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Role of genomics proteomics, and bioinformatics.

  1. 1. Role of Genomics, Proteomics, and Bioinformatics. Presented by:- Lingaraj .V. Anawal M.Pharm 2nd SEM Department of Pharmacology H.S.K College of Pharmacy B.G.K 1
  2. 2. Genome:- It is an organism complete set of DNA. Genomics:- “The branch of molecular biology concerned with the structure, function, evolution, and mapping of genomes”. Executing the sequencing and analysis of entire human genome enables more rapid and effective identification of disease associated genes and provide drug companies with pre validated targets. Proteomics:- It consists of identifying the proteins present in a biological sample or the proteins that are differentially expressed between samples, such as diseased v/s normal tissues. Bioinformatics:- The collection, classification, storage, and analysis of biochemical and biological information using computers especially as applied to molecular genetics and genomics. 2
  3. 3. Structural genomics:- • It is used to describe 3-D structure of every protein encoded by a given genome. • It has potential to inform knowledge of protein function. • It characterize genome structures. • Attempts to determine structure of every protein encoded by genome rather than focusing on one particular protein. 3 Types of Genomics Structural genomics Functional genomics
  4. 4. Example of structural genomics.  Mycobacterium tuberculosis proteome:-  The goal of the TB Structural Genomics Consortium is to determine the structures of potential drug targets in Mycobacterium tuberculosis, the bacterium that causes tuberculosis. The development of novel drug therapies against tuberculosis are particularly important given the growing problem of multi-drug resistant tuberculosis.  The fully sequenced genome of M. tuberculosis has allowed scientists to clone many of these protein targets into expression vectors for purification and structure determination by X-ray crystallography. Studies have identified a number of target proteins for structure determination, including extracellular proteins that may be involved in pathogenesis, iron-regulatory proteins, current drug targets, and proteins predicted to have novel folds. till now structures have been determined for 708 of the proteins encoded by M. tuberculosis. 4
  5. 5. Functional genomics : • Focus on gene transcription, translation, regulation of gene expression and protein-protein interactions. • Goal is to understand relationship between an organism genome and its phenotype. • Measure all gene products like mRNA or proteins within biological sample. Technique used:- DNA Microarray. • Key areas of genomic study:- Development and application of tools for prediction and detection of :-  genes  sequence similarity  gene expression variation ( measurement of mRNA levels through microarray analysis) 5
  6. 6. Role of Genomics in drug development  Drug development is defined in many pharmaceutical companies as the process of taking a new chemical lead through the stages necessary to allow it to be tested in human clinical trials.  Molecular genetics reached human genetics about 1976, when the first human genes were cloned.  Transgenic methods, ‘knock‐outs’ and ‘knock‐in’ began in about 1986, and in about 1996, database searching became a fruitful way to do genomic research.  The term ‘genomics’ describes the discipline in genetics concerned with the study of the complete set of genes (genomes) of organisms. 6
  7. 7. Genomics cont…  The field includes efforts to determine the entire DNA sequence of organisms, fine-scale genetic mapping, studies of intragenomic phenomena such as heterosis, epistasis, pleiotropy and other interactions between loci and alleles within the genome.  The primary goal of genomics research in the pharmaceutical industry in the 1990s was to identify not only new molecular targets but also more of them, and to be first to gain proprietary rights to use those targets.  Genomics explore new opportunities for the drug discovery, especially through technologies like high-throughput sequencing and characterization of expressed human genes.  Knowledge of all the human genes and their functions may allow effective preventive measures. 7
  8. 8. Genomics cont…  The cause of common fatal diseases has been identified by genomics and it shows the potential to identify individuals who are particularly susceptible to a given disease long before that disease becomes apparent.  Pharmacogenomics has its roots in pharmacogenetics. Whereas pharmacogenetics is the study of the linkage between an individual's genotype and that individual's ability to metabolize a foreign compound.  The primary goal is to identify new molecular targets from genome.  Identification of additional gene family members and identification of drug candidates that are much more selective for the target proteins. 8
  9. 9. Genomics cont…  Many new targets for drug research and development, more the targets better the choice of targets to work on. (choice is by using knowledge and tools of genomics understanding the molecular pathway underlying disease)  Understanding the complete linkage of genes, transcription factors, enzymes, receptors, modulators etc… and their aberrations in disease better rational choice of the best target. 9
  10. 10. Examples of genomics Cancer:- Colorectal cancer patients with a particular mutation in the PIK3CA gene are found to benefit from treatment with aspirin post diagnosis. Early detection of cancer:- Cell free circulating DNA is also being explored as a biomarker for cancers. As the tumour cells die they release fragments of their mutated DNA can give insights into the tumor and possible treatments, and even be used to monitor tumor progression. Cystic fibrosis:- Approximently four percent of cystic fibrosis carry G551D mutation in the CTFR gene. Now the drug called ivacaftor has been developed that is extraordinarily effective for such patients. 10
  11. 11. Role of Proteomics in drug development  The term "proteomics" was first coined in 1997 to make an analog with genomics, the study of the genes.  The word "proteome" is a blend of "protein" and "genome", and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student.  Proteomics is a technology platform that is gaining wide spread use in drug discovery and drug development programs.  It provides effective means to identify biomarkers that have the potential to improve decision making surrounding drug efficacy and safety issues based on data derived from the study of key tissues and the discovery and appropriate utilization of biomarkers. 11
  12. 12. Proteomics cont…  With the accumulation of vast amounts of DNA sequences in databases, researchers are realizing that merely having complete sequences of genomes is not sufficient to elucidate biological function.  A cell is normally dependent upon a multitude of metabolic and regulatory pathways for its survival.  There is no strict linear relationship between genes and the protein complement or ‘proteome’ of a cell.  Proteomics is complementary to genomics because it focuses on the gene products, which are the active agents in cells.  Proteomics directly contributes to drug development as almost all drugs are directed against proteins. 12
  13. 13. Proteomics cont…  Proteins are the functional output of the cell and therefore might be expected to provide the most relevant information.  The expression or function of proteins is modulated at many points from transcription to post-translation, which generally cannot be predicted from analysis of nucleic acids alone.  There is poor correlation between the abundance of mRNA transcribed from the DNA and the respective proteins translated from that mRNA and the transcript can be spliced in various ways to yield different protein forms.  The strategy of working forward from the gene has been used a specific genetic lesion is identified and the resultant changes in protein structure, function, or expression are elucidated, so that a drug to counteract or correct such aberrations can be rationally designed. 13
  14. 14. Proteomics cont…  Proteomics has gained much attention as a drug development platform. because disease processes and treatments are often manifesting at the protein level. Drugs ought to produce protein expression effects and, hence the pattern of protein changes after drug application will give information about the mechanism of action, either for therapeutic or toxicological effects.  Various drugs might be compared and grouped according to their signal in metabolistic pathways. Side effects can also be described if additional proteins are involved. The correlation of the dynamic expression of a proteome and the physiological changes related to a healthy or diseased condition can help to:-  Support the understanding of disease mechanisms,  Design new ways for the discovery and validation,  Disease models,  Find new diagnostic markers,  Identify potential therapeutic targets,  Optimise lead compounds for clinical development,  Characterise drug effects,  Study protein toxicology, 14
  15. 15. Proteomics cont…  Proteomics is the systematic high-throughput separation and characterization of proteins within biological systems.  Proteomics measures protein expression directly, not via gene expression, thus achieving better accuracy. Current work uses 2-dimensional polyacrylamide gel electrophoresis(2D- PAGE) and mass spectrometry.  New separation and characterization technologies, such as protein microarray and high throughput chromatography are being developed. 15
  16. 16. Proteomic studies gives information includes  Protein structure and function.  Protein expression levels.  Post translational modifications.  Sub-cellular localization.  Protein-protein interaction.  Protein-nucleotide interaction.  Protein lipid Interaction 16
  17. 17. Process flowchart of proteomics analysis 17Modern methods of drug discovery edited by A. Hillisch and R. Hilgenfield
  18. 18.  Proteomics can be applied as an assay procedure for the potential utility of drug candidates.  This can be achieved by a comparative analysis of reference protein profiles from normal or diseased state with profiles after drug treatment.  This technology can also be integrated with combinatorial chemistry to evaluate comparative SAR of drug analogues.  Pharmaceutical proteomics for target validation split into expression proteomics and cell mapping or interaction proteomics, each having a distinct role in the overall drug discovery process. 18
  19. 19. Functional areas of Proteomics  Fractionation and purification:- (Separation , isolation )  Identification (Primary sequence):- determining primary sequence  Amino acid sequence of the proteins.  Quantitation:- How much of a protein has been detected.  Characterization:- Protein analysis and studies -Sequence homologies. -Post translational modifications. -Functional analysis, including interactions pathways and networks. -Structural analysis and structure-function relationships. 19
  20. 20. Fractionation and purification (Separation , isolation )  Separation and purification of proteins.  Low scale:- Gel and column based fractionation, Differential centrifugation.  Proteins gets separated into bulk fractions/bands based on size/charge.  More advanced method:- Immunoprecipitation and affinity chromatography. Individual protein from mixture.  Most common separation methods for high throughput application are 2D-PAGE , HPLC. 20
  21. 21. 2D-PAGE (Two dimensional polyacrylamide gel electrophoresis)  Protein separation based on mass.  Sample loaded on to a gel slab and an electric current is passed through the gel, the current pulls the proteins down according to their mass, separation by mass alone is not sufficient.  Separation along first dimension of the gel by isoelectric point and second dimension by mass.  Results in a pattern of spots, each represents a protein of specific mass and isoelectric point.  Most effective and widely used.  Does not detect protein at low concentration.  Proteins having similar mass appear at same spot. 21
  22. 22. HPLC (High performance liquid chromatography)  Column filled with packing material that separates proteins by eluting them, detector at the end will identify the proteins.  Gel filtration, ion exchange , affinity, hydrophobic interaction.  This method is much faster , the results are more repeatable.  More sensitive to low concentration proteins. Quantitation  UV absorption at a wavelength of 280nm is a very rapid, simple and effective method of measuring a protein concentration in solution.  Gel based methods can also be used.  Staining techniques:- coomassie blue staining, silver staining are sensitive.  MS will only measure the components that are present in the mixture. 22
  23. 23. Characterization  Protein sequence, structure, and function.  Proteins structure determines its function.  A final 3 dimensional configuration of a protein, active sites and binding sites determines the Protein’s biological or chemical function.  Primary structure:-Sequence of amino acids that forms the protein chain. determines 3D configuration.  Secondary structure:- 3D structure of subsections of protein chain, alpha helices, beta sheets  Tertiary structure:- alpha helices, beta sheets, and other secondary structures assemble into a globular protein structure.  Quaternary structure:- Individual protein chains further assemble into dimers, trimers…etc  Structures may not be complete at tertiary and quaternary stage, PTM changes structures ,influence stability, function, interactions etc. 23
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  26. 26. Difference between Genomics and Proteomics  Genome, which is relatively static, the Proteome is not static; it changes constantly in response to tens of thousands of intra and extracellular environmental signals.  The proteome varies with various factors like health or disease, the nature of each tissue, the stage of cell development, and effects of drug treatments.  The proteome often is defined as “the proteins present in one sample (tissue, organism, and cell culture) at a certain point in time.”  Proteomics runs parallel to genomics in many ways. Genomics starts with the gene and makes inferences about its products (proteins), whereas proteomics begins with the functionally modified protein and works back to the gene responsible for its production.  The sequencing of the human genome has increased interest in proteomics because DNA sequence information provides a static snapshot of the various ways in which the cell might use its proteins, the life of the cell is a dynamic process.  This new data increase the interest of proteomics in the field of science, medicine, and most notably – pharmaceuticals. 26
  27. 27. 27
  28. 28. An overview of proteomics techniques 28
  29. 29. Application of proteomics to drug discovery. Databases Diagnostics Target discovery Target validation Candidate selection Preclinical toxicology Clinical development 29
  30. 30. Application of proteomics to drug discovery and development Preclinical Clinical i. Target identification ii. Target validation iii. Lead candidate selection iv. Mode of action studies v. Toxicology -no observed effect level -screening -mechanism of action i. Diagnostics ii. Markers of response iii. Inclusion and exclusion criteria iv. Subgroup of responders/adverse reaction v. Post-launch differentiation of competitors 30
  31. 31. Database:-  Establishment of subscription databases documenting the protein profiles of specific cell types under different condition.  A series of integrated proteomics/genomics databases has been developed jointly by OGS and Incyte (Palo, Alto, CA, USA) using a range of clinically important human and microbial cell types.  This combined approach is uniquely brings together information linking the genotype and phenotype, via genomic(genes), mRNA(transcription) and Proteomic (translational) data. 31
  32. 32. Diagnostics:-  In this development of novel diagnostics based on the analysis of human body fluids.  Genomics- based on diagnostic approaches may require a sample of the diseased tissue.  Proteomics- based approaches typically do not.  By comparing protein expression profiles obtained from the body fluids of healthy and diseased individuals, changes in the abundance of protein can readily be detected, supporting the development of markers for the diagnosis and monitoring of disease. 32
  33. 33. Target discovery:-  In which sample from normal and diseased tissues are compared directly, is one of the most important pharmaceutical application of proteomics. -Taking oncology as an example;- proteomics studies have been reported in the analysis of neuroblastomas, breast cancer, lung, colon, renal cell carcinoma. 33 Target validation:-  It is ideally requires both gain and loss of function studies to determine whether a change in the expression of given protein leads to a disease phenotype and whether correction of the change reverses it.
  34. 34. Candidate selection:-  Proteomics can play an integral role in the selection and optimization of lead candidates for further development.  It is important to confirm that their efficacy is achieved through the expected mechanism of action and to compare the effects of candidates against a range of markers of efficacy and toxicity. 34
  35. 35. Preclinical toxicology:-  Toxicological screening is an important early stage in the development of a drug when the mode of action has been validated.  Many initially promising drugs fail at this stage, but traditional toxicological screens have been both laborious and difficult to interpret.  Proteomics has great potential in early toxicological screening, since it is anticipated that drug toxicity will produce a limited array of proteomic signatures that can be documented and used to screen the toxicological properties of novel candidates.  LSP are compiling a Molecular Effects of Drugs database, while OGS has launched a collaborative program with Quintiles (Ledbury, UK) to validate this approach by focusing on a range of drugs for which toxicity has already been characterized in detail using standard measures.  The OGS studies, which focus on the P450 enzyme series responsible for metabolizing most drugs show that proteomics can detect and predict the toxic effects of drugs at significantly lower doses than would be required to demonstrate toxicity using standard techniques. 35
  36. 36. Clinical development:- Proteomics has the capacity to reduce the duration of clinical trials, through the development of biomarkers of response.  It also has the ability to reduce the size of patient populations by enabling patients to be subtyped and pre-selected according to stringent molecular criteria.  As a result of these developments, clinical development has the potential to become more efficient and less expensive, while pharmacological therapy in general is likely to become increasingly tailored to the genetic and molecular profiles of specific patient subgroups. 36
  37. 37. BIOINFORMATICS Bioinformatics has become an important part of many areas of biology. “The field of science in which biology, computer science, and information technology merge into a single discipline.” The ultimate goal of the field is to enable the discovery of new biological insights as well as to create a global perspective from which unifying principles in biology can be discerned. “We define it as the creation and development of advanced information and computational technologies for problems in biology, most commonly molecular biology”. It deals with methods for storing, retrieving and analyzing biological data, such as nucleic acid (DNA/ RNA) and protein sequences, structures, functions, pathways and genetic interactions. 37
  38. 38. Role of bioinformatics Drug target identification  One of the major thrusts of current bioinformatics approaches is the prediction and identification of biologically active candidates, and mining and storage of related information.  Drugs are usually only developed when the particular drug target for those drugs actions have been identified and studied.  The number of potential targets for drug discovery process is increasing exponentially.  Mining and warehousing of the human genome sequence using bioinformatics has helped to define and classify the nucleotide compositions of those genes, which are responsible for the coding of target proteins, in addition to identifying new targets that offer more potential for new drugs.  Bioinformatics allows the identification and analysis of more and more biological drug targets. 38
  39. 39. Drug target validation  Bioinformatics also provides strategies and algorithm to predict new drug targets and to store and manage available drug target information.  After the discovery of ‘‘potential’’ drug targets, there is an inappreciable need to establish a strong association between a putative target and disease of interest.  The establishment of such a key association provides justification for the drug development process. This process, known as target validation,  Target validation is an area where bioinformatics is playing a significant role.  Drug target validation helps to moderate the potential for failure in the clinical testing and approval phases. 39
  40. 40. Cost reduction  The current high cost of drug discovery and development is a major cause for concern among pharmaceutical companies.  Along with increasing productivity pharmaceutical companies also aim to reduce the high failure rate in the drug discovery process so that increased number of drugs able to hit the market.  The high cost of various phase of clinical trials acts as limiting factors for number of drugs which can be developed by pharmaceutical companies and hence selecting the compounds with the best chances for approval is critical.  The costs of drug discovery and development generally include total costs from discovery to approval. Some studies have included the costs of failed drugs and the costs for commercialization.  There is also a cost associated with the elongated process, beginning from discovery all the way to final approval.  Advances in bioinformatics accelerate drug discovery process beginning with drug target identification and validation, to assay development, and virtual high throughput screening (HTS) all with the goal of identifying new potential chemical entities.  Bioinformatics provides more efficient target discovery and validation approaches, thus help to ensure that more drug candidates are successful during the approval process and making it more cost-effective. 40
  41. 41. Promote novel/new drug development  There are some collateral costs that bother the pharmaceutical Industry.  These costs include commercialization costs, litigation and drug recall costs, and general costs to society.  Commercialization costs for new drug to be about $250 million per approved drugs, are high mainly because most ‘‘new’’ drugs approved are essentially functional replicas of drugs that already exist.  Most of the copycat drugs are being commercialized to handle the illness for which there are drugs already there is a need of interface that can attract the attention of both physicians and patients who already have access to similar medication.  Bioinformatics can act as proper interface and provides new approaches and opportunities to pharmaceutical companies to efficiently discover potential drug targets and develop novel drugs. 41
  42. 42. Barriers to bioinformatics progress in drug design process  Bioinformatics efforts did not make any impeccable changes in the drug discovery and development process.  This may be because the practice of bioinformatics is relatively new and has only attained prominence in the years following the partial completion of the Human Genome Project(HGP).  Bioinformatics has not made any considerable impact, as projected earlier, on the cost of drugs.  The pharmaceutical industry continues to witness rising costs and withdrawals of drugs from the market after they had been approved and commercialized. Because of multiple documented cases of adverse drug Reactions.  It has been observed that several pharmaceutical industries are facing drug discovery and development related challenges.  These challenges range from high cost of drug discovery to the lengthy and risky trials and approval process and some time withdrawal of previously approved drugs from the market. 42
  43. 43. Reference:-  Modern methods of drug discovery edited by A. Hillisch and R. Hilgenfield.  Use of genomics and proteomics in pharmaceutical drug discovery and development. a review, sharma neha, harikumar s.L. I J of Pharmacy and Pharmaceutical Sci. vol 5 issue 3 2013.  Role of bioinformatics and pharmacogenomics in drug discovery and development process. Pramod Katara. Netw Model Anal Health Inform Bioinforma (2013) 2:225–230.  Aspirin Use, Tumor PIK3CA Mutation, and Colorectal-Cancer Survival. Xiaoyun Liao, M.D., Ph.D et al, N Engl J Med, 2012.  Detection of Circulating Tumor DNA in Early and LateStage Human Malignancies. Sci Transl Med. 2014: 6(224).  A CFTR Potentiator in Patients with Cystic Fibrosis and the G551D Mutation. n engl j med. 2011;365(8):1663-1672. 43
  44. 44. THANK U 44