Przemyslaw Miszta

Lyotropic Cubic Phases for Drug Delivery: Diffusion and Sustained Release from the Mesophase Evaluated by Electrochemical Methods.

Lyotropic liquid crystalline systems are excellent carriers for drugs due to their biocompatibility, stability in aqueous environment, and well-defined structure that allow them to host significantly larger amounts of drugs than carriers such as liposomes or gold nanoparticles. Incorporating the drug within the mesophase gel, or the cubosome/hexosome nanoparticles, decreased its toxic effects toward healthy cells, while appropriate mechanisms can stimulate the release of the drug from the carrier when it approaches the cancerous cell environment. Electrochemical methods-chronocoulometry and voltammetry at micro and normal size electrodes-are used for the first time to simultaneously determine the diffusion coefficients and effective concentrations of a toxic anticancer drug, doxorubicin, in the channels of three liquid-crystalline lipidic cubic phases. This approach was instrumental in demonstrating that the drug diffusion and kinetics of release from the mesophases depend on the aqueous channel size, which in turn is related to the identity and structure of the amphiphilic molecules used for the formation of the mesophase. Structural parameters of the cubic phases with the incorporated drug were characterized by small-angle X-ray scattering (SAXS), and molecular dynamics simulations were applied in order to describe the differences in the distribution of doxorubicin in the cubic phase matrix at acidic and neutral pH. The release of the drug from the phase was retarded at physiological pH, while at lower pH, corresponding to the cancer environment, it was accelerated, provided that suitable amphiphilic molecules were employed for the construction of the liquid crystal drug delivery system.

Quaternary structures of opsin in live cells revealed by FRET spectrometry

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well.

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

Significance Vertebrate rhodopsin (Rh) has been a model system for many G protein-coupled receptors for over a decade. However, due to its thus-far limited repertoire of active ligands, its use in assisting the development of new therapeutic modalities and drugs has been limited. This study elucidates a photocyclic G protein activation by Rh bound with a six-carbon ring retinal (Rh6mr), and thus broadens the diversity of such Rh signaling modulators. Rh6mr does not release its chromophore after light activation, but instead the resulting photoproduct is thermally reisomerized back to its inactive state, abrogating the necessity for a complex retinoid cycle to renew its chromophore. This photocyclic behavior of Rh6mr opens up several avenues for using optogenetic tools based on vertebrate Rhs. Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a one-way reaction that activates transducin (Gt) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C11=C12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged Gt activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates Gt in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure–function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis–to–11,13-dicis isomerization.

Multitarget Strategy to Address Alzheimer's Disease: Design, Synthesis, Biological Evaluation, and Computational Studies of Coumarin-Based Derivatives.

Alzheimers disease (AD) is a major public health challenge that faces an aging global population. Current drug treatment has demonstrated only symptomatic efficacy, leaving an unmet medical need for a new generation of disease‐modifying therapies. Following the multitarget‐directed ligand approach, a small library of coumarin‐based derivatives was designed and synthesized as a follow‐up to our studies on AP2238, aimed at expanding its biological profile. The coumarin substitution pattern at the 6‐ or 7‐position was modified by introducing alkyl chains of variable lengths and with different terminal amino functional groups. 3‐(4‐{[Benzyl(ethyl)amino]methyl}phenyl)‐6‐({5‐[(7‐methoxy‐6H‐indeno[2,1‐b]quinolin‐11‐yl)amino]pentyl}oxy)‐2H‐chromen‐2‐one, bearing the bulkiest amine, emerged as a non‐neurotoxic dual acetylcholinesterase (AChE)/butyrylcholinesterase (BuChE) inhibitor, potentially suitable for the treatment of the middle stage of AD. Furthermore, the introduction of a diethylamino spacer, as in 3‐(4‐{[benzyl(ethyl)amino]methyl}phenyl)‐6‐{[5‐(diethylamino)pentyl]oxy}‐2H‐chromen‐2‐one and 3‐(4‐{[benzyl(ethyl)amino]methyl}phenyl)‐7‐[4‐(diethylamino)butoxy]‐2H‐chromen‐2‐one, led to nanomolar human AChE inhibitors endowed with significant inhibitory activity toward Aβ42 self‐aggregation, whereas the reference compound was completely ineffective. Furthermore, 3‐(4‐{[benzyl(ethyl)amino]methyl}phenyl)‐7‐[4‐(diethylamino)butoxy]‐2H‐chromen‐2‐one also showed promising neuroprotective behavior, which makes it a potential candidate for development into a disease‐modifying agent.

Polyamine Conjugation as a Promising Strategy To Target Amyloid Aggregation in the Framework of Alzheimer’s Disease

Spermine conjugates 2–6, carrying variously decorated 3,5-dibenzylidenepiperidin-4-one as bioactive motives, were designed to direct antiaggregating properties into mitochondria, using a polyamine functionality as the vehicle tool. The study confirmed mitochondrial import of the catechol derivative 2, which displayed effective antiaggregating activity and neuroprotective effects against Aβ-induced toxicity. Notably, a key functional role for the polyamine motif in Aβ molecular recognition was also unraveled. This experimental readout, which was supported by in silico studies, gives important new insight into the polyamine’s action. Hence, we propose polyamine conjugation as a promising strategy for the development of neuroprotectant leads that may contribute to decipher the complex picture of Aβ toxicity.

A novel dominant D109A CRYAB mutation in a family with myofibrillar myopathy affects αB-crystallin structure

Myofibrillar myopathy (MFM) is a group of inherited muscular disorders characterized by myofibrils dissolution and abnormal accumulation of degradation products. So far causative mutations have been identified in nine genes encoding Z-disk proteins, including αB-crystallin (CRYAB), a small heat shock protein (also called HSPB5). Here, we report a case study of a 63-year-old Polish female with a progressive lower limb weakness and muscle biopsy suggesting a myofibrillar myopathy, and extra-muscular multisystemic involvement, including cataract and cardiomiopathy. Five members of the probands family presented similar symptoms. Whole exome sequencing followed by bioinformatic analysis revealed a novel D109A mutation in CRYAB associated with the disease. Molecular modeling in accordance with muscle biopsy microscopic analyses predicted that D109A mutation influence both structure and function of CRYAB due to decreased stability of oligomers leading to aggregate formation. In consequence disrupted sarcomere cytoskeleton organization might lead to muscle pathology. We also suggest that mutated RQDE sequence of CRYAB could impair CRYAB chaperone-like activity and promote aggregation of lens crystallins.

Molecular effects of encapsulation of glucose oxidase dimer by graphene

Knowing the nature of the enzyme–graphene interface is critical for a design of graphene-based biosensors. Extensive contacts between graphene and enzyme could be obtained by employing a suitable encapsulation which does not impede its enzymatic reaction. We have performed molecular dynamics simulations to obtain an insight on many forms of contact between glucose oxidase dimer and the single-layer graphene nano-sheets. The unconnected graphene sheets tended to form a flat stack regardless of their initial positions around the enzyme, whereas the same graphene sheets linked together formed a flower-like shape engendering different forms of wrapping of the enzyme. During the encapsulation no core hydrophobic residues of the enzyme were exposed. Since the polar and charged amino acids populated the enzymes surface we also estimated, using DFT calculations, the interaction energies of individual polar and charged amino acid residues with graphene. It was found that the negatively charged residues can bind to graphene unexpectedly strongly; however, the main effect of encapsulation comes from the overlap of adjacent edges of graphene sheets.

Two desmin gene mutations associated with myofibrillar myopathies in Polish families.

Desmin is a muscle-specific intermediate filament protein which forms a network connecting the sarcomere, T tubules, sarcolemma, nuclear membrane, mitochondria and other organelles. Mutations in the gene coding for desmin (DES) cause skeletal myopathies often combined with cardiomyopathy, or isolated cardiomyopathies. The molecular pathomechanisms of the disease remain ambiguous. Here, we describe and comprehensively characterize two DES mutations found in Polish patients with a clinical diagnosis of desminopathy. The study group comprised 16 individuals representing three families. Two mutations were identified: a novel missense mutation (Q348P) and a small deletion of nine nucleotides (A357_E359del), previously described by us in the Polish population. A common ancestry of all the families bearing the A357_E359del mutation was confirmed. Both mutations were predicted to be pathogenic using a bioinformatics approach, including molecular dynamics simulations which helped to rationalize abnormal behavior at molecular level. To test the impact of the mutations on DES expression and the intracellular distribution of desmin muscle biopsies were investigated. Elevated desmin levels as well as its atypical localization in muscle fibers were observed. Additional staining for M-cadherin, α-actinin, and myosin heavy chains confirmed severe disruption of myofibrill organization. The abnormalities were more prominent in the Q348P muscle, where both small atrophic fibers as well large fibers with centrally localized nuclei were observed. We propose that the mutations affect desmin structure and cause its aberrant folding and subsequent aggregation, triggering disruption of myofibrils organization.

GPCRM: a homology modeling web service with triple membrane-fitted quality assessment of GPCR models

Abstract Due to the involvement of G protein-coupled receptors (GPCRs) in most of the physiological and pathological processes in humans they have been attracting a lot of attention from pharmaceutical industry as well as from scientific community. Therefore, the need for new, high quality structures of GPCRs is enormous. The updated homology modeling service GPCRM (http://gpcrm.biomodellab.eu/) meets those expectations by greatly reducing the execution time of submissions (from days to hours/minutes) with nearly the same average quality of obtained models. Additionally, due to three different scoring functions (Rosetta, Rosetta-MP, BCL::Score) it is possible to select accurate models for the required purposes: the structure of the binding site, the transmembrane domain or the overall shape of the receptor. Currently, no other web service for GPCR modeling provides this possibility. GPCRM is continually upgraded in a semi-automatic way and the number of template structures has increased from 20 in 2013 to over 90 including structures the same receptor with different ligands which can influence the structure not only in the on/off manner. Two types of protein viewers can be used for visual inspection of obtained models. The extended sortable tables with available templates provide links to external databases and display ligand–receptor interactions in visual form.

Approaches for Differentiation and Interconverting GPCR Agonists and Antagonists

Predicting the functional preferences of the ligands was always a highly demanding task, much harder that predicting whether a ligand can bind to the receptor. This is because of significant similarities of agonists, antagonists and inverse agonists which are binding usually in the same binding site of the receptor and only small structural changes can push receptor toward a particular activation state. For G protein-coupled receptors, due to a large progress in crystallization techniques and also in receptor thermal stabilization, it was possible to obtain a large number of high-quality structures of complexes of these receptors with agonists and non-agonists. Additionally, the long-time-scale molecular dynamics simulations revealed how the activation processes of GPCRs can take place. Using both theoretical and experimental knowledge it was possible to employ many clever and sophisticated methods which can help to differentiate agonists and non-agonists, so one can interconvert them in search of the optimal drug.