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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., SweDeliver, COLOTAN

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., SweDeliver, COLOTAN

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., SweDeliver, COLOTAN

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., SweDeliver, COLOTAN

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.

Molecular recognition is a fundamental part of chemical processes, especially those relevant to biology. It refers to the process by which two molecules diffuse and eventually bind with one another to form a complex. This can be broken down into to broad aspects: kinetics and thermodynamics. Kinetics refers to the motion of molecules and the rates of their reactions with each other. Thermodynamics refers to the transfers of energy that drive the reaction when molecules bind together. The work in this dissertation uses computational methods to study both aspects of molecular recognition in a range of systems, and it includes the application of existing methods and development of new tools for simulation and analysis.A strong focus is given to protein-ligand systems, in which the ligand is an inhibitory drug designed to shut down function of its target protein. The concept of developing drugs (inhibitors) that bind with and disrupt the activity of their targets is the basis for much of modern medicine, and has had incredible success. The development of more effective inhibitors is a constant challenge. The physical and chemical principles that predict an inhibitor’s effectiveness have a complex interplay, and an understanding of these principles is challenging and highly sought after. Two projects described here use molecular dynamics (MD) simulations to elucidate these principles through better understanding inhibitor binding thermodynamics. These techniques are applied to a host of inhibitors for the carcinogenic CDK8 protein and to inhibitors of a protease (PLpro) of the recent SARS-CoV2 virus. Novel inhibitors of PLpro are also developed and validated. The other work described herein studies molecular binding kinetics in both natural and engineered systems. Brownian dynamics simulation software is described which has been developed by the author and other group members. Its goal is to provide a robust tool with which researchers can study molecular recognition and

Molecular recognition is a fundamental part of chemical processes, especially those relevant to biology. It refers to the process by which two molecules diffuse and eventually bind with one another to form a complex. This can be broken down into to broad aspects: kinetics and thermodynamics. Kinetics refers to the motion of molecules and the rates of their reactions with each other. Thermodynamics refers to the transfers of energy that drive the reaction when molecules bind together. The work in this dissertation uses computational methods to study both aspects of molecular recognition in a range of systems, and it includes the application of existing methods and development of new tools for simulation and analysis.A strong focus is given to protein-ligand systems, in which the ligand is an inhibitory drug designed to shut down function of its target protein. The concept of developing drugs (inhibitors) that bind with and disrupt the activity of their targets is the basis for much of modern medicine, and has had incredible success. The development of more effective inhibitors is a constant challenge. The physical and chemical principles that predict an inhibitor’s effectiveness have a complex interplay, and an understanding of these principles is challenging and highly sought after. Two projects described here use molecular dynamics (MD) simulations to elucidate these principles through better understanding inhibitor binding thermodynamics. These techniques are applied to a host of inhibitors for the carcinogenic CDK8 protein and to inhibitors of a protease (PLpro) of the recent SARS-CoV2 virus. Novel inhibitors of PLpro are also developed and validated. The other work described herein studies molecular binding kinetics in both natural and engineered systems. Brownian dynamics simulation software is described which has been developed by the author and other group members. Its goal is to provide a robust tool with which researchers can study molecular recognition and

Objective: We study activation of T helper 17 (Th17) and regulatory T (Treg) cells and induction of apoptosis in cells from patients with systemic lupus erythematosus (SLE) compared with controls and effects of atorvastatin and its simulated interactions with other compounds. Methods: Mononuclear cells from 10 patients with SLE and 10 controls were cultured in conditions that induce Th17 and/or Treg cell polarization and/or apoptosis and were studied by FACScan. Gene expression was determined by quantitative real-time reverse transcription–polymerase chain reaction. Cytokines in plasma were determined by enzyme-linked immunosorbent assay. The Search Tool for Interactions of Chemicals (STITCH) was used to retrieve information regarding the binding properties of atorvastatin. Results: Among patients with SLE, the proportion of Th17 (CD4+IL17+) cells was higher compared with controls after activation, with Th17 or Treg polarizing cytokines, phorbol myristate acetate, and ionomycin. In contrast, Treg cells (CD4+CD25+CD127dim/−) frequencies were lower. CD95 stimulation induced relatively more apoptosis in Treg cells and less in Th17 cells, as compared with controls. Addition of atorvastatin normalized Th17/Treg cell balance and apoptosis induction. Accordingly, the ratio of RORC/FoxP3 decreased in patients with SLE. Interleukin 17 and interleukin 6 (IL-6) levels were increased in patients with SLE. Atorvastatin interacted strongly with C-reactive protein (CRP) and also significantly with IL-6. Conclusion: There is a higher proportion of Th17 cells and a lower proportion of Treg cells in patients with SLE after activation. Th17 cells were more resistant than Treg cells to CD95-induced apoptosis in SLE. Atorvastatin normalized these effects. Our findings reveal a novel mechanism behind the imbalance of Th17/Treg cells with implications for treatment in SLE. We determine for the first time simulated interaction between atorvastatin, CRP, and IL-6, implying a novel role of a, QC 20230308

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.

Functional inhibitory peptides of human dipeptidyl peptidase 4 (hDPP4) have been highly anticipated as the active ingredient of functional food for type II diabetes; however, the molecular mechanism of hDPP4 inhibition remains unclear. In this study, we focused on dipeptides and tripeptides, which display structure-function correlations that are relatively easy to analyze, and examined their interactions with hDPP4 on an atomic level using a combination of docking studies and an hDPP4 inhibition assay. First, we performed comprehensive binding mode analysis of the dipeptide library and demonstrated that the formation of a tight interaction with the S1 subsite composing part of the substrate pocket is essential for dipeptides to compete with the substrate and strongly inhibit hDPP4. Next, we synthesized tripeptides by adding various amino acids to the C-terminus of Ile-Pro and Val-Pro, which have especially high inhibitory activity among compounds in the dipeptide library, and measured the hDPP4 inhibitory activity of the tripeptides. When hydrophobic amino acids (Ile, Met, Val, Trp) were added, the inhibitory activity increased several-fold. This phenomenon could be explained as follows: the C-terminal amino acid of the tripeptide formed hydrophobic interactions with Tyr547 and Trp629, which compose the S1′ subsite located relatively outside the substrate pocket, thereby stabilizing the hDPP4-peptide binding. The structural information on the interaction between hDPP4 and peptide inhibitors attained in this study is anticipated to be useful in the development of a more potent hDPP4 competitive inhibitor.

Lung cancer is the leading cause of cancer mortality worldwide, resulting in 88% deaths of all diagnosed patients. Hence, novel therapeutic modalities are urgently needed. Single-stranded oligonucleotide-based aptamers (APTs) are excellent ligands for tumor cell targeting. However, the molecular mechanisms underlying their internalization into living cells have been poorly studied. Towards the application of APTs for active drug targeting to cancer cells, we herein studied the mechanism underlying S15-APT internalization into human non-small cell lung cancer A549 cells. We thus delineated the mode of entry of a model nanomedical system based on quantum dots (QDs) decorated with S15-APTs as a selective targeting moiety for uptake by A549 cells. These APT-decorated QDs displayed selective binding to, and internalization by target A549 cells, but not by normal human bronchial epithelial BEAS2B, cervical carcinoma (HeLa) and colon adenocarcinoma CaCo-2 cells, hence demonstrating high specificity. Flow cytometric analysis revealed a remarkably low dissociation constant of S15-APTs-decorated QDs to A549 cells (Kd = 13.1 ± 1.6 nM). Through the systematic application of a series of established inhibitors of known mechanisms of endocytosis, we show that the uptake of S15-APTs proceeds via a classical clathrin-dependent receptor-mediated endocytosis. This cancer cell-selective mode of entry could possibly be used in the future to evade plasma membranelocalized multidrug resistance efflux pumps, thereby overcoming an important mechanism of cancer multidrug resistance. © Engelberg et al.

One of the main fundamental mechanisms of antibiotic resistance in Gram-negative bacteria comprises an effective change in the membrane permeability to antibiotics. The Gram-negative bacterial complex cell envelope comprises an outer membrane that delimits the periplasm from the exterior environment. The outer membrane contains numerous protein channels, termed as porins or nanopores, which are mainly involved in the influx of hydrophilic compounds, including antibiotics. Bacterial adaptation to reduce influx through these outer membrane proteins (Omps) is one of the crucial mechanisms behind antibiotic resistance. Thus to interpret the molecular basis of the outer membrane permeability is the current challenge. This review attempts to develop a state of knowledge pertinent to Omps and their effective role in antibiotic influx. Further, it aims to study the bacterial response to antibiotic membrane permeability and hopefully provoke a discussion toward understanding and further exploration of prospects to improve our knowledge on physicochemical parameters that direct the translocation of antibiotics through the bacterial membrane protein channels.

Most known drug targets and metabolizing enzymes are located inside cells. Interactions with these proteins are determined by intracellular unbound drug concentrations. Assessing intracellular drug exposure is technically challenging, but essential for predicting pharmacokinetic, pharmacological, and toxicological profiles of new drugs. This thesis aims at establishing and applying a straightforward methodology to measure intracellular unbound drug concentrations. This was achieved by separately measuring cellular drug binding (fu,cell), and total intracellular drug accumulation (Kp). This allowed the calculation of intracellular drug bioavailability (Fic), which represents the fraction of the concentration added to the cells that is unbound in the cell interior. The methodology was initially developed in HEK293 cells, where the Fic of 189 drug-like compounds was measured. Binding to HEK293 cells was governed by compound lipophilicity and was correlated with binding to more complex systems, such as hepatocytes and brain. Due to negligible expression of drug transporters, Fic in this cell line was consistent with pH-dependent subcellular sequestration of lipophilic cations in low pH compartments. The methodology was then applied to study the effects of drug transporters on Fic. The uptake transporter OATP1B1 increased the Fic of its substrates in a concentration-dependent manner. In contrast, the Fic of P-gp substrates was decreased when P-gp was present. In human hepatocytes, the methodology allowed the determination of Fic without prior knowledge of transporter mechanisms or metabolic activity. Finally, the methodology was applied to measure the impact of Fic on target binding and cellular drug response. Intracellular concentrations of active metabolites of pro-drugs targeting the intracellular target thymidylate synthase were in agreement with the level of binding to this target. Further, high Fic was generally required for kinase and protease inhibitors to be act

Most known drug targets and metabolizing enzymes are located inside cells. Interactions with these proteins are determined by intracellular unbound drug concentrations. Assessing intracellular drug exposure is technically challenging, but essential for predicting pharmacokinetic, pharmacological, and toxicological profiles of new drugs. This thesis aims at establishing and applying a straightforward methodology to measure intracellular unbound drug concentrations. This was achieved by separately measuring cellular drug binding (fu,cell), and total intracellular drug accumulation (Kp). This allowed the calculation of intracellular drug bioavailability (Fic), which represents the fraction of the concentration added to the cells that is unbound in the cell interior. The methodology was initially developed in HEK293 cells, where the Fic of 189 drug-like compounds was measured. Binding to HEK293 cells was governed by compound lipophilicity and was correlated with binding to more complex systems, such as hepatocytes and brain. Due to negligible expression of drug transporters, Fic in this cell line was consistent with pH-dependent subcellular sequestration of lipophilic cations in low pH compartments. The methodology was then applied to study the effects of drug transporters on Fic. The uptake transporter OATP1B1 increased the Fic of its substrates in a concentration-dependent manner. In contrast, the Fic of P-gp substrates was decreased when P-gp was present. In human hepatocytes, the methodology allowed the determination of Fic without prior knowledge of transporter mechanisms or metabolic activity. Finally, the methodology was applied to measure the impact of Fic on target binding and cellular drug response. Intracellular concentrations of active metabolites of pro-drugs targeting the intracellular target thymidylate synthase were in agreement with the level of binding to this target. Further, high Fic was generally required for kinase and protease inhibitors to be act

Most known drug targets and metabolizing enzymes are located inside cells. Interactions with these proteins are determined by intracellular unbound drug concentrations. Assessing intracellular drug exposure is technically challenging, but essential for predicting pharmacokinetic, pharmacological, and toxicological profiles of new drugs. This thesis aims at establishing and applying a straightforward methodology to measure intracellular unbound drug concentrations. This was achieved by separately measuring cellular drug binding (fu,cell), and total intracellular drug accumulation (Kp). This allowed the calculation of intracellular drug bioavailability (Fic), which represents the fraction of the concentration added to the cells that is unbound in the cell interior. The methodology was initially developed in HEK293 cells, where the Fic of 189 drug-like compounds was measured. Binding to HEK293 cells was governed by compound lipophilicity and was correlated with binding to more complex systems, such as hepatocytes and brain. Due to negligible expression of drug transporters, Fic in this cell line was consistent with pH-dependent subcellular sequestration of lipophilic cations in low pH compartments. The methodology was then applied to study the effects of drug transporters on Fic. The uptake transporter OATP1B1 increased the Fic of its substrates in a concentration-dependent manner. In contrast, the Fic of P-gp substrates was decreased when P-gp was present. In human hepatocytes, the methodology allowed the determination of Fic without prior knowledge of transporter mechanisms or metabolic activity. Finally, the methodology was applied to measure the impact of Fic on target binding and cellular drug response. Intracellular concentrations of active metabolites of pro-drugs targeting the intracellular target thymidylate synthase were in agreement with the level of binding to this target. Further, high Fic was generally required for kinase and protease inhibitors to be act

Most known drug targets and metabolizing enzymes are located inside cells. Interactions with these proteins are determined by intracellular unbound drug concentrations. Assessing intracellular drug exposure is technically challenging, but essential for predicting pharmacokinetic, pharmacological, and toxicological profiles of new drugs. This thesis aims at establishing and applying a straightforward methodology to measure intracellular unbound drug concentrations. This was achieved by separately measuring cellular drug binding (fu,cell), and total intracellular drug accumulation (Kp). This allowed the calculation of intracellular drug bioavailability (Fic), which represents the fraction of the concentration added to the cells that is unbound in the cell interior. The methodology was initially developed in HEK293 cells, where the Fic of 189 drug-like compounds was measured. Binding to HEK293 cells was governed by compound lipophilicity and was correlated with binding to more complex systems, such as hepatocytes and brain. Due to negligible expression of drug transporters, Fic in this cell line was consistent with pH-dependent subcellular sequestration of lipophilic cations in low pH compartments. The methodology was then applied to study the effects of drug transporters on Fic. The uptake transporter OATP1B1 increased the Fic of its substrates in a concentration-dependent manner. In contrast, the Fic of P-gp substrates was decreased when P-gp was present. In human hepatocytes, the methodology allowed the determination of Fic without prior knowledge of transporter mechanisms or metabolic activity. Finally, the methodology was applied to measure the impact of Fic on target binding and cellular drug response. Intracellular concentrations of active metabolites of pro-drugs targeting the intracellular target thymidylate synthase were in agreement with the level of binding to this target. Further, high Fic was generally required for kinase and protease inhibitors to be act

Most known drug targets and metabolizing enzymes are located inside cells. Interactions with these proteins are determined by intracellular unbound drug concentrations. Assessing intracellular drug exposure is technically challenging, but essential for predicting pharmacokinetic, pharmacological, and toxicological profiles of new drugs. This thesis aims at establishing and applying a straightforward methodology to measure intracellular unbound drug concentrations. This was achieved by separately measuring cellular drug binding (fu,cell), and total intracellular drug accumulation (Kp). This allowed the calculation of intracellular drug bioavailability (Fic), which represents the fraction of the concentration added to the cells that is unbound in the cell interior. The methodology was initially developed in HEK293 cells, where the Fic of 189 drug-like compounds was measured. Binding to HEK293 cells was governed by compound lipophilicity and was correlated with binding to more complex systems, such as hepatocytes and brain. Due to negligible expression of drug transporters, Fic in this cell line was consistent with pH-dependent subcellular sequestration of lipophilic cations in low pH compartments. The methodology was then applied to study the effects of drug transporters on Fic. The uptake transporter OATP1B1 increased the Fic of its substrates in a concentration-dependent manner. In contrast, the Fic of P-gp substrates was decreased when P-gp was present. In human hepatocytes, the methodology allowed the determination of Fic without prior knowledge of transporter mechanisms or metabolic activity. Finally, the methodology was applied to measure the impact of Fic on target binding and cellular drug response. Intracellular concentrations of active metabolites of pro-drugs targeting the intracellular target thymidylate synthase were in agreement with the level of binding to this target. Further, high Fic was generally required for kinase and protease inhibitors to be act

Most known drug targets and metabolizing enzymes are located inside cells. Interactions with these proteins are determined by intracellular unbound drug concentrations. Assessing intracellular drug exposure is technically challenging, but essential for predicting pharmacokinetic, pharmacological, and toxicological profiles of new drugs. This thesis aims at establishing and applying a straightforward methodology to measure intracellular unbound drug concentrations. This was achieved by separately measuring cellular drug binding (fu,cell), and total intracellular drug accumulation (Kp). This allowed the calculation of intracellular drug bioavailability (Fic), which represents the fraction of the concentration added to the cells that is unbound in the cell interior. The methodology was initially developed in HEK293 cells, where the Fic of 189 drug-like compounds was measured. Binding to HEK293 cells was governed by compound lipophilicity and was correlated with binding to more complex systems, such as hepatocytes and brain. Due to negligible expression of drug transporters, Fic in this cell line was consistent with pH-dependent subcellular sequestration of lipophilic cations in low pH compartments. The methodology was then applied to study the effects of drug transporters on Fic. The uptake transporter OATP1B1 increased the Fic of its substrates in a concentration-dependent manner. In contrast, the Fic of P-gp substrates was decreased when P-gp was present. In human hepatocytes, the methodology allowed the determination of Fic without prior knowledge of transporter mechanisms or metabolic activity. Finally, the methodology was applied to measure the impact of Fic on target binding and cellular drug response. Intracellular concentrations of active metabolites of pro-drugs targeting the intracellular target thymidylate synthase were in agreement with the level of binding to this target. Further, high Fic was generally required for kinase and protease inhibitors to be act

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.

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.

Intracellular unbound drug concentrations determine affinity to targets in the cell interior. However, due to difficulties in measuring them, they are often overlooked in pharmacology. Here we present a simple experimental technique for the determination of unbound intracellular drug concentrations in cultured cells that is based on parallel measurements of cellular drug binding and steady-state intracellular drug concentrations. Binding in HEK293 cells was highly correlated with binding in liver-derived systems, whereas binding in plasma did not compare well with cellular binding. Compound lipophilicity increased drug binding, while negative charge and aromatic functional groups decreased binding. Intracellular accumulation of unbound drug was consistent with pH dependent subcellular sequestration, as confirmed by modeling and by inhibition of subcellular pH gradients. The approach developed here can be used to measure intracellular unbound drug concentrations in more complex systems, for example, cell lines with controlled expression of transporters and enzymes or primary cells.

Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC 50 value, is restored at 20% level with 1 μM CA IX inhibitor. © 2012 Elsevier Ltd. All rights reserved.

Nanoscience has arrived. Biological applications of nanoscience are particularly prominent and can be useful in a range of disciplines. Advances in nanoscience are underpinning breakthroughs in biomedical research and are beginning to be adopted by the rheumatology and musculoskeletal science communities. Within these fields, nanoscience can be applied to imaging, drug delivery, implant development, regenerative medicine, and the characterization of nanoscale features of cells, matrices and biomaterials. Nanoscience and nanotechnology also provide means by which the interaction of cells with their environment can be studied, thereby increasing the understanding of disease and regenerative processes. Although its potential is clear, nanoscience research tends to be highly technical, generally targeting an audience of physicists, chemists, materials scientists and engineers, and is difficult for a general audience to follow. This Review aims to step back from the most technical aspects of nanoscience and provide a widely accessible view of how it can be applied to advance the field of rheumatology, with an emphasis on technologies that can have an immediate impact on rheumatology and musculoskeletal research. © 2013 Macmillan Publishers Limited.

Indoleamine 2,3-dioxygenase (IDO) is an interferon-gamma-inducible intracellular enzyme which catalyses the catabolism of tryptophan. The effects of its activity are tryptophan deficiency, excess tryptophan breakdown products (kynurenines) and consumption of reactive oxygen species. Tryptophan deficiency and kynurenine excess have immunodulatory effects including suppressing lymphocyte responses particularly by sensitizing them to apoptosis, which is of interest in many fields of research, particularly transplantation. In several transplant models, increased IDO activity in transplanted cells has been demonstrated to have antirejection properties both in vitro and in vivo. Recently, CTLA4, whether membrane bound or in the form of the costimulation blocking agent, CTLA4Ig, was determined to have much of its effect via increased IDO activity in dendritic cells. This finding, coupled with the capacity of IDO competent dendritic cells to induce T-regulatory cells through high levels of IDO activity, suggest a possible peripheral tolerogenic pathway with important implications for transplantation. Many other areas of transplantation in which IDO activity may be of benefit remain unexplored. In concert with experiments examining increased IDO activity for prolonged graft survival, studies continue to better define the pathophysiologic role of IDO. Understanding more clearly the implications of IDO activity on different cell types is allowing a more focused approach to determining if IDO has a role in generating transplantation tolerance. © 2008 The Authors.

Neurochemical changes were examined in dopamine D1 receptor knockout (D1-/-), dopamine D3 receptor knockout (D3-/-) and dopamine D1/D3 receptor double knockout (D1-/-D3-/-) mice. The level of dopamine D1- and D2-like receptors and gamma-aminobutyric acid (GABAA) receptor was assessed by ligand autoradiography and dopamine D1- and D2 receptor, enkephalin, dynorphin and substance P transcripts measured by in situ hybridization. D1-/- mice had normal GABAA receptor levels, reduced dynorphin and substance P, and increased enkephalin mRNA and dopamine D2-like binding. D1-/-D3-/- mice evidenced decreased dynorphin and substance P but normal enkephalin expression, whereas dopamine D2-like and GABAA receptor binding were increased. Major changes occur in substance P and dynorphin expression in D1-/- mice and these changes are unaffected by loss of dopamine D3 receptors. Upregulated dopamine D2-like binding and enkephalin in D1-/- mice may be due to decreased dopamine turnover. Upregulated enkephalin in D1-/- mice is dependent on functional dopamine D3 receptors. © 2003 Elsevier Science B.V. All rights reserved.

Indoleamine 2,3-dioxygenase (IDO) is an interferon-gamma-inducible intracellular enzyme which catalyses the catabolism of tryptophan. The effects of its activity are tryptophan deficiency, excess tryptophan breakdown products (kynurenines) and consumption of reactive oxygen species. Tryptophan deficiency and kynurenine excess have immunodulatory effects including suppressing lymphocyte responses particularly by sensitizing them to apoptosis, which is of interest in many fields of research, particularly transplantation. In several transplant models, increased IDO activity in transplanted cells has been demonstrated to have antirejection properties both in vitro and in vivo. Recently, CTLA4, whether membrane bound or in the form of the costimulation blocking agent, CTLA4Ig, was determined to have much of its effect via increased IDO activity in dendritic cells. This finding, coupled with the capacity of IDO competent dendritic cells to induce T-regulatory cells through high levels of IDO activity, suggest a possible peripheral tolerogenic pathway with important implications for transplantation. Many other areas of transplantation in which IDO activity may be of benefit remain unexplored. In concert with experiments examining increased IDO activity for prolonged graft survival, studies continue to better define the pathophysiologic role of IDO. Understanding more clearly the implications of IDO activity on different cell types is allowing a more focused approach to determining if IDO has a role in generating transplantation tolerance. © 2008 The Authors.

Neurochemical changes were examined in dopamine D1 receptor knockout (D1-/-), dopamine D3 receptor knockout (D3-/-) and dopamine D1/D3 receptor double knockout (D1-/-D3-/-) mice. The level of dopamine D1- and D2-like receptors and gamma-aminobutyric acid (GABAA) receptor was assessed by ligand autoradiography and dopamine D1- and D2 receptor, enkephalin, dynorphin and substance P transcripts measured by in situ hybridization. D1-/- mice had normal GABAA receptor levels, reduced dynorphin and substance P, and increased enkephalin mRNA and dopamine D2-like binding. D1-/-D3-/- mice evidenced decreased dynorphin and substance P but normal enkephalin expression, whereas dopamine D2-like and GABAA receptor binding were increased. Major changes occur in substance P and dynorphin expression in D1-/- mice and these changes are unaffected by loss of dopamine D3 receptors. Upregulated dopamine D2-like binding and enkephalin in D1-/- mice may be due to decreased dopamine turnover. Upregulated enkephalin in D1-/- mice is dependent on functional dopamine D3 receptors. © 2003 Elsevier Science B.V. All rights reserved.

Background: The in vivo diagnosis of cerebral amyloid angiopathy (CAA) is inferred from clinical and structural imaging features. C-Pittsburgh compound B (PIB) is a PET ligand that binds to beta-amyloid in extracellular plaques and vessel walls. We hypothesized that patients with a clinical diagnosis of CAA-related hemorrhage (CAAH) have increased C-PIB uptake and that the pattern differs from Alzheimer disease (AD). Methodology: Patients with CAAH based on established clinical criteria were studied using C-PIB PET and were compared with age-matched controls and patients with AD. Distribution volume ratio (DVR) parametric maps were created using the cerebellar cortex as a reference region. Result(s): Twelve patients with CAAH of mean age 73.9 (range 58-93) years were compared with 22 normal controls and 13 patients with AD of mean age 71.8 (59-83) and 73.8 (56-90) years, respectively. CAAH PIB median DVR binding was higher in cortical regions (1.69, interquartile range 1.44-1.97) compared with controls (1.32, 1.21-1.44, p = 0.002) but lower than AD (2.04, 1.93-2.26, p = 0.004). The occipital-global uptake ratio was lower among patients with AD than among patients with CAAH (p = 0.008), and the frontal-global uptake ratio was higher (p = 0.012). Conclusion(s): C-Pittsburgh compound B (PIB) binding is moderately increased in most patients with probable cerebral amyloid angiopathy (CAA)-related intracerebral hemorrhage. The distribution may differ from that seen in Alzheimer disease. C-PIB PET may assist in the in vivo diagnosis of CAA and serve as a surrogate marker for future therapeutic studies. © 2010 by AAN Enterprises, Inc.

Background: The in vivo diagnosis of cerebral amyloid angiopathy (CAA) is inferred from clinical and structural imaging features. C-Pittsburgh compound B (PIB) is a PET ligand that binds to beta-amyloid in extracellular plaques and vessel walls. We hypothesized that patients with a clinical diagnosis of CAA-related hemorrhage (CAAH) have increased C-PIB uptake and that the pattern differs from Alzheimer disease (AD). Methodology: Patients with CAAH based on established clinical criteria were studied using C-PIB PET and were compared with age-matched controls and patients with AD. Distribution volume ratio (DVR) parametric maps were created using the cerebellar cortex as a reference region. Result(s): Twelve patients with CAAH of mean age 73.9 (range 58-93) years were compared with 22 normal controls and 13 patients with AD of mean age 71.8 (59-83) and 73.8 (56-90) years, respectively. CAAH PIB median DVR binding was higher in cortical regions (1.69, interquartile range 1.44-1.97) compared with controls (1.32, 1.21-1.44, p = 0.002) but lower than AD (2.04, 1.93-2.26, p = 0.004). The occipital-global uptake ratio was lower among patients with AD than among patients with CAAH (p = 0.008), and the frontal-global uptake ratio was higher (p = 0.012). Conclusion(s): C-Pittsburgh compound B (PIB) binding is moderately increased in most patients with probable cerebral amyloid angiopathy (CAA)-related intracerebral hemorrhage. The distribution may differ from that seen in Alzheimer disease. C-PIB PET may assist in the in vivo diagnosis of CAA and serve as a surrogate marker for future therapeutic studies. © 2010 by AAN Enterprises, Inc.

The interaction of chlorpromazine (CPZ) with artificial membranes (egg-yolk phosphatidylcholine liposomes) has been studied. Measurements of the surface electric potential, which is modified in the presence of the ionized form of the drug, were obtained by electron paramagnetic resonance spectroscopy (EPR) using a positively charged amphiphilic spin-probe. This probe partitions between the aqueous and lipidic phases depending on the surface potential and on the structural state of the membrane. The surface potential was measured as a function of drug concentration in the range where the spectral line-shapes are not affected by the incorporation of the drug. From these experimental results and through an appropriate formalism we obtain information on the binding of the drug to the lipid bilayer and on the ionization of the drug in the lipidic phase. © 1995 Springer-Verlag., SCOPUS: ar.j, info:eu-repo/semantics/published

Ligand-protein inverse docking has recently been introduced as a computer method for identification of potential protein targets of a drug. A protein structure database is searched to find proteins to which a drug can bind or weakly bind. Examples of potential applications of this method in facilitating drug discovery include: (1) identification of unknown and secondary therapeutic targets of a drug, (2) prediction of potential toxicity and side effect of an investigative drug, and (3) probing molecular mechanism of bioactive herbal compounds such as those extracted from plants used in traditional medicines. This method and recent results on its applications in solving various drug discovery problems are reviewed., Singapore-MIT Alliance (SMA)

Imipramine penetration into the lipid core of a membrane was demonstrated through measurements on lipid monolayers (surface pressure and surface potential). The surface pressure measurements allow us to calculate the intrinsic binding constant (partition coefficient) for the lipid-Imipramine interaction. This latter value is in correct agreement with the results obtained by electrophoretic mobility measurements on liposomes. In addition, it was observed that the same mole fraction of "lipid-soluble drug" (Chlorpromazine or Imipramine) incorporated in a given lipidic phase (DPPC) induced the same shift in the transition temperature. Copyright 1999 Academic Press., JOURNAL ARTICLE, info:eu-repo/semantics/published

Imipramine penetration into the lipid core of a membrane was demonstrated through measurements on lipid monolayers (surface pressure and surface potential). The surface pressure measurements allow us to calculate the intrinsic binding constant (partition coefficient) for the lipid-Imipramine interaction. This latter value is in correct agreement with the results obtained by electrophoretic mobility measurements on liposomes. In addition, it was observed that the same mole fraction of "lipid-soluble drug" (Chlorpromazine or Imipramine) incorporated in a given lipidic phase (DPPC) induced the same shift in the transition temperature. Copyright 1999 Academic Press., JOURNAL ARTICLE, info:eu-repo/semantics/published

The aim of the study was to find out whether bisphosphonates transform into insoluble material in human blood and serum in vitro. Samples of fresh blood and serum were incubated with various concentrations of 14C-labelled clodronate, etidronate, pamidronate and tiludronate for 2 h and 8 h at 37 °C. The presence of unfiltrable material in the plasma separated from the blood, and in the serum were studied with 1) 100, 300 and 1,000 kd (kilo Daltons) filter tubes centrifuged at 3,000 g for 60 min, and 2) high-speed centrifugation at 13,000 g for 30 min. The radioactivities in the ultrafiltrates and supernatants were compared to those in the native plasma or serum. All bisphosphonates transformed into unfiltrable material, which was separated from the samples with the 100 and 300 kd filters but not with the 1,000 kd filter. The material was not sedimented with the high-speed centrifugation. The lengthening of the incubation time from 2 h to 8 h increased the unfiltrable fraction, which generally was dependent on the drug concentration in the blood, too. However, the fraction of the unfiltrable material did not seem to increase with time when the drug was incubated with serum instead of blood. Since drug binding to plasma proteins is generally a very rapid process, some factors other than proteins only, e.g. cations or cation residues, present in the blood but not in the serum, should be involved in transforming of bisphosphonates into insoluble material in the blood.