1. Advanced Medicinal Chemistry
Lecture 2:
Finding a Lead
Dr Jeff Stonehouse
AstraZeneca R&D Charnwood

2. The Drug Discovery Process
Target
Identification
3 months to
2 years!
HTS
3-4 months
Active-to-Hit
(AtH)
3 months
Hit-to-Lead
(HtL)
6-9 months
New Lead
Optimisation
Projects (LO)
2 years
Last time we talked about the different targets of interest to the medicinal chemist. This Lecture will focus on the sources available to find a lead compound which can then (hopefully!) be turned into a drug against this selected target.
The Scheme above highlights HTS or High Throughput Screening which is one of the most commonly used methods for generating leads against a target of interest. There are, however, a number of alternative sources of lead compounds…….
Candidate
Drug (CD)

3. Lead Compounds from a Variety of Sources
penicillins
1. Chance Discovery
taxol
2. Natural Products
Viagra
3. Clinical Observation
4. Natural Ligands
5. Existing Drugs
6. High Throughput Screening (HTS)
Here are a selection of sources of lead compounds. This list is by no means exhaustive.
Chance discovery, such as the penicillin mould that killed off bacteria in a petri dish, noticed by Alexander Fleming.
Natural products are a great source of leads. Taxol (paclitaxel) is a big selling drug today for the treatment of cancer. First isolated from the bark of the yew tree, it is now produced by Bristol-Myers-Squibb via a fermentation process.
Viagra (sildenafil citrate) was originally planned as a treatment for angina. The unexpected ‘side effect’ that we are all now aware of opened up a whole new avenue for the compound.
Natural ligands, exisiting drugs and HTS are by far the most commonly used routes to generate lead compound today and will be discussed in more detail.

4. Natural Ligands R=H adrenaline R=Me noradrenaline
Formoterol AstraZeneca
Salbutamol GlaxoSmithKline
Catechol bioisostere (toxicity)
Increased size (selectivity and duration)
Catechol bioisostere (toxicity)
The neurotransmitters adrenaline and noradrenaline are a classic example of the rational design of a drug from a natural ligand.
Adrenaline is known as the ‘fight-or-flight’ hormone which when released into the blood stream causes a number of physiological responses. For example increased heart rate, brochodilation, release of blood sugar, vasodilation and increased blood flow to the muscles. All of these changes prepare the body for fight or flight.
These physiological changes are caused by the activation of a number of alpha and beta adrenoreceptors in the body by the binding of the endogenous ligand adrenaline.
Rationally designed drugs based on adrenaline focus on two main problems:
The catechol unit is easily oxidised in the body and can form toxic by-products which cause side effects.
Adrenaline is non-selective and interacts with a multitude of adrenoreceptors. Bronchodilation (and relief of asthma symptoms) requires selectivity for the beta2-adrenoreceptor.
Long acting inhaled drugs of this type are obtained by increasing the lipophilicity (fattiness) of the side chain such that it remains in the lung tissue for a longer period of time.
Increased size (selectivity and duration)

5. Existing Drugs Also known as the “Me-Too” or “Me-Better” Approach
Viagra
Pfizer
Issues: short duration
Multiple side effects and incompatibility with other drugs
Cialis
Eli Lilly
Levitra
Bayer
BEWARE: Patent Issues!!
The “Me-too” approach is one that is very attractive for the pharmaceutical industry. There is extreme difficulty in getting a new class of drug to market due to insurmountable issues such as severe toxic affects or lack of efficacy. Using a known drug class as a starting point negates these issues as the mechanism of action will be proven and the molecular structure will be unlikely to cause toxicity. This is the advantage of using a molecule that has been through rigorous clinical trials as your lead.
A new class of drug (“first in class”) will usually have some issues associated with it, and this is where the term “me better” is used. The medicinal chemist will alter the lead in such a way as to improve on these issues, thus making the new drug better (“best in class”).
Patents can be an issue in this approach and finding a chemical series that is patentable is a serious consideration.
For example the first in class drug Viagra (sildenafil citrate) has a short duration of action, usually only a few hours. It also shows some side effects including headaches, palpitations, blurring of and ‘blue’ vision and even sudden death in very few cases. There is also an issue with incompatibility with other drugs. When co-administered with protease inhibitors Viagra is not removed as quickly from the body. This can lead to increased incidence and severity of side effects.
Marketed “me-too” drugs are Levitra (Vardenafil) which shows an improved side effect profile and can be co-administered with protease inhibitors. Levitra is very similar in structure to Viagra. Cialis (Tadalafil) has a longer duration of action, lasting up to 36 hours. This drug has been marketed as the “weekend pill”.
36h duration (“the weekend pill”)
Fewer side effects and incompatibility with other drugs

6. 50-70% of new drug projects originate from a HTS
High Throughput Screening (HTS)
“An industrialised process which brings together validated, tractable targets and chemical diversity to rapidly identify novel lead compounds for early phase drug discovery”
50-70% of new drug projects originate from a HTS
How?
validated, tractable targets
target selection for HTS
industrialised process
HTS assay technologies and automation
chemical diversity
sample selection for HTS
HTS (or random screening) is the lead generation legacy of big pharma, and is used extensively as a lead generation tool. Even if there are other avenues of lead generation available (as described previously), HTS will usually be used concurrently.
Over half of new leads are found using HTS.

7. Establishing a HTS ID validated/ tractable targets HT Screen
human & pathogen
genomes
validated/
tractable
targets
target ID
HT Screen
Development
chemical
space
compound
collection
selection
HTS requires 3 things:
A viable target to screen against (see first lecture)
A biological assay and necessary automated equipment (lots of robots)
A source of compounds, usually the company’s compound bank/collection

8. Microtitre Plates – the HTS test tube
For 200K data points:
125 x 1536 well plates
2000 x 96 well plates
500 x 384 well plates
9mm 96 ml
9mm pitch
384LV
25-5ml
4.5mm pitch
384
100-25ml
1536
10-1ml
2.25mm pitch
A company’s compound collection can run to many 100’s of thousands of compounds. The cost and time to screen all of these compounds against a single target of interest is enormous and as such miniaturisation has become a key factor in cutting costs. Smaller plates means that reduced amounts of reagents are used and waste generated. It also allows for a much faster turnaround. This has significantly reduced the cost and time of HTS, but it does still remains extremely expensive.

9. Charnwood HTS Technologies; 1995-2001
Screening can utilise numerous
technologies e.g radioactivity,
fluorescence, luminescence
None are universally applicable, each
with advantages and disadvantages
Biological assays used in HTS should be:
Robust
Reliable
Cost-effective
Simple and user-friendly (‘mix & measure’)
Rapid implementation
Generic
Amenable to automation
Potential for miniaturisation
There are several useful, robust assay technologies used in HTS which are depicted here. Two of the key technologies used are SPA and FLIPR.

10. High throughput radioligand binding assays
Scintillation Proximity Assay – the first true homogeneous HTS screening technology
Molecule too far away to activate bead
I125
Molecule binds
I125
Bound molecule
bead activated
light produced
Nothing bound
bead not activated,
no light
Antibody/receptor
I125
SPA (scintillation proximity assay) is a biological assay used in HTS to identify compounds that bind to a target of interest. It uses a radio-labelled ligand which causes luminescence when a binding event takes place. Compounds that bind to the receptor will displace the radiolabelled ligand and reduce the amount of luminescence. The strength of the binding is proportional to the amount of luminescence emitted and so the extent of binding can be quantified.
I125
Molecule cannot bind
Suitable for I125, 3H, 33P

11. SPA (Scintillation Proximity Assay)
First true homogeneous HTS technology
Allows throughput of ~30K compounds/day in 384 format
Easy to automate, no significant volume of aqueous waste
BUT:
Radioactive (safety headaches)
Long read times (>30min/plate)
Susceptible to quench artefacts
Not applicable to all targets
Another issue with this type of assay is the fact that it only monitors binding ability, which will be confusing for molecules that bind to receptors – is it an agonist, antagonist or inverse agonist?

12. FLIPR – a high throughput fluorimeter
FLIPR is a functional assay and therefore gives information which can determine whether a receptor binder is an agonist, antagonist or inverse agonist. A functional response is measured as fluorescent signalling.
Fluorescent Imaging Plate Reader
Real-time simultaneous imaging of 96- & 384-well plates
Used for HTS Ca2+ flux assays and ion channel screening

13. FLIPR – how it works PC
Cooled CCD Camera
96/384-Tip Pipettor
Drawer Holding
5 Microplates
6 W Argon Ion Laser
Cells loaded with fluorescent dye sensitive to Ca2+ (fluo-3)
CCD camera images base of microtitre plate
Addition of receptor agonist stimulates Ca2+ release, resulting in fluorescence increase
Whole plate is read simultaneously, allowing kinetic analysis
‘Functional’ screen (i.e.whole cell) – greater relevance than simpler screening methods
Throughput is 1000x greater than cuvette-based fluorimeter assay
In a similar manner to SPA assays the extent of activity in a FLIPR assay is quantifiable by the amount of fluorescence emitted.
There are 2 key issues with FLIPR assays:
False positives (compounds that appear active against the target which are not). These are usually compounds (or an impurity present in the compound) that are themselves fluorescent.
False negatives (compounds that appear inactive against the target which are actually active). This is caused by the compound (or an impurity present in the compound) quenching the fluorescent signal. This is a more serious problem than false positives as a this type of compound is missed altogether.

14. Establishing a HTS ID validated/ tractable targets HT Screen
human & pathogen
genomes
validated/
tractable
targets
target ID
HT Screen
Development
chemical
space
compound
collection
selection

15. The AstraZeneca Compound Collection
ASTRA ARCUS
ASTRA PAIN CONTROL
1994
1993
1999
Historical collections from the companies that have eventually formed AstraZeneca were not up to scratch in terms of purity and quality for HTS.
Only 51% of the combined collections from Astra and Zeneca pre-merger were of suitable quality to be added to the AZ compound collection for use in HTS.
Because of the cost and time involved in HTS it is important to have a good quality compound collection. It would not be efficient to occupy wells in the HTS assay with compounds which are not ‘drug-like’ or not pure.
Ca 9% compound overlap
Not a recipe for an optimal screening bank

16. Compound Collection Enhancement
AZ global initiative to boost screening collection
Target: ensure viable Hits from 75% of AZ HTS
Five-year initial lifespan. Two concurrent themes…
Acquisition
300K from 107 available
Stringent filters
Big Medchem input
Accept IP risks
Synthesis
Nominal 500K over 5 years
Target-class focus
Aligned to Research Areas
Early Bioscience input
The AZ compound collection enhancement project was initiated to improve the number and quality of the compounds available for HTS.
There are 2 themes:
Buy a selection of compounds available commercially as compound libraries.
Synthesise compounds at 4 sites across AZ.

17. CCE Structure Chemistry deliberately embedded in Research Areas
HTS
Charnwood
HTS AP
GPCR
Charnwood
Kinase
Alderley
Park
~60 Scientists
Med Chem
Bioscience
Comp Chem
Informatics
Central
Bioscience
Cheminformatics
Protease
Mölndal
Channel
Södertälje
Compound
Management AP
HTS
Mölndal
HTS US
Chemistry deliberately embedded in Research Areas
Not centralised
Benefit of Project exposure
Feeds parallel synthesis skill back into projects
The synthetic effort of the CCE project is located on 4 sites and is split into 4 targets of interest:
G protein coupled receptors
Ion channels
Protease enzyme inhibitors
Kinase enzyme inhibitors
A kinase, which you have not heard of previously, is an enzyme that phosphorylates proteins. This phosphorylation will then potentiate a biological response, leading to a physiological response.
The sites at which these projects are based have been chosen because of the expertise in the target and the relevance to each disease area. For example, the Charnwood site is part of the respiratory and inflammation research area. GPCR targets are of great interest for this particular disease area and have been a source of numerous Charnwood projects.

18. CCE – Library Chemistry
3 most commonly used reactions-
Amide coupling
Reductive amination
Sulphonamide formation
Library or combinatorial chemistry has historically been based upon solid phase peptide synthesis. The advantage of performing chemistry on a solid support is that it is very easy to remove impurities and excess reagents by washing the beads after the reaction. The ‘pure’ compound is then cleaved off the bead at the end of the synthetic sequence. Unfortunately it can be very difficult to get even the simplest reactions to work efficiently on a bead, and analysis of the products is difficult. How do we know if the chemical reaction we have attempted has worked?
At AstraZeneca combinatorial chemistry is performed in solution and products are purified by high throughput high pressure liquid chromatography (HPLC). Although it can be difficult to purify compounds made in solution, experience shows us that there is generally a better success rate for reactions performed this way, when compared to the solid-supported or bead chemistry.

19. CCE – Common Combinatorial Reactions
Amide Coupling
HATU
NMP
Sulphonamide Formation
Reductive Amination

20. Sulphonamide Formation
Mechanism
Amide Coupling
Sulphonamide Formation
Reductive Amination