Liposomal Copermeation Assay Reveals Unexpected Membrane Interactions of Commonly Prescribed Drugs.

The permeation of small molecules across biological membranes is a crucial process that lies at the heart of life. Permeation is involved not only in the maintenance of homeostasis at the cell level but also in the absorption and biodistribution of pharmacologically active substances throughout the human body. Membranes are formed by phospholipid bilayers that represent an energy barrier for permeating molecules. Crossing this energy barrier is assumed to be a singular event, and permeation has traditionally been described as a first-order kinetic process, proportional only to the concentration gradient of the permeating substance. For a given membrane composition, permeability was believed to be a unique property dependent only on the permeating molecule itself. We provide experimental evidence that this long-held view might not be entirely correct. Liposomes were used in copermeation experiments with a fluorescent probe, where simultaneous permeation of two substances occurred over a single phospholipid bilayer. Using an assay of six commonly prescribed drugs, we have found that the presence of a copermeant can either enhance or suppress the permeation rate of the probe molecule, often more than 2-fold in each direction. This can have significant consequences for the pharmacokinetics and bioavailability of commonly prescribed drugs when used in combination and provide new insight into so-far unexplained drug-drug interactions as well as changing the perspective on how new drug candidates are evaluated and tested.

ChannelsDB 2.0: a comprehensive database of protein tunnels and pores in AlphaFold era.

ChannelsDB 2.0 is an updated database providing structural information about the position, geometry and physicochemical properties of protein channels-tunnels and pores-within deposited biomacromolecular structures from PDB and AlphaFoldDB databases. The newly deposited information originated from several sources. Firstly, we included data calculated using a popular CAVER tool to complement the data obtained using original MOLE tool for detection and analysis of protein tunnels and pores. Secondly, we added tunnels starting from cofactors within the AlphaFill database to enlarge the scope of the database to protein models based on Uniprot. This has enlarged available channel annotations ∼4.6 times as of 1 September 2023. The database stores information about geometrical features, e.g. length and radius, and physico-chemical properties based on channel-lining amino acids. The stored data are interlinked with the available UniProt mutation annotation data. ChannelsDB 2.0 provides an excellent resource for deep analysis of the role of biomacromolecular tunnels and pores. The database is available free of charge: https://channelsdb2.biodata.ceitec.cz.

Expanding the squaramide library as mycobacterial ATP synthase inhibitors: Innovative synthetic pathway and biological evaluation.

Mycobacterial ATP synthase is a validated therapeutic target for combating drug-resistant tuberculosis. Inhibition of this enzyme has been featured as an efficient strategy for the development of new antimycobacterial agents against drug-resistant pathogens. In this study, we synthesised and explored two distinct series of squaric acid analogues designed to inhibit mycobacterial ATP synthase. Among the extensive array of compounds investigated, members of the phenyl-substituted sub-library emerged as primary hits. To gain deeper insights into their mechanisms of action, we conducted advanced biological studies, focusing on the compounds displaying a direct binding of a nitrogen heteroatom to the phenyl ring, resulting in the highest potency. Our investigations into spontaneous mutants led to the validation of a single point mutation within the atpB gene (Rv1304), responsible for encoding the ATP synthase subunit a. This genetic alteration sheds light on the molecular basis of resistance to squaramides. Furthermore, we explored the possibility of synergy between squaramides and the reference drug clofazimine using a checkerboard assay, highlighting the promising avenue for enhancing the effectiveness of existing treatments through combined therapeutic approaches. This study contributes to the expansion of investigating squaramides as promising drug candidates in the ongoing battle against drug-resistant tuberculosis.

Small change - big consequence: The impact of C15-C16 double bond in a D­ring of estrone on estrogen receptor activity.

Estrogen receptor alpha (ER) is a key biomarker for breast cancer, and the presence or absence of ER in breast and other hormone-dependent cancers decides treatment regimens and patient prognosis. ER is activated after ligand binding - typically by steroid. 2682 steroid compounds were used in a molecular docking study to identify novel ligands for ER and to predict compounds that may show anticancer activity. The effect of the most promising compounds was determined by a novel luciferase reporter assay. Two compounds, 7 and 12, showing ER inhibitory activity comparable to clinical inhibitors such as tamoxifen or fulvestrant were selected. We propose that the inhibitory effect of compounds 7 and 12 on ER is related to the presence of a double bond in their D-ring, which may protect against ER activation by reducing the electron density of the keto group, or may undergo metabolism leading to an active compound. Western blotting revealed that compound 12 decreased the level of ER in the breast cancer cell line MCF7, which was associated with reduced expression of both isoforms of the progesterone receptor, a well-known downstream target of ER. However, compound 12 has a different mechanism of action from fulvestrant. Furthermore, we found that compound 12 interferes with mitochondrial functions, probably by disrupting the electron transport chain, leading to induction of the intrinsic apoptotic pathway even in ER-negative breast cancer cells. In conclusion, the combination of computational and experimental methods shown here represents a rapid approach to determine the activity of compounds towards ER. Our data will not only contribute to research focused on the regulation of ER activity but may also be useful for the further development of novel steroid receptor-targeted drugs applicable in clinical practice.

αCharges: partial atomic charges for AlphaFold structures in high quality.

The AlphaFold2 prediction algorithm opened up the possibility of exploring proteins' structural space at an unprecedented scale. Currently, >200 million protein structures predicted by this approach are deposited in AlphaFoldDB, covering entire proteomes of multiple organisms, including humans. Predicted structures are, however, stored without detailed functional annotations describing their chemical behaviour. Partial atomic charges, which map electron distribution over a molecule and provide a clue to its chemical reactivity, are an important example of such data. We introduce the web application αCharges: a tool for the quick calculation of partial atomic charges for protein structures from AlphaFoldDB. The charges are calculated by the recent empirical method SQE+qp, parameterised for this class of molecules using robust quantum mechanics charges (B3LYP/6-31G*/NPA) on PROPKA3 protonated structures. The computed partial atomic charges can be downloaded in common data formats or visualised via the powerful Mol* viewer. The αCharges application is freely available at https://alphacharges.ncbr.muni.cz with no login requirement.

Mol* Volumes and Segmentations: visualization and interpretation of cell imaging data alongside macromolecular structure data and biological annotations.

Segmentation helps interpret imaging data in a biological context. With the development of powerful tools for automated segmentation, public repositories for imaging data have added support for sharing and visualizing segmentations, creating the need for interactive web-based visualization of 3D volume segmentations. To address the ongoing challenge of integrating and visualizing multimodal data, we developed Mol* Volumes and Segmentations (Mol*VS), which enables the interactive, web-based visualization of cellular imaging data supported by macromolecular data and biological annotations. Mol*VS is fully integrated into Mol* Viewer, which is already used for visualization by several public repositories. All EMDB and EMPIAR entries with segmentation datasets are accessible via Mol*VS, which supports the visualization of data from a wide range of electron and light microscopy experiments. Additionally, users can run a local instance of Mol*VS to visualize and share custom datasets in generic or application-specific formats including volumes in .ccp4, .mrc, and .map, and segmentations in EMDB-SFF .hff, Amira .am, iMod .mod, and Segger .seg. Mol*VS is open source and freely available at https://molstarvolseg.ncbr.muni.cz/.

Computational Prodrug Design Methodology for Liposome Formulability Enhancement of Small-Molecule APIs.

Encapsulation into liposomes is a formulation strategy that can improve efficacy and reduce side effects of active pharmaceutical ingredients (APIs) that exhibit poor biodistribution or pharmacokinetics when administered alone. However, many APIs are unsuitable for liposomal formulations intended for parenteral administration due to their inherent physicochemical properties─lipid bilayer permeability and water-lipid equilibrium partitioning coefficient. Too high permeability results in premature leakage from liposomes, while too low permeability means the API is not able to pass across biological barriers. There are several options for solving this issue: (i) change of the lipid bilayer composition, (ii) addition of a permeability enhancer, or (iii) modification of the chemical structure of the API to design a prodrug. The latter approach was taken in the present work, and the effect of small changes in the molecular structure of the API on its permeation rate across a lipidic bilayer was systematically explored utilizing computer simulations. An in silico methodology for prodrug design based on the COSMOperm approach has been proposed and applied to four APIs (abiraterone, cytarabine, 5-fluorouracil, and paliperidone). It is shown that the addition of aliphatic hydrocarbon chains via ester or amide bonds can render the molecule more lipophilic and increase its permeability by approximately 1 order of magnitude for each 2 carbon atoms added, while the formation of fructose adducts can provide a more hydrophilic character to the molecule and reduce its lipid partitioning. While partitioning was found to depend only on the size and type of the added group, permeability was found to depend also on the added group location. Overall, it has been shown that both permeability and lipid partitioning coefficient can be systematically shifted into the desired liposome formulability window by appropriate group contributions to the parental drug. This can significantly increase the portfolio of APIs for which liposome or lipid nanoparticle formulations become feasible.

Novel heterocyclic hydroxamates as inhibitors of the mycobacterial zinc metalloprotease Zmp1 to probe its mechanism of function.

Mycobacterial zinc metalloprotease-1 (Zmp1) is an essential enzyme for intracellular survival and pathogenicity of Mycobacterium tuberculosis. However, the exact mechanism of function of this enzyme remains unclear. This paper examines the effect of novel organic molecules on the inhibition of Zmp1. We followed our previous results and synthesised three libraries of new hydroxamates. All compounds were studied for their inhibitory properties towards a recombinant Zmp1 from Mycobacterium tuberculosis by MALDI-TOF MS. Furthermore, a macrophage infection assay was performed to evaluate intracellular antimycobacterial activity. In the whole-cell assay, no direct activity of synthesised heterocyclic hydroxamates was observed against Mycobacterium tuberculosis and Mycobacterium bovis. No acute cellular toxicity was observed against the murine RAW 264.7 macrophage cell line and human MRC-5 lung fibroblast cell line. However, thiazolidinediones 2 showed the dose-dependent inhibition of intracellular survival of Mycobacterium tuberculosis H37Ra. The inhibition was structure-dependent, with the most active derivative 2f inducing an 83.2% reduction of bacterial survival within the macrophage host cell. The promising biological activity confirmed thiazolidinediones 2 as Zmp1 inhibitors that can be used as tool compounds for further exploration of the role of Zmp1 for in vivo pathogenicity. In the long run, thiazolidinediones 2 show the potential to act as a scaffold for Zmp1 inhibitors to target intracellular Mtb as a novel tuberculosis treatment strategy.

OverProt: secondary structure consensus for protein families.

SUMMARY: Every protein family has a set of characteristic secondary structures. However, due to individual variations, a single structure is not enough to represent the whole family. OverProt can create a secondary structure consensus, showing the general fold of the family as well as its variation. Our server provides precomputed results for all CATH superfamilies and user-defined computations, visualized by an interactive viewer, which shows the secondary structure element type, length, frequency of occurrence, spatial variability and ß-connectivity. AVAILABILITY AND IMPLEMENTATION: OverProt Server is freely available at https://overprot.ncbr.muni.cz. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Antitumour drugs targeting tau R3 VQIVYK and Cys322 prevent seeding of endogenous tau aggregates by exogenous seeds.

Emerging experimental evidence suggests tau pathology spreads between neuroanatomically connected brain regions in a prion-like manner in Alzheimer's disease (AD). Tau seeding, the ability of prion-like tau to recruit and misfold naïve tau to generate new seeds, is detected early in human AD brains before the development of major tau pathology. Many antitumour drugs have been reported to confer protection against neurodegeneration, supporting the repurposing of approved and experimental or investigational oncology drugs for AD therapy. In this study, we evaluated whether antitumour drugs that abrogate the generation of seed-competent aggregates of tau Repeat 3 (R3) domain peptides can prevent tau seeding and toxicity in Tau-RD P301S FRET Biosensor cells and Caenorhabditis elegans. We demonstrate that drugs that interact with the N-terminal VQIVYK or the C-terminal region housing the Cys322 prevent R3 dimerisation, abolishing the generation of prion-like R3 seeds. Preformed R3 seeds (fibrils) capped with, or R3 seeds formed in the presence of VQIVYK- or Cys322-targeting drugs have a reduced potency to cause aggregation of naïve tau in biosensor cells and protect worms from aggregate toxicity. These findings indicate that VQIVYK- or Cys322-targeting drugs may act as prophylactic agents against tau seeding.

Optimized SQE atomic charges for peptides accessible via a web application.

BACKGROUND: Partial atomic charges find many applications in computational chemistry, chemoinformatics, bioinformatics, and nanoscience. Currently, frequently used methods for charge calculation are the Electronegativity Equalization Method (EEM), Charge Equilibration method (QEq), and Extended QEq (EQeq). They all are fast, even for large molecules, but require empirical parameters. However, even these advanced methods have limitations-e.g., their application for peptides, proteins, and other macromolecules is problematic. An empirical charge calculation method that is promising for peptides and other macromolecular systems is the Split-charge Equilibration method (SQE) and its extension SQE+q0. Unfortunately, only one parameter set is available for these methods, and their implementation is not easily accessible. RESULTS: In this article, we present for the first time an optimized guided minimization method (optGM) for the fast parameterization of empirical charge calculation methods and compare it with the currently available guided minimization (GDMIN) method. Then, we introduce a further extension to SQE, SQE+qp, adapted for peptide datasets, and compare it with the common approaches EEM, QEq EQeq, SQE, and SQE+q0. Finally, we integrate SQE and SQE+qp into the web application Atomic Charge Calculator II (ACC II), including several parameter sets. CONCLUSION: The main contribution of the article is that it makes SQE methods with their parameters accessible to the users via the ACC II web application ( https://acc2.ncbr.muni.cz ) and also via a command-line application. Furthermore, our improvement, SQE+qp, provides an excellent solution for peptide datasets. Additionally, optGM provides comparable parameters to GDMIN in a markedly shorter time. Therefore, optGM allows us to perform parameterizations for charge calculation methods with more parameters (e.g., SQE and its extensions) using large datasets.

2DProts: database of family-wide protein secondary structure diagrams.

SUMMARY: Secondary structures provide a deep insight into the protein architecture. They can serve for comparison between individual protein family members. The most straightforward way how to deal with protein secondary structure is its visualization using 2D diagrams. Several software tools for the generation of 2D diagrams were developed. Unfortunately, they create 2D diagrams based on only a single protein. Therefore, 2D diagrams of two proteins from one family markedly differ. For this reason, we developed the 2DProts database, which contains secondary structure 2D diagrams for all domains from the CATH and all proteins from PDB databases. These 2D diagrams are generated based on a whole protein family, and they also consider information about the 3D arrangement of secondary structure elements. Moreover, 2DProts database contains multiple 2D diagrams, which provide an overview of a whole protein family's secondary structures. 2DProts is updated weekly and is integrated into CATH. AVAILABILITY AND IMPLEMENTATION: Freely accessible at https://2dprots.ncbr.muni.cz. The web interface was implemented in JavaScript. The database was implemented in Python. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Uncovering of cytochrome P450 anatomy by SecStrAnnotator.

Protein structural families are groups of homologous proteins defined by the organization of secondary structure elements (SSEs). Nowadays, many families contain vast numbers of structures, and the SSEs can help to orient within them. Communities around specific protein families have even developed specialized SSE annotations, always assigning the same name to the equivalent SSEs in homologous proteins. A detailed analysis of the groups of equivalent SSEs provides an overview of the studied family and enriches the analysis of any particular protein at hand. We developed a workflow for the analysis of the secondary structure anatomy of a protein family. We applied this analysis to the model family of cytochromes P450 (CYPs)-a family of important biotransformation enzymes with a community-wide used SSE annotation. We report the occurrence, typical length and amino acid sequence for the equivalent SSE groups, the conservation/variability of these properties and relationship to the substrate recognition sites. We also suggest a generic residue numbering scheme for the CYP family. Comparing the bacterial and eukaryotic part of the family highlights the significant differences and reveals a well-known anomalous group of bacterial CYPs with some typically eukaryotic features. Our workflow for SSE annotation for CYP and other families can be freely used at address https://sestra.ncbr.muni.cz .

Mol* Viewer: modern web app for 3D visualization and analysis of large biomolecular structures.

Large biomolecular structures are being determined experimentally on a daily basis using established techniques such as crystallography and electron microscopy. In addition, emerging integrative or hybrid methods (I/HM) are producing structural models of huge macromolecular machines and assemblies, sometimes containing 100s of millions of non-hydrogen atoms. The performance requirements for visualization and analysis tools delivering these data are increasing rapidly. Significant progress in developing online, web-native three-dimensional (3D) visualization tools was previously accomplished with the introduction of the LiteMol suite and NGL Viewers. Thereafter, Mol* development was jointly initiated by PDBe and RCSB PDB to combine and build on the strengths of LiteMol (developed by PDBe) and NGL (developed by RCSB PDB). The web-native Mol* Viewer enables 3D visualization and streaming of macromolecular coordinate and experimental data, together with capabilities for displaying structure quality, functional, or biological context annotations. High-performance graphics and data management allows users to simultaneously visualise up to hundreds of (superimposed) protein structures, stream molecular dynamics simulation trajectories, render cell-level models, or display huge I/HM structures. It is the primary 3D structure viewer used by PDBe and RCSB PDB. It can be easily integrated into third-party services. Mol* Viewer is open source and freely available at https://molstar.org/.

High-performance macromolecular data delivery and visualization for the web. Corrigendum.

Two citations in the article by Sehnal et al. [(2020), Acta Cryst. D76, 1167-1173] are corrected.

ELIXIR and Toxicology: a community in development.

Toxicology has been an active research field for many decades, with academic, industrial and government involvement. Modern omics and computational approaches are changing the field, from merely disease-specific observational models into target-specific predictive models. Traditionally, toxicology has strong links with other fields such as biology, chemistry, pharmacology and medicine. With the rise of synthetic and new engineered materials, alongside ongoing prioritisation needs in chemical risk assessment for existing chemicals, early predictive evaluations are becoming of utmost importance to both scientific and regulatory purposes. ELIXIR is an intergovernmental organisation that brings together life science resources from across Europe. To coordinate the linkage of various life science efforts around modern predictive toxicology, the establishment of a new ELIXIR Community is seen as instrumental. In the past few years, joint efforts, building on incidental overlap, have been piloted in the context of ELIXIR. For example, the EU-ToxRisk, diXa, HeCaToS, transQST, and the nanotoxicology community have worked with the ELIXIR TeSS, Bioschemas, and Compute Platforms and activities. In 2018, a core group of interested parties wrote a proposal, outlining a sketch of what this new ELIXIR Toxicology Community would look like. A recent workshop (held September 30th to October 1st, 2020) extended this into an ELIXIR Toxicology roadmap and a shortlist of limited investment-high gain collaborations to give body to this new community. This Whitepaper outlines the results of these efforts and defines our vision of the ELIXIR Toxicology Community and how it complements other ELIXIR activities.

CATH: increased structural coverage of functional space.

CATH (https://www.cathdb.info) identifies domains in protein structures from wwPDB and classifies these into evolutionary superfamilies, thereby providing structural and functional annotations. There are two levels: CATH-B, a daily snapshot of the latest domain structures and superfamily assignments, and CATH+, with additional derived data, such as predicted sequence domains, and functionally coherent sequence subsets (Functional Families or FunFams). The latest CATH+ release, version 4.3, significantly increases coverage of structural and sequence data, with an addition of 65,351 fully-classified domains structures (+15%), providing 500 238 structural domains, and 151 million predicted sequence domains (+59%) assigned to 5481 superfamilies. The FunFam generation pipeline has been re-engineered to cope with the increased influx of data. Three times more sequences are captured in FunFams, with a concomitant increase in functional purity, information content and structural coverage. FunFam expansion increases the structural annotations provided for experimental GO terms (+59%). We also present CATH-FunVar web-pages displaying variations in protein sequences and their proximity to known or predicted functional sites. We present two case studies (1) putative cancer drivers and (2) SARS-CoV-2 proteins. Finally, we have improved links to and from CATH including SCOP, InterPro, Aquaria and 2DProt.

High-performance macromolecular data delivery and visualization for the web.

Biomacromolecular structural data make up a vital and crucial scientific resource that has grown not only in terms of its amount but also in its size and complexity. Furthermore, these data are accompanied by large and increasing amounts of experimental data. Additionally, the macromolecular data are enriched with value-added annotations describing their biological, physicochemical and structural properties. Today, the scientific community requires fast and fully interactive web visualization to exploit this complex structural information. This article provides a survey of the available cutting-edge web services that address this challenge. Specifically, it focuses on data-delivery problems, discusses the visualization of a single structure, including experimental data and annotations, and concludes with a focus on the results of molecular-dynamics simulations and the visualization of structural ensembles.

Cytokinin fluoroprobe reveals multiple sites of cytokinin perception at plasma membrane and endoplasmic reticulum.

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.