Hit to lead activities 1

What qualities should be balanced out in a drug

Stability, potency, pk properties, side effects , safety - a balance of all

List the drug discovery journey from target identification to clinical trials

Identify target

Validate target - see if target actual impacts disease, look at bio markers and validate them

Lead identification - identify through chemical libraries, monoclonal antibodies, therapeutic proteins

Optimise lead - through pk formulations, selectivity

Get candidate - needs to be safe, adme, toxicology, look at pharmacology , needs to show efficacy in in vivo and in vitro disease models - look at interactions

Then do IND or CTX applications

To go into clinical trial

What does the drug discovery pathway look from HTS Assay to lead selection

HTS assay, x screening selectivity, activity in other species, secondary phenotypic assays, in vitro DMPK p450 , lead selection

What happens after lead selection to get to candidate selection

Lead selection, reiterative chemistry and in vitro, liability and p450 assay , oral pk, in vivo model, assessment of DMPK chem dev, pharmacology, toxicity and candidate selection

What are the 8 key properties of drugs to consider

Aqueous solubility

Caco 2 permeability

MDR1/MDCK permeability

Microsomal stability

Hep G2 cytotoxicity

Cytotoxicity in suitable cell lines

P450 inhibition

Lipophilicity (Log D)

What is the target value of aqueous solubility

Needs to be over 100 um

Why is aqueous solubility important

It's important for running in vitro assays and in vivo delivery of drug

How much volume of solvent in Ml/ g is needed if drug is very soluble

Less than 1ml/g

How much volume of solvent is needed if drug is practical insoluble

More than 10k ml/g

How much volume of solvent is needed if drug is slightly soluble

100-1000ml/g

What target value is needed for lipophilciity

Target value is 0-3

Why is lipophilicity important

Need some level of aqueous solubility to move towards membrane and diffuse

Lipophilic molecules diffuse through membrane whilst hydrophilic need channels to move through

What does microsomal stability test for

Tests for how fast drug gets cleared out in vivo and clearance

Since liver microsomes contain drug metabolising enzymes on membrane

What is the target value of microsomal stability

Less than 30 uL min-1 mg-1 protein

What is the target value of CYPP450 inhibition

Needs to be less than 10Um

Why do you need to measure cyp450 inhibition

Main enzymes in body, inhibition can cause drug drug interactions, can cause toxicity

Why is caco 2 permeability Papp important

Is caco 2 colon carcinoma cell that mimics the intestinal epithelium

Helps assess permeability of drug, absorption and can see bioavailability of drug

And drug absorption from gut

What is the target value of caco 2 permeability

10-6 cm-1

What is MDR1 MDCK permeability

MDCK transferred with MDR1 which encodes for effluent transporter Pgp

Pgp found in intestines brains kidney

Why is MDR1 MDCK permeability tested

Can see permeability of drug in intestines or brain

And how susceptible it is to effluent transporter Pgp - more susceptible means better effluent less chance of drug resistance

What is the target value of MDR1 MDCK permeability

10-6 cm -1

What is HEP G2 hepatotoxicity used for

Surrogate to show effect of drug in hepatotoxcity

What is the target value of Hep G2 hepatotoxicity

Target value is 50x Ic50/Ec50

Why is cytotoxicity in suitable cell line important

Can look out for likelihood of cellular toxicity in vivo

What is the target of cytotoxicity in suitable cell line

50 x Ic50 or Ec50

What do hit compounds look like

Have a central core - functional groups decorated around it

Are flat rigid, and heteroaromatic

Learn structures of important heterocycles

Why are heteroaliphatic groups important

Improve drug properties of compounds- can introduce polarity to compound

What is template hopping

A compound that's already been patented - can modify it , can convert heteroarom to a different aromatic group - to tune properties

What are the 6 criteria's of hits

Need reproducible in vitro affinity/effiacy

Chemical tractability

No toxicity of compound and metabolites

Needs favourable properties like good pk, aqueous solubility , molecular weight

Patentable structures for chemical novelty

Need evidence that compound can retain its activity - no Dakar positives

REP NCF - reproducible affinity efficacy in vitro, E- evidence of retaining activity no false positives P- patentable novelty, N- no known toxicity of compound and metabolites C- chemical tractability F- favourable properties like log p molecular weight

How was drug discovery techniques used in tyrosine kinase bcr abl inhibitor

Already knew the known target, known protein kinase inhibitor

Was able to use alpha fold to develop models of proteins that haven't been crystallised

Then made a large number of analogues to form imatinib

How can you assess absorption in DMPK druggability properties

Can use caco 2 , iv po pk screening

Caco 2- to assess permeability - therefore absorption and mechanisms of absorption

Po iv pk screening - to assess animal pk , bioavaiblity by looking at pk after drug is injected iv or orally given

How can you assess absorption/distribution in druggability

Can use Pgp on caco 2- can look at the efflux substrate transporter for absorption and impact on cns uptake

What three ways can you assess distribution

RBC distribution

Plasma protein binding

Brain to plasma ratio

What do these three methods to assess distribution allow

Allows for interspecies comparisons , look at biological results

Can look at brain uptake for centrally acting compounds

And potential of cns based adverse events

What five ways can you assess microsomal incubation

Hepatocyte incubation

Microsomal incubation

CYP IC50

PXR activation

Reaction phenotyping

What does the five metabolism assessment factors allow for

Cross species metabolite comparison

Look for cyp or Pgp induction

See if there's drug drug interactions

Look at human clearance

Look at role of polymorphic enzymes

How can you assess for excretion and what does this allow

Can do pk screening with urine analysis - allows to look at animal pk and mechanisms of clearance

How is absorption and bioavailability affected by free drug

Only free drug exerts pharmacological activity can be in equilibrium

Can reversible bind to tissues and plasma and free drug gets cleared

How does caco 2 work

Sample incubated on apical side of monolayer for set amount of time

Then measure permeability on basoalteral side - sample and analyse it

Want a Papp of 10-6 cm-3 for good in vivo absorption

Caco2 cells are used to assess the intestinal absorption of drug candidates. Describe how the caco2 model is used to calculate permeability across the intestinal membrane.

Caco2 carcinoma cells - mimics the intestinal epithelium to determine permeability and absorption of the drug. It's expressed as Papp where over 10^-6 cm-1 suggests good in vivo absorption

This involves compounds being incubated in the apical side and after a period of set time, the drug gets sampled and analysed in the basolateral side to determine how much drug passed through the partially permeable membrane on the epithelium. This can help assess the druggability of a compound where bioavaiblity absorption can be noted and this can determine drugs ability to effectively bind to its target.

How does plasma protein binding affect drug

High binding would affect the dose half life safety

How much fraction unbound of plasma protein do we want

Want over 90% fraction unbound - plasma protein binding expressed as fraction unbound

Too high limits diffusion to site of action

What is the 7 criteria for hit to lead phase

Selectivity over non related targets

Selectivity over related targets 100 fold

Improved affinity/ efficacy

No toxicity or mutagenicity in effiacious dose

Determine patent strategy of lead compound

Needs to be efficacious in animal model - dose response relationship

ppb cyp inhibition herg profiles are acceptable

SEPPSIN

S- selectivity over related targets 100 fold. E- efficacy in animal model shows dose response relation P- PPB cyp induction inhibition is acceptable herg profile is acceptable P- patent strategy to get to lead S- selectivity over non related target I- improved affinity efficacy N- no toxicity mutagenicity at efficacious dose

What other factors are there to consider

Potency

Intrinsic activity

Intrinsic stability

Affinity

Solubility

Permeability

Optimising molecular properties