Dr Dimitrios Vlachakis is a member of the Bioinformatics and Medical Informatics Team at the Biomedical Research Foundation at the Academy of Athens.
He holds a BSc (hons) in Biology with Biochemistry (University of Bedfordshire-UK), a MSc in Molecular Modelling (Cardiff University-Wales-UK), a MPhil in Molecular Biology (Cardiff University-Wales-UK), a PhD in Medicinal Chemistry (Cardiff University-Wales-UK), and a total of 4 years of post-doctoral experience at the institute of biology at NCSR Demokritos, Greece.
His main research is in the field of structural biology and bioinformatics, by focusing on the in silico rational drug design, synthesis and biological evaluation of novel antiviral agents. Only recently Dr Vlachakis introduced and published a novel de novo structure-based drug design approach on the HCV helicases, using 3D structural information. Computer-aided drug design followed by extensive molecular dynamics simulations, helped establish a series of novel compounds, capable of interacting with the putative binding site on the HCV helicase ssRNA channel. Our best-ranking compounds were chemically synthesized and biologically evaluated against the purified enzyme. They exhibited inhibition of the HCV helicase enzyme in submicromolar concentrations and therefore yielded a series of scientific publications along with an international patent. Other research interests include homology/comparative modelling and secondary structure predictions from large-scale genomic sequences, in an attempt to identify suitable pharmacological receptors based on structure, rather than sequence.
He has published on a very diverse set of topics in the areas of biochemistry, molecular biology, computer science and rational drug design. He is the author of a series of monograph scientific books and the co-inventor of scientific international patents on a family of highly potent anti-HCV drug-like compounds and anti-cancer agents. Moreover, he has published more than 25 original research articles and has been on the receiving end of numerous grants and awards, including a NCSRD and two EPSRC fellowships in the fields of computational & structural biology.
Abstract: More than 170 million people worldwide are currently chronically infected with the Hepatitis C virus. Similarly, Dengue fever infects 50 million per year in central Africa. It is the same story for so many other viruses that are members of the flaviviridae family.
Disproportionally to the severity of an infection with almost all members of Flaviviridae, no specific antiviral therapy is available today. There are drugs neither for the treatment nor for the prevention upon infection. Scientists have isolated various proteins and extensively study some of them in an attempt to get information that will eventually be combined to a strategy against the viruses.
Herein, an effort is made to describe the current status of antiviral research within flaviviridae, homology modelling and structure based drug design techniques that may soon lead to new generation of stratagems against flaviviridae. Finally, insights into novel technologies in drug design technology are provided.
Abstract: Hepatitis C and Dengue viruses belong to the family of Flaviviridae1. Viruses in this family are enveloped, have positive-sense RNA and are responsible for a variety of life threatening diseases. Hepatitis C virus is the major etiological agent of post-transfusion hepatitis worldwide1. An estimated 3 % of the world's population is infected with HCV according to the World Health Organization. Infection with HCV will most regularly result in chronic hepatitis, which leads to liver cirrhosis, hepatocellular carcinoma and liver failure. Dengue is currently the most important viral disease transmitted by mosquitoes afflicting humans the world context2. Clinical symptoms range from mild fevers to a severe haemorrhagic disease. To date neither specific antiviral treatments exist nor are there any vaccines available for both infections3. Thus there is an urgent need for new therapies.
The aim of this project is to design compounds that will inhibit the function of the helicases of the hepatitis C virus, the dengue virus, the Japanese Encephalitis Virus and the Yellow Fever virus (all members of the Flavi virus family). These helicases are interesting targets for drug design, firstly for their vital function in the viral cell cycle and secondly for the fact that human cells lack helicases capable of unwinding positive sense double stranded RNA. The 3D structure of the Hepatitis C helicase has been resolved and was obtained from the Protein DataBank (1A1V and 8OHM). The structures of the Dengue, Japanese Encephalitis and the Yellow Fever helicases are unavailable and were obtained by homology modelling, using the helicase of Hepatitis C as template. A set of small libraries of compounds was designed using de novo drug design. The evaluation of these compounds is initially performed in silico and is completed in vitro, as soon as the best in silico scoring compounds are synthesized and tested in the helicase biological enzymatic assay.
The viability of the homology models of the Dengue, Japanese Encephalitis and the Yellow Fever helicases was evaluated by an in silico scoring function and from the fact that the RNA binding motif and the ATP binding motifs were conserved between the model and the template helicase from HepC (figure 1). Examination of the structure of the hepatitis C helicase showed that two residues (ARG393 and CYS431) are probably involved in the interaction with the oligonucleotide and are suitable to be used as
targets for drug discovery. De novo drug design was performed on the helicase as a whole, but with greater focus on the previous two amino acids. The structure based drug design experiment produced a series of potential lead compounds, which were evaluated by docking/scoring methods in silico, they were screened on the two helicases and the more promising ones were selected to be synthesized. The synthesis of these compounds is currently under development.
The establishment of the helicase assay came out using molecular biology techniques for recombinant protein expression. The genes of the Helicase and the NS3 domain (Helicase and Protease) were incorporated into the high copy pGEM-T plasmid in order to amplify them. Two sets of DH5-alpha strains of E.Coli bacteria cells were transformed with the designed vectors and cultured in liquid suspensions to generate stocks of clones. The vectors were extracted from the DH5-alpha strains and the genes were digested and ligated into the pET23b+ expression vector into the BL21lys E.Coli strain. The genes were induced and the protein was produced as revealed by SDS-PAGE. Due to a misplaced stop-codon at the end of the two genes the His-tag of the pET vector was pushed out of frame and protein isolation was impossible. Currently work is ongoing in an attempt to remove the stop-codon, place the His-tag in frame and isolate the proteins by column chromatography.
Abstract: More than 170 million people worldwide are currently chronically infected with the Hepatitis C virus. They are all considered to be at risk of developing cirrhosis. Some of them will develop liver cancer. Hepatitis C has spread all over the world and for every person who has the Aids Virus, 4 have the Hepatitis C Virus. Today, hepatitis C causes ten thousand deaths per year and is the main responsible for more than half of the four thousand liver transplantations that are performed annually.
Infections that mosquitoes carry or more generally arthropod-borne flaviviruses have reached epidemic dimensions in some parts of the world. Dengue fever infects 50 million per year in central Africa. According to the World Health Organisation (WHO) there are 6.5 billion inhabitants on this planet that live in areas of high risk of acquiring dengue. For example only for 2006 the Philippines reported 197 deaths and 14,738 cases of dengue fever. Indonesiaâs Dengue deaths reached 634 and Malaysia has already confirmed 74 deaths due to Dengue virusin the first 9. In Thailand more than 32 000 Thais have been infected with dengue fever and currently Singapore's is going through hits worst dengue fever outbreak ever on record, since the officially reported Dengue cases are nearing 11 000.
Abstract: Molecular modelling is very useful for investigating, comparing, analysing and visualizing chemical structures and for giving qualitative and quantitative information about biological systems. Quantitative information can arise through molecular mechanics and in conjunction with a computer, where the physical properties of the molecules can be evaluated and analysed based on a set of predefined criteria concerning various chemical properties (such as bonding, charges, steric hinderance...). Molecular Modelling can be used to study the geometry, the energy and the chemical properties in silico so efficiently that nowadays it is possible to predict the outcome of chemical reactions, design reactions, determine the unknown three dimensional structures of proteins, screen and design new and effective drugs. In this part, the basic principles and theory of the methods that have been used in this book will be presented.
Abstract: Adipose tissue belongs to the greater classification category of connective tissues. Connective tissues are found throughout the human body. They usually are located around vital organs, thus acting as insulators, protecting them from extreme environmental conditions. One of the major tasks of connective tissues and more particularly adipose tissue is to store energy, especially in the form of lipids. Adipose tissue is highly vascularized tissue as the individual adipocytes contain huge vacuoles filled with lipids. When adipose tissue is stimulated by the nervous system or by hormones the lipid breakdown process accelerates and the surrounding tissues capture the energy that is being released as heat. The heat releasing type of adipose tissue is the brown adipose tissue (Schmidt-Nielson, 1998; Altringham, 1996; Smalley, 1963). As a result the blood that passes through this particular area is warmed up and as the blood circulates it warms the rest of the body as well. Adipose tissue is organised in big depots. These depots can be up to 30 percent of the total size of the body mass. Some adipocytes may have developed different properties (differentiation) according to the task that they have been assigned to. So, adipocytes in the mammary glands differ a bit from adipocytes in the kidneys or the lymph nodes...
Abstract: Protein similarity searches are still based on primary sequence data searching rather than structural information. There are large databases available nowadays, which contain huge amounts of structural and sequence information. However, in many cases, primary sequence comparisons do not cover sequence space adequately, creating an urgent need for a new structure-based similarity search approach. It is universally known that a protein's tertiary structure is more conserved than its primary structure. There are numerous cases of extremely low sequence similarity between homologous viral proteins (~10% sequence identity) that nevertheless have the same function, belong to the same viral family and, more strikingly, when their structures have been determined by X-ray crystallography, have shown the same structural features. Taken together, we propose that the novel structural similarity approach proposed here, in conjunction with classic sequence similarity BLAST searches, could yield results of great interest to science.
Abstract: Simplifying spatially complicated problems in the field of drug design, pharmacology and 3D molecular modelling is becoming very important, owing to the rapid increase in genomic and structural database sizes. The computational load is immense, and novel innovative approaches are sought, in order to perform comprehensive structural studies and 3D searches at only a fraction of the original time required.
Protein docking (PD) and protein-protein interactions (PPI) are two of the most rapidly emerging fields in modern structural bioinformatics. Many studies attempt to justify biological activity and function of small molecules, macromolecules or even molecular complexes using PD and PPI. For example, the majority of the information we have about the molecular processes that take place in the nucleus or the cytoplasm, and affect DNA replication, has been acquired by fast algorithms and machine-learning approaches that investigate protein-protein interactions. Molecular dynamics, genetic and epigenetic networks, systems biology, molecular biology and many other related disciplines use PD and PPI as key research tools. Many databases have been developed in this direction: e.g., the MIPS mammalian protein-protein database, the eF-site molecular surface database, the STRING database of functional protein association networks, BioGRID, VASP, PESDserv and many more. However, the limitation is that these approaches are modelled simulations using graph-theoretical methods, whose sensitivity and specificity is not always trustworthy. Eventually, human input and insight is required, as the application of current algorithms to all available data is impossible owing to hardware- and time- limitations. Here, we present a novel strategy to perform similarity searches and molecular docking experiments using protein molecular surfaces.
Abstract: Since its inception, there has been a controversy on what the letter M in M-theory stands for. M could stand for Magic, Mystery or even Madness (Duff 1997). It is a theory that digs deeper into reality and has the potential to unify separate fields of science. In our world, knowledge is often simplified and available for dissipated systems that are easier to observe and analyze. In cosmology, it is easy to monitor and attribute the behavior of a small set of asteroids using only mathematics. However, in order to tackle more complex systems, we know that neither Newton's laws nor Euclidian mathematics is adequate. Democritus in 460 BC proposed that one cannot keep cutting an object in half forever. There is a point where matter cannot be cut in half. He called that the atom, which in Greek means undividable. Well, what is the quark then? His theory was correct for a long time, and even though today we know it is wrong, we still use it and find it helpful in our quest to understand the world. This also holds true for molecular biology and biochemistry. Vast information is available on reaction chemistry, transcription and translation in the molecular level. However, zoom out a bit and our knowledge comes into question.
Abstract: The study of the natural world, or more simply put, science, is unequivocally based on experiment and observation. The founding of science in the ancient world was synonym to reason and logic over myths and superstitions. The ancient Egyptians were the first to develop astronomy and mathematics, while the Egyptian Edwin Smith papyrus is one of the earliest medical texts on the anatomy of the human brain (Allen et al. 2005). Greek physician Hippocrates, the so-called âfather of medicineâ, is perhaps so well remembered due to his perseverance to separate science from religion, teaching that diseases have natural causes instead of being acts of gods (Hippocrates, On the Sacred Disease).
Abstract: This study pertains to the regulatory network of neurogenin3 (NGN3, approved symbol: NEUROG3), the main regulator of insulin producing cells' formation. In silico regulatory region analyses of known and novel targets of NGN3 revealed the presence of two variants of a regulatory module that appeared conserved at the most phylogenetically distant species with pancreas. Both variants of this module contained binding sites of six transcription factors implicated in pancreas development. Nevertheless, an additional factor was found only into the module of the down-regulated by NGN3 genes. Whole genome analyses confirmed the statistical significance of these regulatory modules. Investigation of protein-protein interactions among the factors bound into these sequences indicated the formation of alternative protein complexes resulting into the up- or down-regulation of the respective genes. Subsequently, an NGN3-guided regulatory network, was modeled, describing the interactions among the analyzed genes with their transcriptional regulators, leading into the differentiation of cells capable of producing insulin.
Abstract: The quest for small drug-like compounds that selectively inhibit the function of biological targets has always been a major focus in the pharmaceutical industry and in academia as well. High-throughput screening of compound libraries requires time, cost and resources. Therefore, the use of alternative methods is necessary for facilitating lead discovery. Computational techniques that dock small molecules into macromolecular targets and predict the affinity and activity of the small molecule are widely used in drug design and discovery, and have become an integral part of the industrial and academic research. In this review, we present an overview of some state-of-the-art technologies in modern drug design that have been developed for expediting the search for novel drug candidates.
Notes: Dalkas G and Vlachakis D are equally FIRST AUTHORS
Abstract: Poly(A)-specific ribonuclease (PARN) is a 3'-exoribonuclease that efficiently degrades poly(A) tails and regulates, in part, mRNA turnover rates. We have previously reported that adenosine- and cytosine-based glucopyranosyl nucleoside analogues with adequate tumour-inhibitory effect could effectively inhibit PARN. In the present study we dissect the mechanism of a more drastic inhibition of PARN by novel glucopyranosyl analogues bearing uracil, 5-fluorouracil or thymine as the base moiety. Kinetic analysis showed that three of the compounds are competitive inhibitors of PARN with K(i) values in the low μM concentration and significantly lower (11- to 33-fold) compared to our previous studies. Detailed kinetic analysis of the most effective inhibitor, the uracil-based nucleoside analogue (named U1), revealed slow-binding behaviour. Subsequent molecular docking experiments showed that all the compounds which inhibited PARN can efficiently bind into the active site of the enzyme through specific interactions. The present study dissects the inhibitory mechanism of this novel uracil-based compound, which prolongs its inhibitory effect through a slow-binding and slow-release mode at the active site of PARN, thus contributing to a more efficient inhibition. Such analogues could be used as leading compounds for further rationale design and synthesis of efficient and specific therapeutic agents. Moreover, our data reinforce the notion that human PARN can be established as a novel molecular target of potential anti-cancer agents through lowering mRNA turnover rates.
Abstract: Arginine/serine (RS) repeats are found in several proteins in metazoans with a wide variety of functions, many of which are regulated by SR protein kinase 1 (SRPK1)-mediated phosphorylation. Lamin B receptor (LBR) is such a protein implicated in chromatin anchorage to the nuclear envelope.
Abstract: Summary: Drugster is a fully interactive pipeline designed to break the command line barrier and introduce a new user-friendly environment to perform drug design, lead and structure optimization experiments through an efficient combination of the PDB2PQR, Ligbuilder, Gromacs and Dock suites. Our platform features a novel workflow that guides the user through each logical step of the iterative 3D structural optimization setup process, by providing a seamless interface to all incorporated packages.
Availability: Drugster can be freely downloaded via our dedicated server system at http://www.bioacademy.gr/bioinformatics/drugster/.
Contact: For support, comments and bug reports please contact: dvlachakis@bioacademy.gr.
Abstract: The viral family of flaviviridae is made up by the genera Flavivirus, Pestivirus and Hepacivirus. It includes numerous important human and animal pathogens. The small, enveloped virions of flaviviridae contain a single positive-sense RNA genome, in a long open reading frame (ORF), which is flanked by untranslated regions (UTRs) at the 5' and 3' ends. Recent studies on subgenomic flaviviridae replicons have revealed that the non-structural (NS) proteins, which are encoded by the C-terminal part of the polyprotein, play a crucial role in viral RNA replication. Accordingly, these proteins are assumed to form replication complexes in conjunction with genomic RNA and possibly with other cellular factors. On the most important non-structural enzymes that plays a key role in the life cycle of flaviviridae viruses is the viral helicase. Sequence alignments of the viral helicases from this family identified several conserved sequence motifs that are important for biological functions.
Herein, an effort is made to summarize the current epidemics associated with the flaviviridae family worldwide, the potential of helices as a promising pharmacological target and the use of nucleoside analogs as simple, efficient and rather versatile antiviral agents.
Abstract: Poly(A)-specific ribonuclease (PARN) is an exoribonuclease/deadenylase that degrades 3Î-end poly(A) tails in almost all eukaryotic organisms. Much of the biochemical and structural information on PARN comes from the human enzyme. However, the existence of PARN all along the eukaryotic evolutionary ladder, requires further and thorough investigation. Although the complete structure of the full-length human PARN, as well as several aspects of the catalytic mechanism still remain elusive, many previous studies indicate that PARN can be used as potent and promising anti-cancer target. In the present study, we attempt to complement the existing structural information on PARN with in-depth bioinformatics analyses, in order to get a hologram of the molecular evolution of PARNs active site. In an effort to draw an outline, which allows specific drug design targeting PARN, an unequivocally specific platform was designed for the development of selective modulators focusing on the unique structural and catalytic features of the enzyme. Extensive phylogenetic analysis based on all the publicly available genomes indicated a broad distribution for PARN across eukaryotic species and revealed structurally important amino acids which could be assigned as potentially strong contributors to the regulation of the catalytic mechanism of PARN. Based on the above, we propose a comprehensive in silico model for the PARNâs catalytic mechanism and moreover, we developed a 3D pharmacophore model, which was subsequently used for the introduction of DNP-poly(A) amphipathic substrate analog as a potential inhibitor of PARN. Indeed, biochemical analysis revealed that DNP-poly(A) inhibits PARN competitively. Our approach provides an efficient intergraded platform for the rational design of pharmacophore models as well as novel modulators of PARN with therapeutic potential.
Abstract: A new assay for the measurement of helicase enzyme activity was developed for the evaluation of the potency of potential inhibitors. This assay involves the use of a DNA or RNA duplex substrate and recombinant purified helicase. The DNA duplex consists of a pair of oligonucleotides, one of which is biotinylated and the other is digoxygenin (DIG)-labelled, both at their respective 5â termini. This DNA duplex is immobilised, via the biotin molecule, on the surface of a neutravidin-coated 96 well plate. Helicase activity results in DNA unwinding upon activation by ATP, leading to the release of the DIG labelled oligonucleotides, which translates in signal (luminescence) reduction with respect to control wells. This signal can be produced and quantified with the aid of a chemiluminescence antibody
Abstract: Therapeutic pelvic radiation is used with or without chemotherapy to treat malignancies of the pelvis that arise from gynecologic, genitourinary, and gastrointestinal organs. Along with the malignant tissue, which is the target of the radiation beam, adjacent organs are commonly included in the radiation field. This results in immediate and late complications. More specifically, the urinary bladder and the gastrointestinal tract are radiation sensitive and commonly symptoms arise from inflammation in these organs. More than 70% of patients who receive pelvic irradiation develop inflammation of the gastrointestinal tract and manifest symptoms of varying severity. More than half of these patients will develop chronic enteritis which will affect their quality of life. The role of the dietitian can be particularly important in the management of the acute and chronic problems of these patients. In the present review, we will attempt to describe the dietitianâs role and critically evaluate the dietary changes needed to improve enteritis in patients who have received therapeutic pelvis irradiation.
Abstract: Bariatric surgery is currently the most effective method of sustainable weight loss among morbidly obese patients. For many patients, the benefits of weight loss, such as decreases in blood glucose, lipids, and blood pressure and increase in physical activity, will outweigh the risks of surgical complications. Proper patient selection, thorough preoperative nutritional education, and postoperative nutritional follow-up contribute to the long-term management of the metabolic abnormalities of obesity. Bariatric surgery is divided into 2 major categories: restrictive procedures and combination of malabsorptive with restrictive procedures. The more dramatic weight loss is generally associated with the combined procedures. There is an increased risk of specific nutritional deficiencies associated with these types of surgery. With proper nutritional supplementation and frequent monitoring of nutritional status, these deficiencies can be successfully managed. This article reviews the most common bariatric surgeries and their typical dietary support and reviews critical nutrition care in regards to total parenteral nutrition for the morbidly obese patient, following bariatric surgery.
Abstract: Malnutrition represents a predictor of low immune response. The dietitian is often called to nutritionally support patients who cope with acute or chronic infections. A thorough nutritional assessment is important in order to recognize individuals with nutritional deficiencies. There are several theories and myths regarding the effect of different nutrients and foods in the management of infections; the aim of this review is to evaluate their role in the prevention and management of acute or chronic infections.
Abstract: BACKGROUND: Usutu virus belongs to the Flaviviridae viral family and constitutes an important pathogen. The viral helicase is an ideal target for inhibitor design, since this enzyme is essential for the survival, proliferation and transmission of the virus. RESULTS: Towards a drug-design approach, the 3D model of the Usutu virus helicase structure has been designed, using conventional homology modelling techniques and the known 3D-structure of the Murray Valley Encephalitis virus helicase, of the same viral family, as template. The model was then subjected to extended molecular dynamics simulations in a periodic box, filled with explicit water molecules for 10 nanoseconds. The reliability of the model was confirmed by obtaining acceptable scores from a variety of in silico scoring tools, including Procheck and Verify3D. CONLCUSION: The 3D model of the Usutu virus helicase exhibits in silico all known structural characteristics of the Flaviviridae viral family helicase enzymes and could provide the platform for further de novo structure-based design of novel anti-Usutu agents.
Abstract: Gromita is a fully integrated and efficient graphical user interface (GUI) to the recently updated molecular dynamics suite Gromacs, version 4. Gromita is a cross-platform, perl/tcl-tk based, interactive front end designed to break the command line barrier and introduce a new user-friendly environment to run molecular dynamics simulations through Gromacs. Our GUI features a novel workflow interface that guides the user through each logical step of the molecular dynamics setup process, making it accessible to both advanced and novice users. This tool provides a seamless interface to the Gromacs package, while providing enhanced functionality by speeding up and simplifying the task of setting up molecular dynamics simulations of biological systems. Gromita can be freely downloaded from http://bio.demokritos.gr/gromita/.
Notes: Sellis D and Vlachakis D are equally FIRST AUTHORS
Abstract: The Amino acid-Polyamine-Organocation (APC) superfamily is the main family of amino acid transporters found in all domains of life and one of the largest families of secondary transporters. Here, using a sensitive homology threading approach and modelling we show that the predicted structure of APC members is extremely similar to the crystal structures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities. All of these proteins, despite having no primary amino acid sequence similarity, share a similar structural core, consisting of two V-shaped domains of five transmembrane domains each, intertwined in an antiparallel topology. Based on this model, we reviewed available data on functional mutations in bacterial, fungal and mammalian APCs and obtained novel mutational data, which provide compelling evidence that the amino acid binding pocket is located in the vicinity of the unwound part of two broken helices, in a nearly identical position to the structures of similar transporters. Our analysis is fully supported by the evolutionary conservation and specific amino acid substitutions in the proposed substrate binding domains. Furthermore, it allows predictions concerning residues that might be crucial in determining the specificity profile of APC members. Finally, we show that two cytoplasmic loops constitute important functional elements in APCs. Our work along with different kinetic and specificity profiles of APC members in easily manipulated bacterial and fungal model systems could form a unique framework for combining genetic, in-silico and structural studies, for understanding the function of one of the most important transporter families.
Abstract: Herein we report a successful application of a computer-aided design approach to identify a novel HCV helicase inhibitor. A de novo drug design methodology was used to generate an initial set of structures that could potentially bind to a putative binding site. Further structure refinement was carried out through docking a series of focused virtual libraries. The most promising compound was synthesised and it exhibited a submicromolar inhibition of the HCV helicase.
Abstract: Poly(A)-specific ribonuclease (PARN) is a cap-interacting deadenylase that mediates, together with other exonucleases, the eukaryotic mRNA turnover and thus is actively involved in the regulation of gene expression. Aminoglycosides and natural nucleotides are the only reported modulators of human PARN activity, so far. In the present study, we show that synthetic nucleoside analogues bearing a fluoro-glucopyranosyl sugar moiety and benzoyl-modified cytosine or adenine as a base can effectively inhibit human PARN. Such nucleoside analogues exhibited substantial inhibitory effects, when tested against various cancer cell lines, as has been previously reported. Kinetic analysis showed that the inhibition of PARN is competitive and could not be released by altering Mg(II) concentration. Moreover, substitution of the 2', 4', or 6'-OH of the sugar moiety with acetyl and/or trityl groups was crucial for inhibitory efficacy. To understand how the nucleosides fit into the active site of PARN, we performed molecular docking experiments followed by molecular dynamics simulations. The in silico analysis showed that these compounds can efficiently dock into the active site of PARN. Our results support the idea that the sugar moiety mediates the stabilization of the nucleoside into the active site through interactions with catalytic amino acid residues. Taken together, our in vitro and in silico data suggest that human PARN is among the molecular targets of these compounds and could act therapeutically by lowering the mRNA turnover rate, thus explaining their known in vivo inhibitory effect at the molecular level.
Abstract: Bovine viral diarrhea virus (BVDV) is a member of the Flaviviridae family of viruses and constitutes a very important pathogen for livestock around the world. The viral helicase is an enzyme essential for the proliferation and transmission of the virus. In this work a 3D-model of the BVDV helicase was produced using homology modelling techniques and the known 3D-structure of the hepatitis C helicase of the Flaviviridae family as template, in an attempt to provide the means for structure-based design of novel anti-BVDV agents.
Abstract: Ssn6, a tetratricopeptide repeat (TPR) containing protein, associates with the Tup1 repressor to form a global transcriptional co-repressor complex, which is conserved across species. The three N-terminal TPR repeats of Ssn6, out of a total of 10, are involved in this particular interaction. Our previously reported 3D-modeling and mutagenesis data suggested that the structural integrity of TPR1 and its correct positioning relatively to TPR2 are crucial for Tup1 binding. In this study, we first investigate the structural stability of the Tup1 binding domain of Ssn6, in pure form, through a combination of CD spectroscopy and limited proteolysis mapping. The obtained data were next combined with molecular dynamics simulations and disorder/order predictions. This combined study revealed that, although competent to fold, in the absence of Tup1, TPR1 is partially unfolded with its helix B being highly dynamic exposing an apolar surface to the solvent. Subsequent CD spectroscopy on this domain complexed with a Tup1 fragment comprising its Ssn6 binding region provided strong evidence for a conformational change consisting of acquisition of alpha-helical structure with simultaneous stabilization of a coiled-coil configuration upon complex formation. We propose that this conformational change occurs largely in the TPR1 of Ssn6 and is in accord with the concept of folding coupled to binding, proposed for other TPR domains. A possible implication of the structural flexibility of Ssn6 TPR1 in Tup1 recognition is discussed and a novel mode of interaction is proposed for this particular TPR-mediated complex.
Abstract: Structure-based drug design methods utilise knowledge of a three dimensional structure of an enzyme/receptor to develop small molecules able to bind to the desired target, generating a specific biological response. These computer-based methodologies are now becoming an integral part of the drug discovery process and, although the principles of molecular recognition are far from being completely understood, some marketed compounds (i.e. Zanamivir, Lopinavir) have been developed with a successful application of structure-based design techniques.
In this presentation we are reporting a successful application of a computer-aided design approach to identify and synthetise a series of novel HCV helicase inhibitors. Initially a putative binding site was identified on the enzyme surface, then a de novo drug design software package was used to generate an initial set of structures that could potentially bind to it. A further structure refinement was carried out by docking a series of virtual libraries derived from the de novo procedure. The best structure identified in silico (AB100) was then prepared and it showed a submicromolar inhibition of the HCV helicase. The results of the replicon assay as well as the enzymatic assay for AB100 and a series of related analogues will be also presented.
Abstract: Einleitung: Das auslösende etiologische Agens bei der Sarkoidose ist immer noch unbekannt allerdings wird, auf Grund der Ãhnlichkeit der Granulome bei Sarkoidose und Tuberkulose, wird eine Beteiligung von Mykobakterien immer wieder diskutiert. Die EAA wird dagegen durch Einatmen organischer Stäube ausgelöst. Beide Erkrankungen zeichnen sich durch eine deutliche Zunahme der Lymphozyten in der BAL aus. Die Mechanismen dieses Einstroms sind noch weitgehend unverstanden. TOLL-like Rezeptoren (TLR) detektieren eine Vielzahl von mikrobiellen Produkten und bilden die erste Linie der Immunabwehr. In dieser Arbeit untersuchten wir die Expression und die Funktion des TLR9 bei BAL Zellen von Patienten mit Sarkoidose und EAA. Da die CXCR3 Liganden CXCL9, CXCL10 und CXCL11 Lymphozytenchemotaxis auslösen, haben wir die in-vitro sowie die CpG induzierte Freisetzung dieser Chemokine untersucht. Methodik: Die TLR9 Expression wurde mittels quantitativer PCR bestimmt, die Cytokinfreisetzung wurde mithilfe kommerziell erhältlicher Antikörperpaare gemessen (R&D Systems, Wiesbaden, BRD). Ergebnisse: In den Zellkulturüberständen von BAL Zellen von Patienten mit Sarkoidose und EAA konnten gegenüber Kontrollen erhöhte Konzentrationen an CXCL9, CXCL10 und CXCL11 nachgewiesen werden. BAL Zellen von Sarkoidosepatienten mit Roentgentyp I bzw. Loefgren Syndrom sowie Patienten mit EAA zeigten gegenüber Kontrollen eine vermehrte TLR9 Expression. Die TLR9 Expression bei Sarkoidosepatienten mit Roentgentypen II bis IV lag dagegen im Bereich der Kontrollen. Bei diesen Patienten findet sich auch die stärkste Vermehrung der Lymphozyten. Stimulation BAL-Zellen von Sarkoidosepatienten mit CpGs der Gruppe A induzierten eine signifikante Zunahme der CXCL10 Freisetzung, die CXCL9 und CXCL11 Freisetzung wurde zwar ebenfalls stimuliert, erreichte aber kein signifikantes Niveau. Zudem zeigte sich eine signifikante Korrelation der CXCL10 Freisetzung mit der TLR9 Expression (rho=0,6, p<0,005). Dieser Zusammenhang lieà sich bei CXCL9 und CXCL11 nicht zeigen. Diskussion: TLR9 Expression auf BAL Zellen findet sich bei den Patientengruppen mit den höchsten Lymphozytenzahlen. TLR9 Liganden induzieren die Freisetzung von CXCL10 und können somit zur Einwanderung CXCR3-positiver Lymphozyten in die Lunge beitragen. Die Ergebnisse zeigen, das die Vermehrung der Lymphozyten durch das angeborene Immunsystem via CXCL10 Freisetzung induziert werden kann.
Abstract: The helicase plays a key role in the life cycle of viruses of the Flaviviridae family. Japanese encephalitis virus is a member of this family and constitutes an important human pathogen (1). In this work, towards a structure-based design of novel anti-japanese encephalitis agents, a 3D-model of the japanese encephalitis helicase virus was produced using homology modelling techniques and the known 3D-structure of the yellow fever helicase of the Flaviviridae family as template.
Abstract: Hepatitis C is a viral infection that affects 170 million people worldwide, including 4million in theUnited States and 8 million in Europe. The virus establishes a chronic infection in 55â85% of cases and 20% of affected individuals develop cirrhosis. At the moment there is neither a vaccine nor an effective antiviral therapy available and efforts to identify a speciï¬c anti-HCV inhibitor have dramatically intensiï¬ed in the last few years. Many research groups have focused their interest on the enzymes involved in the viral replication and, among these enzyme, the viral Helicase/NTPase has proven to be a suitable target for developing novel anti-HCV compounds. Compound 1 is a potent inhibitor of the HCV helicase and, although itsmode of action is still uncertain, it has been proposed that it acts as competitive inhibitor of RNA binding. Starting from this hypothesis, we have prepared a series of novel com-
pounds based on the structure of 1 where the benzimidazole moiety has been replaced by different chemical groups, including the negatively charged carboxylate moiety, which should mimic the phosphate backbone of the nucleic acid. The synthesis, the enzyme inhibition and the biological eval-
uation in replicon of these novel compounds will be presented and analyzed.
Abstract: Purpose: The establishment of the 3D structure of bovine corneal biglycan by means of homologyâbased molecular modelling and the molecular optimisation of the model using molecular dynamics Methods:Molecular modelling experiments were done on a Linux P4 workstation using Molecular Operating Environment (MOE) software. The initial DNA sequence of the gene encoding for bovine corneal biglycan was downloaded from GenBank. The DNA sequences were translated to protein and the blastâP search revealed that the best template was decorin, which has been determined by Xâray crystallography (res. 2.5Ã, PDB entry: 1XKU). The pairâwise alignment was performed using ClustalX and revealed 55% homology identity between the model and the template. The alignment was repeated and manually adjusted within MOE. The homology algorithm of MOE produced a total of 10 different models that were scored and in silico evaluated for their reliability. The best one (as proposed by the MOE algorithm) was further structurally optimised by undergoing molecular dynamics at 300K, 1 atm for 1000 ps with 1 fs per step. The results of the molecular dynamics simulation were collected into a trajectory database by MOE for further analysis. The model was evaluated with Procheck and 95% of its residues were found in the core regions and another 5% in the allowed regions of the Ramachandran plot. Results: The final model of biglycan was structurally similar to the template protein, since it had all of its secondary elements conserved (e.g. the distinct parallel Ãâsheet pattern). The core protein was a horse shoe shaped molecule, with patches of hydrophobic amino acids in its inner cavity. The outer part of the protein consisted mainly of hydrophilic amino acids and exposed polar amino acids providing interaction sites to water molecules and polysaccharide polymers, respectively. Conclusions:The relatively high homology between the decorin and biglycan core proteins confirms that the proposed molecular model for biglycan is reliable. The hydrophilic and hydrophobic patches that were found in the biglycan protein suggest possible interaction sites with water molecules and collagen fibrils in the corneal stroma.
Abstract: The chemokines MIG (CXCL9), IP-10 (CXCL10), and ITAC (CXCL11) bind to CXCR3, a receptor expressed on Th1 cells. Hence, these chemokines might play a role in the recruitment of T cells into the lungs of patients with pulmonary sarcoidosis (SAR) and hypersensitivity pneumonitis (HP). We analysed the release of CXCL9, CXCL10, and CXCL11 by bronchoalveolar lavage (BAL) cells from patients with SAR, HP, usual interstitial pneumonia (UIP), tuberculosis (TB), and controls and correlated the amount of the released chemokines with differential cell count of the BAL. IP-10 and MIG were found to be released by BAL cells from controls and patients. IP-10 release was increased in SAR and HP. MIG release was highest by BAL cells from patients with HP, SAR, and UIP. Although ITAC release was very low in all cases it was significantly increased in all patient groups. IP-10 and MIG disclosed a significant correlation in all groups whereas IP-10 and ITAC correlated only in SAR, MIG and ITAC correlated in SAR, TB and UIP. There was a significant correlation of the percentage of lymphocytes in BAL with IP-10 or MIG in SAR, TB and UIP. In SAR a weak but significant correlation of IP-10 and ITAC with the percentage of CD3+ and CD4+ T cells could be detected. Multiple regression analysis of CXCR3 ligands and differential cell count did not disclose a higher correlation coefficient as revealed by simple correlation analysis. Our data demonstrate that CXCR3 ligands are released by BAL cells and are involved in T cell recruitment into the lung, however, they do not exert a synergistic effect.
Abstract: Hepatitis C and dengue are enveloped Ë n positive-sense RNA viruses. Hepatitis C virus is the major etiological agent of post-transfusion hepatitis worldwide. An estimated 3% of the worldâs population is infected with HCV according to the World Health Organization. Infection with HCV will most
regularly result in chronic hepatitis, which leads to liver cirrhosis, hepatocellular carcinoma and liver failure. Dengue is currently the most important viral disease, transmitted by mosquitoes and afï¬icting humans worldwide. Clinical symptoms range frommild fevers to a severe hemorrhagic disease.
To date, no speciï¬c antiviral treatments exist nor are there any vaccines available for either infections. Thus there is an urgent need for new therapies.
The aim of this project is to design and establish an enzymatic assay that will be used to screen for potential inhibitors of the Helicases of the HCV and dengue viruses as well as the Helicase/protease complex of the dengue virus. Helicases are interesting targets for drug design, ï¬rstly for their vi-
tal function in the viral cell cycle and secondly for the fact that human cells lack helicases capable of unwinding positive sense double stranded RNA. The genes of the HCV Helicase, the dengue virus Helicase and the dengue virus NS3 domain (Helicase and Protease) were incorporated into a pET system
expression vector. The vectors carrying the genes were then transformed into E. coli cells and the genes were expressed (BL21-pLysS strain). It was determined that both Helicases are produced in the cell without the need for induction. This was conï¬rmed by an expression test with variable concentrations of inducer (0â1mMIPTG). Itwas found that the protein was present under all expression systems. However the one induced at 1mMshowed max yield. After induction, the cell suspensions were harvested. SDSâPAGE and His-Tag Western blotting conï¬rmed the existence of the various proteins. Protein isolation was based on the 6(His)-Tags of the three proteins.The proteinswere tested for their functionality using speciï¬c enzymatic assays.
Abstract: BothHepatitisCandDengue are positive-senseRNAviruses. HCVis themajor etiological agent of post-transfusion hepatitis worldwide. According to the World Health Organization, 3% of the worldâs population is suffering from HCV infection. HCV infection leads to chronic hepatitis that may cause liver cirrhosis and may lead to hepatocellular carcinoma and liver failure. Dengue virus can cause from mild fevers to a severe haemorrhagic disease. Dengue virus is currently considered to be the most important human affecting, mosquito-transmitted disease worldwide. There is no effective cure or means of efï¬cient vaccination against either of the diseases, which makes the need for the development of new drugs and therapies much more urgent. The aim of this project is to design the 3D structure of the polymerase protein primarily of the Dengue virus and consequently those of the West Nile virus, the Japanese encephalitis virus and the Yellow fever virus. HCV polymerase (X-ray) was used as template with a homology identity of approximately 17%. The low homology identity percentage was overcome by performing ligand-supported homology modeling techniques and the conserved residue anchoring approach, which can be implemented into MODELLER. The viability of the fourmodel-polymeraseswas evaluated by an in silico scoring function and fromthe fact that the RNA binding motif and the ATP binding motifs were conserved between the models and the HCV template. Superimposition
between HCV and themodel-polymerases showed that seven residues of the active site (all involved in the RNA-binding motif) are conserved and yield an overall RMSd between 0.2 and 3.0 Ë A.
Abstract: This invention is about a highly specific, custom-made three dimensional Pharmacophore model for the catalytic site of the human poly-adenosine ribonuclease (PARN).
For more information please refer to our published article:
D Vlachakis, A Pavlopoulou, G Tsiliki, D Komiotis, C Stathopoulos, N Balatsos, S Kossida (2012) An integrated in silico approach to design specific inhibitors targeting human poly(A)- specific ribonuclease. Plos One In Press: Nov
Abstract: BENZENE DERIVATIVES â Compounds, which according to the invention can display potent inhibition against Hepatitis C Virus (HCV) helicase and potent anti-viral activity against HCV.
For more information please refer to our published article:
S Kandil, S Biondaro, D Vlachakis, A C Cummins, A Coluccia, C Berry, P Leyssen, J Neyts, A Brancale (2009) Discovery of a novel HCV helicase inhibitor by a de novo drug design approach. Bioorg Med Chem Lett 19: 11. 2935-2937
