Structure Modeling of the Norepinephrine Transporter

The norepinephrine transporter (NET) is one of the monoamine transporters. Its X-ray crystal structure has not been obtained yet. Inhibitors of human NET (hNET) play a major role in the treatment of many central and peripheral nervous system diseases. In this study, we focused on the spatial structure of a NET constructed by homology modeling on Drosophila melanogaster dopamine transporter templates. We further examined molecular construction of primary binding pocket (S1) together with secondary binding site (S2) and extracellular loop 4 (EL4). The next stage involved docking of transporter inhibitors: Reboxetine, duloxetine, desipramine, and other commonly used drugs. The procedure revealed the molecular orientation of residues and disclosed ones that are the most important for ligand binding: Phenylalanine F72, aspartic acid D75, tyrosine Y152, and phenylalanine F317. Aspartic acid D75 plays a key role in recognition of the basic amino group present in monoamine transporter inhibitors and substrates. The study also presents a comparison of hNET models with other related proteins, which could provide new insights into their interaction with therapeutics and aid future development of novel bioactive compounds

Enhanced thermal conductivity of polyamide-based nanocomposites containing graphene oxide sheets decorated with compatible polymer brushes

Polyamide-based nanocomposites containing graphene platelets decorated with poly(acrylamide) brushes were prepared and characterized. The brushes were grafted from the surface of graphene oxide (GO), a thermally conductive additive, using atom transfer radical polymerization, which led to the formation of the platelets coated with covalently tethered polymer layers (GO_PAAM), accounting for ca. 31% of the total mass. Polyamide-6 (PA6) nanocomposites containing 1% of GO_PAAM were formed by extrusion followed by injection molding. The thermal conductivity of the nanocomposite was 54% higher than that of PA6 even for such a low content of GO. The result was assigned to strong interfacial interactions between the brushes and PA6 matrix related to hydrogen bonding. Control nanocomposites containing similarly prepared GO decorated with other polymer brushes that are not able to form hydrogen bonds with PA6 revealed no enhancement of the conductivity. Importantly, the nanocomposite containing GO_PAAM also demonstrated larger tensile strength without deteriorating the elongation at break value, which was significantly decreased for the other coated platelets. The proposed approach enhances the interfacial interactions thanks to the covalent tethering of dense polymer brushes on 2D fillers and may be used to improve thermal properties of other polymer-based nanocomposites with simultaneous enhancement of their mechanical properties

Molecular modeling studies on the multistep reactivation process of organophosphate-inhibited acetylcholinesterase and butyrylcholinesterase

Poisoning with organophosphorus compounds used as pesticides or misused as chemical weapons remains a serious threat to human health and life. Their toxic effects result from irreversible blockade of the enzymes acetylcholinesterase and butyrylcholinesterase, which causes overstimulation of the cholinergic system and often leads to serious injury or death. Treatment of organophosphorus poisoning involves, among other strategies, the administration of oxime compounds. Oximes reactivate cholinesterases by breaking the covalent bond between the serine residue from the enzyme active site and the phosphorus atom of the organophosphorus compound. Although the general mechanism of reactivation has been known for years, the exact molecular aspects determining the efficiency and selectivity of individual oximes are still not clear. This hinders the development of new active compounds. In our research, using relatively simple and widely available molecular docking methods, we investigated the reactivation of acetyl- and butyrylcholinesterase blocked by sarin and tabun. For the selected oximes, their binding modes at each step of the reactivation process were identified. Amino acids essential for effective reactivation and those responsible for the selectivity of individual oximes against inhibited acetyl- and butyrylcholinesterase were identified. This research broadens the knowledge about cholinesterase reactivation and demonstrates the usefulness of molecular docking in the study of this process. The presented observations and methods can be used in the future to support the search for new effective reactivators

Emissions from a medium-duty crdi engine fuelled with diesel : biodiesel blends

In the present work, biofuels produced from different raw fatty materials have been proposed as a dominant fuel component in biodiesel–diesel fuel blends. Biofuels were produced from pork lard and rapeseed oil by alkali transesterification using methyl alcohol. Blends of biofuels in volumetric proportions of 60 and 80% of the biocomponent and the remaining part of the conventional fuel were used in a compression ignition engine designed for medium-duty vehicles. The experiments were conducted at two engine rotational speeds (1500 and 3000 rpm, respectively) and a set of load conditions (50, 100, and 200 Nm, respectively). The tests focused on engine efficiency parameters (brake-specific fuel consumption and brake fuel conversion efficiency) as well as exhaust gas emissions (hydrocarbons, carbon monoxide, and carbon dioxide were determined). The obtained results indicate that blends containing biocomponents produced from pork lard were characterized by superior fuel consumption and efficiency results, compared to blends containing biocomponents produced from rapeseed oil. In terms of exhaust emissions, biocomponents produced from pork lard were also characterized by lower emission of all of the examined components compared to rapeseed methyl ester–diesel blends. This study proposes that fuel components obtained from custom (animal) raw-fatty material can be an effective substitute for commonly used rapeseed oil methyl esters.© The Authors. Published by Exeley Inc. with Creative Commons licence (CC BY 4.0).fi=vertaisarvioitu|en=peerReviewed

Efficacy and safety of obinutuzumab-chlorambucil combination in the frontline treatment of elderly patients with chronic lymphocytic leukemia and comorbidities : real‑life data from Polish Adult Leukemia Group (PALG) analysis

Fludarabine- or bendamustine‑based upfront immunochemotherapy is the current standard of care in fit patients with chronic lymphocytic leukemia (CLL). These regimens are poorly tolerated by patients with comorbidities, for whom the obinutuzumab-chlorambucil combination became the recommended first‑line treatment. We aimed to analyze real‑life experience with the obinutuzumab-chlorambucil combination as the frontline treatment in elderly and unfit patients. The retrospective analysis included 86 elderly patients (median age, 74 years) with CLL and a significant burden of comorbidities, treated with obinutuzumab-chlorambucil as the frontline regimen. All patients had a Cumulative Illness Rating Scale score greater than 6 and/or creatinine clearance of 30 to 69 ml/min. Overall response rate at 2 months after treatment completion was 95.3%, with complete remission (CR) rate of 43% and partial remission (PR) rate of 52.3%. Stable disease rate was 4.7%. Progressive disease was not observed after treatment completion. The median progression‑free survival (PFS) was not reached after a median follow‑up of 18 months; estimated PFS at 30 months was 62%. We observed 6 relapses (7%), 3 (3.5%) in patients obtaining CR, and 3 (3.5%) in those with PR after immunochemotherapy. The most frequent adverse events were neutropenia and infusion‑related reactions (IRRs). Grade-3 neutropenia occurred in 11.6% of patients, and grade-3 IRRs, in 2.3%. There were no adverse events of grade 4 or 5. Our data confirm that the obinutuzumab-chlorambucil combination is an effective and well‑tolerated regimen in untreated CLL patients with comorbidities

New tetrahydroacridine hybrids with dichlorobenzoic acid moiety demonstrating multifunctional potential for the treatment of Alzheimers disease

A series of new tetrahydroacridine and 3,5-dichlorobenzoic acid hybrids with different spacers were designed, synthesized, and evaluated for their ability to inhibit both cholinesterase enzymes. Compounds 3a, 3b, 3f, and 3g exhibited selective butyrylcholinesterase (EqBuChE) inhibition with IC50 values ranging from 24 to 607 nM. Among them, compound 3b was the most active (IC50 = 24 nM). Additionally, 3c (IC50 for EeAChE = 25 nM and IC50 for EqBuChE = 123 nM) displayed dual cholinesterase inhibitory activity and was the most active compound against acetylcholinesterase (AChE). Active compound 3c was also tested for the ability to inhibit Aβ aggregation. Theoretical physicochemical properties of the compounds were calculated using ACD Labs Percepta and Chemaxon. A Lineweaver-Burk plot and docking study showed that 3c targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Moreover, 3c appears to possess neuroprotective activity and could be considered a free-radical scavenger. In addition, 3c did not cause DNA damage and was found to be less toxic than tacrine after oral administration; it also demonstrated little inhibitory activity towards hyaluronidase (HYAL), which may indicate that it possesses anti-inflammatory properties. The screening for new in vivo interactions between 3c and known receptors was realized by yeast three-hybrid technology (Y3H)

Analysis of Different Binding Modes for Tiagabine within the GAT-1 Transporter

The recently obtained cryo-electron microscopy structure (PDB code: 7SK2) of the human γ-aminobutyric acid transporter type 1 (hGAT-1) in complex with the antiepileptic drug, tiagabine, revealed a rather unexpected binding mode for this inhibitor in an inward-open state of the transporter. The simultaneously released crystal structures of the modified dopamine transporter with mutations mimicking hGAT-1 indicated an alternative binding mode for the tiagabine analogues that were found to block the transporter in an outward-open state, which is more consistent with the results of previous biological and molecular modeling studies. In view of the above discrepancies, our study compares different hypothetical tiagabine binding modes using classical and accelerated molecular dynamics simulations, as well as MM-GBSA free binding energy (dG) calculations. The results indicate that the most stable and energetically favorable binding mode of tiagabine is the one where the nipecotic acid fragment is located in the main binding site (S1) and the aromatic rings are arranged within the S2 site of the hGAT-1 transporter in an outward-open state, confirming the previous molecular modelling findings. The position of tiagabine bound to hGAT-1 in an inward-open state, partially within the intracellular release pathway, was significantly less stable and the dG values calculated for this complex were higher. Furthermore, analysis of the cryo-electron map for the 7SK2 structure shows that the model does not appear to fit into the map optimally at the ligand binding site. These findings suggest that the position of tiagabine found in the 7SK2 structure is rather ambiguous and requires further experimental verification. The identification of the main, high-affinity binding site for tiagabine and its analogues is crucial for the future rational design of the GABA transporter inhibitors

Modeling of structure of gamma-aminobutyric acid transporters.

Wstęp niniejszej pracy magisterskiej zawiera charakterystykę układu GABA-ergicznego ze szczególnym uwzględnieniem roli wychwytu zwrotnego kwasu gamma-aminomasłowego. Opisano lokalizacje, funkcje oraz znane inhibitory każdego typu transportera GABA (GAT-1, BGT-1, GAT-2, GAT-3). Przedstawiono ogólną budowę oraz molekularny mechanizm działania białek spokrewnionych z tymi transporterami. Część ta opisuje również możliwości wykorzystania inhibitorów wychwytu zwrotnego GABA w leczeniu padaczki, lęku, depresji, a także bólu ostrego i neuropatycznego. W części eksperymentalnej przedstawiono modelowanie homologiczne struktury ludzkich transporterów GABA. Jako matryce wybrano odpowiednie struktury krystaliczne transporterów dopaminowych, serotoninowych, leucynowych, w różnych stanach konformacyjnych. Po dokładnym uliniowieniu sekwencji aminokwasowej wybranych matryc z sekwencjami modelowanych celów, zbudowano różnorodną grupę modeli dla każdego typu ludzkich transporterów GABA, przy użyciu programu Modeller i serwisu SWISS-MODEL. Otrzymane modele oceniono następnie przy pomocy różnych narzędzi: QMEAN, DOPEscore, PROCHECK. Z punktu widzenia analizy oddziaływań ligand-transporter, najważniejsza jest prawidłowa budowa miejsc wiążących. Aby wybrać najlepsze pod tym kątem modele przeprowadzono dokowanie wyselekcjonowanych wcześniej ligandów przy pomocy programów GOLD i Glide. Pozwoliło to na uzyskanie modeli, do których można zadokować ligandy, wyjaśniając ich aktywność lub jej brak, a także selektywność względem danego typu transportera. Uzyskane wyniki są dobrym punktem wyjścia do dalszych badań mających na celu uzyskanie modeli, na podstawie których możliwe będzie projektowanie nowych, selektywnych inhibitorów transporterów GABA.Introduction of the master thesis includes characteristic of the GABA-ergic system with emphasis on role of γ-aminobutyric acid (GABA) uptake. Localization, function and known inhibitors for each type of GABA transporter (GAT-1, BGT-1, GAT-2, GAT-3) were described. General structure and molecular mechanism of action for proteins related to GABA-transporters were presented. This section also describes possibilities of using GABA uptake inhibitors in treatment of epilepsy, anxiety disorders, depression, acute and neuropathic pain. In the experimental part, homology modeling of the structure of human GABA transporters was presented. Suitable crystal structures of dopamine, serotonin and leucine transporters, in various conformational states were used as templates. After careful alignment of templates amino acid sequences with modeled protein sequences, a variety of models for each type of human GABA transporter were obtained using the Modeller and the SWISS-MODEL. Constructed models were next evaluated using various tools like QMEAN, DOPEscore, PROCHECK. From the view point of analysis of ligand-transporter interactions, the most important is proper construction of binding sites. To choose the best models in this respect, docking of previously selected ligands was performed using programs GOLD and Glide. It allowed to obtain models to which it is possible dock ligands, explaining their activity or lack of it, as well as selectivity. Obtained results are a good starting point for further research aimed to obtaining models based on which, it will be possible to design new selective GABA transporters inhibitors

Analysis of binding determinants for different classes of competitive and noncompetitive inhibitors of glycine transporters

Glycine transporters are interesting therapeutic targets as they play significant roles in glycinergic and glutamatergic systems. The search for new selective inhibitors of particular types of glycine transporters (GlyT-1 and GlyT-2) with beneficial kinetics is hampered by limited knowledge about the spatial structure of these proteins. In this study, a pool of homology models of GlyT-1 and GlyT-2 in different conformational states was constructed using the crystal structures of related transporters from the SLC6 family and the recently revealed structure of GlyT-1 in the inward-open state, in order to investigate their binding sites. The binding mode of the known GlyT-1 and GlyT-2 inhibitors was determined using molecular docking studies, molecular dynamics simulations, and MM-GBSA free energy calculations. The results of this study indicate that two amino acids, Gly373 and Leu476 in GlyT-1 and the corresponding Ser479 and Thr582 in GlyT-2, are mainly responsible for the selective binding of ligands within the S1 site. Apart from these, one pocket of the S2 site, which lies between TM3 and TM10, may also be important. Moreover, selective binding of noncompetitive GlyT-1 inhibitors in the intracellular release pathway is affected by hydrophobic interactions with Ile399, Met382, and Leu158. These results can be useful in the rational design of new glycine transporter inhibitors with desired selectivity and properties in the future