Selectivity Determination of a Small Molecule Chemical Probe Using

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Selectivity determination of a small molecule chemical probe using protein microarray and affinity capture techniques Erik C. Hett, Robert E. Kyne, Ariamala Gopalsamy, Michael A. Tones, Hua Xu, Guene L. Thio, Edward Nolan, and Lyn H. Jones ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.6b00089 • Publication Date (Web): 05 Aug 2016 Downloaded from http://pubs.acs.org on August 7, 2016

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ACS Combinatorial Science

Selectivity determination of a small molecule chemical probe using protein microarray and affinity capture techniques Erik C. Hett,†⌠ Robert E. Kyne Jr.,‡⌡ Ariamala Gopalsamy,† Michael A. Tones,ǁ Hua Xu,† Guene L. Thio,∫ Edward Nolan∫ and Lyn H. Jones†* †

Medicine Design, Pfizer, 610 Main Street, Cambridge, Massachusetts, 02139, USA

‡

Medicine Design, Pfizer, East Point Road, Groton, Connecticut, 06340, USA

ǁ

Rare Disease Research Unit, Pfizer, 610 Main Street, Cambridge, Massachusetts, 02139, USA

∫

Protein and Cell Analysis, Life Sciences Solutions, Thermo Fisher Scientific, 5781 Van Allen

Way, Carlsbad, California, 92008, USA ⌠

Present address: Chemical & Molecular Therapeutics, Biogen, 301 Binney St., Cambridge,

Massachusetts, 02142, USA ⌡

Present address: Celgene Corporation, 200 Cambridge Park Drive, Cambridge, Massachusetts,

02140, USA

ACS Paragon Plus Environment

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Abstract Small molecule selectivity is an essential component of candidate drug selection and target validation. New technologies are required to better understand off-target effects, with particular emphasis needed on broad protein profiling. Here we describe the use of a tritiated chemical probe and a 9000 human protein microarray to discern the binding selectivity of an inhibitor of the mRNA decapping scavenger enzyme DcpS. An immobilized m7GTP resin was also used to assess the selectivity of a DcpS inhibitor against mRNA cap-associated proteins in whole cell extracts. These studies confirm the exquisite selectivity of diaminoquinazoline DcpS inhibitors, and highlight the utility of relatively simple protein microarray and affinity enrichment technologies in drug discovery and chemical biology.

Keywords: protein microarray; cap-binding protein; affinity enrichment; chemical probe

Target validation using chemical probes can suffer if the small molecule being used to perturb a relevant phenotype does not possess adequate selectivity, and this can sometimes lead to biased interpretations of the experimental results.1 Additionally, off-target selectivity is an important parameter to optimize in the drug discovery process to avoid potential toxicities. Therefore, techniques are required to assess the broad proteome selectivity of small molecules. Most approaches rely on the screening of a small molecule in panels of selected biochemical and/or binding assays. Cost and capacity limitations usually require that a relatively small number of off-targets are chosen for selectivity screening, such as those that are known to be linked to safety liabilities, or that are similar in binding site structure/function to the ‘on-target’. Here we describe the application of a tritiated chemical probe in conjunction with a human protein microarray system to directly assess the selectivity of a diaminoquinazoline (DAQ) inhibitor of


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the mRNA decapping scavenger enzyme (DcpS), a target of interest for the treatment of spinal muscular atrophy (SMA).2 DcpS is a pyrophosphatase enzyme responsible for the hydrolysis of the residual 7-methylguanylate cap (m7G) structure following 3'-to-5' mRNA degradation.3 The role of DcpS in modulating the turnover of microRNA has also been elucidated recently.4 Additional to the unbiased protein microarray assay, we used a focused affinity enrichment approach to determine the selectivity of the DcpS inhibitor against other mRNA cap-associated proteins in a cellular proteome.

Protein microarrays provide a powerful approach to determine the interaction of small molecules with thousands of proteins. The ProtoArray® human protein microarray is a platform that is comprised of human proteins expressed as N-terminal glutathione S-transferase (GST) fusion proteins, purified under native conditions, and spotted in duplicate on nitrocellulose-coated glass slides.5 The array can be probed using an isotopically-labelled small molecule with isotope detection methods, and the specificity of the interaction is confirmed in competition experiments using an unlabeled small molecule probe. Since the location and identity of every protein in the array is known, the interacting proteins can be readily determined. Previous studies using a DAQ probe (D156156, Figure 1) containing a radioactive reporter I-125 atom, in conjunction with an earlier version of the ProtoArray® containing 5000 proteins, identified DcpS as a potential target of this series2 (our group subsequently demonstrated binding to DcpS in live human primary cells6). This series of molecules was originally discovered from a cell-based reporter screen designed to identify small molecule upregulators of the survival motor neuron protein gene SMN2, which is able to increase SMN protein (deficient in SMA).7 An


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SMN2 upregulator, D156844 (PF-06652474), was used in competition experiments with

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125

I-

D156156 to confirm the specificity of DcpS inhibition.2

D156156

RG3039

D156844 (PF-06652474) H2 10% Pd/C 100%

NEt3, DMF 48% DiBr-DAQ

D155822

3H 2

10% Pd/C 30 mCi 3H-PF-06652474

Figure 1. Structures of diaminoquinazoline DcpS inhibitors RG3039 and D156844 (PF06652474), the I-125 probe D165165, and preparation of the tritiated probe 3H-PF-06652474 used in the protein microarray experiments. However, apart from the presence of the DAQ motif, the probe D156156 does not directly represent the structure and physicochemistry of the most advanced equity from this series (although D156156 was shown to possess SMN2 upregulation activity in the reporter assay). For example, the benzyl piperidine-containing lead molecules PF-06652474 (D156844) and RG3039 (Figure 1) were designed to maximize SMN2 upregulation activity, retain good metabolic stability and high brain exposure in mice, whilst improving selectivity over the off-target dihydrofolate reductase (DHFR).7-8 PF-06652474 and RG3039 were also found to improve survival and the neuromuscular phenotype in mouse models of SMA.9 However, both compounds contain a second basic centre that is absent from D156156. Physicochemistry is an important determinant of protein binding promiscuity10 and we were concerned that the original protein microarray experiment may not have identified all the potential binding partners for PF-


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06652474, which is both dibasic (pKa 7.8 and 8.5) and lipophilic (cLogP 4.1). As a result, to directly assess the broad binding proteome of PF-06652474 we prepared a tritiated derivative (3H-PF-06652474, Figure 1) as the isotopically-labelled probe molecule for use with a more recent iteration of the ProtoArray® human protein microarray (version 5.0) that contains over 9000 proteins to directly measure off-target selectivity.

Synthesis of tritiated probe 3H-PF-06652474 The tritiated probe was prepared using tritium gas and Pd/C catalysis. The procedure was trialed using hydrogen gas to convert the dibromo derivative DiBr-DAQ to PF-06652474 before applying these conditions to the preparation of the tritiated derivative 3H-PF-06652474 (see SI for details). We prepared a probe with two tritium atoms to ensure adequate specific radioactivity could be achieved for the subsequent protein microarray binding assay. Following this relatively simple procedure we were able to prepare a chemical probe suitable for protein microarray experiments that avoided the use of hazardous stannylation and radioactive iodine chemistry, as used previously in the preparation of 125I-D156156.2 Selectivity determination using protein microarrays Radiolabeled small molecule profiling was performed using ProtoArray® Human Protein Microarrays v5.0 at Invitrogen to evaluate the interaction of array proteins with the tritiated probe (3H-PF-06652474) in the presence and absence of PF-06652474. The procedure followed that

detailed

in

the

Invitrogen

ProtoArray®

Application

Guide:

(https://tools.thermofisher.com/content/sfs/manuals/protoarray_applicationsguide_man.pdf).


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In Phase 1, control protein microarrays (which contain only control protein features) were probed with 1 µM 3H-PF-06652474 in a small molecule interaction (SMI) assay buffer with and without 1% casein to determine optimal blocking conditions. Assays were performed in the presence of 3

H-estradiol which binds the estrogen receptor alpha (ERα) printed in each subarray and acts as a

positional mapping guide to identify the array protein hits. The arrays were washed to remove unbound small molecule, exposed on a tritium-sensitive phosphor screen for 14 days and then scanned at 600 dpi. High resolution array images were processed through the ProtoArray® Prospector software (Invitrogen, free to download). The Phase 1 results indicated significant non-specific probe interactions with control proteins and with the array’s nitrocellulose surface in assays using SMI buffer both with and without casein. Additional Phase 1 studies were therefore conducted in which control protein microarrays were probed with a ten-fold lower concentration (100 nM) of 3H-PF-06652474 in SMI assay buffer with 1% casein, 1.0 µM spermidine, or 1X Synthetic Block (Invitrogen) as blocking agents to improve the signal-background ratio. Inclusion of 1.0 µM spermidine produced similar results to those observed in assays both with and without casein. However, the signalbackground ratio from the ERα positive control features in the presence of 100 nM 3H-PF06652474 was improved in the reaction that included 1X Synthetic Block as the blocking agent. Based on these results, profiling 3H-PF-06652474 at a concentration of 100 nM in SMI buffer with 1X Synthetic Block was selected for use in the subsequent Phase 2 and 3 studies. In Phase 2, 3H-PF-06652474 was profiled at 100 nM in duplicate for interactions with more than 9,000 human proteins on ProtoArray® Human Protein Microarrays in the presence and absence of 150 mM NaCl (Figure 2). In Phase 3, 100 nM 3H-PF-06652474 was profiled in duplicate with


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100-fold molar excess of PF-06652474 (Figure S1). The results of these studies identified a single protein that displayed a robust interaction with 3H-PF-06652474 on replicate arrays both with and without 150 mM NaCl. The 3H-PF-06652474 interacting protein was identified as the mRNA decapping scavenger enzyme, DcpS (see SI). Inclusion of the competitor PF-06652474 inhibited the interaction of 3H-PF-06652474 with DcpS by 100%. These results confirmed the exquisite selectivity of PF-06652474 for DcpS.

Figure 2. Images of protein microarrays. Full content ProtoArray® Human Protein Microarrays were incubated in SMI buffer plus 1x Synthetic Block with 3H-estradiol alone or with 3Hestradiol plus 100 nM 3H-PF-06652474, with and without 150 mM NaCl. The image of one array probed with 3H-PF-06652474 in the absence of sodium chloride (the second array from the left) shows the signal from the interaction of 3H-PF-06652474 with the array protein DcpS (red box) (observed in all arrays probed with 3H-PF-06652474). mRNA cap-associated protein selectivity


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Having assessed the broad protein selectivity of PF-06652474 using the protein microarray technology, we wanted to specifically focus on its selectivity for DcpS over other mRNA capbinding proteins, particularly in the physiologically-relevant context of a whole proteome. To achieve this we employed a cap-immobilised resin (7-methyl-GTP Sepharose, Figure 3)11 with peripheral blood mononuclear cells (PBMCs), and used PF-06652474 to compete the enrichment of cap-associated proteins by the resin as a means to determine selectivity. PBMC lysate was pre-incubated with DMSO, m7GTP or PF-06652474 (D156844) and then treated with 7-methylGTP Sepharose to enrich cap-associated proteins. The eluates were separated by SDS-PAGE and then analyzed by immunoblot using antibodies for key cap-associated proteins: DcpS, CBP20, CBP80 and eIF4E. eIF4E is the cap-binding subunit of a complex that is required for capdependent translation initiation.12 CBP20 and CBP80 are components of the cap-binding complex (CBC) that bind to the cap structure synergistically, resulting in transcript processing and translation.13 Inhibition of these additional proteins may therefore lead to phenotypic effects that could complicate the interpretation of target validation cell-based experiments using the DAQ class of molecules. Interestingly, previous work reported inefficiencies of the m7GTPSepharose resin to capture DcpS due to hydrolysis of m7GTP, which leads to reduced resin capacity.14 However, in our hands, the resin worked for the purposes of the above experiment, including the affinity isolation of DcpS from cell lysate (see SI). As shown in Figure 3, PF-06652474 was found to be completely selective for DcpS, and did not compete the enrichment of CBP20, CBP80 or eIF4E using the cap resin in cell lysate (m7GTP was used as a positive control). These results also confirm the selectivity of the DAQ DcpS inhibitor PF-06652474 (D156844).


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Figure 3. a) Structure of the m7GTP-resin reagent used in the pull-down to enrich cap-associated proteins. b) Immunoblots of DcpS, eIF4E, CBP20 and CBP80 showing enrichment from PBMCs, and competition by m7GTP and D156844 (PF-06652474). New methods are required to assess the selectivity of drug candidates and chemical probes. In this work we have refined protein microarray and affinity enrichment techniques to assess the selectivity of a diaminoquinazoline inhibitor of the DcpS enzyme. A significant advantage of using a tritiated probe in the protein microarray assay is that the structure of the parent small molecule of interest is not perturbed, thus enabling direct measurement of specific protein interactions. Although the protein microarray technique can be applied to biotinylated15 or I-125 labelled molecules,2 investment must be made in the development of a tagged probe to ensure the relevant biological effects are retained. Additionally, the facile synthetic preparation of tritiated probes (particularly in this case using catalytic reduction with tritium gas) and subsequent use with the protein microarray (versus other radioactive derivatives) illustrates the ease with which proteome wide profiling can be achieved for small molecules. A recently reported site-selective iron-catalysed hydrogen isotope exchange of pharmaceuticals adds to the synthetic toolkit for tritiated probe preparation.16


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We also used m7GTP resin to assess the selectivity of the diaminoquinazoline DcpS inhibitor for cap-associated proteins in PBMC lysate. 7-Methyl-GTP Sepharose affinity captured DcpS, CBP20, CBP80 and eIF4E from the whole cell proteome, and the isolation of DcpS was exclusively competed by the DAQ inhibitor. Affinity capture of related proteins and competition with the molecule of interest is a simple method for the determination of selectivity. Here we use Western blot analysis to focus on the chemical probe selectivity against a relatively small number of important cap-associated proteins, but the technique could be further developed using mass spectrometry proteomics for unbiased detection of the cap-proteome.14

ASSOCIATED CONTENT Supporting Information. Identification of protein interactor (DcpS) of 3H-PF-06652474 from the protein microarray; determination of percentage competition of DcpS in the protein microarray; experimental details of performing the pull down using m7GTP-Sepharose; synthesis of 3H-PF-06652474 and associated analytical data. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected]. ACKNOWLEDGMENTS


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We thank J. Cherry, R. Ramos-Zayas, K. Schildknegt and A. Narayanan for helpful discussions and PerkinElmer for the preparation of 3H-PF-06652474. REFERENCES 1. (a) Jones, L. H., An industry perspective on drug target validation. Exp. Opin. Drug Discov. 2016, 11, 623-625; (b) Bunnage, M. E.; Chekler, E. L.; Jones, L. H., Target validation using chemical probes. Nat. Chem. Biol. 2013, 9, 195-199. 2. Singh, J.; Salcius, M.; Liu, S.-W.; Staker, B. L.; Mishra, R.; Thurmond, J.; Michaud, G.; Mattoon, D. R.; Printen, J.; Christensen, J.; Bjornsson, J. M.; Pollok, B. A.; Kiledjian, M.; Stewart, L.; Jarecki, J.; Gurney, M. E., DcpS as a therapeutic target for spinal muscular atrophy. ACS Chem. Biol. 2008, 3, 711-722. 3. Liu, H.; Rodgers, N. D.; Jiao, X.; Kiledjian, M., The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases. EMBO J. 2002, 21, 46994708. 4. Meziane, O.; Piquet, S.; Bossé, G. D.; Gagné, D.; Paquet, E.; Robert, C.; Tones, M. A.; Simard, M. J., The human decapping scavenger enzyme DcpS modulates microRNA turnover. Sci. Rep. 2015, 5, 16688. 5. Schweitzer, B.; Meng, L.; Mattoon, D.; Rai, A. J., Immune response biomarker profiling application on ProtoArray protein microarrays. Methods Mol. Biol. 2010, 641, 243-252. 6. (a) Hett, E. C.; Xu, H.; Geoghegan, K. F.; Gopalsamy, A.; Kyne Jr., R. E.; Menard, C. A.; Narayanan, A.; Parikh, M. D.; Liu, S.; Roberts, L.; Robinson, R. P.; Tones, M. A.; Jones, L. H., Rational targeting of active-site tyrosine residues using sulfonyl fluoride probes. ACS Chem. Biol. 2015, 10, 1094-1098; (b) Xu, H.; Gopalsamy, A.; Hett, E. C.; Salter, S.; Aulabaugh, A.; Kyne Jr., R. E.; Pierce, B.; Jones, L. H., Cellular thermal shift and clickable chemical probe assays for the determination of drug-target engagement in live cells. Org. Biomol. Chem. 2016, 14, 6179-6183. 7. Thurmond, J.; Butchbach, M. E. R.; Palomo, M.; Pease, B.; Rao, M.; Bedell, L.; Keyvan, M.; Pai, G.; Mishra, R.; Haraldsson, M.; Andresson, T.; Bragason, G.; Thosteinsdottir, M.; Bjornsson, J. M.; Coovert, D. D.; Burghes, A. H. M.; Gurney, M. E.; Singh, J., Synthesis and biological evaluation of novel 2,4-diaminoquinazoline derivative as SMN2 promoter activators for the potential treatment of spinal muscular atrophy. J. Med. Chem. 2008, 51, 449-469. 8. Singh, J.; Gurney, M. E. Preparation of 2,4-diaminoquinazolines for the treatment of spinal muscular atrophy. WO2008016973, 2008. 9. (a) Meerbeke, J. P. V.; Gibbs, R. M.; Plasterer, H. L.; Miao, W.; Feng, Z.; Lin, M.-Y.; Rucki, A. A.; Wee, C. D.; Xia, B.; Sharma, S.; Jacques, V.; Li, D. K.; Pellizzoni, L.; Rusche, J. R.; Ko, C.-P.; Sumner, C. J., The DcpS inhibitor RG3039 improves motor function in SMA mice. Hum. Mol. Genet. 2013, 22, 4074-4083; (b) Gogliotti, R. G.; Cardona, H.; Singh, J.; Bail, S.; Emery, C.; Kuntz, N.; Jorgensen, M.; Durens, M.; Xia, B.; Barlow, C.; Heier, C. R.; Plasterer, H. L.; Jacques, V.; Kiledjian, M.; Jarecki, J.; Rusche, J.; DiDonato, C. J., The DcpS inhibitor RG3039 improves survival, function and motor unit pathologies in two SMA mouse models. Hum. Mol. Genet. 2013, 22, 4084-4101; (c) Butchbach, M. E.; Singh, J.; Thorsteinsdóttir, M.; Saieva, L.; Slominski, E.; Thurmond, J.; Andrésson, T.; Zhang, J.; Edwards, J. D.; Simard, L. R.; Pellizzoni, L.; Jarecki, J.; Burghes, A. H.; Gurney, M. E., Effects of 2,4-diaminoquinazoline


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derivatives on SMN expression and phenotype in a mouse model for spinal muscular atrophy. Hum. Mol. Genet. 2010, 19, 454-467. 10. Price, D. A.; Blagg, J.; Jones, L. H.; Greene, N.; Wager, T., Physicochemical drug properties associated with in vivo toxicological outcomes: a review. Expert Opin. Drug Metab. Toxicol. 2009, 5, 921-931. 11. Webb, N. R.; Chari, R. V.; DePillis, G.; Kozarich, J. W.; Rhoads, R. E., Purification of the messenger RNA cap-binding protein using a new affinity medium. Biochemistry 1984, 23, 177-181. 12. Merrick, W. C., eIF4F: a retrospective. J. Biol. Chem. 2015, 290, 24091-24099. 13. Gonatopoulos-Pournatzis, T.; Cowling, V. H., Cap-binding complex. Biochem. J. 2014, 457, 231-242. 14. Szczepaniak, S. A.; Zuberek, J.; Darzynkiewicz, E.; Kufel, J.; Jemielity, J., Affinity resins containing enzymatically resistant mRNA cap analogs - a new tool for the analysis of capbinding proteins. RNA 2012, 18, 1421-1432. 15. (a) To, C.; Shilton, B. H.; Di Guglielmo, G. M., Synthetic triterpenoids target the Arp2/3 complex and inhibit branched actin polymerization. J. Biol. Chem. 2010, 285, 27944-27957; (b) Huang, J.; Zhu, H.; Haggarty, S. J.; Spring, D. R.; Hwang, H.; Jin, F.; Snyder, M.; Schreiber, S. L., Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and proteome chips. Proc. Natl. Acad. Sci. USA 2004, 101, 16594-16599. 16. Yu, R. P.; Hesk, D.; Rivera, N.; Pelczer, I.; Chirik, P. J., Iron-catalysed tritiation of pharmaceuticals. Nature 2016, 529, 195-199.


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Protein microarray

Affinity enrichment


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Selectivity Determination of a Small Molecule Chemical Probe Using

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