Hit to Lead, Lead to Candidate

Defining a biochemical hit

Known structure

Preliminary structure activity relationship

Amenable to chemical alteration/production

Interacts with target

Inhibits target activity

Defining a whole-cell antibacterial hit

Biochemical hit plus:

Inhibit bacterial growth

Specific MIC vs specific pathogens

Active against resistant strains

Activity not due to non-specific activity

Types of assays for hit to screening assays

Physiochemical properties

Antibacterial activities

Resistance

Target inhibition

Cytotoxicity

ADME

Human target liabilities

PK, formulation

Tolerability and efficacy in vivo

What is the pKa and what is its relevance?

Acid dissociation constant (pH at which 50% of drug is ionised)

Influences lipophilicity, solubility, protein binding, permeability

Therefore big effect of PK, ADME

Not something that will stop drug development but can be helped by chemists to improve

What are zwitterions?

Something that contains an equal number of positively and negatively charged functional groups

Many approved abx are zwitterions

Helpful for getting through porins

Reasons why solubility is important?

If low solubility present, other assay results not reliable

Will be in aqueous solutions in body so needs to be soluble

What are the two ways to test solubility?

Kinetic (fast and cheap)

Thermodynamic (time and resource-consuming)

Overview of LogD

Distribution coefficient between lipid and aqueous layer

Is important but can be altered

Reasons to include a gram + in a primary MIC panel for gram negative treatment

See if is broad spectrum

If S. aureus doesn’t have the target but is still killed, drug is just cytotoxic

Difference between primary and secondary MIC panel

Primary largely wild-type/efflux-defective

Secondary panel is testing against resistant strains eg carbapenem resistant strains

Hit needs to be unaffected by existing resistance mechanisms

What is the importance of testing MIC with human serum?

If there’s a change in the MIC could be due to plasma protein binding, meaning less efficacious in vivo

NB

What is the aim with MIC90?

Getting a unimodal population

Bimodal distribution means clinical samples have resistant isolates already

Overview of time-kill assay

Time to kill as a function of exposure to different compound concentrations

Informs if compound is bacteriostatic or bactericidal, how rapidly it kills it, if resistant mutants arise in culture

If curve goes down and then back up within 24 hours implies resistant mutants

Important for PKPD modelling for dosing animals

Ways to look at potential resistance

Testing resistance development via examining frequency of mutation, serial passage

WGS to identify mechanisms of resistance in mutants

Example of simple target binding/inhibition assay

Surface plasma resonance

Determine affinity and binding kinetics of a ligand for its receptor

What is the main way cytotoxicity is tested?

HepG2 cells

Measure viability in many ways eg ATP levels

Find out at what concentration the drug kills the eukaryotic cells: ideally want it to be a high concentration

Also check haemolysis

Tests for mutagenicity/carcinogenicity

Ames test—assess if compound causes an increase in mutation frequency. Uses Salmonella his mutants as assay system. Not great for antibiotics

Micronucleus test: assess if compound causes chromosome damage, indicating if is carcinogenic

Need both to be negative

Importance of plasma protein binding for ADME

Only non-protein-bound fraction of antibiotic is actually active

Want it do be below 90% across species

What is microsomal stability assay (ADME related)

Liver microsomes are a set of vesicles that form when cells are disrupted

Have many drug metabolizing enzymes

Want to see how stable compound is in the presence of these enzymes

Use enzymes from variety of species

Has an impact on the half-life

What is the Caco-2 permeability and efflux test (ADME)?

Cell line used to see if compound can cross a layer of cells

Can help predict in vivo absorption of drug; suitability for oral dosing

Main human target liabilities tested

hERG: potassium ion channel, inhibition can cause fatal arrhythmia

NaV1.5: sodium ion channel, important for cardiac muscle

Other channels affecting heart

CYP inhibition/induction (can be an issue with drug alone or can be due to drug-drug interactions)

Target selection: ie if ribosome of bacteria targeted, risk of human mitochondrial ribosomes also being targeted

What is a CEREP assay?

Company assay; screens against 87 human targets, measure affinity of binding

If interactions occur need to individually assess

What is a mouse tolerability study?

Treating with compound to observe for side effects

Determines MTD (maximum tolerated dose) for rest of study

Overview of mouse pharmacokinetic assay

Determining distribution of compound in tissues, half life, excretion etc

Collecting tissues at multiple times post treatment

Main features of lead to CD phase

Optimisation

Continuing the assays, improving on main issues

Candidate drug should be ready for human clinical trials

Candidate criteria beyond hit to lead assays

Preclinical PK profile: acceptable PK in rodent an non-rodent species; sufficient exposure for tox studies; low blood clearance

Rodent toxicology: 7 day study; essentially getting margin of safety