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Small molecule microarray screening methodology based on surface plasmon resonance imaging
⁎Corresponding author at: National Center for Nanoscience and Technology, Beijing 100190, People’s Republic of China. Tel.: +91 8901474914. kasana.chem@gmail.com (Vikramjeet Singh)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
In order to increase the scope and utility of small molecule microarrays (SMMs) we have combined SMMs and SPRi to screen small molecule antagonists against protein targets. Several small molecules, including immunosuppressive drugs (rapamycin and FK506) and reported inhibitors (FOBISIN and Blapsin) of 14-3-3ζ proteins have been used to validate this technology. Furthermore, a small library of isatin derivatives have been synthesized and screened on developed platform against 14-3-3ζ protein. Three molecules, derived from the endogenous intermediate isatin termed, FZIB-35, FZIB-36 and FZIB-38 were identified as novel inhibitors which shows significant interaction with 14-3-3ζ. A mutation in the binding groove of 14-3-3ζ, (K49E), almost abolishes the binding of these compounds to 14-3-3ζ protein. To exclude the probability of false positives, two more purified proteins (PtpA and BirA) were also tested. Furthermore, in order to confirm the binding pocket specificity, competition assay against R18 peptide was also carried out on presented platform. We show that SMMs in combination with SPRi are a powerful method to identify lead compounds in high throughput manner without the need to develop an activity based assay.
Keywords
Small molecule microarray
Surface plasmon resonance
14-3-3ζ protein
Isatin and ligand–protein interaction
1 Introduction
Small molecule microarrays represent valuable tools for high throughput screening (HTS) in drug discovery (Kuruvilla et al., 2002) and enable the discovery of important and unknown protein–ligand interaction resulting in modulation of protein function (Koehler et al., 2003). SMMs in integration with cell based assay and confocal laser scanning microscopy (CLSM) have been also described (Darvas et al., 2004; Molnár et al., 2013). To date, a number of elegant methods have been described for screening of small molecule inhibitors against protein targets. Conventional HTS methods such as TR-FRET, fluorescence polarization and ALPHAscreen face daunting challenges due to a number of limitations such as fluorescence interference, protein labeling, small molecule solubility, and lengthy analysis times. Therefore, an alternative label free detection technology can be significantly advantageous. A great advantage of SPRi over classical SPR technique (Redman, 2007) is throughput, allowing the parallel evaluation of hundreds or thousands of compounds simultaneously (Pillet et al., 2010). Moreover it provides a rapid identification of biomolecular interaction along with their kinetic parameters in real time (Mcdonnell, 2001). A variety of small molecules have been reported on SPRi for measuring protein–ligand interaction and protein–protein inhibition (Jung et al., 2005; Pillet et al., 2011). In this article, a combination of SMMs and SPRi has been used to detect ligand–protein interaction. Different strategies have been described for developing diverse linker systems on solid supports capable of anchoring small molecules (Hackler et al., 2003). A selective immobilization strategy was used for the fabrication of the SMMs through, either amino or hydroxy functional group of small molecules. Compounds were covalently captured on gold chip through simple EDC/NHS chemistry linked via PEG chains. Three different types of experiments were carried out to check the specificity of the ligands to the related target and to exclude false positives. We validate this technology by using the interaction between FKBP12-Rapa-FK506 and some known inhibitors of 14-3-3ζ including the compounds FOBISIN (Zhao et al., 2011) and Blapsin (Yan et al., 2012). 14-3-3 proteins are a family of eukaryotic proteins that can bind to many phosphoserine/phospho-threonine containing signaling proteins such as kinases, phosphatases, and trans-membrane receptors (Aitken, 2006). Hundreds of signaling and disease associated proteins including p53 (Rajagopalan et al., 2010), C-Raf-1 (Molzan et al., 2010), BAD (Jiping et al., 1996), and histone deacetylases (Wang et al., 2000) have been documented to bind to 14-3-3 proteins. The dimeric 14-3-3ζ isoform (Liu et al., 1996), in particular, is one of the most widely expressed and plays a major role in apoptosis. Additionally, a recent investigation identified the ζ isoform as a biomarker with high specificity and sensitivity for the diagnosis and prognosis of head and neck cancer (Macha et al., 2010). Due to the involvement of 14-3-3 proteins in major cellular processes and diseases, current research has shifted toward the discovery of small molecule inhibitors which can provide good therapeutic opportunities. Over the last decade, a number of small molecule antagonists for 14-3-3 proteins have been studied (Yan et al., 2012) including some non-peptidic antagonists which act as inhibitors as well as stabilizers (Milroy et al., 2012). Currently, there is no reported use of SMMs and SPRi in the discovery of new 14-3-3 proteins inhibitors. The main purpose of this research is to evaluate the SPRi technology for the screening of small molecule inhibitors against 14-3-3ζ. Further, a small library of compounds derived from isatin, which contained at least one NH2 or OH group were immobilized and generate small molecule microarrays. Isatin is an endogenous Indole widely distributed in mammalian brain, peripheral tissue, and body fluids (Medvedev et al., 1996). 14-3-3ζ represents one of these targets having specific and comparatively high interaction with isatin (Buneeva et al., 2010). Recently, an isatin derivative has been reported (ID45) against coxsackievirus B3 (CVB3) replication (Zhang et al., 2014). The primary screening of all isatin derivatives results 3 potential hits against 14-3-3ζ. Four different purified proteins, FKBP12, PtpA and BirA including K49E mutant of 14-3-3ζ were tested against screened hits followed by competition approach against R18 peptide (Wang et al., 1999) shows promising inhibitory activity on SPR assay of identified compounds. In order to validate, these compounds further tested in ELISA and able to disrupt 14-3-3ζ interaction with its binding partner PRAS40 protein. Combination of these two advanced technologies, SMMs and SPRi provides rapid screening and kinetics parameters of the tested inhibitors. We believe that this method can be applied for large scale primary screenings at low cost and without the need to develop an activity assay.
2 Material and methods
2.1 Reagents
Unless otherwise noted, material and solvents were obtained from commercial suppliers and used without further purification. Gold coated slides (Plexera), SH-(PEG)n-COOH (M.W. 1000) and SH-(PEG)n-OH (M.W. 346) (Shanghai Yan Yi biotech.). EDC-HCl (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxy succinimide), DMAP (N,N-dimethyl amino pyridine) (Aladdin Chemistry). DMSO, ethanol and ACN (Aldrich). Superblock solution was ordered from Thermo Scientific. FOBISIN 101 and FOBISIN 106 were purchased from Sigma. FKBP12 protein was purchased from Sinobiological Inc. R18 peptide, Blapsin inhibitors, isatin library (34 compounds) and proteins such as 14-3-3ζ, 14-3-3ζ K49E mutant, PtpA BirA, were obtained from Prof. Lixin Zhang’s laboratory (Institute of Microbiology, Chinese Academy of Sciences). Synthesis procedure and NMR of identified inhibitors are presented in supplementary information.
2.2 SMMs protocol
A schematic representation for the screening process of SMMs is provided in Fig. 1. Freshly deposited gold coated standard SPRi chips were cleaned with piranha solution (70% H2SO4/30% H2O2) for 10 min. The chips were extensively rinsed with Millipore water for 30 min. The chips were then immersed in ethanol containing 1 mM solution of SH-(PEG)n-COOH and SH-(PEG)n-OH (1:10) at 4 °C overnight and washed (shaker) in pure ethanol for 30 min before drying with nitrogen. Here we used the standard EDC/NHS chemistry for covalent immobilization of the small molecules on the surface of the chips. The carboxylic group (–COOH) from the SH-PEG-COOH was modified with a 1:1 mixture of EDC (0.39 M)/NHS (0.1 M). N-hydroxy succinimide ester is a robust chemistry widely utilized and able to attack amine and hydroxyl nucleophile groups (Mädler et al., 2009) of small molecules and form stable amide and ester bonds respectively. Compounds at 10 mM concentration in 100% DMSO were spotted into duplicate using a Genetix Qarray mini printer (contact mode printing) produces 250 μM features, covalently immobilized on the sensor chip and blocked by superblock solution to minimize non-specific adsorption of proteins on the surface. A typical array image on a PlexArray® HT system (Plexera) is shown in supplementary Fig. 1. N,N-dimethyl amino pyridine (1 uM) aq. solution was added to the printing solutions to facilitate nucleophile attack to form the desired ester bond. The slides were subsequently washed with DMSO, CAN, DMF, ethanol, PBS and finally with distilled water for 30 min respectively to remove non-specifically adsorbed compounds.
Schematic representation of small molecule microarray.
2.3 SPRi method
All the experiments were carried out using the PlexArray® HT system which is based on surface plasmon resonance imaging (Guan and Cong, 2007). Small molecules containing at least one amino or hydroxy functional group are suitable to be immobilized using this strategy. Purified recombinant proteins, FKBP12, 14-3-3ζ, 14-3-3ζ (K49E), PtpA and BirA were in PBS pH 7.4 containing tween 20 (0.05%) and 10% glycerol. Different concentrations of proteins were used as analyte. A solution of NaOH (10 mM) was used to regenerate the surface and remove bound proteins from the SMMs enabling the sensor chip to be reused for additional analyte injections. All presented data were repeated three times to derive the standard deviations.
2.4 Binding experiments and data analysis
All the stock solutions of small molecules were stored in 100% dimethyl sulphoxide (DMSO) at −20 °C. Protein samples were stored in PBS with 10% glycerol at −20 °C. PBS was used as both assay and running buffer. A typical sample injection cycle consists of 200 s association phase with analyte solution and 300 s dissociation phase with running buffer at 3 ul/s flow rate. Multiple concentrations of each protein 14-3-3ζ (200, 400 and 600 nM) and FKBP12 (25, 50 and 100 nM) were flowed on the SPRi instrument as analyte to get accurate kinetic parameters. Other purified proteins such as 14-3-3ζ (K49E), PtpA and BirA were tested to confirm binding pocket specificity. The highest concentration tested for each protein was 600 nM. For data analysis, we used two software packages: data were analyzed according to our previous work (Singh et al., 2014). The specific binding of protein to the immobilized small molecules was determined by subtracting the nonspecific physical adsorption on reference spots using the Plexera SPR Data Analysis Module.
3 Results
3.1 High throughput screening of inhibitors by SPR imaging assay
The microarrays were then blocked and washed before exposing them to the purified recombinant proteins FKBP12 and 14-3-3ζ. As shown in Fig. 2A, the Rapamycin and FK506 spots bound the FKBP12 protein specifically. Conversely, FOBISIN and Blapsin showed specific binding to 14-3-3ζ, (Fig. 2B). The resultant arrays can be regenerated with 10 mM aqueous NaOH solution and reused several times showing a great reproducibility. Unrelated compounds and surface background were used as negative controls for subtraction to ensure real binding interactions. Rapamycin and FOBISIN 101 were used as negative and positive control against each other. The chemical structures of the 14-3-3ζ (FOBISIN and Blapsin) hits are shown in (Supplementary Fig. 2). The further screening of isatin compounds revealed that three reproducible compounds (FZIB-35, FZIB-36 and FZIB-38) show significant binding (Fig. 3A) to the 14-3-3ζ protein and no response against other proteins including FKBP12, BirA and PtpA. Chemical structures of the isatin hits are shown in Fig. 3B.
Identification of inhibitors by SPRi (A) SPRi graph showing interaction of Rapamycin and FK506 with FKBP12 protein with FOBISIN as a negative control and (B) identification of FOBISIN and Blapsin inhibitors against 14-3-3ζ protein (Rapa was taken as negative control) on SMMs platform.
Identification and structure of inhibitors (A) SPRi graph showing interaction of three identified inhibitors, FZIB-38, FZIB-35 and FZIB-36 including R18 as a positive control and rapamycin as a negative control and (B) chemical structure of identified inhibitors.
3.2 Binding pocket confirmation by SPR imaging assay
In order to test whether these interactions were specific, we first tested different proteins. In the case of the 14-3-3 inhibitors, three unrelated recombinant proteins FKBP12, PtpA, and BirA were tested for binding. These purified proteins were used as analytes and passed at the highest concentration (600 nM) through the SPRi chip to check non-specific binding. All compounds showed no significant binding to PtpA or BirA. FKBP12 once again shows clear binding to the known immunosuppressive drugs Rapamycin and FK506 (Banaszynski et al., 2005). This confirms that FKBP12 was folded properly and it binds specifically to these compounds but not to the unrelated compounds immobilized on the chip (data not shown). As shown in Fig. 4A and B, all known inhibitors including 3 novel hits only bind to the 14-3-3ζ but not to FKBP12, and PtpA or BirA is supporting the specificity of these interactions.
Screening results against mutant and other unrelated proteins. (A) SPR response of all protein targets to known 14-3-3ζ inhibitors and (B) response of new identified inhibitor toward all target proteins. (C) injection of 14-3-3ζ protein followed by K49E mutant of 14-3-3ζ injection shows complete abolishment of binding with known inhibitors and (D) new isatin inhibitors which indicates that inhibitor binds to the specific pocket of 14-3-3ζ.
Structural analysis of 14-3-3ζ has determined that the amphipathic groove is the primary ligand binding site. The amphipathic groove lines up with the surface residue which is conserved between all isoforms of 14-3-3 proteins. Lys-49 is located in the conserved ligand binding site and plays a critical role in ligand interaction ((Zhang et al., 1997). Charge reversal mutation K49E in 14-3-3ζ has shown to decrease its interaction with Raf-1 kinase and thus with R18 peptide (Wang et al., 1998). In order to demonstrate that the interaction of 14-3-3ζ with the aforementioned compounds was via the specific binding pocket, we tested the 14-3-3ζ (K49E) mutant. Two subsequent injections of 14-3-3ζ and 14-3-3ζ (K49E) separated by single regeneration were flowed on a single chip. As shown in Fig. 4C and D the binding of the 14-3-3ζ (K49E) mutant to each inhibitor was dramatically reduced to negligible. This again strongly suggests that, known inhibitors including novel hits represent bona fide inhibitors that bind to the primary ligand binding site.
3.3 Competition assay on SPR imaging
To further confirm that the SMMs combined with SPRi can detect specific binding events of 14-3-3ζ toward their inhibitors, a competition assay based on SPR imaging was developed. R18 is a high affinity peptide antagonist of 14-3-3ζ protein which has strong interaction in the range of 70 nM. We used the R18 peptide as a competitive inhibitor for the immobilized FOBISIN 101, FOBISIN 106, BLAP1, BLAP2, and BLAP3. 14-3-3ζ was injected either alone, or in a mixture with two concentrations of the R18 peptide (Zeta + R18_300 nM and Zeta + R18_600 nM). In all of three injections (Fig. 5A and B), the concentrations of 14-3-3ζ were constant (600 nM). The mixture containing 300 nM R18 peptide shows a dramatic reduction in the signal. The binding signal was almost negligible when the concentration of R18 was increased to 600 nM (Fig. 5C and D). These data together with the lack of binding of the 14-3-3ζ (K49E) mutant to the each inhibitor spots strongly support the ability of these compounds to disrupt functional interactions with relevant physiological partners.
Competition assay of all 14-3-3ζ inhibitors (A) sensorgram showing competition assay against R18 peptide. First injection of 14-3-3ζ followed by two injections of same concentrations in addition to 300 nM and 600 nM of R18 peptide in first and second respectively against known inhibitors and (B) new identified isatin inhibitors to further confirm specific pocket phenomenon. (C) Behavior of 14-3-3ζ to known inhibitors and (D) identified inhibitors in completion assay.
3.4 Verification by ELISA
To validate and see whether new hits screened from SPRi assay has some inhibition activity in solution, compounds were tested in ELISA. ELISA was performed in the same conditions used in the identification of the FOBISIN inhibitor of 14-3-3 protein (see supplementary info.) by Dr. Haian Fu (Zhao et al., 2011) As a whole, ELISA analysis provides further evidence that these inhibitors can interrupt the interaction of 14-3-3 with PRAS40 protein (Fig. 6). However their IC50 values in the low micromolar range, are 3.92, 5.44 and 5.47 for FZIB-38, FZIB35 and FZIB36 respectively. It is important to note that the KD values determined by SMM-SPR method are in general lower than the corresponding IC50 values reported in the literature for known inhibitors also. This could be due to either the enhanced affinity of the immobilized inhibitors on sensor surface or the relatively high concentrations required for protein–protein in vitro inhibition.
Inhibition of 14-3-3ζ-PRAS40 (PPIs) interaction by identified inhibitors in ELISA.
3.5 Kinetics analysis from SPR imaging
Despite the fact that the kinetic parameters can change significantly upon the immobilization of the compounds, we measured the kinetic parameters for all known compounds that bind FKBP12 and 14-3-3ζ (Table 1). Here we used global fitting of a kinetic model in which a 1:1 complex forms between inhibitors and target proteins in data analysis module software. The data fit very well to this model; however, our values for kinetic rate constants determined from our SPRi experiments for Rapamycin and FK506 molecules are significantly different from the ones reported in the literature. This could be due to steric hindrance caused by the immobilization strategy Kinetics for known 14-3-3ζ inhibitors were not available in the literature. For all 14-3-3ζ inhibitors, only IC50 values for protein–protein inhibition have been reported which is based on in vitro (FRET and FP) assays. As far as we know, this was the first time that the kinetic constants for their binding to 14-3-3ζ were determined. The obtained KD values for new identified isatin inhibitors from SPRi assay and IC50 from ELISA presented in (Table 2). The new identified inhibitors show a comparable kinetics from SPRi and IC50 when compared to those from literatures (Zhao et al., 2011; Yan et al., 2012). This difference between KD and IC50 is may be due to that IC50 was obtained for protein–protein inhibition instead of direct measurement ligands affinity toward the target proteins.
| Compounds | Protein | Ka (1/Ms) | Kd (1/s) | KA (1/M) | KD (nM) |
|---|---|---|---|---|---|
| Rapamycin | FKBP12 | 6.6 × 104 | 1.87e−3 | 3.53 × 107 | 28.2 ± 2.3 |
| FK506 | FKBP12 | 4.35 × 104 | 2.35e−3 | 5.73 × 107 | 54.1 ± 2.44 |
| FOBISIN 101 | 14-3-3ζ | 1.18 × 104 | 5.64 × 10−4 | 2.09 × 107 | 47.8 ± 2.81 |
| FOBISIN 106 | 14-3-3ζ | 1.38 × 104 | 4.94 × 10−4 | 2.8 × 107 | 35.8 ± 2.1 |
| BLAP1 | 14-3-3ζ | 1.39 × 104 | 5.54 × 10−4 | 2.5 × 107 | 40 ± 3.92 |
| BLAP2 | 14-3-3ζ | 1.2 × 104 | 6.08 × 10−4 | 1.97 × 107 | 50.8 ± 3.76 |
| BLAP3 | 14-3-3ζ | 7.8 × 103 | 8.54 × 10−4 | 9.14 × 106 | 109 ± 3.74 |
| Compounds | Protein | Ka (1/Ms) | Kd (1/s) | KA (1/M) | KD (nM) | IC50 (μM) |
|---|---|---|---|---|---|---|
| FZIB-38 | 14-3-3ζ | 5.07 × 103 | 2.8 × 10−4 | 1.81 × 107 | 55.3 ± 2.2 | 3.92 |
| FZIB-35 | 14-3-3ζ | 2.94 × 103 | 2.24 × 10−4 | 1.31 × 107 | 76.6 ± 3.8 | 5.44 |
| FZIB-36 | 14-3-3ζ | 1.64 × 103 | 2.93 × 10−4 | 1.24 × 107 | 79.6 ± 4.1 | 5.47 |
4 Discussion
We have demonstrated here that small molecule microarray technology is quite useful in combination with surface plasmon resonance imaging for screening of small molecules modulators against targets of interest. Identification of three novel specific isatin derived compounds that showed potential utility as 14-3-3ζ inhibitors support this methodology. Furthermore, when these compounds were used in ELISA based 14-3-3ζ-PRAS40 binding assay, all three compounds show promising activity suggested that presented methodology has the potential to be used in high throughput manner without the need of development of an activity based assay that in some cases could be difficult to implement. However, during the course of this work, we realize that there is still a lot of room for improvement. Uniformity of spots and signal strength can be increased by trying different length of PEG linker. Photo-cross-linkers that bind randomly to any chemical group in a compound have proved to work well (unpublished data). This will allow the functional immobilization of larger sets of compounds that lack OH or NH2 groups or for which these groups are necessary for binding to their targets. Another approach that facilitates the creation and functionality of SMMs is the use of 3 dimensional surface chemistries instead of the 2 dimensional surfaces utilized in this work. Although, this platform has some drawbacks at present, it has proved to be suitable for screening of FKBP12 and 14-3-3ζ ligands. Although this approach can also be used in conjunction with other existing detection platforms including the use of fluorescence and microscopic readouts, we believe that the real time kinetics information gives this methodology a significant advantage. Low reagent requirements and rapid screening time make SMM technology particularly useful to academic and industrial discovery programs. The specificity and affinity obtained on this SMM platform can avoid long, laborious and costly efforts of primary screening in this field. Further developments on this technology are in progress in our laboratory.
Acknowledgments
This work was financially supported by the following Grants: National Natural Science Foundation of China (Nos. 61077064/60921001) and National Major Scientific Instruments and Equipments Development Project (No. 2011YQ03012405).
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Appendix A
Supplementary material
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.arabjc.2014.12.020.
Appendix A
Supplementary material
Supplementary data 1
Supplementary data 1