Discovery and Characterization of Allosteric WNK Kinase Inhibitors

Oct 7, 2016 - Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triag...
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Discovery and Characterization of Allosteric WNK Kinase Inhibitors Ken Yamada,*,#,†,∥ Ji-Hu Zhang,#,† Xiaoling Xie,#,† Juergen Reinhardt,#,‡ Amy Qiongshu Xie,† Daniel LaSala,§ Darcy Kohls,† David Yowe,† Debra Burdick,†,⊥ Hajime Yoshisue,∥ Hiromichi Wakai,∥ Isabel Schmidt,‡ Jason Gunawan,† Kayo Yasoshima,†,∥ Q. Kimberley Yue,† Mitsunori Kato,†,∥ Muneto Mogi,†,∥ Neeraja Idamakanti,† Natasha Kreder,† Peter Drueckes,‡ Pramod Pandey,† Toshio Kawanami,†,∥ Waanjeng Huang,† Yukiko I. Yagi,∥ Zhan Deng,† and Hyi-Man Park#,†,∥ †

Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts 02139-4133, United States Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, 4002, Switzerland § Novartis Institutes for BioMedical Research, Novartis Pharmaceuticals Corporation, East Hanover, New Jersey 07936-1080, United States ∥ Novartis Institutes for BioMedical Research, Novartis Pharma K.K., Tsukuba, Ibaraki 300-2611, Japan ‡

S Supporting Information *

ABSTRACT: Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1−4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1− 4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.

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activity based screening followed by characterization of atypical kinase inhibitors, such as unusually high selectivity of an AKT inhibitor11 and a LIMK inhibitor,12 or lack of an obvious hinge binding motif for an IGF1R inhibitor.13 In a more planned fashion, an allosteric CHK1 inhibitor14 and EGFR inhibitors15 were discovered by deprioritizing ATP competitive hits by using a high ATP follow-up screen, and an allosteric FAK inhibitor was discovered by screening with unphosphorylated kinase.16 Binding-based discovery also offers an opportunity to find allosteric inhibitors, as evidenced by a fragment-based screen approach for an allosteric PAK inhibitor17 and affinity selection mass spectroscopy for JNK-1 as well as MAPK inhibitors18 and AKT inhibitors.19 More tailored methods to specifically find allosteric inhibitors have also been reported, such as a fluorescently labeled mutant kinase assay for cSrc20 and a fluorescence anisotropy assay for a protein−peptide fragment interaction to find allosteric AURAK inhibitors.21

uman kinases play fundamental roles in cellular signaling and comprise a powerful therapeutic target class.1 However, the majority of human kinases still remain unexploited, with only 45 out of 518 human kinases being targeted by 33 clinically approved inhibitors.2 Moreover, the majority of these are anticancer agents,3 where some degree of broad specificity can be beneficial for overcoming drug resistance or having a broader spectrum of efficacy (Supporting Information, Comment a).4 One of the major obstacles in expanding kinase inhibitor therapeutics is attributed to the lack of selectivity, which is an inherent challenge for the types of inhibitors that target the highly conserved ATP-binding site.5 Therefore, alternative approaches to target less conserved allosteric sites of the kinases have been an active area of research owing to the promise of high selectivity.5−7 Earlier discovery of allosteric kinase inhibitors resulted from cell-based screens, as in the case of MEK inhibitors PD098059,8 U-0126,9 and Bcr-Abl inhibitor GNF-2.10 While having cellular activity from the onset seem advantageous, laborious deconvolution of the responsible biochemical target is often required. More straightforward but perhaps serendipitous discovery of allosteric kinase inhibitors resulted from enzyme © 2016 American Chemical Society

Received: June 13, 2016 Accepted: October 7, 2016 Published: October 7, 2016 3338

DOI: 10.1021/acschembio.6b00511 ACS Chem. Biol. 2016, 11, 3338−3346

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Figure 1. Hit triaging strategy, structure and in vitro profiles of WNK1 kinase inhibitor. (a) Top: HTS was performed on 1.2 million in-house compound collection in HTRF format. (i) Atypical kinase inhibitors (group A) were prioritized, and typical/known kinase inhibitors (group B) were deprioritized using in silico structural filters and known kinase inhibition data. (ii) Inhibitors without a significant shift in a low vs a high ATP concentration assays in HTRF format were prioritized. (iii) In-house kinase panel selectivity was assessed, and selective scaffolds were prioritized. (iv) Scaffolds with some cellular activity on pOSR1 inhibition were selected. Bottom: Structure and biochemical activities of ATP noncompetitive HTS hit compound 1 at low (25 μM) and high (800 μM) ATP concentrations in mobility shift assay format. (b) Enzyme kinetic studies of compound 1 vs ATP in mobility shift assay format. Global fitting of the data to the uncompetitive inhibition equations yielded a Ki of 2.25 ± 0.06 μM. Goodness of the fit was assessed by comparing R2 values from the fit to uncompetitive, noncompetitive, and competitive inhibition equations. R2 values were 0.99, 0.98, and 0.94, respectively. The decrease in both Km and Vmax with increasing inhibitor concentration is consistent with the uncompetitive inhibition, with preference for the enzyme−ATP complex over enzyme alone. (c) Kinase selectivity profile of compound 1 and typical group B hit compound 3.

conformational states. Our strategy for allosteric hit finding consisted of screening at high compound concentration to capture atypical but weak hits as much as possible, prioritization of compounds lacking known kinase inhibition or typical kinase inhibitor motifs, and stringent hit triaging for compounds that do not show an IC50 shift in low vs high ATP concentrations to eliminate ATP-competitive hits. In the HTS campaign, a single-point screen against a 1.2 million in-house compound collection was performed in HTRF format with compound concentration at 50 μM and ATP concentration at 100 μM (2-fold above Km) (Supporting Information, Comment b). All compounds with >30% inhibition were included in the initial hit-list (8,257 hits, 0.8% hit rate). We then sorted these compounds into two groups utilizing available kinase inhibition data and an in silico model to apply biased filters for the subsequent triaging process (Figure 1a). Thus, compounds with known or typical kinase inhibitor structures such as aminopyridines and aminopyrimidines30 (group B) were stringently triaged to include only reference compounds, while atypical kinase inhibitors (group A) were triaged more generously in order to include weak but structurally unique hits totaling 2298 compounds. Hit validation through IC50 determination in HTRF format at low (2 × Km) and high ATP (20 × Km) concentrations (Supporting Information Table 1) revealed eight group A scaffolds that were not ATP competitive. Further prioritization based on a cellular ELISA assay for pOSR1, kinase selectivity profile, and biochemical inhibitory activity in an orthogonal mobility shift assay format narrowed down the hit-to-lead candidates to compound 1 (Figure 1a), which showed the best fit for uncompetitive inhibition in enzyme kinetic analysis31 vs

In search of a next-generation therapy for resistant hypertension,22 we became interested in the inhibition of With-No-Lysine (WNK) kinases 1 and 4, whose gain-offunction mutations are known to cause pseudohypoaldosteronism II (PHA-II), a rare Mendelian form of hypertension.23 Since PHA-II patients respond well to thiazide diuretics,24 direct blockers of sodium chloride cotransporter (NCC) in the kidney, there have been numerous studies on the cellular mechanism of WNK mediated regulation of renal electrolyte handling.25 We have recently shown the direct role of the WNK catalytic activity in the regulation of blood pressure and renal electrolyte handling.26 The results were consistent with the reported WNK phosphorylation cascade in the kidney, where WNK phosphorylates oxidative stress-responsive 1 protein (OSR1) or STE20/SPS1-related proline-alanine-rich protein kinase (SPAK), which in turn phosphorylate NCC and sodium potassium chloride cotransporters (NKCC1/2).27,28 Thus, we envisioned that allosteric inhibitors should enable selective WNK-family inhibition to control blood pressure and excessive electrolyte reuptake in the kidney, offering a unique opportunity to treat resistant hypertension.

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RESULTS AND DISCUSSION While the structural information available at the time based on rat apo-WNK129 revealed a unique catalytic pocket with translocation of the catalytic lysine that might give sufficient opportunity for high selectivity, there was no indication of allosteric pockets to exploit. Nevertheless, we performed a biochemical high-throughput screen (HTS) with the premise that inhibitors may capture allosteric pockets that exist in other 3339

DOI: 10.1021/acschembio.6b00511 ACS Chem. Biol. 2016, 11, 3338−3346

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Figure 2. Structure and in vitro profiling of compound 2. (a) Structure of compound 2 and its biochemical WNK1 inhibition at low and high ATP concentrations in mobility shift assay format and cellular pOSR1 inhibition in HEK293 cells. (b) Kinase selectivity data of compound 2 based on AMBIT KINOMEscan. Green dots indicate less than 35% inhibition, and red dots indicate greater than 35% inhibition at 10 μM screening concentration. Blue dot indicates on target activity for WNK1. Images generated using TREEspot Sof tware Tool and reprinted with permission f rom KINOMEscan, a division of DiscoveRx Corporation, DISCOVERX CORPORATION, 2010. (c) Top: SPR data for compound 2 show additive binding with AMP−PNP under both phosphorylation states. Bottom: Correction for AMP−PNP signal revealed that phosphorylation of WNK1 improves Kd by 20-fold.

Figure 3. Crystal structures of WNK1. (a) Reported structure of rat apo-WNK1 (PDB 3FPQ). The αC-helix (blue), the DLG motif (cyan), and the activation loop (red) are tightly sealed, and there is no allosteric pocket. The unusual location of the catalytic lysine (Lys233) in the β2 strand on the glycine rich loop (green) is also indicated, along with Cys250 in the β3 strand, which is the typical location of the catalytic lysine. (b) Same angle view of human WNK1 with AMP−PNP (yellow) and allosteric inhibitor compound 2 (purple) bound (PDB: 5TF9). The outward movement of the αC-helix (blue) and unwinding of the activation loop (red) creates an allosteric pocket not seen in the rat apo structure. (c) Global view of the ternary structure of compound 2 bound to human WNK1 with AMP−PNP. The allosteric pocket is located adjacent to the ATP binding site, separated by the DLG (cyan) and activation loop (red) and surrounded by the glycine-rich loop (green) and the αC-helix (blue). (d) Close-up of panel c where the aminothiazole moiety makes a hydrogen bond contact with the backbone carbonyl of Val281. (e) Crystal structure of an ATPcompetitive inhibitor WNK463 bound to hWNK1 (PDB: 5DRB) from a similar perspective as in panel c. (f) Close-up of panel e where WNK463 makes the hinge contact and extends into the allosteric back-pocket. Residues shown are catalytic Lys233, gatekeeper Thr301, and Asp368 of DLG and Val281 and Phe283 of the back-pocket.

ATP (Figure 1b, Supporting Information Figure 1a) and noncompetitive inhibition vs peptide substrate (Supporting Information Figure 2a), both in mobility shift assay format. While a prototypical group B inhibitor 3 showed low kinase selectivity with better inhibition of off-target kinases in our inhouse selectivity panel of 31 kinases,32 the group A hit 1 inhibited only WNK1 kinase in the same panel, giving a promising starting point (Figure 1c). Without any cocrystal structures or docking models in hand, we performed a systematic modification of each part of the molecule and quickly noticed a steep SAR in the aminothiazole portion of compound 1. On the other hand, when the 2-fluoro

substituent of compound 1 was substituted with a 4chlorobenzyloxy group, a roughly 50-fold boost in potency was observed to give compound 2 (Figure 2a). As with compound 1, there was no shift in IC50 at high ATP concentration with compound 2, which also dose dependently inhibited sorbitol stimulated pOSR1 in HEK293 cells (Supporting Information Figure 3a, IC50 = 0.570 μM). Importantly, compound 2 showed an exquisite selectivity for WNK1 against a panel of 61 in-house kinases32 (Supporting Information Table 2). The selectivity profile of 2 was confirmed with a more extensive kinase binding panel provided by AMBIT (Figure 2b, Supporting Information Data). It should be noted, 3340

DOI: 10.1021/acschembio.6b00511 ACS Chem. Biol. 2016, 11, 3338−3346

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for the discovery of allosteric inhibitors that preferentially bind to the unphosphorylated form of WNK1. As noted earlier, for such allosteric inhibitors, compound preincubation with unphosphorylated enzyme16 or use of various binding assays17−21 with unphosphorylated enzyme may be preferable. The advantage of our approach is that the inhibitors bind preferentially to the phosphorylated enzyme and are not affected by high physiological ATP concentration. In order to study the effect of WNK inhibition on the cellular electrolyte handling, an NKCC1 assay was established using rubidium flux as a surrogate for cellular potassium uptake. First, the role of WNK1 and OSR1/SPAK in the regulation of the NKCC1 function was confirmed by siRNA knock-down experiments in HeLa cells. Pretreatment of cells with both siRNAs significantly reduced NKCC1 mediated rubidium uptake (Figure 4, Supporting Information Figure 3b). In

however, that compound 2 inhibits WNK1−4 similarly as revealed by radiometric assays (Supporting Information Table 3), as with the previously described ATP-competitive inhibitor WNK463.26 Compound 2 showed an ATP and substrate noncompetitive mode of inhibition in the kinetic study (Supporting Information Figure 1b and Supporting Information Figure 2b). When the binding was examined using surface plasmon resonance (SPR), the additive binding with AMP−PNP, a nonhydrolyzable ATP mimetic, was evident, confirming an ATP noncompetitive binding mode (Figure 2c). Correction for the AMP−PNP signal indicated that compound 2 was binding better to phosphorylated WNK1 (pSer382) compared to unphosphorylated WNK1 (Kd = 0.19 μM vs 4.4 μM). In line with this result, both compounds 1 and 2 showed enhanced protein stabilization in a differential scanning calorimetry (DSC) study with phosphorylated WNK1 vs with unphosphorylated WNK1 (Supporting Information Table 4). At this point, ATP noncompetitive enzyme kinetics and additive binding with AMP−PNP were indicative of an allosteric mode of kinase inhibition. However, cocrystallization of HTS hits with the WNK1 enzyme remained elusive despite numerous attempts. Moreover, the available rat apo crystal structure at the time (PDB: 3FPQ, Figure 3a) showed no allosteric pocket, although small gatekeeper residue Thr301 and translocation of catalytic Lys233 from the β3 to β2 strand (glycine-rich loop) suggested potential access to the back pocket away from the hinge.29 It was only with optimized compound 2 that we were able to obtain a crystal structure to definitively establish the allosteric mode of binding as a ternary complex with WNK1 and AMP−PNP (PDB: 5TF9, Figure 3b−d). The binding of compound 2 is achieved through the formation of an allosteric pocket via an outward movement of the αC-helix and stretching of the activation loop when compared to rat apo-WNK1 (Figure 3b vs a, also in movie format in the Supporting Information). In the ternary complex, the aminothiazole of compound 2 makes a hydrogen bond interaction with the backbone carbonyl of Val281, while the chlorobenzyloxy group penetrates deep in the hydrophobic pocket created between the αC-helix and the activation loop (Figure 3d). The same allosteric back-pocket with Val281 is also occupied by partially allosteric, ATP-competitive inhibitor WNK463 (Figure 3e,f, PDB 5DRB).26 In the case of WNK463, the displacement of Lys233 from the β3 strand as well as the small gatekeeper residue Thr301 creates a tunnel for the compound to extend from a hinge into the allosteric backpocket, which the aminothiazole of compound 2 occupies, between the αC-helix and the DLG loop, flanked by valine 281 (Figure 3f). The allosteric binding mode of compound 2 as revealed in the ternary complex with AMP−PNP and WNK1 is the socalled type III allosteric inhibition mode33 and is somewhat analogous to the MEK inhibitor PD318088.34 Presumably, the compound stabilizes WNK1 in a ternary complex as a “DLG-in, αC-out” conformation, thereby inhibiting the kinase activity. While this movement of αC-helix from the apo-form to allosteric inhibitor bound form is shared with some other kinase inhibitors, the residues surrounding the resultant pockets are not conserved and hence describe the observed exquisite selectivity profile. It should be noted that our screening setup likely probed the phosphorylated WNK1 (WNK1 autophosphorylates at Ser38235). Thus, our screening may not have been suitable

Figure 4. Schematic representation of the WNK(1)-OSR1/SPAKNKCC1 phosphorylation cascade that regulates NKCC1 activity. Both siRNA knock-down of WNK1 alone or in combination with OSR1/ SPAK results in a reduction of rubidium uptake by NKCC1 in HeLa cells, confirming their regulatory role on NKCC1 function. Compound 2 potently inhibits WNK1−4 enzymes in vitro and phosphorylation of the WNK1 substrate OSR1 in HEK293 cells, while it shows no inhibition of the OSR1 enzyme in vitro. Compound 2 reduces bumetanide sensitive rubidium flux by NKCC1 in HT29 cells with an IC50 of 0.24 μM. Bumetanide is a known direct blocker of NKCC1, which showed an IC50 of 1.54 μM in the same assay.

HT29 cells, a direct NKCC1 blocker bumetanide showed dosedependent inhibition of rubidium uptake with an IC50 value of 1.54 μM (Figure 4, Supporting Information Figure 3c). In the same assay, compound 2 dose-dependently inhibited bumetanide sensitive rubidium uptake with an IC50 of 0.24 μM (Figure 4, Supporting Information Figure 3d). In the AMBIT panel, compound 2 did not show any inhibition of OSR1 at 10 μM (Figure 4, Supporting Information Data). We have recently provided direct evidence for the role of WNK kinase catalytic activity in regulating the activity of renal ion transporter NCC via the WNK-OSR1/SPAK-NCC phosphorylation cascade,26 addressing some of the controversy surrounding the exact regulatory mechanism of WNK kinases and renal ion transporter activity.25 By showing the effect of WNK1−4 kinase inhibition as well as WNK1-specific knockdown by siRNA, we provide here compelling evidence for the regulation of NKCC1 activity by WNK(1) kinase catalytic activity via WNK(1)-OSR1/SPAK-NKCC1 phosphorylation cascade. These data are consistent with previous reports27,28 as well as phenotypes of a small molecule WNK-NKCC1 disruptor36 and direct SPAK inhibitor.37 Taken together, our studies indicate that WNK kinases play a fundamental role in the regulation of renal ion transport and consequent cardiovascular homeostasis through volume regulation. 3341

DOI: 10.1021/acschembio.6b00511 ACS Chem. Biol. 2016, 11, 3338−3346

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WNK1 HTRF Assay for Hit Validation. This assay was performed at two different ATP concentrations, 100 μM ATP (2 × Km ATP) and 1 mM ATP (20 × Km ATP), using human WNK1 protein (aa198− 401) produced as described in the Supporting Information. For hit validation, eight compound replicates were made from the original stock dilution plates. The Cybi-WellTM dispenser was used to prepare 1.5 μL of compound dots in the intermediate dilution plates, which were then transferred to the assay plates. For validation at 100 μM ATP, the reactions were carried out and quenched and products detected as described above for screening. For validation at 1 mM ATP, the compound transfer and reagent preparation followed the same standard assay protocol except for using 10× higher ATP in the substrate mixture. The reactions were incubated for 1.5 h at RT, at which time point the signal generation is still in the linear range. A total of 6 μL of the stop and detection reagent was added and incubated at RT for at least 2 h before reading on an Envision Multilabel reader (PerkinElmer). All compounds were tested in duplicate as an eight-point, half-log dilution series from 0.025 to 80 μM. In Silico Filters for Hit-List Triaging. First, a set of physicochemical property filters (e.g., 150 < MW < 700, PSA < 200, −1 < cLogP < 7, reactive motifs, etc.) were applied to deprioritize compounds with unattractive properties. Next, a more stringent activity cutoff (WNK1 HTRF > 35% inhibition at 50 μM compound concentration) was used to exclude an additional 1684 very weak hits. The remaining list was classified into group A (atypical kinase inhibitors) and group B (typical kinase inhibitors), based on the following criteria: (1) A compound that appeared as active (IC50 < 1 μM) for multiple kinase targets in the Novartis kinase panel assay was classified into group B. (2) A list of known kinase inhibitor scaffolds frequently observed in the Novartis corporate collection was compiled, and compounds containing one or more of these scaffolds were put into group B. (3) Several generic kinase inhibitor predictive models were constructed using data from internal and external kinase activity databases, respectively, using the naı̈ve Bayes method with ECFP-6 fingerprints as predictive variables. Any compound scoring very high in any of these predictive models was flagged as group B. WNK1 Mobility Shift Assay. A mobility shift assay using human WNK1 (aa1−491, CarnaBiosciences) was used as an orthogonal enzyme assay to validate hits from the HTRF assay and to perform enzyme kinetic analysis and as the primary assay for medicinal chemistry efforts. A mixture of fluorescein labeled OSR1 peptide substrate (Toray Research Center, Inc.) and ATP was prepared with final concentrations of 10 and 25 μM, respectively, in 7 μL of reaction buffer [20 mM HEPES Na (pH 7.5), 1 mM MnCl2, 0.01% Tween 20, and 2 mM DTT]. Compounds at final concentrations from 0.003 to 100 μM in 1 μL were added to the peptide/ATP mixture at a final DMSO concentration of 10%. The reaction was initiated by the addition of 2 μL of GST-WNK1 (1−491; Carna Biosciences) that was 86% pure at a final concentration of 25 nM in a 384-well plate. After incubation for 3 h at 25 °C, where 15% conversion of the substrate to the product was observed, 10 μL of a stop buffer containing 0.2% coating-3 reagent with 2 mM EDTA was added. For 800 μM ATP, the WNK1 assay was performed for 50 min at 25 °C, where 15% conversion of the substrate to the product was observed. Aliquots from each well were transferred onto a four-sipper chip on a Labchip3000 instrument for electrophoretic separation of the substrate, and the fluorescence intensity was measured under a pressure of −2.0 psi and a voltage of −1650 ΔV. The relative peak heights of the substrate and product were measured, and the peak ratio was calculated using HTS Well Analyzer version 4.1. The ratio was defined as the height of the product peak divided by the sum of the peak heights of the product and the substrate. Calibration was done using the low control, i.e., the enzyme reaction in the presence of 2 mM EDTA, and the high control, i.e., the enzyme reaction in the absence of EDTA. The percentage of inhibition was calculated as follows:

Our approach described herein is conceptually the simplest among the different methods of allosteric hit finding described earlier. It has some distinct advantages such as the straightforward assay setup without a need for special protein engineering, high throughput assay compatibility, and cellular potency of the hits that retain activity at physiological ATP concentration. Arguably, the largest obstacle to allosteric hit finding may be the lure of potent ATP-site binders that most kinase inhibitor screening campaigns will inevitably identify. In summary, we have described a simple and practical approach to the discovery of ATP noncompetitive, allosteric WNK1−4 kinase inhibitor 1. Medicinal chemistry optimization identified potent WNK 1−4 kinase inhibitor 2, suitable for extensive biochemical, biophysical, and cellular characterization. As initially hoped for, the allosteric inhibitor 2 showed an exquisite specificity for WNK-family kinase and selectivity against other kinases. The crystal structure for 2 bound to WNK1 revealed a novel, ligand-induced allosteric binding pocket unique to the WNK family kinases, consistent with the high WNK family selectivity. Last, the activity of compound 2 on WNK substrate OSR1 and downstream transporter NKCC1 provided another line of evidence for the previously reported WNK kinase biology. Our pragmatic approach to discover ATP noncompetitive, allosteric inhibitors with exquisite selectivity should be applicable to other kinase hit discovery efforts, especially for therapeutic modalities where a stringent safety profile is required. Subsequent medicinal chemistry optimization of allosteric WNK inhibitors will be reported in due course.

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METHODS

Reagents. WNK proteins for the mobility shift assay and radiometric WNK family selectivity assay were purchased from CarnaBiosciences; WNK1 (aa1−491, catalog # 05-179), WNK2 (aa166−489, catalog # 05-180), WNK3 (aa1−434, catalog # 05181), and WNK4 (aa1−444, catalog # 05-182). WNK1 (aa198−491) for the HTRF assay and WNK1 S378D (aa206−483) for the SPR assay and X-ray crystallography were produced and purified as described in the Supporting Information. The rabbit anti-pOSR1(Ser325) polyclonal antibody was custom-made by New England Peptide using the antigen peptide Ac−C+RRVPGS(pS)GRLHKTEamide. IgG from production bleeds from rabbits were purified using an affinity column with the nonphosphorylated-OSR1 peptide Ac−C +RRVPGSSGRLHKTE-amide, to yield a phosphorylated-specific OSR1 antibody which was biotinylated according to the manufacturer’s instructions (Thermo Scientific) for the alphaLISA assay. Software. Enzyme kinetics analyses were performed with GraphPad Prism for Windows version 7. Crystallographic images were generated using Pymol versions 1.5.0.4 and 1.8.8.2. SPR data were analyzed by BIAcore T100 Evaluation Software. WNK1 HTRF Assay for Screening. Human WNK1 protein (aa198−401) was produced as described in the Supporting Information, and the HTRF assay was done in a 384-well plate (Greiner) format. The final concentration of WNK1 was 200 nM. The final concentration of generic substrate biotinylated STK3 (Cisbio) was 1 μM. The final concentration of ATP was 100 μM, and that of each screened compound was 50 μM. The assay plates were incubated at RT for 2 h; then Europium cryptate labeled antiphosphopeptide antibody and SA-XL665 were added to final concentrations of 180 ng mL−1 and 62.5 nM, respectively, to stop the reaction and detect the product. The plates were incubated at RT for at least 2 h for equilibration before reading on an Envision Multilabel reader (PerkinElmer), using an excitation of 320 nm, and two emissions at 590 and 665 nm. The screening assay exhibited an average Z′ value of 0.75 and a signal to background ratio of 4.4. The estimated Km for ATP was 56−63 μM.

% inhibition = 100[1 − (r − rlowcontrol)/(rhighcontrol − rlowcontrol)] 3342

DOI: 10.1021/acschembio.6b00511 ACS Chem. Biol. 2016, 11, 3338−3346

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incubated at RT for 80 min in the dark. A total of 12.5 μL of donor beads (10 μg mL−1 final concentration) was added and incubated at RT for another 80 min; then the plate was read in an EnVision Multilabel reader (PerkinElmer). WNK siRNA Knockdown Studies in HeLa Cells. Control siRNA (scrambled), validated human WNK1, WNK3, WNK4, OSR1, and SPAK siRNA’s (Invitrogen) were diluted in Opti-MEM buffer (Invitrogen). Final siRNA concentrations were optimized to 6 pmol/96 well. A reverse transfection approach using lipofectamine RNAiMax (Invitrogen) and 30 000−80 000 HeLa/well was performed in 96 well format. After overnight incubation (37 °C, 5% CO2), cells were washed with cell culture media and processed 24 to 48 h post transfection for atomic absorption spectroscopy (Aurora Biomed, ICR8000). Hyperosmotic stimulation was performed for 20 min using 0.5 M sucrose buffer. Medium was exchanged with Rb+ uptake buffer containing 0.5 mM Ouabain. Bumetanide (1 mM) served as a positive control for NKCC1 blockade. After 30 min, incubation cells were washed with ice-cold wash buffer and lysed in 200 μL of lysis buffer. For each measurement calibration curves using Rb+ standard solutions (Aurora Biomed) were determined. NKCC1 Rb+ Uptake Assay in HT29 Cells. For compound screening, a single channel atomic absorption spectroscopy analyzer (ICR8000, Aurora Biomed) was used. All measurements were done in triplicate, and eight point dose response curves were obtained. Rb+ calibration curves were determined using Rb+ standard solutions (Aurora Biomed). The following buffers were used: cell wash buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM CaCl2, 10 mM HEPES, 10 mM glucose, 10 mM pyruvate, pH 7.4); Rb+ uptake buffer (140 mM NaCl, 5 mM RbCl, 1 mM MgCl2, 10 mM CaCl2, 10 mM HEPES, 10 mM glucose, 10 mM pyruvate, 0.1% BSA); sucrose buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM CaCl2, 10 mM HEPES, 10 mM glucose, 10 mM pyruvate, 0.1% BSA, 0.5 M sucrose); lysis buffer (0.1% SDS in H2O). For compound screening, HT29 cells cultivated in tissue culture were plated in 96 or 384 well assay plates. Cell numbers analyzed were between 30 000 and 80 000 cells/well in a 96 well format and between 7500 and 20 000 cells in a 384 well format. Adherent cells were washed once in cell wash buffer and rested for 5 min at 37 °C. Compound solutions and plates were prepared using a CybiWell and applied and incubated for 20 min. Assay plates were flicked, and compound plus Ouabain (0.5 mM) containing Rb+ loading buffer was applied. Bumetanide served as a positive control for blockade of NKCC1 mediated Rb+ uptake. Cells were incubated for 30 min at 37 °C and thereafter washed with ice-cold wash buffer. Cell lysis buffer was added, and samples were analyzed for Rb+ content. Crystallization, Data Collection, and Structure Determination of WNK1 (206−483). To obtain crystals of WNK1:MnAMP− PNP:compound 2 complex, WNK1 (206−483, S378D, E396A, E397A, K398A) was diluted to 0.5 mg mL−1 in storage buffer and then incubated with 1.5 mM MnCl2, 0.5 mM AMP−PNP, and 50 μM compound 2. The mixture was concentrated 20-fold prior to crystallization. The complex crystals were obtained by the hanging drop vapor diffusion method at 20 °C, with the crystallization well containing 100 mM HEPES (pH 7.5), 22% (w/v) polyethylene glycol 3350, and 200 mM calcium acetate. Prior to data collection, the crystals were transferred to a solution containing 100 mM HEPES (pH 7.5), 25% (w/v) polyethylene glycol 3350, and 10% (v/v) glycerol briefly and then flash frozen in liquid nitrogen. Diffraction data were obtained at an in-house diffraction facility equipped with a FRE (Rigaku) and a RAXIS IV++ (Rigaku). Crystals of the WNK1:MnAMP−PNP:compound 2 complex belong to the monoclinic space group C2 with two complexes in the asymmetric unit. A 2.5 Å data set was collected. The diffraction data were integrated and scaled using HKL2000.38 The structure was determined by molecular replacement with PHASER39 using another WNK1 structure (unpublished results) as a search model. Model building and refinement were performed using COOT40 and PHENIX.41−43 The working R factor and free R factor for the final model were 0.196 and 0.260, respectively. Statistics for the collected data and refined model are summarized in the Supporting Information Table 5. PDB

where r, rlowcontrol, and rhighcontrol are the peak ratios for the tested compound, the low control, and the high control, respectively. For the kinetic analysis, 21 μL of fluorescein-labeled OSR1 peptide (0−40 μM) and ATP (0−800 μM), both at various concentrations, were incubated with 3 μL of DMSO in the reaction buffer containing 20 mM HEPES-Na (pH 7.5), 1 mM MnCl2, 0.01% Tween 20, and 2 mM DTT. The reaction was initiated by the addition of 6 μL of the enzyme at a final concentration of 25 nM in the 384-well plate (Corning). The plate was placed immediately in the Labchip 3000 system, and the samples were sipped onto a four-sipper chip every 3 min from 90 to 250 min. The temperature and humidity in the reaction chamber were maintained at 20 °C and 50%, respectively. The substrates and phosphorylated products were separated and detected on the chip. The initial velocity vs each substrate concentration at each inhibitor concertation was plotted. Data were globally fit to the noncompetitive inhibition equation33 to calculate Ki:

V = VmaxS /(S(1 + I /K i) + K m(1 + I /K i)) where V is measured initial velocity, Vmax is maximal velocity at saturating substrate concentration, S is either ATP or peptide substrate varied in this experiment, and I is inhibitor concentration. In Vitro Radiometric Assays for WNK Family Selectivity Assessment. To address the selectivity of the inhibitors among WNK family kinases, we developed a radiometric kinase assay using WNK1, WNK2, WNK3, and WNK4 proteins (CarnaBioscience). The assay utilized 5 to 10 nM of WNK1−4 protein compared to 25 nM used for mobility shift assay, enabling a more accurate comparison of selectivity among potent inhibitors. Assays measured the incorporation of 33P from [γ-33P]ATP into myelin basic protein (MBP) coated in the wells of 96-well ScintiPlates (PerkinElmer). Each well of the MBP-coated ScintiPlates held 100 μL of a solution containing 20 mM HEPES at pH 7.3, 5 mM MnCl2 (WNK1 and WNK4) or 3 mM MnCl2 (WNK2 and WNK3), 0.01% Tween-20 (WNK1, WNK3, and WNK4) or 0.02% Tween-20 (WNK2), 1 mM TCEP, 2% DMSO, 1 μM ATP (WNK1, WNK3, and WNK4) or 2 μM ATP (WNK2), 1 μCi [γ -33P]ATP (WNK1, WNK2, WNK4) or 0.25 μCi [γ-33P]ATP (WNK3), WNK kinase enzyme (5 nM WNK1, 10 nM WNK2, 5 nM WNK3, or 10 nM WNK4), and the compound at the desired concentration. The plate was sealed and mixed for 20 s at 800 rpm on a benchtop plate shaker. The plate was then placed in a 25 °C shaking incubator at 175 rpm. Reactions were run within the linear range of the assay for each enzyme and stopped by the addition of 50 μL of quench buffer (45 mM EDTA, 0.01% Tween-20, and 20 mM HEPES, pH 7.3). The content of each well was then aspirated, and the well was washed three times with 300 μL of assay wash buffer (150 mM NaCl, 0.02% Tween-20, and 50 mM Tris/HCl at pH 7.4). Incorporation of 33 P into the bound MBP substrate was measured using a MicroBeta TriLux LSC and Luminescence plate counter. Nonspecific background was measured in the absence of WNK enzyme. Effect of WNK Inhibitors on Sorbitol Stimulated OSR1 Phosphorylation in HEK293 Cells. HEK293 cells were reverse transfected with pcDNA-3(+)-flag-OSR1 (Plasmid 0.5 μg, Fugene 6 1.5 μL, 0.4 × 106 HEK293 cells in 1 mL MEM). The cell transfection mixture was then plated at 40 μL/well in a 384 PDL plate (BD) by a Matrix WellMate (ThermoScientific) and incubated at 37 °C for 48 h. On the day of the experiment, 20 μL of medium was aspirated from the cell plate, and 20 μL of 2 × compound in medium was added and incubated at 37 °C for 2.5 h. A total of 20 μL of medium was then replaced with 20 μL of 2 × sorbitol with 1 × compound and incubated at 37 °C. After 1 h, the medium was removed and cell plate gently tapped dry on a lab napkin, followed by the addition of 30 μL of cell lysis buffer (1 tablet of protease inhibitor cocktail, 50 μL of sodium vanadate, and 50 μL of sodium fluoride in 50 mL of AlphaLISA lysis buffer) and stored at −70 °C. pOSR1 was then determined by AlphaLISA (PerkinElmer) according to the manufacturer’s instructions. Briefly, antiflag acceptor beads were diluted in freshly prepared 1 × AlphaLISA buffer (25 ug mL−1). A total of 10 μL of biotin-Rabbit anti-pOSR1 in 1 × AlphaLISA buffer at a final concentration of 5 nM was then added (final concentration of biotin-rabbit anti-pOSR1 1.25 nM) to 2.5 μL of cell lysate in a 384-well OptiPlate (PerkinElmer) and 3343

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ACS Chemical Biology coordinates and accompanying structure factors have been deposited under PDB code 5TF9. Differential Scanning Calorimetry (DSC) Measurements of WNK1 (aa206−483). DSC was used to examine the thermal stability of WNK1 and pWNK1 using a MicroCal VP-Capillary DSC, scanning from 10 to 100 °C with a scan rate of 60 °C per hour. Proteins were dialyzed against a buffer containing 25 mM HEPES (pH 7.3), 100 mM NaCl, and 1 mM TCEP, followed by the addition of 5% (v/v) DMSO or compound solution in DMSO. Sample and blank buffer with 5% (v/v) DMSO solutions were loaded to the sample and reference cells, respectively. DSC data were corrected by baseline subtraction, normalized against concentration, and fitted to get Tm using ORIGIN (MicroCal) software. Surface Plasmon Resonance (SPR) Measurements of WNK1 (aa206−483). SPR measurements were performed to enable detailed studies on binding kinetics as well as to examine additivity of compound binding with AMP−PNP. The measurements were performed on a BIAcore T100 biosensor at 25 °C. WNK1 and pWNK1 proteins (2 mg mL−1) were first buffer exchanged into 1× PBS using Zeba Spin Desalting Columns. EZLink Sulfo-NHS-LC-LCBiotin (1 mg mL−1 in PBS) was then added to the proteins to provide a molar ratio of cross-linking reagent and WNK1 (or pWNK1) of about 1:0.5 to 1:1. The mixtures were incubated on ice for 30 min, and the biotinylation reaction was stopped by the addition of 1× TBS. Unreacted and hydrolyzed biotin reagent was removed by buffer exchange of the mixture into 1× TBS (with 0.5 mM TCEP) using Zeba Spin Desalting Columns. BIAcore running buffer contained 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% Surfactant P20, 0.5 mM TCEP, and 2% (v/v) DMSO. Equivalent amount of biotinylated WNK1 and pWNK1 were immobilized in two different cells of one streptavidin-coated biosensor chip. Compounds were prepared at different dilutions (from 0 to 25 μM) in buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% Surfactant P20, 0.5 mM TCEP, 2.5 mM MnCl2, and 1 mM AMP−PNP, and the final concentration of DMSO was 2%. Binding of the compounds to the immobilized WNK1 and pWNK1 was performed under a flow rate of 50 μL min−1 with 240 s of association time and 1500 s of dissociation time. Sensorgrams without compound were used as a baseline, and baseline subtracted data were analyzed by global fitting to a 1:1 binding model using the BIAcore T100 Evaluation Software. The association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD = kd/ka) were obtained as fitting results.

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synthesis and developed structure−activity relationship; X.X., J.G., and D.K. performed SPR and X-ray crystallography; D.Y., N.I., W.H., Y.I.Y., and H.-M.P. designed and performed the cellular pOSR1 assay; J.R., H.Y., and I.S. designed and performed the NKCC1 assay and siRNA knock-down studies; M.K. performed modeling; K.Yam., M.M., and H.-M.P. designed the hit finding strategy; and K.Yam. and H.-M.P. directed drug discovery experiments. Notes

The authors declare the following competing financial interest(s): All authors were employed by Novartis during the period of their research described in this manuscript. All research was fully funded by Novartis. ⊥ Deceased

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ACKNOWLEDGMENTS The authors thank Drs. J. Elliott and J. Marcinkeviciene for their guidance on this manuscript; Drs. G. Paris and H. Möbitz for their careful reading of the manscript; Mr. K. Gunderson for his assistance on the NMR-based structure confirmation; and Drs. D. Fabbro, G. Iwasaki, and A. B. Jones for the discussions on the allosteric hit finding strategies. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00511. Supporting Figures 1−3, supporting Tables 1−5, supporting data, supporting methods, and comments (PDF) A movie showing the allosteric pocket formation upon compound 2 binding to WNK1 (MPG)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions #

These authors contributed equally to the work. K.Yam. prepared the manuscript; J-H.Z. and Q.K.Y. performed HTS; A.Q.X., D.B., H.W., N.K., and P.P. produced WNK1 protein; Z.D. designed and applied in silico filters for hit-list triaging; Y.I.Y. and H.-M.P. designed and performed the mobility shift assay including enzyme kinetic studies; D.L. designed and performed the radiometric assay; P.D. performed kinase selectivity screen; K.Yam., K.Yas., and T.K. performed chemical 3344

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ACS Chemical Biology

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DOI: 10.1021/acschembio.6b00511 ACS Chem. Biol. 2016, 11, 3338−3346

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DOI: 10.1021/acschembio.6b00511 ACS Chem. Biol. 2016, 11, 3338−3346