BACKGROUND AND PURPOSE:Gamma-glutamyl dipeptides are essentially produced after glutathione (GSH) degradation. While several γ-glutamyl dipeptides have been detected in different organs and body fluids, their biological activity remains elusive. This is the case for γ-Glutamyl-Glycine (γ-Glu-Gly) which has been found in different brain areas. γ-Glutamyl-glycine may bind to ionotropic glutamate receptors. Here, we have investigated whether γ-Glu-Gly could exhibit modulatory actions on excitatory synaptic transmission and plasticity by performing its functional characterization on glutamate, GABA and glycine receptors. EXPERIMENTAL APPROACH:Electrophysiological and calcium imaging experiments were performed on cells heterologously expressing either Glutamate, GABA or Glycine receptors and on cultured hippocampal neurons and acute hippocampal slices. The γ-Glu-Gly production by cultured cells and hippocampal slices was measured by LC-MS analysis, following incubation with modulators of the glutathione metabolic cycle. KEY RESULTS:γ-Glu-Gly exerted partial agonist effects on both NMDA GluN1 and mGlu5 receptors, but without any binding to the glutamate binding domain on both AMPA and NMDA GluN2A receptors. γ-Glu-Gly was devoid of any effect on GABA, glycine and GluN3A receptors. γ-Glu-Gly was able to trigger a long-term increase in synaptic transmission in the CA1 area of mouse hippocampus. CONCLUSION AND IMPLICATIONS:γ-Glu-Gly partially retains the excitatory actions of glutamate on specific receptor sub-types and may trigger plastic events in the hippocampus. Its accumulation under pathological conditions associated with oxidative stress and a high GSH consumption could thus interfere with endogenous synaptic transmission and disrupt natural plasticity, which is a hallmark of neurodegenerative diseases.

BACKGROUND/OBJECTIVES:Antibody-dependent cellular cytotoxicity relies on the interaction between the Fc region of immunoglobulin G1 (IgG1) and the CD16a receptor. While removal of core fucosylation on Fc and introduction of the DFTE mutation set (S239D, H268F, S324T, I332E) are known to enhance CD16a binding, the detailed contributions of these engineered sites in solution remain incompletely defined. METHODS:Here, we employed 1 µs molecular dynamics simulations to map, at atomic resolution, the interaction networks stabilizing pre-formed Fc-CD16a complexes, including afucosylated Fc-wild-type, DFTE-engineered, Fc-fucosylated, and asymmetrically engineered Fc variants. RESULTS:Our results show that only S239D, present on both Fc chains, and H268F on chain A consistently contribute to stabilizing the CD16a interface, while I332E does not form persistent interactions. Glycan-protein contacts are primarily intrachain, with transient interchain glycan-glycan interactions not contributing significantly to complex stability. Fucosylation on Fc significantly reduces binding stability by disrupting peripheral interactions and critical glycan-mediated contacts. Notably, the asymmetric Fc variant, in which the two heavy chains carry distinct sets of substitutions, retains high-affinity binding despite lacking S239D and carrying core fucose, through a novel hydrophobic cluster and reinforced peripheral electrostatic interactions. CONCLUSIONS:Altogether, these findings provide a quantitative framework for how targeted mutations and fucose modifications remodel Fc-CD16a interactions, offering insights for the rational design of next-generation therapeutic antibodies.

The recently characterized honeybee CaV4 channel is a high-voltage-activated Ca2+ channel ortholog to the DSC1 channel identified in Drosophila. While sequence similarities to NaV channels are obvious, permeation properties and current kinetics are more closely aligned with those of CaV channels. CaV4 exhibits a distinctive cation-dependent inactivation pattern, a hallmark of Ca2+ channel behavior, and nonetheless displays sensitivity to a Na+ channel-specific regulator, veratrine. Calcium channel facilitation is a phenomenon whereby the probability of calcium channel opening increases with successive depolarization pulses, resulting in an enhanced Ca2+ influx during repetitive or sustained electrical activity. In this study, we have identified an additional specific property of CaV4 in the form of an atypical voltage-dependent facilitation of the Ca2+ or Ba2+ currents by strong pre-depolarizations or prepulses (pPs). This physiologically relevant phenomenon, known as pP-induced facilitation (PiF), is subject to positive regulation by the amplitude of the pP but to negative regulation by its duration. It produces a hyperpolarizing shift of the I-V curve without any change in the reversal potential and macroscopic or single channel conductance. PiF is thus more pronounced for small depolarizations and almost absent when channels reach their maximal open probability. A mutation that affects the inactivation of the CaV4 channel prevents the occurrence of PiF. This previously undocumented form of facilitation appears exclusive to CaV4 channels. A strong pP may lock CaV4 channels in a pre-open state, rendering them more susceptible to activation and thereby shifting the activation curve toward more negative potentials. This, in turn, would accelerate channel opening and increase current amplitude. Lastly, we show that the inactivation particle of CaV4 (MFLT sequence, equivalent to the IFMT motif in human NaV, or MFMT in Apis NaV channel), in addition to its role in the initiation of the voltage-dependent inactivation, also modulates PiF.

Tumor recurrence is frequently attributed to drug-tolerant cancer cells. We previously demonstrated that downregulation of the Pregnane X Receptor (PXR, NR1I2) reduces chemoresistance and prevents colorectal cancer recurrence in xenograft mouse models. However, there is currently a lack of clinically-suitable PXR antagonists. In this study, we report the design and synthesis of a novel PXR agonist-based PROTAC (JMV7048) which promotes polyubiquitination and degradation of the human PXR protein via E3 CRBN ubiquitin ligase and 26S proteasome pathways. JMV7048 selectively degrades PXR in colon carcinoma, hepatoma, and pancreatic cancer cell lines, with no impact on primary human hepatocytes. Notably, JMV7048 reduces PXR protein expression in drug-tolerant colon cancer cells, sensitizing them to chemotherapy and significantly delaying cancer relapse in xenografted nude mice. These findings suggest that PXR-targeting PROTACs may serve as novel therapeutic agents to enhance the sensitivity of chemo-resistant cancer cells to chemotherapy.

The pacemaker channel (HCN) is responsible for electrical activity in a wide range of excitable cells, including those of invertebrates. Using Xenopus oocytes and HEK cell, we show here that HCN-channel from Apis mellifera is activated by hyperpolarization, modulated by cAMP, and blocked by cesium. Its PNa/PK relative permeability is 1:3, and its unitary conductance is 1.5 pS, which is similar to that of the mammalian HCN2 channel. Moreover, bee h-current is blocked by high concentrations of ZD7288, and organochlorine pesticide chlordecone reduces Ih amplitude in a dose-dependent manner (IC50 value was 9.37 µM) and diminishes HCN conductance, while preserving voltage dependence. In contrast, Deltamethrin exhibits no discernible impact. Molecular docking of Chlordecone in a homology model of bee HCN generated by AlphaFold3 suggests a binding site located at the end of the S6 helix that could explain the conductance inhibition caused by Chlordecone.

Background/Objectives: Tumor-associated antigens are not tumor-specific antigens but proteins that are overexpressed by tumor cells and also weakly expressed at the surface of healthy tissues. Therefore, some side effects are observed when targeted by therapeutic antibodies, a phenomenon named “on-target, off-tumor toxicity”. As tumors generate an acidic microenvironment, we investigated whether we could generate pH-dependent antibodies to increase their tumor specificity. For this proof-of-concept study, we selected the tyrosine kinase receptor AXL because we already developed several antibodies against this target. Methods: To generate a pH-dependent anti-AXL antibody, we performed classical panning of a single-chain variable fragment (scFv) library using phage display at an acidic pH throughout the process. Results: After the third round of panning, 9 scFvs, among the 96 picked clones, bound to AXL at acidic pH and showed very low binding at a neutral pH. After reformatting them into IgG, two clones were selected for further study due to their strong pH-sensitive binding. Using molecular docking and alanine scanning, we found that their binding strongly depended on two histidine residues present on AXL at positions 61 and 116. Conclusions: To conclude, we set-up an easy process to generate pH-dependent antibodies that may increase their tumor-binding specificity and potentially decrease toxicity towards healthy tissues.

DSC1, a Drosophila channel with sequence similarity to the voltage-gated sodium channel (NaV), was identified over 20 years ago. This channel was suspected to function as a non-specific cation channel with the ability to facilitate the permeation of calcium ions (Ca2+). A honeybee channel homologous to DSC1 was recently cloned and shown to exhibit strict selectivity for Ca2+, while excluding sodium ions (Na+), thus defining a new family of Ca2+ channels, known as CaV4. In this study, we characterize CaV4, showing that it exhibits an unprecedented type of inactivation, which depends on both an IFM motif and on the permeating divalent cation, like NaV and CaV1 channels, respectively. CaV4 displays a specific pharmacology with an unusual response to the alkaloid veratrine. It also possesses an inactivation mechanism that uses the same structural domains as NaV but permeates Ca2+ ions instead. This distinctive feature may provide valuable insights into how voltage- and calcium-dependent modulation of voltage-gated Ca2+ and Na+ channels occur under conditions involving local changes in intracellular calcium concentrations. Our study underscores the unique profile of CaV4 and defines this channel as a novel class of voltage-gated Ca2+ channels.

Tumor recurrence is often attributed to drug-tolerant cancer stem cells. We previously demonstrated that down regulation of the Pregnane X Receptor (PXR, NR1I2) decreases chemoresistance of cancer stem cells and prevents colorectal cancer recurrence in xenograft mouse models. These is a lack of PXR antagonists that are appropriate for clinical use. In this study, we report the design and synthesis of a novel PXR agonist-based PROTAC (JMV7048) that induces polyubiquitination and degradation of human PXR protein in an E3 CRBN ubiquitin ligase- and the 26S proteasome-dependent manner. This molecule specifically degrades PXR in colon carcinoma, hepatoma, and pancreatic cancer cell lines, but not in primary cultures of human hepatocytes. Crucially, JMV7048 decreased PXR protein expression in colon cancer stem cells and sensitized them to chemotherapy significantly delaying cancer relapse in vivo. PROTACs targeting PXR protein could thus become novel therapeutic agents to enhance cancer cell sensitivity to chemotherapy. ### Competing Interest Statement The authors have declared no competing interest.

Cav2.1 channels are expressed throughout the brain and are the predominant Ca2+ channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four variations (p.A405T in I-II loop and p.R1359W, p.R1667W and p.S1799L in IIIS4, IVS4, and IVS6 helices, respectively) identified in patients exhibiting a wide spectrum of disorders including ataxia symptoms. Functional analysis using two major Cav2.1 splice variants (Cav2.1+e47 and Cav2.1−e47) in Xenopus laevis oocytes, revealed a lack of effect upon A405T substitution and a significant loss-of-function caused by R1359W, whereas R1667W and S1799L caused both channel gain-of-function and loss-of-function, in a splice variant-dependent manner. Structural analysis revealed the loss of interactions with S1, S2, and S3 helices upon R1359W and R1667W substitutions, but a lack of obvious structural changes with S1799L. Computational modeling suggests that biophysical changes induced by Cav2.1 pathogenic mutations might affect action potential frequency in Purkinje cells.

The intensive use of insecticides, combined with other factors such as habitat loss, might explain worldwide decrease of insect populations documented in the past twenty years. However, due to the involvement of pest species in crop destruction and in vector-borne diseases, insecticides will probably continue to be required still for decades. The most commercially successful insecticides are neurotoxicants acting on ion channels of the central nervous system affecting thereby cellular excitability and synaptic transmission and causing insect paralysis and fatality. In this article, we provide an overview of the insecticides acting on voltage-gated sodium channels, GABA-gated chloride channels and nicotinic acetylcholine receptors. We summarize the current knowledge on those ion channels from the honeybee Apis mellifera and discuss the possible mode of action of neurotoxic insecticides.

Protein-polymer bioconjugates have shown great promise in biomedical and life science applications including drug delivery and diagnosis. The current bioconjugation strategies suffer from lack of efficiency and versatility. In this article, poly(styrene-alt-maleic anhydride) copolymers were first prepared by RAFT polymerization and characterized by different analytical techniques. Then, the poly(styrene-alt-maleic anhydride) precursors were functionalized with primary amine such as azidopropylamine and amino poly(ethylene glycol). The reaction of amino compounds with maleic anhydride was found to be a highly efficient, a versatile, and a facile chemical ligation reaction for the synthesis of macromolecules with quantitative yield under mild conditions. The main benefit is the incorporation of a wide range of functionality by easily changing the primary amine compound. For the amphiphilic graft copolymers based on poly(ethylene glycol), aggregation behavior in water was investigated. In a second part, azido-functionalized polystyrene copolymers were used to prepare a new protein-polymer bioconjugate by copper-free click chemistry reaction.

Abstract ADP-ribosylation factor 1 (Arf1) is a crucial regulator in vesicle-mediated membrane trafficking and involved in the activation of signaling molecules. However, the molecular mechanisms underlying Arf1-dependent cancer development and progression are far from understood. We report here that Arf1 is the most amplified gene in Arf gene family in breast cancer, and knocking it down leads to an induction of autophagy via the AMPK pathway. To effectively target Arf1, we rationally designed and synthesized constrained ATC-based (4-amino-(methyl)-1,3-thiazole-5-carboxylic acid) γ-dipeptides, which can block Arf1 activation more efficiently than other commercially available Arf1 inhibitors through targeting the Arf1-GDP/ARNO complex at the Golgi. The anticancer effects of these γ-dipeptides were evaluated in triple native breast cancer MDA-MB-231 cells and their bone-seeking and lung-seeking sublines, showing reduced cell viability along with significant increased autophagy following treatment in both 2D and 3D cell cultures. Among these new inhibitors, the γ-dipeptide 10b displayed strongest cytotoxicity. The orthotopic NSC mouse model for breast cancer further showed the γ-dipeptides 10b exhibited a superior anticancer effects by inducing autophagic cell death in breast tumor, with no significant systemic toxicity. These findings reveal that Arf1-targeting γ-dipeptides developed by our collaborative team may represent a promising targeted therapeutic to improve treatment of breast cancer. Citation Format: Leilei He, Alain Chavanieu, Yen Vo-Hoang, Yong Teng. Novel Arf1-targeting ã-dipeptides counteract triple negative breast cancer by inducing autophagic death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1229.

Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship.

Peptidic toxins that target specifically mammalian channels and receptors can be found in the venom of animals. These toxins are rarely used directly as tools for biochemical experiments, and need to be modified via the attachment of chemical groups (e.g., radioactive or fluorescent moieties). Ideally, such modifications should maintain the toxin specificity and affinity for its target. With the goal of obtaining fluorescent derivatives of BeKm-1, a toxin from the scorpion species Buthus eupeus that selectively inhibits the voltage-gated potassium ion channel hERG, we produced four active analogues using a model of BeKm-1 docking to the outer mouth of the channel. In these BeKm-1 analogues, the natural peptide was linked to the fluorescent cyanine 5 (Cy5) probe via four different linkers at Arg1 or Arg/Lys27. All analogues retained their specificity towards the hERG channel in electrophysiological experiments but displayed a lesser affinity. These results validate our strategy for designing toxin analogues and demonstrate that different chemical groups can be attached to different residues of BeKm-1.

Metastasis is the major cause of cancer-related death in breast cancer patients, which is controlled by specific sets of genes. Targeting these genes may provide a means to delay cancer progression and allow local treatment to be more effective. We report for the first time that ADP-ribosylation factor 1 (ARF1) is the most amplified gene in ARF gene family in breast cancer, and high-level amplification of ARF1 is associated with increased mRNA expression and poor outcomes of patients with breast cancer. Knockdown of ARF1 leads to significant suppression of migration and invasion in breast cancer cells. Using the orthotopic xenograft model in NSG mice, we demonstrate that loss of ARF1 expression in breast cancer cells inhibits pulmonary metastasis. The zebrafish-metastasis model confirms that the ARF1 gene depletion suppresses breast cancer cells to metastatic disseminate throughout fish body, indicating that ARF1 is a very compelling target to limit metastasis. ARF1 function largely dependents on its activation and LM11, a cell-active inhibitor that specifically inhibits ARF1 activation through targeting the ARF1-GDP/ARNO complex at the Golgi, significantly impairs metastatic capability of breast cancer cell in zebrafish. These findings underline the importance of ARF1 in promoting metastasis and suggest that LM11 that inhibits ARF1 activation may represent a potential therapeutic approach to prevent or treat breast cancer metastasis.

ABSTRACT Introduction: Fragment-based approaches have played an increasing role alongside high-throughput screening in drug discovery for 15 years. The label-free biosensor technology based on surface plasmon resonance (SPR) is now sensitive and informative enough to serve during primary screens and validation steps. Areas covered: In this review, the authors discuss the role of SPR in fragment screening. After a brief description of the underlying principles of the technique and main device developments, they evaluate the advantages and adaptations of SPR for fragment-based drug discovery. SPR can also be applied to challenging targets such as membrane receptors and enzymes. Expert opinion: The high-level of immobilization of the protein target and its stability are key points for a relevant screening that can be optimized using oriented immobilized proteins and regenerable sensors. Furthermore, to decrease the rate of false negatives, a selectivity test may be performed in parallel on the main target bearing the binding site mutated or blocked with a low-off-rate ligand. Fragment-based drug design, integrated in a rational workflow led by SPR, will thus have a predominant role for the next wave of drug discovery which could be greatly enhanced by new improvements in SPR devices.

HAK/KUP/KT K(+) transporters have been widely associated with K(+) transport across membranes in bacteria, fungi, and plants. Indeed some members of the plant HAK/KUP/KT family contribute to root K(+) uptake, notably at low external concentrations. Besides such role in acquisition, several studies carried out in Arabidopsis have shown that other members are also involved in developmental processes. With the publication of new plant genomes, a growing interest on plant species other than Arabidopsis has become evident. In order to understand HAK/KUP/KT diversity in these new plant genomes, we discuss the evolutionary trends of 913 HAK/KUP/KT sequences identified in 46 genomes revealing five major groups with an uneven distribution among angiosperms, notably between dicotyledonous and monocotyledonous species. This information evidenced the richness of crop genomes in HAK/KUP/KT transporters and supports their study for unraveling novel physiological roles of such transporters in plants.

The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC50 of Lip and HD1 were 33.27 and 128.61 μg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.

Shaker K+ channels form the major K+ conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements, including a C-linker region and a cyclic nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the sixth transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis (Arabidopsis thaliana) Shaker subunits, K+ channel in Arabidopsis thaliana2 (KAT2) and Arabidopsis thaliana K+ rectifying channel1 (AtKC1). These two subunits contribute to K+ transport in planta by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane, whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified two contiguous amino acids, valine-381 and serine-382, located in the C-linker carboxy-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine-amino acid stretch 312TVRAASEFA320 that composes the first C-linker α-helix located just below the pore is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD three-dimensional model, based on animal HCN (for Hyperpolarization-activated, cyclic nucleotide-gated K+) channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function.