ABSTRACT
Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics.
Subject(s)
Amidohydrolases/metabolism , Anti-Bacterial Agents/metabolism , Antineoplastic Agents/metabolism , Hydroxamic Acids/metabolism , Mitochondria/enzymology , Amidohydrolases/genetics , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Division/physiology , Cell Line, Tumor/drug effects , Enzyme Inhibitors/metabolism , Humans , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Membrane Potentials/physiology , Mice , Mitochondria/drug effects , Molecular Sequence Data , Molecular Structure , Neoplasm Transplantation , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Sequence Alignment , Transplantation, HeterologousABSTRACT
Peptide deformylases (PDFs) have been investigated as potential specific targets for antibiotics, but the possible existence of a functional human PDF (HsPDF) presents a potential hurdle to the design of specific drugs. We have expression cloned a HsPDF that has deformylase activity, although it is a slower and catalytically less active enzyme than bacterial or plant PDFs. A cobalt-substituted form of HsPDF (but not nickel or zinc) is active, and the enzyme appears to be active at a pH between 6.0 and 7.2, a temperature range of 25-50 degrees C, and in a low KCl ionic strength buffer. Actinonin inhibits HsPDF activity with an IC50 of 43 nM and kills Daudi and HL60 human cancer cell lines with an LC50 of 5.3 and 8.8 microM, respectively. The inhibition of HsPDF may provide an explanation for the mechanism by which actinonin is cytotoxic against various human tumor cell lines.
