Assignment on: “Target identification in drug discovery” ens Submited to: ‘Submited by: Dr. Durg Vijay Singh S.M. Shayez Karim Central University of South Bihar Saima Firdaus Patna- 800014 (BIHAR), INDIA Shweta Kumari Swati KumariIntroduction: ‘dentifying the biological origin of a disease, and the potential targets for intervention, is the first step in the discovery of a medicine.” > A target is a broad term which can be applied to a range of biological entities which may include for example proteins, genes and RNA. Target identification is the process of identifying the direct molecular target — for example protein or nucleic acid — of a small molecule. In clinical pharmacology, target, v identification is aimed at finding the efficacy target of a drug/pharmaceutical or other xenobiotic. > There is need to find a protein (e.g. receptor) or gene associated with a disease with which a potential drug interacts ~ the so-called targets. Note: Not every target is equally capable of affecting the course of a disease, ‘Sra sence ren ng. 9010. Shain dbs EAL> The drug discovery process starts with the identification of a molecular target and the next is the Target Validation. During target validation, its a tested in the body. The target sociation with a specific disease and its ability to regulate biological processe validation confirms that interactions with the target produce the desired change in the behavior of diseased cells. ve ILis the critical step in Drug discovery process. Identification of new drug targets, target validation, biochemical assay development followed by LEAD identification provides very important input in the development of new potential drug candidate Drug discovery GENOMICS, PROTEOMICS & BIOPHARM. Potentially producing many more targets and “personalized” targets HIGH THROUGHPUT SCREENING Screening up to 100,000compounds a day for activity against a tar Identify disease _ VIRTUAL SCREENING Using a compu predict activity Isolate COMBINATORIAL CHEMISTRY Rapidly producing vast numbers of compounds a MOLECULAR MODELING Computer graphics & models help improve activity Preclinical testing IN VITRO & IN SILICO ADME MODELS Tissue and computer models begin torreplace animal testing Figure: steps of drug discovery Characteristics of Drug Target 1. A specific drug target might have the following characteristic 2. The drug target is a biomolecule(s), normally a protein that could exist in isolated or complex modality.3. The biomolecules have special sites that match other molecules (commonly small molecules with special structures). These molecules could be endogenous or extraneous substances such as chemical molecules (drugs). 4. The bio-molecular structure might change when the biomolecule binds to small molecules and the changes in structure normally are reversible, Following the change in the biomolecule’s structure various physiological responses ‘occur and induce regulation of the cell, organ, tissue, or body status. 6. The physiological responses triggered by the changes in biomolecule structure play a major role in complex regulation and have a therapeutic effect on pathological conditions. 7. The expression, activity, and structure of the biomolecule might change over the duration of the pathological process. 8. Small molecules binding to the biomolecules are drugs. a nal Drug Discovery v/s New Strategies in Drug discovery > The path to identifying and validating the drug candidate molecule is not a one size- fits- all-diseases strategy. > The first stage in drug discovery process is to understand the disease mechanism, using cellular and genetic approaches, in order to identify potential drug targets. = ii toxicity functional in silico toxicity latabasic (in vitro & assays genomics, structure-based assays eorcey — invive) in vitro& structural design (in vitro & a in vivo) genomics, (novel or in vivo) {in vivo), proteomics known compound) animal and HTS research bioinformatics (in vitro & (invive) ; in vivo) a Figure: a) Traditional Drug Discovery. b) New Strategies in Drug discovery> Target identification and mechanism of action studies play an important role in small- molecule discovery. « A good target needs to be efficacious, safe, meet clinical and commercial needs and, above all, be ‘druggable’. A ‘druggable’ target is accessible to the putative drug molecule, be that a small molecule or larger biological and upon binding, elicit a biological response which may be measured both in vitro and in vivo. % Good target identification and validation enables increased confidence in the relationship between target and disease and allows us to explore whether target modulation will lead to mechanism-based side effects. a —— — — = ae aa mn — cone x‘ / om es Sg. ff eran Lccome a Bae —. Caner) — Coae rl Nz siose = once ao arene coma eee oer a sooner Figure Torget ID and validation is « mufunctional proces. INC, inmunhitochersry > Current therapy is based upon less than 500 molecular targets of about 10000 possible targets: a, 45% of which are G-protein coupled receptors b. 28% are enzymes c. 11% are hormones and factorsd. 5% are ion channels fe. 2% are nuclear receptors meEnzymes Channels Receptors 1 Receptors (GPCRS) unknown ‘Figure: Current Drug Targets few target classes; based on 483 drugs in Goodinan and Gi ns "The Pharmacological busi of therapeutics Souce: Jurgen Drews, et alDag Disvovery: A Historical Pespective. Science 287, 1960 (2000):DOr 10.1126/scienes 287 5460.19650 > Target identification can be approached by direct biochemical methods, genetic interactions, or computational inference, Combinations these approaches may be required to fully characterize mechanisms of small-molecule action. Accordingly, the Broad Institute uses a multi-faceted approach to the target identification problem in the context of genome-based drug discovery, including: a. Quantitative proteomics based on mass spectrometry b. Genetic complementation of small-molecule effects using RNA interference ‘Computational inference by connect ity analysis using reference compounds > A forward genetics (or classical genetics) approach is characterized by identifying, often under experimental selection pressure, a phenotype of interest, followed by identification ‘of the gene (or genes) responsible for the phenotype. Modem molecular biological methods, particularly genetic engineering approaches, have given rise to reverse genetics (sometimes equated with molecular genetics), in which a specific gene of interest is targeted for mutation, deletion or functional ablation (for ‘example, with RNAiI 1), followed by a broad search for the resulting phenotype.a, = 2 Figure. Mechanism-of-action and target identification in chemical genetics. (a) Target-based approaches (reverse chemical genetics) begin with target validation, in which a role is established for a protein in a pathway or disease, followed by a biochemical assay (0 find candidate small molecules; mechanism-of- aetion studies are still required to validate cellular activities of candidates and evaluate possible side cffects.(b) Phenotype-based approaches (lorward chemical genetics) begin with a phenotype in a model system and an assay for small molecules that cam pertush this phenotype: candidate small molecules must then undergo targetidentification and mechanism-of-action studies to determine the protein responsible for ‘phenotypic change. Drugs Target at a molecular level > The main molecular targets for drugs are proteins (mainly enzymes, receptors, and ‘transport proteins) and nucleic acids (DNA and RNA). > These are large molecules (macromolecules) that have molecular weights measured in the order of several thousand atomic mass units. They are much bigger than the typical drug, which has a molecular weight in the order of a few hundred atomic mass units. The interaction of a drug with a macromolecular target involves a process known as binding.There is usually a specific area of the macromolecule where this takes place, known as the binding site. > Typically, this takes the form of a hollow or canyon on the surface of the macromolecule allowing the drug to sink into the body of the larger molecule. Some drugs react with the binding site and become permanently attached via a covalent bond. > However, most drugs interact through weaker forms of interaction known as intermolecular bonds .These include: © Electrostatic or ionic bonds, Hydrogen bonds, © Van der Waals interactions, ° > Dipole-dipole interactions, and ° Hydrophobic interactions (tis also possible for these interactions to take place within a molecule, in which case they are called intra molecular bonds.) Note: None of these bonds is as s rong as the covalent bonds that makeup the skeleton of a molecule, and so they can be formed and then broken again. This means that, equilibrium takes place between the drug being bound and unbound to its target. > The binding forces are strong enough to hold the drug for a certain period of time to let it have an effect on the target, but weak enough to allow the drug to depart once it has done its job. The length of time the drug remains at its target will then depend on the number of intermolecular bonds involved in holding it there. > Drugs that have a large number of interactions are likely to remain bound longer than those that have only a few. The relative strength of the different intermolecular binding forces is also an important factor. Functional groups present in the drug can be important in forming inter-molecular bonds with the target binding site. If they do so, they are called binding groups. > However, the carbon skeleton of the drug also plays an important role in binding the drug to its target through van der Waals interactions. As far as the target binding site is concerned, it too contains functional groups and carbon skeletons which can formintermolecular bonds with ‘visiting’ drugs. The specific regions where this takes place are known as binding regions. > The study of how drugs interact with their targets through binding interactions and produce a pharmacological effect is known as pharmacodynamics. Approaches to target identificatios ‘There are three distinct and complementary approaches for discovering the protein target of a small molecule: 1. Direct biochemical methods - Direct methods involve labeling the protein or small molecule of interest, incubation of the two populations and direct detection of binding, usually following some type of wash procedure. 2. Genetic interaction methods — Genetic manipulation can also be used to identify protein targets by modulating presumed targets in cells, thereby changing small-molecule sensitivity. Comparative genomics strategies aim to compare simultaneously two or more genomes in order to identify similarities and differences, and hence identify potential drug targets.3. Computational inference methods — Target hypotheses, in contrast, can be generated by computational inference, using pattern recognition to compare small-molecule effects to those of known reference molecules or genetic perturbations, Target identification Lead finding Lead optimization Tools for target identification and validation: Reliable technologies for addressing target identification and validation are the foundation of successful drug development. Microarrays have been well utilized in genomicyproteomics approaches for gene/protein expression profiling and tissue/cell- scale target validation. > Antisense technologies (including RNA interference technology) enable sequence-based gene knockdown at the RNA level. Zinc finger proteins are a DNA transcription- targeting version of knockdown.> Chemical genomics and proteomics are emerging tools for generating phenotype changes, thus leading to target and hit identifications. Target identification with proteomics is performed by comparing the protein expression levels in normal and diseased tissues. > NMR-based screening, as well as activity-based protein profiling, are trying to meet the requirement of high-throughput target identification, Microarrays: ‘+ Target identification seeks to identify new targets, normally proteins (or DNA/RNA), whose modulation might inhibit or reverse disease progression ‘© Current technologies enable researchers to attempt to correlate changes in gene (genomics) and protein (proteomics) expression with human disease, in the hope of finding new targets ‘+ Assess gene and protein expression (via nucleic acid or protein microarrays) to identify novel targets, and can also be used to validate the found targets at the tissue or cell scale (via tissue or cell microarrays) Nucleic acid microarrays Today, nucleic-acid microarrays, which primarily use short oligonucleotides (15-25 nt), long oligonucleotides (50-120 nt) and PCR-amplified cDNAs (100-3000 base pairs) as array elements, are overwhelmingly dominant because of the relatively easy synthesis and the chemical robustness of DNA. Data generated from genome sequencing projects in several organisms has provided the ‘opportunity to build comprehensive maps of transcriptional regulation. Array-based gene expression analysis (immobilized DNA probes hybridizing to RNA or cDNA targets) has enabled parallel monitoring of cellular transcription at the level of the genome. ‘Thus, nucleic-acid microarrays have had a significant impact on our understanding of normal and abnormal cell biochemistry and, thus, on the choice of targets for drug design,In oncology, data generated from high density oligonucleotide microarrays from Affymetrix containing 62 907 probe sets have been analyzed and compared, to identify 97 genes as physiological targets of the retinoblastoma protein pathway, deregulation of which is a hallmark ‘of human cancer. Further characterization of these genes should provide insights into how this pathway controls proliferation, thus providing potential therapeutic targets. Protein microarray Because most drug targets are proteins, protein and peptide microarrays are set to have an important impact on drug discovery. Protein arrays, an emerging yet very promising technology, are now being used to examine enzyme-substrate, DNA-protein and protein-protein interactions. By profiling the differential expression of proteins using antibody arrays and correlating the changes to a disease phenotype, putative targets (and biomarkers) to a particular disease can be identified, although to date, such microarrays have not been used to their full potential because of difficulties with the technology. ‘This strategy is based on the mechanism of the reaction between the ligands and proteins, thus demonstrating the approach as an activity-hased and high-throughput method. Further application of this method may lie on targeting known drugs or biological active compounds. and cell microarrays An alternative to the use of whole-tissue specimens is the use of live cell microarrays, which can be used to identify potential drug targets by functionally characterizing large numbers of gene products in cell-based assays.TA won sea Proce “Ra smicoaray 1 ae NA, Protsin nf? nc tnger protons (chemical genomiesiprotsomies SS C ‘Teesuelet merearay Core Opn n ees Bay Figure: Tools for target identification and validation. Antisense technology: A key strategy in target validation is to determine what happens, with respect to phenotype and/or the expression of other genes in cells or model organisms, if a gene of interest is either deleted or its activity is inhibited Gene knockout mimics the activity of a drug that completely inhibits the normal funetion of the ‘gene’s product, Antisense oligonucleotides‘Complementary to a portion of a target mRNA molecule, oligonucleotides are the original type of molecule used for blocking protein synthesis of the target mRNA, and thus achieving the knockdown of the target gene One example is the identification of COX17 as a therapeutic target for non-small cell lung cancer (NSCLC), nce RNA interference (RNAi) is another type of technology involving sequence-specific RNA for use in gene knockdown. This approach avoided some limits such as selectivity of mutational targeting, complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification, and should make possible the systematic identification of components within each pathway, thus leading to potential therapeutic targets. .c finger proteins Zine finger proteins (ZFPs) have remarkable versatility for recognizing different sequences of DNA, and variations in the amino acid sequence of the C2H2 domains allow them to be targeted to different locations in the genome. Each zine finger is a short stretch of 30 amino acids, containing two conserved cysteines and two conserved histidines. 1 These proteins have been used as the DNA-binding domains of novel transcription factors (ZFP TFS), ZEP TFs can be applied to potential new drug target validation in two ways. a. One direct way involves designing ZFPS to validate the phenotypic effects of activating or repressing a gene. b. Alternatively, libraries of ZFP TFs might be used to screen cells for desired phenotypic changes.Rather than finding drugs for targets in the conventional pharmaceutical approach, forward chemical genomics, in a sense, finds targets for known drugs Ks goal is to discover the specifie molecular targets and pathways that are modulated by particular chemical molecules (i.e. study the biochemistry underling the phenotype changes induced by chemicals). ‘Tagged libra roach Various kind of chemicals have been used to generate novel chemotypes and once an effect is found, the next step is to identify the biological target using an affinity matrix made of the immobilized hit compound. High-throughput NMR-based screening is also utilized for fast identification of drug—target interaction, | tot & ® 4a p ot Taoped brary scorearh. ana pment che ee Affinity Chromatography as a Classical Method for TarDespite the large number of target identification techniques described to date, affinity chromatography remains the most widely used method. The typical project begins with structure-activity relationship (SAR) studies in which various functional groups of the small molecule of interest are modified or removed to determine which one(s) are dispensable for drug activity The primary limitation of affinity chromatography is the need to derivatize the small molecules of interest. SAR studies are time-consuming and requite extensive medicinal chemistry expertise that is often lacking in the academic laboratories performing forward chemical genetics and phenotypic small molecule screens. A recent major advance in the affinity chromatography approach for target identification takes advantage of the quantitative capability of SILAC to drastically increase the sensitivity of this approach for target identification. affinity responsive target stability (DART! DARTS is the method for target identification that relies on drug-induced protease resistance, Drug affinity responsive target stability (DARTS) is a relatively quick and straightforward approach to identify potential protein targets for small molecules. It relies on the protection against proteolysis conferred on the target protein by interaction with a small molecule. The basis for DARTS is that a protein becomes stabilized upon binding to a small molecule compound or other ligand, which leads to decreased susceptibility of the target protein to degradation by proteases. This decreased proteolysis is specific to the target protein(s) and ‘occurs for both high and low affinity compounds. Moreover, DARTS works especially well using extremely complex protein samples such as whole cell lysates where non-specific protein- ligand interactions are minimized due to the large number and variety of proteins in the mixture, DARTS is advantageous because any small molecule can be used in its native form, meaning noStructure-Activity Relationship (SAR) studies or chemical modifications to the ligand are necessary for target identification. DART- Overview: First, the source of protein must be chosen. Generally any cell type that is sensitive to the biological effects of the small molecule can be used. Second, any small molecule believed to bind proteins should be suitable for DARTS (including drug-like small molecules that are not susceptible to proteolysis, and peptide ligands that are themselves susceptible to proteolysis but whose binding to target proteins would protect them from proteolysis), but the concentration range of small molecule to use is also an important consideration. Given that the targets of most small molecules being used for DARTS are unknown, the binding affinities will also not be known. Therefore, one can only estimate the binding affinity to the most relevant target(s) based upon the ECS0 of the compound, although this correlation may not be valid for all compounds and biological systems. Since the EC50 gives only @ rough estimate of binding affinity, it is done initially using a concentration of the compound that is 10-fold higher than the EC50. Using a concentration of compound that is significantly higher than the KD will help ensure maximal protection of the target protein from proteolysis by saturating the protein with ligand. It is particularly useful for the initial identification of the protein targets of small molecules, but can also be used to validate potential protein-ligand interactions predicted or identified by other means and to estimate the affinity of interactions, DART ws Affinity Chromatography: DARTS offers an unprecedented ability to identify new proteins targeted by small molecules. It is similar to affinity chromatography in that both are affinity based methods that start withcomplex protein samples and selectively enrich the target protein(s) while depleting all non- target proteins, However, whereas affinity chromatography utilizes positive enrichment by selectively pulling ‘out the target proteins and leaving behind non-targets, DARTS uses negative enrichment by digesting away non-target proteins while leaving behind the target proteins that are rendered protease-resistant v ¥ ¥ Shenliang Wang, Tae Bo Sim, Yang-Suk Kim and Young-Tae Chang ; Tools for target identification and validation; www.science direct; Current Opinion in Chemical Biology 2004, 8:371-377. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Screening, Bioinformatics, ‘Chemoinformatics, and Drug Design. ISBN: 978-3-527-3273 1-7 Selzer, P.M., Brutsche, S., Wiesner, P., Schmid, P., and Mullner, H. (2000) Target-based drug discovery for the development of novel antiinfectives. Int. J. Med. 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