Purinergic approach to effective glioma treatment with temozolomide reveals enhanced anti-cancer effects mediated by P2X7 receptor.

The purinergic signaling pathway is the oldest evolutionary transmitter system that regulates a wide array of physiological and pathophysiological processes in central nervous system. However, the question of how the purinergic compounds interact with administrated drugs is rarely addressed. We aimed to clarify the interplay between purinergic signaling and chemotherapeutic drug temozolomide (TMZ) in human glioma cell line. We applied an initial retinoic acid-induced differentiation of A172 glioma cells and tested the P2X7 receptor expression in undifferentiated and differentiated gliomas. We compared the P2X7 receptor agonists/antagonists influence and their co-action with TMZ in both cell types through assessment of cell proliferation, viability and migrative properties. Molecular docking allowed to indicate the potential binding site for TMZ in the structure of hP2X7 receptor. Differentiated cells turned out to be more susceptible to ATP and TMZ alone but also to the concerted action of TMZ and ATP. Enhanced effects triggered by ATP and TMZ treatment include the decreased by 70% viability, and reduced migration ability of differentiated A172 glioma cells. Noteworthy, these results can be achieved already at low non-toxic ATP concentration and at reduced to 125 µM effective concentration of TMZ. Therefore, ATP molecules must be present and maintained at appropriate concentration in glioma cells microenvironment to achieve their co-action with TMZ and enhanced anti-cancer activity. All that, in turn, could shorten the therapy, increase its efficacy and limit the side effects for the patient. Our purinergic approach creates a promising perspective for developing novel combined oncological therapies.

Structural Insights into ATP-Sensitive Potassium Channel Mechanics: A Role of Intrinsically Disordered Regions.

Commonly used techniques, such as CryoEM or X-ray, are not able to capture the structural reorganizations of disordered regions of proteins (IDR); therefore, it is difficult to assess their functions in proteins based exclusively on experiments. To fill this gap, we used computational molecular dynamics (MD) simulation methods to capture IDR dynamics and trace biological function-related interactions in the Kir6.2/SUR1 potassium channel. This ATP-sensitive octameric complex, one of the critical elements in the insulin secretion process in human pancreatic ß-cells, has four to five large, disordered fragments. Using unique MD simulations of the full Kir6.2/SUR1 channel complex, we present an in-depth analysis of the dynamics of the disordered regions and discuss the possible functions they could have in this system. Our MD results confirmed the crucial role of the N-terminus of the Kir6.2 fragment and the L0-loop of the SUR1 protein in the transfer of mechanical signals between domains that trigger insulin release. Moreover, we show that the presence of IDRs affects natural ligand binding. Our research takes us one step further toward understanding the action of this vital complex.

Photo-Switchable Sulfonylureas Binding to ATP-Sensitive Potassium Channel Reveal the Mechanism of Light-Controlled Insulin Release.

ATP-sensitive potassium (KATP) channels are present in numerous organs, including the heart, brain, and pancreas. Physiological opening and closing of KATPs present in pancreatic ß-cells, in response to changes in the ATP/ADP concentration ratio, are correlated with insulin release into the bloodstream. Sulfonylurea drugs, commonly used in type 2 diabetes mellitus treatment, bind to the octamer KATP channels composed of four pore-forming Kir6.2 and four SUR1 subunits and increase the probability of insulin release. Azobenzene-based derivatives of sulfonylureas, such as JB253 inspired by well-established antidiabetic drug glimepiride, allow for control of this process by light. The mechanism of that phenomenon was not known until now. In this paper, we use molecular docking, molecular dynamics, and metadynamics to reveal structural determinants explaining light-controlled insulin release. We show that both trans- and cis-JB253 bind to the same SUR1 cavity as antidiabetic sulfonylurea glibenclamide (GBM). Simulations indicate that, in contrast to trans-JB253, the cis-JB253 structure generated by blue light absorption promotes open structures of SUR1, in close similarity to the GBM effect. We postulate that in the open SUR1 structures, the N-terminal tail from Kir6.2 protruding into the SUR1 pocket is stabilized by flexible enough sulfonylureas. Therefore, the adjacent Kir6.2 pore is more often closed, which in turn facilitates insulin release. Thus, KATP conductance is regulated by peptide linkers between its Kir6.2 and SUR1 subunits, a phenomenon present in other biological signaling pathways. Our data explain the observed light-modulated activity of photoactive sulfonylureas and widen a way to develop new antidiabetic drugs having reduced adverse effects.

Interactions of Sea Anemone Toxins with Insect Sodium Channel-Insights from Electrophysiology and Molecular Docking Studies.

Animal venoms are considered as a promising source of new drugs. Sea anemones release polypeptides that affect electrical activity of neurons of their prey. Voltage dependent sodium (Nav) channels are the common targets of Av1, Av2, and Av3 toxins from Anemonia viridis and CgNa from Condylactis gigantea. The toxins bind to the extracellular side of a channel and slow its fast inactivation, but molecular details of the binding modes are not known. Electrophysiological measurements on Periplaneta americana neuronal preparation revealed differences in potency of these toxins to increase nerve activity. Av1 and CgNa exhibit the strongest effects, while Av2 the weakest effect. Extensive molecular docking using a modern SMINA computer method revealed only partial overlap among the sets of toxins' and channel's amino acid residues responsible for the selectivity and binding modes. Docking positions support earlier supposition that the higher neuronal activity observed in electrophysiology should be attributed to hampering the fast inactivation gate by interactions of an anemone toxin with the voltage driven S4 helix from domain IV of cockroach Nav channel (NavPaS). Our modelling provides new data linking activity of toxins with their mode of binding in site 3 of NavPaS channel.

Structural Determinants of Insulin Release: Disordered N-Terminal Tail of Kir6.2 Affects Potassium Channel Dynamics through Interactions with Sulfonylurea Binding Region in a SUR1 Partner.

Inward rectifying potassium ion channels (KATP), sensitive to the ATP/ADP concentration ratio, play an important, control role in pancreatic ß cells. The channels close upon the increase of this ratio, which, in turn, triggers insulin release to blood. Numerous mutations in KATP lead to severe and widespread medical conditions such as diabetes. The KATP system consists of a pore made of four Kir6.2 subunits and four accompanying large SUR1 proteins belonging to the ABCC transporters group. How SUR1 affects KATP function is not yet known; therefore, we created simplified models of the Kir6.2 tetramer based on recently determined cryo-EM KATP structures. Using all-atom molecular dynamics (MD) with the CHARMM36 force field, targeted MD, and molecular docking, we revealed functionally important rearrangements in the Kir6.2 pore, induced by the presence of the SUR1 protein. The cytoplasmic domain of Kir6.2 (CTD) is brought closer to the membrane due to interactions with SUR1. Each Kir6.2 subunit has a conserved, functionally important, disordered N-terminal tail. Using molecular docking, we found that the Kir6.2 tail easily docks to the sulfonylurea drug binding region located in the adjacent SUR1 protein. We reveal, for the first time, dynamical behavior of the Kir6.2/SUR1 system, confirming a physiological role of the Kir6.2 disordered tail, and we indicate structural determinants of KATP-dependent insulin release from pancreatic ß cells.

How strong are hydrogen bonds in the peptide model?

Detailed knowledge of intramolecular hydrogen bonds, including their nanomechanics, in a peptide secondary structure is crucial for understanding mechanisms of numerous biochemical processes. Single-molecule force spectroscopy has become a powerful tool to study directly the mechanical properties of single biopolymers and monitoring the hydrogen bonds. However, the interpretation of such experiments, due to their poor temporal resolution relative to the rate of intramolecular dynamics, requires the support of molecular simulations. In this work, we provide a methodology for determining the kinetic and energetic characteristics of hydrogen bonds in a template model of the protein secondary structure. Our approach, based on the steered molecular dynamics method, employs dynamic force spectroscopy calculations and uses two advanced theoretical models of force-induced unbinding. A systematic analysis of the simulated data with these models allowed for quantitative characterization of a single hydrogen bond in the α-helix of the AAKA(AEAAKA)5AC peptide model and detailed explanation of the mechanism of the α-helix unfolding. The methodology proposed here may be extended to other molecular structures stabilized by internal hydrogen bonds.

Spacial models of malfunctioned protein complexes help to elucidate signal transduction critical for insulin release.

Mutations in gene KCNJ11 encoding the Kir6.2 subunit of the ATP-sensitive potassium channel (KATP), a representative of a quite complex biosystem, may affect insulin release from pancreatic beta-cells. Both gain and loss of channel activity are observed, which lead to varied clinical phenotypes ranging from neonatal diabetes to congenital hyperinsulinism. In order to understand the mechanisms of the channel function better we mapped, based on the literature review, known medically relevant Kir6.2/SUR1 mutations into recently (2017) determined CryoEM 3D structures of this complex. We used a clustering algorithm to find hots spots in the 3D structure, thus we may hypothesize about their nano-mechanical role in the channel gating and the insulin level control. We also adapted a simple model of the channel gating to cover all currently known factors that can influence the KATP biosystem functions.

Canine Adipose-Derived Stem Cells: Purinergic Characterization and Neurogenic Potential for Therapeutic Applications.

The presented results evidence that canine adipose-derived stem cells (ADSCs) represent the premature population of stem cells with great biological potential and properties. ADCS are easy to obtain and culture, able to differentiate into the neurogenic lineage as well as it is easy to control their proliferation rate with nucleotides and nucleosides or analogues. We report that in vitro cultured canine ADSCs response to adenosine- and ATP-mediated stimulation. Differences in canine ADSCs and human mesenchymal stem cells in ecto-nucleotidase activity have been observed. The ecto-nucleotidase activity changes during ADSCs in vitro transdifferentiation into neurogenic lineage are fast and simple to analyze. Therefore, the simple analysis of ecto-enzymes activity allows for verification of the stem cells quality: their stemness or initiation of the differentiation process. The biological potential of the cells isolated from canine fat, as well as the good quality control of this cell culture, make them a promising tool for both experimental and therapeutic usage. J. Cell. Biochem. 118: 58-65, 2017. © 2016 Wiley Periodicals, Inc.

Total hip arthroplasty in HIV-seropositive patients--case study and review of literature.

Hip arthroplasty is widely used in the treatment of osteoarthritis, especially in patients with steroid-induced osteonecrosis of the femoral head. However, this procedure in HIV-seropositive patients has not been described in the Polish literature and is not considered a standard treatment. This paper presents a case study of an HIV-seropositive patient who underwent hip arthroplasty for steroid-induced avascular necrosis of the femoral head without significant complications. Based on this case, we propose an approach to the management of this group of patients.

Molecular basis of lateral force spectroscopy nano-diagnostics: computational unbinding of autism related chemokine MCP-1 from IgG antibody.

Monocyte-chemoattractant protein-1 (MCP-1), also known as CCL2, is a potent chemoattractant of T cells and monocytes, involved in inflammatory and angio-proliferative brain and retinal diseases. Higher expression of MCP-1 is observed in metastatic tumors. Unusual levels of MCP-1 in the brain may be correlated with autism. Immunochemistry where atomic force microscope (AFM) tips functionalized with appropriate antibodies against MCP-1 are used could in principle support medical diagnostics. Useful signals from single molecule experiments may be generated if interaction forces are large enough. The chemokine-antibody unbinding force depends on a relative motion of the interacting fragments of the complex. In this paper the stability of the medically important MCP-1- immunoglobulin G antibody Fab fragment complex has been studied using steered molecular dynamics (SMD) computer simulations with the aim to model possible arrangements of nano-diagnostics experiments. Using SMD we confirm that molecular recognition in MCP1-IgG is based mainly on six pairs of residues: Glu39A - Arg98H, Lys56A - Asp52H, Asp65A - Arg32L, Asp68A - Arg32L, Thr32A - Glu55L, Gln61A - Tyr33H. The minimum external force required for mechanical dissociation of the complex depends on a direction of the force. The pulling of the MCP-1 antigen in the directions parallel to the antigen-antibody contact plane requires forces about 20 %-40 % lower than in the perpendicular one. Fortunately, these values are large enough that the fast lateral force spectroscopy may be used for effective nano-diagnostics purposes. We show that molecular modeling is a useful tool in planning AFM force spectroscopy experiments.

Immune thrombocytopenia in patients with chronic lymphocytic leukemia treated with cladribine-based regiments or chlorambucil--follow-up of PALG-CLL randomized trials.

OBJECTIVES: The relationship between treatments of chronic lymphocytic leukemia (CLL) with cladribine (2-CdA) or chlorambucil and immune thrombocytopenia (IT) has not been yet determined. METHODS: The records of 777 patients in two randomized Polish Adult Leukemia Group (PALG)-CLL programs treated with these agents were retrospectively analyzed. RESULTS: Immune thrombocytopenia occurred in 55 of 777 (7.1%) patients. No significant differences in IT prevalence were seen between patients on chlorambucil or 2-CdA-based regiments (P = 0.33). IT developed at a median time of 0.499 yr (0.06-4.8) from the start of CLL therapy. This time was significantly longer in patients treated with chlorambucil (2.03 yr, 95% CI: 0.06-4.22) in relation to patients treated with 2-CdA-based regiments (0.52 yr, 95%CI: 0.34-0.69, P = 0.049). Overall survival (OS) of patients with IT and those without IT were similar (2.65 yr vs. 3.2 yr P = 0.23) but the severity of bleeding was more pronounced in the 2-CdA group. The responses to IT therapy were 35%, 54% and 75% for steroids, chemotherapy and splenectomy, respectively. CONCLUSIONS: In this study, an unexpectedly high percentage of IT incidence was demonstrated in patients with CLL requiring chemotherapy. Although no marked differences were seen in IT frequency in patients treated with 2-CdA-based regiments compared to chlorambucil regimen, the clinical course of hemorrhagic diathesis was more severe in 2-CdA group. Also, the time elapsed from study screening to IT diagnosis was significantly shorter in the 2-CdA group than in the chlorambucil group suggesting a causative relationship. The appearance of IT did not influence the median time of OS.

Molecular jamming--the cystine slipknot mechanical clamp in all-atom simulations.

A recent survey of 17 134 proteins has identified a new class of proteins which are expected to yield stretching induced force peaks in the range of 1 nN. Such high force peaks should be due to forcing of a slip-loop through a cystine ring, i.e., by generating a cystine slipknot. The survey has been performed in a simple coarse grained model. Here, we perform all-atom steered molecular dynamics simulations on 15 cystine knot proteins and determine their resistance to stretching. In agreement with previous studies within a coarse grained structure based model, the level of resistance is found to be substantially higher than in proteins in which the mechanical clamp operates through shear. The large stretching forces arise through formation of the cystine slipknot mechanical clamp and the resulting steric jamming. We elucidate the workings of such a clamp in an atomic detail. We also study the behavior of five top strength proteins with the shear-based mechanostability in which no jamming is involved. We show that in the atomic model, the jamming state is relieved by moving one amino acid at a time and there is a choice in the selection of the amino acid that advances the first. In contrast, the coarse grained model also allows for a simultaneous passage of two amino acids.

Insights into catalytic activity of industrial enzyme Co-nitrile hydratase. Docking studies of nitriles and amides.

Nitrile hydratase (NHase) is an enzyme containing non-corrin Co3+ in the non-standard active site. NHases from Pseudonocardia thermophila JCM 3095 catalyse hydration of nitriles to corresponding amides. The efficiency of the enzyme is 100 times higher for aliphatic nitriles then aromatic ones. In order to understand better this selectivity dockings of a series of aliphatic and aromatic nitriles and related amides into a model protein based on an X-ray structure were performed. Substantial differences in binding modes were observed, showing better conformational freedom of aliphatic compounds. Distinct interactions with postranslationally modified cysteines present in the active site of the enzyme were observed. Modeling shows that water molecule activated by a metal ion may easily directly attack the docked acrylonitrile to transform this molecule into acryloamide. Thus docking studies provide support for one of the reaction mechanisms discussed in the literature.

Cladribine in a weekly versus daily schedule for untreated active hairy cell leukemia: final report from the Polish Adult Leukemia Group (PALG) of a prospective, randomized, multicenter trial.

Cladribine (2-chlorodeoxyadenosine, 2-CdA) treatment-associated infections may shorten potentially long-term survival in hairy cell leukemia (HCL). In search of the optimal mode of 2-CdA administration, 132 patients with untreated HCL were randomized to receive either standard 5-day 2-CdA protocol or a novel schedule of 6 weekly 2-CdA infusions suggested to be less toxic. Analysis of treatment response confirmed similar complete remission rates, overall response rates, progression-free survival, and overall survival in both 2-CdA protocols. However, we did not observe lower toxicity in the weekly schedule. Of special interest, no significant differences were found in the rate of grade 3/4 infections (18% for daily and 26% for weekly protocol, difference -8.2%; 95% confidence interval [CI] -23.2% to 6.9%; P = .28) and the rate of septic deaths (3% for daily and 2% for weekly protocol, difference 1.4%; 95% CI -4.3% to 7.0%; P = .64). In conclusion, HCL treatment with weekly 2-CdA infusions is equally effective but no safer than the standard 5-day 2-CdA protocol.

Comparison of cladribine plus prednisone with chlorambucil plus prednisone in patients with chronic lymphocytic leukemia. Final report of the Polish Adult Leukemia Group (PALG CLL1).

BACKGROUND: We previously published an early report of a randomized, multicenter trial on the efficacy and toxicity of cladribine (2-CdA) + prednisone (P) compared with chlorambucil (Chl) + P in previously untreated patients with progressive or symptomatic chronic lymphocytic leukemia. Here we present the final report of this study. MATERIAL/METHODS: Of 229 patients, 126 received 2-CdA+P and 103 Chl+P. Patients with no response or progression after three courses or who relapsed earlier than 12 months after completing one treatment were switched to the other. Patients who relapsed later were retreated with the same schedule as before. RESULTS: Thirty-three patients were retreated with 2-CdA+P and 19 with Chl+P. Overall response (and complete response) rates were 35% (6%) and 47% (16%), respectively. In 50 patients initially treated with Chl+P and then with 2-CdA+P, complete response (CR) was achieved in 12 (24%) and overall response (OR) in 32 (64%). In 28 patients originally treated with 2-CdA+P and then with Chl+P, CR in 1 (3%, p=0.01) and OR in 6 (21%, p=0.003) were obtained. We found no statistically significant difference in overall survival time in patients treated initially with 2-CdA+P and Chl+P aged 60 years (4.63 and 5.27 years, respectively, p=0.45), 60-70 years (3.29 and 3.14 years, p=0.79), and >70 years (1.53 and 1.93 years, p=0.11). CONCLUSIONS: 2-CdA+P is significantly more effective as a second-line treatment and re-treatment than Chl+P. However, we found a trend to longer survival in elderly patients treated with Chl+P.

Complex cytogenetics in a case of probably work related MDS/AML.

In the majority of cases of overt acute myeloid leukemia (AML), there is no knowledge about a preleukemic phase of myelodysplastic syndrome (MDS). A few recent case-control studies suggest an association between MDS and several occupations. We report a unique case of MDS/AML related to the patient's work condition with numerous cytostatic agents. The karyotype revealed a spectrum of genetic rearrangements identified by conventional cytogenetics, fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). We suggest that amplification of chromosomal material may play a greater role in leukemogenesis than has been recognized previously.