L7+8 - Drug Discovery Flashcards
Lipsinki Rule of 5
MW < 500
logP < 5
< 5 hydrogen bond donors
<10 hydrogen bond acceptors
aromatic groups, rotatable bonds, polar surface area and toxic groups should also be assessed
High-Throughput Screening
100,000 - 1,000,000s of compounds in library
Used to find start points for drugs discovery
Can used various types of assay to assess ligand
Compound Library Design
Choose representative compounds
Follow Rule of 3 but even more limited, eg MW < 350, logP < 3, < 3 hydrogen bonds
Focused Screening
1,000 - 10,000 compounds
Compounds generally selected for screening after computational analysis of receptor/target (requires crystal structure)
Rational Design
Uses computer modelling and knowledge to select attractive compounds for synthesis or purchase
Can be done for hit/lead optimisation
Does not always work as crystal structures are solved in complex with a single ligand, and the receptor can adopt different conformations, does not account for induced fit, or enthalpy/entropy
Ligand Efficiency (LE) =
LE = dG / N
dG = Gibbs Free energy N = number of non-hydrogen atoms
LE = (1.4(-log IC50)) / N
Fragment Screening
MW = 100-250, logP < 3, <3 Hydrogen bonds, < 3 Rotatable bonds
Small library, essentially ‘screens shapes’ in the ligand binding pocket
Pros:
small library covers large chemical space, potentially a better fitting compound, better start point for drug discovery
Cons:
Fragments are low potency, requires specialised assays, needs crystal structure
GPCR Screening Assays
Radioligand Binding Assay
Ca2+-sensitive Assays
B-arrestin Assays
Radioligand Binding Assay
Displacement Assay used to examine competitive ligand binding activity
35S GTP gammaS - assesses binding of Gy subunit, can investigate the effect of agonists, antagonists, PAMs or NAM
Ca2+-Sensitive Assay
Fluo3 binds Ca2+ and fluoresces, can be used to detec intracellular Ca2+
B-Arrestin Assays
B-galactosidase knockin to B-arrestin
- thus, B-arrestin activation induces B-Gal activity, its reaction releases chemiluminescence, which can be measured
Z’
'Zed Prime' Measure of rubustness of assays > 0.6 = excellent 0.3 - 0.6 = Workable < 0.3 = bad
Hit Optimisation
Synthesis and test analogues of identified hit from HTS
Confirm efficacy and theraputic effect
Most developable compounds -> LEADS
Lead Optimisation
Optimise potency and Physicochemical properties
Optimise ADME/DMPK
Assess in animal models of disease
Reduce off-target effects
Metabolism assays
Liver Microsomes - isolated from rough ER of hepatocytes, contains the enzymes for Phase I and II metabolism; requires cofactors
Liver Hepatocytes - Contains all components of a cell required for metabolism; expensive
CCR5 and HIV
HIV binds to CD4 proteins on immune cells, GP120 binds to CCR5, GP41 penetrates membrane
CCR5 is Gi/o-coupled
Maraviroc
Pfizer Drug, used radioligand HTS to discover hits, used Ca2+ to further assess activity
Was found to be an antagonist, suitable for oral administration, with good DMPK.
Crystal Structure of CCR5 was solved in complex with maraviroc
Preclinical Studies
Test in 2 animals (eg rat and primate)
Test Efficacy (assess theraputic affect and mechanism)
Pharmacokinetics (dose prediction, and PK predictions)
Test safety/toxicity (too toxic, stop project)
Drug Safety
Assess for Actute, Subacute, Chronic individuals, and multiple-dose toxicity
Assess for DNA damage/cellular damage
Behavioural Analysis in animals
Blood sample testing - look for markers of tissue damage/and abnormalities
Histopathology - organ damage, necrosis, hypertrophy
Cardiovascular Safety
BP, HR, arrhythmia?
Torsades de Pointe - hERG Channels, prolonged QT interval
TDP can be assessed by radioligand binding (dispacement using a known hERG channel antagonists)
Test cardic channels for changes in activity - eg hERG, hNa+v 1.5, hKCNQ1, L-type Ca2+ channels
In Vivo - Piglets administered drug while alive, or perfused isolated heart preperation
Reproductive Toxicity
Thalidomide
Teratogenicity
In vitro tests - Xenopus lavis, cell adhesion, division, differentiation, gene expression
In vivo tests - Rabbits, rat, mice, primates, zebra fish
Phase I Clinical Trials
General safety (in health people)
50-100
PK/PD observations, Dose ranging
Phase II Clinical Trials
Dose and efficacy studies in disease patients-
400-800, produces vast majority of the labelling information you get on the ‘big sheet’
Phase III Clinical Trials
Longer-term safety studies
300-3000
Requires extra control measure for statistical validity - placebo control, active control, double blind, randomised
Regulatory Approval and Registration Phase
Submission of ALL data (animal, human, cellular), even synthesis procedures, tablet formulations, self life
Sent to FDA - America, EMEA - Europe, Ministry of Health, Labour and Welfare - Japan
Usually takes around 18 months for approval
Phase IV Clinical Trials
Very long term safety, is after drug is approved - feedback from doctors
Cost effectiveness
Very rare/unforseen side-effects (withdrawl or use restrictions)
Translational Biomarkers
Biochemical feature that can be used to measure progress of disease and treatment effectiveness non-invasively
Eg Interleukin-1 = pro-inflammatory cytokine
Interleukin-10 = anti-inflammatory cytokine
Phase 0 Clinical Trials
First in human trials, using sub-theraputic doses for early proof of concept to allow for an early yes or no decision to move forward with full trials or not.
10-15 disease patients, usually measure translational biomarkers/something non-invasive
PET Emission Analysis
Positron Emission - occurs by Beta-decay in nucleus of atom, releases positron and neutrino; positron and electron collide and emit anti-parallel photons (beta-waves)
Photons can be detected using PET imager, but required co-detection of both anti-parallel photos in order to locate position of emmision in body/brain
BOLD MRI
Blood Oxygen Level Dependant Magnetic Resonance Imaging
