Hydrophobic Ring Research Articles

Inhibition of bacterial undecaprenyl pyrophosphate synthase by small fungal molecules.

Viridicatumtoxin and spirohexaline, small fungal molecules with a tetracyclic scaffold and an additional spirobicyclic ring in common, were found to inhibit bacterial undecaprenyl pyrophosphate (UPP) synthase with IC50 values of 4 and 9 μm, respectively. These molecules showed weak inhibitory activity against catalytically related enzymes such as bacterial octaprenyl pyrophosphate synthase and yeast dehydrodolichyl pyrophosphate synthase, indicating that the compounds preferentially inhibit UPP synthase. They showed antimicrobial activity, particularly against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, molecular modeling strongly suggested that the hydrophobic spirobicyclic ring of viridicatumtoxin interacts with three hydrophobic clefts of the active site in MRSA UPP synthase.

The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2

BackgroundThe cytochrome P450 enzyme CYP199A4 can efficiently demethylate 4-methoxybenzoic acid. The substrate is positioned in the enzyme active site with the methoxy group ideally positioned for demethylation. This occurs through interactions of hydrophobic benzene ring with aromatic phenylalanine residues and the charged carboxylate group with polar and basic amino acids. MethodsIn vitro substrate binding and kinetic turnover assays coupled with HPLC and GC–MS analysis and whole-cell oxidation turnovers. ResultsModification of the carboxylate group to an amide or aldehyde resulted in substrate binding, as judged by the almost total shift of the spin state to the high-spin form, but binding was three orders of magnitude weaker. Changing the carboxylate to phenol alcohol, ketone, ester and nitro groups and boronic, sulfinic and sulfonic acids resulted in a dramatic reduction in the binding affinity. Even phenylacetic acids were mediocre substrates for CYP199A4, despite maintaining a carboxylate group. The weaker binding of all of these substrates results in lower levels of turnover activity and product formation compared to 4-methoxybenzoic acid. ConclusionSubstrate binding to CYP199A4 is tightly regulated by interactions between the 4-methoxybenzoic acid and the amino acids in the active site. The benzoic acid carboxylate moiety is critical for optimal substrate binding and turnover activity with CYP199A4. General significanceAn understanding of how the CYP199A4 enzyme has evolved to be highly selective for para-substituted benzoic acids. This provides valuable insight into how other, as yet structurally uncharacterised, monooxygenase enzymes may bind benzoic acid substrates.

Computational Study of Anthracycline Interactions with Membrane-Embedded P-Glycoprotein

P-glycoprotein (P-gp), an ATP-dependent efflux pump, is responsible for multi-drug resistance when overexpressed in cancer cells due to its broad substrate specificity. A clear understanding of the P-gp binding pathway and specificity is necessary for the design of effective anti-tumor agents. Daunorubicin and idarubicin are chemotherapeutic agents susceptible to P-gp-mediated efflux. Although both drugs are structurally similar, idarubicin is four times more lipophilic and is resistant to P-gp-mediated efflux. A combination of microsecond timescale atomistic molecular dynamics (MD) simulations and adaptive biasing force simulations were used to sample conformations of the protein and drugs in a lipid bilayer. Our simulations show that a lipid-solvated cavity in P-gp more accurately reproduces the inward-facing conformation found in the crystal structures. More importantly the substrate portal, the gate between the cavity and the membrane inner leaflet, remains open to drug entry when it has been previously observed to close in simulation. Unrestrained MD simulations of daunorubicin embedded in a lipid bilayer show drug diffusion from bulk lipid to this substrate portal. Potential of mean force (PMF) calculations of the drugs along the membrane normal show minima in the hydrophobic core of the membrane. Though the drug centers-of-mass are at similar membrane depths, the methoxy functional group in daunorubicin results in the drug orienting close to the head group region of the membrane. Idarubicin, which lacks the methoxy group, orients its hydrophobic ring system towards the membrane center. Continuing studies are aimed at determining the consequences of drug orientation in the membrane as they enter the protein cavity through the substrate portal as well as characterizing the drugs in the active site.

Effects of Benzoic Acid and its Analogues on Osmotic Fragility in Guinea Pig Erythrocytes In Vitro

In our previous report, benzoic acid and some of its chemical analogues were shown to increase osmotic fragility (OF) in isolated rat red blood cells (RBCs). However, there have been no reports on the effects of benzoic acid and its derivatives on OF in the RBCs of other animals. To clarify these inter-species differences, the effects of those chemicals on OF were examined in guinea pig RBCs. The isolated RBCs were exposed to the chemicals at a concentration of 0.1-100 mM for 1 h. OF was measured by determining the 50% hemolysis of RBCs using a 0.1-0.8% NaCl solution. Benzoic acid and some of its derivatives decreased OF in a dose dependent manner. Replacement of the carboxylic group with another group or the introduction of another element on the benzene ring also affected OF. These effects were dependent on both the type of molecule and the position at which they were introduced into the benzene nucleus. The introduction of chlorine, bromine or iodine tended to increase OF, even at low concentrations. With regard to the action of the tested chemicals, the hydrophobic benzene ring is thought to enter the phospholipid layer while the hydrophilic carboxylic group remains at the membrane surface, perturbing the RBC membrane. Inter-species differences between rat and guinea pig were confirmed in the response of the RBC membrane to benzoic acid and its derivatives. These differences are speculated to be due to differences in the lipid composition of the RBC membrane.

Flexible Ion Barrier

The central vasculature of plant roots is protected by a hydrophobic ring of endodermal cells that are enclosed by lamellae of suberin. Barberon et al. demonstrate that endodermal suberization plasticity facilitates ion homeostasis, with antithetical regulation of suberin deposition and degradation by the phytohormones abscisic acid and ethylene.

Effect of pH and Salt on Adsorption of Double-Stranded DNA on Graphene Oxide.

Graphene oxide (GO) has a large surface-to-volume ratio and hydrophobic hexagonal rings that can interact with biomolecules. Single-stranded DNA adsorbs strongly to the surface of GO via hydrophobic interactions. GO has been used in optical biosensors and biomedical platforms for the detection of DNA, proteins, and small molecules. This study was designed to measure the adsorption of double-stranded DNA (dsDNA) onto GO according to DNA length, salt concentration, and pH of the reaction. Results showed that dsDNA molecules were adsorbed progressively as the pH changed from 6.0 to 4.0. At high pH, dsDNA adsorption was enhanced by the presence of MgCl2 rather than NaCl. Desorption of DNA from GO, with triton X-100 led to the rapid release of DNA from GO in the presence of MgCl2.

Rosin: Recent advances and potential applications in novel drug delivery system

Gum exudates are among the oldest natural polymer. Natural gums have remained one of the major areas of interest for the researchers for their applicability in the drug delivery system because of their wide availability, inexpensiveness and availability in a variety of structures with varied properties. They can be easily modified chemically, biochemically and are highly stable, safe, non-toxic, gel forming and in addition are biodegradable. Rosin is one of the natural gums obtained as resinous constituent of the oleoresin exuded by various species of pine, mostly conifers. Rosin consists primarily of abietic- and pimaric-type resin acids (or rosin acids) with characteristic hydrophobic hydrophenanthrene rings which impart them excellent film-forming properties. Rosin and its derivatives are biopolymers that are increasingly used for their pharmaceutical applications. In the pharmaceutical field, it has been investigated for film-forming and coating properties, microencapsulation and as a matrix material in the tablets for sustained and controlled drug release. This article reviews the literature on rosin and its derivative and describes the varied applications of the rosin and their future exploitation in novel drug delivery systems.

Correlating molecular character of NIR imaging agents with tissue-specific uptake.

Near-infrared (NIR) fluorescent contrast agents are emerging in optical imaging as sensitive, cost-effective, and nonharmful alternatives to current agents that emit harmful ionizing radiation. Developing spectrally distinct NIR fluorophores to visualize sensitive vital tissues to selectively avoid them during surgical resection of diseased tissue is of great significance. Herein, we report the synthetic variation of pentamethine cyanine fluorophores with modifications of physicochemical properties toward prompting tissue-specific uptake into sensitive tissues (i.e., endocrine glands). Tissue-specific targeting and biodistribution studies revealed localization of contrast agents in the adrenal and pituitary glands, pancreas, and lymph nodes with dependence on molecular characteristics. Incorporation of hydrophobic heterocyclic rings, alkyl groups, and halogens allowed a fine-tuning capability to the hydrophobic character and dipole moment for observing perturbation in biological activity in response to minor structural alterations. These NIR contrast agents have potential for clinical translation for intraoperative imaging in the delineation of delicate glands.

Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking

The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase.

Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg2+ or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode.

The Ingenious Structure of Central Rotor Apparatus in VoV1; Key for Both Complex Disassembly and Energy Coupling between V1 and Vo

Vacuolar type rotary H+-ATPases (VoV1) couple ATP synthesis/hydrolysis by V1 with proton translocation by Vo via rotation of a central rotor apparatus composed of the V1-DF rotor shaft, a socket-like Vo-C (eukaryotic Vo-d) and the hydrophobic rotor ring. Reconstitution experiments using subcomplexes revealed a weak binding affinity of V1-DF to Vo-C despite the fact that torque needs to be transmitted between V1-DF and Vo-C for the tight energy coupling between V1 and Vo. Mutation of a short helix at the tip of V1-DF caused intramolecular uncoupling of VoV1, suggesting that proper fitting of the short helix of V1-D into the socket of Vo-C is required for tight energy coupling between V1 and Vo. To account for the apparently contradictory properties of the interaction between V1-DF and Vo-C (weak binding affinity but strict requirement for torque transmission), we propose a model in which the relationship between V1-DF and Vo-C corresponds to that between a slotted screwdriver and a head of slotted screw. This model is consistent with our previous result in which the central rotor apparatus is not the major factor for the association of V1 with Vo (Kishikawa and Yokoyama, J Biol Chem. 2012 24597-24603).

Hydrophobic interaction between contiguous residues in the S6 transmembrane segment acts as a stimuli integration node in the BK channel.

Large-conductance Ca(2+)- and voltage-activated K(+) channel (BK) open probability is enhanced by depolarization, increasing Ca(2+) concentration, or both. These stimuli activate modular voltage and Ca(2+) sensors that are allosterically coupled to channel gating. Here, we report a point mutation of a phenylalanine (F380A) in the S6 transmembrane helix that, in the absence of internal Ca(2+), profoundly hinders channel opening while showing only minor effects on the voltage sensor active-resting equilibrium. Interpretation of these results using an allosteric model suggests that the F380A mutation greatly increases the free energy difference between open and closed states and uncouples Ca(2+) binding from voltage sensor activation and voltage sensor activation from channel opening. However, the presence of a bulky and more hydrophobic amino acid in the F380 position (F380W) increases the intrinsic open-closed equilibrium, weakening the coupling between both sensors with the pore domain. Based on these functional experiments and molecular dynamics simulations, we propose that F380 interacts with another S6 hydrophobic residue (L377) in contiguous subunits. This pair forms a hydrophobic ring important in determining the open-closed equilibrium and, like an integration node, participates in the communication between sensors and between the sensors and pore. Moreover, because of its effects on open probabilities, the F380A mutant can be used for detailed voltage sensor experiments in the presence of permeant cations.

Exploration of the structural requirements of HIV-protease inhibitors using pharmacophore, virtual screening and molecular docking approaches for lead identification

Pharmacoinformatics approaches are widely used in the field of drug discovery as it saves time, investment and animal sacrifice. In the present study, pharmacore-based virtual screening was adopted to identify potential HIV-protease ligands as anti-HIV agents. Pharmacophore is the 3D orientation and spatial arrangement of functional groups that are critical for binding at the active site cavity. Virtual screening retrieves potential hit molecules from databases based on imposed criteria. A set of 30 compounds were selected with inhibition constant as training set from 129 compounds of dataset set and subsequently the pharmacophore model was developed. The selected best model consists of hydrogen bond acceptor and donor, hydrophobic and aromatic ring, features critical for HIV-protease inhibitors. The model exhibits high correlation (R=0.933), less rmsd (1.014), high cross validated correlation coefficient (Q2=0.872) among the ten models examined and validated by Fischer's randomization test at 95% confidence level. The acceptable parameters of test set prediction, such as Rpred2=0.768 and rm(test)2=0.711 suggested that external predictivity of the model was significant. The pharmacophore model was used to perform a virtual screening employing the NCI database. Initial hits were sorted using a number of parameters and finally seven compounds were proposed as potential HIV-protease molecules. One potential HIV-protease ligand is reportedly confirmed as an active agent for anti-HIV screening, validating the current approach. It can be postulated that the pharmacophore model facilitates the selection of novel scaffold of HIV-protease inhibitors and can also allow the design of new chemical entities.

Detection of UV-induced mutagenic thymine dimer using graphene oxide.

In this paper, we report for the first time that graphene oxide (GO) can interact with mutagenic DNA but not intact DNA. After UV-irradiated fluorophore-linked DNA containing thymine repeats was mixed with GO, a decrease in fluorescence was observed in a time-dependent manner. In contrast, no fluorescence change was observed with intact DNA, indicating that UV irradiation of DNA resulted in the formation of mutagenic bases. Because GO is known to act as a fluorescence quencher, the decreased fluorescence implies adsorption of the UV-irradiated DNA onto GO. It appears that the decreased fluorescence might result from the greater accessibility of hydrophobic methyl groups and phenyl rings of thymine dimers to GO and from deformed DNA structures with less effective charge shielding under salt-containing conditions. Using this affinity of GO for mutagenic DNA, we could detect UV-irradiated DNA at concentrations as low as 100 pM. We were also able to analyze the ability of phototoxic drugs to catalyze the formation of mutagens under UV irradiation with GO. Because our method is highly sensitive and feasible and does not require the pretreatment of DNA, we propose that it could accelerate the screening of potential phototoxic drug candidates that would be able to sensitize mutagenic dsDNA.

Human PrimPol mutation associated with high myopia has a DNA replication defect.

PrimPol is a primase-polymerase found in humans, and other eukaryotes, involved in bypassing lesions encountered during DNA replication. PrimPol employs both translesion synthesis and repriming mechanisms to facilitate lesion bypass by the replisome. PrimPol has been reported to be a potential susceptibility gene associated with the development of myopia. Mutation of tyrosine 89 to aspartic acid (PrimPolY89D) has been identified in a number of cases of high myopia, implicating it in the aetiology of this disorder. Here, we examined whether this mutation resulted in any changes in the molecular and cellular activities associated with human PrimPol. We show that PrimPolY89D has a striking decrease in primase and polymerase activities. The hydrophobic ring of tyrosine is important for retaining wild-type extension activity. We also demonstrate that the decreased activity of PrimPolY89D is associated with reduced affinities for DNA and nucleotides, resulting in diminished catalytic efficiency. Although the structure and stability of PrimPolY89D is altered, its fidelity remains unchanged. This mutation also reduces cell viability after DNA damage and significantly slows replication fork rates in vivo. Together, these findings establish that the major DNA replication defect associated with this PrimPol mutant is likely to contribute to the onset of high myopia.

Ion Conductivity of the Bacterial Translocation Channel SecYEG Engaged in Translocation

While engaged in protein transport, the bacterial translocon SecYEG must maintain the membrane barrier to small ions. The preservation of the proton motif force was attributed to (i) cation exclusion, (ii) engulfment of the nascent chain by the hydrophobic pore ring, and (iii) a half-helix partly plugging the channel. In contrast, we show here that preservation of the proton motif force is due to a voltage-driven conformational change. Preprotein or signal peptide binding to the purified and reconstituted SecYEG results in large cation and anion conductivities only when the membrane potential is small. Physiological values of membrane potential close the activated channel. This voltage-dependent closure is not dependent on the presence of the plug domain and is not affected by mutation of 3 of the 6 constriction residues to glycines. Cellular ion homeostasis is not challenged by the small remaining leak conductance.

Exploring structural requirement, pharmacophore modeling, and de novo design of LRRK2 inhibitors using homology modeling approach

A mutation in the gene, encoding leucine rich repeat kinase 2 (LRRK2), is a genetic cause of Parkinson’s disease (PD). LRRK2 is a dimeric multidomain protein, largely regulates guanosine triphosphate (GTP). G2019S and I2020T, the mutation encodes in the kinase domain of LRRK2 increase the GTPase activity, are the important regulators in pathogenesis of PD. To design potent LRRK2 inhibitors, pharmacophore modeling approach was employed with a wide chemical diversity of compound’s database. The best hypothesis consists of hydrogen-bond acceptor and donor as well as hydrophobic aliphatic and ring aromatic features. The model was validated by the test and decoy sets followed by Fischer’s randomization test. The validated model was used to screen the database of compounds, which were designed through de novo approach. Homology model of the kinase domain of LRRK2 was built initially using the crystal structure of Janus kinase 3. The designed molecules were further screened for ADMET properties, and ligand–receptor interaction of top hits was analyzed by molecular docking studies to explore potent LRRK2 inhibitors.

Synthesis of the Aluminophosphate ICP-1 by Self-Assembly of 1,3-Diphenylguanidine: Insights into Supramolecular Aggregation

1,3-Diphenylguanidine (DPG) has distinguishable polar and apolar groups, aromatic rings that can self-assemble through π–π type interactions, and high conformational flexibility. These features make it a potential self-assembling structure-directing agent in the synthesis of hybrid host–guest aluminophosphates. Computational simulations show that the molecule has a strong tendency to self-assemble in aqueous solution. Large supramolecular organic aggregates are produced, with the hydrophobic aromatic rings located in the center of the aggregates, stabilized by π–π type interactions, and the hydrophilic guanidine groups on the external surface in close contact with water molecules. With this organic molecule, a new 1-D AlPO framework material (ICP-1) was formed. Its structure, characterized by a combination of single-crystal and powder diffraction techniques, consists of AlP2O8H chains connected to the polar groups of the organic DPG molecules through a complex H-bonding network. This material has an extremely high organic content, close to that of typical mesoporous materials. However, DPG molecules are part of the ICP-1 network, rather than guest molecules in the pores, so removal of DPG results in a collapse of the structure, limiting its potential applications. Nevertheless, this work demonstrates the potential of using self-assembling organic molecules for producing very open-framework materials.

Preliminary anticonvulsant and toxicity screening of substituted benzylidenehydrazinyl-N-(6-substituted benzo[d]thiazol-2-yl)propanamides.

Keeping in view the structural requirements suggested in the pharmacophore model for anticonvulsant activity, a new series of 3-(2-(substitutedbenzylidene)hydrazinyl)-N-(substituted benzo[d]thiazol-2-yl)-propanamides were synthesized with aromatic hydrophobic aryl ring (A), NH–C=O as hydrogen bonding domain (HBD), nitrogen atom as electron donor (D), and phenyl as distal aryl ring (C). Synthesized compounds were characterized by FTIR, 1H NMR, 13C NMR, mass spectroscopy, and elemental analysis. Preliminary in vivo anticonvulsant screening (phase I) was performed by two most adopted seizure models, maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ). Based on anticonvulsant screening results, two compounds, 5h and 5p, were found to be most active; they exhibited activity comparable to standard drugs phenytoin (PHY) and carbamazepine (CBZ). These active compounds were subjected to phase II and phase III screening, where they displayed much higher protective index (PI) in comparison to the standard drugs. In phase IV screening, the bioavailability of active compounds was assessed on oral administration. Further, preliminary safety profiles of 5h and 5p were evaluated by the neurotoxicity testing and liver enzyme estimation.

Molecular Modeling of Paclitaxel Interacting with Membranes

Cell membranes define a confined space for the cell and form an essential barrier from the surrounding environments. They also provide active/passive transport systems for some essential nutrients and small molecules. But it is unclear how most of hydrophobic drugs such as paclitaxel penetrate through the cell membranes. Paclitaxel has been shown to aggregate both in hydrophilic and hydrophobic environments. Additionally it has been suggested to contribute to pore formation in the membrane. Here we investigated interactions between paclitaxel and a model cell membrane of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using molecular dynamics simulations. The change of free energy across the bilayer interface calculated by adaptive biasing force method showed good agreement with experimental data. We performed 300ns long simulations of POPC membranes with randomly inserted drugs up to 0.1 mole fraction. We compared the results with a separate set of simulations where the drugs were initially inserted at lattice points with a single orientation. Interestingly, the main baccatinic core and the three more hydrophobic phenyl rings showed different preferential positioning in the membrane along the z-axis. This was consistent with the rotation and orientation of the drug. The clustering of the drug molecules in the membrane, order parameters of lipid tails, and water penetration along with the drug clusters were analyzed. Modeling the transport of hydrophobic drugs into the cell through computational investigations will provide insights of the drug delivery process at a molecular level.