*5.2. Inhibitors of NKCC1*

It has been established that the recovery process from many neurological disorders including stroke would highly benefit from inhibition of NKCC1 activity [94]. Suppression of NKCC1 activity through bumetanide is neuroprotective and improves post-stroke neurophysiological status [142,150]. Thus, bumetanide has the potential to influence many CNS disorders [94]. Several studies have demonstrated the contribution of NKCC1 in the development and progression of post-stroke edema and cell death, thus targeting NKCC1 could be a potential neuroprotective target [22,26,32,65,111,151]. In fact, a pharmacological study using bumetanide demonstrated a significantly reduced neuronal Na+ overload and cell death. Bumetanide also simultaneously reduced infarct volume and brain edema [104]. Another rat model study showed that bumetanide administered after focal cerebral ischemia in rats (given 7 days post-ischemia, and continued for 21 days), improved behavioral recovery and promoted neurogenesis 4 weeks post-havoc [141]. Low concentrations of bumetanide (2 to 10 μM) are capable of inhibiting NKCCs in vitro with no significant effect on the KCCs; a high concentration has been shown to inhibit the activities of both NKCC1 and KCC2 [152,153]. The expression of NKCC1 is common at the luminal side of endothelial cells of the BBB, thereby allowing easy interaction between the transporter and its inhibitor (bumetanide) when administered intravenously, which subsequently decreased edema in MCA occlusion model of stroke in rats [154]. Bumetanide acts through docking to the binding site at the trans-membrane region of NKCC1. Docking to this region allows for the inhibition of NKCC1 activity and reduced [Cl−]i in neurons [94]; a more likely mechanism through which the drug confers neuroprotection and neuronal recovery following stroke episodes [94,155].

Simard et al. [156] and Walcott et al. [42] in their respective reviews highlighted the implication of the constitutive expression of NKCC1 and SUR1-regulated NCCa-ATP (SUR1/TRPM4) channel on the cascade of events that are involved in the pathogenesis of cerebral ischemia and the impact of combinatorial therapy of bumetanide and glibenclamide in ameliorating the havoc. Bhuiyan and colleagues [106] reported that bumetanide downregulated the WNK-Cab39-NKCC1 signaling pathway, consequently reducing the susceptibility of hypertensive rats to ischemic brain damage. Furthermore, in a recent

study a synergistic treatment with mild hypothermia (33.5 ◦C for 30 min) and inhibitor DAPT (50 μM) attenuated the overexpression of NKCC1 mRNA following global cerebral ischemia injury in rats [20].

In animal stroke models, bumetanide administration pre- and post-stroke induction led to the down-regulation of NKCC1 expression. Other observations include a reduction in edema, infarction volume, and ischemic necrotic cell death especially in the early stage of ischemic damage, promotion of neurogenesis, and improved sensorimotor recovery [17,97,109,141,157,158]. In another rat model study, ET-1 was used to induce focal ischemia but post treatment with bumetanide selectively inhibited NKCC1 expression in the cortex and promoted synaptic plasticity in the denervated cervical spinal cord following cerebral ischemia [111]. Similarly, Xu et al. [141] demonstrated that chronic treatment with bumetanide promotes neurogenesis and behavioral recovery after ET-1-induced stroke in rats. In addition, bumetanide (10 μM) was used in another study to block NKCC1 in order to facilitate decreased [Cl−]i in hippocampal tissue cultured from rats either during oxygen-glucose deprivation for 120 min or post-exposure. The drug improved neuronal viability during the acute ischemic episode which suggested its critical role in the modulation of transmembrane chloride transport [27].

Indeed, bumetanide appears to be a promising pharmacological inhibitor of NKCC1; it possess some demerits that may limit its application as an anti-stroke drug to some extent [23,94]. Alongside bumetanide, a novel inhibitor STS66 (a prodrug of bumetanide) also exhibits promising potential as a pharmacological inhibition of NKCC1 and has been demonstrated to also reduced ischemic infarction, swelling and neurological deficits in mice model of transient ischemic stroke [124]. Interestingly, STS66 can penetrate BBB more easily and appears to be more efficient in eliciting the aforementioned anti-stroke properties [124], which is one of the various reasons it has been recently proposed as a better therapeutic drug in stroke management when compared with bumetanide [23,65]. A finding contrary to the common hypothesis on the efficacy of bumetanide was recently reported [18]. In this study, post treatment with bumetanide (40 mg/kg) following Intracerebral hemorrhage induction in male Sprague Dawley rats failed to improve behavior or lessen injury neither did the drug normalized ion concentrations after late dosing [18].

In spite of the positive outcomes demonstrated clinically by administration of bumetanide to patients with psychiatric/neurological conditions [159–166]; the drug has exhibited strong diuretic effect resulting from the inhibition of NKCC2 expression in the kidney which may pose serious challenges to issues on drug compliance and health concerns [167–169], thereby limiting the therapeutic applications of bumetanide. Hence, selective inhibition of NKCC1 in lieu of renal NKCC2 may attenuate the diuretic glitches. In this regard, Savardi et al. [170] recently discovered ARN23746, a selective inhibitor of NKCC1 in lieu of NKCC2 and KCC2 in vivo. The reports from the study demonstrated that the pharmacokinetic profile of ARN23746 is better when compared with that of bumetanide in vitro and in vivo. Briefly, the study demonstrated that ARN23746 (10 μM) restored aberrantly high [Cl−]i to the physiological level in mature hippocampal neuronal cultures of Ts65Dn mouse model Down syndrome (DS) coupled with rescued cognitive impairment in Ts65Dn with no significant diuretic effect in either the WT or Ts65Dn mice [170]. Furthermore, the researchers demonstrated that intraperitoneal administration of ARN23746 recovered social and repetitive behaviors associated with the main symptoms of autism spectrum disorder (ASD) in valproic acid (VPA) mouse model of ASD. In addition, neither diuretic effect nor overt toxicity of the compound were present in the ARN23746 treated mice [170]. ARN23746 has great potential for further development into a clinically-relevant drug for the treatment of DS, ASD [170], and possibly several other neurological conditions characterized by impaired Cl− homeostasis including stroke.

## *5.3. Activator of KCC3*

The KCCs, especially KCC2 and KCC3, are popular due to increased findings on human disease-causing mutations [68,121,122]. Hence, the discovery of small molecules that modulate these cotransporters' activities is prioritized within the field. Discovery of such modulators may aid development of therapeutic drugs for the management of KCCrelated diseases as well as other pathological conditions including stroke [171]. Currently, the loop diuretics bumetanide and furosemide are the only FDA-approved drugs that modulate the KCCs [172]. In a mouse model study, bumetanide is demonstrated to be involved in the stimulation of KCC3 expression and subsequent extrusion of [Cl−]i in the sensory neurons [114]. In addition, Adragna and co-workers [87] in a cell culture study substituted Thr<sup>991</sup> and Thr<sup>1048</sup> residues with alanine at the carboxyl terminus of KCC3a protein, which prevented inhibitory phosphorylation at the substituted sites and subsequently triggered increased expression of KCC3a mRNA. Interestingly, the flux condition accompanied a down-regulation of NKCC1 expression, facilitated by the addition of ouabain (0.1 mM), and bumetanide (10 μM) to the flux media [87].

However, Delpire and Weaver [171] recently expressed their concerns for the need to develop modulators of KCC activity to provide insights into KCC modulation as a therapeutic strategy for neurological conditions such as stroke. Unfortunately, these FDA approved drugs (bumetanide and furosemide) are poor inhibitors of KCCs, with a higher potency for NKCC1 or NKCC2 (IC50 = 0.5–5.0 μM) in comparison to KCC (IC50 = 50–500 mM) [33,171]. In fact, drugs that can act as weak inhibitors might be better alternatives as complete inhibition mimics a loss-of-function, which could presumably be harmful for the nervous system. As the KCC isoforms have different expression patterns and physiological functions, target specificity in the deployed pharmacological approach is also an issue [172]. In a large screening effort targeted against KCC2, Delpire et al. [173] was able to identify inhibitory compounds more potent (3–4x) than the two loop diuretics. However, these compounds are not ideal drug candidates due to the following reasons: (1) non-specificity to KCC2 as they concurrently inhibit KCC3, and (2) poor pharmacokinetic properties [173,174]. Meanwhile, the ability of loop diuretics to reach the CNS/PNS remains obscure [171,172]. Perhaps a better pharmacological approach would be to develop therapeutic compounds that are specific modulators of the KCC3. Fortunately, our recently developed novel molecular compound, ZT-1a, is a SPAK kinase inhibitor that specifically stimulates KCC3 and inhibits NKCC1 by decreasing their SPAK-dependent phosphorylation/signaling pathway in cultured cells and in vivo rat and mouse brains [70] (also see Figure 2). In addition, the systematic administration of ZT-1a ameliorated phosphorylation of co-transporters and cerebral edema following ischemia, protect against brain damage and improve neurological functions after stroke episode [70].

#### **6. Conclusions and Future Directions**

The CCCs play crucial roles in regulating neuronal functions. The cotransporters are key mediators of several and important cellular functions such as cell volume regulation, trans-epithelial ion transport, and maintenance of [Cl−]i. Modulation of NKCC1 and KCC3 expressions by their upstream regulator, WNK-SPAK/OSR1 is implicated in the development and progression of stroke. There are several demonstrations that phosphorylation of NKCC1 and KCC3 via the WNK-SPAK/OSR1 signaling may lead to activation of NKCC1 and inhibition of KCC3 either during or post-stroke episode. In fact, the role of NKCC1 and KCC3 as well as their regulatory proteins in stroke pathogenesis suggests that they are potential targets for the treatment of stroke. The pharmacological strategies that were discussed in this review possess potential therapeutic efficacies for stroke management. Novel compounds must successfully address concerns regarding off-target effects due to the many isoforms and physiological function related to the WNK-SPAK/OSR1-CCC pathway. As advances in stroke therapy may also benefit other neurological impairments, we strongly suggest consistent follow-up actions on currently available pharmacological treatments for stroke through detailed research studies to aid further development of therapeutic drugs with a better pharmacokinetic profile. Hence, we holistically advocate for increased focus on human clinical research on this topic as informed by its paucity to that regards.

**Author Contributions:** S.S.J. and J.Z. were responsible for writing the whole passage. S.S.J., N.F.M.A., and J.Z. were responsible for checking and revision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was in part supported by a Commonwealth PhD Scholarship (S.S.J.) and the University of Exeter Medical School start-up fund (J.Z.) and NIH Grants R01 NS109358 (J.Z.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no competing interests.

#### **References**

