Biochemical approaches underpinning drug discovery

How are target pathways used in drug development?

to generate structure-activity relationships (SAR) and direct isoform selectivity

primary assay

used to test compounds to the identified target, to screen those which could potentially be used as a drug

primary assay output

must be rapid and measurable eg radiolabelled phosphate, antibody detection or fluorescence

primary kinase screening assay - radiolabelling

labelled ATP transfers labelled phosphate to substrate

primary kinase screening assay - FRET

substrate tagged with donor molecule and phosphorylated allowing it to bind a 2nd molecule labelled with an acceptor group

primary kinase screening assay - NADH coupled

conversion of ATP to ADP for substrate phosphorylation coupled with PEP → pyruvate which is reduced by NADH to lactate. NADH depletion read, correlates with kinase action

When are primary cell assays carried out?

if desired cutoffs from primary isolated enzyme assays are met

what do primary cell assays involve?

primary pharmacodynamic markers which demonstrate target engagements in a cellular setting

what are primary cell assays used for?

to see if compound SAR can be translated from test tube to cellular setting

what primary cell assays are used for kinases

phosphoantibody based readout eg fluorescent in cell western/western blots

what are orthogonal/secondary assays?

assay which differentiates between compounds that generate false positives from compounds actually active against the target

what are orthogonal assays coupled to?

luciferase, alkaline phosphatase secretion, GFP inductions etc

outline the steps of a luciferase reporter assay

Target gene regulatory region cloned upstream of luciferase. Vector DNA introduced to cells → transcription. Cells broken up and luciferin added. Cells containing target gene fluoresce

orthogonal markers

linked markers regulated specifically by target protein and inhibited by variations of target

CXCL12

orthogonal marker induced by IKK alpha and inhibited by IKKbeta - can be fused to a .luc-reporter

what assay is used to measure cell death resistance in cancer?

apoptotic assay

what assay is used to measure the sustaining of proliferative signalling in cancer

growth/viability assay

what assay is used to measure the activation of invasion and metastasis in cancer?

migration/invasion assays

what assays are used to measure replicative immortality in cancer?

clonogenic assay

apoptotic assay

Caspase reagent added to well plate containing blank/control/assay samples. Samples incubated and fluorescence measured

clonogenic assay

tumour disaggregated → single cells and clumps, cultured and plated. Colony number evaluated after 2 weeks

MTT assay

Cells plated and incubated. MTT reagent added and incubated until purple precipitate present. Detergent added and sample left in dark, then has absorbance read

what are MTT assays used for?

assessing cell metabolic activity (colorimetric)

migration assay

cells plated on ECM/collagen and wound area created. Collagen gel layered on top and timelapse images collected

cancer drugs inhibiting evasion of growth suppressors

cyclin dependent kinase inhibitors

cancer drugs which stop evasion of immune destruction

immune activating anti-CTLA4 mAb

cancer drugs which prevent replicative immortality

telomerase inhibitors

cancer drugs which prevent activation of invasion and metastasis

HGF/c-Met inhibitors

what cancer drugs inhibit the induction of angiogenesis?

VEGF signalling inhibitors

what cancer drugs prevent genome instability and mutation?

PARP inhibitors

what cancer drugs cause cancer cell death

proapoptotic BH3 mimetics

what cancer drugs regulate cell metabolism

aerobic glycolysis inhibitors

Lipinski’s rule of 5 for oral absorption and general cell permeability

• <5 H bond donors

• <10 H bond acceptors (no. of N and O atoms in molecule)

• molecular weight <500

• partitioning coefficient <5

in vivo imaging system

cells labelled with luciferase, animals flushed with luciferin output. Greater cancer mass = greater output

parallel use of test tube assay and in vivo model assays

test for potential toxicity

maximal tolerated dose

in vivo models. Should only ethically do this experiment once