Essay 1: paragraph flashcards

1. You have been assigned a new project in a pharmaceutical company to screen novel small molecule inhibitors of breast cancer targets. (a) Discuss FOUR parameters you would consider in your measurement. (50% of marks) (b) Discuss ONE approach or technique you would use to measure this parameter. Your answer should include the principle, ONE advantage and ONE limitation of the technique. (50% of marks)

INTRODUCTION: Why is a multi-parameter screening strategy essential when developing small-molecule inhibitors for breast cancer?

• Breast cancer drug discovery increasingly focuses on:

  • early, multi-parameter screening

  • ensure that small-molecule inhibitors not only potent but also translatable to patients

• Industrial screening campaign measurements must:

  • assess target engagement

  • cellular activity

  • functional anti-cancer effects

  • developability in parallel.

• Four key parameters are considered:

  • biochemical potency and selectivity

  • cellular target engagement

  • phenotypic anti-proliferative efficacy

  • early ADME-toxicity properties

• Together,

  • reduce late-stage attrition

  • prioritise compounds with genuine therapeutic properties.

1st PARAGRAPH: PARAMETER 1: BIOCHEMICAL POTENCY - Why is biochemical potency and selectivity a critical first parameter in screening breast cancer inhibitors?

• Biochemical potency:

  • confirms direct inhibition of the intended molecular target

  • distinguishes on-target effects from non-specific cytotoxicity.

• Selectivity profiling against related enzymes:

  • essential

  • as off-target inhibition increases the risk of toxicity and clinical failure

• Potent but non-selective compounds:

  • often fail later due to safety issues

• Biochemical potency is:

  • necessary to validate target engagement

  • but insufficient on its own to guarantee cellular or therapeutic efficacy

2nd PARAGRAPH: TECHNIQUE (Part b): TR-FRET - How does a TR-FRET assay measure biochemical inhibition, and what are one advantage and one limitation?

• Time-resolved FRET Kinase assay

• This measures:

  • inhibition of target-mediated phosphorylation

  • by detecting energy transfer between donor and acceptor fluorophore when substrate is bound.

  • energy transfer occurs when a labelled substrate binds the active target

  • inhibition reduces signal by preventing substrate interaction

• Advantage:

  • assay is homogeneous (no wash steps)

  • high-throughput and suitable for screening large compound libraries

• Limitation:

  • measures catalytic inhibition only

  • may miss compounds that bind allosterically or act through non-competitive mechanisms

  • requires orthogonal follow-up assays.

3rd PARAGRAPH: PARAMETER 2: CELLULAR TARGET ENGAGEMENT - Why must cellular target engagement be measured in addition to biochemical potency?

• Biochemical potency fails to translate due to:

  • compounds having poor cell permeability

  • high intracellular ATP can out-compete inhibitors

• Measuring cellular target engagement:

  • confirms compounds reach and bind the target in cells → in disease-relevant cellular environment

  • links biochemical inhibition to cellular context

• This parameter:

  • reduces false positives from biochemical assays

  • ensures downstream phenotypic effects are mechanistically linked to intended target.

4th PARAGRAPH: PARAMETER 3: PHENOTYPIC ANTI-CANCER EFFICACY - Why is phenotypic anti-cancer efficacy an essential screening parameter?

• Phenotypic efficacy assesses:

  • whether target inhibition produces desired biological outcome

• Relevant outcomes include:

  • reduced proliferation

  • induction of apoptosis or cell death

• Cancer-relevant models:

  • ensures that compounds phenocopy genetic suppression of the target.

• 3-D or more physiologically relevant models are valuable:

  • better reflect tumour behaviour and architecture

  • reduce over-estimation of drug potency

  • therapeutic response than simple monolayers.

5TH PARAGRAPH - PARAMETER 4: ADME-TOX AND DEVELOPABILITY - Why are early ADME-toxicity measurements critical in breast cancer drug discovery?

• Effective cellular inhibitors can even fail due to poor pharmacokinetics or toxicity

• Many compounds fail due to:

  • rapid clearance

  • poor bioavailability

• Early ADME-toxicity assessment evaluates:

  • metabolic stability

  • permeability

  • likelihood of achieving sufficient exposure in vivo.

• Identifying liabilities early:

  • prevents progression of non-developable compounds that are unlikely to be dosed safely

  • or effectively in patients

CONCLUSION: What is the overall benefit of integrating multiple screening parameters in a pharmaceutical discovery project?

• By Integrating biochemical, cellular, phenotypic and ADME-toxicity parameters:

  • pharmaceutical screening campaign prioritises:

    • compounds with both mechanistic validity

    • translational potential

• The strategy combines:

  • mechanistic validity

  • cellular relevance

  • functional efficacy

  • drug-like properties

• This strategy:

  • maximises efficiency

  • reduces late-stage attrition

  • aligning pre-clinical discovery with the demands of modern precision oncology.