Interaction of the novel anthracycline antitumor agent N-benzyladriamycin-14-valerate with the C1-regulatory domain of protein kinase C: structural requirements, isoform specificity, and correlation with drug cytotoxicity.
Anthracycline antibiotics like doxorubicin (DOX) are known to exert their antitumor effects primarily via DNA intercalation and topoisomerase II inhibition. By contrast, the noncross-resistant cytoplasmically localizing DOX analogue, N-benzyladriamycin-14-valerate (AD 198), only weakly binds DNA and does not inhibit topoisomerase II, yet it displays superior antitumor activity, strongly suggesting a distinct cytotoxic mechanism. In recent modeling studies, we reported a structural similarity between AD 198 and commonly accepted ligands for the C1-domain of protein kinase C (PKC), and we hypothesized that the unique biological activity of AD 198 may derive, in part, through this kinase. Consistent with this hypothesis, the present biochemical studies demonstrate that AD 198 competes with [3H]phorbol-12,13-dibutyrate ([3H]PDBu) for binding to phorbol-responsive PKC isoforms, the isolated C1b domain of PKC-delta (delta C1b), and the nonkinase phorbol ester receptor, beta2-chimaerin. In NIH/3T3 cells, AD 198 competitively blocks PKC activation by C1-ligands. Importantly, neither DOX nor N-benzyladriamycin, the principal AD 198 metabolite, inhibits basal or phorbol-stimulated PKC activity or appreciably competes for [3H]PDBu binding. In CEM cells, structure activity studies with 14-acyl congeners indicate that the rapid induction of apoptosis correlates with competition for [3H]PDBu binding, strongly implicating phorbol-binding proteins in drug activity. Collectively, these studies support the conclusion that AD 198 is a C1-ligand and that C1-ligand receptors are selective drug targets. These studies provide the impetus for continuing efforts to understand the molecular basis for the unique biological activity of AD 198 and provide for the design of analogues with improved affinity for C1-domains and potentially greater antitumor activity.
Roaten
J Brent
JB
Department of Pharmacology, University of Tennessee College of Medicine, Memphis, Tennessee 38163, USA.
Kazanietz
Marcelo G
MG
Caloca
Maria Jose
MJ
Bertics
Paul J
PJ
Lothstein
Leonard
L
Parrill
Abby L
AL
Israel
Mervyn
M
Sweatman
Trevor W
TW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001921:Brain / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000031:analogs & derivatives / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000970:Antineoplastic Agents; D020033:Protein Isoforms; D011994:Recombinant Proteins; D004317:Doxorubicin; C046808:N-benzyladriamycin-14-valerate; D011493:Protein Kinase C