Disrupting the Constitutive, Homodimeric Protein-Protein Interface in in CK2β Using a Biophysical Fragment-Based Approach
What is CK2β protein?
- CK2β is Serine/threonine-selective protein kinase.
- CK2β forms Heterotetrameric complex.(α2/β2)
- Main act of CK2β is Cell cycle control, DNA repair, regulation of the circadian rhythm, and other cellular processes.
- De-regulation of CK2 has been linked to tumorigenesis.
- Disruption of CK2β dimer interfere CK2β function.
Purpose of this article
- Using Fragment-based screening instead of High-throughput screening, to find small molecule which interfere CK2β function.
Steps for screening small molecule
Fluorescence based Thermal Shift Screening
Ligand-Observed NMR spectroscopy
Mass spectrometry
Homodissociation ITC
Structure-Activity Relationship
Structure features of CK2β Dimer interface
- Dimerization largely derived by hydrophobic interactions.
- Salt-bridge, Hydrogen bonds stabilize dimer structure.
- Tm will change by binding Fragments whether stabilized or destabilized.
- Dehomooligomeric transition cause Negative thermal shift.
- 60 destabilizing fragment hits show ∆Tm < -1.5℃.
- However, possibility of small proportion of CK2β monomer provides a rapid route for unfolding.
- Also bind to and distort the CK2β dimer interface without completely causing subunit dissociation.
- Additional experimental evidence must be provided.
- Monitoring differences in the properties of the ligand spectra upon interaction with the macromolecular target.
- STD, waterLOGSY, CPMG are commonly used.
- Validated 45 of the 60 destabilizing hits, 40 fragments showed binding in all three NMR experiment.
- No correlation between the degree of binding observed in the NMR experiment and ∆Tm values.
- ∆Tm cannot be used as a measure of its binding affinity and degree of binding in NMR assays.
- 18 out of 40 compounds induced a higher population of monomeric CK2β
- 1 and 2 (5-substituted pyrazole core) had the greatest effects on dimer disruption.
- 3, 4 (pyrazole scaffold), 5-7 (quinolone), 8-11 (naphthol) show monomerization effect.
- No correlation between monomerization and thermal shift.
- Through mutation of Zinc-coordinating cysteine residue, Zinc finger is associated with Dimerization.
- Fragments do not cause metal sequestration on Zinc finger while disrupting dimer.
- Fragments cause distorting and weakening dimer interface.
- Double mutation of Pro110 and Val143 to weaken binding affinity of dimer CK2β.
- Out of 18 fragments, 2 showed most potent in mediating dimerization disruption.
- Using structural analogs of 4, 16 compounds.
- Functional group substitution induce different Kd values.
- Mutant CK2β is potentially useful to develop fragments, disrupting dimer CK2β.
Critical aspect
If the sequence of the steps change may have different result.