Modulation of brain cation-Cl− cotransport via the SPAK kinase inhibitor ZT-1a

The SLC12A cation-Cl− cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide (“ZT-1a”). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.


I.
General Methods for Chemistry.
Reagents and solvents were obtained from commercial sources and used without further purification.
Reactions were monitored by thin layer chromatography (TLC) on glass plates coated with silica gel with fluorescent indicator. Compounds were purified either by chromatography on silica gel or by preparative high performance liquid chromatography (HPLC). Silica gel chromatography was performed on the Teledyne Synthesis of 1a-1j.

5-chloro-2-hydroxybenzoyl chloride (I-1a)
The mixture of 5-chloro-2-hydroxy-benzoic acid (103.5 mg, 0.6 mmol) in 1.5 mL of thionyl chloride was refluxed at 80°C for 2 h. The resulting solution was cooled to room temperature, and excess thionyl chloride was removed under vacuum to afford I-1a as gummy yellow solid, I-1a was directly used in the next step.

Plasmids, protein expression and purification
DNA clones were from the Division of Signal Transduction Therapy (University of Dundee). The SPAK proteins were expressed in E. Coli and purified as described previously 1 . IC 50 values were calculated with GraphPad Prism using non-linear regression analysis.

ZT-1a pharmacokinetic (PK) properties in sham control and ischemic stroke mice
ZT-1a content in brain homogenates and plasma was assayed at the University of Pittsburgh Small Molecule Biomarker Core, using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
C57BL/6j mice (male, 11 weeks old) at 3 hr post Sham surgery or tMCAO were intraperitoneally injected with 5 mg/kg ZT-1a. At 2 hr post injection, blood was collected into heparin-treated EP tubes by a cardiac puncture. After a brief cardiac perfusion with ice-cold normal saline, brain tissues (contralateral and ipsilateral hemispheres) were collected. Plasma samples were prepared by centrifugation of whole blood for 10 min at 1,500g using a refrigerated centrifuge. Brain tissues and plasma samples were diluted, and protein was precipitated with acetonitrile prior to detection of ZT-1a with a triple quad mass spectrometer. Glyburide was used as the internal standard. Samples were then injected by autosampler and ZT-1a was eluted from a Waters Acquity UPLC BEH C18, 1.7 um, 2.1x100 mm reversed-phase column with a water (with 0.1% formic acid) and acetonitrile (with 0.1% formic acid) gradient. Detection and quantitation of ZT-1a were achieved in the positive mode with a Thermo Fisher TSQ Quantum Ultra mass spectrometer interfaced via an electrospray ionization (ESI) probe with the Water UPLC Acquity solvent delivery system.

Human fibroblasts
Primary fibroblast cell culture was established as described previously 3 . Cells (2x10 5 cells per coverslip) were cultured on glass coverslips coated with poly-D-lysine in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.1 mg/mL penicillin/streptomycin Cultures were incubated at 37 o C in an incubator with 5% CO 2 and atmospheric air. Passages 9-21 were used in the study.

Cell volume measurements
Cell volume change was determined using the dye Calcein AM as a marker of intracellular water Supplemental Fig. 1 Concentration-response experiments testing effects of Closantel analogues on phosphorylation of KCC3 Thr991/Thr1048. HEK293 cells were transfected with cDNA encoding wild type N-terminally FLAG-tagged KCC3. 36 h post-transfection, cells were exposed 30 min to either control isotonic conditions or hypotonic low Clconditions, then treated in the same conditions with the specified inhibitors (1b, 1c, 1f, 1d, 1h and 1g) at the indicated concentrations for an additional 30 min.
Lysates were subjected to SDS-PAGE and Western blotting with the indicated antibodies. See Supplementary Fig. 2 for immunoblot quantitation.
Supplemental Fig. 6 Evidence that SPAK associates with WNK1, and interaction is disrupted by ZT-1a and its analogs. Non-transfected HEK293 cell lysates were incubated with the RFQV peptide (SEEGKPQLVGRFQVTSSK), AFQV peptide (SEEGKPQLVGAFQVTSSK), STOCK1S-50699, Closantel or ZT-1a for 30 min on ice and subjected to SPAK antibody immunoprecipitation. Immunoprecipitates were subjected to immunoblot probed with antibody to total WNK1 and antibody to total SPAK. Left panels: representative immunoblots. Right panels, summarized data quantitated by densitometry (n=3, means ± SEM; **, Data are mean ± SD, n = 3. c and f Similar reductions and recoveries of rCBF in the ischemic ipsilateral hemispheres (IL) were detected in the ZT-1a-treated (c) or Closantel-treated ischemic mice (f), and neither differed from vehicle-injected mice. Data are mean ± SEM, n = 3.
Supplemental Fig. 12 Kaplan-Meier survival curve of naïve and ischemic stroke mice treated with Closantel or ZT-1a.