Jinwei Zhang

GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor.

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for some forms of Parkinsons disease. Here we report the discovery and characterization of 2-arylmethyloxy-5-subtitutent-N-arylbenzamides with potent LRRK2 activities exemplified by GSK2578215A which exhibits biochemical IC(50)s of around 10 nM against both wild-type LRRK2 and the G2019S mutant. GSK2578215A exhibits exceptionally high selectivity for LRRK2 across the kinome, substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.3-1.0 μM in cells and in mouse spleen and kidney, but not in brain, following intraperitoneal injection of 100mg/kg.

The WNK-SPAK/OSR1 pathway: Master regulator of cation-chloride cotransporters

A two-tiered kinase cascade is a potentially important therapeutic target for hypertension and neurological diseases of hyperexcitability. Gloss Intracellular chloride concentration is critical in the maintenance of cellular volume and the regulation of neuronal activity and blood pressure. Because of its therapeutic importance, several drugs exist that target cotransporters of the regulated Cl− transport, especially the cotransporters in the kidney. In this Review, with five figures and 115 references, Alessi et al. describe the genetic and biochemical experiments that led to the discovery of the molecular components involved in this essential physiological regulatory pathway that controls intracellular Cl− concentration. Knowledge of the proteins, how they interact, and the regulatory mechanisms that control their activity presents novel targets for controlling hypertension and intervening in neurological diseases associated with excess neuronal activity. The WNK-SPAK/OSR1 kinase complex is composed of the kinases WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase) or the SPAK homolog OSR1 (oxidative stress–responsive kinase 1). The WNK family senses changes in intracellular Cl− concentration, extracellular osmolarity, and cell volume and transduces this information to sodium (Na+), potassium (K+), and chloride (Cl−) cotransporters [collectively referred to as CCCs (cation-chloride cotransporters)] and ion channels to maintain cellular and organismal homeostasis and affect cellular morphology and behavior. Several genes encoding proteins in this pathway are mutated in human disease, and the cotransporters are targets of commonly used drugs. WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate Cl−-importing, Na+-driven CCCs or inhibit the Cl−-extruding, K+-driven CCCs. These coordinated and reciprocal actions on the CCCs are triggered by an interaction between RFXV/I motifs within the WNKs and CCCs and a conserved carboxyl-terminal docking domain in SPAK and OSR1. This interaction site represents a potentially druggable node that could be more effective than targeting the cotransporters directly. In the kidney, WNK-SPAK/OSR1 inhibition decreases epithelial NaCl reabsorption and K+ secretion to lower blood pressure while maintaining serum K+. In neurons, WNK-SPAK/OSR1 inhibition could facilitate Cl− extrusion and promote γ-aminobutyric acidergic (GABAergic) inhibition. Such drugs could have efficacy as K+-sparing blood pressure–lowering agents in essential hypertension, nonaddictive analgesics in neuropathic pain, and promoters of GABAergic inhibition in diseases associated with neuronal hyperactivity, such as epilepsy, spasticity, neuropathic pain, schizophrenia, and autism.

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin‐like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65‐phosphorylated ubiquitin (ubiquitinPhospho‐Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho‐Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site‐directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho‐Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho‐Ser65 to Parkin disrupts the interaction between the Ubl domain and C‐terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho‐Ser65. Our results thus suggest that a major role of ubiquitinPhospho‐Ser65 is to promote PINK1‐mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho‐Ser65‐binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho‐Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho‐Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho‐Ser65.

The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K + -Cl − co-transporters

Precise homoeostasis of the intracellular concentration of Cl− is achieved via the co-ordinated activities of the Cl− influx and efflux. We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na+–K+ ion co-transporters), also promote inhibition of the KCCs (K+–Cl− co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr1048)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic high K+ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 in KCC1/KCC3 and Thr6 in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser96). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl− influx, we propose that the targeting of WNK–SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl− extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.

Characterization of TAE684 as a potent LRRK2 kinase inhibitor

Leucine-rich repeat kinase 2 (LRRK2) is linked to Parkinsons disease and may represent an attractive therapeutic target. Here we report a 2,4-dianilino-5-chloro-pyrimidine, TAE684, a previously reported inhibitor of anaplastic lymphoma kinase (ALK), is also a potent inhibitor of LRRK2 kinase activity (IC(50) of 7.8nM against wild-type LRRK2, 6.1nM against the G2019S mutant). TAE684 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1-0.3μM in cells and in mouse spleen and kidney, but not in brain, following oral doses of 10mg/kg.

WNK1-regulated inhibitory phosphorylation of the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons

Immature neurons need WNK1-dependent phosphorylation of KCC2 to prevent a premature switch in the Cl− gradient and the effect of GABA. Keeping immature neurons excited After birth, signaling by the neurotransmitter GABA in the brain switches from excitatory to inhibitory. GABA mediates both the excitatory and inhibitory responses by binding to ligand-gated ion channels that conduct Cl−. Whether opening these channels triggers hyperpolarization (inhibition) or depolarization (excitation) depends on the concentration of Cl− in neurons. Friedel et al. identified phosphorylation events in the K+-Cl− cotransporter KCC2, which depended on the activity of the kinase WNK1, inhibited KCC2 activity, and contributed to the depolarizing effect of GABA-mediated signaling in immature rat neurons by maintaining high internal Cl− concentration. This regulatory mechanism has implications for the normal developmental excitatory-to-inhibitory GABA switch and neurodevelopmental diseases, such as autism, epilepsy, and spasticity. Activation of Cl−-permeable γ-aminobutyric acid type A (GABAA) receptors elicits synaptic inhibition in mature neurons but excitation in immature neurons. This developmental “switch” in the GABA function depends on a postnatal decrease in intraneuronal Cl− concentration mediated by KCC2, a Cl−-extruding K+-Cl− cotransporter. We showed that the serine-threonine kinase WNK1 [with no lysine (K)] forms a physical complex with KCC2 in the developing mouse brain. Dominant-negative mutation, genetic depletion, or chemical inhibition of WNK1 in immature neurons triggered a hyperpolarizing shift in GABA activity by enhancing KCC2-mediated Cl− extrusion. This increase in KCC2 activity resulted from reduced inhibitory phosphorylation of KCC2 at two C-terminal threonines, Thr906 and Thr1007. Phosphorylation of both Thr906 and Thr1007 was increased in immature versus mature neurons. Together, these data provide insight into the mechanism regulating Cl− homeostasis in immature neurons, and suggest that WNK1-regulated changes in KCC2 phosphorylation contribute to the developmental excitatory-to-inhibitory GABA sequence.

Characterisation of the Cullin‐3 mutation that causes a severe form of familial hypertension and hyperkalaemia

Deletion of exon 9 from Cullin‐3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin‐RING‐ubiquitin‐ligase complexes. Bound to KLHL3, CUL3‐RBX1 ubiquitylates WNK kinases, promoting their ubiquitin‐mediated proteasomal degradation. Since WNK kinases activate Na/Cl co‐transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin‐RING‐ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto‐ubiquitylates and loses interaction with two important Cullin regulators: the COP9‐signalosome and CAND1. A novel knock‐in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases.

Enhanced electricity production by use of reconstituted artificial consortia of estuarine bacteria grown as biofilms.

Microbial fuel cells (MFCs) can convert organic compounds directly into electricity by catalytic oxidation, and although MFCs have attracted considerable interest, there is little information on the electricity-generating potential of artificial bacterial biofilms. We have used acetate-fed MFCs inoculated with sediment, with two-chamber bottles and carbon cloth electrodes to deliver a maximum power output of ~175 mW · m(-2) and a stable power output of ~105 mW · m(-2). Power production was by direct transfer of electrons to the anode from bacterial consortia growing on the anode, as confirmed by cyclic voltammetry (CV) and scanning electron microscopy (SEM). Twenty different species (74 strains) of bacteria were isolated from the consortium under anaerobic conditions and cultured in the laboratory, of which 34% were found to be exoelectrogens in single-species studies. Exoelectrogenesis by members of the genera Vibrio , Enterobacter , and Citrobacter and by Bacillus stratosphericus was confirmed, by use of culture-based methods, for the first time. An MFC with a natural bacterial consortium showed higher power densities than those obtained with single strains. In addition, the maximum power output could be further increased to ~200 mW · m(-2) when an artificial consortium consisting of the best 25 exoelectrogenic isolates was used, demonstrating the potential for increased performance and underlying the importance of artificial biofilms for increasing power output.

Inflammation-dependent cerebrospinal fluid hypersecretion by the choroid plexus epithelium in posthemorrhagic hydrocephalus

The choroid plexus epithelium (CPE) secretes higher volumes of fluid (cerebrospinal fluid, CSF) than any other epithelium and simultaneously functions as the blood–CSF barrier to gate immune cell entry into the central nervous system. Posthemorrhagic hydrocephalus (PHH), an expansion of the cerebral ventricles due to CSF accumulation following intraventricular hemorrhage (IVH), is a common disease usually treated by suboptimal CSF shunting techniques. PHH is classically attributed to primary impairments in CSF reabsorption, but little experimental evidence supports this concept. In contrast, the potential contribution of CSF secretion to PHH has received little attention. In a rat model of PHH, we demonstrate that IVH causes a Toll-like receptor 4 (TLR4)- and NF-κB-dependent inflammatory response in the CPE that is associated with a ∼3-fold increase in bumetanide-sensitive CSF secretion. IVH-induced hypersecretion of CSF is mediated by TLR4-dependent activation of the Ste20-type stress kinase SPAK, which binds, phosphorylates, and stimulates the NKCC1 co-transporter at the CPE apical membrane. Genetic depletion of TLR4 or SPAK normalizes hyperactive CSF secretion rates and reduces PHH symptoms, as does treatment with drugs that antagonize TLR4–NF-κB signaling or the SPAK–NKCC1 co-transporter complex. These data uncover a previously unrecognized contribution of CSF hypersecretion to the pathogenesis of PHH, demonstrate a new role for TLRs in regulation of the internal brain milieu, and identify a kinase-regulated mechanism of CSF secretion that could be targeted by repurposed US Food and Drug Administration (FDA)-approved drugs to treat hydrocephalus.

WNK Kinase Signaling in Ion Homeostasis and Human Disease

WNK kinases, along with their upstream regulators (CUL3/KLHL3) and downstream targets (the SPAK/OSR1 kinases and the cation-Cl- cotransporters [CCCs]), comprise a signaling cascade essential for ion homeostasis in the kidney and nervous system. Recent work has furthered our understanding of the WNKs in epithelial transport, cell volume homeostasis, and GABA signaling, and uncovered novel roles for this pathway in immune cell function and cell proliferation.