Intro/Target Selection & Lead Identification

Question 1

1) What are the types of new drugs?

Answer 1

1. New drug for novel use: New discovery about a disease state that may have no treatment before or this drug represents a new approach in treatment
2. A new drug that represents a new generation of another group of drugs already in use.
3. A new drug that is just another variation of a known drug (me-too)

Question 2

2) What are the different approaches to target selection?

Answer 2

1. Hypothesis driven (traditional approach): identify disease -> understand disease ->devise approach -> identify drugs that fit approach
2. Genomic approach:
   - Analysing the entire genetic information of an organism in the context of healthy vs disease state
   - Understanding genotype-phenotype correlation to identify disease target genes
   - Eg. Molecular basis of CML: Philadelphia chromosome encodes constitutively active tyrosine kinase, BCR-ABL. Imatinib blocks ATP binding and thus inhibits BCR-ABL kinase activity.
3. Post genomics approach-proteomics: Separation and characterization of proteins in an organism. Compare protein expression in normal vs disease state to identify novel target proteins.

Question 3

3) How do you know that a gene/protein is targetable?

Answer 3

RNAi screens can be done. This is carried out by ‘knocking down’ individual genes to find genes that regulate key disease processes. This allows identification of targets by function. There are in vitro and in vivo approaches to this.

Question 4

4) How is target validation carried out?

Answer 4

1. In vitro: cell based assays to verify that the target gene/protein is involved in disease progression at the cellular level. Cell lines can be engineered to display loss of function (using siRNA/shRNA) or gain of function (overexpression). This allows analysis of specific roles of the target.
2. In vivo assays: Animal disease models that can be used to verify that your target gene/protein contributes to disease progression in a more complex environment. To study loss of function: inject mice with tumor cells, use shRNA to knock down a potential oncogene and observe tumor progression. To study gain of function, overexpress normal mice with potential oncogene and observe if tumors form.

Question 5

5) Define a 1) HIT 2) LEAD 3) preclinical development candidate.

Answer 5

• HIT A compound that interacts with the chosen target at a given concentration (usually in the micromolar range)
• LEAD A compound with drug-like properties, initial SAR and a promising IP position
• Preclinical Development Candidate NCE with optimized pharmacological and pharmacokinetic properties and a secure IP position

Question 6

6) What are the methods of lead identification? What are their benefits/drawbacks?

Answer 6

(full answer on p2)

1. Rational drug design: Understanding structure activity relationship. This requires coordination of structural biology and organic chemistry, and drug design is based on the pharmacophore of endogenous ligand/substrate. Designed to bind active site and block receptor activation or enzyme activity. Eg. BCR-ABL inhibition: Identification of c-ABL autoinhibitory mechanism. Myristoylation of N-Term of c-ABL causes binding of myristate moiety into deep hydrophobic pocket of kinase domain. This results in a 90 degree bending of the a-1 helix of the C-term and autoinhibition. BCR-ABL lacks N-term myristoylation site, but it can be replaced with allosteric inhibitors.
2. High throughput screening: Often relies on cell free/cell based assays– Target-specific effects are measured quantitatively by a reporter assay Ex: fluorescence, luminescence, cell shape, cell metabolism, color formation. Drug candidates are evaluated for ability to block activity. Formats: 96-well and 384-well plates (high-throughput), 1,536-well plates (ultra high-throughput (UHTS)). SAR is difficult to do without knowing the exact molecular target and mode of drug interaction.

Question 7

7) What are the cell based/cell free approach to HTS? What is a robust screening assay?

Answer 7

Cell free: based on isolated target molecule (can be whole or active fragment, such as an enzyme). Treat the drug candidates and see if the enzyme activity is inhibited. (inhibition of fluorescence). Examples are binding and enzymatic activity assays.

Cell based: Cell based reporter gene assay. Engineer cell lines, activate molecular target and treat with drug candidates. Disadvantage is that the drug hit may be acting either directly on target or indirectly be interfering pathway (up or downstream of target) Z’=1–(3s +3B)/(μs-μB) Z’>0.8 (very good) Z’>0.6 (good) Z’<0.5 (not robust for screening)

Question 8

8) How does fragment based screening work?

Answer 8

Run diverse set of structures and identify those that bind to the target (does not have to be a perfect fit. Custom building the drug based on fragments that bind.

Question 9

9) How are compound libraries created? What are the requirements for a good library?

Answer 9

– Acquisition from external vendors

– Generation from chemical library synthesis • Random libraries

• Focused libraries

– Generation from medicinal chemistry efforts

• Targeted synthesis • Combinatorial synthesis
• A good library should be

– Large

– Diverse

– Examples of libraries: FDA-approved drugs, Natural product libraries

– Containing only “lead-like” or “drug-like”compounds

• Non-reactive
• No known toxic moieties
• Following Lipinski’s Rule-of-5
• Aqueous soluble

Question 10

1) What is Lipinski’s rule of 5?

Answer 10

1. Fewer than 5 hydrogen bond donors
2. Fewer than 10 hydrogen-bond acceptors
3. A molecular weight of less than 500 daltons
4. A partitioning coefficient (logP) of less than 5

Question 11

2) What is lipophilicity and how is it calculated?

Answer 11

Lipophilicity is the ability of a compound to partition between lipophilic organic phase (octanal) and polar aqueous phase (water)

LogP =[Conc]octonal/[Conc]water

LogP <1: poor permeability

1-3: moderate permeability

3-5: high permeability

>5: high permeability