Previously, we designed and synthesized a potent NF-kappaB inhibitor, DHMEQ. Although DHMEQ showed potent anti-inflammatory and anticancer activities in animals, its molecular target has not been elucidated. In the present study, its target protein was found to be p65 and other Rel homology proteins. We found that (-)-DHMEQ bound to p65 covalently with a 1:1 stoichiometry by conducting SPR and MALDI-TOF MS analyses. MS analysis of the chymotrypsin-digested peptide suggested the binding of (-)-DHMEQ to a Cys residue. Formation of Cys/(-)-DHMEQ adduct in the protein was supported by chemical synthesis of the adduct. Substitution of specific Cys in p65 and other Rel homology proteins resulted in the loss of (-)-DHMEQ binding. (-)-DHMEQ is the first NF-kappaB inhibitor that was proven to bind to the specific Cys by chemical methodology. These findings may explain the highly selective inhibition of NF-kappaB and the low toxic effect of (-)-DHMEQ in cells and animals.
The novel piperidine compound, DTCM-glutarimide, was found to be a new inhibitor of macrophage activation, inhibiting AP-1 activity. It also inhibited graft rejection in mice, and thus may be a candidate for an anti-inflammatory agent.
NF-κB is a transcription factor for the immune activation and tissue stability, but excess activation of NF-κB often causes inflammation and cancer. An NF-κB component RelB is involved in B-cell maturation and autoimmunity. In the present research we studied the role of the RelB DNA binding domain on cellular stability and importin affinity. We prepared a RelB protein mutated at Arg141 to Ala and Tyr142 to Ala (AA mutant) having no DNA binding activity. The stability of this mutant protein was greatly reduced compared with that of the wild-type protein. We also constructed a nuclear localization signal-inactivated mutant of RelB, and found that this mutant was also unstable in the cells. Thus, RelB destabilization was caused by the loss of DNA binding possibly because of the change in cellular localization. The mutation also decreased the affinity to importin-α5 decreasing the nuclear localization. Our newly discovered NF-κB inhibitor (-)-DHMEQ binds to a specific Cys residue in RelB to inhibit DNA binding and also decreased the stability and importin affinity. These findings would indicate that the DNA binding activity of this transcription factor is a crucial for its stability and intracellular localization.
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