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In Philadelphia Chromosome (Ph) positive ALL and CML the fusion between BCR and ABL leads to the BCR/ABL fusion proteins, which induces the leukemic phenotype because of the constitutive activation of multiple signaling pathways down-stream to the aberrant BCR/ABL fusion tyrosine kinase. Targeted inhibition of BCR/ABL by ABL-kinase inhibitors induces apoptosis in BCR/ABL transformed cells and leads to complete remission in Ph positive leukemia patients. However, a large portion of patients with advanced Ph+ leukemia relapse and acquire resistance. Kinase domain (KD) mutations interfering with inhibitor binding represent the major mechanism of acquired resistance in patients with Ph+ leukemia. Tetramerization of BCR/ABL through the N-terminal coiled-coil region (CC) of BCR is essential for the ABL-kinase activation. Targeting the CC-domain forces BCR/ABL into a monomeric conformation, reduces its kinase activity and increases the sensitivity for Imatinib. Here we show that i.) targeting the tetramerization by a peptide representing the Helix-2 of the CC efficiently reduced the autophosphorylation of both WT BCR/ABL and its mutants; ii.) Helix-2 inhibited the transformation potential of BCR/ABL independently of the presence of mutations; iii.) Helix-2 efficiently cooperated with Imatinib as revealed by their effects on the transformation potential and the factor-independence related to BCR/ABL with the exception of mutant T315I. These findings suggest that BCR/ABL harboring the T315I mutation have a transformation potential which is at least partially independent from its kinase activity. Targeted inhibition of BCR/ABL by small molecule inhibitors reverses the transformation potential of BCR/ABL. We definitively proved that targeting the tetramerization of BCR/ABL mediated by the N-terminal coiled-coil domain (CC) using competitive peptides, representing the Helix-2 of the CC, represents a valid therapeutic approach for treating Ph+ leukemia. To further develop competitive peptides for targeting BCR/ABL, we created a membrane permeable Helix-2 peptide (MPH-2) by fusing the Helix-2 peptide with a peptide transduction tag. In this study, we report that the MPH-2: (i) interacted with BCR/ABL in vivo; (ii) efficiently inhibited the autophosphorylation of BCR/ABL; (iii) suppressed the growth and viability of Ph+ leukemic cells; and (iv) was efficiently transduced into mononuclear cells (MNC) in an in vivo mouse model. The T315I mutation confers resistance against all actually approved ABL-kinase inhibitors and competitive peptides. It seems not only to decrease affinity for kinase inhibitors but to confer additional features to the leukemogenic potential of BCR/ABL. To determine the role of T315I in resistance to the inhibition of oligomerization and in the leukemogenic potential of BCR/ABL, we investigated its influence on loss-of-function mutants with regard to the capacity to mediate factor-independence. Thus we studied the effects of T315I on BCR/ABL mutants lacking functional domains in the BCR portion indispensable for the oncogenic activity of BCR/ABL such as the N-terminal coiled coil (CC), the tyrosine phosphorylation site Y177 and the serine/threonine kinase domain (ST), as well as on the ABL portion of BCR/ABL (#ABL-T315I) with or without the inhibitory SH3 (delta SH3-ABL) domain. Here we report that i.) T315I restored the capacity to mediate factor independence of oligomerization_deficient p185BCR/ABL; ii.) resistance of p185-T315I against inhibition of the oligomerization depends on the phosphorylation at Y177; iii.) autophosphorylation at Y177 is not affected by the oligomerization inhibition, but phosphorylation at Y177 of endogenous BCR parallels the effects of T315I; iv.) the effects of T315I are associated with an intact ABL_kinase activity; v.) the presence of T315I is associated with an increased ABL_kinase activity also in mutants unable to induce Y177 phosphorylation of endogenous BCR; vi.) there is no direct relationship between the ABL-kinase activity and the capacity to mediate factor_independence induced by T315I as revealed by the #ABL-T315I mutant, which was unable to induce Y177 phosphorylation of BCR only in the presence of the SH3 domain. In contrast to its physiological counterpart c-ABL, the BCR/ABL kinase is constitutively activated, inducing the leukemic phenotype. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl binding pocket in a process called “capping”, which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, BCR/ABL “escapes” this auto-inhibition. Allosteric inhibition by myristate “mimics”, such as GNF-2, is able to inhibit unmutated BCR/ABL, but not the BCR/ABL that harbors the “gatekeeper” mutation T315I. Here we investigated the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. We demonstrate that inhibition of oligomerization was able not only to increase the efficacy of GNF-2 on unmutated BCR/ABL, but also to overcome the resistance of BCR/ABL-T315I to allosteric inhibition. These results strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. Taken together these data suggest that the inhibition of tetramerization inhibits BCR/ABL-mediated transformation and can contribute to overcome Imatinib-resistance. The study provides the first evidence that an efficient peptide transduction system facilitates the employ-ment of competitive peptides to target the oligomerization interface of BCR/ABL in vivo. Further the data show that T315I confers additional leukemogenic activity to BCR/ABL, which might explain the clinical behavior of patients with BCR/ABL -T315I-positive blasts. In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors.
The aim of the study was to investigate the role of the CX3C chemokine FKN in the role of platelet adhesion. The presence of the FKN receptor CX3CR1 in platelets is demonstrated and G-protein dependent activation of platelets with soluble FKN results in the increased adhesion of platelets to collagen and fibrinogen under flow 228 and adhesion of leucocytes to firmly attached platelets 231. Whether membrane-bound FKN is capable to promote the direct adhesion of platelets in flowing blood analogue to leucocytes was completely unknown. The adhesion mechanisms of FKN in mediating the adhesion of leucocytes under flow are well characterised and represent a novel unique mechanism of leucocyte capture and firm adhesion: FKN is responsible for immediate arrest of flowing CX3CR1 expressing leucocytes without the participation of additional adhesion receptors and ligands. This is in contrast to the classical leucocyte adhesion pathways, which are multistep processes involving leucocyte arrest, rolling and subsequent cell activation prior to firm arrest. In leucocytes, the FKN – CX3CR1 axis is sufficient to allow rapid arrest of leucocytes at low shear flow conditions 67, 101, 115, 122, 261. The set of data from this study demonstrates that immobilised FKN was capable to mediate the adhesion of platelets under low shear conditions, whereas there was no interaction in the absence of shear flow. In the presence of vWf in the adhesion matrix, FKN mediated the potent increased adhesion of platelets. This was in parts due to the activation of flowing platelets via CX3CR1 and the augmented translocation of platelets on FKN via the vWf receptor GPIbα. With respect to platelet activation, the function of endothelial FKN was comparable to leucocytes: in both cell types, the FKN dependent activation is mediated by its cognate receptor CX3CR1. This is in contrast to the adhesive capacity: in leucocytes, FKN dependent adhesion is mediated by CX3CR1, whereas in platelets, the adhesive capacity was mostly mediated by the vWf receptor GPIbα with only minor contribution from CX3CR1. In platelets, activation and adhesion by FKN were mediated by two distinct receptors, whereas in leucocytes, CX3CR1 is solely responsible for FKN dependent activation and adhesion. The presented results point out to a role of platelets in early stage of atherosclerosis. The in vivo expression of both, FKN and vWf is regulated by TNF-α, which is released in early stages of inflammation. The presence of vWf and FKN in the endothelial lining of blood vessels during these conditions is sufficient to initiate the capturing and translocation of platelets on the tunica interna. The rolling of platelets on the endothelium can induce endothelial damage and inflammation of the vessel, which might advance to the generation of clinically significant atherosclerotic plaques and fibrous atheroma.
We developed the Pharmacophore Alignment Search Tool (PhAST), a text-based technique for rapid hit and lead structure searching in large compound databases. For each molecule, a two-dimensional graph of potential pharmacophoric points (PPPs) is created, which has an identical topology as the original molecule with implicit hydrogen atoms. Each vertex is coloured by a symbol representing the corresponding PPP. The vertices of the graph are canonically labelled. The symbols associated with the vertices are combined to a so-called PhAST-Sequence beginning with the vertex with the lowest canonical label. Due to the canonical labelling the created PhAST-Sequence is characteristic for each molecule. For similarity assessment, PhAST-Sequences are compared using the sequence identity in their global pairwise alignment. The alignment score lies between 0 (no similarity) and 1 (identical PhAST-Sequences). In order to use global pairwise sequence alignment, a score matrix for pharmacophoric symbols was developed and gap penalties were optimized. PhAST performed comparably and sometimes superior to other similarity search tools (CATS2D, MOE pharmacophore quadruples) in retrospective virtual screenings using the COBRA collection of drugs and lead structures. Most importantly, the PhAST alignment technique allows for the computation of significance estimates that help prioritize a virtual hit list.
Shape complementarity is a compulsory condition for molecular recognition. In our 3D ligand-based virtual screening approach called SQUIRREL, we combine shape-based rigid body alignment with fuzzy pharmacophore scoring. Retrospective validation studies demonstrate the superiority of methods which combine both shape and pharmacophore information on the family of peroxisome proliferator-activated receptors (PPARs). We demonstrate the real-life applicability of SQUIRREL by a prospective virtual screening study, where a potent PPARalpha agonist with an EC50 of 44 nM and 100-fold selectivity against PPARgamma has been identified...
The representation of small molecules as molecular graphs is a common technique in various fields of cheminformatics. This approach employs abstract descriptions of topology and properties for rapid analyses and comparison. Receptor-based methods in contrast mostly depend on more complex representations impeding simplified analysis and limiting the possibilities of property assignment. In this study we demonstrate that ligand-based methods can be applied to receptor-derived binding site analysis. We introduce the new method PocketGraph that translates representations of binding site volumes into linear graphs and enables the application of graph-based methods to the world of protein pockets. The method uses the PocketPicker algorithm for characterization of binding site volumes and employs a Growing Neural Gas procedure to derive graph representations of pocket topologies. Self-organizing map (SOM) projections revealed a limited number of pocket topologies. We argue that there is only a small set of pocket shapes realized in the known ligand-receptor complexes.
For a virtual screening study, we introduce a combination of machine learning techniques, employing a graph kernel, Gaussian process regression and clustered cross-validation. The aim was to find ligands of peroxisome-proliferator activated receptor gamma (PPAR-y). The receptors in the PPAR family belong to the steroid-thyroid-retinoid superfamily of nuclear receptors and act as transcription factors. They play a role in the regulation of lipid and glucose metabolism in vertebrates and are linked to various human processes and diseases. For this study, we used a dataset of 176 PPAR-y agonists published by Ruecker et al. ...
A new method to bridge the gap between ligand and receptor-based methods in virtual screening (VS) is presented. We introduce a structure-derived virtual ligand (VL) model as an extension to a previously published pseudo-ligand technique [1]: LIQUID [2] fuzzy pharmacophore virtual screening is combined with grid-based protein binding site predictions of PocketPicker [3]. This approach might help reduce bias introduced by manual selection of binding site residues and introduces pocket shape information to the VL. It allows for a combination of several protein structure models into a single "fuzzy" VL representation, which can be used to scan screening compound collections for ligand structures with a similar potential pharmacophore. PocketPicker employs an elaborate grid-based scanning procedure to determine buried cavities and depressions on the protein's surface. Potential binding sites are represented by clusters of grid probes characterizing the shape and accessibility of a cavity. A rule-based system is then applied to project reverse pharmacophore types onto the grid probes of a selected pocket. The pocket pharmacophore types are assigned depending on the properties and geometry of the protein residues surrounding the pocket with regard to their relative position towards the grid probes. LIQUID is used to cluster representative pocket probes by their pharmacophore types describing a fuzzy VL model. The VL is encoded in a correlation vector, which can then be compared to a database of pre-calculated ligand models. A retrospective screening using the fuzzy VL and several protein structures was evaluated by ten fold cross-validation with ROC-AUC and BEDROC metrics, obtaining a significant enrichment of actives. Future work will be devoted to prospective screening using a novel protein target of Helicobacter pylori and compounds from commercial providers.
Two methods for the fast, fragment-based combinatorial molecule assembly were developed. The software COLIBREE® (Combinatorial Library Breeding) generates candidate structures from scratch, based on stochastic optimization [1]. Result structures of a COLIBREE design run are based on a fixed scaffold and variable linkers and side-chains. Linkers representing virtual chemical reactions and side-chain building blocks obtained from pseudo-retrosynthetic dissection of large compound databases are exchanged during optimization. The process of molecule design employs a discrete version of Particle Swarm Optimization (PSO) [2]. Assembled compounds are scored according to their similarity to known reference ligands. Distance to reference molecules is computed in the space of the topological pharmacophore descriptor CATS [3]. In a case study, the approach was applied to the de novo design of potential peroxisome proliferator-activated receptor (PPAR gamma) selective agonists. In a second approach, we developed the formal grammar Reaction-MQL [4] for the in silico representation and application of chemical reactions. Chemical transformation schemes are defined by functional groups participating in known organic reactions. The substructures are specified by the linear Molecular Query Language (MQL) [5]. The developed software package contains a parser for Reaction-MQL-expressions and enables users to design, test and virtually apply chemical reactions. The program has already been used to create combinatorial libraries for virtual screening studies. It was also applied in fragmentation studies with different sets of retrosynthetic reactions and various compound libraries.
There is a renewed interest in pseudoreceptor models which enable computational chemists to bridge the gap of ligand- and receptor-based drug design. We developed a pseudoreceptor model for the histamine H4 receptor (H4R) based on five potent antagonists representing different chemotypes. Here we present the selection of potential ligand binding pockets that occur during molecular dynamics (MD) simulations of a homology-based receptor model. We present a method for prioritizing receptor models according to their match with the consensus ligand-binding mode represented by the pseudoreceptor. In this way, ligand information can be transferred to receptor-based modelling. We use Geometric Hashing to match three-dimensional points in Cartesion space. This allows for the rapid translation- and rotation-free comparison of atom coordinates, which also permits partial matching. The only prerequisite is a hash table, which uses distance triplets as hash keys. Each time a distance triplet occurring in the candidate point set which corresponds to an existing key, the match is represented by a vote of the respective key. Finally, the global match of both point sets can be easily extracted by selection of voted distance triplets. The results revealed a preferred ligand-binding pocket in H4R, which would not have been identified using an unrefined homology model of the protein. The key idea was to rely on ligand information by pseudoreceptor modelling.
A generic drug product (World Health Organization (WHO) terminology: multisource product) is usually marketed and manufactured after the expiry date of the innovator’s patent. Generic drugs are less expensive than the innovator products because generic manufacturers do not have to amortize the investment costs of research, development, marketing, and promotion. Multisource products must contain the same active pharmaceutical ingredients (APIs) as the original formulation and have to be shown to be interchangeable with the original formulation. Multisource products have to be shown bioequivalent to the innovator counterpart with respect to pharmacokinetic and pharmacodynamic properties. Multisource products are therefore identical in dose, strength, route of administration, safety, efficacy, and intended use. Bioequivalence can be demonstrated by in vitro dissolution, pharmacokinetic, pharmacodynamic or clinical studies. Since 2000, the U.S. Food and Drug Administration (FDA) allows the approval of certain multisource products solely on the basis of in vitro studies, i.e. by waiving in vivo studies in humans (“Biowaiver”), based on the Biopharmaceutics Classification Scheme (BCS). The BCS characterizes APIs by their solubility and permeability in the gastrointestinal tract (GIT). The different BCS Classes I-IV (Class I: high solubility, high permeability; Class II: low solubility, high permeability; Class III: high solubility, low permeability and Class IV: low solubility, low permeability) result from all possible combinations of high and low solubility with high and low permeability. Since the adoption of the BCS by the FDA in 1995, the BCS criteria have been under continuous development. In 2006, the WHO has released the most recent bioequivalence guidance including relaxed criteria for bioequivalence studies based on modified BCS criteria. According to this guidance, APIs belonging to the BCS classes I – and under defined conditions - II and III – are eligible for a biowaiver-based approval. The principal objective of this work was to characterize the first-line anti tuberculosis APIs, isoniazid, pyrazinamide, ethambutol dihydrochloride and rifampicin, according to their physicochemical, biopharmaceutical, pharmacokinetic and pharmacological properties and to classify them according to the BCS. Ethambutol dihydrochloride and isoniazid were classified as borderline BCS class I/III APIs. Pyrazinamide was classified as a BCS class III and rifampicin as a BCS class II API. Based on the BCS classification and the additional criteria defined in the WHO bioequivalence guidance, the possibility of biowaiver-based approval for immediate release (immediate release) solid oral dosage forms containing the first-line antituberculosis drugs was evaluated. A biowaiver-based approval with defined constraints was recommended for immediate release solid oral dosage forms containing isoniazid (interaction with reducing sugars), pyrazinamide and ethambutol dihydrochloride (relative narrow therapeutic index). Rifampicin was classified as a BCS class II API, and it was concluded that rifampicin containing solid oral immediate release drug products as well as Scale-Up and Post-Approval Changes (SUPAC) changes should not be approved by a biowaiver on the following basis: (i) its solubility and dissolution are highly variable due to polymorphism and instability, (ii) concomitant intake of food and antacids reduces its absorption and bioavailability, (iii) no in vitro predictive dissolution test has been found which correlates to in vivo absorption and (iv) several publications reporting cases of non-bioequivalent and bioinequivalent rifampicin products have been located in the literature. Thus, it is recommended that bioequivalence of rifampicin containing solid oral immediate release drug products should be established by in vivo pharmacokinetic studies in humans. This risk-benefit benefit assessment of a biowaiver-based approval was presented as a poster at the American Association of Pharmaceutical Scientists (AAPS) 2005 and subsequently published as “Biowaiver Monographs” in the Journal of Pharmaceutical Sciences. Based on the assessment of the dissolution properties of the antituberculosis drugs for a biowaiver approval, quality control dissolution methodologies for the International Pharmacopoeia (Pharm. Int.) were developed, presented at the WHO expert meeting and adopted in the Pharm. Int. (http://www.who.int/medicines/publications/pharmprep/OMS_TRS_948.pdf). Additionally, preliminary biowaiver recommendations were also developed for four firstline antimalarial drugs listed on the WHO Essential Medicines List (EML): Quinine, as both the hydrochloride and sulphate, and proguanil hydrochloride were classified as borderline BCS class I/III APIs. Since quinine is a narrow therapeutic index drug and many cases of non-bioequivalence have been reported in the literature, a biowaiverbased approval was not recommended. For solid oral immediate release dosage forms containing proguanil a biowaiver-based approval was recommended under the condition that they dissolve very rapidly. Primaquine phosphate was classified as a BCS class I API. Therefore, a biowaiver-based approval was recommended for immediate release solid oral dosage forms containing primaquine phosphate. Mefloquine hydrochloride was classified as a basic, BCS class IV/II API, making it ineligible for the biowaiver. Additionally, reports of non-bioequivalence and a narrow therapeutic index were found in the scientific literature. Consequently, bioequivalence of solid oral immediate release dosage forms containing mefloquine hydrochloride should be established by in vivo pharmacokinetic studies. The results for quinine hydrochloride and sulphate, proguanil hydrochloride, primaquine diphosphate and mefloquine hydrochloride were presented as a poster at the Pharmaceutical Sciences World Congress (PSWC) 2007 and published as a WHO Collaborating Center Report in June 2006. The aim of this project was to collect, evaluate, generate and publish relevant information for a biowaiver-based approval of essential medicines in order to provide a summary to local regulatory authorities. This information complements the selected list of essential medicines by providing information about the biopharmaceutical properties and pharmaceutical quality of solid oral immediate release dosage forms containing these APIs. The aim of the biowaiver project, inspired by the WHO and brought in life by the International Pharmaceutical Federation (FIP), is to enable access to essential medicines in standardized quality at an affordable price. In this work, a significant contribution to this aim in the form of four biowaiver monographs for the antituberculosis drugs and several reports on the antimalarials has been achieved.
The transcription factor p63 is part of the p53 protein family, which consists of three members, p53, p63 and p73. P63 shares structural similarity with all family members, but is associated to different biological functions than p53 or p73. While p53 is mainly linked to tumor suppression and p73 is connected with neuronal development, p63 has been connected to critical biological roles within ectodermal development and skin stem cell biology as well as supervision of the genetic stability of oocytes. Due to its gene structure p63 is expressed as at least six different isoforms, three of them containing a N-terminal transactivation domain. The isoforms that are of biological relevance both have a C-terminal inhibitory domain that negatively regulates the transcriptional activity. This inhibitory domain is supposed to contain two individual components of which one is internally binding and masking the transactivation domain while the other one can be sumoylated. To further investigate this domain a mutational analysis with the help of transactivation assays in SAOS2 cells was carried out to identify the critical amino acids within the inhibitory domain and the impact on transcriptional activity of TAp63alpha, the p63-isoform which is essential for the integrity of the female germline. The results of these experiments show that a stretch of approximately 13 amino acids seems to be important for the regulation of transcriptional activity in TAp63alpha, due to the increased transcriptional activity occurring in this region after mutation. Additional experiments showed that this mechanism is distinct from sumoylation, which seems to have only implications for the intracellular level of TAp63alpha. As a conclusion, the C-terminus of the Tap63alpha is essential for two different mechanisms, which control the transcriptional activity of the protein. Both regulatory elements are independent from each other and can now be restricted to certain amino acids. Activation of the wild type protein might take place in the identified region via post-translational modification. Furthermore an inhibition assay was carried out to test if the same region might have implications on the second biological relevant isoform deltaNp63alpha. The results show that the same amino acids which show an impact on transcriptional activity in Tap63alpha lead to a significant change in functional behaviour of deltaNp63alpha. There is a possibility that both proteins are regulated with opposite effects via the same mechanisms, based at the C-terminus of the p63alpha-isoforms. In both cases a modification of these residues could lead to a more opened conformation of the protein with consequences on promoter binding, which can be even important for deltaNp63alpha with respect to promoter squelching. Both alpha-isoforms seem to be regulated via the C-terminus and to elucidate if that is also the case for TAp63gamma a deletion analysis was carried out. The results show that there are also amino acids within the C-terminus of TAp63gamma, which have implications on the transcriptional activity of the protein. Therefore the C-terminus seems to play a major role for regulation of diverse p63 isoforms.
Protein kinases are targets for drug development. Dysregulation of kinase activity leads to various diseases, e.g. cancer, inflammation, diabetes. Human polo-like kinase 1 (Plk1), a serine/threonine kinase, is a cancer-relevant gene and a potential drug target which attracts increasing attention in the field of cancer therapy. Plk1 is a key player in mitosis and modulates entry into mitosis and the spindle checkpoint at the meta-/anaphase transition. Plk1 overexpression is observed in various human tumors, and it is a negative prognostic factor for cancer patients. The same catalytical mechanism and the same co-substrate (ATP) lead to the problem of inhibitor selectivity. A strategy to solve this problem is represented by targeting the inactive conformation of kinases. Kinases undergo conformational changes between active and inactive conformation and thus an additional hydrophobic pocket is created in the inactive conformation where the surrounding amino acids are less conserved. A "homology model" of the inactive conformation of Plk1 was constructed, as the crystal structure in its inactive conformation is unknown. A crystal structure of Aurora A kinase served as template structure. With this homology model a receptor-based pharmacophore search was performed using SYBYL7.3 software. The raw hits were filtered using physico-chemical properties. The resulting hits were docked using Gold3.2 software, and 13 candidates for biological testing were manually selected. Three compounds of the 13 tested exhibit anti-proliferative effects in HeLa cancer cells. The most potent inhibitor, SBE13, was further tested in various other cancer cell lines of different origins and displayed EC50 values between 12 microM and 39 microM. Cancer cells incubated with SBE13 showed induction of apoptosis, detected by PARP (Poly-Adenosyl-Ribose-Polymerase) cleavage, caspase 9 activation and DAPI staining of apoptotic nuclei.
Background: Microarray analysis still remains a powerful tool to identify new components of the transcriptosome and it has helped to increase the knowledge of targets triggered by stress conditions such as hypoxia and nitric oxide. However, analysis of transcriptional regulatory events remain elusive due to the contribution of altered mRNA stability to gene expression patterns, as well as changes in the half-life of mRNAs, which influence mRNA expression levels and their turn over rates. To circumvent these problems, we have focused on the analysis of newly transcribed (nascent) mRNAs by nuclear run on (NRO), followed by microarray analysis. Result: We identified 188 genes that were significantly regulated by hypoxia, 81 genes were affected by nitric oxide, and 292 genes were induced by the co-treatment of macrophages with both NO and hypoxia. Fourteen genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6 and Fxyd5) were common to hypoxia and/or nitric oxide treatments, but with different levels of expression. We observed that 166 transcripts were regulated only when cells were co-treated with hypoxia and NO but not with either treatment alone, pointing to the importance of a crosstalk between hypoxia and NO. In addition, both array and proteomics data supported a consistent repression of hypoxia regulated targets by NO. Conclusion: By eliminating the interference of steady state mRNA in gene expression profiling, we increased the sensitivity of mRNA analysis and identified previously unknown hypoxia-induced targets. Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signalling.
Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA's secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent ({approx}1 µs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.
In previous investigations an impact of cellular copper homeostasis on ageing of the ascomycete Podospora anserina has been demonstrated. Here we provide new data indicating that mitochondria play a major role in this process. Determination of copper in the cytosolic fraction using total reflection X-ray fluorescence spectroscopy analysis and eGfp reporter gene studies indicate an age-related increase of cytosolic copper levels. We show that components of the mitochondrial matrix (i.e. eGFP targeted to mitochondria) become released from the organelle during ageing. Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension. In addition, we demonstrate that increased copper concentrations in the culture medium lead to the appearance of senescence biomarkers in human diploid fibroblasts (HDFs). Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs. These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans.
The title compound, C25H22O5, was obtained by a dehydrogenative carbonylation reaction. It crystallizes with one half-molecule in the asymmetric unit. The molecules have crystallographic C2 symmetry and the two atoms of the carbonyl group are located on the rotation axis. The methoxy groups are coplanar with the benzene ring to which they are attached [C-C-O-C = 1.0 (6)°]. The two furan rings are inclined at 17.3 (3)° with respect to each other and the dihedral angle between the furan ring and the benzene ring is 75.83 (12)°. The crystal structure is stabilized by C-H...O hydrogen bonds. Key indicators: single-crystal X-ray study; T = 183 K; mean ( σ(C–C) = 0.006 Å; R factor = 0.081; wR factor = 0.195; data-to-parameter ratio = 13.4.
The Mg centre in the title compound, [MgBr2(C2H7N)3], is pentacoordinated in a trigonal-bipyramidal mode with the two Br atoms in axial positions and the N atoms of the dimethylamine ligands in equatorial positions. The MgII centre is located on a crystallographic twofold rotation axis. The crystal structure is stabilized by N—H⋯Br hydrogen bonds. The N atom and H atoms of one dimethylamine ligand are disordered over two equally occupied positions.
In the title compound, C11H14O4, an intermediate for the synthesis of a new kind of estrogen receptor modulator, all non-H atoms lie on a common plane (r.m.s. deviation = 0.0472 Å). All C-C bonds in the side chain are in a trans conformation, and the hydroxyl group is also trans to the methylene chain. In the crystal structure, molecules form centrosymmetric dimers showing a head-to-head arrangement which is stabilized by O-H...O hydrogen bonds. A weak C-H...O contact is also present.
The complete molecule of the title compound, C18H24N2O2, is generated by a crystallographic inversion centre. The torsion angles in the hexamethylene chain are consistent with an antiperiplanar conformation, whereas the conformation of the O—CH2—CH2—CH2 unit is gauche. The three-dimensional crystal packing is stabilized by N—H⋯O and N—H⋯N hydrogen bonding.