Zelquistinel Is an Orally Bioavailable Novel NMDA Receptor Allosteric Modulator That Exhibits Rapid and Sustained Antidepressant-Like Effects.

BACKGROUND: The role of glutamatergic receptors in major depressive disorder continues to be of great interest for therapeutic development. Recent studies suggest that both negative and positive modulation of N-methyl-D-aspartate receptors (NMDAR) can produce rapid antidepressant effects. Here we report that zelquistinel, a novel NMDAR allosteric modulator, exhibits high oral bioavailability and dose-proportional exposures in plasma and the central nervous system and produces rapid and sustained antidepressant-like effects in rodents by enhancing activity-dependent, long-term synaptic plasticity. METHODS: NMDAR-mediated functional activity was measured in cultured rat brain cortical neurons (calcium imaging), hNR2A or B subtype-expressing HEK cells, and synaptic plasticity in rat hippocampal and medial prefrontal cortex slices in vitro. Pharmacokinetics were evaluated in rats following oral administration. Antidepressant-like effects were assessed in the rat forced swim test and the chronic social deficit mouse model. Target engagement and the safety/tolerability profile was assessed using phencyclidine-induced hyperlocomotion and rotarod rodent models. RESULTS: Following a single oral dose, zelquistinel (0.1-100 µg/kg) produced rapid and sustained antidepressant-like effects in the rodent depression models. Brain/ cerebrospinal fluid concentrations associated with zelquistinel antidepressant-like activity also increased NMDAR function and rapidly and persistently enhanced activity-dependent synaptic plasticity (long-term potentiation), suggesting that zelquistinel produces antidepressant-like effects by enhancing NMDAR function and synaptic plasticity. Furthermore, Zelquistinel inhibited phencyclidine (an NMDAR antagonist)-induced hyperlocomotion and did not impact rotarod performance. CONCLUSIONS: Zelquistinel produces rapid and sustained antidepressant effects by positively modulating the NMDARs, thereby enhancing long-term potentiation of synaptic transmission.

Positive N-Methyl-D-Aspartate Receptor Modulation by Rapastinel Promotes Rapid and Sustained Antidepressant-Like Effects.

BACKGROUND: Modulation of glutamatergic synaptic transmission by N-methyl-D-aspartate receptors can produce rapid and sustained antidepressant effects. Rapastinel (GLYX-13), initially described as a N-methyl-D-aspartate receptor partial glycine site agonist, exhibits rapid antidepressant effect in rodents without the accompanying dissociative effects of N-methyl-D-aspartate receptor antagonists. METHODS: The relationship between rapastinel's in vitro N-methyl-D-aspartate receptor pharmacology and antidepressant efficacy was determined by brain microdialysis and subsequent pharmacological characterization of therapeutic rapastinel concentrations in N-methyl-D-aspartate receptor-specific radioligand displacement, calcium mobilization, and medial prefrontal cortex electrophysiology assays. RESULTS: Brain rapastinel concentrations of 30 to 100 nM were associated with its antidepressant-like efficacy and enhancement of N-methyl-D-aspartate receptor-dependent neuronal intracellular calcium mobilization. Modulation of N-methyl-D-aspartate receptors by rapastinel was independent of D-serine concentrations, and glycine site antagonists did not block rapastinel's effect. In rat medial prefrontal cortex slices, 100 nM rapastinel increased N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents and enhanced the magnitude of long-term potentiation without any effect on miniature EPSCs or paired-pulse facilitation responses, indicating postsynaptic action of rapastinel. A critical amino acid within the NR2 subunit was identified as necessary for rapastinel's modulatory effect. CONCLUSION: Rapastinel brain concentrations associated with antidepressant-like activity directly enhance medial prefrontal cortex N-methyl-D-aspartate receptor activity and N-methyl-D-aspartate receptor-mediated synaptic plasticity in vitro. At therapeutic concentrations, rapastinel directly enhances N-methyl-D-aspartate receptor activity through a novel site independent of the glycine coagonist site. While both rapastinel and ketamine physically target N-methyl-D-aspartate receptors, the 2 molecules have opposing actions on N-methyl-D-aspartate receptors. Modest positive modulation of N-methyl-D-aspartate receptors by rapastinel represents a novel pharmacological approach to promote well-tolerated, rapid, and sustained improvements in mood disorders.

The NMDA receptor modulator zelquistinel durably relieves behavioral deficits in three mouse models of autism spectrum disorder.

Autism spectrum disorders (ASD) are complex neurodevelopmental disorders characterized by deficient social communication and interaction together with restricted, stereotyped behaviors. Currently approved treatments relieve comorbidities rather than core symptoms. Since excitation/inhibition balance and synaptic plasticity are disrupted in ASD, molecules targeting excitatory synaptic transmission appear as highly promising candidates to treat this pathology. Among glutamatergic receptors, the NMDA receptor has received particular attention through the last decade to develop novel allosteric modulators. Here, we show that positive NMDA receptor modulation by zelquistinel, a spirocyclic ß-lactam platform chemical, relieves core symptoms in two genetic and one environmental mouse models of ASD. A single oral dose of zelquistinel rescued, in a dose-response manner, social deficits and stereotypic behavior in Shank3Δex13-16-/- mice while chronic intraperitoneal administration promoted a long-lasting relief of such autistic-like features in these mice. Subchronic oral mid-dose zelquistinel treatment demonstrated durable effects in Shank3Δex13-16-/-, Fmr1-/- and in utero valproate-exposed mice. Carry-over effects were best maintained in the Fmr1 null mouse model, with social parameters being still fully recovered two weeks after treatment withdrawal. Among recently developed NMDA receptor subunit modulators, zelquistinel displays a promising therapeutic potential to relieve core symptoms in ASD patients, with oral bioavailability and long-lasting effects boding well for clinical applications. Efficacy in three mouse models with different etiologies supports high translational value. Further, this compound represents an innovative pharmacological tool to investigate plasticity mechanisms underlying behavioral deficits in animal models of ASD.

Zelquistinel acts at an extracellular binding domain to modulate intracellular calcium inactivation of N-methyl-d-aspartate receptors.

Stinels are a novel class of N-methyl-d-aspartate glutamate receptor (NMDAR) positive allosteric modulators. We explored mechanism of action and NR2 subtype specificity of the stinel zelquistinel (ZEL) in HEK 293 cells expressing recombinant NMDARs. ZEL potently enhanced NMDAR current at NR2A (EC50 = 9.9 ± 0.5 nM) and NR2C-containing (EC50 = 9.7 ± 0.6 nM) NMDARs, with a larger ceiling enhancement at NR2B-NMDAR (EC50 = 35.0 ± 0.7 nM), while not affecting NR2D-containing NMDARs. In cells expressing NR2A and NR2C-containing NMDARs, ZEL exhibited an inverted-U dose-response relation, with a low concentration enhancement and high concentration suppression of NMDAR currents. Extracellular application of ZEL potentiated NMDAR receptor activity via prolongation of NMDAR currents. Replacing the slow Ca2+ intracellular chelator EGTA with the fast chelator BAPTA blocked ZEL potentiation of NMDARs, suggesting an action on intracellular Ca2+-calmodulin-dependent inactivation (CDI). Consistent with this mechanism of action, removal of the NR1 intracellular C-terminus, or intracellular infusion of a calmodulin blocking peptide, blocked ZEL potentiation of NMDAR current. In contrast, BAPTA did not prevent high-dose suppression of current, indicating this effect has a different mechanism of action. These data indicate ZEL is a novel positive allosteric modulator that binds extracellularly and acts through a unique long-distance mechanism to reduce NMDAR CDI, eliciting enhancement of NMDAR current. The critical role that NMDARs play in long-term, activity-dependent synaptic plasticity, learning, memory and cognition, suggests dysregulation of CDI may contribute to psychiatric disorders such as depression, schizophrenia and others, and that the stinel class of drugs can restore NMDAR-dependent synaptic plasticity by reducing activity-dependent CDI.

Extracellular application of the N-methyl-D-aspartate receptor allosteric modulator rapastinel acts remotely to regulate Ca2+ inactivation at an intracellular locus.

BACKGROUND: A novel N-methyl-D-aspartate receptor (NMDAR) allosteric modulator, rapastinel (RAP, formerly GLYX-13), elicits long-lasting antidepressant-like effects by enhancing long-term potentiation (LTP) of synaptic transmission. RAP elicits these effects by binding to a unique site in the extracellular region of the NMDAR complex, transiently enhancing NMDAR-gated current in pyramidal neurons of both hippocampus and medial prefrontal cortex. METHODS: We compared efficacy of RAP in modulating Schaffer collateral-evoked NMDAR-currents as a function of kinetics of the Ca2+ chelator in the intracellular solution, using whole-cell patch-clamp recordings. The intracellular solution contained either the slow Ca2+ chelator EGTA [3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecane-1,14-dioic acid, 0.5 mmol/l] or the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA {2,2',2″,2‴-[ethane-1,2-diylbis(oxy-2,1-phenylenenitrilo)] tetraacetic acid, 5 mmol/l}. NMDAR-gated currents were pharmacologically isolated by bath application of the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptor antagonist 6-nitro-2,3-dioxo-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (10 µmol/l) plus the GABA receptor blocker bicuculline (20 µmol/l). RESULTS: When the slow Ca2+ chelator EGTA was in the intracellular solution, RAP elicited significant enhancement of NMDAR-gated current at a 1 µmol/l concentration, and significantly reduced current at 10 µmol/l. In contrast, when recording with the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA, NMDAR current increased in magnitude by 84% as BAPTA washed into the cell, and the enhancement of NMDAR current by 1 µmol/l RAP was completely blocked. Interestingly, the reduction in NMDAR current from 10 µmol/l RAP was not affected by the presence of BAPTA in the recording pipette, indicating that this effect is mediated by a different mechanism. CONCLUSION: Extracellular binding of RAP to the NMDAR produces a novel, long-range reduction in affinity of the Ca2+ inactivation site on the NMDAR C-terminus accessible to the intracellular space. This action underlies enhancement in NMDAR-gated conductance elicited by RAP.

A peripheral adrenoceptor-mediated sympathetic mechanism can transform stress-induced analgesia into hyperalgesia.

BACKGROUND: Stress has paradoxical effects on pain, causing stress-induced analgesia but also exacerbating pain via poorly understood mechanisms. Adrenergic neurotransmission is integral in pathways that regulate the response to both pain and stress. Hyperalgesia is often associated with enhanced adrenergic sensitivity of primary afferents, but sympathetic nervous system outflow has not been demonstrated to exacerbate pain perception after stress. METHODS: Rats or C57/BL6 wild-type mice treated with α-2 receptor antagonists or α-2A receptor knockout mice were exposed to ultrasonic noise stress or footshock stress and subsequently tested for hotplate paw withdrawal latencies. The sensory sensitivity of α-2A knockout mice to electrical and chemical stimuli was tested neurophysiologically and behaviorally. The effects of sympatholytic treatments were investigated. RESULTS: Noise and footshock stressors induced thermal hyperalgesia in rats pretreated systemically with α-2 antagonists. Wild-type mice pretreated with α-2 antagonists and α-2A knockout mice also exhibited thermal hyperalgesia induced by noise stress. Local spinal or intraplantar injection of an α-2 antagonist counteracted stress-induced analgesia without causing hyperalgesia. The α-2A knockout mice had decreased thresholds for peripheral sensitization with sulprostone and for windup of the dorsal horn neuronal response to repetitive electrical stimuli. Stress-induced hyperalgesia was abolished and the sensitization was attenuated by sympathectomy or systemic administration of an α-1-adrenergic antagonist. CONCLUSIONS: Sympathetic postganglionic nerves can enhance pain sensation via a peripheral α-1-adrenoceptor mechanism when sympathetic outflow is disinhibited. The net effect of stress on pain sensation reflects a balance between descending spinal inhibition and sympathetic outflow that can shift toward pain facilitation when central and peripheral α-2-adrenoceptor inhibitory mechanisms are attenuated.

Rapastinel, an NMDAR positive modulator, produces distinct behavioral, sleep, and EEG profiles compared with ketamine.

Rapastinel, a positive NMDAR modulator, produces rapid-acting and long-lasting antidepressant-like effects; however, unlike ketamine, the abuse potential for rapastinel is minimal. Ketamine has also been shown to induce psychotomimetic/dissociative side effects, aberrant gamma oscillations, and effects similar to sleep deprivation, which may potentially limit its clinical use. In this study, we compared the side effect profile and potential sleep-altering properties of rapastinel (3, 10, and 30 mg/kg) to ketamine (30 mg/kg) in rodents. In addition, we investigated corresponding changes in transcriptomics and proteomics. Rapastinel exhibited no effect on locomotor activity and prepulse inhibition in mice, while ketamine induced a significant increase in locomotor activity and a significant decrease in prepulse inhibition, which are indications of a psychosis-like state. The effects of rapastinel on sleep architecture were minimal, and rapastinel did not alter gamma frequency oscillations. In contrast, ketamine administration resulted in a greater latency to slow wave and REM sleep, disrupted duration of sleep, and affected duration of wakefulness during sleep. Further, ketamine increased cortical oscillations in the gamma frequency range, which is a property associated with psychosis. Rapastinel induced similar plasticity-related changes in transcriptomics to ketamine in rats but differed in several gene ontology classes, some of which may be involved in the regulation of sleep. In conclusion, rapastinel demonstrated a lower propensity than ketamine to induce CNS-related adverse side effects and sleep disturbances.

Alpha-1-adrenergic receptor agonist activity of clinical alpha-adrenergic receptor agonists interferes with alpha-2-mediated analgesia.

BACKGROUND: The use of alpha-2 adrenergic agonists for analgesia is limited due to a narrow therapeutic window. Definition of the role of alpha receptor subtypes in alpha agonist mediated analgesia may identify strategies to separate the analgesic from sedative and cardiovascular effects. METHODS: Analgesic activity of brimonidine, clonidine, and tizanidine was investigated in wild-type C57B/6, alpha-2A, and alpha-2C knockout mice with allodynia induced by N-methyl-D-aspartate or sulprostone. The alpha receptor selectivity of the alpha agonists was assessed using functional in vitro recombinant assays. RESULTS: Brimonidine, clonidine, and tizanidine reduced N-methyl-D-aspartate- and sulprostone-induced allodynia in wild-type mice, but not alpha-2A knockout mice. In alpha-2C knockout mice, brimonidine and tizanidine reduced allodynia in both models, whereas clonidine only reduced N-methyl-D-aspartate-induced allodynia. In vitro, clonidine and tizanidine activated alpha-1 and alpha-2 receptors with similar potencies, whereas brimonidine was selective for alpha-2 receptors. In alpha-2C knockout mice with sulprostone-induced allodynia, blockade of clonidine's alpha-1 receptor agonist activity restored clonidine's analgesic efficacy. In wild-type mice, the analgesic potency of intrathecal clonidine and tizanidine was increased 3- to 10-fold by coadministration with the alpha-1A-selective antagonist 5-methylurapidil without affecting sedation. Following intraperitoneal administration, the therapeutic window was negligible for clonidine and tizanidine, but greater for brimonidine. 5-Methylurapidil enhanced the therapeutic window of intraperitoneal clonidine and tizanidine approximately 10-fold. CONCLUSIONS: Alpha-1A receptor agonist activity can counterbalance alpha-2 receptor agonist-induced analgesia. Greater alpha-2 selectivity may enhance the therapeutic window of alpha-2 agonists in the treatment of pain.

Broad modulation of neuropathic pain states by a selective estrogen receptor beta agonist.

The effects of estrogens on pain perception remain controversial. In animal models, both beneficial and detrimental effects of non-selective estrogens have been reported. ERb-131 a non-steroidal estrogen receptor beta ligand was evaluated in several pain animal models involving nerve injury or sensitization. Using functional and binding assays, ERb-131 was characterized as a potent and selective estrogen receptor beta agonist. In vivo, ERb-131 was devoid of estrogen receptor alpha activity as assessed in a rat uterotrophic assay. ERb-131 alleviated tactile hyperalgesia induced by capsaicin, and reversed tactile allodynia caused by spinal nerve ligation and various chemical insults. Moreover, ERb-131 did not influence the pain threshold of normal healthy animals. Thus, estrogen receptor beta agonism is a critical effector in attenuating a broad range of anti-nociceptive states.

CRM1-dependent function of a cis-acting RNA export element.

Viruses often contain cis-acting RNA elements, which facilitate the posttranscriptional processing and export of their messages. These elements fall into two classes distinguished by the presence of either viral or cellular RNA binding proteins. To date, studies have indicated that the viral proteins utilize the CRM1-dependent export pathway, while the cellular factors generally function in a CRM1-independent manner. The cis-acting element found in the woodchuck hepatitis virus (WHV) (the WHV posttranscriptional regulatory element [WPRE]) has the ability to posttranscriptionally stimulate transgene expression and requires no viral proteins to function. Conventional wisdom suggests that the WPRE would function in a CRM1-independent manner. However, our studies on this element reveal that its efficient function is sensitive to the overexpression of the C terminus of CAN/Nup214 and treatment with the antimicrobial agent leptomycin B. Furthermore, the overexpression of CRM1 stimulates WPRE activity. These results suggest a direct role for CRM1 in the export function of the WPRE. This observation suggests that the WPRE is directing messages into a CRM1-dependent mRNA export pathway in somatic mammalian cells.