Protein Kinases as Drug Targets

Protein Kinases as Drug Targets

Instructor: Brian Law, Ph.D.

1. Kinases as Receptors

• Kinases serve as crucial receptors in various signaling pathways due to their ability to catalyze phosphorylation on proteins.
• Their role spans across numerous cellular processes including growth, differentiation, and metabolism.

2. The Anatomy of a Protein Kinase

• General Structure: Protein kinases exhibit a complex structure composed of various domains that are essential for their function.
• The P Loop: A generic motif found in kinases, frequently denoted as GXGXXG. For instance, in Cdk2, critical residues like T14, Y15, and others are part of this sequence.
• Protein kinases are often modular, allowing for interaction with substrate proteins and ATP.
• Enzymatic Reaction Formula:
  $ext{Protein} + ext{ATP} <br /> ightarrow ext{P-Protein} + ext{ADP}$
    which illustrates how kinases facilitate the transfer of a phosphate group.
• Commonly targeted residues include serine, threonine, and tyrosine.

3. ATP-Competitive Inhibitors

• Advantages:
    - ATP-competitive inhibitors are relatively straightforward to design because they mimic the natural substrate ATP, allowing them to bind at the ATP binding site of kinases.
• Disadvantages:
    - ATP binding sites across various kinases are highly conserved evolutionarily.
    - There are a large number of kinases within the human genome (the kinome).
    - Many kinases exhibit functional redundancy, especially in higher mammals which complicates targeting strategies.

4. Variations on ATP-Competitive Inhibitors

• Type I Inhibitors: Specifically bind to the active conformation of kinases, resembling ATP in structural properties.
• Allosteric Inhibitors: These bind to sites distinct from the ATP binding site, thus providing additional functional selectivity.
• Irreversible Inhibitors: Form covalent bonds with cysteine residues near the ATP binding site, enhancing selectivity by permanently modifying the target kinase.
• Gatekeeper Residue: Refers to a specific amino acid that dictates the size of the specificity pocket, significantly influencing inhibitor binding.

5. Assessment of Kinase Selectivity/Off-Target Effects

• Selectivity Measurements:
    - Assays are developed to evaluate the binding affinity of compounds to various kinases in a high-throughput manner.
    - Example of a binding assay using phage display technology shows the competitive binding of a test compound (e.g., inhibitor) against a biotinylated ligand (e.g., SB202190) for P38 MAP kinase.
• Exchange of Binding data:
    - The efficiency of phage-tagged kinases binding to ligands can be monitored through quantitative PCR, allowing researchers to evaluate multiple inhibitors simultaneously.

6. Therapeutic Resistance to Kinase Inhibitors

• Mechanisms of Resistance:
    - Resistance can arise through various mechanisms including:
      - Mutations in the inhibitor binding site that do not impair ATP binding.
      - Overexpression of specific kinases that negate inhibitor effects.
      - Activation of alternative signaling pathways due to redundancy.
      - Drug efflux mechanisms via transport proteins that expel inhibitors from cells.
• Example 1: Gleevec (Imatinib) targeting BCR-Abl in chronic myeloid leukemia patients demonstrates resistance when key mutations occur in the kinase domain leading to treatment failure.
• Example 2: Inhibition of EGFR (Epidermal Growth Factor Receptor) and resistance mechanisms allowing alternative pathways (like c-Met) to sustain cell survival despite inhibition.
• Example 3: Herceptin (trastuzumab) interaction with HER2 highlights resistance formed through receptor masking and evasion of therapeutic action.

Summary Points:

1. A wealth of structural knowledge exists in designing ATP-competitive inhibitors, though absolute specificity is challenging due to the kinome's complexity.
2. Alternative binding pocket interactions may enhance inhibitor affinity.
3. Various strategic design considerations for inhibitors could considerably improve selectivity against particular kinases.
4. Combination therapies may be paramount for overcoming the therapeutic resistance posed by mutation-driven pathways prevalent in various cancers.