Identification of a novel GSK3ß inhibitor involved in abrogating KRas dependent pancreatic tumors in Wnt/beta-catenin and NF-kB dependent manner.

Pancreatic cancer is an aggressive malignancy with a poor survival rate because it is difficult to diagnose the disease during its early stages. The currently available treatments, which include surgery, chemotherapy and radiation therapy, offer only limited survival benefit. Pharmacological interventions to inhibit Glycogen Synthase Kinase-3beta (GSK3ß) activity is an important therapeutic strategy for the treatment of pancreatic cancer because GSK3ß is one of the key factors involved in the onset, progression as well as in the acquisition of chemoresistance in pancreatic cancer. Here, we report the identification of MJ34 as a potent GSK3ß inhibitor that significantly reduced growth and survival of human mutant KRas dependent pancreatic tumors. MJ34 mediated GSK3ß inhibition was seen to induce apoptosis in a ß-catenin dependent manner and downregulate NF-kB activity in MiaPaCa-2 cells thereby impeding cell survival and anti-apoptotic processes in these cells as well as in the xenograft model of pancreatic cancer. In vivo acute toxicity and in vitro cardiotoxicity studies indicate that MJ34 is well tolerated without any adverse effects. Taken together, we report the discovery of MJ34 as a potential drug candidate for the therapeutic treatment of mutant KRas-dependent human cancers through pharmacological inhibition of GSK3ß.

Discovery of a novel and highly selective JAK3 inhibitor as a potent hair growth promoter.

JAK-STAT signalling pathway inhibitors have emerged as promising therapeutic agents for the treatment of hair loss. Among different JAK isoforms, JAK3 has become an ideal target for drug discovery because it only regulates a narrow spectrum of γc cytokines. Here, we report the discovery of MJ04, a novel and highly selective 3-pyrimidinylazaindole based JAK3 inhibitor, as a potential hair growth promoter with an IC50 of 2.03 nM. During in vivo efficacy assays, topical application of MJ04 on DHT-challenged AGA and athymic nude mice resulted in early onset of hair regrowth. Furthermore, MJ04 significantly promoted the growth of human hair follicles under ex-vivo conditions. MJ04 exhibited a reasonably good pharmacokinetic profile and demonstrated a favourable safety profile under in vivo and in vitro conditions. Taken together, we report MJ04 as a highly potent and selective JAK3 inhibitor that exhibits overall properties suitable for topical drug development and advancement to human clinical trials.

Subunit-specific inhibition of BK channels by piperine.

Piperine is the principal alkaloid present in black pepper and is well-known for its diverse pharmacological effects, including inhibition of different ion channels. Large conductance Ca2+-activated K+ channels (BK) are widely expressed across several tissues and play a vital role in many physiological functions. In this study, we investigated the pharmacological effects of piperine on various BK channel subunit compositions (BKα, BKαß1,4, BKαγ1,3) expressed in HEK293T cells. Piperine in zero Ca2+ reversibly inhibited currents from the pore-forming BKα channels in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of 4.8 µM. Elevating the internal Ca2+ concentration from 0 to 100 µM significantly attenuated the inhibitory effects of piperine on BKα channels. The mutation G311S in the pore domain failed to alter the modulatory effects of piperine, whereas deletion of the entire cytoplasmic domain from BKα channels ablated its inhibitory effects. Addition of either BKß1 or ß4 regulatory subunits did not alter the efficacy of piperine on BKα channels. Interestingly, co-expression of either BKγ1 or BKγ3 subunits greatly diminished the ability of piperine to inhibit BKα channels. Our findings demonstrate that piperine is a potent natural modulator of BKα/BKαß1,4 subunits but not BKαγ1,3 subunits. The mechanism of piperine modulation appeared to be allosteric and differs from that of other BK pore blockers (paxilline, peptide toxins, and quaternary ammonium compounds). Together, our results unravel the potential of piperine to inhibit BK channels, providing a new tool to explore mechanisms underlying the effects of regulatory subunits.

Differential regulation of BK channels by fragile X mental retardation protein.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein prominently expressed in neurons. Missense mutations or complete loss of FMRP can potentially lead to fragile X syndrome, a common form of inherited intellectual disability. In addition to RNA regulation, FMRP was also proposed to modulate neuronal function by direct interaction with the large conductance Ca2+- and voltage-activated potassium channel (BK) ß4 regulatory subunits (BKß4). However, the molecular mechanisms underlying FMRP regulation of BK channels were not studied in detail. We have used electrophysiology and super-resolution stochastic optical reconstruction microscopy (STORM) to characterize the effects of FMRP on pore-forming BKα subunits, as well as the association with regulatory subunits BKß4. Our data indicate that, in the absence of coexpressed ß4, FMRP alters the steady-state properties of BKα channels by decreasing channel activation and deactivation rates. Analysis using the Horrigan-Aldrich model revealed alterations in the parameters associated with channel opening (L0) and voltage sensor activation (J0). Interestingly, FMRP also altered the biophysical properties of BKαß4 channels favoring channel opening, although not as dramatically as BKα. STORM experiments revealed clustered multi-protein complexes, consistent with FMRP interacting not only to BKαß4 but also to BKα. Lastly, we found that a partial loss-of-function mutation in FMRP (R138Q) counteracts many of its functional effects on BKα and BKαß4 channels. In summary, our data show that FMRP modulates the function of both BKα and BKαß4 channels.

Physiological Roles and Therapeutic Potential of Ca2+ Activated Potassium Channels in the Nervous System.

Within the potassium ion channel family, calcium activated potassium (KCa) channels are unique in their ability to couple intracellular Ca2+ signals to membrane potential variations. KCa channels are diversely distributed throughout the central nervous system and play fundamental roles ranging from regulating neuronal excitability to controlling neurotransmitter release. The physiological versatility of KCa channels is enhanced by alternative splicing and co-assembly with auxiliary subunits, leading to fundamental differences in distribution, subunit composition and pharmacological profiles. Thus, understanding specific KCa channels' mechanisms in neuronal function is challenging. Based on their single channel conductance, KCa channels are divided into three subtypes: small (SK, 4-14 pS), intermediate (IK, 32-39 pS) and big potassium (BK, 200-300 pS) channels. This review describes the biophysical characteristics of these KCa channels, as well as their physiological roles and pathological implications. In addition, we also discuss the current pharmacological strategies and challenges to target KCa channels for the treatment of various neurological and psychiatric disorders.

Functional validation of Ca2+-binding residues from the crystal structure of the BK ion channel.

BK channels are dually regulated by voltage and Ca2+, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca2+ concentration is sensed by a large cytoplasmic region in the channel known as "gating ring", which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca2+ coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca2+. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca2+ in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca2+ sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca2+ coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca2+ binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca2+ binding sites regulating the Ca2+ dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Differential efficacy of GoSlo-SR compounds on BKα and BKαγ1-4 channels.

Large conductance, voltage and Ca2+ activated K+ channels (BK channels) are abundantly expressed throughout the body and are important regulators of smooth muscle tone and neuronal excitability. Their dysfunction is implicated in various diseases including overactive bladder, hypertension and erectile dysfunction. Therefore, BK channel openers bear significant therapeutic potential to treat the above diseases. GoSlo-SR compounds were designed to be potent and efficacious BK channel openers. Although their structural activity relationships, activation in both BKα and BKαß channels and the hypothetical mode of action of these compounds has been studied in detail in recent years, their effectiveness to open the BKαγ channels still remains unexplored. In this study, we have examined the efficacy of 3 closely related GoSlo-SR openers, GoSlo-SR-5-6 (SR-5-6), GoSlo-SR-5-44 (SR-5-44) and GoSlo-SR-5-130 (SR-5-130) using inside out patches on BKα channels coexpressed with 4 different LRRC (γ1-4) subunits in HEK293 cells. Our data suggests that the activation effects due to SR-5-6 were not significantly affected in the presence of γ1-4 subunits. Interestingly, the effects of more efficacious BK channel opener SR-5-44 were altered by different γ subunits. In cells expressing BKα channels, the shift in V1/2 (ΔV1/2) induced by SR-5-44 (3 µM) was -76 ± 3 mV, whereas it was significantly reduced by ∼70 % in BKαγ1 channels (ΔV1/2= -23 ± 3, p < 0.001, ANOVA). In BKαγ2 channels the ΔV1/2 was -36 ± 1 mV, which was less than that observed in BKαγ3 and BKαγ4 channels where the ΔV1/2 was -47 ± 5 mV, and -82 ± 5 mV, respectively. Additionally, the excitatory effects of a 'ß specific' BK channel opener, SR-5-130 were only partially restored in the patches containing BKαγ1-4 channels. Together this data highlights that subtle modifications in GoSlo-SR structures alter their effectiveness on BK channels with accessory γ subunits and this study might provide a scaffold for the development of more tissue specific BK channel openers.

Molecular mechanisms underlying the effect of the novel BK channel opener GoSlo: involvement of the S4/S5 linker and the S6 segment.

GoSlo-SR-5-6 is a novel large-conductance Ca(2+)-activated K(+) (BK) channel agonist that shifts the activation V1/2 of these channels in excess of -100 mV when applied at a concentration of 10 µM. Although the structure-activity relationship of this family of molecules has been established, little is known about how they open BK channels. To help address this, we used a combination of electrophysiology, mutagenesis, and mathematical modeling to investigate the molecular mechanisms underlying the effect of GoSlo-SR-5-6. Our data demonstrate that the effects of this agonist are practically abolished when three point mutations are made: L227A in the S4/S5 linker in combination with S317R and I326A in the S6C region. Our data suggest that GoSlo-SR-5-6 interacts with the transmembrane domain of the channel to enhance pore opening. The Horrigan-Aldrich model suggests that GoSlo-SR-5-6 works by stabilizing the open conformation of the channel and the activated state of the voltage sensors, yet decouples the voltage sensors from the pore gate.

Development of GoSlo-SR-5-69, a potent activator of large conductance Ca2+-activated K+ (BK) channels.

We have designed, synthesised and characterised the effects of a number of novel anthraquinone derivatives and assessed their effects on large conductance, Ca(2+) activated K(+) (BK) channels recorded from rabbit bladder smooth muscle cells using the excised, inside/out configuration of the patch clamp technique. These compounds are members of the GoSlo-SR family of compounds, which potently open BK channels and shift the voltage required for half maximal activation (V1/2) negatively. The efficacy of the anilinoanthraquinone derivatives was enhanced when the size of ring D was increased, since the cyclopentane and cyclohexane derivatives shifted the V1/2, by -24 ± 6 mV and -54 ± 8 mV, respectively, whereas the cycloheptane and cyclooctane derivatives shifted the V1/2 by -61 ± 6 mV and -106 ± 6 mV. To examine if a combination of hydrophobicity and steric bulking of this region further enhanced their ability to open BK channels, we synthesised a number of naphthalene and tetrahydro-naphthalene derivatives. The tetrahydro-2-naphthalene derivative GoSlo-SR-5-69 was the most potent and efficacious of the series since it was able to shift the activation V1/2 by greater than -100 mV when applied at a concentration of 1 µM and had an EC50 of 251 nM, making it one of the most potent and efficacious BK channel openers synthesised to date.