ABSTRACT
The intrinsically disordered HIV-1 Tat protein binds the viral RNA transactivation response structure (TAR), which recruits transcriptional cofactors, amplifying viral mRNA expression. Limited Tat transactivation correlates with HIV-1 latency. Unfortunately, Tat inhibitors are not clinically available. The small molecule didehydro-cortistatin A (dCA) inhibits Tat, locking HIV-1 in persistent latency, blocking viral rebound. We generated chemical derivatives of dCA that rationalized molecular docking of dCA to an active and specific Tat conformer. These revealed the importance of the cycloheptene ring and the isoquinoline nitrogen's positioning in the interaction with specific residues of Tat's basic domain. These features are distinct from the ones required for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known ligand of dCA. Besides, we demonstrated that dCA activity on HIV-1 transcription is independent of CDK8. The binding of dCA to Tat with nanomolar affinity alters the local protein environment, rendering Tat more resistant to proteolytic digestion. dCA thus locks a transient conformer of Tat, specifically blocking functions dependent of its basic domain, namely the Tat-TAR interaction; while proteins with similar basic patches are unaffected by dCA. Our results improve our knowledge of the mode of action of dCA and support structure-based design strategies targeting Tat, to help advance development of dCA, as well as novel Tat inhibitors.IMPORTANCE Tat activates virus production, and limited Tat transactivation correlates with HIV-1 latency. The Tat inhibitor dCA locks HIV in persistent latency. This drug class enables block-and-lock functional cure approaches, aimed at reducing residual viremia during therapy and limiting viral rebound. dCA may also have additional therapeutic benefits since Tat is also neurotoxic. Unfortunately, Tat inhibitors are not clinically available. We generated chemical derivatives and rationalized binding to an active and specific Tat conformer. dCA features required for Tat inhibition are distinct from features needed for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known target of dCA. Furthermore, knockdown of CDK8 did not impact dCA's activity on HIV-1 transcription. Binding of dCA to Tat's basic domain altered the local protein environment and rendered Tat more resistant to proteolytic digestion. dCA locks a transient conformer of Tat, blocking functions dependent on its basic domain, namely its ability to amplify viral transcription. Our results define dCA's mode of action, support structure-based-design strategies targeting Tat, and provide valuable information for drug development around the dCA pharmacophore.
Subject(s)
Anti-HIV Agents/metabolism , HIV-1/drug effects , Heterocyclic Compounds, 4 or More Rings/metabolism , Isoquinolines/metabolism , tat Gene Products, Human Immunodeficiency Virus/metabolism , Anti-HIV Agents/chemical synthesis , Cyclin-Dependent Kinase 8/metabolism , HeLa Cells , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Humans , Isoquinolines/chemical synthesis , Molecular Docking Simulation , Protein BindingABSTRACT
Marizomib (NPI-0052) is a naturally derived irreversible proteasome inhibitor that potently induces apoptosis via a caspase-8 and ROS-dependent mechanism in leukemia cells. We aim to understand the relationship between the irreversible inhibition of the proteasome and induction of cell death in leukemia cells by using analogs of marizomib that display reversible and irreversible properties. We highlight the importance of sustained inhibition of at least two proteasome activities as being key permissive events for the induction of the apoptotic process in leukemia cells. These data provide the basis for the development of new approaches to generate more effective anti-proteasome therapies.
Subject(s)
Apoptosis/drug effects , Lactones/pharmacology , Protease Inhibitors/pharmacology , Pyrroles/pharmacology , Caspase 8/metabolism , Humans , Lactones/chemistry , Leukemia/drug therapy , Leukemia/metabolism , Leukemia/pathology , Oxidative Stress/drug effects , Protease Inhibitors/chemistry , Pyrroles/chemistry , Reactive Oxygen Species/metabolism , Tumor Cells, CulturedABSTRACT
The present study was undertaken to compare the cellular transport characteristics of [(3)H]NPI-0052 (1R,4R,5S)-4-(2-chloroethyl)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione (marizomib; salinosporamide A) and [(3)H]NPI-0047 (1R,4R, 5S)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-4-ethyl-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione in RPMI 8226 multiple myeloma and PC-3 prostate adenocarcinoma cells to determine whether these properties explain differences in the cytotoxic potencies of these chemical analogs. The results indicate that marizomib, which possesses a chemical-leaving group, is more cytotoxic to both cell lines and inhibits proteasome activity more completely at lower concentrations than NPI-0047, a nonleaving-group analog. Moreover, it was found that both compounds accumulate in these cells by simple diffusion and the same carrier-mediated transport system. Although the rate of uptake is similar, the cellular efflux, which does not seem to be mediated by a major ATP-binding cassette (ABC)-efflux transporter, is more rapid for NPI-0047 than for marizomib. Experiments revealed that the irreversible binding of marizomib to the proteasome is responsible for its slower efflux, longer duration of action, and greater cytotoxicity compared with NPI-0047. The discovery that major ABC transporters of the multidrug resistance-associated protein family do not seem to be involved in the accumulation or removal of these agents suggests they may not be affected by multidrug resistance mechanisms during prolonged administration.
Subject(s)
Antineoplastic Agents/pharmacology , Lactones/pharmacology , Proteasome Endopeptidase Complex/drug effects , Pyrroles/pharmacology , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP-Binding Cassette Transporters/metabolism , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Humans , Lactams/metabolismABSTRACT
Expedient access to a highly functionalized 2-pyrrolidinone (8), the gamma-lactam core of 20S proteasome inhibitor (-)-salinosporamide A (marizomib; NPI-0052; 1), using a regio- and stereoselective epoxide formation/reductive oxirane ring-opening strategy is presented. Notably, the sequential construction of the C-4, C-3, and C-2 stereocenters of 1 in a completely stereocontrolled fashion is a key feature of streamlining the synthesis of intermediate 12. A related strategy is also discussed.
Subject(s)
Lactones/chemical synthesis , Pyrroles/chemical synthesis , Epoxy Compounds , Ethylene Oxide , Proteasome Inhibitors , Pyrrolidinones , StereoisomerismABSTRACT
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
Large-scale fermentation of the marine actinomycete Salinispora tropica for production of salinosporamide A (NPI-0052; 1) clinical trials materials provided crude extracts containing minor secondary metabolites, including salinosporamide B (2) and a new congener, 3. Spectroscopic characterization revealed that 3 is identical to antiprotealide, a molecular hybrid of 20S proteasome inhibitors 1 and omuralide (4) not previously described as a natural product. Analysis of crude extracts from shake flask cultures of three wild-type S. tropica strains confirmed the production of antiprotealide at 1.1, 0.8, and 3.0 mg/L. Thus, antiprotealide is a natural product metabolite of S. tropica.
Subject(s)
Actinobacteria/chemistry , Biological Products/chemistry , Biological Products/isolation & purification , Lactams/chemistry , Lactams/isolation & purification , Lactones/chemistry , Lactones/isolation & purification , Pyrroles/chemistry , Pyrroles/isolation & purification , Animals , Biological Products/pharmacology , Drug Screening Assays, Antitumor , Humans , Inhibitory Concentration 50 , Lactams/pharmacology , Lactones/pharmacology , Marine Biology , Molecular Structure , Proteasome Endopeptidase Complex , Pyrroles/pharmacology , RabbitsABSTRACT
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 marine actinomycete strain NPS8920 produces a new class of 4-oxazolidinone antibiotics lipoxazolidinone A, B and C. Lipoxazolidinone A possesses good potency (1-2 microg/mL) against drug-resistant pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Strain NPS8920 exhibits different morphologies in both agar and submerged cultures. The ability of strain NPS8920 to sporulate on saline-based agar media but not on deionized water-based agar medium supported that strain NPS8920 is a marine actinomycete. While strain NPS8920 does not require seawater for growth, the production of lipoxazolidinones by strain NPS8920 can only be detected in the seawater-based media. The optimal production of lipoxazolidinones was observed in the natural seawater-based medium. Strain NPS8920 produced 10-20% of lipoxazolidinones in the synthetic sea salt Instant Ocean-based medium and no production in the sodium chloride-based and deionized water-based media.
Subject(s)
Actinobacteria/growth & development , Actinobacteria/metabolism , Anti-Bacterial Agents/metabolism , Oxazolidinones/metabolism , Seawater/microbiology , Culture Media/chemistry , Molecular StructureABSTRACT
Marine actinomycete strain NPS008920, a member of the new genus Marinispora, was isolated from a sediment sample collected in Cocos Lagoon, Guam. In natural sea water containing media, the strain produced a series of novel 2-alkylidene-5-alkyl-4-oxazolidinones, lipoxazolidinone A (1), B (2), and C (3). Compounds 1- 3 showed broad spectrum antimicrobial activity similar to that of the commercial antibiotic linezolid (Zyvox), a 2-oxazolidinone. Hydrolysis of the amide bond of the 4-oxazolidinone ring of 1 resulted in loss of antibacterial activity. The 2-alkylidene-4-oxazolidinone represents a new antibiotic pharmacophore and is unprecedented in nature.
Subject(s)
Actinobacteria/chemistry , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Oxazolidinones/isolation & purification , Oxazolidinones/pharmacology , Anti-Bacterial Agents/chemistry , Guam , Haemophilus influenzae/drug effects , Marine Biology , Molecular Structure , Oxazolidinones/chemistry , 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
A Streptomyces sp. (NPS008187) isolated from a marine sediment collected in Alaska was found to produce three new pyrrolosesquiterpenes, glyciapyrroles A (1), B (2), and C (3), along with the known diketopiperazines cyclo(leucyl-prolyl) (4), cyclo(isoleucyl-prolyl) (5), and cyclo(phenylalanyl-prolyl) (6). The structures of 1, 2, and 3 were established using spectroscopic methods.
Subject(s)
Pyrroles/isolation & purification , Sesquiterpenes/isolation & purification , Streptomyces/chemistry , Alaska , Geologic Sediments , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Peptides, Cyclic/chemistry , Peptides, Cyclic/isolation & purification , Pyrroles/chemistry , Sesquiterpenes/chemistryABSTRACT
Salinosporamide A (1, NPI-0052) is a potent proteasome inhibitor in development for treating cancer. In this study, a series of analogues was assayed for cytotoxicity, proteasome inhibition, and inhibition of NF-kappaB activation. Marked reductions in potency in cell-based assays accompanied replacement of the chloroethyl group with unhalogenated substituents. Halogen exchange and cyclohexene ring epoxidation were well tolerated, while some stereochemical modifications significantly attenuated activity. These findings provide insights into structure-activity relationships within this novel series.