L7+8 - Drug Discovery

Question 1

Lipsinki Rule of 5

Answer 1

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

Question 2

High-Throughput Screening

Answer 2

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

Question 3

Compound Library Design

Answer 3

Choose representative compounds

Follow Rule of 3 but even more limited, eg MW < 350, logP < 3, < 3 hydrogen bonds

Question 4

Focused Screening

Answer 4

1,000 - 10,000 compounds

Compounds generally selected for screening after computational analysis of receptor/target (requires crystal structure)

Question 5

Rational Design

Answer 5

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

Question 6

Ligand Efficiency (LE) =

Answer 6

LE = dG / N

dG = Gibbs Free energy
N = number of non-hydrogen atoms

LE = (1.4(-log IC50)) / N

Question 7

Fragment Screening

Answer 7

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

Question 8

GPCR Screening Assays

Answer 8

Radioligand Binding Assay
Ca2+-sensitive Assays
B-arrestin Assays

Question 9

Radioligand Binding Assay

Answer 9

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

Question 10

Ca2+-Sensitive Assay

Answer 10

Fluo3 binds Ca2+ and fluoresces, can be used to detec intracellular Ca2+

Question 11

B-Arrestin Assays

Answer 11

B-galactosidase knockin to B-arrestin

- thus, B-arrestin activation induces B-Gal activity, its reaction releases chemiluminescence, which can be measured

Question 12

Z’

Answer 12

'Zed Prime' 
Measure of rubustness of assays
> 0.6 = excellent
0.3 - 0.6 = Workable
< 0.3 = bad

Question 13

Hit Optimisation

Answer 13

Synthesis and test analogues of identified hit from HTS
Confirm efficacy and theraputic effect
Most developable compounds -> LEADS

Question 14

Lead Optimisation

Answer 14

Optimise potency and Physicochemical properties
Optimise ADME/DMPK
Assess in animal models of disease
Reduce off-target effects

Question 15

Metabolism assays

Answer 15

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

Question 16

CCR5 and HIV

Answer 16

HIV binds to CD4 proteins on immune cells, GP120 binds to CCR5, GP41 penetrates membrane

CCR5 is Gi/o-coupled

Question 17

Maraviroc

Answer 17

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

Question 18

Preclinical Studies

Answer 18

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)

Question 19

Drug Safety

Answer 19

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

Question 20

Cardiovascular Safety

Answer 20

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

Question 21

Reproductive Toxicity

Answer 21

Thalidomide
Teratogenicity
In vitro tests - Xenopus lavis, cell adhesion, division, differentiation, gene expression
In vivo tests - Rabbits, rat, mice, primates, zebra fish

Question 22

Phase I Clinical Trials

Answer 22

General safety (in health people)
50-100
PK/PD observations, Dose ranging

Question 23

Phase II Clinical Trials

Answer 23

Dose and efficacy studies in disease patients-

400-800, produces vast majority of the labelling information you get on the ‘big sheet’

Question 24

Phase III Clinical Trials

Answer 24

Longer-term safety studies
300-3000
Requires extra control measure for statistical validity - placebo control, active control, double blind, randomised

Question 25

Regulatory Approval and Registration Phase

Answer 25

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

Question 26

Phase IV Clinical Trials

Answer 26

Very long term safety, is after drug is approved - feedback from doctors
Cost effectiveness
Very rare/unforseen side-effects (withdrawl or use restrictions)

Question 27

Translational Biomarkers

Answer 27

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

Question 28

Phase 0 Clinical Trials

Answer 28

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

Question 29

PET Emission Analysis

Answer 29

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

Question 30

BOLD MRI

Answer 30

Blood Oxygen Level Dependant Magnetic Resonance Imaging