Disrupting
Protein-Protein Interaction(PPI)
constitutively
Sample
Protein Kinase CK2
Overexpressed in various cancer types.
Heterotetrameric complex (α2/β2).
Small-molecule PPI inhibitors
fragment-based screening cascade
Ser/Thr protein kinase.
2 Catalytic subunits(CK2α,α) attached
to dimer of regulatory subunit(CK2β,β)
The unique molecular architecture of
the CK2 holoenzyme could be exploited in the design of
inhibitors that do not target the ATP site and thus provide a
more specific mode of action, prompting the discovery of
various non-ATP-competitive inhibitors against CK2.
fragment-based drug discovery(FBDD)
- Fluorescence-based thermal shift(FTS) screening
❗The melting temperature (Tm) of proteins decreased in the presence of the small-molecule oligomeric disruptor, supporting the use of negative thermal shifts to identify molecules that cause a dehomooligomeric transition
Both SPD304 and 6-hydroxydopa, which promoted subunit disassembly by binding to a non-native form of their protein target, lowered the melting temperature of their protein complexes.
Could be expected to decrease the protein’s stability.
8000 fragments-> 60 destabilizing fragments.
fragments that cause negative thermal shifts signify preferential fragment binding to the unfolded form of the protein.
Ligands that increase the Tm of a protein cause stabilization of the protein−ligand complex.
- NMR Spectroscopy
❗ligand-observed NMR assays depend on monitoring differences in the properties of the ligand spectra upon interaction with the macromolecular target.
The extent of thermal destabilization cannot be used as a measure of its binding affinity, and its degree of binding in NMR assays.
(Same binding, different binding enthalpies/ΔTm)
[3 experiments in ligand-observed NMR]
1.Saturation transfer difference(STD)
2.Water-ligand observed via gradient spectroscopy (waterLOGSY)
3.Carr-Purcell-Meiboom-Gill(CPMG)
- Native MS
The 5-substituted pyrazole is the most important core in mediating dimer disruption.
Fragments may be potentially mediating the disruption of dimeric CK2β by mainly engaging in hydrophobic interactions with residues at the dimer interface.
- Dimer Interface of CK2β
Hydrophobic residues(Pro110, Val112, Tyr113, Leu124, Val143, Tyr144) are important to dimer stability.
Salt-bridge interactions between Arg111, Asp112 are stabilizing the dimer CK2β.
- Homodissociation ITC
CK2β P110D/V143D mutant decreased the catalytic activity of CK2α, highlighting that the modulation of CK2α catalytic activity by CK2β is highly dependent on a proper dimeric architecture of CK2β.
Dimerdisrupting fragments could promote CK2β degradation and an attenuation of CK2α catalytic activity through favoring the formation of the CK2β monomer.
ITC assay confirming the mechanism of fragment-induced dimer-disruption and examine structure-activity relationships governing dimerization affinity.
polar interactions and hydrophobic or aromatic stacking interactions contribute to weakening dimeric association in the CK2β double mutant.
Replacing the methylene linker in 16 with an NH group caused approximately 2−fold increase in dimer-disrupting, suggesting a role for hydrogen bonding interactions in effecting subunit disassembly.
Substitutions at the meta position of the phenyl ring were generally more favorable for dimer disruption than para substitutions.
❗Orthogonal biophysical experiments involving native MS and homodissociation ITC support a mechanism that is consistent with fragment-induced dimeric disruption.