Drug Discovery: Case Study 3 - Protein Kinases as Drug Targets

The human kinome

-518 kinases encoded in the human genome

- 40 of which are atypical protein kinases (lack homology with the major subfamilies)

- 478 classical protein kinases: 388 serine/threonine kinases and 90 tyrosine kinases

Protein kinases

- can phosphorylate specific serine/threonine or tyrosine amino acid residues

- uses ATP to deposit a phosphate group onto protein

How is specificity of protein kinase action mediated

Protein kinases recognise certain amino acid consensus sequences around the serine/threonine (S/T) or around the tyrosine (Y) to be modified

PKA consensus phosphorylation site

R-R-X-S/T-aromatic

- two arginines before phosphorylation site

- aromatic amino acid after phosphorylation site

GSK3 consensus phosphorylation site

S-X-X-X-pS

- serine before phosphorylation site

- downstream requires an already phosphorylated serine

What do protein kinases do?

- alter enzymatic activity

- alter ability to interact with other cellular components = can promote binding (act as a scaffold)

- can alter localisation in a cell

- can alter stability

- can alter what proteins interact with it

Why are protein kinases of interest to pharmacologists

- protein kinase levels or mutants are often observed in disease pathologies such as cancer

- Activity is often upregulated therefore pharmacologists are looking for protein kinase inhibitors

what proteins dephosphorylate proteins

- protein phosphatases

cyclosporin

Immunosuppressant

- inhibits a protein phosphatase = promotes activity of the phosphorylated form of a protein

- discovery lead to attempts to make protein phosphatase inhibitors, none of which have yet been approved

Which protein phosphatase inhibitor has advanced the most through clinical trials

LB-100

- targets PP2A

- catalytic inhibitor

What therapeutic areas are being targeted by inhibition of protein kinases

- cancer

- immune system

- degenerative disease

v-Src vs c-Src

c-Src = non-receptor tyrosine kinase signalling protein that can regulate cell proliferation, differentiation and apoptosis

v-Src = constituently active mutant form of c-Src expressed by a tumour-causing retrovirus (RSV) which activates cells to divide uncontrollably and to evade pro-apoptotic death signals

How is v-Src constitutively active and how does this lead to cancer

- protein encoded by the virus has a truncated C-terminal region which means it loses a tyrosine at position 527

- tyrosine at this position is critical - when phosphorylated, it binds to another part of the protein and holds the protein in its inactive conformation - to become active it requires phosphorylation of a different residue and dephosphorylation of tyrosine 527

- in the viral version the src is constitutively active as it isn't held in the inactive form = uncontrolled proliferation = abhorrent tyrosine kinase activity drives the cancer

What are the problems with targeting protein kinases with drugs

- small molecules bind to the active site of protein kinases (ATP binding site)

- ATP is present at high concentrations in cells so it outcompetes the small molecule inhibitors

= need to develop highly specific high affinity molcules

- if you bind to the active site of a protein kinase the possibility is there wont be very good specificity as active sites for protein kinases are well conserved

Imatinib

tyrosine kinase inhibitor

- imatinib was the first tyrosine kinase inhibitor (TKI) to be developed and approved for clinical use in cancer treatment

Different types of protein kinase inhibition

Type I - bind to the active form of the protein kinase

Type II - bind preferentially to the inactive form of the protein kinase but still in the active site

Type III - bind to sites adjacent to the active site and cause inhibition (allosteric)

Type IV - binds any other site other than the active site (allosteric)

*type III and IV are the most desirable as they are the most specific and wont be outcompeted

Other approaches to protein kinase inhibition that are not small molecule inhibitors

- biologic agents

e.g. monoclonal antibodies, nanobodies = biotherapeutics

- some protein kinases (RTKs) span the plasma membrane so can be targeted by biologic agents

Examples of monoclonal antibodies used to target protein kinases

1. Trastuzumab (herceptin) - binds to and causes down-regulation of (HER2) EGF receptor

2. Bevacizumab (Avastin) - binds to the VEGR-receptor ligand vascular endothelial growth factor preventing receptor activation

RTKs

- single TM spanning polypeptides which dimerise upon ligand binding

- following dimerisation the C terminal of each monomer phosphorylates the other monomer of the dimer pair on specific tyrosine residues = autophosphorylation

- allows the RTK to act as a scaffold for IC signalling pathways to be assembled

Receptor tyrosine kinase therpeutics - HER2

- HER2 is an RTK closely related to EGFR

- no specific ligand has been identified for HER2 so its believed that it must heterodimerise with EGFR to become active

- HER2 is overexpressed in 30% of breast cancers = more proteins to partner with active receptors = upregulation of growth factor signalling that gives cells the advantage that allows them to proliferate and cause cancer

Drug that targets HER2

- Lapatinib = HER2 selective tyrosine kinase inhibitor

When are non-specific therapeutics generally accepted for use to fight cancer

- when the cancer is so aggressive that without the drug it will be untreatable

- off target effects are less of a concern

Trastuzumad deruxtecan (Enhertu)

= antibody-drug conjugate

-approved for treatment of breast cancers and gastric/oesophageal adenocarcinomas

- monoclonal antibody (trastuzumab) is covalently linked to the topoisomerase I inhibitor deruxtecan

- monoclonal antibody binds to the HER2 receptors on the surface of cancer cells and delivers the deruxtecan to the cell interior, where it can bind topoisomeriase I leading to DNA damage and cell destruction

- linker regin is cleaved to release the active drug