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.

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  • 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.

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  • 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.

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  • 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.

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  • 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.

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Critical aspect

If the sequence of the steps change may have different result.