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

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)

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.

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)

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

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.

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

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 96 300-100ml 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.

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.

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

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?

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

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.

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

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

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.

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.

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.

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

Sulphonamide Formation Mechanism Amide Coupling Sulphonamide Formation Reductive Amination