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Drug Discovery Part I: Screening for Compounds Jim Wells ...

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<strong>Drug</strong> <strong>Discovery</strong><br />

<strong>Part</strong> I: <strong>Screening</strong> <strong>for</strong> <strong>Compounds</strong><br />

<strong>Jim</strong> <strong>Wells</strong><br />

& Michelle Arkin<br />

UCSF<br />

Pharmaceuticals:<br />

How do we �nd these things?<br />

Lipitor<br />

Gleevec<br />

Brief history of drug discovery<br />

Human to molecular target<br />

“Age of Botanicals”, Human screening<br />

Founding of FDA (1906) (Safety, Efficacy)<br />

Animal-based screening (anesthetics)<br />

Bacterial cell screening (penicillin)<br />

Tissue screening (GPCR)<br />

Target-based screening (HTS)<br />

Mechanism/structure-based (HIV)<br />

Molecular and cell-based (kinase)<br />

Genomics-based patient pro�ling


Modern drug discovery: target to human<br />

Classes of <strong>Drug</strong> Molecules<br />

•Small organic molecules (10,000 MW, injectable)<br />

•Vaccines<br />

9 steps from target to pill<br />

Cell<br />

Protein<br />

Animal efficacy<br />

Animal Tox<br />

Human Safety<br />

Efficacy<br />

Registration<br />

NDA �ling<br />

Disease target


Target ID: what’s causing disease<br />

Target validation:<br />

What’s causing the disease?<br />

Infectious agent or host imbalance?<br />

Molecular target leading to disease<br />

• Bacteria: �nd target not in humans<br />

• Host imbalance:<br />

Replace underactive protein (insulin)<br />

Inhibit overactive protein (kinase)<br />

The genome (genetic space) is huge<br />

and lots to search<br />

• ~20,000 human genes that<br />

code <strong>for</strong> proteins<br />

• Which one is causing the disease


Target validation:<br />

Is the target “druggable”?<br />

•Are there known small molecules that<br />

bind the target or a homolog?<br />

Gleevec binding to BCR-ABL<br />

•Does the target have a site that looks like it<br />

will bind a small molecule (cavity, energetic<br />

hot-spot)?<br />

Small molecules like certain targets<br />

Target % of sm drugs Affinity<br />

(-ΔG per<br />

C,N,O)<br />

GPCR 45 0.5-0.7 kcal<br />

Kinases 4 0.35-0.5 kcal<br />

Protease 2 0.25-0.4 kcal<br />

P-P targets 0


Goals <strong>for</strong> oral drugs (chemical properties,<br />

Lipinski Rules)<br />

• Low Molecular Weight (


Hit Identi�cation: getting on the board<br />

Ways to identify a hit<br />

• Start with a natural substrate and<br />

make drug-like<br />

• Start with someone else’s hit<br />

(“patent bust”)<br />

• Design a denovo hit by Structurebased<br />

Design<br />

• <strong>Screening</strong> (HTS or Fragment<br />

methods)<br />

You have to test A LOT of compounds<br />

to �nd a drug


Start with libraries of drug-like molecules<br />

Functional<br />

groups<br />

~ 500,000<br />

compounds<br />

How to handle that many compounds?<br />

• Miniaturization (1 – 100 uL)<br />

• Automation<br />

• Parallel pipetting<br />

• Robotics<br />

• Plate readers<br />

Demo here<br />

• freezers<br />

• Demo liquid handler<br />

• Demo plates


Assay <strong>for</strong>mats: Biochemical<br />

• Use a puri�ed protein and an<br />

activity you can visualize<br />

substrate<br />

• Use when<br />

enzyme<br />

enzyme<br />

• You know what protein you<br />

want to inhibit<br />

• You can express and purify the<br />

protein<br />

• Selectivity among similar<br />

proteins is important<br />

Assay <strong>for</strong>mats: Cell-based<br />

• Screen in whole cells when<br />

• Molecular target known,<br />

but not isolable from cell<br />

• Goal is to alter a cellular<br />

phenotype<br />

• Pathway is known, best<br />

target is not known<br />

Assay <strong>for</strong>mats: Cell-based<br />

• Results read out by<br />

• Whole well<br />

�uorescence/<br />

luminescence<br />

• Cell-by-cell properties


Demo<br />

• Biochemical assays<br />

- Light-box fluorophore<br />

High-content cells screens:<br />

Quantitative microscopy<br />

• Demo<br />

High-content screens:<br />

Quantitative microscopy


% inhibition<br />

Assay quality and Hit selection<br />

Next Steps?<br />

• Retest<br />

• Determine mechanism<br />

• Optimize!<br />

compound #<br />

A hit is just the �rst step to discovering a<br />

drug<br />

Demo<br />

• Chemist in a hood


Introduction to <strong>Drug</strong> <strong>Discovery</strong><br />

Thanks to Kate Augustyn, Steven Chen, and<br />

Kenny Ang<br />

References<br />

High-throughput screening assays <strong>for</strong> the identi�cation of<br />

chemical probes. Inglese J, et al. Nat Chem Biol. 2007 Aug;<br />

3(8):466-79<br />

Small-molecule inhibitors of protein-protein interactions:<br />

progressing towards the dream. Arkin MR, <strong>Wells</strong> JA. Nat<br />

Rev <strong>Drug</strong> Discov. 2004 Apr;3(4):301-17.<br />

The development of imatinib as a therapeutic agent <strong>for</strong><br />

chronic myeloid leukemia. Deininger M, Buchdunger E,<br />

Druker BJ. Blood. 2005 Apr 1;105(7):2640-53<br />

Making better drugs: Decision gates in non-clinical drug<br />

development. Pritchard JF, Jurima-Romet M, Reimer ML,<br />

Mortimer E, Rolfe B, Cayen MN. Nat Rev <strong>Drug</strong> Discov. 2003<br />

Jul;2(7):542-53<br />

<strong>Drug</strong> <strong>Discovery</strong><br />

<strong>Part</strong> I: <strong>Screening</strong> <strong>for</strong> <strong>Compounds</strong><br />

Thanks to<br />

Kate Augustyn<br />

Steven Chen<br />

Kenny Ang

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