ABSTRACT
N-Hydroxylating monooxygenases are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on d-Lys, although it binds l-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA, and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producing more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the flavin adenine dinucleotide (FAD) domain that precludes binding of the nicotinamide cofactor. This "occluded" structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. Biological implications of these findings are discussed.
Subject(s)
Bacterial Proteins , Lysine , Mixed Function Oxygenases , Nocardia/enzymology , Oxygen Consumption/physiology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/genetics , Flavin-Adenine Dinucleotide/metabolism , Hydroxylation , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , NADP/chemistry , NADP/genetics , NADP/metabolism , Nocardia/genetics , Protein Structure, TertiaryABSTRACT
The mechanism of Mycobacterium smegmatis G (MbsG), a flavin-dependent l-lysine monooxygenase, was investigated under steady-state and rapid reaction conditions using primary and solvent kinetic isotope effects, substrate analogs, pH and solvent viscosity effects as mechanistic probes. The results suggest that l-lysine binds before NAD(P)H, which leads to a decrease in the rate constant for flavin reduction. l-lysine binding has no effect on the rate of flavin oxidation, which occurs in a one-step process without the observation of a C4a-hydroperoxyflavin intermediate. Similar effects were determined with several substrate analogs. Flavin oxidation is pH independent while the kcat/Km and kred/KD pH profiles for NAD(P)H exhibit single pKa values of â¼6.0, with increasing activity as the pH decreases. At lower pH, the enzyme becomes more uncoupled, producing more hydrogen peroxide and superoxide. Hydride transfer is partially rate-limiting at neutral pH and becomes more rate-limiting at low pH. An inverse solvent viscosity effect on kcat/Km for NAD(P)H was observed at neutral pH whereas a normal solvent viscosity effect was observed at lower pH. Together, the results indicate a unique mechanism where a rate-limiting and pH-sensitive conformational change occurs in the reductive half-reaction, which affects the efficiency of lysine hydroxylation.
Subject(s)
Bacterial Proteins/metabolism , Dinitrocresols/metabolism , Lysine/metabolism , Mixed Function Oxygenases/metabolism , Mycobacterium smegmatis/chemistry , NADP/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biocatalysis , Flavins , Hydrogen Peroxide/metabolism , Hydrogen-Ion Concentration , Kinetics , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Mycobacterium smegmatis/enzymology , Oxidation-Reduction , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Superoxides/metabolismABSTRACT
Flavin-dependent monooxygenase (FMO) from Methylophaga sp. strain SK1 catalyzes the NADPH- and oxygen-dependent hydroxylation of a number of xenobiotics. Reduction of the flavin cofactor by NADPH is required for activation of molecular oxygen. The role of a conserved tryptophan at position 47 was probed by site-directed mutagenesis. FMOW47A resulted in an insoluble inactive protein; in contrast, FMOW47F was soluble and active. The spectrum of the flavin in the mutant enzyme was redshifted, indicating a change in the flavin environment. The kcat values for NADPH, trimethylamine, and methimazole, decreased 5-8-fold. Primary kinetic isotope effect values were higher, indicating that hydride transfer is more rate-limiting in the mutant enzyme. This is supported by a decrease in the rate constant for flavin reduction and in the solvent kinetic isotope effect values. Results from molecular dynamics simulations show reduced flexibility in active site residues and, in particular, the nicotinamide moiety of NADP+ in FMOW47F. This was supported by thermal denaturation experiments. Together, the data suggests that W47 plays a role in maintaining the overall protein flexibility that is required for conformational changes important in hydride transfer.
Subject(s)
Flavins/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Piscirickettsiaceae/enzymology , Tryptophan/metabolism , Amino Acid Sequence , Catalytic Domain , Flavin-Adenine Dinucleotide/metabolism , Mixed Function Oxygenases/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , NADP/metabolism , Piscirickettsiaceae/chemistry , Piscirickettsiaceae/genetics , Piscirickettsiaceae/metabolism , Protein Stability , Sequence Alignment , Tryptophan/chemistry , Tryptophan/geneticsABSTRACT
UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in microorganisms by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The enzyme has gained attention recently as a promising target for the design of new antifungal, antitrypanosomal, and antileishmanial agents. Here we report the first crystal structure of UGM complexed with its redox partner NAD(P)H. Kinetic protein crystallography was used to obtain structures of oxidized Aspergillus fumigatus UGM (AfUGM) complexed with NADPH and NADH, as well as reduced AfUGM after dissociation of NADP(+). NAD(P)H binds with the nicotinamide near the FAD isoalloxazine and the ADP moiety extending toward the mobile 200s active site flap. The nicotinamide riboside binding site overlaps that of the substrate galactopyranose moiety, and thus NADPH and substrate binding are mutually exclusive. On the other hand, the pockets for the adenine of NADPH and uracil of the substrate are distinct and separated by only 6 Å, which raises the possibility of designing novel inhibitors that bind both sites. All 12 residues that contact NADP(H) are conserved among eukaryotic UGMs. Residues that form the AMP pocket are absent in bacterial UGMs, which suggests that eukaryotic and bacterial UGMs have different NADP(H) binding sites. The structures address the longstanding question of how UGM binds NAD(P)H and provide new opportunities for drug discovery.
Subject(s)
Aspergillus fumigatus/enzymology , Intramolecular Transferases/chemistry , NADP/chemistry , Binding Sites , Crystallography, X-Ray , Enzyme Activation , Intramolecular Transferases/genetics , Intramolecular Transferases/metabolism , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , NADP/metabolism , Oxidation-ReductionABSTRACT
The histone deacetylase inhibitor (HDACi), panobinostat (Pano), is approved by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) for treatment of relapsed/refractory multiple myeloma (MM). Despite regulatory approvals, Pano is used on a limited basis in MM due largely to an unfavorable toxicity profile. The MM treatment landscape continues to evolve, and for Pano to maintain a place in that paradigm it will be necessary to identify treatment regimens that optimize its effectiveness, particularly those that permit dose reductions to eliminate unwanted toxicity. Here, we propose such a regimen by combining Pano with LTI6426, a first-in-class orally bioavailable protein disulfide isomerase (PDI) inhibitor. We show that LTI6426 dramatically enhances the anti-MM activity of Pano in vitro and in vivo using a proteasome inhibitor resistant mouse model of MM and a low dose of Pano that exhibited no signs of toxicity. We go on to characterize a transcriptional program that is induced by the LTI6426/Pano combination, demonstrating a convergence of the two drugs on endoplasmic reticulum (ER) stress pathway effectors ATF3 (Activating Transcription Factor 3), DDIT3/CHOP (DNA Damage Inducible Transcript 3, a.k.a. C/EBP Homologous Protein), and DNAJB1 (DnaJ homolog subfamily B member 1, a.k.a. HSP40). We conclude that LTI6426 may safely enhance low-dose Pano regimens and that ATF3, DDIT3/CHOP, and DNAJB1 are candidate pharmacodynamic biomarkers of response to this novel treatment regimen.
Subject(s)
Multiple Myeloma , Animals , HSP40 Heat-Shock Proteins , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Humans , Hydroxamic Acids/pharmacology , Hydroxamic Acids/therapeutic use , Mice , Molecular Targeted Therapy , Multiple Myeloma/genetics , Panobinostat/pharmacology , Protein Disulfide-Isomerases/therapeutic useABSTRACT
Multiple myeloma (MM) and mantle cell lymphoma (MCL) are blood cancers that respond to proteasome inhibitors. Three FDA-approved drugs that block the proteasome are currently on the market, bortezomib, carfilzomib, and ixazomib. While these proteasome inhibitors have demonstrated clinical efficacy against refractory and relapsed MM and MCL, they are also associated with considerable adverse effects including peripheral neuropathy and cardiotoxicity, and tumor cells often acquire drug resistance. TIR-199 belongs to the syrbactin class, which constitutes a novel family of irreversible proteasome inhibitors. In this study, we compare TIR-199 head-to-head with three FDA-approved proteasome inhibitors. We demonstrate that TIR-199 selectively inhibits to varying degrees the sub-catalytic proteasomal activities (C-L/ß1, T-L/ß2, and CT-L/ß5) in three actively dividing MM cell lines, with Ki50 (CT-L/ß5) values of 14.61⯱â¯2.68â¯nM (ARD), 54.59⯱â¯10.4â¯nM (U266), and 26.8⯱â¯5.2â¯nM (MM.1R). In most instances, this range was comparable with the activity of ixazomib. However, TIR-199 was more effective than bortezomib, carfilzomib, and ixazomib in killing bortezomib-resistant MM and MCL cell lines, as judged by a low resistance index (RI) between 1.7 and 2.2, which implies that TIR-199 indiscriminately inhibits both bortezomib-sensitive and bortezomib-resistant MM and MCL cells at similar concentrations. Importantly, TIR-199 reduced the tumor burden in a MM mouse model (pâ¯<â¯0.01) confirming its potency in vivo. Given the fact that there is still no cure for MM, the further development of TIR-199 or similar molecules that belong to the syrbactin class of proteasome inhibitors is warranted.
Subject(s)
Amides/pharmacology , Azoles/pharmacology , Bortezomib/therapeutic use , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , Multiple Myeloma/pathology , Proteasome Inhibitors/pharmacology , Tumor Burden/drug effects , Amides/administration & dosage , Amides/chemistry , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Azoles/administration & dosage , Azoles/chemistry , Bortezomib/administration & dosage , Cell Line, Tumor , Drug Synergism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Multiple Myeloma/drug therapy , Peptides, Cyclic/chemistry , Proteasome Inhibitors/administration & dosage , Proteasome Inhibitors/chemistry , Xenograft Model Antitumor AssaysABSTRACT
Protein disulfide isomerase (PDI, PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies, although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4, CHOP, and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors.
Subject(s)
Bortezomib/pharmacology , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Proteasome Inhibitors/pharmacology , Protein Disulfide-Isomerases/antagonists & inhibitors , Bortezomib/chemical synthesis , Bortezomib/chemistry , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Molecular Structure , Procollagen-Proline Dioxygenase/metabolism , Proteasome Inhibitors/chemical synthesis , Proteasome Inhibitors/chemistry , Protein Disulfide-Isomerases/metabolism , Structure-Activity RelationshipABSTRACT
Histone deacetylase inhibitors (HDACi) are largely ineffective in the treatment of solid tumors. In this study, we describe a new class of protein disulfide isomerase (PDI) inhibitors that significantly and synergistically enhance the antitumor activity of HDACi in glioblastoma and pancreatic cancer preclinical models. RNA-sequencing screening coupled with gene silencing studies identified ATF3 as the driver of this antitumor synergy. ATF3 was highly induced by combined PDI and HDACi treatment as a result of increased acetylation of key histone lysine residues (acetylated histone 3 lysine 27 and histone 3 lysine 18) flanking the ATF3 promoter region. These chromatin marks were associated with increased RNA polymerase II recruitment to the ATF3 promoter, a synergistic upregulation of ATF3, and a subsequent apoptotic response in cancer cells. The HSP40/HSP70 family genes DNAJB1 and HSPA6 were found to be critical ATF3-dependent genes that elicited the antitumor response after PDI and HDAC inhibition. In summary, this study presents a synergistic antitumor combination of PDI and HDAC inhibitors and demonstrates a mechanistic and tumor suppressive role of ATF3. Combined treatment with PDI and HDACi offers a dual therapeutic strategy in solid tumors and the opportunity to achieve previously unrealized activity of HDACi in oncology. SIGNIFICANCE: This study uses a first-in-class PDI inhibitor entering clinical development to enhance the effects of epigenetic drugs in some of the deadliest forms of cancer.
Subject(s)
Activating Transcription Factor 3/metabolism , Brain Neoplasms/drug therapy , Glioblastoma/drug therapy , Histone Deacetylase Inhibitors/pharmacology , Pancreatic Neoplasms/drug therapy , Protein Disulfide-Isomerases/antagonists & inhibitors , Acetylation , Activating Transcription Factor 3/genetics , Animals , Cell Line, Tumor , Drug Synergism , Gene Silencing , HSP40 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/genetics , Histones/metabolism , Humans , Mice , Mice, Nude , Mice, SCID , Promoter Regions, Genetic , RNA Polymerase II/metabolism , Up-RegulationABSTRACT
Siderophore A (SidA) from Aspergillus fumigatus is a flavin-containing monooxygenase that hydroxylates ornithine (Orn) at the amino group of the side chain. Lysine (Lys) also binds to the active site of SidA; however, hydroxylation is not efficient and H2 O2 is the main product. The effect of pH on steady-state kinetic parameters was measured and the results were consistent with Orn binding with the side chain amino group in the neutral form. From the pH dependence on flavin oxidation in the absence of Orn, a pKa value >9 was determined and assigned to the FAD-N5 atom. In the presence of Orn, the pH dependence displayed a pKa value of 6.7 ±0.1 and of 7.70 ±0.10 in the presence of Lys. Q102 interacts with NADPH and, upon mutation to alanine, leads to destabilization of the C4a-hydroperoxyflavin (FADOOH ). Flavin oxidation with Q102A showed a pKa value of ~8.0. The data are consistent with the pKa of the FAD N5-atom being modulated to a value >9 in the absence of Orn, which aids in the stabilization of FADOOH . Changes in the FAD-N5 environment lead to a decrease in the pKa value, which facilitates elimination of H2 O2 or H2 O. These findings are supported by solvent kinetic isotope effect experiments, which show that proton transfer from the FAD N5-atom is rate limiting in the absence of a substrate, however, is significantly less rate limiting in the presence of Orn and or Lys.
Subject(s)
Aspergillus fumigatus/enzymology , Flavin-Adenine Dinucleotide/chemistry , Fungal Proteins/chemistry , Mixed Function Oxygenases/chemistry , Siderophores/chemistry , Oxidation-ReductionABSTRACT
Multiple Myeloma (MM) is highly sensitive to disruptions in cellular protein homeostasis. Proteasome inhibitors (PIs) are initially effective in the treatment of MM, although cures are not achievable and the emergence of resistance limits the durability of responses. New therapies are needed for refractory patients, and those that combat resistance to standard of care agents would be particularly valuable. Screening of multiple chemical libraries for PI re-sensitizing compounds identified E61 as a potent enhancer of multiple PIs and MM specific activity. Using a tandem approach of click chemistry and peptide mass fingerprinting, we identified multiple protein disulfide isomerase (PDI) family members as the primary molecular targets of E61. PDIs mediate oxidative protein folding, and E61 treatment induced robust ER and oxidative stress responses as well as the accumulation of ubiquitinylated proteins. A chemical optimization program led to a new structural class of indene (exemplified by lead E64FC26), which are highly potent pan-style inhibitors of PDIs. In mice with MM, E64FC26 improved survival and enhanced the activity of bortezomib without any adverse effects. This work demonstrates the potential of E64FC26 as an early drug candidate and the strategy of targeting multiple PDI isoforms for the treatment of refractory MM and beyond.
Subject(s)
Antineoplastic Agents/pharmacology , Indenes/pharmacology , Multiple Myeloma/drug therapy , Proteasome Inhibitors/pharmacology , Protein Disulfide-Isomerases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Animals , Combinatorial Chemistry Techniques , High-Throughput Screening Assays , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/enzymology , Multiple Myeloma/pathology , Tumor Cells, CulturedABSTRACT
Curative responses in the treatment of multiple myeloma (MM) are limited by the emergence of therapeutic resistance. To address this problem, we set out to identify druggable mechanisms that convey resistance to proteasome inhibitors (PIs; e.g., bortezomib), which are cornerstone agents in the treatment of MM. In isogenic pairs of PI sensitive and resistant cells, we observed stark differences in cellular bioenergetics between the divergent phenotypes. PI resistant cells exhibited increased mitochondrial respiration driven by glutamine as the principle fuel source. To target glutamine-induced respiration in PI resistant cells, we utilized the glutaminase-1 inhibitor, CB-839. CB-839 inhibited mitochondrial respiration and was more cytotoxic in PI resistant cells as a single agent. Furthermore, we found that CB-839 synergistically enhanced the activity of multiple PIs with the most dramatic synergy being observed with carfilzomib (Crflz), which was confirmed in a panel of genetically diverse PI sensitive and resistant MM cells. Mechanistically, CB-839 enhanced Crflz-induced ER stress and apoptosis, characterized by a robust induction of ATF4 and CHOP and the activation of caspases. Our findings suggest that the acquisition of PI resistance involves adaptations in cellular bioenergetics, supporting the combination of CB-839 with Crflz for the treatment of refractory MM.