Principles in Pharmacology - Lecture 7

Life 207 - Principles in pharmacology Week 3 Lecture 7 09/10/18- Identifying drug targets 2 Protein based methods The term “proteomics” refers to the study of the proteome, and is largely concerned with the investigation of how proteins interact within the cell. Proteomics is also used in a more specific sense to describe the use of mass spectrometry combined with bioinformatics to study proteins. Proteomics doesn’t just look at the abundance of proteins we have it looks at the interactions these proteins have and have they change and respond to different stimulus. Mass Spectrometry: Mass spec is used to analyse complex mixtures. It now enables us to study large biomolecules such as fragments of proteins The key to using MS in proteomics is the coupling of fragment data to bioinformatics database searches Proteins are broken down into predictable fragments, resulting in a fingerprint. This fingerprint region can be used to identify the protein. Manipulation of expression: Recombinant expression is generally reserved for the overexpression of protein. Other techniques for the manipulation of expression levels, both in vitro and in vivo are also important for drug development. It has increased our understanding when we remove a protein from an animal or an tissue. This includes: – RNA suppression i.e. antisense, RNA interference (RNAi) this can be done quickly in a lab. – Transgenic (in vivo) techniques. E.g. gene “knock-outs”, knock-ins” knock out is where you take a protein out, knock in is where you put a gene in Functional Assays: Assays range from standard assays used for validation, to high throughput methods used for lead compounds. This high throughput allows us to assay and screen large amount of compounds quickly. Life 207 - Principles in pharmacology With functional proteins we analyse any protein or DNA sequence we want, and it is a very robust method. They are essential in the pharmacology industry. High throughput screening: It was developed in response to the increasing size of complexity of compound libraries, and the continuing need to maintain growth in drug piplines. HTS require: -compound libraries - suitable assay methods - Robotic workflow - Computerised data handling systems This allows us to screen huge numbers of compounds very quickly and efficiently to look for a lead compound. We use robots to be highly specific, and can also pipette much quicker. HTS requires: - Functional targets = easiest in cell-based assays. This could be a cell line, mouse or human cell. The choice of the expression systems is often crucial. - Low assay costs, the cheaper it is the more amount we can do - Ability to miniaturise and automate. The more we can assay, and the smaller volumes we can use the cheaper it is. Therefore we can use a 1536 well plate. This is miniaturisation which minimises cost. They do this by using these higher density plates. - High sensitivity, to identify binding in a um range, reduced volumes requires higher sensitivity. Techniques used in HTS - Ligand bidning assays – a ligand can either be an agonist or antagonist - Reporter – gene assays (stable put into our cell a reporter gene that will light up when the gene is activated) - Cell based fluorescent assays - Cell proliferation assays – this is used in cancer - Cell viability assays Example: Detection of calcium influx using FLIPR (flurescence imaging plate reader) - screen an orphan GPCR ACOR12 expressed in HEK cells (human embryonic kidney cell line) - Constant peak is +ve control. Life 207 - Principles in pharmacology Identifying lead compounds: - A random chemical librarby - ½ to 1 million compounds used in screens to find the initial lead compound. - These would usually be with compounds that are similar with similar functional groups. - HTS in combination with random compound libraries is used to sift through the library, and combinational chemistry refines that initial lead compound - As the project progresses from lead to drug candidate, compounds libraries must move from diversity to similarity Structure based methods: - Structure based drug design can be used as an alternative or supplement to HTS in the identification of lead compounds - Atomic structure of the target proteins are determined using x ray crystallography, NMR or modelling we would use modelling if we cannot get our proteins from crystallography or NMR. - These are then used to design potential lead compounds, which can then be tested and used as templates for combinatorial libraries. Basics of structure determination: - All structural methods seek to define the conformation of biological macromolecules at the atomic level - For crystal structures determined using x rays the quality of a structure is defined by the resolution. Below -2.5A is general considered atomic resolution - For modelling studies the quality can only be assessed by the degree to which the model predicts function X-ray crystallography: - X-ray crystallography is based on the diffraction of x rays from ordered protein crystals - There are three key requirements to determine an X ray structure: - Milligram quantities of pure proteins - Ordered crystals - A solution to the “phase problem”