Hit to Lead Activities and Lead Optimisation

What are hit compounds?

Compounds which show some interactions/modulation of the target (may not have favourable physicochemical properties)

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What is a drug?

Not the most potent, nor the most stable, nor the best absorbed, nor the least active against cardiac ion channels. But provides the best balance (between discovery and knowledge of drugs)

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Outline the process for drug discovery

(Diagram). Target identification (select disease), lead identification (target validation, chemical libraries). Lead optimisation (safety, efficacy). Development of candidate (toxicology, ADME, safety, pharmacology). Clinical trials

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What happens if the potency of a drug is compromised?

Increase the dose to compensate for a low potency (but cannot compromise stability, safety, efficacy

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What do in vitro assays provide? (1)

ADME properties, physicochemical and PK measurements. E.g. aqueous solubility - important for in vitro assays in vivo delivery of drug. Log D - measure lipophilicity, movement across membranes such as BBB

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What do in vitro assays provide? (2)

Hep G2 hepatotoxicity - surrogate for effects of toxicity on human liver. Cytotoxicity in suitable cell line - likelihood of cellular toxicity in vivo. ADME. In vitro - outside biological system

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Why are in vitro assays used?

Used to determine effectiveness of drug. Don't want to take experiment straight into an animal (unknown toxicity profile). Generate sufficient proof of concept data (justify experimental use in animals - licence)

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Describe features of ADME properties

Absorption (via GIT). Distribution (only free drug can cross biological membrane, not protein bound, higher volume of distribution is generally better). Metabolism (liver, determine half life of drug). Excretion (cytotoxicity screening)

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Why are hit compounds usually small compounds?

Might have to change properties, add substitutions, functional groups (adds to molecular weight)

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What do hit compounds look like?

Functional groups are usually arranged around a central core or scaffold. Core is usually - flat, rigid, (hetero)aromatic (N, O, S) - contained in majority of compounds

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Describe the importance of heterocycles found in drugs? (1)

E.g. pyridine, quinoline, imidazole purine etc. Rapid synthesis of related analogues. Altering template allows tuning of properties. Heteroatoms induce polarity (effect on lipophilicity). Heterocycles in DNA bases

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Describe the importance of heterocycles found in drugs? (2)

Able to alter drug like properties e.g. introduce NH group, makes compound basic and hydrophilic (reduce lipophilicity). Synthetic tractability, patentability, identify to modify core structure, add heterocycle

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What is the criteria for hits? (1)

Reproducible in vitro affinity/efficacy. Favourable properties (cLogP, pKa, aqueous solubility, molecular weight etc.). Chemical tractability. Evidence that related compounds retain activity (not a false positive)

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What is the criteria for hits? (2)

Patentable structures/strategy to enhance chemical novelty. No significant toxicity alerts from compound or known metabolites (e.g. paracetamol, liver toxicity, quinone)

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Describe features of analogue synthesis

Work with all the information available (minimal active fragment, X-ray crystallography etc.). Work with that you know is likely to lead to further active compounds. Develop pharmacophore

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Describe features of using a pharmacophore

Convert hit into a more generic format. Use techniques e.g. QSAR (how substituents influence activity of compound). If analogues don't improve activity there is no use. Might get more information by synthesising a few analogues

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Describe features of developing a pharmacophore

Development of pharmacophore - need a few analogues to determine activity. Rational drug design approach, computational techniques, use in silico technique (development pharmacophore if there are few analogues). Determines if model is valid

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Describe features of dual D2/5HT 2 antagonist (1)

Dopamine - intramolecular H bonding. Reason for poor in vivo efficacy - drug is too lipophilic. Make analogues to improve drug properties (lipophilicity). Replacement of aromatic rings with heterocycles, improves polarity of compound

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Describe features of dual D2/5HT 2 antagonist (2)

Cl substitution - electronegative element, improves polarity

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Describe features of tyrosine kinase BCR-ABL inhibitor (1)

Used in leukaemia, improves life expectancy by a few years, relatively safe. 1000 analogues made (modifications of known protein kinase C inhibitor). In vitro system - very small quantities. In vivo system - larger quantities of compounds needed

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Describe features of tyrosine kinase BCR-ABL inhibitor (2)

X-ray crystallography confirmed binding and accounts for Imatinib resistance

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Describe the assessment of DMPK properties - A

Determine drugability of the molecule. Caco-2 (epithelial, drugs cross membrane). IV/PO/PK screening. Determine - moderate to high absorption, mechanism of absorption, bioavailability, animal PK, (dose)

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Describe the assessment of DMPK properties - A/D

P-gp via Caco-2. Efflux transporter substrate for absorption and impact on CNS uptake

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Describe the assessment of DMPK properties - D (1)

RBC distribution. Plasma protein binding. Brain to plasma ratio (drug for AD, need to know if it crosses BBB, most drugs are for non-CNS indication/doesn't cross BBB).

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Describe the assessment of DMPK properties - D (2)

Interspecies comparison to better interpret biological and tox results. Brain uptake for centrally acting compounds or potential for CNS based adverse events

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Describe the assessment of DMPK properties - M (1)

Microsomal incubation, hepatocyte incubation, CYP IC50, PXR activation, reaction phenotyping. Projection of human clearance. Cross-species metabolite comparison (coverage of human metabolites). Potential drug-drug interactions

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Describe the assessment of DMPK properties - M (2)

Potential for CYP or P-gp induction. Role of polymorphic enzymes

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Describe the assessment of DMPK properties - E

PK screening with urine analysis. Animal PK and mechanism of clearance. Determine if drug is excreted changed or unchanged, determine metabolites, in vitro/in vivo combination. Want drug to remain unchanged when reaching urine

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Describe features of absorption and bioavailability (1)

Only free drug exerts pharmacological activity. Free drug is in equilibrium across system. Drug reversibility bound to tissues and in plasma. Only free drug will be cleared. Protein bound to drug changed to free drug - reversible process

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Describe features of absorption and bioavailability (2)

Drug being cleared - not a reversible process

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Describe features of Caco-2 - evaluation of intestinal absorption (1)

Absorption can occur throughout GIT, mostly in SI. Compounds incubated on apical (gut) side of monolayer, after a set time, basolateral side is sampled and analysed. Permeability (P app >10^-6 cm^-1, good in vivo absorption)

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Describe features of Caco-2 - evaluation of intestinal absorption (2)

Intestinal absorption model, caco-2 cell layer, add drug, measure drug concentration on basolateral side

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Describe features of plasma protein binding (1)

Important to know extent of drug-protein binding in plasma. High binding will affect dose, drug half life and may have safety implications. PPB expressed as fu (fraction unbound). Too high will limit diffusion to site of action. Fu >90% preferred

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Describe features of plasma protein binding (2)

High protein binding, low concentration of free drug, need to increase dose, fraction unbound increases (but may need to use maximum tolerated dose). Preferable to have a drug which is not highly protein bound

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Describe features of plasma protein binding (3)

Free-drug gives toxicity (no drug bound to protein). Highly protein bound drug, dynamic equilibrium with free drug, drug bound to protein not coming off easily, next dose, binding sites occupied, fraction unbound is higher, gives toxicity, kinetics

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What is the criteria for hit to lead phase? (1)

Improved affinity/efficacy. Selective over related targets (>100 fold). PPB, CYP inhibition/induction and hERG profiles acceptable. Selectivity over non-related targets. Efficacy in animal model - dose response relationship

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What is the criteria for hit to lead phase? (2)

No toxicity or mutagenicity at efficacious dose. Patent strategy determined for lead compounds

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Which techniques are used between lead generation and lead optimisation (find leads)? (1)

In vitro pharmacology (biochemical assay, selective assay, cellular assay. PK/PD, in vivo PK - need quantifiable biomarker to indicate if the patient is improving or not responding

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Which techniques are used between lead generation and lead optimisation (find leads)? (2)

Multiple dosing - determine how body behaves with multiple dosing e.g. drug accumulation, elimination, absorption (adjust formulation to CR to prevent accumulation/reduce dosing rather than make another compound

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Which techniques are used between lead generation and lead optimisation (find leads)? (3)

Different species (support species selection). In vivo efficacy (single/repeat dose to confirm in vivo pharmacologic effects. Preliminary safety (non-GLP) - MTD studies - to help design pivotal tox studies

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Which techniques are used between lead generation and lead optimisation (find leads)? (4)

Consider different populations, population behaviours (e.g. mental health). Drugs with narrow therapeutic index, administered in clinical setting

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What are the steps towards lead optimisation?

Target discovery, target validation, assay development, screening for hits and leads, lead optimisation

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Describe the balancing act of lead optimisation? (1)

Oral drugs journey from the gut to target includes interactions with water, membranes and proteins. Differing environments mean we spend more time optimising molecular properties and balance these with potency/selectivity

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Describe the balancing act of lead optimisation? (2)

Solubility (disintegration, disaggregation, dissolution). Gut membrane permeation. Hepatic/renal Cl - drug made more polar during metabolism for excretion in urine. PPB, tissue distribution.

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Which factors are considered for lead optimisation? (1)

Potency, distribution, affinity, intrinsic activity, intrinsic stability, solubility, permeability.

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Which factors are considered for lead optimisation? (2)

High log P - highly protein bound, drug might be less soluble (reduce hydrophilicity). Increase hydrophilicity - might be soluble but might not cross membrane, easily excreted due to already being polar, insufficient drug levels in tissues

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Which factors are considered for lead optimisation? (3)

Consider stability - metabolic and chemical. E.g. enteric coating - stable in acidic pH, drug released in SI

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What is lead optimisation? (1)

Refining the chemical structure of a confirmed hit to improve its drug characteristics. Synthesis of analogue series. Testing the series to correlate changes in chemical structure to biological and pharmacological data to establish SAR

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What is lead optimisation? (2)

Potency, bioavailability, stability, selectivity. Optimisation cycle is repeated until the candidate molecule is selected

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What are the top 10 tactics for lead optimisation? (1)

Start with a good lead (low mwt, log P, potent, selective, novel, functionally active). Look before you leap (library/lab research). Chemistry show allow rapid diversification (multiple sites of variation/chemistry suitable for parallel follow up)

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What are the top 10 tactics for lead optimisation? (2)

Optimise lipophilic interactions (log P/potency plots and ligand efficiency - spot outliers). Optimise polar interactions (look for specific H bonds and meaningful loss (or gains) in potency

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What are the top 10 tactics for lead optimisation? (3)

Heteroatom insertion (aryl/heterocycle switch or CH2/O/N switch). Bioisosteres (amide reversal, isoelectronic and/or isometric replacement). Optimise dipole (F or CF3, N/C-F switch)

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What are the top 10 tactics for lead optimisation? (4)

Conformational control (break a ring, make a ring, pre-organisation can be very beneficial to potency, make changes to lock preferred conformation/interacts with binding site)

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What are the top 10 tactics for lead optimisation? (5)

Challenge your own hypotheses, invest in alternative templates/series (get out of the box)

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What is Lipinski's Rule of 5?

Poor absorption/permeation and solubility are likely when - number of H bond donors (NH, OH) >5, number of H bond acceptors >10, MW >500, clogP >5. ~90% of oral drugs adhere to this rule

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Do antibacterial drugs follow Lipinski's Rule of 5?

MW > 500

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Describe features of the Rule of 5 - H bond donors/acceptors

Too many H bond donors/acceptors make desolvation too difficult preventing absorption across gut wall. H bond donors (NH, OH) are 2-3x worse than acceptors (O, N)

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Describe features of the Rule of 5 - clogP >5 (1)

Lipophilic compounds have poor solubility (poor absorption). CYPs metabolise lipophilic compounds (poor bioavailability). Keep clogP <5

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Describe features of the Rule of 5 - clogP >5 (2)

Ionised compounds cannot pass through membrane (poorly absorbed). Unionised compounds can pass through membrane (better absorption). E.g. neostigmine, physostigmine - no ionisable group

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Describe features of the Rule of 5 - MW > 500

For MW > 500, there is no middle ground. Few NH/OH/N/O - too lipophilic. Lots of NH/OH/N/O - cannot desolvate. As MW increases, sites of metabolism increases and as MW increases membrane penetration decreases

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Describe features of ionisable compounds - membrane partitioning

Only unionised drug can cross membrane. Ionised drug must first lose charge. Dependent upon pKa and permeation rate of unionised form

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What is lipophilicity? (1)

Physical property for predicting biological activity of drugs (body is made up of fatty compartments, cell membranes, and aqueous compartments, inter-intra cellular fluid). Drugs need to pass between different compartments to get to site of action

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What is lipophilicity? (2)

Suitable balance between lipophilicity and hydrophilicity is essential for the drug to have the right transport properties to be effective

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Describe the impact of lipophilicity and bioavailability between olanzapine and clozapine

Clozapine has a dose of 300 mg BD with a bioavailability of 55% and a log P of 3.72. Olanzapine (introduction of a heterocycle), change in dose to 5-10 mg OD with a bioavailability of 100% and log P of 2.94

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What is promiscuity?

Drug binds to many drug target sites. Lipophilicity will give potency but not just for target. Hydrophobic interactions are mostly not directional, much less specific than polar interactions. Lipophilic amines are bad in this case

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Describe the opposing factors in lead optimisation (diagram)

Decrease in log P (drug not absorbed across gut, unless paracellular, but increase in solubility). Increase in log D (increase Vd, bases/neutral/PPB, increase CYP metabolism). Increase in log P (increase QT interval effects, bases with pKa >7)

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Is there a preferred lipophilicity range for oral drugs?

Balance between properties. Molecular mass of 300-500 and log P of around 3 (sweet spot)

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Describe features of PK screening (1)

PK of drug candidates in animals are often predictive of PK in human. Test compound dosed in rodents, dogs or primates. Drug levels measured by HTS methods. PK parameters optimised based on therapeutic target (onset of action, DOA, bioavailability)

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Describe features of PK screening (2)

Graph, drug D – long half-life, less frequent dosing, concentration still close to maximum concentration at 24h. (Clinical management - S/E could be persistent).

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Describe features of PK screening (3)

Ensure that structural features of chemical compounds are different in order to get distinct PK profiles

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Describe features of PK screening (4)

Sufficient chemical diversity – have back up candidates if one compound causes toxicity (lack of diversity – if one compound fails, the rest of the compounds will fail)

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Describe features of genetic toxicology - philosophy

Flexible approach. Scaled down regulatory assays often used (best prediction for later in development)

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Describe features of genetic toxicology - options

In silico analysis can be useful as first e.g. Derek Nexus/Leadscope. Include in vitro cell based assays for gene mutation and chromosomal damage

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Describe features of genetic toxicology screening - typical screening assays (1)

Ames assay (bacterial gene mutation) - normally 2 of 5 standard strains used (TA98 and TA100) or mini-ames test (modified bacterial mutation test)

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Describe features of genetic toxicology screening - typical screening assays (2)

In vitro micronucleus in mammalian cells (detection of chromosomal damage/aneuploidy). Conducted in human lymphocytes or in cell lines such as CHO/L5178Y/TK6. Screens for other genetic toxicology endpoints and also cytotoxicity may be used

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Outline the integration of safety and efficacy to improve the probability of technical success (1)

Concept - target ID, in vitro screening - lead identification - lead optimisation - candidate selective - definitive toxicology - clinical development - launch - post-launch activities

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Outline the integration of safety and efficacy to improve the probability of technical success (2)

Proactive - deal with issues before they arise (from concept to candidate selection). Reactive - solve issues that arise in later stages of development (definitive toxicology, clinical development, return to hits and leads)

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Describe features of candidate selection - interpretation and use of data (1)

Pharmacology (optimal properties, translatable model). DMPK (drug-like properties, DDI risks, PK projections). Toxicology (De-risk GLP development, understand impact of compound). Integrated picture

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Describe features of candidate selection - interpretation and use of data (2)

Investigate MOA, PK/PD modelling, TK/TD modelling, required exposure multiples

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Describe features of advancing the molecule to get a candidate molecule

Bring data/expertise together for decision making. Collation of individual scientific results. Assess experimental results against product target profile. Choose best molecule to advance. Consider pharmacology, ADME properties, early toxicology

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Hit to Lead Activities and Lead Optimisation

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