Bay41-4109-induced aberrant polymers of hepatitis b capsid proteins are removed via STUB1-promoted p62-mediated macroautophagy

The hepatitis B virus (HBV) core protein (HBc) functions in multiple steps of the viral life cycle. Heteroaryldihydropyrimidine compounds (HAPs) such as Bay41-4109 are capsid protein allosteric modulators that accelerate HBc degradation and inhibit the virion secretion of HBV, specifically by misleading HBc assembly into aberrant non-capsid polymers. However, the subsequent cellular fates of these HAP-induced aberrant non-capsid polymers are not well understood. Here, we discovered that that the chaperone-binding E3 ubiquitin ligase protein STUB1 is required for the removal of Bay41-4109-induced aberrant non-capsid polymers from HepAD38 cells. Specifically, STUB1 recruits BAG3 to transport Bay41-4109-induced aberrant non-capsid polymers to the perinuclear region of cells, thereby initiating p62-mediated macroautophagy and lysosomal degradation. We also demonstrate that elevating the STUB1 level enhances the inhibitory effect of Bay41-4109 on the production of HBeAg and HBV virions in HepAD38 cells, in HBV-infected HepG2-NTCP cells, and in HBV transgenic mice. STUB1 overexpression also facilitates the inhibition of Bay41-4109 on the cccDNA formation in de novo infection of HBV. Understanding these molecular details paves the way for applying HAPs as a potentially curative regimen (or a component of a combination treatment) for eradicating HBV from hepatocytes of chronic infection patients.

In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

The Caenorhabditis elegans DEG-3/DES-2 Channel Is a Betaine-Gated Receptor Insensitive to Monepantel

Natural plant compounds, such as betaine, are described to have nematocidal properties. Betaine also acts as a neurotransmitter in the free-living model nematode Caenorhabditis elegans, where it is required for normal motility. Worm motility is mediated by nicotinic acetylcholine receptors (nAChRs), including subunits from the nematode-specific DEG-3 group. Not all types of nAChRs in this group are associated with motility, and one of these is the DEG-3/DES-2 channel from C. elegans, which is involved in nociception and possibly chemotaxis. Interestingly, the activity of DEG-3/DES-2 channel from the parasitic nematode of ruminants, Haemonchus contortus, is modulated by monepantel and its sulfone metabolite, which belong to the amino-acetonitrile derivative anthelmintic drug class. Here, our aim was to advance the pharmacological knowledge of the DEG-3/DES-2 channel from C. elegans by functionally expressing the DEG-3/DES-2 channel in Xenopus laevis oocytes and using two-electrode voltage-clamp electrophysiology. We found that the DEG-3/DES-2 channel was more sensitive to betaine than ACh and choline, but insensitive to monepantel and monepantel sulfone when used as direct agonists and as allosteric modulators in co-application with betaine. These findings provide important insight into the pharmacology of DEG-3/DES-2 from C. elegans and highlight the pharmacological differences between non-parasitic and parasitic nematode species.

Impact of membrane lipid polyunsaturation on dopamine D2 receptor ligand binding and signaling

The heterogenous and dynamic constitution of the membrane fine-tunes signal transduction. In particular, the polyunsaturated fatty acid (PUFA) tails of phospholipids influence the biophysical properties of the membrane, production of second messengers, or membrane partitioning. Few evidence mostly originating from studies of rhodopsin suggest that PUFAs directly modulate the conformational dynamic of transmembrane proteins. However, whether such properties translate to other G protein-coupled receptors remains unclear. We focused on the dopamine D2 receptor (D2R), a main target of antipsychotics. Membrane enrichment in n-3, but not n-6, PUFAs potentiates ligand binding. Molecular dynamics simulations show that the D2R preferentially interacts with n-3 over n-6 PUFAs. Furthermore, even though this mildly affects signalling in heterologous systems, in vivo n-3 PUFA deficiency blunts the effects of D2R ligands. These results suggest that n-3 PUFAs act as allosteric modulators of the D2R and provide a putative mechanism for their potentiating effect on antipsychotic efficacy.

Druggable Lipid Binding Sites in Pentameric Ligand-Gated Ion Channels and Transient Receptor Potential Channels

Lipids modulate the function of many ion channels, possibly through direct lipid-protein interactions. The recent outpouring of ion channel structures by cryo-EM has revealed many lipid binding sites. Whether these sites mediate lipid modulation of ion channel function is not firmly established in most cases. However, it is intriguing that many of these lipid binding sites are also known sites for other allosteric modulators or drugs, supporting the notion that lipids act as endogenous allosteric modulators through these sites. Here, we review such lipid-drug binding sites, focusing on pentameric ligand-gated ion channels and transient receptor potential channels. Notable examples include sites for phospholipids and sterols that are shared by anesthetics and vanilloids. We discuss some implications of lipid binding at these sites including the possibility that lipids can alter drug potency or that understanding protein-lipid interactions can guide drug design. Structures are only the first step toward understanding the mechanism of lipid modulation at these sites. Looking forward, we identify knowledge gaps in the field and approaches to address them. These include defining the effects of lipids on channel function in reconstituted systems using asymmetric membranes and measuring lipid binding affinities at specific sites using native mass spectrometry, fluorescence binding assays, and computational approaches.

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.

Allosteric ligands control the activation of a class C GPCR heterodimer by acting at the transmembrane interface

G protein-coupled receptors (GPCRs) are among the most promising drug targets. They often form homo- and heterodimers with allosteric cross-talk between receptor entities, which contributes to fine tuning of transmembrane signaling. Specifically controlling the activity of GPCR dimers with ligands is a good approach to clarify their physiological roles and to validate them as drug targets. Here, we examined the mode of action of positive allosteric modulators (PAMs) that bind at the interface of the transmembrane domains of the heterodimeric GABAB receptor. Our site-directed mutagenesis results show that mutations of this interface impact the function of the three PAM tested. The data support the inference that they act at the active interface between both transmembrane domains, the binding site involving residues of the TM6s of the GABAB1 and the GABAB2 subunit. Importantly, the agonist activity of these PAMs involves a key region in the central core of the GABAB2 transmembrane domain, which also controls the constitutive activity of the GABAB receptor. This region corresponds to the sodium ion binding site in class A GPCRs that controls the basal state of the receptors. Overall, these data reveal the possibility of developing allosteric compounds able to specifically modulate the activity of GPCR homo- and heterodimers by acting at their transmembrane interface.

Neurosteroid modulation of GABAA receptor function by independent action at multiple specific binding sites

: Neurosteroids are endogenous modulators of GABAA receptors that mediate anxiety, pain, mood and arousal. The 3-hydroxyl epimers, allopregnanolone (3α-OH) and epi-allopregnanolone (3β-OH) are both prevalent in mammalian brain and produce opposite effects on GABAA receptor function, acting as positive and negative allosteric modulators respectively. This Perspective provides a model to explain the actions of 3α-OH and 3β-OH neurosteroids. The model is based on evidence that the neurosteroid epimers bind to an overlapping subset of specific sites on GABAA receptors, with their net functional effect on channel gating being the sum of their independent effects at each site.