Drug discovery siva

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Drug Discovery Process- Fundamental methodological approach : 

Drug Discovery Process- Fundamental methodological approach Prepared & Presented by Dr. Siva Reddy Challa Professor & HOD KVSR Siddharha College of Pharm.Sciences Vijayawada

Small Molecule Research Vs Large molecule research: 

S mall Molecule Research Vs Large molecule research Small molecule Research : a small molecule is a low molecular weight organic compound. The upper molecular weight limit for a small molecule is approximately 800 Daltons which allows for the possibility to rapidly diffuse across cell membranes so that they can reach intracellular sites of action. Eighty percent of traded drugs have molecular weights under 450 daltons. Small molecules are often considered superior to "large molecule" biologics because they can be taken orally.

Organization of Drug discovery : 

Organization of Drug discovery

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Role of PK-PD labs in Drug discovery

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Drug Candidate safety testing Animal Studies - relevant species - transgenic KO/KI mice - conditional KOs - agonists/antagonists - antibodies - antisense - RNAi Studies of Disease Mechanisms Human Studies Phases I,II, III Target -receptor; -ion channel; -transporter; -enzyme; - signalling molecule Lead Search -Develop assays (use of automation) -Chemical diversity -Highly iterative process Molecula r Studies Lead optimization -selectivity -efficacy in animal models -tolerability: AEs mechanism- based or structure-based? -pharmacokinetics -highly iterative process Drug Approval and Registration Target selection & validation Discovery Development

Glenmark R & D: Drugs in pipeline : 

Glenmark R & D: Drugs in pipeline

Choosing a Disease: 

Choosing a Disease Pharmaceutical companies are commercial enterprises Pharmaceutical companies will, therefore, tend to avoid products with a small market (i.e. a disease which only affects a small subset of the population)

Choosing a Disease: 

Choosing a Disease Pharmaceutical companies will also avoid products that would be consumed by individuals of lower economic status (i.e. a disease which only affects third world countries )

Choosing a Disease (cont.): 

Choosing a Disease (cont.) Most research is carried out on diseases which afflict “first world” countries: (e.g. cancer, cardiovascular diseases, depression, diabetes, flu, migraine, obesity).

Identifying a Drug Target: 

Identifying a Drug Target Drug Target = specific macromolecule, or biological system, which the drug will interact with Sometimes this can happen through incidental observation…

Selectivity is Important!: 

Selectivity is Important! e.g. targeting a bacterial enzyme, which is not present in mammals, or which has significant structural differences from the corresponding enzyme in mammals

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Having the genetic code for the production of an enzyme or a receptor may enable us to over-express that protein and determine its structure and biological function. If it is deemed important to the disease process, inhibitors (of enzymes), or antagonists or agonists of the receptors can be prepared through a process called rational drug design.

Types of drug designs : 

Types of drug designs Structure-based Drug design (rational drug design) (When The Structure Of Target protein is KNOWN) Structure-based drug design (or direct drug design) relies on knowledge of the three dimensional structure of the biological target obtained through methods such as x-ray crystallography or NMR spectroscopy. If an experimental structure of a target is not available, it may be possible to create a homology model of the target based on the experimental structure of a related protein. Using the structure of the biological target, candidate drugs that are predicted to bind with high affinity and selectivity to the target may be designed

Types of drug designs : 

Types of drug designs Ligand-based Drug design (indirect drug design) (When The Structure Of Target protein is NOT KNOWN) Ligand-based drug design (or indirect drug design) relies on knowledge of other molecules that bind to the biological target of interest. These other molecules may be used to derive a pharmacophore model that defines the minimum necessary structural characteristics a molecule must possess in order to bind to the target. In other words, a model of the biological target may be built based on the knowledge of what binds to it and this model in turn may be used to design new molecular entities that interact with the target.

Types of drug designs : 

Fragment-based drug design 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. Types of drug designs

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The Human Genome Project and related technologies has generated thousands of novel potential drug targets. Validating those targets and their drugability and generating therapeutic options are now the rate limiting steps in drug development.

PROTEOMICS : 

Differential display of protein expression in diseased and normal tissue. PROTEOMICS May be a better approach to target identification than microarrays of gene expression Not all expressed genes produce proteins

Genomics /Proteomics : 

Genomics /Proteomics

X-ray Crystallography: 

X-ray Crystallography

X-ray Crystallography: 

X-ray Crystallography X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information.

Strategies used for target identification and target validation: 

Strategies used for target identification and target validation Understanding the pathophysiology of the disease and identify if there is any altered gene expression of a gene using Microarray technology See if there is an overexpressiveness of a gene corresponds to a selected target protein and see if overexpression of that gene or protein correlates with development of disease of our interest for which drugs are to be discovered. Establish the strong association between target gene or protein and disease of our interest. Identify the structure of protein and predict the functions of that protein based on the 3D structures of the same using various protein library data bases.

Strategies used for target identification and target validation: 

Strategies used for target identification and target validation Isolated tissues with a target protein is taken and see if the target protein is modulated (stimulated or blocked) with the various ligands, how the actions are varying. If actions are stimulated, analyze whether the actions are clinically relevant to disease of out interest. If you identify agonist, if you give agonist, actions should be stimulatory and the same actions should be blocked by the antagonist. Analyze whether the stimulatory actions are relevant to decrease the disease of our interest or inhibitory actions are relevant to decrease the disease process. Test if the target protein is blocked by antibodies which are specific to that target protein.

Methods used for target identification and target validation: 

Methods used for target identification and target validation ELISA technique Western Blotting Northern Blotting Microarray technology Polymerase Chain Reaction (PCR) studies Patch Clamping technique Cell line studies Cloning of target protein Hybridization and Immobilization Transgenic animals Knock out animals

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Microarray technology

ELISA (Enzyme linked immunosorbitant assay): 

ELISA ( Enzyme linked immunosorbitant assay )

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ELISA ELISA

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Western Blotting

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The western blot (sometimes called the protein immunoblot) is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. Western Blotting

Relation ship of HTS & VS: 

Relation ship of HTS & VS

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High through-put screening (HTS)

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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 which modulate a particular biomolecular pathway

Docking in drug discovery: 

Docking in drug discovery Docking is a Computational simulation of a candidate ligand binding to a receptor. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using for example scoring functions. Docking is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets in order to in turn predict the affinity and activity of the small molecule. Hence docking plays an important role in the rational design of drugs.

Lipinski's Rule of Five : 

Partition coefficient log P in -0.4 to +5.6 range Molar refractivity from 40 to 130 Molecular weight from 160 to 500 Number of atoms from 20 to 70 (includes H-bond donors [e.g.;OHs and NHs] and H-bond acceptors [e.g.; Ns and Os] Polar surface area no greater than 140 Ǻ2 Lipinski's Rule of Five

Lipinski's Rule of Five designing a drug like candidate : 

Lipinski's Rule of Five designing a drug like candidate Lipinski's rule states that, in general, an orally active drug has no more than one violation of the following criteria: Not more than 5 hydrogen bond donors (nitrogen or oxygen atoms with one or more hydrogen atoms) Not more than 10 hydrogen bond acceptors (nitrogen or oxygen atoms) A molecular mass less than 500 daltons An octanol-water partition coefficient log P not greater than 5

IC50 value determination in in-vitro Pharmacology Lab (potency determination lab): 

IC 50 value determination in in-vitro Pharmacology Lab (potency determination lab) According to the FDA, IC50 represents the concentration of a drug that is required for 50% inhibition in vitro. It is commonly used as a measure of antagonist drug potency in pharmacological research. Lower the IC50 value, greater the potency of the drug . Sometimes, IC 50 is also converted to the pIC 50 scale (-log IC 50 ), in which higher values indicate exponentially greater potency.

Methods to determine IC50 value in-vitro Pharmacology Lab: 

Methods to determine IC 50 value in-vitro Pharmacology Lab Competition binding assays (CARRIED OUT IN RADIOACTIVE LAB) In this type of assay, a single concentration of radioligand (usually an agonist) is used in every assay tube. The ligand is used at a low concentration, usually at or below its Kd value. The level of specific binding of the radioligand is then determined in the presence of a range of concentrations of other competing non-radioactive compounds (usually antagonists), in order to measure the potency with which they compete for the binding of the radioligand.

Methods to determine IC-50 value in-vitro Pharmacology Lab: 

Methods to determine IC-50 value in-vitro Pharmacology Lab Competition binding assays In this situation the IC50 is the concentration of competing ligand which displaces 50% of the specific binding of the radioligand. The IC50 value is converted to an absolute inhibition constant Ki using the Cheng-Prusoff equation where Ki is the binding affinity of the inhibitor, IC50 is the functional strength of the inhibitor,

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Functional antagonist assay The IC 50 of a drug can be determined by constructing a dose-response curve and examining the effect of different concentrations of antagonist on reversing agonist activity. IC 50 values can be calculated for a given antagonist by determining the concentration needed to inhibit half of the maximum biological response of the agonist. Methods to determine IC-50 value in-vitro Pharmacology Lab

Tissue Homogenization for binding studies : 

Tissue Homogenization for binding studies

HPLC or LC-MS Method developments for NCEs and Estimation of Bioavailability studies for NCEs in DMPK Lab: 

HPLC or LC-MS Method developments for NCEs and Estimation of Bioavailability studies for NCEs in DMPK Lab NCEs which show low IC 50 values in in-vitro lab, they will be sent to DMPK Lab. Getting ready with HPLC method developments for the estimation of NCEs in serum samples. Dosing of the NCE into rat model and estimate the total PK profile (like AUC, t max, C max etc) Based on the data, sending the NCEs with good bioavailabilty to the in-vivo pharmacology lab.

ED50 value determination in In-vivo Pharmacology Lab (Efficacy determination lab): 

ED 50 value determination in In-vivo Pharmacology Lab (Efficacy determination lab)

Fundamentals in basic Pharmacological research : 

Fundamentals in basic Pharmacological research Do the all potent componds produce efficacy? Why should we calculate the ED 50 value? Requirements for testing the efficacy? Whether all the protocols are approved by IAEC ? What kind of disease animal models should be used to test the efficacy? What is the grouping of animals ?

Fundamentals in basic Pharmacological research : 

Fundamentals in basic Pharmacological research How do you solubilise the compound? If the compound is not soluble, What should we do? How much starting dose, we should give? After dosing, at what time point, we should take the read outs of efficacy? At what time points, we should collect the blood samples? Whether to collect the tissue samples also?

STATISTICAL ANALYSIS OF THE DATA : 

STATISTICAL ANALYSIS OF THE DATA Student t test One way ANOVA followed by post hoc test Two way ANOVA followed by post hoc test

Toxicolological Studies in Toxicology Lab : 

Toxicolological Studies in Toxicology Lab NCEs which have low ED 50 values, will be sent to the toxicology lab. -------------------------------------------------- Acute toxicity studies Subchronic toxicity studies Chronic studies ------------------------------------ Rat studies and Dog/Monkey studies

Thank You for your patience : 

Thank You for your patience