Week 1 Part 1 Flashcards
Developing a new drug from original idea to launch a finished product takes how many years?
12-15 years
What is the cost of making a drug?
Excess of 1 billion dollars
Where can idea of a target come from?
Academic and clinical research
Commercial sector
Why does a drug discovery programme initiate?
Disease/clinical condition without suitable medical procedure available
Where does initial research happen?
In academia
Generates data to develop hypothesis that inhibition/activation of a protein/pathway will result in a therapeutic effect in a disease state
What is the outcome of activity?
selection of a target which may require further validation to go into lead discovery phase in order to justify a drug discovery effort
What is lead discovery?
Intensive research
Find a drug-like small molecule or biological therapeutic
This progresses into preclinical and if successful —> clinical development
Finally be a marketed medicine
Why does drug fail?
They do not work
They are not safe
What is an important step in developing a new drug?
Target identification and validation
What is the definition of a target?
Broad term
Applied to a range of biological entities
E.g. proteins, genes, RNA
What does a good target need to be?
Efficacious
Safe
Meet clinical and commercial needs
Be druggable
What is a druggable target accessible to?
Putative drug molecule
Be a small molecule or a lager biological
Upon binding, elicit a biological response
Measured both in vitro and in vivo
What does a good target identification and validation enable?
Increases confidence in relation between target and disease
Increased target identification
Data mining of available biomedical data
Use of bioinformatics approach - selecting and prioritising potential disease targets
Include publication and patent information
Gene expression data
Transgenic phenotyping
Compound profiling data
Look for genetic associations
Is there a link between genetic polymorphism and risk of disease
Is the polymorphism functional ?
What is an example of phenotypes in human where mutation can nullify or over activate the receptor?
Voltage-gated sodium channel Nav1.7
Incur a pain phenotype
Insensitivity or oversensitivity
What do you use phenotypic screening for?
Identify disease relevant targets in
Target validation
Once identified, the target needs to be fully prosecuted
What is the technique for target validation?
In-vitro tools through use of animal models
Modulation of a desired target in disease patients
What is Anti-sense technology?
Use RNA-like chemically modifies oligonucleotide that is complementary to region of target MRNA molecule
What does binding of antisense oligonucleotide + target MRNA prevent?
Binding of translational machinery
Blocking synthesis of encoded proteins
Transgenic animal for target validation
Involves whole animal
Allows the observation of phenotypic endpoint to explain the functional consequence of gene manipulation
Use of transgenic animal is expensive and time consuming
The use of small interfering RNA (siRNA) for target validation
Short double stranded fragments
SiRNA are then separated into single strands
Integrated into an active RNA induced silencing complex (RISC)
After integration, siRNA base-pair to their target MRNA and induce cleavage of MRNA
Prevent it from being used as a translation template
Monoclonal antibodies for target validation
Interact with a larger region of target molecule surface
Allows for a better discrimination between closely related targets
Provide higher affinity
Can be selected to bind unique epitopes
Example of efficacy of mAb in vivo - function neutralising anti-TrkA antibody MNAC13 - reduce both neuropathic pain and inflammatory hypersensitivity
What is a classic target validation tool?
Small bioactive molecule that interacts with and functionally modulates effector protein
Chemical genomics of target validation?
Study of genomic responses to chemical compounds
Goal: rapid identification of novel drugs
Brings together diversity-oriented chemical libraries and high information content cellular assays along with informatics and lining tools necessary for storing and analysing data generated
What can be developed during hit identification and lead discovery phase of drug discovery process?
Compound screening assays
Definition of hit molecule
Compound which has desired activity in compound screen and whose activity is confirmed upon retesting
What are examples of screening paradigms that exist for hit molecule?
High throughout screening (HTS) Focused or knowledge-based screening Pharmacores and molecular modelling Fragment screening Tissue based approach
High-throughput screening
Screening of entire compound library directly against the drug target
Or in a more complex assay system (cell based assay)
Whose activity is dependent upon target
What do you observe in a HTS?
Observe purified protein which are the target and put HTS that may have potential characteristic of interest
Setting up HTS
Compounds may be derived from medicinal chemistry and SAR
Or large pharmaceutical companies
What is the purpose of HTS?
Identify through a robust reproducible assay a drug/compound that may be of interest
What is usually preferred in HTS?
Fluorescence based assay in terms of how quickly they can give you results on a time-scale basis
What is a lot of HTS ?
Automated
Massive array set hon
Whereby assay solution can repeated many times with all of these different compounds
6 months after HTS
Move into a process where it is identified as hits
Filter down compounds - look for one that actually produced a desired effect
Look at affinity for compound for a receptor
Generate dose-response curves
What is focused or knowledge based screening?
Selecting from chemical library smaller subsets of molecules that are likely to have activity at target protein
Based on the knowledge of target protein and literature or patent precedent for chemical classes
What is pharma core and molecular modelling?
Virtual screens of compound data base
What is fragment screening?
Generation of very small molecular compound libraries
Screened at high concentration
Accompanied by the generation of protein structure to enable compound progression
What is tissue-based approach?
Looks for a response more aligned with final desired in-vivo effect
G protein coupled receptors
It is very unlikely that only one receptor is causing all of the symptoms
When ligand + GPCR = conformational change - act as a guanine nucleotide exchange factor (GEF)
The GPCR can activate G protein by exchanging the GDP bound for a GTP
The G proteins alpha subunit together with the bound GTP can dissociate from beta and gamma subunit to further affect intracellular signalling proteins
Fluorescent imaging plate reader (FlipR) assay
Fast and easy method for detecting activation through changes in intracellular calcium concentration
Instrument which are used to detect biological, chemical or physical events of a sample in microtiter
What does coupling receptors to Gq protein stimulate?
Inatracellular calcium flux upon binding
Functional response: calcium-sensitive due and fluorescence plate reader
Fluo-3 dye Ester is loaded into cell and cleaved by?
Cell esterases to activate dye
What does Fluo-3 result in?
Increased fluorescent emission at 520nm
What is amenable for High throughput?
Grow your own cells
Functional assay
High throughout tells you nothing about
How the receptor will operate in a native environment
What is lead discovery phase?
Development of biological assay
Identification of molecules with activity at drug target
Where has cell based assays been applied to?
Membrane receptors
Ion channels
Nuclear receptors
Generate functional readout as a consequence of compound activity
What is biochemical assay
Applied to both receptor and enzyme target
Measure affinity of test compound for target protein
Used to identify hit and candidate molecules
What is the choice of assay formation?
Dependent on biology of drug target protein
Equipment infrastructure
The experience of the scientist
Whether an inhibitor or activator molecule
Is sought for
scale of the compound screen
What are requirements for
Assay format?
Pharmacological relevance of assay Reproducibility of assay Assay cost Assay quality Effects of compound in the assay
What is assay capable of identifying?
Compounds with the desired potency and mechanism of action
Pharmacological relevance of assay
Determine whether assay pharmacology is predictive of disease state
Assay cost
Compound screening assay performed in microtitre plates
Academia: performed in 96-well or 384-well
Industry: formatted in 384-well or 1536-well microtitre
Assay quality
Determined according to Z’ factor
Statistical parameter - consider signal window in assay
Considers the variance around both Hugh and low signals in the assay
What is Z factor?
Standard means of measuring assay quality on a plate bases
Ranged from 0 to 1
Assay with a Z factor greater than > 0.4
Robust for compound screening
Many groups prefer to work with assays with a Z factor…
> 0.6
Effects of compound in the assay
Cell-based assays are intolerant to solvent concentration of greater than 1% DMSO
Biochemical assays can be performed in solvent concentration of up to 10% DMSO
What is the compound concentration for hit discovery ?
Run at 1-10 mM
Compound libraries contain small molecular weight molecules that obey chemical parameters such as
Lipinski rule of 5
What is clogP?
Measure it lipophilicity which affects absorption into the body
Molecules with these features are “drug-like”
Clinically marketed drugs
Molecular weight of less than 350 and a clogP of < 3
Why is it important to initiate drug programme with small simple molecule?
Lead optimisation
Improve potency and selectivity
What is the first and second step in initial refinement process?
1: compounds known by library curator removed from further consideration
2: generate a dose-response curve in the primary assay for each hit
Why are reversible compounds favoured?
Their effects can be more easily ‘washed-out’ following a drug withdrawal
What is lead series?
Medicinal chemists would look to cluster compounds into groups
Hit-to-lead phase
Refine each hit series
Produce more potent and selective compound which possess PK properties
Examine efficacy in any in-vivo models that are available
Involves intensive SAR investigations around each core compound structure
Measurements made to establish magnitude of activity and selectivity of compound
Carrie our systemically
Structural information about target is known
What is solubility and permeability assessments crucial for?
Rule in and out the potential of a compound to be a drug
What is a formulation strategy?
Design a tablet such that it dissolves in a particular region of the gut at a PH in which the compound is more soluble
What is 5-hydroxytryptamine (5-HT)?
Group of GPCR and ligand gayer ion
channel found in CNS and PNS
Mediate both excitatory and inhibitory neurotransmissiojn
5-HT3
Ligand-gated Na+ and K+ cation channel
Depolarising plasma membrane
Excitatory
What is SOC criteria?
Biological activity
Sample integrity
Potential to optimise
Assay availability
What can lead molecule be?
Optimised
Turned into a potential drug
What is lead optimisation phase?
Maintain favourable properties in lead compounds
Improve on deficiencies in lead structure
Compounds at this stage may have met initial goals of Lead optimisation phase
ready for final characterisation before declared as preclinical candidate
Team continue to explore synthetically in order to produce potential back up incase compound fails
Once a candidate is reached, the attrition rate of compound entering the clinical phase is high
Only 1 in 10 candidates reach the market
Once a candidate reaches clinical
Stage, it becomes increasingly difficult to kill the project, why?
At this stage the project has become public knowledge and thus termination can influence confidence in the company and shareholder value
What may help in this endeavour
Carry more studies - improved toxicology screens
Establish predictive translational models based on a thorough disease understanding
Identifying biomarkers
What is calcium imaging?
Show calcium status of an isolated cells, tissue or medium
