Introduction

In drug discovery and development, ion channels are the third largest class of drug targets [1], behind only receptors and enzymes. The drugs that modulate these proteins act as (1) agonists or antagonists of receptors, (2) activators or inhibitors of enzymes, or (3) openers or blockers of ion channels. Hence, ion channels are important drug targets [2,3,4,5,6], comprising 19% of all human proteins with corresponding drugs approvals [7], largely for the treatment of diseases of the nervous, cardiovascular, and endocrine systems. In the pursuit of new classes of drugs for human diseases, ion channels represent a promising group of proteins in the discovery and development of new therapeutics.

Functionally, ion channels are membrane proteins forming pores that selectively allow ions to cross organellar and cell plasma membranes. Ion channels conduct different ionic species and thus are classified as sodium (Na+), potassium (K+), calcium (Ca2+), nonselective cation, or chloride (Cl−) anion channels. Ion channels are expressed in both excitable and nonexcitable cells and transport their respective ion species relying on an open channel state and the electrochemical gradient across the cellular membrane. Ion channels are gated either by changes in membrane potential voltages (voltage-operated ion channels), by second messengers, or receptor ligands [8]. Ion channels can be activated depending on the replenishment of intracellular calcium stores and are involved in the sensing of physical stimuli such as cold, heat, pH, mechanical stress, and ultimately pain [9]. Intracellular calcium, ATP, pH, and many other factors may regulate basic ion channel activity, while mutations of ion channels may cause the ion channel dysfunction underlying many known channelopathies [10, 11]. Thus, ion channels play important roles in both physiology and pathophysiology.

An important pathophysiology is brain ischemia. According to the World Health Organization [12, 13], stroke [14, 15] is a leading cause of mortality and immobility in adults worldwide. Stroke prevalence has consistently increased in recent years, accompanied by high mortality rates. Except for tissue plasminogen activator, which has a limited therapeutic window [16], there are no other effective therapeutic measures against stroke, resulting in high social and economic impacts worldwide [17].

Neonates can undergo hypoxic–ischemic brain injury, and hypoxic–ischemic encephalopathy (HIE) is a subsequent related early-onset brain and neuropsychological disorder in children [18, 19]. HIE manifests as a group of brain impairments, such as neurodevelopmental delay, cognitive and motor dysfunctions, and permanent neuropsychological handicaps in the form of cerebral palsy with or without epilepsy, learning and intellectual disabilities. Neonatal hypoxic–ischemic brain injury and its related brain disorders, HIE and cerebral palsy, cause significant lifetime health care costs, and lifelong disability causes a noticeable burden worldwide. For years, studies examined the mechanism of HI brain injury and the related HIE, but these research efforts have not yet yielded positive results for therapeutic treatment. Currently, the limited clinical management for HI brain injury is using ventilation and inducing mild hypothermia [20]. In both adults and children, hypoxia-ischemia injury causes a substantial health and economic burden.

Mechanistically, cerebral ischemia triggers calcium overload and intracellular ionic imbalance and eventually leads to neuronal cell death [14, 15]. During cerebral ischemia, glutamate, an excitatory neurotransmitter, is released from neurons to act on glutamate receptor channels and trigger calcium overload and neuronal cell death [14, 15]. Mediated by glutamate NMDA and AMPA receptor channels, glutamate excitotoxicity has been the traditionally accepted mechanism [21, 22] and has been in the spotlight for stroke research for decades. Theoretically, inhibition of calcium overload is deemed to be neuroprotective. Consistent with this idea, numerous in vitro and in vivo studies have demonstrated that blocking calcium-mediated glutamate receptor channels during oxygen and glucose deprivation and cerebral ischemia reduces intracellular calcium overload and subsequently ischemic brain damage. Despite overwhelming positive experimental results, subsequent human clinical trials of anti-excitotoxic therapies (AET) did not show the expected therapeutic outcomes [23]. As a result, stroke researchers started probing for alternative pathways instead of the traditional excitotoxicity glutamate mechanism. This effort led to many new studies showing that a nonglutamate mechanism of cerebral ischemia can trigger intracellular ionic imbalance and ischemic neuronal cell death [21, 22]. Therefore, neuronal cell death and ischemic brain damage during cerebral ischemia have been recognized as the result of both glutamate-mediated excitotoxicity mechanisms and the newly identified nonglutamate mechanisms of cerebral ischemia and stroke [21, 22]. The nonglutamate mechanisms of cerebral ischemia are thought to partially explain the unsatisfactory outcomes of previous AET clinical trials. It is therefore critical to investigate these new mechanisms and their therapeutic potential for stroke. As with drug development for the traditional excitotoxicity pathway, new therapies must be developed strictly based on the guidelines set by the Stroke Therapy Academic Industry Roundtable Protocol [24] for testing potential stroke therapies using in vivo models of stroke with multiple species. It is also essential to consider the effects of a potential drug on both the glutamate and nonglutamate mechanisms of stroke. The nonglutamate mechanisms of cerebral ischemia that have emerged in recent years involve ATP-sensitive potassium (KATP) channels [25,26,27,28], transient receptor potential (TRP) channels [29,30,31], volume-regulated anion channels [32], acid-sensing ion channels [33], hemichannels [34], sodium-calcium exchangers [35], or other nonselective cation channels [36]. The sequence of activation of nonglutamate mechanisms must be considered. For instance, KATP channels cause anoxic depolarization at the early stage of ischemic events, followed by traditional glutamate excitotoxicity and calcium overload; after glutamate excitotoxicity, TRPM7 activity can be triggered, and TRPM2 ion channels can be prompted in later phase of cerebral ischemia and initiated during ischemia-induced neuroinflammation, accelerating the devastating ischemia cascade. Thus, in the treatment of stroke, the nonglutamate mechanisms of cerebral ischemia are attractive targets for neuroprotection and bring attention to the related ion channels.

Screening of chemical libraries for new drug targets, including ion channels, is a major strategy in drug discovery. Two important sources of drug leads as screening targets include natural products and synthetic libraries [37], the former of which has been a focus of our group’s chemists. Naturally occurring secondary metabolites produced by organisms have evolved to interact with biomolecules and are well established leads for drug development as potent ion channel blockers [37]. For instance, the development of Prialt® (ziconotide approved by the FDA in 2004), a peptide from the toxin of the cone snail Conus magus, for the management of chronic pain stands as a representative example of a therapeutically important calcium ion channel blocker. Here, we discuss waixenicin A, a potent ion channel blocker targeting TRPM7 that was isolated from the marine soft coral Sarcothelia edmondsoni. The effective in vitro and in vivo activity of waixenicin A gives it great potential as a lead for the development of treatments against brain disorders related to the overactivity of the TRPM7 protein.

TRP channels

TRP channels were initially described in Drosophila photoreceptors [38], as the mutant trp gene initiates a transient response to light [38] and is involved in phototransduction [39, 40]. Located on the cell membrane of various cell types, TRP channels are a superfamily of cation channels [41,42,43]. The family of TRP channels has nearly 30 mammalian homologs [43]. The classification of TRP channels has been based on their homologous sequences, and they have been separated into six subfamilies: (1) TRPC (canonical), (2) TRPV (vanilloid), (3) TRPM (melastatin), (4) TRPA (ankyrin), (5) TRPML (mucolipin), and (6) TRPP (polycystin) channels. Different physical and chemical stimuli can activate different TRP channels, which in turn play various physiological and pathological roles in different cells and tissues [41,42,43].

The melastatin subfamily TRPM channels have eight members, TRPM1 to TRPM8. TRPM7 [41, 44] is broadly expressed in many cell types and tissues, including neurons and the brain. TRPM7 is a bifunctional protein with a channel domain fused to an active kinase domain [44, 45]. The TRPM7 channel pore is selective for divalent cations, such as magnesium and calcium, and trace metal ions with a selectivity profile of Zn2+ ≈ Ni2+ » Ba2+ > Co2+ > Mg2+ ≥ Mn2+ ≥ Sr2+ ≥ Cd2+ ≥ Ca2+ ions [46].

Associated with important functions in physiology and pathophysiology, TRPM7 channel activity can be modulated by distinct intracellular and extracellular factors [47], such as Mg2+- and Mg2+-complexed nucleotides (such as Mg·ATP and Mg·GTP) [48, 49], extracellular pH [50, 51], osmolarity [52], and halides [53]. TRPM7 participates in a diverse range of cell biology, such as cellular and systemic magnesium homeostasis [54, 55] and cell growth, proliferation and adhesion [44, 56]. Overexpression of TRPM7 reduces cell viability [44, 57, 58]. TRPM7 is crucial for embryonic development [59] and skeleton formation [60]. Since TRPM7 channel activity is inversely correlated with the amount of cellular Mg·ATP [48], it was hypothesized [44] and subsequently shown that ischemia activates TRPM7 channels [61]. During ischemic conditions, low concentrations of Mg2+-nucleotides [48] and acidic conditions [51, 62] may thus contribute to TRPM7 activity and lead to ischemic cell death and brain damage.

Under physiological conditions, TRPM7 plays an important role in cell growth and neurite outgrowth [44, 56, 63]. We have recently shown that a specific TRPM7 inhibitor, waixenicin A (Fig. 1), suppressed TRPM7 currents in vitro and enhanced neurite outgrowth and maturation in primary cultures of mouse hippocampal neurons [63].

Fig. 1
figure 1

Structures of various inhibitors of TRPM7 including natural products and compounds from screens: waixenicin A is a selective and potent TRPM7 inhibitor; other non-selective and less potent TRPM7 inhibitors include carvacrol, xyloketal B, spermine, FTY720, sphingosine, AA861, MK886, NS8593 and NDGA. (IC50 = half maximal inhibitory concentration toward TRPM7; ND = not determined).

Full size image

Under pathophysiological conditions, TRPM7 also plays key roles in ischemia- and/or hypoxia-induced neuronal cell death [29, 30, 61]. Studies on the role of TRPM7 in anoxia and cerebral ischemia were initially conducted using virally mediated shRNA when a specific TRPM7 inhibitor was not available [29, 61]. As a “proof-of-principle” study, we were able to show that suppression of TRPM7 using virally mediated shRNA reduced ischemic neuronal cell death and preserved behavioral outcomes in vivo using a rat model of global ischemia [29]. Using the nonselective TRPM7 inhibitor carvacrol (Fig. 1), we were later able to show that pharmacological inhibition of TRPM7 also reduced neonatal hypoxic–ischemic brain injury in mice and preserved related behavioral outcomes [30]. We also tested another marine compound, xyloketal B [64, 65] (Fig. 1), as an inhibitor of TRPM7-mediated currents and attenuator of mouse neonatal hypoxic–ischemic brain injury and adult brain damage in a focal stroke model. Recently, we were able to demonstrate that waixenicin A, a specific TRPM7 ion channel inhibitor, attenuated hypoxic–ischemic brain injury and preserved both short-term and long-term behavioral outcomes and affected the related TRPM7 signaling in hypoxic–ischemic brain injury [66]. While our data implicated a clear impact of the TRPM7 channel domain in ischemia, recent data confirmed a critical role of the TRPM7 kinase domain in arterial thrombosis and ischemic stroke in vivo [67]. It has been shown that the TRPM7 kinase domain regulates store-operated calcium entry (SOCE) in DT40 B cells in vitro [68]. In a follow-up study, a single point mutation rendering the TRPM7 kinase domain inactive in vivo led to SOCE suppression in mouse platelets through impaired phosphatidylinositol-4,5-bisphosphate (PIP2) signaling, ultimately impairing platelet aggregation [67]. Such genetic impairment of the TRPM7 kinase domain has also been shown to negatively impact SOCE in mouse T lymphocytes in vivo [69]. Notably, it is currently unknown whether waixenicin A interferes with the activity of the TRPM7 kinase in addition to its effect on channel conductance; this is a very real possibility that, if found to be true, would show a broadened impact of this compound as a potential drug in brain injury, including SOCE-mediated inflammatory processes.

Finally, and in relation to TRPM7 and its impact on brain cancer, we were able to show that carvacrol and xyloketal B inhibited TRPM7 currents along with cell survival in glioma (GBM) cell lines in vitro, albeit at quite high concentrations [70, 71]. Furthermore, our most recent data demonstrate that waixenicin A inhibited GBM cell proliferation, migration, and invasion in vitro and inhibited GBM cell functions in vivo, as well as the related signaling (unpublished results).

Thus, we have now demonstrated the potential of the specific TRPM7 inhibitor waixenicin A in three in vitro [63] and three in vivo studies [66]. Therefore, we offer additional details on the promise that waixenicin A may hold in drug development by targeting TRPM7 for brain disorders and diseases.

Development of the TRPM7 inhibitor waixenicin A

Several inhibitors of TRPM7 are now known, including the natural products carvacrol and xyloketal B, which exhibit in vivo activity [30, 64, 65]. Other reported inhibitors of TRPM7 with in vitro potency of <10 μM include spermine, FTY720, sphingosine, docebenone (AA861), MK886, NS8593, and nordihydroguaiaretic acid (NDGA) [72]. Although all of these small molecules are reported to be inhibitors of TRPM7 (Fig. 1), inhibitory concentration curves (IC50) were not determined for some of them, with inhibition reported only at one or two concentrations for xyloketal B, AA861, MK886, and NDGA. Of these TRPM7 inhibitors, FTY720 and sphingosine were effective at IC50 values of 0.7 and 0.6 µM, respectively. The focus here is on the natural product waixenicin A, which has shown the greatest potency in vitro (with an IC50 value as low as 16 nM [73]) and in vivo activity against TRPM7.

Waixenicin A [63, 73] is a marine compound isolated from the Hawaiian soft coral S. edmondsoni. Using our groups’ established TRPM7 medium-throughput cell-based assay measuring TRPM7-mediated Mn2+ quenching of the Fura-2 fluorophore [73], the Horgen and Fleig groups identified an extract of S. edmondsoni as a dose-dependent inhibitor of TRPM7. Following bioassay-directed isolation, they showed that waixenicin A, a xenicane diterpene, inhibits cell proliferation through an Mg2+-dependent blockade of TRPM7 [73]. Subsequent in vivo studies proved that administration of waixenicin A in a cancer mouse model induced hypomagnesemia via insufficient magnesium absorption in the colon [74], showing proof-of-principle of Waixenicin A effectiveness in suppressing TRPM7 activity on a systemic level.

Waixenicin A has been extensively tested on various heterologously overexpressed TRPM channels in HEK293 cells [63, 73] and in primary neurons [63] for its selectivity for TRPM7 inhibition. This natural product demonstrates exceptional selectivity, failing to inhibit a number of related TRPM channels, including its closest homolog, TRPM6 [69], and in fact, it has no effect against zebrafish TRPM7 [75]. Furthermore, waixenicin A has been extensively tested in vitro as an inhibitor of TRPM7 currents and calcium signaling with electrophysiology and calcium imaging, and through IC50 and in vivo dose determinations [63, 66, 73]. Notably, in numerous laboratories around the world, waixenicin A has demonstrated in vitro effects that are consistent with the TRPM7 inhibition of biological effects in broad applications, such as the inhibition of malaria parasite invasion into erythrocytes [76] and a range of anticancer activities [77, 78], reduced hyperglycemia-induced neuronal damage [79], and inhibition of store-operated calcium entry in lymphocytes [68] and neuronal outgrowth [63].

In addition to waixenicin A demonstrating high selectivity against TRPM7 and consistent in vitro efficacy, it shows exceptional in vivo potency for reducing brain injury in mice. The effective in vivo testing dose was 37 µg/kg body weight by intraperitoneal injection using an hypoxic–ischemic brain injury model [66]. This outcome aligns with the high index of waixenicin A for blood brain barrier (BBB) permeation, as determined by a central nervous system multiparameter optimization (CNS MPO) analysis [80], leading to a brain/plasma equilibration rate of −3.1 for this compound, which predicts sufficient BBB penetration for CNS activity. Aside from the excellent predicted BBB properties of waixenicin A, hypoxic and ischemic conditions damage and open the BBB to enable drugs to more easily enter into brain tissue and cells. Finally, the neonatal hypoxic–ischemic brain injury model is based on p7 mouse pups, which are in a developmental stage with an incomplete BBB [81]. Since waixenicin A shows potent and specific inhibitory effects on TRPM7, it is important to determine the structure–activity relationship of waixenicin A and congeners including simplified derivatives as a next step and evaluate active candidates in critical in vivo models, including a neonatal hypoxic–ischemic brain injury model [66], adult middle cerebral artery occlusion model [28], and GBM xenograft models.

Conclusions and future direction

Waixenicin A, a potent and specific TRPM7 inhibitor, holds great promise as a drug lead for further development in therapeutics for brain diseases and disorders. Our group has demonstrated both in vitro and in vivo models showing that waixenicin A promotes neurite outgrowth, reduces brain damage in ischemia and hypoxia, and suppresses GBM cell functions. Thus, TRPM7 is an excellent drug development lead compound, and waixenicin A is an excellent candidate for further biological studies and directed medicinal chemistry research.