Primate Monocytes - CD14, CD16 - Ziegler-Heitbrock

A small molecule ubiquitination inhibitor blocks NF-kappa B-dependent cytokine expression in cells and rats

Abstract

A small molecule inhibitor of NF-kappaB-dependent cytokine expression was discovered that blocked TNFalpha-induced IkappaBalpha degradation in MM6 cells but not the degradation of beta-catenin in Jurkat cells. Ro106-9920 blocked LPS-dependent expression of TNFalpha, IL-1betaand IL-6 in fresh human peripheral blood mononuclear cells with IC(50) values below 1 {micro)M. Ro106-9920 also blocked TNFalpha production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require betaTRCP, but associates with IkappaBalpha and will ubiquitinate IkappaBalphaS32/36E (IkappaBalphaee) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell free system as a time-dependent inhibitor of IkappaBalphaee ubiquitination with an IC(50) value of 2.3 0.09 {micro}M. The E3 ligase activity is inhibited by cysteine alkylating reagents, supported by E2UBCH7 and requires cIAP2 or a cIAP2 associated protein for activity. These activities are inconsistent to what has been reported for SCF(superbetaTRCP}, the putative E3 for IkappaBalpha ubiquitination. Ro106-9920 was observed to be selective for IkappaBalphaee ubiquitination over E1, E2UBCH7, nonspecific ubiquitination of cellular proteins and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized, but essential, ubiquitination activity associated with LPS and TNFalpha induced IkappaBalpha degradation and NF-kappaB activation.