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Department of Pharmacology

 

Can Current Molecular Docking Methods Accurately Predict RNA Inhibitors?

Thu, 18/07/2024 - 11:00

J Chem Inf Model. 2024 Jul 18. doi: 10.1021/acs.jcim.4c00235. Online ahead of print.

ABSTRACT

Ribonucleic acids (RNAs), particularly the noncoding RNAs, play key roles in cancer, making them attractive drug targets. While conventional methods such as high throughput screening are resource-intensive, computational methods such as RNA-ligand docking can be used as an alternative. However, currently available docking methods are fine-tuned to perform protein-ligand and protein-protein docking. In this work, we evaluated three commonly used docking methods─AutoDock Vina, HADDOCK, and HDOCK─alongside RLDOCK, which is specifically designed for RNA-ligand docking. Our evaluation was based on several criteria including cognate docking, blind docking, scoring potential, and ranking potential. In cognate docking, only RLDOCK showed a success rate of 70% for the top-scoring docked pose. Despite this, all four docking methods did not achieve an overall success rate exceeding 50% amidst our attempt to refine the top-scoring docked poses using molecular dynamics simulations. Meanwhile, all four docking methods showed poor performance in scoring potential evaluation. Although AutoDock Vina achieved an area under the receiver operating characteristic curve of 0.70, it showed poor performance in terms of Matthews' correlation coefficient, precision, enrichment factors, and normalized enrichment factors at 1, 2, and 5%. These results highlight the growing need for further optimization of docking methods to assess RNA-ligand interactions.

PMID:39023229 | DOI:10.1021/acs.jcim.4c00235

α-Synuclein Oligomers Displace Monomeric α-Synuclein from Lipid Membranes

Tue, 25/06/2024 - 11:00

ACS Nano. 2024 Jun 25. doi: 10.1021/acsnano.3c10889. Online ahead of print.

ABSTRACT

Parkinson's disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerization and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OαS-membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD.

PMID:38916260 | DOI:10.1021/acsnano.3c10889

Intestinal barrier function in the naked mole-rat: an emergent model for gastrointestinal insights

Tue, 25/06/2024 - 11:00

Am J Physiol Gastrointest Liver Physiol. 2024 Jun 25. doi: 10.1152/ajpgi.00080.2024. Online ahead of print.

ABSTRACT

The intestinal barrier plays a crucial role in homeostasis, both by facilitating absorption of nutrients and fluids, and providing a tight shield to prevent the invasion by either pathogen or commensal microorganisms. Intestinal barrier malfunction is associated with systemic inflammation, oxidative stress, and decreased insulin sensitivity, which may lead to the dysregulation of other tissues. Therefore, a deeper understanding of physiological aspects related to an enhanced barrier function is of significant scientific and clinical relevance. The naked mole-rat has many unusual biological features, including attenuated colonic neuron sensitivity to acid and bradykinin, and resistance to chemical-induced intestinal damage. However, insight into their intestinal barrier physiology is scarce. Here, we observed notable macroscopic and microscopic differences in intestinal tissue structure between naked mole-rats and mice. Moreover, naked mole-rats showed increased number of larger goblet cells and elevated mucus content. In measuring gut permeability, naked mole-rats showed reduced permeability compared to mice, measured as transepithelial electrical resistance, especially in ileum. Furthermore, intestinal ion secretion induced by serotonin, bradykinin, histamine, and capsaicin was significantly reduced in naked mole-rats compared to mice, despite the expression of receptors for all these agonists. In addition, naked mole-rats exhibited reduced pro-secretory responses to the non-selective adenylate cyclase activator forskolin. Collectively, these findings indicate that naked mole-rats possess a robust and hard-to-penetrate gastrointestinal barrier, that is resistant to environmental and endogenous irritants. Naked mole-rats may therefore provide valuable insights into the physiology of the intestinal barrier and set the stage for the development of innovative and effective therapies.

PMID:38915279 | DOI:10.1152/ajpgi.00080.2024

Dissecting the roles of dynamin and clathrin in platelet pinocytosis

Thu, 13/06/2024 - 11:00

Biochem Biophys Res Commun. 2024 Jun 10;725:150250. doi: 10.1016/j.bbrc.2024.150250. Online ahead of print.

ABSTRACT

Platelets endocytose many molecules from their environment. However, this process of pinocytosis in platelets is poorly understood. Key endocytic regulators such as dynamin, clathrin, CDC42 and Arf6 are expressed in platelets but their roles in pinocytosis is not known. Stimulated platelets form two subpopulations of pro-aggregatory and procoagulant platelets. The effect of stimulation on pinocytosis is also poorly understood. In this study, washed human platelets were treated with a range of endocytosis inhibitors and stimulated using different activators. The rate of pinocytosis was assessed using pHrodo green, a pH-sensitive 10 kDa dextran. In unstimulated platelets, pHrodo fluorescence increased over time and accumulated as intracellular puncta indicating constituently active pinocytosis. Stimulated platelets (both pro-aggregatory and procoagulant) had an elevated pinocytosis rate compared to unstimulated platelets. Dynamin inhibition blocked pinocytosis in unstimulated, pro-aggregatory and procoagulant platelets indicating that most platelet pinocytosis is dynamin dependent. Although pinocytosis was clathrin-independent in unstimulated and procoagulant populations, clathrin partially contributed to pinocytosis in pro-aggregatory platelets.

PMID:38870846 | DOI:10.1016/j.bbrc.2024.150250

Influence of point mutations on PR65 conformational adaptability: Insights from molecular simulations and nanoaperture optical tweezers

Fri, 31/05/2024 - 11:00

Sci Adv. 2024 May 31;10(22):eadn2208. doi: 10.1126/sciadv.adn2208. Epub 2024 May 31.

ABSTRACT

PR65 is the HEAT repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem repeat protein. Its conformational mechanics plays a crucial role in PP2A function by opening/closing substrate binding/catalysis interface. Using in silico saturation mutagenesis, we identified PR65 "hinge" residues whose substitutions could alter its conformational adaptability and thereby PP2A function, and selected six mutations that were verified to be expressed and soluble. Molecular simulations and nanoaperture optical tweezers revealed consistent results on the specific effects of the mutations on the structure and dynamics of PR65. Two mutants observed in simulations to stabilize extended/open conformations exhibited higher corner frequencies and lower translational scattering in experiments, indicating a shift toward extended conformations, whereas another displayed the opposite features, confirmed by both simulations and experiments. The study highlights the power of single-molecule nanoaperture-based tweezers integrated with in silico approaches for exploring the effect of mutations on protein structure and dynamics.

PMID:38820156 | DOI:10.1126/sciadv.adn2208

Investigating the Interactions of the Cucumber Mosaic Virus 2b Protein with the Viral 1a Replicase Component and the Cellular RNA Silencing Factor Argonaute 1

Sat, 25/05/2024 - 11:00

Viruses. 2024 Apr 25;16(5):676. doi: 10.3390/v16050676.

ABSTRACT

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

PMID:38793558 | DOI:10.3390/v16050676

Application of the Cellular Thermal Shift Assay (CETSA) to validate drug target engagement in platelets

Mon, 20/05/2024 - 11:00

Platelets. 2024 Dec;35(1):2354833. doi: 10.1080/09537104.2024.2354833. Epub 2024 May 20.

ABSTRACT

Small molecule drugs play a major role in the study of human platelets. Effective action of a drug requires it to bind to one or more targets within the platelet (target engagement). However, although in vitro assays with isolated proteins can be used to determine drug affinity to these targets, additional factors affect target engagement and its consequences in an intact platelet, including plasma membrane permeability, intracellular metabolism or compartmentalization, and level of target expression. Mechanistic interpretation of the effect of drugs on platelet activity requires comprehensive investigation of drug binding in the proper cellular context, i.e. in intact platelets. The Cellular Thermal Shift Assay (CETSA) is a valuable method to investigate target engagement within complex cellular environments. The assay is based on the principle that drug binding to a target protein increases that protein's thermal stability. In this technical report, we describe the application of CETSA to platelets. We highlight CETSA as a quick and informative technique for confirming the direct binding of drugs to platelet protein targets, providing a platform for understanding the mechanism of action of drugs in platelets, and which will be a valuable tool for investigating platelet signaling and function.

PMID:38767506 | DOI:10.1080/09537104.2024.2354833

Targeting the <em>Plasmodium falciparum</em> UCHL3 ubiquitin hydrolase using chemically constrained peptides

Mon, 13/05/2024 - 11:00

Proc Natl Acad Sci U S A. 2024 May 21;121(21):e2322923121. doi: 10.1073/pnas.2322923121. Epub 2024 May 13.

ABSTRACT

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

PMID:38739798 | DOI:10.1073/pnas.2322923121

In the Murine and Bovine Maternal Mammary Gland Signal Transducer and Activator of Transcription 3 is Activated in Clusters of Epithelial Cells around the Day of Birth

Thu, 09/05/2024 - 11:00

J Mammary Gland Biol Neoplasia. 2024 May 9;29(1):10. doi: 10.1007/s10911-024-09561-5.

ABSTRACT

Signal transducers and activators of transcription (STAT) proteins regulate mammary development. Here we investigate the expression of phosphorylated STAT3 (pSTAT3) in the mouse and cow around the day of birth. We present localised colocation analysis, applicable to other mammary studies requiring identification of spatially congregated events. We demonstrate that pSTAT3-positive events are multifocally clustered in a non-random and statistically significant fashion. Arginase-1 expressing cells, consistent with macrophages, exhibit distinct clustering within the periparturient mammary gland. These findings represent a new facet of mammary STAT3 biology, and point to the presence of mammary sub-microenvironments.

PMID:38722417 | DOI:10.1007/s10911-024-09561-5

Proactive vaccination using multiviral Quartet Nanocages to elicit broad anti-coronavirus responses

Mon, 06/05/2024 - 11:00

Nat Nanotechnol. 2024 May 6. doi: 10.1038/s41565-024-01655-9. Online ahead of print.

ABSTRACT

Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle's branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 'Kraken' RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection.

PMID:38710880 | DOI:10.1038/s41565-024-01655-9

Coordinating activation of endo-lysosomal two-pore channels and TRP mucolipins

Wed, 10/04/2024 - 11:00

J Physiol. 2024 Apr 10. doi: 10.1113/JP283829. Online ahead of print.

ABSTRACT

Two-pore channels and TRP mucolipins are ubiquitous endo-lysosomal cation channels of pathophysiological relevance. Both are Ca2+-permeable and regulated by phosphoinositides, principally PI(3,5)P2. Accumulating evidence has uncovered synergistic channel activation by PI(3,5)P2 and endogenous metabolites such as the Ca2+ mobilizing messenger NAADP, synthetic agonists including approved drugs and physical cues such as voltage and osmotic pressure. Here, we provide an overview of this coordination.

PMID:38598430 | DOI:10.1113/JP283829

Intracellular pathways of calcitonin gene-related peptide-induced relaxation of human coronary arteries: A key role for Gβγ subunit instead of cAMP

Sun, 07/04/2024 - 11:00

Br J Pharmacol. 2024 Apr 7. doi: 10.1111/bph.16372. Online ahead of print.

ABSTRACT

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) is a potent vasodilator. While its signalling is assumed to be mediated via increases in cAMP, this study focused on elucidating the actual intracellular signalling pathways involved in CGRP-induced relaxation of human isolated coronary arteries (HCA).

EXPERIMENTAL APPROACH: HCA were obtained from heart valve donors (27 M, 25 F, age 54 ± 2 years). Concentration-response curves to human α-CGRP or forskolin were constructed in HCA segments, incubated with different inhibitors of intracellular signalling pathways, and intracellular cAMP levels were measured with and without stimulation.

RESULTS: Adenylyl cyclase (AC) inhibitors SQ22536 + DDA and MDL-12330A, and PKA inhibitors Rp-8-Br-cAMPs and H89, did not inhibit CGRP-induced relaxation of HCA, nor did the guanylyl cyclase inhibitor ODQ, PKG inhibitor KT5823, EPAC1/2 inhibitor ESI09, potassium channel blockers TRAM-34 + apamin, iberiotoxin or glibenclamide, or the Gαq inhibitor YM-254890. Phosphodiesterase inhibitors induced a concentration-dependent decrease in the response to KCl but did not potentiate relaxation to CGRP. Relaxation to forskolin was not blocked by PKA or AC inhibitors, although AC inhibitors significantly inhibited the increase in cAMP. Inhibition of Gβγ subunits using gallein significantly inhibited the relaxation to CGRP in human coronary arteries.

CONCLUSION: While CGRP signalling is generally assumed to act via cAMP, the CGRP-induced vasodilation in HCA was not inhibited by targeting this intracellular signalling pathway at different levels. Instead, inhibition of Gβγ subunits did inhibit the relaxation to CGRP, suggesting a different mechanism of CGRP-induced relaxation than generally believed.

PMID:38583945 | DOI:10.1111/bph.16372

Comprehensive machine learning boosts structure-based virtual screening for PARP1 inhibitors

Sat, 06/04/2024 - 11:00

J Cheminform. 2024 Apr 7;16(1):40. doi: 10.1186/s13321-024-00832-1.

ABSTRACT

Poly ADP-ribose polymerase 1 (PARP1) is an attractive therapeutic target for cancer treatment. Machine-learning scoring functions constitute a promising approach to discovering novel PARP1 inhibitors. Cutting-edge PARP1-specific machine-learning scoring functions were investigated using semi-synthetic training data from docking activity-labelled molecules: known PARP1 inhibitors, hard-to-discriminate decoys property-matched to them with generative graph neural networks and confirmed inactives. We further made test sets harder by including only molecules dissimilar to those in the training set. Comprehensive analysis of these datasets using five supervised learning algorithms, and protein-ligand fingerprints extracted from docking poses and ligand only features revealed one highly predictive scoring function. This is the PARP1-specific support vector machine-based regressor, when employing PLEC fingerprints, which achieved a high Normalized Enrichment Factor at the top 1% on the hardest test set (NEF1% = 0.588, median of 10 repetitions), and was more predictive than any other investigated scoring function, especially the classical scoring function employed as baseline.

PMID:38582911 | DOI:10.1186/s13321-024-00832-1

Manipulating Myc for reparative regeneration

Fri, 05/04/2024 - 11:00

Front Cell Dev Biol. 2024 Mar 21;12:1357589. doi: 10.3389/fcell.2024.1357589. eCollection 2024.

ABSTRACT

The Myc family of proto-oncogenes is a key node for the signal transduction of external pro-proliferative signals to the cellular processes required for development, tissue homoeostasis maintenance, and regeneration across evolution. The tight regulation of Myc synthesis and activity is essential for restricting its oncogenic potential. In this review, we highlight the central role that Myc plays in regeneration across the animal kingdom (from Cnidaria to echinoderms to Chordata) and how Myc could be employed to unlock the regenerative potential of non-regenerative tissues in humans for therapeutic purposes. Mastering the fine balance of harnessing the ability of Myc to promote transcription without triggering oncogenesis may open the door to many exciting opportunities for therapeutic development across a wide array of diseases.

PMID:38577503 | PMC:PMC10991803 | DOI:10.3389/fcell.2024.1357589