Symmetric activation and modulation of the human calcium-sensing receptor

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca2+ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near–full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.

GABAA Receptor Subunit Composition Drives Its Sensitivity to the Insecticide Fipronil

Fipronil (FPN) is a worldwide-used neurotoxic insecticide, targeting, and blocking GABAA receptors (GABAARs). Beyond its efficiency on insect GABAARs, FPN causes neurotoxic effects in humans and mammals. Here, we investigated the mode of action of FPN on mammalian α6-containing GABAARs to understand its inhibitory effects on GABA-induced currents, as a function of the synaptic or extrasynaptic localization of GABAARs. We characterized the effects of FPN by electrophysiology using Xenopus oocytes which were microtransplanted with cerebellum membranes or injected with α6β3, α6β3γ2S (synaptic), and α6β3δ (extrasynaptic) cDNAs. At micromolar concentrations, FPN dose-dependently inhibited cerebellar GABA currents. FPN acts as a non-competitive antagonist on ternary receptors. Surprisingly, the inhibition of GABA-induced currents was partial for extra-synaptic (α6β3δ) and binary (α6β3) receptors, while synaptic α6β3γ2S receptors were fully blocked, indicating that the complementary γ or δ subunit participates in FPN-GABAAR interaction. FPN unexpectedly behaved as a positive modulator on β3 homopentamers. These data show that FPN action is driven by the subunit composition of GABAARs—highlighting the role of the complementary subunit—and thus their localization within a physiological synapse. We built a docking model of FPN on GABAARs, which reveals two putative binding sites. This is consistent with a double binding mode of FPN on GABAARs, possibly one being of high affinity and the other of low affinity. Physiologically, the γ/δ subunit incorporation drives its inhibitory level and has important significance for its toxicity on the mammalian nervous system, especially in acute exposure.

GSK-3β manipulates ferroptosis sensitivity by dominating iron homeostasis

Ferroptosis is a newly characterized form of non-apoptotic-programmed cell death, which is driven by the lethal accumulation of iron-catalyzed lipid peroxides. Uncontrolled ferroptosis is implicated in the pathogenesis of a group of human diseases, while targeted induction of ferroptosis provides a potent therapeutic design for cancers. During the past decade, the fundamental regulatory circuits of ferroptosis have been identified. In this study, we show that the multifaceted Ser/Thr protein kinase GSK-3β acts as a positive modulator of the ferroptosis program. Pharmacological inhibition of GSK-3β by selective inhibitor LY2090314 or genetic KD of GSK-3β by shRNA potently promotes ferroptotic resistance. GSK-3β KD antagonizes the expression of iron metabolic components including DMT1, FTH1, and FTL, leading to the disruption of iron homeostasis and decline in intracellular labile free iron level. Taken together, our findings elaborate an indispensable role of GSK-3β in determining ferroptotic sensitivity by dominating cellular iron metabolism, which provides further insight into GSK-3β as a target for cancer chemotherapy.

A Facile Approach to Bis(isoxazoles), Promising Ligands of the AMPA Receptor

A convenient synthetic approach to novel functionalized bis(isoxazoles), the promising bivalent ligands of the AMPA receptor, was elaborated. It was based on the heterocyclization reactions of readily available electrophilic alkenes with the tetranitromethane-triethylamine complex. The structural diversity of the synthesized compounds was demonstrated. In the electrophysiological experiments using the patch clamp technique on Purkinje neurons, the compound 1,4-phenylenedi(methylene)bis(5-aminoisoxazole-3-carboxylate) was shown to be highly potent positive modulator of the AMPA receptor, potentiating kainate-induced currents up to 70% at 10−11 M.

IFI44L as a Forward Regulator Enhancing Host Antituberculosis Responses

Interferon-induced protein 44-like (IFI44L) gene is a type I interferon-stimulated gene (ISG) that plays a critical role in antiviral activity and constitutes a promising diagnostic marker. However, its precise role and function in tuberculosis have not been unveiled. This study showed that IFI44L acts as an antimicrobial target and positive modulator in human macrophages. Knockdown of IFI44L led to increased Mycobacterium tuberculosis intracellular survival. Moreover, IFI44L was significantly upregulated, and it restricted the intracellular survival of M. tuberculosis H37Rv strains at 72 h after rifampicin treatment. Individuals with cutaneous tuberculosis (CTB) were found to have significantly higher IFI44L expression after 6 months of rifampicin therapy than after only 1 month. These results demonstrated that IFI44L induced positive regulation and clearance of M. tuberculosis from human macrophages. This antimicrobial activity of IFI44L makes it a possible target for therapeutic applications against M. tuberculosis.

ZDHHC11 Positively Regulates NF-κB Activation by Enhancing TRAF6 Oligomerization

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a RING domain ubiquitin ligase that plays an important role in nuclear factor-κB (NF-κB) signaling by regulating activation of the TAK1 and IKK complexes. However, the molecular mechanisms that regulate TRAF6 E3 activity remain unclear. Here, we found that ZDHHC11, a member of the DHHC palmitoyl transferase family, functions as a positive modulator in NF-κB signaling. ZDHHC11 overexpression activated NF-κB, whereas ZDHHC11 deficiency impaired NF-κB activity stimulated by IL-1β, LPS, and DNA virus infection. Furthermore, Zdhhc11 knockout mice had a lower level of serum IL6 upon treatment with LPS and D-galactosamine or HSV-1 infection than control mice. Mechanistically, ZDHHC11 interacted with TRAF6 and then enhanced TRAF6 oligomerization, which increased E3 activity of TRAF6 for synthesis of K63-linked ubiquitination chains. Collectively, our study indicates that ZDHHC11 positively regulates NF-κB signaling by promoting TRAF6 oligomerization and ligase activity, subsequently activating TAK1 and IKK complexes.

An Emerging Role for Sigma-1 Receptors in the Treatment of Developmental and Epileptic Encephalopathies

Developmental and epileptic encephalopathies (DEEs) are complex conditions characterized primarily by seizures associated with neurodevelopmental and motor deficits. Recent evidence supports sigma-1 receptor modulation in both neuroprotection and antiseizure activity, suggesting that sigma-1 receptors may play a role in the pathogenesis of DEEs, and that targeting this receptor has the potential to positively impact both seizures and non-seizure outcomes in these disorders. Recent studies have demonstrated that the antiseizure medication fenfluramine, a serotonin-releasing drug that also acts as a positive modulator of sigma-1 receptors, reduces seizures and improves everyday executive functions (behavior, emotions, cognition) in patients with Dravet syndrome and Lennox-Gastaut syndrome. Here, we review the evidence for sigma-1 activity in reducing seizure frequency and promoting neuroprotection in the context of DEE pathophysiology and clinical presentation, using fenfluramine as a case example. Challenges and opportunities for future research include developing appropriate models for evaluating sigma-1 receptors in these syndromic epileptic conditions with multisystem involvement and complex clinical presentation.

Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 reveal a novel binding site for positive modulators

Kv3 ion-channels constitute a class of functionally distinct voltage gated ion channels characterized by their ability to fire at a high frequency. Several disease relevant mutants, together with biological data, suggest the importance of this class of ion channels as drug targets for CNS disorders, and several drug discovery efforts have been reported. Despite the increasing interest for this class of ion channels, no structure of a Kv3 channel has been reported yet. We have determined the cryo-EM structure of Kv3.1 at 2.6 Å resolution using full length wild type protein. When compared to known structures for potassium channels from other classes, a novel domain organization is observed with the cytoplasmic T1 domain, containing a well resolved Zinc site and displaying a rotation by 35°. This suggests a distinct cytoplasmic regulation mechanism for the Kv3.1 channel. A high resolution structure was obtained for Kv3.1 in complex with a novel positive modulator Lu AG00563. The structure reveals a novel ligand binding site for the Kv class of ion channels located between the voltage sensory domain and the channel pore, a region which constitutes a hotspot for disease causing mutations. The discovery of a novel binding site for a positive modulator of a voltage gated potassium channel could shed light on the mechanism of action for these small molecule potentiators. This finding could enable structure-based drug design on these targets with high therapeutic potential for the treatment of multiple CNS disorders.

Leptin, Both Bad and Good Actor in Cancer

Leptin is an important regulator of basal metabolism and food intake, with a pivotal role in obesity. Leptin exerts many different actions on various tissues and systems, including cancer, and is considered as a linkage between metabolism and the immune system. During the last decades, obesity and leptin have been associated with the initiation, proliferation and progression of many types of cancer. Obesity is also linked with complications and mortality, irrespective of the therapy used, affecting clinical outcomes. However, some evidence has suggested its beneficial role, called the “obesity paradox”, and the possible antitumoral role of leptin. Recent data regarding the immunotherapy of cancer have revealed that overweight leads to a more effective response and leptin may probably be involved in this beneficial process. Since leptin is a positive modulator of both the innate and the adaptive immune system, it may contribute to the increased immune response stimulated by immunotherapy in cancer patients and may be proposed as a good actor in cancer. Our purpose is to review this dual role of leptin in cancer, as well as trying to clarify the future perspectives of this adipokine, which further highlights its importance as a cornerstone of the immunometabolism in oncology.

Regulation of auditory fear discrimination by the novel Kv3 voltage-gated potassium channel modulator AUT00206

Psychiatric diseases like anxiety-related disorders and schizophrenia are characterized by impaired cognition and emotional regulation linked to corticolimbic disinhibition. Restoring the balance between excitation and inhibition in corticolimbic circuits may therefore ameliorate certain features of these disorders, such as inappropriately attributing affective salience to innocuous cues. Corticolimbic activity is tightly controlled by parvalbumin-expressing GABAergic interneurons, which also regulate fear discrimination. The voltage-gated potassium channels Kv3.1 and Kv3.2 are highly expressed in these neurons, therefore Kv3.1/3.2 modulation may have potential for treating disorders associated with cognitive and emotional dysregulation. We determined the effects of the novel Kv3.1/3.2 positive modulator AUT00206 on fear discrimination. Female rats underwent limited or extended auditory fear discrimination training that we previously showed leads to discrimination or generalization, respectively, based on passive fear responding (i.e. freezing). We also assessed darting as an active fear response. We found that limited training resulted in discrimination based on freezing, which was unaffected by AUT00206. In contrast, we found that extended training resulted in generalization based on freezing and the emergence of discrimination based on darting. Importantly, AUT00206 had dissociable effects on fear discrimination and expression with extended training. While AUT00206 mitigated generalization without affecting expression based on freezing, it reduced expression without affecting discrimination based on darting. Our results indicate that Kv3.1/3.2 modulation regulates the attribution of affective significance to threat- and safety-related cues in a response-specific manner. This suggests that targeting Kv3.1 and Kv3.2 channels may provide a promising avenue for treating cognitive and emotional dysregulation in psychiatric disease.