Hit-to-lead activities 2

what is lead optimisation?

refining chemical structure of a confirmed hit to improve its drug like characteristics

what does lead optimisation involve?

• series of analogue testing

• testing the series to determine the structure activity relationship → assess potency, bioavailability, stability, selectivity etc

what is analogue synthesis?

• work with all the information you have (e.g. x ray crystallography) to produce further active compounds

• develop the pharmacophore

how was ziprasidone 5-HT₂ antagonist developed?

known ligand for serotonin receptors taken, pharmacophore developed → screening showed poor in vivo efficacy → was optimised to ziprasidone

how many optimisation cycles are required?

might have to go through a number of cycles

what are some things to keep in mind to aid lead optimisation? (6)

• start with a good lead → not always the most potent/active but rather has a good balance of MW, logP, potency, activity etc

• chemistry is important → tractability, diversification, steps involved and how easy it is to make analogue

• consider logP

• insertion of heteroaromatic groups will increase polarity :. better drug like properties

• biosiosteres (= functional groups that behave similarly within biological system)

• structural simplification → don’t want unnecessarily complex structures, also reduces MW

what is lipinski’s rule of 5?

• MW less than 500 Da

• not more than 5 H bond donors

• not more than 10 H bond acceptors

• LogP value less than 5

how useful are lipinski’s rules?

• for assessing bioavailability of oral drugs in humans :. measure of drug likeness

• however, many commercial drugs break these rules e.g. antibacterials (because they target bacteria, not human cells)

• good for considering final drug product, however there are better rules available for hit to lead activities stage

why is H bond donors and accceptors important for Lipinski’s rule of 5?

• too many H bond donors and acceptors make desolvation (partitioning into membrane after dissolving in water) difficult :. affects absorption across gut membrane

• donors have more influence than acceptors

why is cLogP important for Lipinski’s rule of 5?

• lipophilic compounds have poor aqueous solubility :. poor absorption

• lipophilic compounds have more interactions with CYP enzyme system

• if >5 = poor absorption because compounds do not partition out of membrane into aqueous cellular environment

why is molecular weight important for Lipinski’s rule of 5?

• if >500, absorption may be affected since larger molecules are harder to cross membranes :. limited diffusion

• opportunity of modification is very limited

• selectivity issues and higher conc of drug required due to absorption issues :. off target toxicity many be observed

give an example of where balancing lipophilicity was important for lead optimisation

olanzapine (antipsychotic) → developed from clozapine

• clozapine = higher logP value (3.7) :. was not very water soluble

• olanzapine altered its structure so it made the phenyl ring into a thiophene ring which improves polarity :. improves solubility

• due to this, bioavailability went from 55% in clozapine to ~100% in olanzapine

• daily dosing regimen decreased due to more exposure to drug

why are lipophilic drugs often known as being promiscuous?

• promiscuous = many off target side effects

• lipophilicity can often increase affinity to target but also increase off target interactions

• hydrophobic interactions are less specific than polar interactions

what can you do to try reduce the promiscuity of lipophilic drugs?

maintain logP less than 3

how is drug safety considered in lead optimisation?

before clinical studies in humans, it is necessary to demonstrate safety in vitro and in vivo

what assumptions about drug safety are used in in vitro and in vivo safety testing?

• in vitro assays predict in vivo effects

• effects of chemicals in laboratory animals apply to humans (extrapolate effects)

• use of high doses in animals is valid for predicting possible toxicity in humans

these assumptions are broadly true but we cannot be certain that a chemical will show no toxic effects in humans

what are the 5 sources of toxicity?

• mechanism based pharmacology (toxicity linked to target activation)

• formation of reactive metabolites

• activation of other receptors e.g. hERG

• interactions with other substances

• idiosyncratic (= an unpredictable, characteristic, adverse reaction of an individual,) toxicity

how can toxicities be avoided?

making a very potent compound

• dose required will decrease

• this concept does not apply to mechanism based since this is to due to target activation and more potent drugs will allow tighter binding to target

when can mechanism based pharmacology toxicity be a big problem?

for drugs designed for completely novel targets rather than new drugs for a known mechanism

• usually not predictable from in vitro tests, can sometimes predict from animal models

give an example of mechanism based pharmacology toxicity

beta agonists (e.g. salbutamol) → activation of B2 receptors in the lunch causing dilation of airways

• inhalation :. most of the drug stays in the lungs

• if patient takes too many doses, the levels in systemic circulation rise :. can act on B2 receptors in the heart causing palpitations

give an example of toxicity caused by the formation of reactive metabolites

paracetamol

• phase 1 oxidation causes production of NAPQI

• NAPQI normally reacts with glutathione :. becomes more polar and excreted from urine

• in toxicity, NAPQI reacts with proteins causing toxic effects→ hepatotoxicity

what type of electrophile is glutathione?

scavenger electrophile → reacts with reactive metabolites and removes them from the system

how can you avoid toxicities from occurring due to reactive metabolites?

• avoid functional groups known to show reactive metabolites

• ‘Ames’ test to detect mutagenicity

how can you test for functional groups to show reactive metabolites?

look for binding to proteins or glutathione (detect by mass spec)

how does the Ames test work?

• use a genetically modified bacterium that cannot synthesise histidine

• expose bacteria to drug

• if the bacteria mutates and regains the ability to produce histidine, they will form colonies on an agar plate

• number of colonies is compared to a control group to determine if the substance causes an increase in mutations

• can also be carried out in the presence of liver enzymes to simulate how the substance is metabolised in the body

describe how you can avoid activation of other receptors/enzymes i.e. off target toxicity

• screen against other systems

  • before nomination to preclinical studies, the compound will be tested in many other assays in vitro to look for activity

• potency is important to have a safe therapeutic window

  • if you can increase potency at the target receptor, a lower dose is needed :. lower risk of off target side effects

why is mimicking hERG interaction important during lead optimisation?

• every drug is screen for this according to MRHA regulations

• hERG = potassium cardiac channel

• activation causes prolongation of QT wave (hyperpolarisation)

• can lead to fatal arrhythmias (irregular heart heat)

what is the pharmacophore for hERG?

• aromatic ring

• alkyl spacer

• basic centre

drugs are screened for activity based on this (all basic aromas)

give an example of a drug that activates hERG and how this activity was reduced

farnesyltransferase inhibitors

• changes lipophilic aromatic ring to a polar one reduces hERG activity by >10x

why is the cytochrome P450 system important?

• majority of top 200 drugs are primarily metabolised by CYP450 enzymes

• :. compounds which inhibit/induce CYPs have potential to interact with many other drugs

give an example of a drug that causes hERG toxicity

terfenadine

• now banned → was on the market for OTC hayfever treatment

• found to cause life threatening cardiac arrythmias when co-administered with e.g. erythromycin

• other drug e.g. erythromycin causes inhibition of CYP450 → prevents metabolism of terfenadine :. [drug] in blood increases :. can bind to hERG :. arrhythmia

which PK parameters are screened during lead optimisation? what are these useful for?

• C_max , T_max , half life, volume of distribution, clearance

• useful to optimising onset of action, duration of action and bioavailability

what are the concepts of being proactive vs reactive in selecting a pre-clinical candidate?

• proactive: deal with issues before they arise

• reactive: solve issues that arise in later stages of development

why is it harder to make modifications after selecting a pre-clinical candidate?

already carried out screening/testing earlier :. modifications would require repeats of these tests which isn’t preferable

what are the criteria for preclinical candidates?

• preferred crystalline form identified

• compound sufficiently stable to allow a shelf life of >2 yrs

• scale up of lead compound to 100g demonstrated (ned this much in animal studies)

• full PK and metabolite profiling in 2 species (1 lower species e.g rodents and 1 higher species e.g. primate/dog)

• predicted human half life and dose

• no toxicity in extended animal study