ABSTRACT
Many marketed drugs contain fluorine, reflecting its ability to modulate a variety of biological responses. The unique 20S proteasome inhibition profile of fluorosalinosporamide compared to chlorinated anticancer agent salinosporamide A (NPI-0052) is exemplary and relates to each halogen's leaving group potential. Crystal structures of fluoro-, hydroxy-, and bromosalinosporamide in complex with the yeast 20S proteasome core particle (CP) provide mechanistic insights into ligand binding and leaving group elimination and the ability to fine-tune the duration of proteasome inhibition. Fluorosalinosporamide/CP crystal structures determined over time offer striking snapshots of the ligand trapped with an intact fluoroethyl group in anticipation of fluoride elimination, followed by complete nucleophilic displacement of fluoride to give the highly stabilized cyclic ether found for salinosporamide A and bromosalinosporamide. This two-step reaction pathway is consistent with a mechanism for partially reversible proteasome inhibition by fluorosalinosporamide. Proteasome catalyzed fluoride displacement provides preliminary insights into the active site Thr1N pK(a).
Subject(s)
Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Hydrocarbons, Fluorinated/metabolism , Hydrocarbons, Fluorinated/pharmacology , Lactones/metabolism , Lactones/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Bromides/chemistry , Buffers , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Hydrocarbons, Fluorinated/chemistry , Hydrogen-Ion Concentration , Lactones/chemistry , Ligands , Models, Molecular , Molecular Conformation , Proteasome Endopeptidase Complex/chemistry , Saccharomyces cerevisiae/enzymology , Water/chemistryABSTRACT
A series of chlorinated bisindole pyrroles, lynamicins A-E (1-5), was discovered from a novel marine actinomycete, NPS12745, which was isolated from a marine sediment collected off the coast of San Diego, California. Close to full length 16S rRNA sequence analysis indicated that NPS12745 is a novel strain of a recently described marine actinomycete with the proposed genus name Marinispora. The antimicrobial spectrum of these compounds was evaluated against a panel of 11 pathogens, which demonstrated that these substances possess broad-spectrum activity against both Gram-positive and Gram-negative organisms. Significantly, compounds 1-5 were active against drug-resistant pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium.
Subject(s)
Actinobacteria/chemistry , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Hydrocarbons, Chlorinated/isolation & purification , Hydrocarbons, Chlorinated/pharmacology , Indoles/isolation & purification , Indoles/pharmacology , Pyrroles/isolation & purification , Pyrroles/pharmacology , Actinobacteria/genetics , Anti-Bacterial Agents/chemistry , California , Drug Resistance, Bacterial/drug effects , Enterococcus faecium/drug effects , Hydrocarbons, Chlorinated/chemistry , Indoles/chemistry , Marine Biology , Methicillin Resistance/drug effects , Microbial Sensitivity Tests , Molecular Structure , Pyrroles/chemistry , Staphylococcus aureus/drug effects , Vancomycin Resistance/drug effectsABSTRACT
Salinosporamide A ( 1 (NPI-0052)) is a potent, monochlorinated 20S proteasome inhibitor in clinical trials for the treatment of cancer. To elucidate the role of the chlorine leaving group (LG), we synthesized analogues with a range of LG potentials and determined their IC 50 values for inhibition of chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) activities of 20S proteasomes. Proteasome activity was also determined before and after attempted removal of the inhibitors by dialysis. Analogues bearing substituents with good LG potential exhibited the greatest potency and prolonged duration of proteasome inhibition, with no recovery after 24 h of dialysis. In contrast, activity was restored after
Subject(s)
Lactams/chemical synthesis , Lactams/pharmacology , Lactones/chemical synthesis , Lactones/pharmacology , Proteasome Inhibitors , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Animals , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydrolysis , Kinetics , Lactams/chemistry , Lactones/chemistry , Models, Molecular , Molecular Structure , Proteasome Endopeptidase Complex/metabolism , Protein Subunits/antagonists & inhibitors , Protein Subunits/metabolism , Pyrroles/chemistry , Rats , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A novel enantioselective total synthesis of 20S proteasome inhibitor Salinosporamide A (NPI-0052; 1) is presented. Key features include intramolecular aldol cyclization of 6 to simultaneously generate the three chiral centers of advanced intermediate 5, cyclohexene ring addition using B-2-cyclohexen-1-yl-9-BBN, and inversion of the C-5 stereocenter by oxidation followed by enantioselective enzymatic reduction.
Subject(s)
Lactones/chemical synthesis , Pyrroles/chemical synthesis , Crystallography, X-Ray , Lactones/chemistry , Models, Molecular , Molecular Conformation , Pyrroles/chemistry , StereoisomerismABSTRACT
Feeding sodium butyrate (0.25-1 mg/ml) to cultures of Salinispora tropica NPS21184 enhanced the production of salinosporamide B (NPI-0047) by 319% while inhibiting the production of salinosporamide A (NPI-0052) by 26%. Liquid chromatography mass spectrometry analysis of the crude extract from the strain NPS21184 fed with 0.5 mg/ml sodium [U-(13)C(4)]butyrate indicated that butyrate was incorporated as a contiguous four-carbon unit into NPI-0047 but not into NPI-0052. Nuclear magnetic resonance analysis of NPI-0047 and NPI-0052 purified from the sodium [U-(13)C(4)]butyrate-supplemented culture extract confirmed this incorporation pattern. The above finding is the first direct evidence to demonstrate that the biosynthesis of NPI-0047 is different from NPI-0052, and NPI-0047 is not a precursor of NPI-0052.
Subject(s)
Butyric Acid/chemistry , Lactams/chemistry , Lactones/chemistry , Pyrroles/chemistry , Lactams/metabolism , Lactones/metabolism , Magnetic Resonance Spectroscopy , Micromonosporaceae/metabolism , Pyrroles/metabolismABSTRACT
We examined the effects of halogens on the production of salinosporamide A (NPI-0052) by the obligate marine actinomycete Salinispora tropica NPS465, specifically the production of analogs containing halogens other than chlorine. Adding NaF, NaBr and NaI directly to the production medium prepared in seawater containing -3% NaCl did not induce the production of the corresponding analogs. Replacing seawater with 2-3% NaI in the production medium enhanced the production of NPI-0052 by 2.1 fold. Replacing seawater with 2-3% NaBr in the production medium suppressed the production of NPI-0052 but induced the production of a brominated analog at very low yield. Using a stepwise enrichment of bromide in the seed cultures in order to reduce the chloride ion carried over to the production medium, the production of the brominated analog was enhanced by 4 fold. We also demonstrated that the growth of this obligate marine actinomycete is dependent upon sodium concentration, not chloride concentration.