*5.1. Inhibitors of WNK-SPAK/OSR1 Pathway*

A quick recap of the following crucial information from previous sections, which may contain pharmacological strategies for managing stroke: (1) WNK-SPAK/OSR1 modulates the activities of CCCs through a well-coordinated reciprocal pattern of regulation [53,60,67,69], (2) activation of WNK-SPAK/OSR1 signaling pathway stimulates NKCC1 and inhibits KCC3 expressions [19,53,67,72], and (3) development and progression of stroke have been implicated with phosphorylated WNK-SPAK/OSR1 signaling pathway and subsequent upand down-regulations of NKCC1 and KCC3 expressions, respectively [22,140]. Therefore, we can safely presume that molecular compounds that act as pharmacological or genetic antagonists of WNK-SPAK/OSR1 kinases are likely potential drug candidates for the treatment of stroke. Recent reports have shown that drugs that are potent blockers of WNK-SPAK/OSR1 signaling pathway reduce phosphorylation of NKCC1 and KCC1 which enables cellular chloride expulsion, subsequently mitigating cerebral edema and other

neurological anomalies following ischemia. This then protects against brain damage and enhances post-stroke brain functions [32,65,70,86,92,94,124,141].

The WNK-SPAK/OSR1 pathway constitutes potential therapeutic targets in Cl− dysregulation [23]. The loop diuretic, bumetanide protects the brain from damage by mediating GABAergic signaling in NKCC1 expression following ischemic injury [22,23,26,142]. Furthermore, treatment with the drug reversed the impact of GABA-mediated depolarization, which may promote functional recovery after stroke via neuron repair/protection as adjudged by its effect of GABAA receptor antagonist and WNK3 knockout [94,142]. Pharmacologically targeting the WNK-SPAK/OSR1 kinase pathway could be a strategy to restore GABAergic inhibition [23]. Indeed, genetic or pharmacological inhibition of WNK-SPAK/OSR1 activity would lead to cotransporter dephosphorylation: inhibition of NKCC1 and activation KCC3, which would enhance [Cl−]i extrusion [67]. Furthermore, enhancement of [Cl−]i extrusion in the neurons would facilitate GABAA receptor-mediated hyperpolarization and thus inhibit neuronal activity through combined NKCC1 inhibition and KCC3 stimulation [19,22,53,65,67] (Figure 2).

Recent studies have indicated that decline in NKCC1 protein expression along with WNK3 knockdown, contributes to lessened post-stroke brain injury and accelerated neurobehavioral recovery [32,86,92]. These reports and more have immensely motivated the development of novel therapeutic strategies that have targeted WNK-SPAK/OSR1 signaling pathways to improve post-stroke physiological functions [70,106,124]. A recent rat model study demonstrated an upregulation of WNK-SPAK/OSR1-NKCC1 signaling pathway in the brains of spontaneously induced-hypertensive rats and subsequently augmented susceptibility to ischemic damage [106]. However, intraperitoneal administration of bumetanide (10 mg/kg) post-reperfusion blocked the WNK-Cab39-NKCC1 signaling pathway and subsequently mitigated post-ischemic infarction and cell swelling and improved neurological functions in animals [106]. Loop diuretics are often used to inhibit NKCCs. Inhibition of NKCC2 promote diuresis in the kidney and reduces pressure due to excess fluid in the lungs. Hence, loop diuretics are a treatment option hypertension and pulmonary edema. Although some loop diuretics inhibit KCCs, they do so very poorly. Researchers have explored the loop diuretics bumetanide and furosemide as novel treatment options for brain disorders [65]. However, a number of unfavorable physiochemical characteristics associated with the use of bumetanide [23,41,65] call for better alternatives in the management of neurological diseases including stroke. Recently, Huang and coworkers [124] reported that a novel NKCC1 inhibitor (STS66) is superior to bumetanide in ameliorating ischemic brain injury following transient MCA occlusion and large-vessel ischemic stroke models. In the study, ischemic injury stimulated WNK-SPAK-NKCC1 cascades in brains of AngiotensinII (AngII)-induced hypertensive mice. However, STS66 treatment completely blocked this pathway and by implication mitigated ischemic infarction, cerebral edema, and neuronal death as well as neurological deficits in both stroke models with hypertension comorbidity [124].

We recently proposed that improved understanding of cooperative interactions among different phospho-sites of cotransporters and the molecular mechanisms involved in their physiological regulations could provide insights to inform potential pharmacological interventions [71]. Recently, we conducted a large-scale phospho-proteomics study with the application of immunoblot and phospho-antibodies immunoprecipitation techniques to investigate the regulatory mechanisms of a broad kinase inhibitor, staurosporine and Nethylmalemide (NEM), a modulator of both kinase and phosphatase activities on phosphorylation of specific KCC2 and NKCC1 in HEK293 cells and immature cultured hippocampal neurons [71]. Our analyses revealed dephosphorylation of Thr203, Thr207, and Thr212 of NKCC1 and Thr<sup>1007</sup> of KCC2 following application of the two agents. The two compounds resulted in dephosphorylation of sites Thr233 and Ser373, phosphorylation sites located within the T-loop and S-loop of SPAK. Hence, the study suggests the inhibitory effect of staurosporine and NEM on WNK-SPAK/OSR1 signaling pathway in the regulation of NKCC1 and KCC2 is in a reciprocal pattern [71]. We are of the opinion that the underlying

information from this study will be highly important for future development of integrative therapeutic strategies in the management of neurological diseases such as cerebral stroke.

Importantly, evolving roles of WNK-SPAK/OSR1 signaling in stroke as discussed in this review, points to additional possible applications of WNK-SPAK/OSR1 modulation in neurological diseases. In view of this, a promising strategy could involve exploitation of the unique structure of these kinases to enhance protein specificity [53,70]. Immense efforts to inhibit WNKs or SPAK/OSR1 for the treatment of human diseases such as hypertension have led to the discoveries of small molecule inhibitors. WNK kinase inhibitors include WNK463 [143], PP121 [144], and SPAK inhibitors such as STOCK1S-14279, Closantel [145], Rafoxanide [146], Verteporfin [74], STOCK1S-50699, and STOCK2S-26016, [147], as well as HK01 [148] and 20I [149] (also see Figures 3 and 4). Unfortunately, none of these compounds is an ideal drug candidate for the treatment of brain disorders due to their relatively low penetrability through the blood-brain barrier (BBB). Recently, we employed a scaffold-hybrid strategy in our laboratory to develop a novel compound ZT-1a. ZT-1a is a non-ATP-competitive SPAK blocker, which specifically inhibits this signaling pathway by decreasing SPAK-dependent phosphorylation of NKCC1 and KCCs in cell cultures as well as in vivo mouse and rat brains [70]. In brief, treatment with ZT-1a (2.5–5.0 mg/kg) abated post-stroke related brain injuries and improved neurological features/functions. The data from the study suggests that ZT-1a or related compounds that are CCC modulators could be a therapeutic strategy for neurodegenerative disorders such as cerebral stroke [70]. Hence, we holistically advocate for follow-up with detailed research studies on the development of more WNK-SPAK/OSR1 inhibitors with favorable pharmacokinetic properties for clinical use.
