Electrophysiological and pharmacological characterization of a novel and potent neuronal Kv7 channel opener SCR2682 for antiepilepsy.

Voltage-gated Kv7/KCNQ/M potassium channels play an essential role in the control of membrane potential and neuronal excitability. Activation of the neuronal Kv7/KCNQ/M-current represents an attractive therapeutic strategy for treatment of hyperexcitability-related neuropsychiatric disorders such as epilepsy, pain, and depression, which is an unmet medical need. In this study, we synthesized and characterized a novel compound, N-(4-(2-bromo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-2,6-dimethylphenyl)-3,3-dimethylbutanamide (SCR2682) 2,6-dimethyl-4-(piperidin-yl) phenyl)-amide derivative, that exhibits selective and potent activation of neuronal Kv7/KCNQ/M-channels. Whole-cell patch-clamp recordings of human embryonic kidney 293 cells expressing Kv7.2/Kv7.3 channels show that SCR2682 selectively activates the channel current in a dose-dependent manner with an EC50 of 9.8 ± 0.4 nM, which is ∼100-fold more potent than a U.S. Food and Drug Administration-approved antiepileptic drug (retigabine) for treatment of partial epilepsy. SCR2682 shifts voltage-dependent activation of the Kv7.2/7.3 current toward more negative membrane potential, to about -37 mV (V1/2). SCR2682 also activates the native M-current in rat hippocampal or cortical neurons, causing marked hyperpolarization and potent inhibition of neuronal firings. Mechanistically, mutating the tryptophan residue 236 located at the fifth transmembrane segment of Kv7.2 abolishes the chemical activation of the channel by SCR2682. Furthermore, intraperitoneal or intragastric administration of SCR2682 results in a dose-dependent inhibition of seizures by maximal electroshock. Taken together, our findings demonstrate that a novel small molecule, SCR2682, selectively and potently activates neuronal Kv7 channels and reverses epileptic seizures in rodents. Thus, SCR2682 may warrant further evaluation for clinical development of antiepileptic therapy.-Zhang, F., Liu, Y., Tang, F., Liang, B., Chen, H., Zhang, H., Wang, K. Electrophysiological and pharmacological characterization of a novel and potent neuronal Kv7 channel opener SCR2682 for antiepilepsy.

Identification and characterization of a series of novel HCN channel inhibitors.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a critical role in controlling pacemaker activity in both heart and nervous system. Developing HCN channel inhibitors has been proposed to be an important strategy for the treatment of pain, heart failure, arrhythmias, and epilepsy. One HCN channel inhibitor, ivabradine, has been clinically approved for the treatment of angina pectoris and heart failure. In this study, we designed and synthesized eight alkanol amine derivatives, and assessed their effects on HCN channels expressed in COS7 cells using a whole-cell patch clamp method. Among them, compound 4e displayed the most potent inhibitory activity with an IC50 of 2.9 ± 1.2 µM at - 120 mV on HCN2 channel expressed in COS7 cells. Further analysis revealed that application of compound 4e (10 µM) caused a slowing of activation and a hyperpolarizing shift (ΔV1/2 = - 30.2 ± 2.9 mV, n = 5) in the voltage dependence of HCN2 channel activation. The inhibitory effect of compound 4e on HCN1 and HCN4 channel expressed in COS7 cells was less potent with IC50 of 17.2 ± 1.3 and 7.3 ± 1.2 µM, respectively. Besides, we showed that application of compound 4e (10 µM) inhibited Ih and action potential firing in acutely dissociated mouse small dorsal root ganglion neurons. Our study provides a new strategy for the design and development of potent HCN channel inhibitors.

Synthesis and Characterization of Novel Acyl-Glycine Inhibitors of GlyT2.

It has been demonstrated previously that the endogenous compound N-arachidonyl-glycine inhibits the glycine transporter GlyT2, stimulates glycinergic neurotransmission, and provides analgesia in animal models of neuropathic and inflammatory pain. However, it is a relatively weak inhibitor with an IC50 of 9 µM and is subject to oxidation via cyclooxygenase, limiting its therapeutic value. In this paper we describe the synthesis and testing of a novel series of monounsaturated C18 and C16 acyl-glycine molecules as inhibitors of the glycine transporter GlyT2. We demonstrate that they are up to 28 fold more potent that N-arachidonyl-glycine with no activity at the closely related GlyT1 transporter at concentrations up to 30 µM. This novel class of compounds show considerable promise as a first generation of GlyT2 transport inhibitors.

Photoswitchable Inhibitor of a Glutamate Transporter.

Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a critical role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacological tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with ∼200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiological roles of glutamate transporters in the brain, as well as to study the molecular interactions of transporters with ligands.

Synthesis and Evaluation of Potent KCNQ2/3-Specific Channel Activators.

KQT-like subfamily (KCNQ) channels are voltage-gated, noninactivating potassium ion channels, and their down-regulation has been implicated in several hyperexcitability-related disorders, including epilepsy, neuropathic pain, and tinnitus. Activators of these channels reduce the excitability of central and peripheral neurons, and, as such, have therapeutic utility. Here, we synthetically modified several moieties of the KCNQ2-5 channel activator retigabine, an anticonvulsant approved by the U.S. Food and Drug Administration. By introducing a CF3-group at the 4-position of the benzylamine moiety, combined with a fluorine atom at the 3-position of the aniline ring, we generated Ethyl (2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)amino)phenyl)carbamate (RL648_81), a new KCNQ2/3-specific activator that is >15 times more potent and also more selective than retigabine. We suggest that RL648_81 is a promising clinical candidate for treating or preventing neurologic disorders associated with neuronal hyperexcitability.

HCN Channels Modulators: The Need for Selectivity.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, the molecular correlate of the hyperpolarization-activated current (If/Ih), are membrane proteins which play an important role in several physiological processes and various pathological conditions. In the Sino Atrial Node (SAN) HCN4 is the target of ivabradine, a bradycardic agent that is, at the moment, the only drug which specifically blocks If. Nevertheless, several other pharmacological agents have been shown to modulate HCN channels, a property that may contribute to their therapeutic activity and/or to their side effects. HCN channels are considered potential targets for developing drugs to treat several important pathologies, but a major issue in this field is the discovery of isoform-selective compounds, owing to the wide distribution of these proteins into the central and peripheral nervous systems, heart and other peripheral tissues. This survey is focused on the compounds that have been shown, or have been designed, to interact with HCN channels and on their binding sites, with the aim to summarize current knowledge and possibly to unveil useful information to design new potent and selective modulators.

The synthesis and BK channel-opening activity of N-acylaminoalkyloxime derivatives of dehydroabietic acid.

A series of N-acylaminoalkyloxime derivatives of dehydroabietic acid were synthesized and evaluated for BK channel-opening activities in an assay system of CHO-K1 cells expressing hBKα channels. The structure-activity relationship study revealed that a non-covalent interaction between the S atom of the 2-thiophene and the carbonyl O atom may contribute to conformation restriction for interaction with the ion channel. This research could guide the design and synthesis of novel abietane-based BK channel opener.

Identification and electrophysiological evaluation of 2-methylbenzamide derivatives as Nav1.1 modulators.

Voltage-gated sodium channels (Nav) are crucial to the initiation and propagation of action potentials (APs) in electrically excitable cells, and during the past decades they have received considerable attention due to their therapeutic potential. Here, we report for the first time the synthesis and the electrophysiological evaluation of 16 ligands based on a 2-methylbenzamide scaffold that have been identified as Nav1.1 modulators. Among these compounds, N,N'-(1,3-phenylene)bis(2-methylbenzamide) (3a) has been selected and evaluated in ex-vivo experiments in order to estimate the activation impact of such a compound profile. It appears that 3a increases the Nav1.1 channel activity although its overall impact remains moderate. Altogether, our preliminary results provide new insights into the development of small molecule activators targeting specifically Nav1.1 channels to design potential drugs for treating CNS diseases.

Development of a new photochromic ion channel blocker via azologization of fomocaine.

Photochromic blockers of voltage gated ion channels are powerful tools for the control of neuronal systems with high spatial and temporal precision. We now introduce fotocaine, a new type of photochromic channel blocker based on the long-lasting anesthetic fomocaine. Fotocaine is readily taken up by neurons in brain slices and enables the optical control of action potential firing by switching between 350 and 450 nm light. It also provides an instructive example for "azologization", that is, the systematic conversion of an established drug into a photoswitchable one.

Synthesis of purine and 7-deazapurine nucleoside analogues of 6-N-(4-Nitrobenzyl)adenosine; inhibition of nucleoside transport and proliferation of cancer cells.

Human equilibrative nucleoside transporter 1 (hENT1) is a prototypical nucleoside transporter protein ubiquitously expressed on the cell surface of almost all human tissue. Given the role of hENT1 in the transport of nucleoside drugs, an important class of therapeutics in the treatment of various cancers and viral infections, efforts have been made to better understand the mechanisms by which hENT1 modulates nucleoside transport. To that end, we report here the design and synthesis of novel tool compounds for the further study of hENT1. The 7-deazapurine nucleoside antibiotic tubercidin was converted into its 4-N-benzyl and 4-N-(4-nitrobenzyl) derivatives by alkylation at N3 followed by a Dimroth rearrangement to the 4-N-isomer or by fluoro-diazotization followed by SN Ar displacement of the 4-fluoro group by a benzylamine. The 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin antibiotics were prepared by the alkylation approach. Cross-membrane transport of labeled uridine by hENT1 was inhibited to a weaker extent by the 4-nitrobenzylated tubercidin and sangivamycin analogues than was observed with 6-N-(4-nitrobenzyl)adenosine. Type-specific inhibition of cancer cell proliferation was observed at micromolar concentrations with the 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin, and also with 4-N-benzyltubercidin. Treatment of 2',3',5'-O-acetyladenosine with aryl isocyanates gave the 6-ureido derivatives but none of them exhibited inhibitory activity against cancer cell proliferation or hENT1.

Total synthesis of (29S,37S)-isomer of malevamide E, a potent ion-channel inhibitor.

The first total synthesis of (29S,37S)-malevamide E (1), a potent ion channel inhibitor, has been achieved in a convergent fashion involving Julia-Kocienski olefination, Urpi acetal aldol and Shiina macrolactonization reactions as the key steps. The strategy developed herein is amenable for the synthesis of the other possible isomers in search for the correct stereoisomer of the naturally occurring molecule.

[The new glutamic acid derivative RGPU-135 (glutaron, neuroglutamin) modulates ion currents in mollusk neurons].

The new glutamic acid derivative RGPU-135 (3-phenylglutamic acid hydrochloride, glutaron, neuroglutamin) produces dose-dependent and reversible modulation of transmembrane sodium, potassium and, to a greater extent, calcium ion currents in neurons of Lymnaea stagnalis and Planorbarius corneus mollusks at concentrations of 1, 10, 100, and 1000 microM. At concentrations within 1 - 10 microM micromole, Ca and K currents are activated rather insignificantly; at 100 pmole, the amplitude of calcium currents is increased by 5 - 10%; and at 1000 microM, the Na and K ion currents are suppressed by 5 - 12%. RGPU-135 does not influence the membrane surface charge potential and the gating of ion channels. The effects of RGPU-135 were quickly reversible, which indicated the relatively weak drug binding to the membrane structures and ion channels.

A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain.

Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including Na(V)1.7 and Na(V)1.8 sodium channels and Ca(V)3.2 T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactivation of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics.

Partial structures of ketoconazole as modulators of the large conductance calcium-activated potassium channel (BK(Ca)).

A series of partial structures of ketoconazole has been synthesized and tested for activity on the large conductance calcium-activated potassium channel (BK) in bovine smooth muscle cells. This has provided openers and blockers of the channel. The results suggest that the phenyl and phenoxy moieties are important for interaction with BK, whereas the imidazole group is unimportant. The properties of the phenoxy moiety seem to determine whether the compounds act to open or block the channel.