A millisecond coarse-grained simulation approach to decipher allosteric cannabinoid binding at the glycine receptor α1.

Glycine receptors (GlyR) are regulated by small-molecule binding at several allosteric sites. Cannabinoids like tetrahydrocannabinol (THC) and N-arachidonyl-ethanol-amide (AEA) potentiate the GlyR response but their mechanism of action is not fully established. By combining millisecond coarse-grained (CG) MD simulations powered by Martini 3 with backmapping to all-atom representations, we have characterized the cannabinoid-binding site(s) at the zebrafish GlyR-α1 active state with atomic resolution. Based on hundreds of thousand ligand-binding events, we find that cannabinoids bind to the transmembrane domain of the receptor at both intrasubunit and intersubunit sites. For THC, the intrasubunit binding mode predicted in simulation is in excellent agreement with recent cryo-EM structures, while intersubunit binding recapitulates in full previous mutagenesis experiments. Intriguingly, AEA is predicted to bind at the same intersubunit site despite the strikingly different chemistry. Statistical analyses of the ligand-receptor interactions highlight potentially relevant residues for GlyR potentiation, offering experimentally testable predictions. The predictions for AEA have been validated by electrophysiology recordings of rationally designed mutants. The results highlight the existence of multiple cannabinoid-binding sites for the allosteric regulation of GlyR and put forward an effective strategy for the identification and structural characterization of allosteric binding sites.

Extracellular Vesicles Induce Nuclear Factor-κB Activation and Interleukin-8 Synthesis through miRNA-191-5p Contributing to Inflammatory Processes: Potential Implications in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Extracellular vesicles (EVs) play a pivotal role in a variety of physiologically relevant processes, including lung inflammation. Recent attention has been directed toward EV-derived microRNAs (miRNAs), such as miR-191-5p, particularly in the context of inflammation. Here, we investigated the impact of miR-191-5p-enriched EVs on the activation of NF-κB and the expression of molecules associated with inflammation such as interleukin-8 (IL-8). To this aim, cells of bronchial epithelial origin, 16HBE, were transfected with miR-191-5p mimic and inhibitor and subsequently subjected to stimulations to generate EVs. Then, bronchial epithelial cells were exposed to the obtained EVs to evaluate the activation of NF-κB and IL-8 levels. Additionally, we conducted a preliminary investigation to analyze the expression profiles of miR-191-5p in EVs isolated from the plasma of patients diagnosed with chronic obstructive pulmonary disease (COPD). Our initial findings revealed two significant observations. First, the exposure of bronchial epithelial cells to miR-191-5p-enriched EVs activated the NF-kB signaling and increased the synthesis of IL-8. Second, we discovered the presence of miR-191-5p in peripheral blood-derived EVs from COPD patients and noted a correlation between miR-191-5p levels and inflammatory and functional parameters. Collectively, these data corroborate and further expand the proinflammatory role of EVs, with a specific emphasis on miR-191-5p as a key cargo involved in this process. Consequently, we propose a model in which miR-191-5p, carried by EVs, plays a role in airway inflammation and may contribute to the pathogenesis of COPD.

A weak coupling mechanism for the early steps of the recovery stroke of myosin VI: A free energy simulation and string method analysis.

Myosin motors use the energy of ATP to produce force and directed movement on actin by a swing of the lever-arm. ATP is hydrolysed during the off-actin re-priming transition termed recovery stroke. To provide an understanding of chemo-mechanical transduction by myosin, it is critical to determine how the reverse swing of the lever-arm and ATP hydrolysis are coupled. Previous studies concluded that the recovery stroke of myosin II is initiated by closure of the Switch II loop in the nucleotide-binding site. Recently, we proposed that the recovery stroke of myosin VI starts with the spontaneous re-priming of the converter domain to a putative pre-transition state (PTS) intermediate that precedes Switch II closing and ATPase activation. Here, we investigate the transition from the pre-recovery, post-rigor (PR) state to PTS in myosin VI using geometric free energy simulations and the string method. First, our calculations rediscover the PTS state agnostically and show that it is accessible from PR via a low free energy transition path. Second, separate path calculations using the string method illuminate the mechanism of the PR to PTS transition with atomic resolution. In this mechanism, the initiating event is a large movement of the converter/lever-arm region that triggers rearrangements in the Relay-SH1 region and the formation of the kink in the Relay helix with no coupling to the active site. Analysis of the free-energy barriers along the path suggests that the converter-initiated mechanism is much faster than the one initiated by Switch II closure, which supports the biological relevance of PTS as a major on-pathway intermediate of the recovery stroke in myosin VI. Our analysis suggests that lever-arm re-priming and ATP hydrolysis are only weakly coupled, so that the myosin recovery stroke is initiated by thermal fluctuations and stabilised by nucleotide consumption via a ratchet-like mechanism.

The Nicotinic Acetylcholine Receptor and Its Pentameric Homologs: Toward an Allosteric Mechanism of Signal Transduction at the Atomic Level.

The nicotinic acetylcholine receptor has served, since its biochemical identification in the 1970s, as a model of an allosteric ligand-gated ion channel mediating signal transition at the synapse. In recent years, the application of X-ray crystallography and high-resolution cryo-electron microscopy, together with molecular dynamic simulations of nicotinic receptors and homologs, have opened a new era in the understanding of channel gating by the neurotransmitter. They reveal, at atomic resolution, the diversity and flexibility of the multiple ligand-binding sites, including recently discovered allosteric modulatory sites distinct from the neurotransmitter orthosteric site, and the conformational dynamics of the activation process as a molecular switch linking these multiple sites. The model emerging from these studies paves the way for a new pharmacology based, first, upon the occurrence of an original mode of indirect allosteric modulation, distinct from a steric competition for a single and rigid binding site, and second, the design of drugs that specifically interact with privileged conformations of the receptor such as agonists, antagonists, and desensitizers. Research on nicotinic receptors is still at the forefront of understanding the mode of action of drugs on the nervous system.

The effect of probe density coverage on the detection of oenological tannins in quartz crystal microbalance with dissipation monitoring (QCM-D) experiments.

BACKGROUND: Polyphenols are a group of compounds found in grapes, musts, and wines. Their levels are crucial for grape ripening, proper must fermentation, and final wine characteristics. Standard chemical analysis is commonly used to detect these compounds, but it is costly, time consuming, and requires specialized laboratories and operators. To address this, this study explores a functionalized acoustic sensor for detecting oenological polyphenols. RESULTS: The method involves utilizing a quartz crystal microbalance with dissipation monitoring (QCM-D) to detect the target analyte by using a gelatin-based probe layer. The sensor is functionalized by optimizing the probe coverage density to maximize its performance. This is achieved by using 12-mercaptododecanoic acid (12-MCA) to immobilize the probe onto the gold sensor surface, and dithiothreitol (DTT) as a reducing and competitive binding agent. The concentration of 12-MCA and DTT in the solutions is varied to control the probe density. QCM-D measurements demonstrate that the probe density can be effectively adjusted using this approach, ranging from 0.2 × 1013 to 2 × 1013 molecules cm-2 . This study also investigates the interaction between the probe and tannins, confirming the ability of the sensor to detect them. Interestingly, the lower probe coverage achieves higher detection signals when normalized to probe immobilization signals. Moreover, significant changes in mechanical properties of the functionalization layer are observed after the interaction with samples. CONCLUSION: The combination of QCM-D with gelatin functionalization holds great promise for future applications in the wine industry. It offers real-time monitoring capabilities, requires minimal sample preparation, and provides high sensitivity for quality control purposes. © 2024 Society of Chemical Industry.

Structural mechanisms of α7 nicotinic receptor allosteric modulation and activation.

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that plays an important role in cholinergic signaling throughout the nervous system. Its unique physiological characteristics and implications in neurological disorders and inflammation make it a promising but challenging therapeutic target. Positive allosteric modulators overcome limitations of traditional α7 agonists, but their potentiation mechanisms remain unclear. Here, we present high-resolution structures of α7-modulator complexes, revealing partially overlapping binding sites but varying conformational states. Structure-guided functional and computational tests suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. We extend the study using a time-resolved cryoelectron microscopy (cryo-EM) approach to reveal asymmetric state transitions for this homomeric channel and also find that a modulator with allosteric agonist activity exploits a distinct channel-gating mechanism. These results define mechanisms of α7 allosteric modulation and activation with implications across the pentameric receptor superfamily.

Chronic Rapamycin administration via drinking water mitigates the pathological phenotype in a Krabbe disease mouse model through autophagy activation.

Krabbe disease (KD) is a rare disorder arising from the deficiency of the lysosomal enzyme galactosylceramidase (GALC), leading to the accumulation of the cytotoxic metabolite psychosine (PSY) in the nervous system. This accumulation triggers demyelination and neurodegeneration, and despite ongoing research, the underlying pathogenic mechanisms remain incompletely understood, with no cure currently available. Previous studies from our lab revealed the involvement of autophagy dysfunctions in KD pathogenesis, showcasing p62-tagged protein aggregates in the brains of KD mice and heightened p62 levels in the KD sciatic nerve. We also demonstrated that the autophagy inducer Rapamycin (RAPA) can partially reinstate the wild type (WT) phenotype in KD primary cells by decreasing the number of p62 aggregates. In this study, we tested RAPA in the Twitcher (TWI) mouse, a spontaneous KD mouse model. We administered the drug ad libitum via drinking water (15 mg/L) starting from post-natal day (PND) 21-23. We longitudinally monitored the mouse motor performance through grip strength and rotarod tests, and a set of biochemical parameters related to the KD pathogenesis (i.e. autophagy markers expression, PSY accumulation, astrogliosis and myelination). Our findings demonstrate that RAPA significantly enhances motor functions at specific treatment time points and reduces astrogliosis in TWI brain, spinal cord, and sciatic nerves. Utilizing western blot and immunohistochemistry, we observed a decrease in p62 aggregates in TWI nervous tissues, corroborating our earlier in-vitro results. Moreover, RAPA treatment partially removes PSY in the spinal cord. In conclusion, our results advocate for considering RAPA as a supportive therapy for KD. Notably, as RAPA is already available in pharmaceutical formulations for clinical use, its potential for KD treatment can be rapidly evaluated in clinical trials.

Alchemical Free Energy Calculations on Membrane-Associated Proteins.

Membrane proteins have diverse functions within cells and are well-established drug targets. The advances in membrane protein structural biology have revealed drug and lipid binding sites on membrane proteins, while computational methods such as molecular simulations can resolve the thermodynamic basis of these interactions. Particularly, alchemical free energy calculations have shown promise in the calculation of reliable and reproducible binding free energies of protein-ligand and protein-lipid complexes in membrane-associated systems. In this review, we present an overview of representative alchemical free energy studies on G-protein-coupled receptors, ion channels, transporters as well as protein-lipid interactions, with emphasis on best practices and critical aspects of running these simulations. Additionally, we analyze challenges and successes when running alchemical free energy calculations on membrane-associated proteins. Finally, we highlight the value of alchemical free energy calculations calculations in drug discovery and their applicability in the pharmaceutical industry.

Untargeted Lipidomic Approach for Studying Different Nervous System Tissues of the Murine Model of Krabbe Disease.

Krabbe disease is a rare neurodegenerative disease with an autosomal recessive character caused by a mutation in the GALC gene. The mutation leads to an accumulation of psychosine and a subsequent degeneration of oligodendrocytes and Schwann cells. Psychosine is the main biomarker of the disease. The Twitcher mouse is the most commonly used animal model to study Krabbe disease. Although there are many references to this model in the literature, the lipidomic study of nervous system tissues in the Twitcher model has received little attention. This study focuses on the comparison of the lipid profiles of four nervous system tissues (brain, cerebellum, spinal cord, and sciatic nerve) in the Twitcher mouse compared to the wild-type mouse. Altogether, approximately 230 molecular species belonging to 19 lipid classes were annotated and quantified. A comparison at the levels of class, molecular species, and lipid building blocks showed significant differences between the two groups, particularly in the sciatic nerve. The in-depth study of the lipid phenotype made it possible to hypothesize the genes and enzymes involved in the changes. The integration of metabolic data with genetic data may be useful from a systems biology perspective to gain a better understanding of the molecular basis of the disease.

ChemFlow_py: a flexible toolkit for docking and rescoring.

The design of accurate virtual screening tools is an open challenge in drug discovery. Several structure-based methods have been developed at different levels of approximation. Among them, molecular docking is an established technique with high efficiency, but typically low accuracy. Moreover, docking performances are known to be target-dependent, which makes the choice of the docking program and corresponding scoring function critical when approaching a new protein target. To compare the performances of different docking protocols, we developed ChemFlow_py, an automated tool to perform docking and rescoring. Using four protein systems extracted from DUD-E with 100 known active compounds and 3000 decoys per target, we compared the performances of several rescoring strategies including consensus scoring. We found that the average docking results can be improved by consensus ranking, which emphasizes the relevance of consensus scoring when little or no chemical information is available for a given target. ChemFlow_py is a free toolkit to optimize the performances of virtual high-throughput screening (vHTS). The software is publicly available at https://github.com/IFMlab/ChemFlow_py .

Mode Characterization and Sensitivity Evaluation of a Surface Acoustic Wave (SAW) Resonator Biosensor: Application to the Glial-Fibrillary-Acidic-Protein (GFAP) Biomarker Detection.

Biosensors based on surface acoustic waves (SAWs) offer unique advantages due to their high sensitivity, real-time response capability, and label-free detection. The typical SAW modes are the Rayleigh mode and the shear-horizontal mode. Both present pros and cons for biosensing applications and generally need different substrates and device geometries to be efficiently generated. This study investigates and characterizes SAW resonator biosensors on lithium niobate in terms of modes generated and biosensing performance. It reveals the simultaneous presence of two typical SAW modes, the first around 1.6 GHz and the second around 1.9 GHz, differently polarized and clearly separated in frequency, which we refer to as slow and fast modes. The two modes are studied by numerical simulations and biosensing experiments with the glial-fibrillary-acidic-protein (GFAP) biomarker. The slow mode is generally more sensitive to changes in surface properties, such as temperature and mass changes, by a factor of about 1.4 with respect to the fast mode.

Tanshinone IIA and Cryptotanshinone Counteract Inflammation by Regulating Gene and miRNA Expression in Human SGBS Adipocytes.

Inflammation of the adipose tissue contributes to the onset and progression of several chronic obesity-related diseases. The two most important lipophilic diterpenoid compounds found in the root of Salvia milthorrhiza Bunge (also called Danshen), tanshinone IIA (TIIA) and cryptotanshinone (CRY), have many favorable pharmacological effects. However, their roles in obesity-associated adipocyte inflammation and related sub-networks have not been fully elucidated. In the present study, we investigated the gene, miRNAs and protein expression profile of prototypical obesity-associated dysfunction markers in inflamed human adipocytes treated with TIIA and CRY. The results showed that TIIA and CRY prevented tumor necrosis factor (TNF)-α induced inflammatory response in adipocytes, by counter-regulating the pattern of secreted cytokines/chemokines associated with adipocyte inflammation (CCL2/MCP-1, CXCL10/IP-10, CCL5/RANTES, CXCL1/GRO-α, IL-6, IL-8, MIF and PAI-1/Serpin E1) via the modulation of gene expression (as demonstrated for CCL2/MCP-1, CXCL10/IP-10, CCL5/RANTES, CXCL1/GRO-α, and IL-8), as well as related miRNA expression (miR-126-3p, miR-223-3p, miR-124-3p, miR-155-5p, and miR-132-3p), and by attenuating monocyte recruitment. This is the first demonstration of a beneficial effect by TIIA and CRY on adipocyte dysfunction associated with obesity development and complications, offering a new outlook for the prevention and/or treatment of metabolic diseases.

A Fast and Reliable Method Based on QCM-D Instrumentation for the Screening of Nanoparticle/Blood Protein Interactions.

The interactions that nanoparticles have with blood proteins are crucial for their fate in vivo. Such interactions result in the formation of the protein corona around the nanoparticles, and studying them aids in nanoparticle optimization. Quartz crystal microbalance with dissipation monitoring (QCM-D) can be used for this study. The present work proposes a QCM-D method to study the interactions on polymeric nanoparticles with three different human blood proteins (albumin, fibrinogen and γ-globulin) by monitoring the frequency shifts of sensors immobilizing the selected proteins. Bare PEGylated and surfactant-coated poly-(D,L-lactide-co-glycolide) nanoparticles are tested. The QCM-D data are validated with DLS and UV-Vis experiments in which changes in the size and optical density of nanoparticle/protein blends are monitored. We find that the bare nanoparticles have a high affinity towards fibrinogen and γ-globulin, with measured frequency shifts around -210 Hz and -50 Hz, respectively. PEGylation greatly reduces these interactions (frequency shifts around -5 Hz and -10 Hz for fibrinogen and γ-globulin, respectively), while the surfactant appears to increase them (around -240 Hz and -100 Hz and -30 Hz for albumin). The QCM-D data are confirmed by the increase in the nanoparticle size over time (up to 3300% in surfactant-coated nanoparticles), measured by DLS in protein-incubated samples, and by the trends of the optical densities, measured by UV-Vis. The results indicate that the proposed approach is valid for studying the interactions between nanoparticles and blood proteins, and the study paves the way for a more comprehensive analysis of the whole protein corona.

Crosslinked Chitosan Nanoparticles with Muco-Adhesive Potential for Intranasal Delivery Applications.

Intranasal drug delivery is convenient and provides a high bioavailability but requires the use of mucoadhesive nanocarriers. Chitosan is a well-established polymer for mucoadhesive applications but can suffer from poor cytocompatibility and stability upon administration. In this work, we present a method to obtain stable and cytocompatible crosslinked chitosan nanoparticles. We used 2,6-pyridinedicarboxylic acid as a biocompatible crosslinker and compared the obtained particles with those prepared by ionotropic gelation using sodium tripolyphosphate. Nanoparticles were tested to evaluate the size and the surface charge, as well as their stability in storage conditions (4 °C), at the nasal cavity temperature (32 °C), and at the body temperature (37 °C). The crosslinked chitosan nanoparticles showed a size around 150 nm and a surface charge of 10.3 mV ± 0.9 mV, both compatible with the intranasal drug administration. Size and surface charge parameters did not significantly vary over time, indicating the good stability of these nanoparticles. We finally tested their cytocompatibility in vitro using SHSY5Y human neuroblastoma and RPMI 2650 human nasal epithelial cells, with positive results. In conclusion, the proposed synthetic system shows an interesting potential as a drug carrier for intranasal delivery.

Accelerated epithelial layer healing induced by tactile anisotropy in surface topography.

Mammalian cells respond to tactile cues from topographic elements presented by the substrate. Among these, anisotropic features distributed in an ordered manner give directionality. In the extracellular matrix, this ordering is embedded in a noisy environment altering the contact guidance effect. To date, it is unclear how cells respond to topographical signals in a noisy environment. Here, using rationally designed substrates, we report morphotaxis, a guidance mechanism enabling fibroblasts and epithelial cells to move along gradients of topographic order distortion. Isolated cells and cell ensembles perform morphotaxis in response to gradients of different strength and directionality, with mature epithelia integrating variations of topographic order over hundreds of micrometers. The level of topographic order controls cell cycle progression, locally delaying or promoting cell proliferation. In mature epithelia, the combination of morphotaxis and noise-dependent distributed proliferation provides a strategy to enhance wound healing as confirmed by a mathematical model capturing key elements of the process.

Mechanotransduction Impairment in Primary Fibroblast Model of Krabbe Disease.

Krabbe disease (KD) is a genetic disorder caused by the absence of the galactosylceramidase (GALC) functional enzyme. No cure is currently available. Here, we investigate the mechanotransduction process in primary fibroblasts collected from the twitcher mouse, a natural KD murine model. Thanks to mechanotransduction, cells can sense their environment and convert external mechanical stimuli into biochemical signals that result in intracellular changes. In GALC-deficient fibroblasts, we show that focal adhesions (FAs), the protein clusters necessary to adhere and migrate, are increased, and that single-cell migration and wound healing are impaired. We also investigate the involvement of the autophagic process in this framework. We show a dysregulation in the FA turnover: here, the treatment with the autophagy activator rapamycin boosts cell migration and improves the clearance of FAs in GALC-deficient fibroblasts. We propose mechanosensing impairment as a novel potential pathological mechanism in twitcher fibroblasts, and more in general in Krabbe disease.

Wordom update 2: A user-friendly program for the analysis of molecular structures and conformational ensembles.

We present the second update of Wordom, a user-friendly and efficient program for manipulation and analysis of conformational ensembles from molecular simulations. The actual update expands some of the existing modules and adds 21 new modules to the update 1 published in 2011. The new adds can be divided into three sets that: 1) analyze atomic fluctuations and structural communication; 2) explore ion-channel conformational dynamics and ionic translocation; and 3) compute geometrical indices of structural deformation. Set 1 serves to compute correlations of motions, find geometrically stable domains, identify a dynamically invariant core, find changes in domain-domain separation and mutual orientation, perform wavelet analysis of large-scale simulations, process the output of principal component analysis of atomic fluctuations, perform functional mode analysis, infer regions of mechanical rigidity, analyze overall fluctuations, and perform the perturbation response scanning. Set 2 includes modules specific for ion channels, which serve to monitor the pore radius as well as water or ion fluxes, and measure functional collective motions like receptor twisting or tilting angles. Finally, set 3 includes tools to monitor structural deformations by computing angles, perimeter, area, volume, ß-sheet curvature, radial distribution function, and center of mass. The ring perception module is also included, helpful to monitor supramolecular self-assemblies. This update places Wordom among the most suitable, complete, user-friendly, and efficient software for the analysis of biomolecular simulations. The source code of Wordom and the relative documentation are available under the GNU general public license at http://wordom.sf.net.

Stabilized Reversed Polymeric Micelles as Nanovector for Hydrophilic Compounds.

Small hydrophilic drugs are widely used for systemic administration, but they suffer from poor absorption and fast clearance. Their nanoencapsulation can improve biodistribution, targeted delivery, and pharmaceutical efficacy. Hydrophilics are effectively encapsulated in compartmented particles, such as liposomes or extracellular vesicles, which are biocompatible but poorly customizable. Polymeric vectors can form compartmental structures, also being functionalizable. Here, we report a system composed of polymeric stabilized reversed micelles for hydrophilic drugs encapsulation. We optimized the preparation procedure, and calculated the critical micellar concentration. Then, we developed a strategy for stabilization that improves micelle stability upon dilution. We tested the drug loading and delivery capabilities with creatine as a drug molecule. Prepared stabilized reversed micelles had a size of around 130 nm and a negative z-potential around -16 mV, making them functional as a drug carrier. The creatine cargo increased micelle size and depended on the loading conditions. The higher amount of loaded creatine was around 60 µg/mg of particles. Delivery tests indicated full release within three days in micelles with the lower cargo, while higher loadings can provide a sustained release for longer times. Obtained results are interesting and encouraging to test the same system with different drug cargoes.

Illumination of a progressive allosteric mechanism mediating the glycine receptor activation.

Pentameric ligand-gated ion channel mediate signal transduction at chemical synapses by transiting between resting and open states upon neurotransmitter binding. Here, we investigate the gating mechanism of the glycine receptor fluorescently labeled at the extracellular-transmembrane interface by voltage-clamp fluorometry (VCF). Fluorescence reports a glycine-elicited conformational change that precedes pore opening. Low concentrations of glycine, partial agonists or specific mixtures of glycine and strychnine trigger the full fluorescence signal while weakly activating the channel. Molecular dynamic simulations of a partial agonist bound-closed Cryo-EM structure show a highly dynamic nature: a marked structural flexibility at both the extracellular-transmembrane interface and the orthosteric site, generating docking properties that recapitulate VCF data. This work illuminates a progressive propagating transition towards channel opening, highlighting structural plasticity within the mechanism of action of allosteric effectors.

ChemFlow─From 2D Chemical Libraries to Protein-Ligand Binding Free Energies.

The accurate prediction of protein-ligand binding affinities is a fundamental problem for the rational design of new drug entities. Current computational approaches are either too expensive or inaccurate to be effectively used in virtual high-throughput screening campaigns. In addition, the most sophisticated methods, e.g., those based on configurational sampling by molecular dynamics, require significant pre- and postprocessing to provide a final ranking, which hinders straightforward applications by nonexpert users. We present a novel computational platform named ChemFlow to bridge the gap between 2D chemical libraries and estimated protein-ligand binding affinities. The software is designed to prepare a library of compounds provided in SMILES or SDF format, dock them into the protein binding site, and rescore the poses by simplified free energy calculations. Using a data set of 626 protein-ligand complexes and GPU computing, we demonstrate that ChemFlow provides relative binding free energies with an RMSE < 2 kcal/mol at a rate of 1000 ligands per day on a midsize computer cluster. The software is publicly available at https://github.com/IFMlab/ChemFlow.