Hassan Pajouhesh

T-Type Calcium Channel Blockers That Attenuate Thalamic Burst Firing and Suppress Absence Seizures

Two high-affinity T-type calcium channel blockers attenuate neural activity in the thalamus and suppress seizures in a genetic model of absence epilepsy. To Soothe a Seizure Some epileptic children and adolescents experience “absence” seizures hundreds of times a day. Although apparently mild, these seizures—so named because they involve a sudden, brief absence of consciousness—can be dangerous if they occur during swimming or driving, for example. Unfortunately, the drugs available for treating such seizures are not completely effective. Tringham et al. sought to address this problem by rational drug design. Although the root cause of such seizures is not known, they are associated with abnormal, highly synchronous neuronal activity in certain brain regions. Voltage-gated ion channels, which have crucial functions in generating and propagating neuronal signals, likely play a key role. Several lines of evidence link one type of ion channel, low voltage–activated T-type calcium channels, to absence seizures. Using the structure of an N-type calcium channel blocker as a starting point, the researchers designed and screened small, focused libraries of compounds in a high-throughput assay that monitored calcium influx via a recombinant T-type channel. Two high-affinity T-type calcium channel blockers, termed Z941 and Z944, were identified; Z944 was highly selective for T-type channels and exhibited a preference for inactivated channels (the likely configuration in hyperexcited neurons). In a rat model of absence epilepsy, both compounds markedly reduced the time spent in seizures and the number of seizures per hour. In contrast to current first-line drugs for treating absence seizures, Z941 and Z944 also reduced the average seizure duration and cycle frequency. Both compounds were well tolerated in rats. Given its in vitro and in vivo activities, Z944 will progress to phase 1 clinical studies to test its safety in humans. Further studies will be needed to determine whether its marked effects in the rat model of absence epilepsy translate to the more complicated human condition. Absence seizures are a common seizure type in children with genetic generalized epilepsy and are characterized by a temporary loss of awareness, arrest of physical activity, and accompanying spike-and-wave discharges on an electroencephalogram. They arise from abnormal, hypersynchronous neuronal firing in brain thalamocortical circuits. Currently available therapeutic agents are only partially effective and act on multiple molecular targets, including γ-aminobutyric acid (GABA) transaminase, sodium channels, and calcium (Ca2+) channels. We sought to develop high-affinity T-type specific Ca2+ channel antagonists and to assess their efficacy against absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. Using a rational drug design strategy that used knowledge from a previous N-type Ca2+ channel pharmacophore and a high-throughput fluorometric Ca2+ influx assay, we identified the T-type Ca2+ channel blockers Z941 and Z944 as candidate agents and showed in thalamic slices that they attenuated burst firing of thalamic reticular nucleus neurons in GAERS. Upon administration to GAERS animals, Z941 and Z944 potently suppressed absence seizures by 85 to 90% via a mechanism distinct from the effects of ethosuximide and valproate, two first-line clinical drugs for absence seizures. The ability of the T-type Ca2+ channel antagonists to inhibit absence seizures and to reduce the duration and cycle frequency of spike-and-wave discharges suggests that these agents have a unique mechanism of action on pathological thalamocortical oscillatory activity distinct from current drugs used in clinical practice.

Multiple Cationic Amphiphiles Induce a Niemann-Pick C Phenotype and Inhibit Ebola Virus Entry and Infection

Ebola virus (EBOV) is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC50 1.6 to 8.0 µM) at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann–Pick C1 protein (NPC1), a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents) inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.

A screen of approved drugs and molecular probes identifies therapeutics with anti–Ebola virus activity

Several FDA-approved drugs, including bepridil and sertraline, may be therapeutics against filovirus infections. Drug repurposing for Ebola virus The recent Ebola virus outbreak has highlighted the lack of therapies for filovirus infection. FDA-approved drugs serve as a source of medications with a proven safety record. Johansen et al. have now screened about 2600 FDA-approved drugs and molecular probes and found 80 that have some efficacy against Zaire ebolavirus in vitro. These drugs were mechanistically diverse, and several drugs, including a calcium channel blocker and an antidepressant, could protect against infection in a mouse model. Although new therapy development is ongoing, these repurposed drugs can rapidly move to human testing and may serve on the frontline against Ebolavirus infection. Currently, no approved therapeutics exist to treat or prevent infections induced by Ebola viruses, and recent events have demonstrated an urgent need for rapid discovery of new treatments. Repurposing approved drugs for emerging infections remains a critical resource for potential antiviral therapies. We tested ~2600 approved drugs and molecular probes in an in vitro infection assay using the type species, Zaire ebolavirus. Selective antiviral activity was found for 80 U.S. Food and Drug Administration–approved drugs spanning multiple mechanistic classes, including selective estrogen receptor modulators, antihistamines, calcium channel blockers, and antidepressants. Results using an in vivo murine Ebola virus infection model confirmed the protective ability of several drugs, such as bepridil and sertraline. Viral entry assays indicated that most of these antiviral drugs block a late stage of viral entry. By nature of their approved status, these drugs have the potential to be rapidly advanced to clinical settings and used as therapeutic countermeasures for Ebola virus infections.

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

&NA; 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 NaV1.7 and NaV1.8 sodium channels and CaV3.2 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. A novel organic compound stabilizes slow‐inactivated sodium and calcium channels to reduce the excitability of nociceptors and dorsal horn neurons and attenuate neuropathic pain signaling.

Structure–activity relationships of diphenylpiperazine N-type calcium channel inhibitors

A novel series of compounds derived from the previously reported N-type calcium channel blocker NP118809 (1-(4-benzhydrylpiperazin-1-yl)-3,3-diphenylpropan-1-one) is described. Extensive SAR studies resulted in compounds with IC(50) values in the range of 10-150 nM and selectivity over the L-type channels up to nearly 1200-fold. Orally administered compounds 5 and 21 exhibited both anti-allodynic and anti-hyperalgesic activity in the spinal nerve ligation model of neuropathic pain.

A Fluorescence-Based High-Throughput Screening Assay for the Identification of T-Type Calcium Channel Blockers

T-type voltage-gated Ca(2+) channels have been implicated in contributing to a broad variety of human disorders, including pain, epilepsy, sleep disturbances, cardiac arrhythmias, and certain types of cancer. However, potent and selective T-type Ca(2+) channel modulators are not yet available for clinical use. This may in part be due to their unique biophysical properties that have delayed the development of high-throughput screening (HTS) assays for identifying blockers. One notable challenge is that at the normal resting membrane potential (V(m)) of cell lines commonly utilized for drug screening purposes, T-type Ca(2+) channels are largely inactivated and thus cannot be supported by typical formats of functional HTS assays to both evoke and quantify the Ca(2+) channel signal. Here we describe a simple method that can successfully support a fluorescence-based functional assay for compounds that modulate T-type Ca(2+)channels. The assay functions by exploiting the pore-forming properties of gramicidin to control the cellular V(m) in advance of T-type Ca(2+) channel activation. Using selected ionic conditions in the presence of gramicidin, T-type Ca(2+) channels are converted from the unavailable, inactivated state to the available, resting state, where they can be subsequently activated by application of extracellular K(+). The fidelity of the assay has been pharmacologically characterized with sample T-type Ca(2+) channel blockers whose potency has been determined by conventional manual patch-clamp techniques. This method has the potential for applications in high-throughput fluorometric imaging plate reader (FLIPR(R), Molecular Devices, Sunnyvale, CA) formats with cell lines expressing either recombinant or endogenous T-type Ca(2+) channels.

Structure-activity relationships of trimethoxybenzyl piperazine N-type calcium channel inhibitors

We previously reported the small organic N-type calcium channel blocker NP078585 that while efficacious in animal models for pain, exhibited modest L-type calcium channel selectivity and substantial off-target inhibition against the hERG potassium channel. Structure-activity studies to optimize NP078585 preclinical properties resulted in compound 16, which maintained high potency for N-type calcium channel blockade, and possessed excellent selectivity over the hERG (~120-fold) and L-type (~3600-fold) channels. Compound 16 shows significant anti-hyperalgesic activity in the spinal nerve ligation model of neuropathic pain and is also efficacious in the rat formalin model of inflammatory pain.

Modular, efficient synthesis of asymmetrically substituted piperazine scaffolds as potent calcium channel blockers.

A novel approach to the synthesis of substituted piperazines and their investigation as N-type calcium channel blockers is presented. A common scaffold exhibiting high activity as N-type blockers is N-substituted piperazine. Using recently developed titanium and zirconium catalysts, we describe the efficient and modular synthesis of 2,5-asymmetrically disubstituted piperazines from simple amines and alkynes. The method requires only three isolation/purification protocols and no protection/deprotection steps for the diastereoselective synthesis of 2,5-dialkylated piperazines in moderate to high yield. Screening of the synthesized piperazines for N-type channel blocking activity and selectivity shows the highest activity for a compound with a benzhydryl group on the nitrogen (position 1) and an unprotected alcohol-functionalized side chain.

Stereospecific synthesis and absolute configuration of the (2S, 3S, 4S)-isomer of 2-methyl-2-(carboxycyclopropyl)glycine (MCCG)

Abstract The conformationally restricted metabotropic glutamate receptor antagonist (2 S ,3 S ,4 S )-2-methyl-2-(carboxycyclopropyl)glycine 1 (MCCG) has been synthesized in a stereoselective manner (>99% ee) with the (2 S ,3 S ,4 S ) absolute configuration of this molecule being confirmed by X-ray crystallographic analysis. Subsequent physico–chemical studies were undertaken and the data are at odds with those of the commercially available product.