Differentiation of Hdm2-mediated p53 ubiquitination and Hdm2 autoubiquitination activity by small molecular weight inhibitors.
The oncoprotein hdm2 ubiquitinates p53, resulting in the rapid degradation of p53 through the ubiquitin (Ub)-proteasome pathway. Hdm2-mediated destabilization and inactivation of p53 are thought to play a critical role in a number of human cancers. We have used an in vitro enzyme assay, monitoring hdm2-catalyzed Ub transfer from preconjugated Ub-Ubc4 to p53, to identify small molecule inhibitors of this enzyme. Three chemically distinct types of inhibitors were identified this way, each with potency in the micromolar range. All three types of compounds display selective inhibition of hdm2 E3 ligase activity, with little or no effect on other Ub-using enzymes. Most strikingly, these compounds do not inhibit the autoubiquitination activity of hdm2. Steady-state analysis reveals that all three classes behave as simple reversible inhibitors of the enzyme and that they are noncompetitive with respect to both substrates, Ub-Ubc4 and p53. Studies of the effects of combinations of two inhibitory molecules on hdm2 activity indicate that the three types of compounds bind in a mutually exclusive fashion, suggesting a common binding site on hdm2 for all of these inhibitors. These compounds establish the feasibility of selectively blocking hdm2-mediated ubiquitination of p53 by small molecule inhibitors. Selective inhibitors of hdm2 E3 ligase activity could provide a novel mechanism for the development of new chemotherapeutics for the treatment of human cancers.
Lai
Zhihong
Z
Department of Chemical Enzymology, Bristol-Myers Squibb Company, Experimental Station, Wilmington, DE 19880, USA. zhihong.lai@bms.com
Yang
Tao
T
Kim
Young B
YB
Sielecki
Thais M
TM
Diamond
Melody A
MA
Strack
Peter
P
Rolfe
Mark
M
Caligiuri
Maureen
M
Benfield
Pamela A
PA
Auger
Kurt R
KR
Copeland
Robert A
RA
D019921:Biotinylation; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002384:Catalysis; D056827:Endosomal Sorting Complexes Required for Transport; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000378:metabolism; D008954:Models, Biological; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D010453:Peptide Synthases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D000075702:Nedd4 Ubiquitin Protein Ligases; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D010453:Peptide Synthases