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Background/Objectives: Microneedle(MN)-based drug delivery is one of the potential approaches to overcome the limitations of oral and hypodermic needle delivery. An in silico model has been developed for hollow microneedle (HMN)-based drug delivery in the skin and its subsequent absorption in the blood and tissue compartments in the presence of interstitial flow. The drug's reversible specific saturable binding to its receptors and the kinetics of reversible absorption across the blood and tissue compartments have been taken into account. Methods: The governing equations representing the flow of interstitial fluid, the transport of verapamil in the viable skin and the concentrations in the blood and tissue compartments are solved using combined Marker and Cell and Immersed Boundary Methods to gain a quantitative understanding of the model under consideration. Results: The viscoelastic skin is predicted to impede the transport of verapamil in the viable skin and, hence, reduce the concentrations of all forms in the blood and the tissue compartments. The findings reveal that a higher mean concentration in the viable skin is not always associated with a longer MN length. Simulations also predict that the concentrations of verapamil in the blood and bound verapamil in the tissue compartment rise with decreasing tip diameters. In contrast, the concentration of free verapamil in the tissue increases with increasing injection velocities. Conclusions: The novelty of this study includes verapamil metabolism in two-dimensional viscoelastic irregular viable skin and the nonlinear, specific, saturable, and reversible binding of verapamil in the tissue compartment. The tip diameter and the drug's injection velocity are thought to serve as regulatory parameters for the effectiveness and efficacy of MN-mediated therapy if the MN is robust enough to sustain the force needed to penetrate a wider tip into the skin. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 envelope (E) protein forms a five-helix bundle in lipid bilayers whose cation-conducting activity is associated with the inflammatory response and respiratory distress symptoms of COVID-19. E channel activity is inhibited by the drug 5-(N,N-hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid-state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. 13 C, 15 N-labeled HMA is combined with fluorinated or 13 C-labeled ETM. Conversely, fluorinated HMA is combined with 13 C, 15 N-labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA-protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid-facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift perturbation data, which showed the largest perturbation for N-terminal residues. This difference suggests that HMA has higher affinity for the protein-lipid interface than the channel pore. These results give insight into the inhibition mechanism of HMA for SARS-CoV-2 E.

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.

Introduction: The administration of antibiotics can expose the digestive microbiota of humans and animals to sub-inhibitory concentrations, potentially favouring the selection of resistant bacteria. The minimal selective concentration (MSC) is a key indicator to understand this process. The MSC is defined as the lowest concentration of an antibiotic that promotes the growth of a resistant strain over a susceptible isogenic strain. It represents the lower limit of the sub-minimal inhibitory concentration (MIC) selective window, where resistant mutants can be selected. Previous studies focused on determining the MSC under standard culture conditions, whereas our research aimed to determine the MSC in a model that approximates in vivo conditions. Methods: We investigated the MSC of oxytetracycline (OTC) in Mueller-Hinton broth (MHB) and sterilised intestinal contents (SIC) from the jejunum, caecum and rectum (faeces) of pigs, using two isogenic strains of Escherichia coli (one susceptible and one resistant to OTC). Additionally, the MIC of OTC against the susceptible strain was determined to assess the upper limit of the sub-MIC selective window. Results: Our study took a novel approach, and the results indicated that MIC and MSC values were lower in MHB than in SIC. In the latter, these values varied depending on the intestinal segment, with distal compartments exhibiting higher MIC and MSC values. Moreover, the sub-MIC selective window of OTC in SIC narrowed from the jejunum to the rectum, with a significantly closer MSC to MIC in faecal SIC. Discussion: The results suggest that OTC binds to digestive contents, reducing the fraction of free OTC. However, binding alone does not fully explain our results, and interactions between bacteria and intestinal contents may play a role. Furthermore, our findings provide initial estimates of low concentrations facilitating resistance selection in the gut. Finally, this research enhances the understanding of antimicrobial resistance selection, emphasising the intricate interplay between antibiotics and intestinal content composition in assessing the risk of resistance development in the gut. [ABSTRACT FROM AUTHOR]

IntroductionThe administration of antibiotics can expose the digestive microbiota of humans and animals to sub-inhibitory concentrations, potentially favouring the selection of resistant bacteria. The minimal selective concentration (MSC) is a key indicator to understand this process. The MSC is defined as the lowest concentration of an antibiotic that promotes the growth of a resistant strain over a susceptible isogenic strain. It represents the lower limit of the sub-minimal inhibitory concentration (MIC) selective window, where resistant mutants can be selected. Previous studies focused on determining the MSC under standard culture conditions, whereas our research aimed to determine the MSC in a model that approximates in vivo conditions.MethodsWe investigated the MSC of oxytetracycline (OTC) in Mueller-Hinton broth (MHB) and sterilised intestinal contents (SIC) from the jejunum, caecum and rectum (faeces) of pigs, using two isogenic strains of Escherichia coli (one susceptible and one resistant to OTC). Additionally, the MIC of OTC against the susceptible strain was determined to assess the upper limit of the sub-MIC selective window.ResultsOur study took a novel approach, and the results indicated that MIC and MSC values were lower in MHB than in SIC. In the latter, these values varied depending on the intestinal segment, with distal compartments exhibiting higher MIC and MSC values. Moreover, the sub-MIC selective window of OTC in SIC narrowed from the jejunum to the rectum, with a significantly closer MSC to MIC in faecal SIC.DiscussionThe results suggest that OTC binds to digestive contents, reducing the fraction of free OTC. However, binding alone does not fully explain our results, and interactions between bacteria and intestinal contents may play a role. Furthermore, our findings provide initial estimates of low concentrations facilitating resistance selection in the gut. Finally, this research enhances the understanding of antimicrobial resistance selection, emphasising the intricate interplay between antibiotics and intestinal content composition in assessing the risk of resistance development in the gut.

[Display omitted] The role of human serum albumin (HSA) in the transport of molecules predicates its involvement in the determination of drug distribution and metabolism. Optimization of ADME properties are analogous to HSA binding thus this is imperative to the drug discovery process. Currently, various in silico predictive tools exist to complement the drug discovery process, however, the prediction of possible ligand-binding sites on HSA has posed several challenges. Herein, we present a strong and deeper-than-surface case for the prediction of HSA-ligand binding sites using multi-cavity molecular descriptors by exploiting all experimentally available and crystallized HSA-bound drugs. Unlike previously proposed models found in literature, we established an in-depth correlation between the physicochemical properties of available crystallized HSA-bound drugs and different HSA binding site characteristics to precisely predict the binding sites of investigational molecules. Molecular descriptors such as the number of hydrogen bond donors (nHD), number of heteroatoms (nHet), topological polar surface area (TPSA), molecular weight (MW), and distribution coefficient (LogD) were correlated against HSA binding site characteristics, including hydrophobicity, hydrophilicity, enclosure, exposure, contact, site volume, and donor/acceptor ratio. Molecular descriptors nHD, TPSA, LogD, nHet, and MW were found to possess the most inherent capacities providing baseline information for the prediction of serum albumin binding site. We believe that these associations may form the bedrock for establishing a solid correlation between the physicochemical properties and Albumin binding site architecture. Information presented in this report would serve as critical in provisions of rational drug designing as well as drug delivery, bioavailability, and pharmacokinetics. [ABSTRACT FROM AUTHOR]

In the present work we studied the interactions of a newly synthesized drug candidate, 2-(2-hydroxy-4-methoxybenzylidene)-1-(1H-benzimidazol-2-yl)hydrazine (2H4MBBH), with human serum albumin (HSA) by fluorescence spectroscopy. 2H4MBBH-HSA binding parameters were assessed by fluorescence quenching strategy. As made clear by the concentration data, 2H4MBBH unequivocally quenched the instrinsic HSA fluorescence. The calculated Stern-Volmer quenching constant Ksv, the Ka of 2H4MBBH-HSA complexes, as well as the thermodynamic parameters ∆H°, ∆S° and ∆G°, showed that the H-bonding forces play major part in the interaction of 2H4MBBH with HSA. These calculations point to a quenching component based on 2H4MBBH-HSA static complex formation rather than energetic collisions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Accurate predictions of ligand binding affinities would greatly accelerate the first stages of drug discovery campaigns. However, using highly accurate interatomic potentials based on quantum mechanics (QM) in free energy methods has been so far largely unfeasible due to their prohibitive computational cost. Here, we present an efficient method to compute QM free energies from simulations using cheap reference potentials, such as force fields (FFs). This task has traditionally been out of reach due to the slow convergence of computing the correction from the FF to theQMpotential. To overcome this bottleneck, we generalize targeted free energy methods to employ multiple maps--implemented with normalizing flow neural networks (NNs)--that maximize the overlap between the distributions. Critically, the method requires neither a separate expensive training phase for the NNs nor samples from the QM potential. We further propose a one-epoch learning policy to efficiently avoid overfitting, and we combine our approach with enhanced sampling strategies to overcome the pervasive problem of poor convergence due to slow degrees of freedom. On the drug-like molecules in the HiPen dataset, the method accelerates the calculation of the free energy difference of switching from an FF to a DFTB3 potential by three orders of magnitude compared to standard free energy perturbation and by a factor of eight compared to previously published nonequilibrium calculations. Our results suggest that our method, in combination with efficient QM/MM calculations, may be used in lead optimization campaigns in drug discovery and to study protein-ligand molecular recognition processes. [ABSTRACT FROM AUTHOR]

Gaussian accelerated molecular dynamics (GaMD) is a robust computational method for simultaneous unconstrained enhanced sampling and free energy calculations of biomolecules. It works by adding a harmonic boost potential to smooth biomolecular potential energy surface and reduce energy barriers. GaMD greatly accelerates biomolecular simulations by orders of magnitude. Without the need to set predefined reaction coordinates or collective variables, GaMD provides unconstrained enhanced sampling and is advantageous for simulating complex biological processes. The GaMD boost potential exhibits a Gaussian distribution, thereby allowing for energetic reweighting via cumulant expansion to the second order (i.e., "Gaussian approximation"). This leads to accurate reconstruction of free energy landscapes of biomolecules. Hybrid schemes with other enhanced sampling methods, such as the replica exchange GaMD (rex-GaMD) and replica exchange umbrella sampling GaMD (GaREUS), have also been introduced, further improving sampling and free energy calculations. Recently, new "selective GaMD" algorithms including the ligand GaMD (LiGaMD) and peptide GaMD (Pep-GaMD) enabled microsecond simulations to capture repetitive dissociation and binding of small-molecule ligands and highly flexible peptides. The simulations then allowed highly efficient quantitative characterization of the ligand/peptide binding thermodynamics and kinetics. Taken together, GaMD and its innovative variants are applicable to simulate a wide variety of biomolecular dynamics, including protein folding, conformational changes and allostery, ligand binding, peptide binding, protein-protein/nucleic acid/carbohydrate interactions, and carbohydrate/nucleic acid interactions. In this review, we present principles of the GaMD algorithms and recent applications in biomolecular simulations and drug design.

The drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55–100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.

The development of efficient drug formulations for Parkinson's disease (PD) treatment is challenged by achieving pharmacokinetic profiles, reduced side effects, and better permeability through the blood–brain barrier (BBB). As nanoparticles may facilitate the delivery of drugs in the brain due to their high-loading capacity and ability to cross biological barriers, we designed two different types of selenium nanoparticles (SeNPs) that may increase the transport of drugs across the BBB and may act as antioxidants at the site of action. The SeNPs were functionalized with polyvinylpyrrolidone (PVP) and polysorbate 20 (Tween) and characterized in terms of their size, size distribution, shape, surface charge, and colloidal stability in relevant biological media. Their drug-loading capacity was tested using dopamine and l-DOPA as therapeutically active agents for PD. Thermodynamic analysis revealed that binding processes occurred spontaneously through hydrogen bond/van der Waals interactions or electrostatic interactions. The strongest interaction was observed between PVP-SeNPs and l-DOPA or dopamine, which was characterized by a binding constant several orders of magnitude higher than for Tween-SeNPs. However, the addition of human transferrin as a model plasma protein significantly reduced this difference, which indicates the crucial role of protein corona formation in the design of drug nanodelivery systems. In vitro evaluation by cell-free and cellular transwell models showed efficient internalization of SeNP-loaded l-DOPA/dopamine by human endothelial brain cells, while facilitated BBB permeability for l-DOPA, and dopamine was achieved using PVP-SeNPs. Overall, the high potential of SeNPs as drug-delivery vehicles in PD treatment was demonstrated. [ABSTRACT FROM AUTHOR]

Solid-state NMR (ss-NMR) is a powerful tool to investigate noncrystallizable, poorly soluble molecular systems, such as membrane proteins, amyloids, and cell walls, in environments that closely resemble their physical sites of action. Rotational-echo double resonance (REDOR) is an ss-NMR methodology, which by reintroducing heteronuclear dipolar coupling under magic angle spinning conditions provides intramolecular and intermolecular distance restraints at the atomic level. In addition, REDOR can be exploited as a selection tool to filter spectra based on dipolar couplings. Used extensively as a spectroscopic ruler between isolated spins in site-specifically labeled systems and more recently as a building block in multidimensional ss-NMR pulse sequences allowing the simultaneous measurement of multiple distances, REDOR yields atomic-scale information on the structure and interaction of proteins. By extending REDOR to the determination of 1H–X dipolar couplings in recent years, the limit of measurable distances has reached ~15–20 Å, making it an attractive method of choice for the study of complex biomolecular assemblies. Following a methodological introduction including the most recent implementations, examples are discussed to illustrate the versatility of REDOR in the study of biological systems. [ABSTRACT FROM AUTHOR]

Reduction of the rapid delayed rectifier potassium current (IKr) via drug binding to the human Ether-à-go-go-Related Gene (hERG) channel is a well recognised mechanism that can contribute to an increased risk of Torsades de Pointes. Mathematical models have been created to replicate the effects of channel blockers, such as reducing the ionic conductance of the channel. Here, we study the impact of including state-dependent drug binding in a mathematical model of hERG when translating hERG inhibition to action potential changes. We show that the difference in action potential predictions when modelling drug binding of hERG using a state-dependent model versus a conductance scalingmodel depends not only on the properties of the drug and whether the experiment achieves steady state, but also on the experimental protocols. Furthermore, through exploring the model parameter space, we demonstrate that the state-dependent model and the conductance scalingmodel generally predict different action potential prolongations and are not interchangeable, while at high binding and unbinding rates, the conductance scaling model tends to predict shorter action potential prolongations. Finally, we observe that the difference in simulated action potentials between the models is determined by the binding and unbinding rate, rather than the trapping mechanism. This study demonstrates the importance of modelling drug binding and highlights the need for improved understanding of drug trapping which can have implications for the uses in drug safety assessment. [ABSTRACT FROM AUTHOR]

The latest biological findings observe that the motionless "lock‐and‐key" theory is not generally applicable and that changes in atomic sites and binding pose can provide important information for understanding drug binding. However, the computational expenditure limits the growth of protein trajectory‐related studies, thus hindering the possibility of supervised learning. A spatial‐temporal pre‐training method based on the modified equivariant graph matching networks, dubbed ProtMD which has two specially designed self‐supervised learning tasks: atom‐level prompt‐based denoising generative task and conformation‐level snapshot ordering task to seize the flexibility information inside molecular dynamics (MD) trajectories with very fine temporal resolutions is presented. The ProtMD can grant the encoder network the capacity to capture the time‐dependent geometric mobility of conformations along MD trajectories. Two downstream tasks are chosen to verify the effectiveness of ProtMD through linear detection and task‐specific fine‐tuning. A huge improvement from current state‐of‐the‐art methods, with a decrease of 4.3% in root mean square error for the binding affinity problem and an average increase of 13.8% in the area under receiver operating characteristic curve and the area under the precision‐recall curve for the ligand efficacy problem is observed. The results demonstrate a strong correlation between the magnitude of conformation's motion in the 3D space and the strength with which the ligand binds with its receptor. [ABSTRACT FROM AUTHOR]

Polyphenols have attained pronounced attention due to their ability to provide numerous health benefits and prevent several chronic diseases. In this study, we designed, synthesized and analyzed a water-soluble molecule presenting a good antioxidant activity, namely catechol hydrazinyl-thiazole (CHT). This molecule contains 3′,4′-dihydroxyphenyl and 2-hydrazinyl-4-methyl-thiazole moieties linked through a hydrazone group with very good antioxidant activity in the in vitro evaluations performed. A preliminary validation of the CHT developing hypothesis was performed evaluating in silico the bond dissociation enthalpy (BDE) of the phenol O-H bonds, compared to our previous findings in the compounds previously reported by our group. In this paper, we report the binding mechanism of CHT to human serum albumin (HSA) using biophysical methods in combination with computational studies. ITC experiments reveal that the dominant forces in the binding mechanism are involved in the hydrogen bond or van der Waals interactions and that the binding was an enthalpy-driven process. NMR relaxation measurements were applied to study the CHT–protein interaction by changing the drug concentration in the solution. A molecular docking study added an additional insight to the experimental ITC and NMR analysis regarding the binding conformation of CHT to HSA. [ABSTRACT FROM AUTHOR]

Electron paramagnetic resonance (EPR) spectroscopy is an advantageous technique to monitor solubility of drugs in an aqueous solution. In the presence of a drug carrier, the bound and unbound drug fractions can be determined in the same sample simultaneously. To enhance the solubility of hydrophobic drugs, a transporter protein of bovine serum albumin (BSA) can be used directly or in the form of nanoparticle. Moreover, a cationic BSA can be used to enhance anionic drug loading. Here, drugs with different water solubility, salicylic acid (high), ibuprofen (low) and chlorambucil (none) were spin labeled and studied with EPR spectroscopy. Remarkably, it has been shown that albumin nanoparticles are much more effective than albumin proteins in dissolving hydrophobic drugs in water. Furthermore, different drug loading methods were compared, and different from other techniques drug release can be monitored directly from the NPs pellet dissolution by EPR spectroscopy. [ABSTRACT FROM AUTHOR]

Human schistosomiasis is a disease which globally affects over 229 million people. Three major species affecting humans are Schistosoma mansoni, S. haematobium and S. japonicum. Previous treatment of S. mansoni includes the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The OXA activating enzyme was identified and crystallized, as being a S. mansoni sulfotransferase (SmSULT). S. haematobium and S. japonicum possess homologs of SmSULT (ShSULT and SjSULT) begging the question; why does oxamniquine fail to kill S. haematobium and S. japonicum adult worms? Investigation of the molecular structures of the sulfotransferases indicates that structural differences, specifically in OXA contact residues, do not abrogate OXA binding in the active sites as previously hypothesized. Data presented argue that the ability of SULTs to sulfate and thus activate OXA and its derivatives is linked to the ability of OXA to fit in the binding pocket to allow the transfer of a sulfur group.

In the present study, the lactoferrin (LF) that had been extracted from camel milk (through ion-exchange chromatography) has been loaded onto hydrogel particles of calcium alginate (Alg), while its effects on osteoblast cells line MG63 have been thoroughly evaluated. The extracted LF has been purified by the application of HPLC and SDS-PAGE techniques. Subsequent to being exposed to Alg (two concentrations of 0.2% and 0.5% in three pHs 6, 7, and 8), the interactions between LF and Alg have been studied by multiple spectroscopic as well as the zeta potential, and DLS particle size distribution. Fluorescence spectroscopy results have displayed the quenched emissions of proteins upon their interactions with Alg. In accordance with the obtained results, the zeta potential value of LF has faced a decrease through the formation of LF–Alg complex. Moreover, an outcome of 89.9% of loading has been perceived in both concentrations of Alg that the utilization of SEM electron microscopy. Once the loading had been confirmed, the effect of the particles that have been loaded on the MG63 cell line was put under investigation through the employment of MTT test. Taken together, it has been indicated by the achieved results that LF and Alg alone cannot cause any toxicity on the MG63 cells, while significantly increase their growth by being added to the cell culture media. The results have suggested that throughout all of the samples with evident increased cell growth, the highest interaction between LF and Alg has been observed to be at pH 8. [ABSTRACT FROM AUTHOR]

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type-like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles. [ABSTRACT FROM AUTHOR]

Gaussian accelerated molecular dynamics (GaMD) is a robust computational method for simultaneous unconstrained enhanced sampling and free energy calculations of biomolecules. It works by adding a harmonic boost potential to smooth biomolecular potential energy surface and reduce energy barriers. GaMD greatly accelerates biomolecular simulations by orders of magnitude. Without the need to set predefined reaction coordinates or collective variables, GaMD provides unconstrained enhanced sampling and is advantageous for simulating complex biological processes. The GaMD boost potential exhibits a Gaussian distribution, thereby allowing for energetic reweighting via cumulant expansion to the second order (i.e., "Gaussian approximation"). This leads to accurate reconstruction of free energy landscapes of biomolecules. Hybrid schemes with other enhanced sampling methods, such as the replica‐exchange GaMD (rex‐GaMD) and replica‐exchange umbrella sampling GaMD (GaREUS), have also been introduced, further improving sampling and free energy calculations. Recently, new "selective GaMD" algorithms including the Ligand GaMD (LiGaMD) and Peptide GaMD (Pep‐GaMD) enabled microsecond simulations to capture repetitive dissociation and binding of small‐molecule ligands and highly flexible peptides. The simulations then allowed highly efficient quantitative characterization of the ligand/peptide binding thermodynamics and kinetics. Taken together, GaMD and its innovative variants are applicable to simulate a wide variety of biomolecular dynamics, including protein folding, conformational changes and allostery, ligand binding, peptide binding, protein–protein/nucleic acid/carbohydrate interactions, and carbohydrate/nucleic acid interactions. In this review, we present principles of the GaMD algorithms and recent applications in biomolecular simulations and drug design. This article is categorized under:Structure and Mechanism > Computational Biochemistry and BiophysicsMolecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo MethodsMolecular and Statistical Mechanics > Free Energy Methods [ABSTRACT FROM AUTHOR]

Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the hinged lid-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water-protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors.

Photolyases are flavoenzymes responsible for light-driven repair of carcinogenic crosslinks formed in DNA by UV exposure. They possess two non-covalently bound chromophores: flavin adenine dinucleotide (FAD) as a catalytic center and an auxiliary antenna chromophore that harvests photons and transfers solar energy to the catalytic center. Although the energy transfer reaction has been characterized by time-resolved spectroscopy, it is strikingly important to understand how well natural biological systems organize the chromophores for the efficient energy transfer. Here, we comprehensively characterized the binding of 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF) to Xenopus (6–4) photolyase. In silico simulations indicated that a hydrophobic amino acid residue located at the entrance of the binding site dominates translocation of a loop upon binding of 8-HDF, and a mutation of this residue caused dysfunction of the efficient energy transfer in the DNA repair reaction. Mutational analyses of the protein combined with modification of the chromophore suggested that Coulombic interactions between positively charged residues in the protein and the phenoxide moiety in 8-HDF play a key role in accommodation of 8-HDF in the proper direction. This study provides a clear evidence that Xenopus (6–4) photolyase can utilize 8-HDF as the light-harvesting chromophore. The obtained new insights into binding of the natural antenna molecule will be helpful for the development of artificial light-harvesting chromophores and future characterization of the energy transfer in (6–4) photolyase by spectroscopic studies. [ABSTRACT FROM AUTHOR]

We used NMR to show that the antipsychotic phenothiazine drugs promazine and promethazine bind to GDP-KRAS. Promazine also binds to oncogenic GDP-KRAS(G12D), and to wild type GppNHp-KRAS. A panel of additional phenothiazines bind to GDP-KRAS but with lower affinity than promazine or promethazine. Binding is most dependent on substitutions at C-2 of the tricyclic phenothiazine ring. Promazine was used to generate an NMR-driven HADDOCK model of the drug/GDP-KRAS complex. The structural model shows the tricyclic phenothiazine ring of promazine associates with the hydrophobic pocket p1 that is bordered by the central β sheet and Switch II in KRAS. Binding appears to stabilize helix 2 in a conformation that is similar to that seen in KRAS bound to other small molecules. Association of phenothiazines with KRAS may affect normal KRAS signaling that could contribute to multiple biological activities of these antipsychotic drugs. Moreover, the phenothiazine ring represents a new core scaffold on which to design modulators of KRAS activity. [ABSTRACT FROM AUTHOR]

Heme‐nitric oxide/oxygen binding (H‐NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H‐NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H‐NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H‐NOX and coiled‐coil domains. Here, we report the full NMR structure for CO‐ligated Sw H‐NOX in the presence and absence of stimulator compound IWP‐051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi‐empirical quantum potential energy approach. Although IWP‐051 binding is weak, a single binding conformation was found at the interface of the two H‐NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix‐connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure‐based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H‐NOX proteins. The largest dynamical loop lies at the interface between Sw H‐NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction. PDB Code(s): 6OCV and 6WQE; [ABSTRACT FROM AUTHOR]

Background: SARS-CoV-2 has the Spike glycoprotein (S) which is crucial in attachment with host receptor and cell entry leading to COVID-19 infection. The current study was conducted to explore drugs against Receptor Binding Domain (RBD) of SARS-CoV-2 using in silico pharmacophore modelling and virtual screening approach to combat COVID-19. Methods: All the available sequences of RBD in NCBI were retrieved and multiple aligned to get insight into its diversity. The 3D structure of RBD was modelled and the conserved region was used as a template to design pharmacophore using LigandScout. Lead compounds were screened using Cambridge, Drugbank, ZINC and TIMBLE databases and these identified lead compounds were screened for their toxicity and Lipinski's rule of five. Molecular docking of shortlisted lead compounds was performed using AutoDock Vina and interacting residues were visualized. Results: Active residues of Receptor Binding Motif (RBM) in S, involved in interaction with receptor, were found to be conserved in all 483 sequences. Using this RBM motif as a pharmacophore a total of 1327 lead compounds were predicted initially from all databases, however, only eight molecules fit the criteria for safe oral drugs. Conclusion: The RBM region of S interacts with Angiotensin Converting Enzyme 2 (ACE2) receptor and Glucose Regulated Protein 78 (GRP78) to mediate viral entry. Based on in silico analysis, the lead compounds scrutinized herewith interact with S, hence, can prevent its internalization in cell using ACE2 and GRP78 receptor.The compounds predicted in this study are based on rigorous computational analysis and the evaluation of predicted lead compounds can be promising in experimental studies.

Human schistosomiasis is a disease which globally affects over 229 million people. Three major species affecting humans are Schistosoma mansoni, S. haematobium and S. japonicum. Previous treatment of S. mansoni includes the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The OXA activating enzyme was identified and crystallized, as being a S. mansoni sulfotransferase (Sm SULT). S. haematobium and S. japonicum possess homologs of Sm SULT (Sh SULT and Sj SULT) begging the question; why does oxamniquine fail to kill S. haematobium and S. japonicum adult worms? Investigation of the molecular structures of the sulfotransferases indicates that structural differences, specifically in OXA contact residues, do not abrogate OXA binding in the active sites as previously hypothesized. Data presented argue that the ability of SULTs to sulfate and thus activate OXA and its derivatives is linked to the ability of OXA to fit in the binding pocket to allow the transfer of a sulfur group. Image 1 • OXA can kill S. mansoni but not S. haematobium or S. japonicum. • S. mansoni whole worm homogenates activate OXA, while S. haematobium and S. japonicum homogenates do not. • Differences in SULT amino acid contacts do not abrogate OXA binding in S. haematobium but may affect binding in S. japonicum. • The ability of OXA or its derivative to fit in the binding pocket determines whether sulfation takes place and parasite killing results. [ABSTRACT FROM AUTHOR]

Purpose: We have developed a 3D brain unit network model to understand the spatial-temporal distribution of a drug within the brain under different (normal and disease) conditions. Our main aim is to study the impact of disease-induced changes in drug transport processes on spatial drug distribution within the brain extracellular fluid (ECF). Methods: The 3D brain unit network consists of multiple connected single 3D brain units in which the brain capillaries surround the brain ECF. The model includes the distribution of unbound drug within blood plasma, coupled with the distribution of drug within brain ECF and incorporates brain capillaryblood flow, passive paracellular and transcellular BBB transport, active BBB transport, brain ECF diffusion, brain ECF bulk flow, and specific and nonspecific brain tissue binding. All of these processes may change under disease conditions. Results: We show that the simulated disease-induced changes in brain tissue characteristics significantly affect drug concentrations within the brain ECF. Conclusions: We demonstrate that the 3D brain unit network model is an excellent tool to gain understanding in the interdependencies of the factors governing spatial-temporal drug concentrations within the brain ECF. Additionally, the model helps in predicting the spatial-temporal brain ECF concentrations of existing drugs, under both normal and disease conditions. [ABSTRACT FROM AUTHOR]

Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia and gastrointestinal stromal tumors. Binding of drugs to proteins influence their pharmacokinetic and pharmacodynamics action. In the blood, the drug is distributed in the body in the free form or bound to plasma protein. Albumin and α-1 glycoprotein (AGP) are plasma proteins with the highest affinity for drug substances. Drugs which are weak acids mainly bind to plasma albumin, while drugs that are bases have affinity for α-1 glycoprotein. The main goal of this study is to quantitatively evaluate the interaction between imatinib mesylate (IMT) and α-1 glycoprotein to characterize the nature and forces underlying the formation of a molecular complex. Relaxation experiments provide quantitative information about the relationship between the binding affinity and structure of IMT. Thus, association constant was determined as K a = 873.36 M−1. The ITC data revealed that the binding was an entropy driven process and the association constant K a = 3.22 × 103 M−1, with a 1:1 stoichiometry. The results obtained by NMR and ITC were complemented with a molecular docking study. • Quantitatively evaluate the interaction between imatinib mesylate (IMT) and α-1 glycoprotein (AGP) • The energetics of binding of IMT to AGP was assessed by ITC, NMR and molecular docking. • 1:1 binding stoichiometry • Structure of the conformation with the highest binding energy of IMT:AGP complex. [ABSTRACT FROM AUTHOR]

Colonic mucus is a key factor in the colonic environment because it may affect drug absorption. Due to the similarity of human and canine gastrointestinal physiology, dogs are an established preclinical species for the assessment of controlled release formulations. Here we report the development of an artificial colonic mucus model to mimic the native canine one. In vitro models of the canine colonic environment can provide insights for early stages of drug development and contribute to the implementation of the 3Rs (refinement, reduction, and replacement) of animal usage in the drug development process. Our artificial colonic mucus could predict diffusion trends observed in native mucus and was successfully implemented in microscopic and macroscopic assays to study macromolecular permeation through the mucus. The traditional Transwell set up was optimized with the addition of a nylon filter to ensure homogenous representation of the mucus barrier in vitro. In conclusion, the canine artificial colonic mucus can be used to study drug permeation across the mucus and its flexibility allows its use in various set ups depending on the nature of the compound under investigation and equipment availability. [Display omitted] [ABSTRACT FROM AUTHOR]

ABCB1/P-glycoprotein is an ATP binding cassette transporter that is involved in the clearance of xenobiotics, and it affects the disposition of many drugs in the body. Conformational flexibility of the protein within the membrane is an intrinsic part of its mechanism of action, but this has made structural studies challenging. Here, we have studied different conformations of P-glycoprotein simultaneously in the presence of ivacaftor, a known competitive inhibitor. In order to conduct this, we used high contrast cryo-electron microscopy imaging with a Volta phase plate. We associate the presence of ivacaftor with the appearance of an additional density in one of the conformational states detected. The additional density is in the central aqueous cavity and is associated with a wider separation of the two halves of the transporter in the inward-facing state. Conformational changes to the nucleotide-binding domains are also observed and may help to explain the stimulation of ATPase activity that occurs when transported substrate is bound in many ATP binding cassette transporters.

Tripterygium wilfordii Hook F. (TWHF) is a Chinese traditional medicine with cardiac toxicities. However, the mechanism of acute cardiac toxicity is not very clear. By using patch clamp techniques, we found that 0.05 mg/ml and 0.1 mg/ml of the aqueous crude extract of TWHF inhibit 21.4 ± 1.6% and 86.7 ± 5.7% (n = 5) of hERG current Amplitudes (IhERG) respectively. We further found that Celastrol, one of main components of TWHF, inhibits hERG with an IC50 of 0.83 μM. Additional mutagenesis studies show that mutations of T623A, S624A and F656A significantly alter the inhibition and S624A has the strongest effect, supported by our docking model. Our data suggest that inhibition of hERG channel activity by Celastrol contributed to TWHF cardiotoxicity.

Binding strength of the anti-diabetic drugs chlorpropamide (CPM) and tolbutamide (TBM) with model protein bovine serum albumin (BSA) shows strong modulation in presence of colloidal gold nanoparticles (AuNP). Intrinsic tryptophan fluorescence of both the native BSA and BSA-AuNP conjugate quenched in presence of the drugs. Stern-Volmer quenching constant (KSV) of CPM binding to BSA-AuNP conjugate at different temperatures is almost twice (6.76~14.76 × 103 M−1) than the corresponding values in native BSA (3.21~5.72 × 103 M−1). However, the calculated KSV values with TBM show certain degree of reduction in presence of AuNP (6.46× 103 M−1), while comparing with native BSA (8.83 × 103 M−1). The binding mode of CPM towards BSA-AuNP conjugate is mainly through hydrophobic forces; whereas, TBM binding is identified to be Van der Waal's and hydrogen bonding type of interaction. Fluorescence lifetime analysis confirms static type of quenching for the intrinsic tryptophan fluorescence of BSA as well as BSA-AuNP conjugate with addition of CPM and TBM at different concentrations. The α-helical content in the secondary structure of BSA is decreased to 48.32% and 45. 28% in presence of AuNP, when the concentration of CPM is 0.08 mM and 0.16 mM in comparison with that of native protein (50.13%). On the other hand, the intensity of sugar induced advanced glycated end (AGE) product fluorescence is decreased by 55% and 80% at 0.13 nM and 0.68 nM AuNP, respectively. Change in the binding strength of the drugs with transport protein and reduced AGE product formation in presence of AuNP could lead to a major development in the field of nanomedicine and associated drug delivery techniques. [ABSTRACT FROM AUTHOR]

Valproic acid (VPA) is an anticonvulsant drug that is also used to treat migraines and bipolar disorder. Its proposed biological targets include human voltage-gated sodium channels, among other membrane proteins. We used the prokaryotic NavMs sodium channel, which has been shown to be a good exemplar for drug binding to human sodium channels, to examine the structural and functional interactions of VPA. Thermal melt synchrotron radiation circular dichroism spectroscopic binding studies of the full-length NavMs channel (which includes both pore and voltage sensor domains), and a pore-only construct, undertaken in the presence and absence of VPA, indicated that the drug binds to and destabilizes the channel, but not the poreonly construct. This is in contrast to other antiepileptic compounds that have previously been shown to bind in the central hydrophobic core of the pore region of the channel, and that tend to increase the thermal stability of both pore-only constructs and full-length channels. Molecular docking studies also indicated that the VPA binding site is associated with the voltage sensor, rather than the hydrophobic cavity of the pore domain. Electrophysiological studies show that VPA influences the block and inactivation rates of the NavMs channel, although with lower efficacy than classical channel-blocking compounds. It thus appears that, while VPA is capable of binding to these voltage-gated sodium channels, it has a very different mode and site of action than other anticonvulsant compounds. [ABSTRACT FROM AUTHOR]

We recently reported that aripiprazole binds strongly to human albumin. In continuing our investigations, we investigated the mechanism responsible for the binding and the related interactions of aripiprazole with α1-acid glycoprotein (AGP). The extrinsic Cotton effects for the binding of aripiprazole and its derivatives to AGP were generated, but the magnitudes of the induced circular dichroism intensities did not correlate with those for the binding affinities. It therefore appears that the binding mode of aripiprazole with AGP is somewhat complicated, compared with that of albumin. Isothermal titration calorimetry data obtained for the binding of aripiprazole with AGP were different from that for albumin systems in that the 3 driving reactions, entropy-driven, enthalpy-driven, and the entropy-enthalpy mixed type, were all found for the AGP system, but not albumin. Moreover, the weak binding mode of aripiprazole with the 2 proteins were supported by a molecular docking model analysis. The concentration of albumin in plasma is about 50 times higher than those of AGP, but AGP levels in plasma are increased by about 10 times under inflammatory disease. Therefore, the involvement of these 2 plasma proteins should be considered in more depth for understanding the pharmacokinetics of aripiprazole. [ABSTRACT FROM AUTHOR]

The development of drugs that target the brain is very challenging. A quantitative understanding is needed of the complex processes that govern the concentration–time profile of a drug (pharmacokinetics) within the brain. So far, there are no studies on predicting the drug concentration within the brain that focus not only on the transport of drugs to the brain through the blood–brain barrier (BBB), but also on drug transport and binding within the brain. Here, we develop a new model for a 2D square brain tissue unit, consisting of brain extracellular fluid (ECF) that is surrounded by the brain capillaries. We describe the change in free drug concentration within the brain ECF, by a partial differential equation (PDE). To include drug binding, we couple this PDE to two ordinary differential equations that describe the concentration–time profile of drug bound to specific as well as non-specific binding sites that we assume to be evenly distributed over the brain ECF. The model boundary conditions reflect how free drug enters and leaves the brain ECF by passing the BBB, located at the level of the brain capillaries. We study the influence of parameter values for BBB permeability, brain ECF bulk flow, drug diffusion through the brain ECF and drug binding kinetics, on the concentration–time profiles of free and bound drug. [ABSTRACT FROM AUTHOR]

Abstract Previously, we reported on the high-affinity binding of aripiprazole (ARP), an antipsychotic drug, to human albumin and the role of the chlorine atom of ARP on this binding. In this study, we investigated the binding mode of ARP to human albumin in detail using ARP derivatives and several animal-derived albumins. ARP bound strongly to human and dog albumin. The circular dichroism (CD) spectra of ARP bound to human and dog albumin were also similar. Deschloro-ARP bound less strongly to all of the albumin species compared to ARP, and the shapes of CD spectra were similar for all albumin species. CD spectra of dimethyl-ARP, for which chlorine atoms were substituted methyl groups, were quite similar to that of deschloro-ARP. In displacement experiments, competitive binding was observed between ARP and deschloro-ARP. These results suggest that the chlorine atoms in ARP are involved in the binding modes of ARP for human and dog albumins, whereas ARP and deschloro-ARP appear to share the same binding region in site II. The aforementioned results imply that compounds having a chlorine atom bind more strongly to plasma proteins, resulting in a long blood retention time. Therefore, findings reported here may provide the basically useful data for drug design. [ABSTRACT FROM AUTHOR]

Abstract Cytochrome P450 (CYP) is a class of heme-containing enzymes which mainly catalyze a monooxygenation reaction of various chemicals, and hence CYP plays a key role in the drug metabolism. Although CYP2C19 isoform is a minor hepatic CYP, it metabolizes clinically important drugs such as omeprazole and S ‑mephenytoin. In this work, the interaction of purified CYP2C19 WT (CYP2C19) with seven drugs (phenytoin, S ‑mephenytoin, omeprazole, lansoprazole, cimetidine, propranolol, and warfarin) was investigated using spectroscopic methods. The binding of each drug and the induced structural change in the heme distal environment were evaluated. Ferric form of CYP2C19 was revealed to contain a six-coordinate low-spin heme with a water molecule as a sixth ligand in a distal site, and the addition of each drug caused varied minor fraction of five-coordinate heme. It was suggested that the ligated water molecule was partly moved away from the heme distal environment and that the degree of water removal was dependent on the type of drugs. The effect on the coordination was varied with the studied drugs with wide variation in the dissociation constants from 2.6 μM for lansoprazole to 5400 μM for warfarin. Phenytoin and S ‑mephenytoin showed that binding to CYP2C19 occurred in a stepwise manner and that the coordination of a water molecule was facilitated in the second binding step. In the ferrous CO-bound state, ν(Fe CO) stretching mode was clearly observed at 471 cm−1 in the absence of drugs. The Raman line was greatly up-shifted by omeprazole (487 cm−1) and lansoprazole (477 cm−1) but was minimally affected by propranolol, phenytoin, and S ‑mephenytoin. These results indicate that slight chemical modification of a drug greatly affects the heme distal environments upon binding. Graphical Abstract Crystal structure of drug-free CYP2C19. Showing the substrate recognition sites. Unlabelled Image Highlights • Although cytochrome P450 2C19 (CYP2C19) isoform is a minor hepatic CYP, it metabolizes clinically important drugs. • In this work, the interaction of purified CYP2C19 WT with seven drugs was investigated using spectroscopic methods. • The binding of these drugs induced a variety of structural changes in the heme distal environment. • Drug binding also produced different degree of water removal from the heme iron which is dependent on the type of drug. • In addition, it was found that slight chemical modifications of the drug could affect the drug specificities of the enzyme. [ABSTRACT FROM AUTHOR]