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Drug discovery hit to lead

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Drug discovery, hit to lead by DR ANTHONY MELVIN CRASTO

Published in: Health & Medicine

Drug discovery hit to lead

  1. 1. HIT TO LEAD Hit to lead (H2L) also known as lead generation is a stage in early drug discovery where small molecule hits from a high throughput screen (HTS) are evaluated and undergo limited optimization to identify promising lead compounds These lead compounds undergo more extensive optimization in a subsequent step of drug discovery called lead optimization (LO). The drug discovery process generally follows the following path that includes a hit to lead stage: target validation (TV) → assay development → high-throughput screening → hit to lead (H2L) → lead optimization (LO) → preclinical drug development → clinical drug development
  2. 2. The hit to lead stage starts with confirmation and evaluation of the initial screening hits and is followed by synthesis of analogs (hit expansion). Typically the initial screening hits display binding affinities for their biological target in the micromolar (10−6 molar concentration) range. Through limited H2L optimization, the affinities of the hits are often improved by several orders of magnitude to the nanomolar (10−9 M) range. The hits also undergo limited optimization to improve metabolic half life so that the compounds can be tested in animal models of disease and also to improve selectivity against other biological targets binding that may result in undesirable side effects. On average, only one in every 5,000 compounds that enters drug discovery to the stage of pre-clinical
  3. 3. Hit confirmation After hits are identified from a high throughput screen, the hits are confirmed and evaluated using the following methods: Confirmatory testing: compounds that were found active against the selected target are re-tested using the same assay conditions used during the HTS to make sure that the activity is reproducible. Dose response curve: the compound is tested over a range of concentrations to determine the concentration that results in half maximal binding or activity (IC50 or EC50 value respectively). Orthogonal testing: confirmed hits are assayed using a different assay which is usually closer to the target physiological condition or using a different technology. Secondary screening: confirmed hits are tested in a functional cellular assay to determine efficacy.
  4. 4. Synthetic tractability: medicinal chemists evaluate compounds according to their synthesis feasibility and other parameters such as up-scaling or cost of goods. Biophysical testing: nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), dynamic light scattering (DLS), surface plasmon resonance (SPR), dual polarisation interferometry (DPI), microscale thermophoresis (MST) are commonly used to assess whether the compound binds effectively to the target, the kinetics,thermodynamics, and stoichiometry of binding, any associated conformational change and to rule out promiscuous binding. Hit ranking and clustering: Confirmed hit compounds are then ranked according to the various hit confirmation experiments. Freedom to operate evaluation: hit structures are checked in specialized databases to determine if they are patentable
  5. 5. Hit expansion Following hit confirmation, several compound clusters will be chosen according to their characteristics in the previously defined tests. An Ideal compound cluster will contain members that possess: high affinity towards the target (less than 1 µM) selectivity versus other targets significant efficacy in a cellular assay druglikeness (moderate molecular weight and lipophilicity usually estimated as ClogP). Affinity, molecular weight and lipophilicity can be linked in single parameter such asligand efficiency and lipophilic efficiency. low to moderate binding to human serum albumin low interference with P450 enzymes and P-glycoproteins low cytotoxicity
  6. 6. metabolic stability high cell membrane permeability high water solubility (above 10 µM) chemical stability synthetic tractability patentability The project team will usually select between three and six compound series to be further explored. The next step will allow the testing of analogous compounds to determine aquantitative structure-activity relationship (QSAR). Analogs can be quickly selected from an internal library or purchased from commercially available sources ("SAR by catalog"). Medicinal chemists will also start synthesizing related compounds using different methods such as combinatorial chemistry, high-throughput chemistry, or more classical organic chemistry synthesis.
  7. 7. Lead optimization phase The objective of this drug discovery phase is to synthesize lead compounds, new analogs with improved potency, reduced off-target activities, and physiochemical/metabolic properties suggestive of reasonable in vivo pharmacokinetics. This optimization is accomplished through chemical modification of the hit structure, with modifications chosen by employing knowledge of the structure-activity relationship (SAR) as well as structure-based design if structural information about the target is available. Lead optimization is concerned with experimental testing and confirmation of the compound based on animal efficacy models and ADMET (in vitro and in situ) tools.
  8. 8. Application of ADME-Tox tools has increased the success rate of drug development as well as helped in reducing the cost and time factors. The use of in silico and in vitro ADME-Tox has found universal acceptance. In silico tools provide a much higher throughput, but they suffer from adequate predictability, which limits their use. However, the reliance onin silico models is due to their ability to predict which compounds should be synthesized based on confirmed hits and structural modifications since it helps in selecting a drug-like compound. The best way to implement the ADME-Tox property prediction is the integration of both in silico and in vitro approaches to supplement each other for the production of candidate drugs.
  9. 9. CHEMINFORMATICS Cheminformatics (also known as chemoinformatics, chemioinformatics and chemical informatics) is the use of computer and informational techniques applied to a range of problems in the field of chemistry. These in silico techniques are used in, for example, pharmaceutical companies in the process of drug discovery. These methods can also be used in chemical and allied industries in various other forms. History The term chemoinformatics was defined by F.K. Brown in 1998: Chemoinformatics is the mixing of those information resources to transform data into information and information into knowledge for the intended purpose of making better decisions faster in the area of drug lead identification and optimization. Since then, both spellings have been used, and some have evolved to be established as Cheminformatics, while European Academia settled in 2006 for Chemoinformatics. The recent establishment of the Journal of Cheminformatics is a strong push towards the shorter variant. Basics Cheminformatics combines the scientific working fields of chemistry, computer science and information science for example in the areas of topology, chemical graph theory,information retrieval and data mining in the chemical space. Cheminformatics can also be applied to data analysis for various industries like paper and pulp, dyes and such allied industries.
  10. 10. Drug design, sometimes referred to as rational drug design or simply rational design, is the inventive process of finding new medications based on the knowledge of a biological target. The drug is most commonly an organic small molecule that activates or inhibits the function of a biomolecule such as a protein, which in turn results in a therapeutic benefit to the patient. In the most basic sense, drug design involves the design of molecules that are complementary in shape and charge to the biomolecular target with which they interact and therefore will bind to it. Drug design frequently but not necessarily relies oncomputer modeling techniques. This type of modeling is often referred to as computer-aided drug design. Finally, drug design that relies on the knowledge of the three-dimensional structure of the biomolecular target is known as structure-based drug design. In addition to small molecules, biopharmaceuticals and especially therapeutic antibodies are an increasingly important class of drugs and computational methods for improving the affinity, selectivity, and stability of these protein-based therapeutics have also been
  11. 11. The phrase "drug design" is to some extent a misnomer. A more accurate term is ligand design (i.e., design of a molecule that will bind tightly to its target).[4] Although design techniques for prediction of binding affinity are reasonably successful, there are many other properties, such as bioavailability, metabolic half-life, side effects, etc., that first must be optimized before a ligand can become a safe and efficacious drug. These other characteristics are often difficult to predict with rational design techniques. Nevertheless, due to high attrition rates, especially during clinical phases of drug development, more attention is being focused early in the drug design process on selecting candidate drugs whose physicochemical properties are predicted to result in fewer complications during development and hence more likely to lead to an approved, marketed drug. Furthermore, in vitro experiments complemented with computation methods are increasingly used in early drug discovery to select compounds with more favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles
  12. 12. Drug discovery cycle highlighting both ligand-based (indirect) and structure- based (direct) drug design strategies.
  13. 13. Drug development is the process of bringing a new pharmaceutical drug to the market once a lead compound has been identified through the process of drug discovery. It includes pre- clinical research on microorganisms and animals, clinical trials on humans, and may include the step of obtaining regulatory approval to market the drug.
  14. 14. Timeline showing the various drug approval tracks and research phases
  15. 15. Drug metabolism also known as xenobiotic metabolism is the biochemical modification of pharmaceutical substances or xenobioticsrespectively by living organisms, usually through specialized enzymatic systems. Drug metabolism often converts lipophilic chemical compounds into more readily excreted hydrophilic products. The rate of metabolism determines the duration and intensity of a drug's pharmacological action. Cytochrome P450 oxidases are important enzymes in xenobioticmetabolism.
  16. 16. Phases I and II of the metabolism of a lipophilic xenobiotic.
  17. 17. Mechanism Involved enzyme[8] Co-factor[8] Location[8] methylation methyltransferase S-adenosyl-L-methionine liver, kidney, lung, CNS sulphation sulfotransferases 3'-phosphoadenosine-5'-phosphosulfate liver, kidney, intestine acetylation •N-acetyltransferases •bile acid-CoA:amino acid N- acyltransferases acetyl coenzyme A liver, lung, spleen, gastric mucosa, RBCs, lymphocytes glucuronidation UDP-glucuronosyltransferases UDP-glucuronic acid liver, kidney, intestine, lung, skin, prostate, brain glutathione conjugation glutathione S-transferases glutathione liver, kidney glycine conjugation acetyl Co-enzyme As glycine liver, kidney
  18. 18. High-content screening (HCS), also known as high-content analysis (HCA) or cellomics, is a method that is used in biological research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a cell in a desired manner. Hence high content screening is a type of phenotypic screenconducted in cells. Phenotypic changes may include increases or decreases in the production of cellular products such as proteins and/or changes in the morphology (visual appearance) of the cell. High content screening includes any method used to analyze whole cells or components of cells with simultaneous readout of several parameters. Hence the name "high content screening". Unlike high-content analysis, high-content screening implies a level of throughput which is why the term "screening" differentiates HCS from HCA, which may be high in content but low in throughput. In high content screening, cells are first incubated with the substance and after a period of time, structures and molecular components of the cells are analyzed. The most common analysis involves labeling proteins with fluorescent tags, and finally changes in cell phenotype are measured using automated image analysis. Through the use of fluorescent tags with different absorption and emission maxima, it is possible to measure several different cell components in parallel. Furthermore, the imaging is able to detect changes at a subcellular level (e.g., cytoplasm vs. nucleus vs. other organelles). Therefore a large number of data points can be collected per cell. In addition to fluorescent labeling, various label free assays have been used in high content screening
  19. 19. High-throughput screening (HTS) is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Using robotics, data processing and control software, liquid handling devices, and sensitive detectors, High-throughput screening allows a researcher to quickly conduct millions of chemical, genetic, or pharmacological tests. Through this process one can rapidly identify active compounds, antibodies, or genes that modulate a particular biomolecular pathway. The results of these experiments provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology.
  20. 20. High-throughput screening robots
  21. 21. Fragment-based lead discovery (FBLD) also known as fragment-based drug discovery (FBDD) is a method used for finding lead compounds as part of the drug discoveryprocess. It is based on identifying small chemical fragments, which may bind only weakly to the biological target, and then growing them or combining them to produce a lead with a higher affinity. FBLD can be compared with high-throughput screening (HTS). In HTS, libraries with up to millions of compounds, with molecular weights of around 500 Da, are screened, and nanomolar binding affinities are sought. In contrast, in the early phase of FBLD, libraries with a few thousand compounds with molecular weights of around 200 Da may be screened, and millimolar affinities can be considered useful.
  22. 22. In drug development, pre-clinical development, also named preclinical studies and nonclinical studies, is a stage of research that begins before clinical trials (testing in humans) can begin, and during which important feasibility, iterative testing and drug safety data is collected. The main goals of pre-clinical studies are to determine the safe dose for First-in-man study and start to assess product's safety profile. Products may include new or iterated or like-kind medical devices, drugs, gene therapy solutions, etc. On average, only one in every 5,000 compounds that enters drug discovery to the stage of pre- clinical development becomes an approved drug
  23. 23. Drug design, sometimes referred to as rational drug design or simply rational design, is the inventive process of finding new medications based on the knowledge of a biological target.[1] The drug is most commonly an organic small molecule that activates or inhibits the function of a biomolecule such as a protein, which in turn results in a therapeutic benefit to the patient. In the most basic sense, drug design involves the design of molecules that are complementary in shape and charge to the biomolecular target with which they interact and therefore will bind to it. Drug design frequently but not necessarily relies oncomputer modeling techniques.This type of modeling is often referred to as computer-aided drug design. Finally, drug design that relies on the knowledge of the three-dimensional structure of the biomolecular target is known as structure-based drug design. In addition to small molecules, biopharmaceuticals and especially therapeutic antibodies are an increasingly important class of drugs and computational methods for improving the affinity, selectivity, and stability of these protein-based therapeutics have also been
  24. 24. Diagram showing how structure-based drug design affects enzyme function
  25. 25. Small molecule design consists of assembly, docking, and scoring. Since Rosetta already has a docking algorithm (RosettaLigand) and a scoring framework, ...
  26. 26. Computational drug design
  27. 27. Structure Based Drug Design: The iterative process whereby compounds generated by molecular modeling are synthesized and crystallized with their
  28. 28. A schematic illustration of the method of fragment-based screening and drug design. Initially, a fragment is identified in a screening effort. At the subsequent stages the fragment may be modified for more efficient binding and further "grown" to cover the whole ligand binding site.
  29. 29. Flow chart for structure based drug design
  30. 30. principle of the INPHARMA NOEs.
  31. 31. SUMMARY
  32. 32. Target Type What is meant by the term ‘challenging target’? Initial fragment screening campaigns focused on kinases These have clearly defined ATP pockets and are considered more druggable Typical hit rate: 5-10% Protein-protein interactions are more difficult to target as these do not have clearly defined pockets. These tend to have ‘hot-spots’ on the protein surface where binding occurs Typical hit rate: 0.1-4% CYP121 (Metalloprotein) Hit rate 3.9% CDK (Kinase) Hit rate 8.7% RAD51-BRCA2 (Protein-Protein Interaction) Hit rate 0.2 % Few slides of Case study
  33. 33. Fragment Elaboration This is the most frequent method of increasing potency for a fragment and a number of successful fragment campaigns have been carried out using this strategy Typically a single fragment in a binding pocket is ‘grown’ using chemical synthesis to pick up further interactions with the protein. This is the case that is the most likely to arise where a single fragment binds to protein or multiple fragments bind to a specific area of the binding pocket Structural information on how the ligand binds to the protein is key to guiding fragment development Enzyme Enzyme Fragment A Fragment Growing
  34. 34. Fragment Growing –Kinases (CDK2) Human Kinome ATP ADP General phosphorylation reaction catalysed by kinases The first targets that were screened using a fragment based approach were kinases. In many cases a key chemotype mimicking the aminopurine ring typically comes out these fragment screens Typically the hit-rate for kinases are high due to the nature of the ATP binding pocket A major problem in targeting kinases is selectivity (over 500 in human genome)
  35. 35. CDK2 Fragment Library 500 Fragments Primary Screening X-Ray crystallography (Cocktails of 4 fragments) X-Ray Crystallography Isothermal titration calorimetry (ITC) 500 Fragments >30 Fragments 4 Fragments With companies such as Astex the screening is carried out using X-ray crystallography where the fragment are screened in cocktails With this type of screening it is important to ensure when cocktailing there is sufficient fragment difference to ensure that when the hits are deconvoluted that the fragment can be identified In some cases fragment libraries containing Br modified fragments is used Fragment Growing – CDK2 (Astex) The fragment library was composed of a focused kinase set, a drug fragment set and compounds identified by virtual screening against a structure of CDK 2 Small fragment library size Fragment Screening Cascade - CDK2 Fragment Screening – X-ray crystallography How are these fragments binding to CDK2?
  36. 36. Fragment Growing – CDK Series 3 IC50 185 mM LE 0.57 IC50 3 mM LE 0.42 IC50 97 mM LE 0.39 IC50 3 nM LE 0.45 IC50 47 nM LE 0.40 AT7519 Fragment growing of the initial indazole hit led to a compound with a 50 fold increase in potency. Removal of the phenyl ring of the indazole offered a new startpoint and this was subsequently elaborated to a compound with a IC50 of 47 nM with only a small drop in LE (AT7519) Interestingly the piperidine is protruding out of the pocket toward solvent and the two chlorine atoms in the 2 and 6 position of the phenyl ring fill small hydrophobic pockets on the protein AT7519 is currently in Phase II clinical trials and has shown good indications against a range of human tumor cell lines The structure of AT7519 makes amenable to scale-up which is important in the later stage clinical trials Series 3
  37. 37. THANKS amcrasto@gmail.co m

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