Key Interaction Sites Research Articles

The molecular structure of an axle-less F1-ATPase

F1Fo ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F1-ATPase catalytic cores can hydrolyse ATP, passing through a series of conformational states involving rotation of the central γ rotor subunit and the opening and closing of the catalytic β subunits. Cooperativity in F1-ATPase has long thought to be conferred through the γ subunit, with three key interaction sites between the γ and β subunits being identified. Single molecule studies have demonstrated that the F1 complexes lacking the γ axle still “rotate” and hydrolyse ATP, but with less efficiency. We solved the cryogenic electron microscopy structure of an axle-less Bacillus sp. PS3 F1-ATPase. The unexpected binding-dwell conformation of the structure in combination with the observed lack of interactions between the axle-less γ and the open β subunit suggests that the complete γ subunit is important for coordinating efficient ATP binding of F1-ATPase.

Cryo-EM structure of small-molecule agonist bound delta opioid receptor-Gi complex enables discovery of biased compound

Delta opioid receptor (δOR) plays a pivotal role in modulating human sensation and emotion. It is an attractive target for drug discovery since, unlike Mu opioid receptor, it is associated with low risk of drug dependence. Despite its potential applications, the pharmacological properties of δOR, including the mechanisms of activation by small-molecule agonists and the complex signaling pathways it engages, as well as their relation to the potential side effects, remain poorly understood. In this study, we use cryo-electron microscopy (cryo-EM) to determine the structure of the δOR-Gi complex when bound to a small-molecule agonist (ADL5859). Moreover, we design a series of probes to examine the key receptor-ligand interaction site and identify a region involved in signaling bias. Using ADL06 as a chemical tool, we elucidate the relationship between the β-arrestin pathway of the δOR and its biological functions, such as analgesic tolerance and convulsion activities. Notably, we discover that the β-arrestin recruitment of δOR might be linked to reduced gastrointestinal motility. These insights enhance our understanding of δOR’s structure, signaling pathways, and biological functions, paving the way for the structure-based drug discovery.

The molecular structure of an axle-less F1-ATPase.

F1Fo ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F1-ATPase catalytic cores can hydrolyse ATP, passing through a series of conformational states involving rotation of the central γ rotor subunit and the opening and closing of the catalytic β subunits. Cooperativity in F1-ATPase has long thought to be conferred through the γ subunit, with three key interaction sites between the γ and β subunits being identified. Single molecule studies have demonstrated that the F1 complexes lacking the γ axle still "rotate" and hydrolyse ATP, but with less efficiency. We solved the cryogenic electron microscopy structure of an axle-less Bacillus sp. PS3 F1-ATPase. The unexpected binding-dwell conformation of the structure in combination with the observed lack of interactions between the axle-less γ and the open β subunit suggests that the complete γ subunit is important for coordinating efficient ATP binding of F1-ATPase.

Gaining Insights into Key Structural Hotspots within the Allosteric Binding Pockets of Protein Kinases.

Protein kinases are essential regulators of cell function and represent one of the largest and most diverse protein families. They are particularly influential in signal transduction and coordinating complex processes like the cell cycle. Out of the 518 human protein kinases identified, 478 are part of a single superfamily sharing catalytic domains that are related in sequence. The dysregulation of protein kinases due to certain mutations has been associated with various diseases, including cancer. Although most of the protein kinase inhibitors identified as type I or type II primarily target the ATP-binding pockets of kinases, the structural and sequential resemblances among these pockets pose a significant challenge for selective inhibition. Therefore, targeting allosteric pockets that are beside highly conserved ATP pockets has emerged as a promising strategy to prevail current limitations, such as poor selectivity and drug resistance. In this article, we compared the binding pockets of various protein kinases for which allosteric (type III) inhibitors have already been developed. Additionally, understanding the structure and shape of existing ligands could aid in identifying key interaction sites within the allosteric pockets of kinases. This comprehensive review aims to facilitate the design of more effective and selective allosteric inhibitors.

Combating HTLV-1 infections with Taxus baccata phytoconstituents: Molecular mechanisms potential anti-ATLL agents

Taxus baccata is recognized as a traditional herb with antiviral and anticancer properties, making it a valuable candidate for anti-proliferative and antiviral agents, particularly in the absence of a cure for HTLV-1 infection and related diseases. The alkaloid extract of Taxus baccata was evaluated for its impact on HTLV-1-MT2 cell proliferation and HTLV-1 protease activity, presenting a promising avenue for therapeutic applications akin to HIV-PR inhibitors. Given the pressing need for effective treatments for HTLV-1-associated conditions, our study delved into the alkaloid extract's effects through immunofluorescence assays on HTLV-1 protease both in vitro and in silico. Confirmation of Taxus baccata extraction was achieved through immunofluorescence, infrared spectroscopy (IR), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC) analysis, with paclitaxel serving as a control. Furthermore, the anticancer properties of the alcoholic and alkaloid extracts were explored through in vitro assays using various cell lines, including HTLV-1-MT2, A549, HT29, and MCF7, alongside flow cytometry assessments. Notably, treatment with the alkaloid extract significantly impacted the survival of HTLV-1-MT2 cells (-2.44±0.012), alcoholic extract (11.17±0.13) and paclitaxel (0.00±0.18) were evaluated. GC-MS analysis identified Dimethyl malate, Lichexanthone, and Glycinexylidide as bioactive compounds within the plant, with investigations into their molecular interactions with HTLV-1 protease conducted. Molecular dynamics studies revealed key interaction sites between the compounds and HTLV-1 protease (PDB ID:4YDF), particularly highlighting the binding sequence of the dimethyl malate ligand within the protease A chain (Ala59). Collectively, the alkaloid compounds from Taxus baccata exhibit potential inhibitory effects on HTLV-1 oncovirus proliferation and transmission.

Abstract P52: Combination of 5-fluorouracil and Menadione Exerts Synergistic Effect on Colorectal Cancer Cells by Targeting Wnt/β-catenin Signaling Pathway

Abstract Colorectal cancer is the third most common type of cancer and the second most in terms of mortality rate worldwide. 5-fluorouracil (5-FU), the fluoropyrimidine drug, that interferes with DNA replication and repair processes, is the first-line-of-treatment against colorectal cancer. However, cells gain resistance over time and relapses occur upon continuous 5-FU therapy. Interestingly, 5-FU promotes stemness in colorectal cancer via Wnt signaling pathway which is critical for its progression. Studies have pointed out the possibility of using a Wnt signaling inhibitor along with 5-FU as a better strategy to overcome the poor outcomes of current 5-FU based therapies. We have previously shown that menadione (vitamin K3) suppresses Wnt signaling pathway in colorectal cancer cells. In the present study, we attempted to evaluate if the combination of menadione and 5-FU can exert a synergistic effect in the human colorectal cancer cell lines HCT116 and SW620. Analysis of the cell viability assay results using CompuSyn software confirmed that the combination indeed acts synergistically. The combination efficiently downregulated the expression of the Wnt signal transducer protein, β-catenin, and the expression of other proteins in the pathway such as phospho-GSK3β in the cell lines HCT116 and SW620. Docking of the individual drugs and their combination on β-catenin using Autodock Vina showed that 5-FU and menadione in combination bind to key protein-protein interaction (PPI) sites of β-catenin whereby they may inhibit β-catenin-TCF interaction or activate the β-catenin destruction complex. Taken together, our data possibly explains that the synergistic action of 5-FU and menadione in colorectal cancer cells may be due to the simultaneous binding of the two drugs to multiple PPI sites thereby blocking the activity of β-catenin. Citation Format: Vidya P Warrier, Sankaran Venkatachalam, Sakthivel Ramasamy, M Michael Gromiha, Devarajan Karunagaran. Combination of 5-fluorouracil and Menadione Exerts Synergistic Effect on Colorectal Cancer Cells by Targeting Wnt/β-catenin Signaling Pathway [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P52.

Abstract 4327: Development of bioluminescent biosensors to monitor the interaction betweenGSDMD and caspase-1 in living cells

Abstract Gasdermin D (GSDMD) is the key executioner of pyroptosis, an inflammatory form of programmed cell death implicated in various inflammation-driven diseases, including cancer, heart failure and strokes. The interaction between GSDMD and caspase-1 (CASP1) orchestrates the critical pathway of GSDMD-mediated pyroptosis. During pyroptosis, pyroptosis stimuli activate CASP1, which subsequently binds to and cleave GSDMD between GSDMD’s N- and C-terminal domains. The free N-terminal domains oligomerize and translocate to the cell membrane, forming pores and releasing proinflammatory cytokines and causing cellular lysis. Key interaction sites have been identified for GSDMD recognition and engagement by CASP1; therefore, blocking functionally significant residues presents a new therapeutic strategy for inhibiting CASP1-induced GSDMD cleavage and pyroptosis. Currently, there is no small molecule capable of specifically disrupting the GSDMD and CASP1 interaction. In response, the ongoing pursuit of identifying potential inhibitors is a driving force for developing a biosensor capable of monitoring this interaction and their roles in downstream pyroptotic effects. In this study, we utilized the NanoLuc Binary Technology (NanoBiT) to construct several bioluminescent biosensors comprising two NanoLuc luciferase enzyme fragments (LgBiT and SmBiT) fused to GSDMD and CASP1, respectively, in various combinations. Further validation of these biosensors by luciferase assays in living cells identified a GSDMD-CASP1 biosensor with highest luciferase activity. This newly developed biosensor paves the way for high-throughput screens to identify novel small molecule inhibitors disrupting the GSDMD-CASP1 interplay. These small molecule inhibitors will effectively impede GSDMD-mediated pyroptosis, holding promise for novel therapies of various human diseases. Citation Format: Tynan Kelly. Development of bioluminescent biosensors to monitor the interaction betweenGSDMD and caspase-1 in living cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4327.

Exploring the synthesis, molecular structure and biological activities of novel Bis-Schiff base derivatives: A combined theoretical and experimental approach

A library of six novel bis-Schiff base derivatives (2a-f) were synthesized, characterized through modern spectroscopic techniques and screened for their α-glucosidase and α-amylase inhibitory activities (in vitro). In the series, compound 2a (IC50 = 5.64 ± 2.22 µM) and 2f (IC50 = 22.78 ± 2.37 µM) were the most potent α-amylase inhibitors while the remaining four compounds showed significant to less activity. In case of α-glucosidase inhibition, four compounds 2e (IC50 = 2.83 ± 0.18 µM), 2c (IC50 = 7.03 ± 0.15 µM), 2f (IC50 = 9.99 ± 0.20 µM), and 2d (IC50 = 14.68 ± 0.21 µM) displayed excellent inhibitory activity while two compounds showed good inhibitory activity, comparing with the standard acarbose drug. The DFT assay was used to measure the FMO of the synthesized molecules, which indicated their stability, bioactivity and charge transfer. The MEP analysis revealed the distribution of electrostatic potential on the molecular surface of 2a-f, which is helpful for understanding reactivity and interactions. The AIM study showed low hydrogen bond energy and non-covalent interactions. This implies that these molecules may have weak hydrogen bonding and non-covalent interactions, which could affect their chemical behavior. The molecular docking study has provided valuable insights into the interactions between the synthesized derivatives of 2,4-dihydroxyacetophenone with α-glucosidase and α-amylase proteins. The results not only shed light on the binding affinity and key interaction sites but also offer potential avenues for the design of novel drug candidates to control postprandial glucose level in diabetic patients.

Ala307Thr variation modulates FSHR structure and impairs its binding affinity for FSH: Implications in polycystic ovarian syndrome.

Follicle-stimulating hormone receptor (FSHR) belongs to the family of G-protein coupled receptors and acts as a cognate receptor for follicle-stimulating hormone (FSH). Among the various polymorphic changes reported in FSHR, rs6165 polymorphism leading to Ala307Thr variation in the extracellular domain of the FSHR (FSHRED ) is widely reported. Therefore we attempted to evaluate the functional implications of this variation by studying its effects on FSHRED structure as well as FSH binding. Our atomic-scale investigations reveal that the hinge region, a key hormone interaction site in the extracellular domain of Wt FSHR, exhibits significantly more flexibility compared with the variant structure. Moreover, the Wt receptor in complex with FSH was observed to form a pocket-like structure in its hinge region whereas such a structure was not detected in the variant. The study further reveals that the key residue, sTyr335, required for FSH recognition and FSHR activation, exhibits lower binding free energy in the variant structure as compared to the Wt. In conclusion, our results point out that Ala307Thr variation leads to structural and conformational anomalies in FSHRED which may alter its FSH binding and affect its activation.

Piperazine Derivatives Containing the α-Ketoamide Moiety Discovered as Potential Anti-Tomato Spotted Wilt Virus Agents.

A total of 35 piperazine derivatives were designed and synthesized, and their activities against tomato spotted wilt virus (TSWV) were evaluated systematically. Compounds 34 and 35 with significant anti-TSWV activity were obtained. Their EC50 values were 62.4 and 59.9 μg/mL, prominently better than the control agents ningnanmycin (113.7 μg/mL) and ribavirin (591.1 μg/mL). To explore the mechanism of the interaction between these compounds and the virus, we demonstrated by agrobacterium-mediated, molecular docking, and microscale thermophoresis (MST) experimental methods that compounds 34 and 35 could inhibit the infection of TSWV by binding with the N protein to prevent the assembly of the virus core structure ribonucleoprotein (RNP), and it also meant that the arginine at 94 of the N protein was the key site of interaction between the compounds and the TSWV N target. Therefore, this study demonstrated the potential for forming antiviral agents from piperazine derivatives containing α-ketoamide moieties.

Comparison of Intermolecular Interactions of Irreversible and Reversible Inhibitors with Bruton's Tyrosine Kinase via Molecular Dynamics Simulations.

Bruton's tyrosine kinase (BTK) is a key protein from the TEC family and is involved in B-cell lymphoma occurrence and development. Targeting BTK is therefore an effective strategy for B-cell lymphoma treatment. Since previous studies on BTK have been limited to structure-function analyses of static protein structures, the dynamics of conformational change of BTK upon inhibitor binding remain unclear. Here, molecular dynamics simulations were conducted to investigate the molecular mechanisms of association and dissociation of a reversible (ARQ531) and irreversible (ibrutinib) small-molecule inhibitor to/from BTK. The results indicated that the BTK kinase domain was found to be locked in an inactive state through local conformational changes in the DFG motif, and P-, A-, and gatekeeper loops. The binding of the inhibitors drove the outward rotation of the C-helix, resulting in the upfolded state of Trp395 and the formation of the salt bridge of Glu445-Arg544, which maintained the inactive conformation state. Met477 and Glu475 in the hinge region were found to be the key residues for inhibitor binding. These findings can be used to evaluate the inhibitory activity of the pharmacophore and applied to the design of effective BTK inhibitors. In addition, the drug resistance to the irreversible inhibitor Ibrutinib was mainly from the strong interaction of Cys481, which was evidenced by the mutational experiment, and further confirmed by the measurement of rupture force and rupture times from steered molecular dynamics simulation. Our results provide mechanistic insights into resistance against BTK-targeting drugs and the key interaction sites for the development of high-quality BTK inhibitors. The steered dynamics simulation also offers a means to rapidly assess the binding capacity of newly designed inhibitors.

Personalized Treatment for Infantile Ascending Hereditary Spastic Paralysis Based on In Silico Strategies.

Infantile onset hereditary spastic paralysis (IAHSP) is a rare neurological disease diagnosed in less than 50 children worldwide. It is transmitted with a recessive pattern and originates from mutations of the ALS2 gene, encoding for the protein alsin and involved in differentiation and maintenance of the upper motoneuron. The exact pathogenic mechanisms of IAHSP and other neurodevelopmental diseases are still largely unknown. However, previous studies revealed that, in the cytosolic compartment, alsin is present as an active tetramer, first assembled from dimer pairs. The C-terminal VPS9 domain is a key interaction site for alsin dimerization. Here, we present an innovative drug discovery strategy, which identified a drug candidate to potentially treat a patient harboring two ALS2 mutations: one truncation at lysine 1457 (not considered) and the substitution of arginine 1611 with a tryptophan (R1611W) in the C-terminus VPS9. With a protein modeling approach, we obtained a R1611W mutant model and characterized the impact of the mutation on the stability and flexibility of VPS9. Furthermore, we showed how arginine 1611 is essential for alsin’s homo-dimerization and how, when mutated to tryptophan, it leads to an abnormal dimerization pattern, disrupting the formation of active tetramers. Finally, we performed a virtual screening, individuating an already therapy-approved compound (MK4) able to mask the mutant residue and re-establishing the alsin tetramers in HeLa cells. MK4 has now been approved for compassionate use.

Identification of a Phage Display-Derived Peptide Interacting with the N-Terminal Region of Factor VII Activating Protease (FSAP) Enables Characterization of Zymogen Activation.

Factor VII Activating protease (FSAP) has a protective effect in diverse disease conditions as inferred from studies in FSAP–/– mice and humans deficient in FSAP activity due to single-nucleotide polymorphism. The zymogen form of FSAP in plasma is activated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear whether this activation mechanism is specific and amenable to manipulation. Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide, NNKC9/41, that activates pro-FSAP in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30–35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity.

Phosphatidylinositol phosphates modulate interactions between the StarD4 sterol trafficking protein and lipid membranes

There is substantial evidence for extensive nonvesicular sterol transport in cells. For example, lipid transfer by the steroidogenic acute regulator-related proteins (StarD) containing a StarT domain has been shown to involve several pathways of nonvesicular trafficking. Among the soluble StarT domain–containing proteins, StarD4 is expressed in most tissues and has been shown to be an effective sterol transfer protein. However, it was unclear whether the lipid composition of donor or acceptor membranes played a role in modulating StarD4-mediated transport. Here, we used fluorescence-based assays to demonstrate a phosphatidylinositol phosphate (PIP)-selective mechanism by which StarD4 can preferentially extract sterol from liposome membranes containing certain PIPs (especially, PI(4,5)P2 and to a lesser degree PI(3,5)P2). Monophosphorylated PIPs and other anionic lipids had a smaller effect on sterol transport. This enhancement of transport was less effective when the same PIPs were present in the acceptor membranes. Furthermore, using molecular dynamics (MD) simulations, we mapped the key interaction sites of StarD4 with PIP-containing membranes and identified residues that are important for this interaction and for accelerated sterol transport activity. We show that StarD4 recognizes membrane-specific PIPs through specific interaction with the geometry of the PIP headgroup as well as the surrounding membrane environment. Finally, we also observed that StarD4 can deform membranes upon longer incubations. Taken together, these results suggest a mechanism by which PIPs modulate cholesterol transfer activity via StarD4.

Left on a Doorstep: The Role of Infant Abandonment in Preserving and Exposing Family Secrets in Australia 1834–1954

Based on a database of Australian cases from 1834–1954, this article argues that abandonment was an intentional strategy intended to maximise a child's chances of survival while preserving its family's reputation. However, abandonment had the potential to expose family secrets, bringing them into the public gaze and subjecting them to interrogation. Abandonment was also used for revenge, exposing the identity of putative fathers in a demand for financial support. Through this analysis the article positions abandonment as a key site of interaction between the individual and society, and the private and the public in relation to the politics of secrecy.

Lipid photopharmacology reveals a DAG-bound, sensitized state of TRPC3

Coordination of membrane lipids preserves structural integrity and subsidizes regulatory functions of channel complexes. Transient receptor potential canonical 3 (TRPC3) channel is a tetrameric and non-selective cation channel, which is directly activated by diacylglycerols (DAGs). Recent single-particle cryo-EM studies identified two key lipid interaction sites named L1 and L2, which can accommodate DAGs. To delve into the role of L1 and L2 in TRPC3 regulation, we combined electrophysiology with site-directed mutagenesis guided by molecular dynamics (MD) simulations.

Isolation and screening of umami peptides from preserved egg yolk by nano-HPLC-MS/MS and molecular docking

The material basis leading to the rich umami flavor of preserved egg yolk is poorly understood. This study used nano-high-performance liquid chromatography − tandem mass spectrometry (nano-HPLC-MS/MS) to isolate, identify, and screen umami peptides from preserved egg yolk. Five novel umami peptides—AGFMPLP, APYSGY, PPMF, SLSSLMK, and VAMNPVDHPH—were identified. Molecular docking showed that Phe527 on the taste receptor T1R1/T1R3 (T1R1, taste receptor type 1 member 1; T1R3, taste receptor type 1 member 3) was the key interaction site. Hydrogen bonding, electrostatic interactions, and hydrophobic interactions were the main binding forces between T1R1/T1R3 and umami peptides. These results contribute to understanding the umami peptides in preserved egg yolk and the interaction mechanism between umami peptides and umami receptors.

Molecular Structure and Vibrational Spectra of Water Molecules Sorbed in Poly(2-methoxyethylacrylate) Revealed by Molecular Dynamics Simulation

Molecular dynamics (MD) simulations of water sorption in poly(2-methoxyethylacrylate) (PMEA) are carried out to elucidate the hydrogen bonding (H-bonding) structures of the water molecules and the side chains of PMEA. A PMEA model incorporating lone-pair virtual sites on the carbonyl and methoxy oxygens of the side chain of PMEA, which are the key interaction sites in a biocompatible polymer, is newly developed. The PMEA model well reproduces the experimentally observed features in the infrared spectra of the hydrated polymer, as well as the radial distribution function of the water molecules in contact with the polymer, as calculated by ab initio MD simulations. The MD simulation results reveal that water molecules tend to form H-bonds with the carbonyl oxygen and the methoxy oxygen of the side chain of PMEA simultaneously, which enhance the "head-to-tail" stacking structure of the side chains at a low concentration range of water. Further penetration of water into the PMEA structure gradually increases the water-water H-bonding state and promotes the formation of water clusters even below the equilibrium water content.

Introducing ADNP and SIRT1 as new partners regulating microtubules and histone methylation.

Activity-dependent neuroprotective protein (ADNP) is essential for brain formation and function. As such, de novo mutations in ADNP lead to the autistic ADNP syndrome and somatic ADNP mutations may drive Alzheimer's disease (AD) tauopathy. Sirtuin 1 (SIRT1) is positively associated with aging, the major risk for AD. Here, we revealed two key interaction sites for ADNP and SIRT1. One, at the microtubule end-binding protein (EB1 and EB3) Tau level, with EB1/EB3 serving as amplifiers for microtubule dynamics, synapse formation, axonal transport, and protection against tauopathy. Two, on the DNA/chromatin site, with yin yang 1, histone deacetylase 2, and ADNP, sharing a DNA binding motif and regulating SIRT1, ADNP, and EB1 (MAPRE1). This interaction was linked to sex- and age-dependent altered histone modification, associated with ADNP/SIRT1/WD repeat-containing protein 5, which mediates the assembly of histone modification complexes. Single-cell RNA and protein expression analyses as well as gene expression correlations placed SIRT1-ADNP and either MAPRE1 (EB1), MAPRE3 (EB3), or both in the same mouse and human cell; however, while MAPRE1 seemed to be similarly regulated to ADNP and SIRT1, MAPRE3 seemed to deviate. Finally, we demonstrated an extremely tight correlation for the gene transcripts described above, including related gene products. This correlation was specifically abolished in affected postmortem AD and Parkinson's disease brain select areas compared to matched controls, while being maintained in blood samples. Thus, we identified an ADNP-SIRT1 complex that may serve as a new target for the understanding of brain degeneration.

Molecular mechanism of CaCCinh-A01 inhibiting TMEM16A channel

TMEM16A is a calcium-activated chloride channel that is associate with several diseases, including pulmonary diseases, hypertension, diarrhea and cancer. The CaCCinh-A01 (A01) is widely recognized as an efficient blocker of TMEM16A and has been used as a tool drug to inhibit TMEM16A currents in the laboratory. A01 also has excellent pharmacokinetic properties and can be developed as a drug to target TMEM16A. However, the molecular mechanism how A01 inhibits TMEM16A is still elusive, which slows down its drug development process. Here, calculations identified that the binding pocket of A01 was located above the pore, and it was also discovered that the binding of A01 to TMEM16A not only blocked the pore but also led to its collapse. The interaction model analysis predicted that R515/K603/E623 were crucial residues for the binding between TMEM16A and A01, and the site-directed mutagenesis studies confirmed the above results. The binding mode and quantum chemical calculations showed that the carboxyl and the amide oxygen atom of A01 were the key interaction sites between TMEM16A and A01. Therefore, our study proposed the inhibitory mechanism of TMEM16A current by A01 and revealed how A01 inhibits TMEM16A at the molecular level. These findings will shed light on both the development of A01 as a potential drug for TMEM16A dysfunction-related disorders and drug screening targeting the pocket.