High Throughput Screening (HTS) & Drug Discovery Lectures 1 & 2 PDF

Introduction To High Throughput Screening (HTS) & Drug Discovery Assoc. Prof. Abdullah Gibriel  Drug Discovery & FDA Approval Drug discovery: is the process by which new medications, drugs or new chemical entities (NCE) are discovered prior to being finally app...

Introduction To High Throughput Screening (HTS) & Drug Discovery Assoc. Prof. Abdullah Gibriel  Drug Discovery & FDA Approval Drug discovery: is the process by which new medications, drugs or new chemical entities (NCE) are discovered prior to being finally approval by the FDA. With the devastating increase in diseases, virus spread, and patients, it has become essential to invent new medicines and drugs. The process of drug discovery is challenging and a costly affair. It takes about 12 to 15 years and costs over $1 billion dollars to develop a new drug and introduce the finished product in the market. Drug discovery involves various steps from disease identification, identification and validation of related target(s), development of hits & lead discovery that can interact with the targets to treat diseases, preclinical and clinical trials and then finally marketing.  High Throughput Screening (HTS) FACT 1: Recent understanding of the molecular pathogenesis and mechanisms of various diseases have dramatically increased the number of protein targets for drug discovery and treatment. FACT 2: New technologies have increased the number of drugs that can be tested for activity against those target proteins. HTS is one of the main avenues/approaches to invent/discover drugs and NCE faster. Nowadays, HTS is the standard and first adopted method for rapid and quick drug discovery in various pharmaceutical industries. HTS is an automated process by which millions of chemical compounds/entities are rapidly assayed/tested/screened for their biological or biochemical activity to modify the properties of a selected biological targets.  HTS process ensures that the time taken to screen compound libraries is reduced and the whole drug discovery process is speeded up. HTS is essential to eliminate the time that would have been wasted on investigating compounds that have little or no desired effect on the biological target. HTS aims to find potential candidate compounds that efficiently affect a biological target. These so-called candidate compounds are referred to as ‘hits.’ Those hits are potential ligands for receptors, enzymes, ion-channels or other pharmacological targets of interest. Typically, HTS assays are performed in "automation-friendly" microtiter plates with a 96, 384, 1536 or 3456 well format The greater the number and diversity of compounds screened, the more successful screen is likely to be. Through HTS, 50,000-100,000 different compounds can be screened per day!!  Knowledge gained from one drug target can be transferred to related targets. It is essential to understand that HTS method alone cannot wholly evaluate a potential drug as detailed toxicity, pharmacokinetics & bioavailability studies are further needed for this evaluation. As an example, HTS process successfully identified a potent pan-SRC (tyrosine kinase) inhibitor known as ‘Dasatinib, BMS-354825’ for the biological target of diabetes. This drug is a tyrosine kinase inhibitor (TKI) which exerts antihyperglycemic effects that can reverse or prevent type 1 & 2 diabetes mellitus by correcting insulin resistance and β cell dysfunction.  HTS Short History  High throughput screening was invented by Gyula Takátsy (a Hungarian medical doctor) in 1951; he made the first microtiter plate using Lucite and creating 6 rows of 12 wells in it.  Takátsy carried out research on the variability of antigenic structure and the pathology and epidemiology of influenza virus mutations, as well Dr.Gyula Takátsy as vaccine production and control.  The microtiter plate has further grown to include standardized 96,384,1536, 3456 well formats, with additional 3072 well nanoplate formats. Microwell plates and automated procedures have become standard in countless laboratories. Microplates  Commonly used terms in drug discovery & HTS High throughput screen (HTS): an optimised, miniaturised automated assay format that enables the testing of 50,000 - 100,000 chemically diverse compounds per day. Assay: a test system/experiment in which biological activity can be detected/ Robust: fast, powerful & quick experiment Multiplexing: The ability of a test/assay to screen/detect/investigate activity of more than one target/gene through running only one test/experiment. Sensitive: an assay that can detect minimal amount of a target/gene/molecule present in a sample. The smaller the amount that can be detected the higher the sensitivity of the assay.  Specific: an assay that can detect only a specific molecule/gene/target even if present within a pool of other molecules/genes/target. Reproducible/Reliable: An assay that produces the same results even if run during another different time given that targets, molecules, genes and/or the conditions are kept the same. Hit: is any compound/molecule with known chemical structure that is confirmed to have binding activity to the target that are concentration dependent and appears on HTS results. Progressible hit: a representative of a compound series with confirmed and promising biological activities with acceptable mechanism of action and some limited structure-activity relationship information. Lead: a compound with potential (as measured by potency, selectivity, physico-chemical properties, absence of toxicity or novelty) to progress to a full drug development program. Lead is the compound with therapeutic or pharmacological activity but suboptimal structure that still requires modification. This process is commonly referred as lead generation or hit to lead (H2L). The lead is further optimized and thus can then go through preclinical and clinical trials and if approved gets marketed. Pharmacophore: the ensemble of steric and electronic features that is necessary to ensure the optimal supra‐molecular interactions with a specific biological target structure and to trigger (or to block) its biological response. In other simple words, it is the minimal structure required with essential features for activity. For example pyrazole rine was confirmed to have powerful anti-cancer activity. Libraries: are referred as sets of compounds produced by combinatorial chemistry. Depending on how the solid-phase are handled, these compounds may be either mixtures or individual compounds.  HTS and assay method include various steps such as - target identification & validation, - Highthroughput compound library selection/generation with extreme care and precision. - Hit to lead generation/discovery (primary y& secondary assays, sample preparation, reagent preparation, compound management etc etc..) - Assay development, During HTS, various factors that are essential to be considered; - Quality and number of validated targets, - Diversity and number of compounds, - Capability to screen compounds in a cost effective way. - Capability to screen compounds in a timely manner using robust informative assays HTS main aim is to rapidly screen for and to identify progressible hits rather than to discover and to develop the final lead molecule itself that would be a promising drug for tearing diseases in the future..  Biological target identification & Validation There are presently around 500 targets that are being utilized by various pharmaceutical companies. Among those targets are; - Cell membranes receptors, mostly G-protein coupled receptors (GPCRs) Receptor tyrosine kinases (RTKs) which represent nearly 45% of biological targets. - Enzymes which represent nearly 28% of biological targets. - Hormones which represent nearly 11% of biological targets. - Ion channels (Ics) which represent nearly 5% of biological targets. - Nuclear receptors (NRs) which represent nearly 2% of biological targets. - DNA which represent nearly 2% of biological targets.  HTS process precisely focuses on single mechanism contributing to identification of target specific compounds. The HTS assays helps to screen various types of libraries such as - genomics, proteins, combinatorial chemistry or peptide libraries.  Biological target identification & Validation  Compound library selection/generation strategy/approaches Which strategy is best for hit identification? When a target is identified, a decision has to be made about which chemicals to screen, in order to identify potential lead compounds. Random screening: - All drug molecules/moieties can be included to be screened for biological activity against target. - Estimated no. of possible drug molecules would be extremely high reaching almost ± 1040!!! This is simply not possible. Focused screening: - A limited number of compounds are pre-selected for screening. - Compounds previously identified as hitting specific classes of targets (e.g. kinases) and compounds with similar structures or newly designed to fit the target are most probably chosen/selected. - Has proved successful as a hit generation strategy. Screening Approach Screening Methodology Comment  HTS for compound libraries (Hit to lead approach) through primary & secondary screening processes Today, in most of the drug discovery labs, the collection of libraries has increased from 400,000 to more than 1 million compounds. HTS can be utilized to screen for all kind of novel biological active compounds (libraries): - Natural products - Combinatorial Libraries (peptides, chemicals…) - Biological libraries In order to screen those numbers of compound libraries, automated 384, 1536 or 3456 well plates or higher density single compound test formats are used through the primary screening process. Ideally, the primary screen is responsible and designed for rapid identification of hits from this library of compounds. The aim is to achieve a minimum number of false positives and maximum number of confirmed hits.  These primary screens run in multiples of single compound concentrations. The results of the primary screening method are expressed in terms of percent activity as a negative (0 percent) and a positive (100 percent) control. The achieved Hits are further retested, generally independently from the first assay. After retesting, if a compound displays the same activities, it is accepted as a confirmed hit, and the process go through secondary screening for lead optimization to decide and further filter the substances that will make it on to clinical trials. TYPES OF HTS screens/assays: Functional and Non-functional. - Functional/mechanistic: Study exactly how the compound interacts with target (mechanism of action) - Non-functional/non-functional: To find out if the compound interacts with target or not with no detailed mechanism of action. Traditional screening vs. high-throughput screening (HTS) Traditional methods were time consuming as compared to high-throughput discovery. Screening ability of HTS increased 200 times as compared to traditional screening, leading to an increase of efficacy and accuracy, in High-throughput screening method. Moreover in HTS method minimal amounts of test compounds are used.  Advantages of HTS High sensitivity Minimization of High speed of of assay (single assay assay molecule (microtiter (Automation). detection). plate assay). Low complexity Clear message Low background of assay (Yes/No signal. (specific answer). interaction). Cost Fast data effectiveness for Reproducibility. processing of large screened results. compound  Importance & applications of HTS Highly efficient, fast, Efficient tool in accurate and studying biomolecular dependable in interactions and compound screening. pathways. Useful in toxicology, Useful in DNA to study mechanism sequencing. of action of various drugs and toxins. Study drug-drug Useful in Useful in interactions and the cytotoxicity genotoxicity effects of drugs on assays. assays. metabolizing enzymes.  Limitations of HTS 1-High cost if screening for small number of compounds. 2-Contamination of samples might be possible. This should be avoided to ensure accurate results. 3-Possibility of false positive results when screening large compound libraries (waste time and resources). 4-Data analysis & interpretations requires patience, professionalism and true expertise. 5-Limited synthetic chemistry approaches/experiments for lead optimization and development  Assay Technology in HTS are either Cell based assays (Phenotypic assays) (cell growth, cytotoxicity etc, etc) Biochemical/Enzyme based test (Mainly enzymatic activity testing)  Cell-based assays refer to any of a number of different experiments based on the use of live cells Mimic more closely the in-vivo situation and performed in cell cultures. Cell - based assays have become an important test as they can provide information about bioavailability, cytotoxicity, apoptosis, oxidative stress, cytotoxicity, cell proliferation, cell adhesion, migration, invasion and various biochemical pathways. Different cell lines are being used in cell based assays Such as; HUMAN CELL LINES DU145, PC3, Lncap (Prostate cancer) MCF-7, MDA-MB-438, T47D (Breast cancer) THP-1 (Acute Myeloid Leukemia) Cell-based assays offer a more accurate representation of the real-life model since live cells are used. The cell based assays are linked to a detector/indicator that shows/quantifies the interaction between the ligand and the target.  Biochemical/Enzyme based assays Involve the use of cell-free in-vitro systems to examine effect of compound library on particular targets. The biochemical based assay systems consist of targets/enzymes/receptors or mimetics of receptors (components that mimic active parts of receptors) to directly investigate whether the library of compounds will be active against specified targets. This target has usually been isolated from a cell and is no longer part of it. Referred to as mechanism-based assay. Reports either binding of a ligand to a receptor (whether this binding activates or inactivates downstream cascade signaling) or no effect at all on the tested/examined target. Three main types of biochemical assays based on the method of detection. the colorimetric or chromogenic assays, fluorometric or fluorogenic assays, and luminescent assays.  Assays do not require purification of the target protein. Cell - based assays enable the analysis of sample compound activity in an environment that is similar to the one in which a drug would really act Can immediately select against compounds /potential drugs that are generally cytotoxic, or that cannot permeate cellular membranes to reach intracellular sites Hit/lead compounds identified by cell based assays have passed important validation steps, saving time and costs in drug development Cell-based assays visualize all possible drug-target interactions e.g. activators, target interactions, inhibition, etc etc… It also provides a platform for toxicity studies. Types of HTS Assays are segregated into cell-based assays and biochemical assays. Biochemical assays are further segregated into homogeneous and heterogeneous assays. Homogeneous assay is a single step process (simple assay with minimal steps). It reduces cost with minimal robotic complexity required in automation. It is characterized by the interaction between the surrounding environment and the analyte. One of its drawback is that there are interference in measurements as it is carried out in the presence of other assay components. Heterogeneous assays It is a bit more complicated than the homogeneous assay method as it involves a few additional steps such as filtration, centrifugation etc to ensure that the component(s) to be measured are separated from rest of the components, which may cause interference. * A thumb rule always followed is that when homogeneous assay fails, heterogeneous assay measurement method is generally carried out to avoid any interference from other components present in the sample.  DETECTION METHODS IN HTS: Spectroscopy Mass Spectrometry Chromatography Calorimetry X-ray diffraction Microscopy Radioactive method  SPECTROSCOPY IN HTS: Fluorescence Spectroscopy Total internal reflection fluorescence (TIRF) Nuclear magnetic resonance (NMR) Absorption and luminescence Fourier transformed infrared(FTIR) Light scattering CHROMATOGRAPHY IN HTS: Gas chromatography (GC) Thin layer chromatography (TLC) Flash chromatography Liquid chromatography (HPLC) Ion Exchange chromatography Reverse phase chromatography Hydrophobic interaction chromatography Affinity chromatography  CALORIMETRY IN HTS: Isothermal titration Calorimetry (ITC) Differential scanning Calorimetry (DSC) MICROSCOPY IN HTS: Scanning Tunnelling Microscopy Atomic Force Microscopy Confocal Microscopy