs, Topo I/II inhibition assays with 1-12 were performed. Some of the compounds showed strong inhibition against these enzymes at 100 ??M, but there was no clear correlation between cytotoxicity and Topo I/II inhibition ability. Topo I/II inhibition mode assays were also performed, which verified that these compounds are topoisomerase suppressors, not poisons. Based on these results, we conclude that although DNA binding and/or topoisomerase inhibition may contribute to the observed cytotoxicity of 1-12, other mechanisms of action are also likely to be important.     

A vinblastine fluorescent probe for pregnane X receptor in a time-resolved fluorescence resonance energy transfer assay

The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows the binding of many drugs and drug leads to it, possibly causing undesired drug–drug interactions. Therefore, it is crucial to evaluate whether lead compounds bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. One fluorescent PXR probe is currently commercially available; however, because its chemical structure is not publicly disclosed, it is not optimal for studying ligand–PXR interactions. Here we report the characterization of BODIPY FL–vinblastine, generated by labeling vinblastine with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), as a high-affinity ligand for human PXR with a K_d value of 673 nM. We provide evidence that BODIPY FL–vinblastine is a unique chemical entity different from either vinblastine or the fluorophore BODIPY FL in its function as a high-affinity human PXR ligand. We describe a BODIPY FL–vinblastine-based human PXR time-resolved fluorescence resonance energy transfer assay, which was used to successfully test a panel of human PXR ligands. The BODIPY FL–vinblastine-based biochemical assay is suitable for high-throughput screening to evaluate whether lead compounds bind to PXR.     

Design, synthesis and biological evaluation of piperazine analogues as CB1 cannabinoid receptor ligands

After the CB1 receptor antagonist SR141716 (rimonabant) was previously reported to modulate food intake, CB1 antagonism has been considered as a new therapeutic target for the treatment of obesity. Several series of urea, carbamate, amide, sulfonamide and oxalamide derivatives based on 1-benzhydrylpiperazine scaffold were synthesized and tested for CB1 receptor binding affinity. The SAR studies to optimize the CB1 binding affinity led to the potent urea derivatives. After the additional SAR studies to optimize the substituents of diphenyl rings, the combination of 2-chlorophenyl and 4-chlorophenyl turned out to be the most potent scaffold. The CB2 binding affinity assay as well as functional assay was also conducted on these compounds. Herein we wish to introduce several novel CB1 antagonists with IC(50) values less than 100 nM for the CB1 receptor binding.