Allosteric inhibition of dengue virus RNA-dependent RNA polymerase by Litsea cubeba phytochemicals: a computational study.

RNA-dependent RNA polymerase (RdRp) is considered a potential drug target for dengue virus (DENV) inhibition and has attracted attention in antiviral drug discovery. Here, we screened 121 natural compounds from Litsea cubeba against DENV RdRp using various approaches of computer-based drug discovery. Notably, we identified four potential compounds (Ushinsunine, Cassameridine, (+)-Epiexcelsin, (-)-Phanostenine) with good binding scores and allosteric interactions with the target protein. Moreover, molecular dynamics simulation studies were done to check the conformational stability of the complexes under given conditions. Additionally, we performed post-simulation analysis to find the stability of potential drugs in the target protein. The findings suggest Litsea cubeba-derived phytomolecules as a therapeutic solution to control DENV infection.Communicated by Ramaswamy H. Sarma.

Discovery of allosteric regulators with clinical potential to stabilize alpha-L-iduronidase in mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal disease caused by lowered activity of the enzyme alpha-L-iduronidase (IDUA). Current therapeutic options show limited efficacy and do not treat some important aspects of the disease. Therefore, it may be advantageous to identify strategies that could improve the efficacy of existing treatments. Pharmacological chaperones are small molecules that protect proteins from degradation, and their use in combination with enzyme replacement therapy (ERT) has been proposed as an alternative therapeutic strategy. Using the SEE-Tx® proprietary computational drug discovery platform, a new allosteric ligand binding cavity in IDUA was identified distal from the active site. Virtual high-throughput screening of approximately 5 million compounds using the SEE-Tx® docking platform identified a subset of small molecules that bound to the druggable cavity and functioned as novel allosteric chaperones of IDUA. Experimental validation by differential scanning fluorimetry showed an overall hit rate of 11.4%. Biophysical studies showed that one exemplary hit molecule GT-01803 bound to (Kd = 22 µM) and stabilized recombinant human IDUA (rhIDUA) in a dose-dependent manner. Co-administration of rhIDUA and GT-01803 increased IDUA activity in patient-derived fibroblasts. Preliminary in vivo studies have shown that GT-01803 improved the pharmacokinetic (PK) profile of rhIDUA, increasing plasma levels in a dose-dependent manner. Furthermore, GT-01803 also increased IDUA enzymatic activity in bone marrow tissue, which benefits least from standard ERT. Oral bioavailability of GT-01803 was found to be good (50%). Overall, the discovery and validation of a novel allosteric chaperone for rhIDUA presents a promising strategy to enhance the efficacy of existing treatments for MPS I. The compound's ability to increase rhIDUA activity in patient-derived fibroblasts and its good oral bioavailability underscore its potential as a potent adjunct to ERT, particularly for addressing aspects of the disease less responsive to standard treatment.

Positive Allosteric Modulation of NMDARs Prevents the Altered Surface Dynamics Caused by Patients' Antibodies.

OBJECTIVES: A positive allosteric modulator of the NMDAR, SGE-301, has been shown to reverse the alterations caused by the antibodies of patients with anti-NMDAR encephalitis (NMDARe). However, the mechanisms involved beyond receptor modulation are unclear. In this study, we aimed to investigate how this modulator affects NMDAR membrane dynamics. METHODS: Cultured hippocampal neurons were treated with SGE-301 or vehicle, alongside with immunoglobulins G (IgG) from patients with NMDARe or healthy controls. NMDAR surface dynamics were assessed with single-molecule imaging by photoactivated localization microscopy. RESULTS: NMDAR trajectories from neurons treated with SGE-301 were less confinement, with increased diffusion coefficients. This effect mainly occurred at synapses because extrasynaptic diffusion and confinement were minimally affected by SGE-301. Treatment with patients' IgG reduced NMDAR surface dynamics and increased their confinement. Remarkably, SGE-301 incubation antagonized patients' IgG effects in both synaptic and extrasynaptic membrane compartments, restoring diffusion and confinement values similar to those from neurons exposed to control IgG. DISCUSSION: We demonstrate that SGE-301 upregulates NMDAR surface diffusion and antagonizes the pathogenic effects of patients' IgG on NMDAR membrane organization. These findings suggest a potential therapeutic strategy for NMDARe.

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

Akt Inhibitor Advancements: From Capivasertib Approval to Covalent-Allosteric Promises.

Akt kinase is vital in cell growth, survival, metabolism, and migration. Dysregulation of Akt signaling is implicated in cancer and metabolic disorders. In the context of cancer, overactive Akt promotes cell survival and proliferation. This has spurred extensive research into developing Akt inhibitors as potential therapeutic agents to disrupt aberrant Akt signaling. Akt inhibitors are classified into three main types: ATP-competitive, allosteric, and covalent-allosteric inhibitors (CAAIs). ATP-competitive inhibitors compete with ATP for binding to Akt, allosteric inhibitors interact with the Pleckstrin homology (PH) domain, and covalent-allosteric inhibitors form covalent bonds, making them more potent and selective. Notably, capivasertib (AZD5363), a potent ATP-competitive Akt inhibitor, received FDA approval in November 2023 for use in combination with the estrogen receptor degrader fulvestrant to treat breast cancer. Challenges remain, including improving selectivity, identifying biomarkers to tailor treatments, and enhancing therapeutic efficacy while minimizing adverse effects. Particularly covalent-allosteric inhibitors hold promise for future more effective and personalized treatments.

Bitter taste receptor activation by cholesterol and an intracellular tastant.

Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.

CB1 Receptor Negative Allosteric Modulators as a Potential Tool to Reverse Cannabinoid Toxicity.

While the opioid crisis has justifiably occupied news headlines, emergency rooms are seeing many thousands of visits for another cause: cannabinoid toxicity. This is partly due to the spread of cheap and extremely potent synthetic cannabinoids that can cause serious neurological and cardiovascular complications-and deaths-every year. While an opioid overdose can be reversed by naloxone, there is no analogous treatment for cannabis toxicity. Without an antidote, doctors rely on sedatives, with their own risks, or 'waiting it out' to treat these patients. We have shown that the canonical synthetic 'designer' cannabinoids are highly potent CB1 receptor agonists and, as a result, competitive antagonists may struggle to rapidly reverse an overdose due to synthetic cannabinoids. Negative allosteric modulators (NAMs) have the potential to attenuate the effects of synthetic cannabinoids without having to directly compete for binding. We tested a group of CB1 NAMs for their ability to reverse the effects of the canonical synthetic designer cannabinoid JWH018 in vitro in a neuronal model of endogenous cannabinoid signaling and also in vivo. We tested ABD1085, RTICBM189, and PSNCBAM1 in autaptic hippocampal neurons that endogenously express a retrograde CB1-dependent circuit that inhibits neurotransmission. We found that all of these compounds blocked/reversed JWH018, though some proved more potent than others. We then tested whether these compounds could block the effects of JWH018 in vivo, using a test of nociception in mice. We found that only two of these compounds-RTICBM189 and PSNCBAM1-blocked JWH018 when applied in advance. The in vitro potency of a compound did not predict its in vivo potency. PSNCBAM1 proved to be the more potent of the compounds and also reversed the effects of JWH018 when applied afterward, a condition that more closely mimics an overdose situation. Lastly, we found that PSNCBAM1 did not elicit withdrawal after chronic JWH018 treatment. In summary, CB1 NAMs can, in principle, reverse the effects of the canonical synthetic designer cannabinoid JWH018 both in vitro and in vivo, without inducing withdrawal. These findings suggest a novel pharmacological approach to at last provide a tool to counter cannabinoid toxicity.

The allostery and modification of hGHRH molecules and specific dimer produced significant fertility effect by proliferating and activating in-situ ovarian mesenchymal stem cells.

The negative coordination of growth hormone secretagogue receptor (GHS-R) and growth hormone-releasing hormone receptor (GHRH-R) involves in the repair processes of cellular injury. The allosteric U- or H-like modified GHRH dimer Grinodin and 2Y were comparatively evaluated in normal Kunming mice and hamster infertility models induced by CPA treatment. 1-3-9 µg of Grinodin or 2Y per hamster stem-cell-exhaustion model was subcutaneously administered once a week, respectively inducing 75-69-46 or 45-13-50 % of birth rates. In comparison, the similar mole of human menopausal gonadotropin (hMG) or human growth hormone (hGH) was administered once a day but caused just 25 or 20 % of birth rates. Grinodin induced more big ovarian follicles and corpora lutea than 2Y, hMG, hGH. The hMG-treated group was observed many distorted interstitial cells and more connective tissues and the hGH-treated group had few ovarian follicles. 2Y had a plasma lifetime of 21 days and higher GH release in mice, inducing lower birth rate and stronger individual specificity in reproduction as well as only promoting the proliferation of mesenchymal-stem-cells (MSCs) in the models. In comparison, Grinodin had a plasma lifetime of 30 days and much lower GH release in mice. It significantly promoted the proliferation and activation of ovarian MSCs together with the development of follicles in the models by increasing Ki67 and GHS-R expressions, and decreasing GHRH-R expression in a dose-dependent manner. However, the high GH and excessive estrogen levels in the models showed a dose-dependent reduction in fertility. Therefore, unlike 2Y, the low dose of Grinodin specifically shows low GHS-R and high GHRH-R expressions thus evades GH and estrogen release and improves functions of organs, resulting in an increase of fertility.

Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. AIM OF THE STUDY: This study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. MATERIALS AND METHODS: SW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. RESULTS: Wighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a "closed" conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. CONCLUSION: Wighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Understanding the Modulatory Role of E2012 on the γ-Secretase-Substrate Interaction.

Allosteric modulation plays a critical role in enzyme functionality and requires a deep understanding of the interactions between the active and allosteric sites. γ-Secretase (GS) is a key therapeutic target in the treatment of Alzheimer's disease (AD), through its role in the synthesis of amyloid ß peptides that accumulate in AD patients. This study explores the structure and dynamic effects of GS modulation by E2012 binding, employing well-tempered metadynamics and conventional molecular dynamics simulations across three binding scenarios: (1) GS enzyme with and without L458 inhibitor, (2) the GS-substrate complex together with the modulator E2012 in two different binding modes, and (3) E2012 interacting with a C99 substrate fragment. Our findings reveal that the presence of L458 induces conformational changes that contribute to stabilization of the GS enzyme dynamics, previously reported as a key factor that allowed the resolution of the cryo-EM structure and the enhanced binding of E2012. Furthermore, we identified the most favorable binding site for E2012 within the GS-substrate complex, uncovering significant modulatory effects and a complex network of interactions that influence the position of the substrate for catalysis. In addition, we explore a potential substrate-modulator binding before the formation of the enzyme-substrate complex. The insights gained from our study emphasize the importance of these interactions in the development of potential therapeutic interventions that target the functionality of the GS enzyme in AD.

Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers.

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.

mGlu2 and mGlu3 receptor negative allosteric modulators attenuate the interoceptive effects of alcohol in male and female rats.

RATIONALE: The subjective effects of alcohol are associated with alcohol use disorder (AUD) vulnerability and treatment outcomes. The interoceptive effects of alcohol are part of these subjective effects and can be measured in animal models using drug discrimination procedures. The newly developed mGlu2 and mGlu3 negative allosteric modulators (NAMs) are potential therapeutics for AUD and may alter interoceptive sensitivity to alcohol. OBJECTIVES: To determine the effects of mGlu2 and mGlu3 NAMs on the interoceptive effects of alcohol in rats. METHODS: Long-Evans rats were trained to discriminate the interoceptive stimulus effects of alcohol (2.0 g/kg, i.g.) from water using both operant (males only) and Pavlovian (male and female) drug discrimination techniques. Following acquisition training, an alcohol dose-response (0, 0.5, 1.0, 2.0 g/kg) experiment was conducted to confirm stimulus control over behavior. Next, to test the involvement of mGlu2 and mGlu3, rats were pretreated with the mGlu2-NAM (VU6001966; 0, 3, 6, 12 mg/kg, i.p.) or the mGlu3-NAM (VU6010572; 0, 3, 6, 12 mg/kg, i.p.) before alcohol administration (2.0 g/kg, i.g.). RESULTS: In Pavlovian discrimination, male rats showed greater interoceptive sensitivity to 1.0 and 2.0 g/kg alcohol compared to female rats. Both mGlu2-NAM and mGlu3-NAM attenuated the interoceptive effects of alcohol in male and female rats using Pavlovian and operant discrimination. There may be a potential sex difference in response to the mGlu2-NAM at the highest dose tested. CONCLUSIONS: Male rats may be more sensitive to the interoceptive effects of the 2.0 g/kg alcohol training dose compared to female rats. Both mGlu2-and mGlu3-NAM attenuate the interoceptive effects of alcohol in male and female rats. These drugs may have potential for treatment of AUD in part by blunting the subjective effects of alcohol.

Channeling Nicotinamide Phosphoribosyltransferase (NAMPT) to Address Life and Death.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in NAD+ biosynthesis via salvage of NAM formed from catabolism of NAD+ by proteins with NADase activity (e.g., PARPs, SIRTs, CD38). Depletion of NAD+ in aging, neurodegeneration, and metabolic disorders is addressed by NAD+ supplementation. Conversely, NAMPT inhibitors have been developed for cancer therapy: many discovered by phenotypic screening for cancer cell death have low nanomolar potency in cellular models. No NAMPT inhibitor is yet FDA-approved. The ability of inhibitors to act as NAMPT substrates may be associated with efficacy and toxicity. Some 3-pyridyl inhibitors become 4-pyridyl activators or "NAD+ boosters". NAMPT positive allosteric modulators (N-PAMs) and boosters may increase enzyme activity by relieving substrate/product inhibition. Binding to a "rear channel" extending from the NAMPT active site is key for inhibitors, boosters, and N-PAMs. A deeper understanding may fulfill the potential of NAMPT ligands to regulate cellular life and death.

Phosphorylation-dependent allosteric regulation of Cx43 gap junction inhibitor potency.

Physiological and pathological processes such as homeostasis, embryogenesis, development, tumorigenesis, and cell movement depend on the intercellular communication through gap junctions (GJIC). Connexin (Cx)-based GJ channels are formed of two apposing hemichannels in the contiguous cells and provide a direct pathway for electrical and metabolic intercellular communication. The main modulators of GJ conductance are transjunctional voltage, intracellular pH, Ca2+, Mg2+, and phosphorylation. Chemical modulators of GJIC are being used in cases of various intercellular communication-dependent diseases. In this study, we used molecular docking, dual whole-cell patch-clamp, and Western blotting to investigate the impact of connexin phosphorylation on GJ chemical gating by α-pinene and other GJ inhibitors (octanol, carbenoxolone, mefloquine, intracellular pH, glycyrrhetinic acid, and sevoflurane) in HeLa cells expressing exogenous Cx43 (full length and truncated at amino acid 258) and other connexins typical of heart and/or nervous system (Cx36, Cx40, Cx45, and Cx47), and in cells expressing endogenous Cx43 (Novikoff and U-87). We found that Ca2+-regulated kinases, such as Ca2+/calmodulin-dependent kinase II, atypical protein kinase C, cyclin-dependent kinase, and Pyk2 kinase may allosterically modulate the potency of α-pinene through phosphorylation of Cx43 C-terminus. The identified new phenomenon was Cx isoform-, inhibitor-, and cell type-dependent. Overall, these results suggest that compounds, the potency of which depends on receptor phosphorylation, might be of particular interest in developing targeted therapies for diseases accompanied by high kinase activity, such as cardiac arrhythmias, epilepsy, stroke, essential tremor, inflammation, and cancer.

Synthesis and Structure-Activity Relationships for Glutamate Transporter Allosteric Modulators.

Excitatory amino acid transporters (EAATs) are essential CNS proteins that regulate glutamate levels. Excess glutamate release and alteration in EAAT expression are associated with several CNS disorders. Previously, we identified positive allosteric modulators (PAM) of EAAT2, the main CNS transporter, and have demonstrated their neuroprotective properties in vitro. Herein, we report on the structure-activity relationships (SAR) for the analogs identified from virtual screening and from our medicinal chemistry campaign. This work identified several selective EAAT2 positive allosteric modulators (PAMs) such as compounds 4 (DA-023) and 40 (NA-014) from a library of analogs inspired by GT949, an early generation compound. This series also provides nonselective EAAT PAMs, EAAT inhibitors, and inactive compounds that may be useful for elucidating the mechanism of EAAT allosteric modulation.

Allosteric Modulation of the YAP/TAZ-TEAD Interaction by Palmitoylation and Small-Molecule Inhibitors.

The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, it translocates into the nucleus, where it associates with TEAD transcription factors. This partnership is instrumental in regulating the transcription of progrowth and antiapoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes autopalmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences the binding of YAP and TAZ to TEAD in distinct manners. This discovery has significant implications for the design and deployment of future molecular interventions targeting this interaction.

An allosteric inhibitor of sirtuin 2 blocks hepatitis B virus covalently closed circular DNA establishment and its transcriptional activity.

296 million people worldwide are predisposed to developing severe end-stage liver diseases due to chronic hepatitis B virus (HBV) infection. HBV forms covalently closed circular DNA (cccDNA) molecules that persist as episomal DNA in the nucleus of infected hepatocytes and drive viral replication. Occasionally, the HBV genome becomes integrated into host chromosomal DNA, a process that is believed to significantly contribute to circulating HBsAg levels and HCC development. Neither cccDNA accumulation nor expression from integrated HBV DNA are directly targeted by current antiviral treatments. In this study, we investigated the antiviral properties of a newly described allosteric modulator, FLS-359, that targets sirtuin 2 (SIRT2), an NAD+-dependent deacylase. Our results demonstrate that SIRT2 modulation by FLS-359 and by other tool compounds inhibits cccDNA synthesis following de novo infection of primary human hepatocytes and HepG2 (C3A)-NTCP cells, and FLS-359 substantially reduces cccDNA recycling in HepAD38 cells. While pre-existing cccDNA is not eradicated by short-term treatment with FLS-359, its transcriptional activity is substantially impaired, likely through inhibition of viral promoter activities. Consistent with the inhibition of viral transcription, HBsAg production by HepG2.2.15 cells, which contain integrated HBV genomes, is also suppressed by FLS-359. Our study provides further insights on SIRT2 regulation of HBV infection and supports the development of potent SIRT2 inhibitors as HBV antivirals.

Cobalamins Function as Allosteric Activators of an Angelman Syndrome-Associated UBE3A/E6AP Variant.

Genetic aberrations of the maternal UBE3A allele, which encodes the E3 ubiquitin ligase E6AP, are the cause of Angelman syndrome (AS), an imprinting disorder. In most cases, the maternal UBE3A allele is not expressed. Yet, approximately 10 percent of AS individuals harbor distinct point mutations in the maternal allele resulting in the expression of full-length E6AP variants that frequently display compromised ligase activity. In a high-throughput screen, we identified cyanocobalamin, a vitamin B12-derivative, and several alloxazine derivatives as activators of the AS-linked E6AP-F583S variant. Furthermore, we show by cross-linking coupled to mass spectrometry that cobalamins affect the structural dynamics of E6AP-F583S and apply limited proteolysis coupled to mass spectrometry to obtain information about the regions of E6AP that are involved in, or are affected by binding cobalamins and alloxazine derivatives. Our data suggest that dietary supplementation with vitamin B12 can be beneficial for AS individuals.

Modulation of mGlu5 reduces rewarding associative properties of nicotine via changes in mesolimbic plasticity: Relevance to comorbid cigarette smoking in psychosis.

RATIONALE: Antipsychotic medications that are used to treat psychosis are often limited in their efficacy by high rates of severe side effects. Treatment success in schizophrenia is further complicated by high rates of comorbid nicotine use. Dopamine D2 heteroreceptor complexes have recently emerged as targets for the development of more efficacious pharmaceutical treatments for schizophrenia. OBJECTIVE: The current study sought to explore the use of the positive allosteric modulator of the mGlu5 receptor 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) as a treatment to reduce symptoms related to psychosis and comorbid nicotine use. METHODS: Neonatal treatment of animals with the dopamine D2-like receptor agonist quinpirole (NQ) from postnatal day (P)1-21 produces a lifelong increase in D2 receptor sensitivity, showing relevance to psychosis and comorbid tobacco use disorder. Following an 8-day conditioning paradigm, brain tissue in the mesolimbic pathway was analyzed for several plasticity markers, including brain derived neurotrophic factor (BDNF), phosphorylated p70 ribosomal S6 kinase (phospho-p70S6K), and cadherin-13 (Cdh13). RESULTS: Pretreatment with CDPPB was effective to block enhanced nicotine conditioned place preference observed in NQ-treated animals. Pretreatment was additionally effective to block the nicotine-induced increase in BDNF and sex-dependent increases in cadherin-13 in the ventral tegmental area (VTA), as well as increased phospho-p70S6K in the nucleus accumbens (NAcc) shell found in NQ-treated animals. CONCLUSION: In conjunction with prior work, the current study suggests positive allosteric modulation of the mGlu5 receptor, an emerging target for schizophrenia therapeutics, may be effective for the treatment of comorbid nicotine abuse in psychosis.