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Journal of Biological Education Fabry disease as a model for: studying rare diseases, facing a new pharmacological approach, training free programs and website resources Giuseppina Andreottia§,…, MariaVittoria Cubellisb§ a Istituto di Chimica Biomolecolare – CNR, Pozzuoli 80078, Italy b Dipartimento di Biologia, Università Federico II, Napoli 80126, Italy §To whom correspondence should be addressed Giuseppina Andreotti, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Comprensorio Olivetti, Edificio 70, Via Campi Flegrei 34 I-80078 Pozzuoli (Napoli) Phone +39081-8675241 Fax 39-081-8675340; Email [email protected] Maria Vittoria Cubellis, Dipartimento di Biologia, Complesso di Monte Sant'Angelo, VIA Cinthia Napoli 80126; Tel.: +39 081-679118; Fax: +39 081-679233; Email: [email protected] Fabry disease as a model for: studying rare diseases, facing a new pharmacological approach, training free programs and website resources Abstract Recent advances in cell and molecular biology has been producing enormous amount of results in terms of genetic analysis and profiling and a urgent demand for rapid and accurate tools for the analysis of the results has been contemporarily raising. In the last two decades we recorded the development of large number of websites, web-applications and databases expressly created by multidisciplinary experts. These are essential tools for handling and analyzing biological data but a specific formation is needed and actually educational materials and courses in the bioinformatics area have been continuously increasing. On the other hand rare diseases have been catching increasing attention either from the scientific community and from the common people. The experience herein delineated is an interdisciplinary activity, designed for students in several fields (biology, medicine, chemistry) and give the opportunity to face chemical, biological and medical issues at molecular level, without wet-job, provided that these aspects are developed by others, in separate but complementary contexts. We think that it will be a valuable tool for biology students to increase their understanding of basic bioinformatic concepts. It is essentially an exercise to practice modern technologies and some steps were intentionally oversimplified. The outcomes of such kind of experience may give useful and interesting scientific results, but the clinical implications have to considered only an exercise and only professional subjects can utilize such results for practical and diagnostic purposes. Starting from a rare disease, Fabry disease, a journey through several and different websites, webapplications and databases can be realized mimicking an in silico scientific project, aiming at outlining a molecular diagnosis of a illness, identifying new molecules for developing new drugs, choosing the best therapeutic approach. Keywords: bioinformatics education; bioinformatics tools; rare disease; pharmacological chaperone; laboratory guide Introduction Fabry disease: a rare disease A rare disease is any disease that has a low prevalence and affects a small percentage of the population (with definitions ranging from 1/1000 to 1/200000 in the different regions in the world). There are more than 6000 rare diseases. On the whole, rare diseases may affect 30 million European Union citizens. 80% of rare diseases are of genetic origin, and are often chronic and lifethreatening. Rare disease is an important field of interest for the translational research but also for the application of personalized medicine approach. This approach is based on the integration of several individual information: the genome variation, physiology and cellular phenotype, the interaction with the personal environment and others (Palau 2012). Fabry disease is X−linked and relatively frequent, 1−9 in 100000 (OMIM: 30150) (ref, METTEREI UNA REVIEW RECENTE SUL FABRY DISEASE). At molecular level, Fabry disease is characterized by mutations of the gene enconding lysosomal alpha-galactosidase (AGAL) that causes low levels of this enzymatic activity leading to the accumulation (within the lysosomes) of the unprocessed substrate (Gb3, globotriaosylceramide, and its derivatives). It is one of the most common lysosomal storage diseases. Different mutations of the gene encoding AGAL result in a wide phenotypic spectrum, with respect to age at onset, rate of disease progression, severity of clinical manifestations. Patients with the late onset form of Fabry disease retain some AGAL activity and are asymptomatic until adult age when they develop cardiac and kidney problems. Since the age of onset can be late and its complications, cardiac manifestations, stroke and chronic renal disease, are very similar to those of other very common disorders, Fabry disease could have been under-diagnosed and an estimate as high as 1 in 3100 live births has been put forward. At present, the treatments of Fabry disease are symptomatic and life-long. So far the Enzymatic Replacement Therapy (ERT) was the only approved therapy. This therapy requires intravenous infusions of purified AGAL produced by genetically engineered cell lines every 2 weeks. Pharmacological chaperone: a promising strategy for the treatment of some genetic diseases The therapeutic approach with pharmacological chaperones (PCs) relies on small molecules that bind and stabilize the enzyme, preventing its early degradation by intracellular machineries and increasing its total intracellular levels. It exploits small molecules which can be administered orally, can reach 'difficult' tissues such as the brain and presumably have low cost. They act as life jackets or chaperones for enzymes that become unstable and are degraded upon mutation, although they retain the essential residues needed for their enzymatic activity. The same mutant proteins are able to comply their duty if they are given the chance to survive long enough and to get to the site where they are needed. At molecular level this therapeutic approach depends on the fact that the complex enzyme-ligand is thermodynamically more stable than the enzyme alone. The ligand may be any kind of small molecules provided that it binds to the enzyme, included competitive inhibitors (which occupies the active site) but also allosteric ligands (which bind to sites other than the active one) (Ringe and Petsko, 2009). The treatment of metabolic diseases with competitive inhibitors as chemical chaperons at subinhibitory intracellular concentrations was first proposed by Fan et al in 1999. The approach is getting known as pharmacological chaperone therapy and despite its novelty, it has produced a few drugs which are in clinical trials or ready for the market. Fabry disease is an example of disorders which can benefit from PC therapy. A large variability of genotypes and phenotypes is observed and only a fraction of missense mutations respond to PC therapy. The effects of missense mutations and hence their responsiveness to drugs, depends on the site where they occur in the protein. Treatment with pharmacological chaperones, in particular 1deoxy-galactonojirimycin (DGJ, migalastat hydrochloride or AT1001) has already passed phase three clinical trials (Markham 2016). Learning objectives Basically a student should gain familiarity with several websites and will see an important although not exhaustive application of all of them. More specifically, a student should be able to: (1) analyze a DNA/protein sequence (using BLAST); (2) search for a protein with known structure (using PDB); (3) visualize a protein structure and highlight specific aminoacid/region/ecc with RasMol; (4) search for properties of enzymes (using BRENDA). Moreover the student will be introduced to the docking (with SwissDock) and with a rare disease. Finally a student will be introduced to a quite novel usage of the term “chaperone”, namely the pharmacological chaperone, that is so far poorly studied and mostly confused with the most popular molecular chaperone. Materials and methods Requirements It is an in silico experience so that only a computer with an internet connection is required. In order to follow this exercise, students should have learned the basic biochemistry topics such as proteins/enzymes (structure and function), DNA, carbohydrates, and also basic concepts of thermodynamic and cell biology. The experience The experience will start from the results of a DNA analysis on a hypothetical patient. By utilizing website resource (BLAST) the nucleotide sequence will be compared to control database and then the encoded protein will be identified. Then the protein structure will be searched (PDB) and visualized (RasMol). A public and comprehensive database for enzymes (BRENDA) will be searched for the identification of specific molecules potentially able to bind to the above identified protein and the efficacy of the binding will be checked (SwissDock). Eventually a web-application designed to help clinicians to choose the best therapeutic approach will be considered (FABRY_CEP). Bioniformatic tools Bioinformatics tools have the advantages of enabling fast, low-cost and reliable analysis of biological data with user-friendly interfaces. These will be used for this experience: BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi PDB, http://www.rcsb.org/pdb/home/home.do (Berman et al. 2000) RasMol, http://www.openrasmol.org/ (Bernstein 2000; Sayle and Milner-White 1995) BRENDA, http://www.brenda-enzymes.org/index.php SwissDock, http://www.swissdock.ch/ FABRY_CEP, http://www1.na.icb.cnr.it/project/fabry_cep/ (Cammisa et al. 2013) Clinical case Mister X was suspected to have a rare genetic disease and his doctor prescribed a genetic test to check for Fabry disease, in order to provide a more informed and tailored drug prescription. The sequence of the gene analyzed was the following (BISOGNEREBBE SPECIFICARE SU COSA E STATA FATTA QUESTA SEQUENZA….): atgcagctgaggaacccagaactacatctgggctgcgcgcttgcgcttcgcttcctggccctcgtttcctgggacatccctggggctagagcact ggacaatggattggcaaggacgcctaccatgggctggctgcactgggagcgcttcatgtgcaaccttgactgccaggaagagccagattcctg catcagtgagaagctcttcatggagatggcagagctcatggtctcagaaggctggaaggatgcaggttatgagtacctctgcattgatgactgttg gatggctccccaaagagattcagaaggcagacttcaggcagaccctcagcgctttcctcatgggattcgccagctagctaattatgttcacagca aaggactgaagctagggatttatgcagatgttggaaataaaacctgcgcaggcttccctgggagttttggatactacgacattgatgcccagacct ttgctgactggggagtagatctgctaaaatttgatggttgttactgtgacagtttggaaaatttggcagatggttataagcacatgtccttggccctga ataggactggcagaagcattgtgtactcctgtgagtggcctctttatatgtggccctttcaaaagcccaattatacagaaatccgacagtactgcaa tcactggcgaaattttgctgacattgatgattcctggaaaagtataaagagtatcttgcactggacatcttttaaccaggagagaattgttgatgttgc tggaccagggggttggaatgacccagatatgttagtgattggcaactttggcctcagctggaatcagcaagtaactcagatggccctctgggcta tcatggctgctcctttattcatgtctaatgacctccgacacatcagccctcaagccaaagctctccttcaggataaggacgtaattgccatcaatcag gaccccttgggcaagcaagggtaccagcttagacagggagacaactttgaagtgtgggaacgacctctctcaggcttagcctgggctgtagct atgataaaccggcaggagattggtggacctcgctcttataccatcgcagttgcttccctgggtaaaggagtggcctgtaatcctgcctgcttcatca cacagctcctccctgtgaaaaggaagctagggttctatgaatggacttcaaggttaagaagtcacataaatcccacaggcactgttttgcttcagct agaaaatacaatgcagatgtcattaaaagacttactttaaaa Results and discussion Step 1. DNA analysis In order to analyze the DNA sequence obtained the program blastn will be used (figure 1A). 1) Open the website and choose blastn (it searches nucleotide databases using a nucleotide query). 2) Paste the given sequence into the Enter Query Sequence box. 3) Choose the Organism “Homo sapiens”. 4) Start the search. The output is a list of all the significant alignments with DNA sequences (each sequence has been determined within a specific scientific project then deposited by the authors in this bank, all the details can be achieved by the Accession number) (figure 1B). In particular, all the sequence producing significant alignments belong to human alpha-galactosidase. For each alignment many parameters are shown. For the sake of simplicity, we can focus our attention on those alignments for which the query sequence was totally considered (100% query cover), no gaps were introduced (in order to match the sequences), and a high identity was recorded. Looking at the alignment with “Homo sapiens galactosidase alpha (GLA), mRNA” it is possible to observe that all the nucleotides matched except one: at position 730 of the query sequence (the patient’s sequence) a C was identified instead of a G as in the control sequence (Homo sapiens galactosidase alpha) (figure 1C). Step 2. Looking for structural data In order to understand the effect of this mutation on the encoded protein it is necessary to consider the aminoacidic sequence since a change in one DNA base pair in the coding region may results in the substitution of one aminoacid for another in the protein made by a gene (missense mutation) or introduce a stop codon so that this type of mutation results in a shortened protein that may function improperly or not at all (nonsense mutation). We can search for the structure of human AGAL by using the program (figure 2A). 1) Choose blastx (it searches protein databases using a translated nucleotide query). 2) Paste the given sequence into the Enter Query Sequence box. 3) Choose the Organism “Homo sapiens”. 4) Choose the database Protein Data Bank proteins (pdb). 5) Start the search. The output is a list of all the significant alignments with the relative accession number (figure 2B). In this case the best score was obtained with “Chain A, Structure Of Human Alpha-galactosidase”. In this specific case, the substitution G->C we noticed above, produce a missense mutation and the protein sequence resulted modified in fat the coding triplet CAC introduce the residue Histidine instead of the Aspartic acid found in control sequence (in the position 244). So far we have verified that the patient has a mutated alpha-galactosidase and in particular the mutation found was p.D244H. But we need to know more about alpha-galactosidase structure. Was it already determined? The output of blastx also indicate a Sequence ID: 1R46 (figure 2C). It is now possible to find more information about this structure running a search for 1R46 in the PDB (figure 3A). 1) Open the website. 2) Paste the given ID in the search box and Go. We can learn (figure 3B) that: 1) this structure refers to the human alpha-galactosidase; 2) it is designed as a hydrolase; 3) the structure was deposited in this bank in 2003; 4) the authors were Garman and Garboczi; and 5) this structure was determined by X-ray diffraction. From the published paper we can learn many other details for example “The catalytic mechanism of the enzyme is revealed by the location of two aspartic acid residues (D170 and D231), which act as a nucleophile and an acid/base, respectively” (Garman and Garboczi 2004). Step 3. Structural analysis of the alpha-galactosidase We can download and save the data (choosing the PDB Format, the dowloaded file will be saved as 1r46.pdb). In order to visualize the three-dimensional structure it is necessary to use a specific program. One of the simplest is RasMol, that is also free. Having previously loaded RasWin (from the RasMol website), the Windows version of the software RasMol, the file 1r46.pdb will be easily opened (figure 4A). We can read all the details of this structure (total atoms, bonds, secondary structural elements and so on). We then: 1) select colours and then 2) select chain, we can see that AGAL has a quaternary structure, in fact it is a homodimer, that means it is composed of two identical polypeptide chains (blue and light blue in figure 2B). Moreover it is a glycoprotein bearing oligosaccharides (green) linked on specific aminoacidic residues. We can visualize the aminoacids responsible for the catalytic activity, namely residue 231 and 170 by writing “select 231 or 170” in the dialog box. But where is located the mutated aminoacid? Does it belong to the catalytic site? Does it hamper the enzymatic activity? To address these questions we can write in the dialog box as follow: color green, spacefill, select 244, color red, spacefill (figure 4C). In so doing we will highlight clearly (within each chain) the aminoacids responsible for the catalytic activity (red) and the mutated aminoacid 244 (green) and we can realize that this aminoacid is situated far from the active site suggesting that the mutation could not hinder the catalytic activity. Actually it has been demonstrated that this mutation alter the protein stability but does not modify the catalytic properties of AGAL. This is one of those missense mutations amenable for treatment with PC (see step 5). Step 4. How to develop a pharmacological chaperone What is the function of AGAL in the cells? We can find this and many other information within BRENDA by running a search for “alpha-GAL EC 3.2.1.22” and selecting Homo sapiens in the appropriate dialog box. Looking at the menu on the left, among Enzyme-Ligand interactions, Bibliography/Links/Disease, ecc, we can learn that an AGAL deficiency cause the Fabry-Anderson disease (figure 5A). AGAL is a hydrolyse whose natural substrate is the globotriaosylceramide (figure 5B). We can notice that Gb3 contains a galactose moiety (figure 6) which is specifically recognized by the enzyme (through the aminoacids lining its catalytic site). It has been widely demonstrated that the binding of an inhibitor to an enzyme stabilizes the enzyme so that it comes naturally to look for PC among competitive inhibitors of an enzyme. In this same database we can see that 1-deoxy-galactonojirimycin (DGJ or migalastat) is an inhibitor of AGAL (figure 5B). We can observe that it resembles the galactose moiety of the Gb3 (as shown in figure 6). Does DGJ bind to AGAL? Is it possible to verify it? Where is the binding site? How strong is the binding? In other words we need to run a molecular docking, that allow the prediction of the preferred orientation of one molecule to a second when bound to each other to form a stable complex. For this purpose we can use SwissDock, and our target is 1R46 while our ligand is migalastat. We also need to specify a Job name and an email address before starting the docking (figure 7A). It will take several hours to obtain the results. The output shows the binding site and also the calculated binding energy (a negative binding energy indicates a favorable thermodynamic interaction) (figure 7B). The obtained results clearly indicate that DGJ might bind strongly to AGAL so this molecule can be considered a potential PC. Indeed DGJ is a PC for Fabry disease. It took several years and hundreds of experiments to asses this point. One of the first step in that direction was to test the molecule in vitro (having identified the candidate molecule from a commercially available compounds it will be quite easy to pass to the experimental test, otherwise it will be necessary to synthesize the molecule and this will require a huge effort of preliminary experimental job). This experiments are outside the scope of this exercise but in brief it is necessary to treat appropriate cells (for examples fibroblasts) with the molecule and verify (by enzymatic assay and western blotting) that the treatment provoke an increase of the intracellular level of the target protein (figure 8). Many other experiments allowed the identification of DGJ as a candidate for clinical trials and recently has been approved for the treatment of Fabry disease in the EU in patients with amenable mutations. Step 5. How to identify the AGAL mutations amenable for PC therapy? Up to now xx missense different mutation have been identified for AGAL. There is a wide heterogeneity of genotypes but this explains only part of the heterogeneity observed in phenotypes because clinical phenotype, age of onset and course of Fabry disease are highly variable, even within the same family. At present the treatments of Fabry disease are symptomatic and life-long. Enzyme replacement (ERT) has already been introduced into medical practice and is considered “the clinical gold standard”. This therapy requires intravenous infusions of purified AGAL produced by genetically engineered cell lines every 2 weeks. In theory PCs represent a useful alternative to ERT because they do not cause adverse immunological reactions and can be administered orally. In practice therapy with PCs is limited because only some mutations can be rescued by these drugs. It has been observed that usually, but not necessarily, late-onset forms, as well as mutations that do not occur in the catalytic domain of AGAL, respond to 1-deoxy-galactonojirimycin. The decision to use it in therapy has to be taken on a case-by-case basis after precise genotyping because DGJ is not effective on all alphagalactosidase (AGAL) mutants. Fabry_CEP is a user-friendly web-application designed to help clinicians Choose Eligible Patients for the therapy with pharmacological chaperones (figure 9). It provides a database and a predictive tool to evaluate the responsiveness of lysosomal alpha-galactosidase mutants to a small molecule drug, namely 1-Deoxy-galactonojirimycin. The user can introduce any missense/nonsense mutation in the coding sequence (in the query data box the mutation both substitute nucleotide or substitute aminoacid can be added), learn whether it is has been tested and gain access to appropriate reference literature. In the absence of experimental data structural, functional and evolutionary analysis provides a prediction and the probability that a given mutation is responsive to the drug. In the analyzed case, p.D244H, we can read that the mutation does not occurred in the active site, does not interfere with disulphide bridge formation and it has been sperimetally proved to be responsive to the treatment with DGJ. Acknowledgment This work was supported by Telethon-Italy under Grant GGP12108. References MANCA REVIEW RECENTE ED ESAUSTIVA X FABRY DISEASE Berman H.M., Westbrook J., Feng Z., Gilliland G., Bhat T.N., Weissig H., Shindyalov I.N., Bourne P.E. 2000. “The Protein Data Bank.” Nucleic Acids Research 28: 235-242. Bernstein H.J. 2000. Recent changes to RasMol, recombining the variants. Trends in Biochemical Sciences 25(9): 453-455. Cammisa M., Correra A., Andreotti G., Cubellis M.V. 2013. “Fabry_CEP: a tool to identify Fabry mutations responsive to pharmacological chaperones.” Orphanet Journal of Rare Diseases 8:111. Fan J.Q., Ishii S., Asano N., Suzuki Y. 1999. “Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor.” Nature Medicine 5: 112-115. Garman S.C. and Garboczi D.N. 2004. “The molecular defect leading to Fabry disease: structure of human alpha-galactosidase.” Journal of Molecular Biology 337(2): 319-335. Markam A. 2016. “Migalastat: First Global Approval.” Drugs 76(11): 1147-52. Palau F. 2012. “Personalized medicine in rare diseases.” Personalized Medicine 9(2): 137-141. Ringe D. and Petsko G.A. 2009. “Q&A: What are pharmacological chaperones and why are they interesting?” Journal of Biology 8: 80. Sayle R.A. and Milner-White E. J. 1995. “RasMol: Biomolecular graphics for all.” Trends in Biochemical Sciences 20(9): 374-376. Figure captions Figure 1. DNA analysis by using bastn. Panel A, input data. Panel B, list of all the significant alignments with DNA sequences. Panel C, DNA sequence comparison between patient’s DNA and Homo sapiens galactosidase alpha. Figure 2. DNA analysis by using bastx. Panel A, input data. Panel B, list of all the significant alignments with protein sequences. Panel C, protein sequence comparison between patient’s DNA and Homo sapiens galactosidase alpha. Figure 3. Structural analysis of the alpha-galactosidase (1R46) by using PDB. Panel A, input data. Panel B, output information. Figure 4. Structural details of alpha-galactosidase (1R46) by using RasMol. Panel A, global overview. Panel B, quaternary structure. Panel C, Catalytic residues and localization of the mutation found in the patient. Figure 5. Gaining info about AGAL by using BRENDA. Panel A, input data. Panel B and C, selected output information showing details about : disease, natural substrate, inhibitors. Figure 6. Chemical formulas of: Gb3 (A), galactose (B), DGJ (C). Figure 7. Molecular docking by using SwissDock. Panel A, input data. Panel B, output information. Figure 8. An illustrative scheme for testing in vitro potential pharmacological chaperone. Figure 9. Gaining info about specific mutations in AGAL by using Fabry_CEP.