Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands - Article Example

?Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands INTRODUCTION Identification of ligands thatcome into contact with nuclear receptors is usually a major biological problem. Besides, it a critical first step in the process of drug discovery (Shi 685). A number of experimental techniques are usually used to screen possible ligand materials against nuclear receptors, however, none of the current assay screening techniques permits screening without modification of either the protein and/or the ligand in a high-throughput fashion. As a partial solution to this problem differential scanning fluorimetry (DSF) is normally used. This technique allows for the identification of conditions that enhance the stability of proteins. DSF is used for this process as it allows possible ligands that have not been modified to be screened as 10µL reactions in 96-well format against partially purified protein, revealing particular interactors. The researchers aim to find out whether differential scanning fluorimetry permits screening without modification of either the protein and/or the ligand in a high-throughput fashion using a commercially-available nuclear receptor ligand candidate chemical library. The researchers attempt to identify interactors of the human estrogen receptor ? ligand binding domain (ER? LBD). They will also investigate whether compounds that come into contact with the ligand stabilize the protein and lead to an increase of the thermal denaturation point. This process will be monitored using the SYPRO dye which is able to detect even slight environmental changes. BACKGROUND INFORMATION Nuclear receptors (NRs) are transcription factors that exist in all metazoa that control cellular activities. Incidentally, the activities of NRs are regulated by interactions that occur between small-molecule ligands and a structurally-conserved ligand-binding domain (LBD) of the NR. This interaction induce structural changes resulting in new interaction surfaces for the recruitment of transcriptional co-activators and/or co-repressors. A lot of attention has been focused on the identification of ligands that bind to NRs and this is pushed by two main factors: i. Identification of natural ligands of a given NR is important in comprehending the role of the NR within the organism, particularly when the ligand of the NR is not clearly known or understood; ii. Identification of the ligands is important in the development of drugs and has led to a rise of different techniques each with known strengths and weaknesses (DeSantis et al. 2). Differential scanning technologies permit identification of conditions that improve the stability of proteins. The conversion from native to denatured protein is measured in relation to temperature changes. Two differential scanning techniques that can be used are scattering (Differential Scanning Light Scattering) or the fluorescence of an environmentally-sensitive dye (Differential Scanning Fluorescence – DSF). The techniques allow for the variation of a number of variables such as pH, salt, addition of small molecules (Schulman and Heyman 642). Cell conditions that result into a shift of proteins to higher denaturation temperature are identified as “stabilizing” conditions. This knowledge has been used to create conditions that are optimal for crystal growth (Vedadi et al., 2006). Other studies have used this technique to establish that peptides can shift the denaturation point of proteins to a higher temperature. This paper attempts to show that DSF can be used to successfully identify known interactors of the estrogen receptor ? (ER?) from a commercially available compound library. MATERIALS AND METHODS i. Reagents and instruments His6-hER? LDB (302-552) expression vector Terrific Broth IPTG EDTA-Free Protease Inhibitor Tablets Branson Sonifier 450 Ni-NTA Superflow ?-estradiol pET15b Sypro Orange, 5000X CFX96 Real-Time PCR System Nuclear Receptor Ligand Library BML-2802 ii. Methods The first step in this study was the production and purification of estrogen receptor. In this process, BL21 (DE3) E. coli cells were transformed using an expression vector provided among the reagents. The second stage involved the production of protein “melting” curves. This was achieved using the RT-PCR machine was programmed to equilibrate samples at 25°C for 5 minutes and then increase temperature to 95°C at a rate of 1°C/minute, taking a fluorescence reading every 0.2°C using a LED/photodiode set matched to the excitation and emission wavelengths of SYPRO orange. The last stage involved ligand titrations in which A series of Estradiol (E2) dilutions was created in 10%(v/v) DMS on Analysis Buffer ranging from 1 to 500?M. Purified ER? LBD was assayed against the E2 dilution series at two different protein concentrations RESULTS AND DISCUSSION Upon purification by a single affinity column, the purification product containing human ER? LBD produced a denaturation curve with a melting point (Tm) of ~41°C (representative curve in Figure 1a-b). Addition of ?-estradiol (E2) produced a denaturation curve with two transitions, at approximately 41°C and 59°C. Addition of ?-estradiol (E2) produced a denaturation curve with two transitions, at approximately 41°C and approximately 59°C. When the denaturation experiment is run on the products of the “empty vector” purification, the resultant curve displays a melting curve that closely matches the melting point seen for apo ER?LBD and the first peak of ER? LBD plus E2 (Figure 1e-f), but does not change upon addition of E2. Titrations of the E2 yielded an apparent EC50 that is well above the sub-nanomolar reported values for ER/E2 associations. Both ligands demonstrated similar protein concentration - EC50 correlations. When viewed over a broader ligand concentration range, all three ligands are detected at 10µM, but also demonstrate subtle increases of the maximum Tm at higher concentrations of ligand. DSF requires relatively small amounts of both ligand and protein and has the ability to detect (but not differentiate) interactors that can potentially activate or repress transcription of the genes under the regulation of the NR. It is demonstrated DSF has the ability to identify all known interactors of ER?contained in a commercially-available compound library with a high degree of reproducibility and minimal-to-no background. The detection limit of this technique appears to be determined by combination of both the affinity of the protein-ligand interaction, as well as the concentration of the protein used in the assay. Lower protein concentration actually improves the detection limit, but quality of the signal sets a practical lower limit on how dilute a protein can be used. CONCLUSION For many nuclear receptors, especially among non-vertebrates, no ligands are known, making it impossible to incorporate ligands to improve the stability and behavior of a NR LBD. This experiment showed that DSF tolerates relatively low protein concentration and preparations that are not completely pure, making it especially amenable to qualitative identification of ligands of orphan NRs. The researchers contend that this technique has the potential to be applicable to other NR LBDs than can be at least partially purified. Works Cited DeSantis, Kara, Reed, Aaron, Rahhal, Raneen, and Reinking, Jeff. Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands. Nuclear Receptor Signaling, 10: 2012. 1-5. Schulman, Ian G. and Heyman, Robert A. The flip side: Identifying small molecule regulators of nuclear receptors Chem Biol 11: 2004. 639-46. Shi, Yin. Orphan nuclear receptors, excellent targets of drug discovery Comb Chem High Throughput Screen 9:2006. 683-9. Read More