ABSTRACT
BACKGROUND: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. METHODS: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. RESULTS: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models. CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.
Subject(s)
Alcohol Oxidoreductases , DNA-Binding Proteins , Melanoma , Humans , Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/metabolism , Alcohol Oxidoreductases/genetics , Animals , Mice , Melanoma/drug therapy , Melanoma/pathology , Melanoma/metabolism , Melanoma/genetics , Cell Line, Tumor , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Cell Proliferation/drug effects , Antineoplastic Agents/pharmacology , Epithelial-Mesenchymal Transition/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Tuberculosis (TB) is a global lethal disease caused by Mycobacterium tuberculosis (Mtb). The flavoenzyme decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1) plays a crucial part in the biosynthesis of lipoarabinomannan and arabinogalactan for the cell wall of Mtb and represents a promising target for anti-TB drug development. Therefore, there is an urgent need to discover DprE1 inhibitors with novel scaffolds, improved bioactivity and high drug-likeness. Recent studies have shown that artificial intelligence/computer-aided drug design (AI/CADD) techniques are powerful tools in the discovery of novel DprE1 inhibitors. This review provides an overview of the discovery of DprE1 inhibitors and their underlying mechanism of action and highlights recent advances in the discovery and optimization of DprE1 inhibitors using AI/CADD approaches.
Subject(s)
Antitubercular Agents , Artificial Intelligence , Humans , Antitubercular Agents/pharmacology , Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/metabolism , Mycobacterium tuberculosis/drug effects , Drug Design , Computer-Aided Design , Drug Development/methods , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Tuberculosis/drug therapy , Animals , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Drug Discovery/methodsABSTRACT
ABSTRACT: Multiple myeloma (MM) cells are addicted to MYC and its direct transactivation targets IRF4 for proliferation and survival. MYC and IRF4 are still considered "undruggable," as most small-molecule inhibitors suffer from low potency, suboptimal pharmacokinetic properties, and undesirable off-target effects. Indirect inhibition of MYC/IRF4 emerges as a therapeutic vulnerability in MM. Here, we uncovered an unappreciated tumor-suppressive role of C-terminal binding protein 2 (CTBP2) in MM via strong inhibition of the MYC-IRF4 axis. In contrast to epithelial cancers, CTBP2 is frequently downregulated in MM, in association with shortened survival, hyperproliferative features, and adverse clinical outcomes. Restoration of CTBP2 exhibited potent antitumor effects against MM in vitro and in vivo, with marked repression of the MYC-IRF4 network genes. Mechanistically, CTBP2 impeded the transcription of MYC and IRF4 by histone H3 lysine 27 deacetylation (H3K27ac) and indirectly via activation of the MYC repressor IFIT3. In addition, activation of the interferon gene signature by CTBP2 suggested its concomitant immunomodulatory role in MM. Epigenetic studies have revealed the contribution of polycomb-mediated silencing and DNA methylation to CTBP2 inactivation in MM. Notably, inhibitors of Enhance of zeste homolog 2, histone deacetylase, and DNA methyltransferase, currently under evaluation in clinical trials, were effective in restoring CTBP2 expression in MM. Our findings indicated that the loss of CTBP2 plays an essential role in myelomagenesis and deciphers an additional mechanistic link to MYC-IRF4 dysregulation in MM. We envision that the identification of novel critical regulators will facilitate the development of selective and effective approaches for treating this MYC/IRF4-addicted malignancy.
Subject(s)
Alcohol Oxidoreductases , Co-Repressor Proteins , Interferon Regulatory Factors , Multiple Myeloma , Proto-Oncogene Proteins c-myc , Animals , Humans , Mice , Alcohol Oxidoreductases/metabolism , Alcohol Oxidoreductases/antagonists & inhibitors , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Interferon Regulatory Factors/metabolism , Interferon Regulatory Factors/genetics , Multiple Myeloma/metabolism , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction/drug effects , Tumor Suppressor Proteins/metabolism , Co-Repressor Proteins/antagonists & inhibitors , Co-Repressor Proteins/metabolismABSTRACT
Mycobacterium tuberculosis is one of the global leading causes of death due to a single infectious agent. Pretomanid and delamanid are new antitubercular agents that have progressed through the drug discovery pipeline. These compounds are bicyclic nitroimidazoles that act as pro-drugs, requiring activation by a mycobacterial enzyme; however, the precise mechanisms of action of the active metabolite(s) are unclear. Here, we identify a molecular target of activated pretomanid and delamanid: the DprE2 subunit of decaprenylphosphoribose-2'-epimerase, an enzyme required for the synthesis of cell wall arabinogalactan. We also provide evidence for an NAD-adduct as the active metabolite of pretomanid. Our results highlight DprE2 as a potential antimycobacterial target and provide a foundation for future exploration into the active metabolites and clinical development of pretomanid and delamanid.
Subject(s)
Antitubercular Agents , Mycobacterium tuberculosis , Nitroimidazoles , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Molecular Targeted Therapy , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Alcohol Oxidoreductases/antagonists & inhibitors , Nitroimidazoles/pharmacology , Nitroimidazoles/therapeutic use , Cell Wall/metabolism , Drug Resistance , Prodrugs/chemistry , Prodrugs/metabolism , Spectrophotometry , NAD/metabolism , KineticsABSTRACT
Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-ß-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Antiparasitic Agents/pharmacology , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Ivermectin/analogs & derivatives , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Alcohol Oxidoreductases/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Binding Sites , Dose-Response Relationship, Drug , Drug Discovery , Ivermectin/pharmacology , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Structure-Activity RelationshipABSTRACT
Non-alcoholic steatohepatitis (NASH) is a clinically important spectrum of non-alcoholic fatty liver disease (NAFLD) in humans. NASH is a stage of NAFLD progression wherein liver steatosis accompanies inflammation and pro-fibrotic events. Presently, there are no approved drugs for NASH, which has become a leading cause of liver transplant worldwide. To discover novel drug targets for NASH, we analyzed a human transcriptomic NASH dataset and found Aldo-keto reductase family 1 member B10 (AKR1B10) as a significantly upregulated gene in livers of human NASH patients. Similarly murine Akr1b10 and Aldo-keto reductase family 1 member B8 (Akr1b8) gene, which is a murine ortholog of human AKR1B10, were also found to be upregulated in a mouse model of diet-induced NASH. Furthermore, pharmacological inhibitors of AKR1B10 significantly reduced the pathological features of NASH such as steatosis, inflammation and fibrosis in mouse. In addition, genetic silencing of both mouse Akr1b10 and Akr1b8 significantly reduced the expression of proinflammatory cytokines from hepatocytes. These results, thus, underscore the involvement of murine AKR1B10 and AKR1B8 in the pathogenesis of murine NASH and raise an intriguing possibility of a similar role of AKR1B10 in human NASH.
Subject(s)
Alcohol Oxidoreductases/metabolism , Aldo-Keto Reductases/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/genetics , Aldo-Keto Reductases/antagonists & inhibitors , Aldo-Keto Reductases/genetics , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Cell Line , Cytokines/metabolism , Disease Models, Animal , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Liver/metabolism , Liver/pathology , Liver Cirrhosis/etiology , Liver Cirrhosis/prevention & control , Male , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/complications , Non-alcoholic Fatty Liver Disease/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Sulindac/therapeutic useABSTRACT
As an anti-tuberculosis target, DprE1 contains two flexible loops (Loop I and Loop II) which have never been exploited for developing DprE1 inhibitors. Here Leu317 in Loop II was discovered as a new functional site to combat drug-resistance in Mycobacterium strains. Based on TCA1, LZDT1 was designed to optimize the hydrophobic interaction with Leu317. A subsequent biochemical and cellular assay displayed increased potency of LZDT1 in inhibiting DprE1 and killing drug-sensitive/-resistant Mycobacterium strains. The improved activity of LZDT1 and its analogue LZDT2 against multidrug resistant tuberculosis was particularly highlighted. For LZDT1, its enhanced interaction with Leu317 also impaired the drug-insensitivity of DprE1 caused by Cys387 mutation. A new nonbenzothiazole lead (LZDT10) with reduced Cys387-dependence was further produced by optimizing interactions with Leu317, improvement directions for LZDT10 were discussed as well. Our research underscores the value of potential functional sites in disordered loops, and affords a feasible way to develop these functional sites into opportunities for drug-resistance management.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Mycobacterium tuberculosis/drug effects , Tuberculosis, Multidrug-Resistant/drug therapy , Alcohol Oxidoreductases/metabolism , Antitubercular Agents/chemical synthesis , Antitubercular Agents/chemistry , Bacterial Proteins/metabolism , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Molecular Structure , Mycobacterium tuberculosis/metabolism , Structure-Activity Relationship , Tuberculosis, Multidrug-Resistant/metabolismABSTRACT
Psoriasis is a chronic immune-mediated disease characterized by excessive proliferation of epidermal keratinocytes and increased immune cell infiltration to the skin. Although it is well-known that psoriasis pathogenesis is driven by aberrant production of proinflammatory cytokines, the mechanisms underlying the imbalance between proinflammatory and anti-inflammatory cytokine expression are incompletely understood. In this study, we report that the transcriptional coregulators CtBP1 and 2 can transactivate a common set of proinflammatory genes both in the skin of imiquimod-induced mouse psoriasis model and in human keratinocytes and macrophages stimulated by imiquimod. We find that mice overexpressing CtBP1 in epidermal keratinocytes display severe skin inflammation phenotypes with increased expression of T helper type 1 and T helper type 17 cytokines. We also find that the expression of CtBPs and CtBP-target genes is elevated both in human psoriatic lesions and in the mouse imiquimod psoriasis model. Moreover, we were able to show that topical treatment with a peptidic inhibitor of CtBP effectively suppresses the CtBP-regulated proinflammatory gene expression and thus attenuates psoriatic inflammation in the imiquimod mouse model. Together, our findings suggest to our knowledge previously unreported strategies for therapeutic modulation of the immune response in inflammatory skin diseases.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Anti-Inflammatory Agents/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Psoriasis/drug therapy , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Anti-Inflammatory Agents/therapeutic use , Cell Proliferation/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease Models, Animal , HaCaT Cells , Humans , Imiquimod/immunology , Keratinocytes/drug effects , Keratinocytes/immunology , Keratinocytes/pathology , Mice , Mice, Transgenic , Psoriasis/genetics , Psoriasis/immunology , Psoriasis/pathology , Transcriptional Activation/drug effects , Transcriptional Activation/immunologyABSTRACT
Metabolism of polyunsaturated fatty acids results in the formation of hydroxylated fatty acids that can be further oxidized by dehydrogenases, often resulting in the formation of electrophilic, α,ß-unsaturated ketone containing fatty acids. As electrophiles are associated with redox signaling, we sought to investigate the metabolism of the oxo-fatty acid products in relation to their double bond architecture. Using an untargeted liquid chromatography mass spectrometry approach, we identified mono- and di-saturated products of the arachidonic acid-derived 11-oxoeicosatetraenoic acid (11-oxoETE) and mono-saturated metabolites of 15-oxoETE and docosahexaenoic acid-derived 17-oxodocosahexaenoinc acid (17-oxoDHA) in both human A549 lung carcinoma and umbilical vein endothelial cells. Notably, mono-saturated oxo-fatty acids maintained their electrophilicity as determined by nucleophilic conjugation to glutathione while a second saturation of 11-oxoETE resulted in a loss of electrophilicity. These results would suggest that prostaglandin reductase 1 (PTGR1), known only for its reduction of the α,ß-unsaturated double bond, was not responsible for the saturation of oxo-fatty acids at alternative double bonds. Surprisingly, knockdown of PTGR1 expression by shRNA confirmed its participation in the formation of 15-oxoETE and 17-oxoDHA mono-saturated metabolites. Furthermore, overexpression of PTGR1 in A549 cells increased the rate and total amount of oxo-fatty acid saturation. These findings will further facilitate the study of electrophilic fatty acid metabolism and signaling in the context of inflammatory diseases and cancer where they have been shown to have anti-inflammatory and anti-proliferative signaling properties.
Subject(s)
Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/metabolism , A549 Cells , Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Arachidonic Acids/chemistry , Arachidonic Acids/metabolism , Chromatography, Liquid , Docosahexaenoic Acids/chemistry , Docosahexaenoic Acids/metabolism , Electrochemistry , Fatty Acids, Monounsaturated/chemistry , Fatty Acids, Monounsaturated/metabolism , Gene Knockdown Techniques , Human Umbilical Vein Endothelial Cells , Humans , Oxidation-Reduction , Signal Transduction , Tandem Mass Spectrometry , Up-RegulationABSTRACT
We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc-siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.
Subject(s)
Adenosine/chemistry , Cytidine/chemistry , Glycols/chemistry , Guanosine/chemistry , Oligoribonucleotides/chemistry , RNA, Double-Stranded/chemistry , RNA, Small Interfering/chemistry , Acetylgalactosamine , Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Base Pairing , COS Cells , Chlorocebus aethiops , Dimethylformamide/analogs & derivatives , Dimethylformamide/chemistry , Ethylamines/chemistry , Female , Hepatocytes/cytology , Hepatocytes/metabolism , Hydrogen Bonding , Mice , Mice, Inbred C57BL , Oligoribonucleotides/genetics , Oligoribonucleotides/metabolism , Organophosphorus Compounds/chemistry , Prealbumin/antagonists & inhibitors , Prealbumin/genetics , Prealbumin/metabolism , Primary Cell Culture , RNA Stability , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolismABSTRACT
Retinol dehydrogenase 12 (RDH12) is expressed in photoreceptor inner segments and catalyses the reduction of all-trans retinal (atRAL) to all-trans retinol (atROL), as part of the visual cycle. Mutations in RDH12 are primarily associated with autosomal recessive Leber congenital amaurosis. To further our understanding of the disease mechanisms, HEK-293 cell lines expressing wildtype (WT) and mutant RDH12 were created. The WT cells afforded protection from atRAL-induced toxicity and oxidative stress. Mutant RDH12 cells displayed reduced protein expression and activity, with an inability to protect cells from atRAL toxicity, inducing oxidative and endoplasmic reticulum (ER) stress, with upregulation of sXBP1, CHOP, and ATF4. Pregabalin, a retinal scavenger, attenuated atRAL-induced ER stress in the mutant RDH12 cell lines. A zebrafish rdh12 mutant model (rdh12u533 c.17_23del; p.(Val6AlafsTer5)) was generated through CRISPR-Cas9 gene editing. Mutant fish showed disrupted phagocytosis through transmission electron microscopy, with increased phagosome size at 12 months post-fertilisation. Rhodopsin mislocalisation and reduced expression of atg12 and sod2 indicated early signs of a rod-predominant degeneration. A lack of functional RDH12 results in ER and oxidative stress representing key pathways to be targeted for potential therapeutics.
Subject(s)
Alcohol Oxidoreductases/metabolism , Apoptosis , Endoplasmic Reticulum Stress , Mutation , Oxidative Stress , Retinal Diseases/pathology , Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/genetics , Animals , Autophagy , CRISPR-Cas Systems , HEK293 Cells , Humans , Retinal Diseases/etiology , Retinal Diseases/metabolism , ZebrafishABSTRACT
Inhibition of hydroxy acid oxidase 1 (HAO1) is a strategy to mitigate the accumulation of toxic oxalate that results from reduced activity of alanine-glyoxylate aminotransferase (AGXT) in primary hyperoxaluria 1 (PH1) patients. DNA-Encoded Chemical Library (DECL) screening provided two novel chemical series of potent HAO1 inhibitors, represented by compounds 3-6. Compound 5 was further optimized via various structure-activity relationship (SAR) exploration methods to 29, a compound with improved potency and absorption, distribution, metabolism, and excretion (ADME)/pharmacokinetic (PK) properties. Since carboxylic acid-containing compounds are often poorly permeable and have potential active glucuronide metabolites, we undertook a brief, initial exploration of acid replacements with the aim of identifying non-acid-containing HAO1 inhibitors. Structure-based drug design initiated with Compound 5 led to the identification of a nonacid inhibitor of HAO1, 31, which has weaker potency and increased permeability.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , DNA/chemistry , Small Molecule Libraries/chemistry , Alcohol Oxidoreductases/metabolism , Animals , Binding Sites , Crystallography, X-Ray , DNA/metabolism , Drug Design , Half-Life , Humans , Hyperoxaluria, Primary/metabolism , Hyperoxaluria, Primary/pathology , Indoles/chemistry , Indoles/metabolism , Male , Mice , Molecular Docking Simulation , Small Molecule Libraries/metabolism , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/metabolism , Transaminases/genetics , Transaminases/metabolismABSTRACT
In this study, we report the design and synthesis of a series of novel thiophene-arylamide compounds derived from the noncovalent decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) inhibitor TCA1 through a structure-based scaffold hopping strategy. Systematic optimization of the two side chains flanking the thiophene core led to new lead compounds bearing a thiophene-arylamide scaffold with potent antimycobacterial activity and low cytotoxicity. Compounds 23j, 24f, 25a, and 25b exhibited potent in vitro activity against both drug-susceptible (minimum inhibitory concentration (MIC) = 0.02-0.12 µg/mL) and drug-resistant (MIC = 0.031-0.24 µg/mL) tuberculosis strains while retaining potent DprE1 inhibition (half maximal inhibitory concentration (IC50) = 0.2-0.9 µg/mL) and good intracellular antimycobacterial activity. In addition, these compounds showed good hepatocyte stability and low inhibition of the human ether-à-go-go related gene (hERG) channel. The representative compound 25a with acceptable pharmacokinetic property demonstrated significant bactericidal activity in an acute mouse model of tuberculosis. Moreover, the molecular docking study of template compound 23j provides new insight into the discovery of novel antitubercular agents targeting DprE1.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Amides/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Drug Design , Thiophenes/chemistry , Thiophenes/pharmacology , Alcohol Oxidoreductases/chemistry , Alcohol Oxidoreductases/metabolism , Animals , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacokinetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Drug Resistance, Bacterial/drug effects , Humans , Mice , Microbial Sensitivity Tests , Molecular Docking Simulation , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Protein Conformation , Structure-Activity Relationship , Thiophenes/metabolism , Thiophenes/pharmacokinetics , Tissue DistributionABSTRACT
Exploitation of naturally occurring genetic mutations could empower the discovery of novel aspects of established cancer genes. We report here that TRPS1, a gene linked to the tricho-rhino-phalangeal syndrome (TRPS) and recently identified as a potential breast cancer driver, promotes breast carcinogenesis through regulating replication. Epigenomic decomposition of TRPS1 landscape reveals nearly half of H3K9me3-marked heterochromatic origins are occupied by TRPS1, where it encourages the chromatin loading of APC/C, resulting in uncontrolled origin refiring. TRPS1 binds to the genome through its atypical H3K9me3 reading via GATA and IKAROS domains, while TRPS-related mutations affect its chromatin binding, replication boosting, and tumorigenicity. Concordantly, overexpression of wild-type but not TRPS-associated mutants of TRPS1 is sufficient to drive cancer genome amplifications, which experience an extrachromosomal route and dynamically evolve to confer therapeutic resistance. Together, these results uncover a critical function of TRPS1 in driving heterochromatin origin firing and breast cancer genome evolution.
Subject(s)
Evolution, Molecular , Heterochromatin/metabolism , Repressor Proteins/metabolism , Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , DNA Replication , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Histones/metabolism , Humans , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Mice , Mice, SCID , Mutagenesis, Site-Directed , Protein Binding , RNA Interference , RNA, Small Interfering/metabolism , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/genetics , Transplantation, HeterologousABSTRACT
INTRODUCTION: In recent years, the advent of multidrug-resistant tuberculosis (MDR-TB), the extensively-resistant TB (XDR-TB), and the total drug-resistant-TB (TDR-TB) have led the community to develop new antitubercular molecules. The decaprenylphosphoryl-ß-D-ribose-2'-epimerase-1 (DprE1) is an established target to developed new anti-TB drugs. This enzyme is required to synthesize the cell wall of Mycobacterium tuberculosis (Mtb). AREA COVERED: This patent review focuses on the granted patents and patent applications related to the chemical entities developed as DprE1 inhibitors for TB treatment from the publication year of the BTZ-043 compound patent application (2007) till 30 September 2020. EXPERT OPINION: The DprE1 has many advantages in the development of new antitubercular molecules, for example, its location in the periplasm of the Mtb cell wall and its absence in the human body. This indicates that the DprE1 inhibitors are selective for Mtb, and their toxic and side effects on the human body may be negligible or small. Accordingly, the use of DprE1 inhibitors may be benefic for patients with drug-resistant bacteria that require long-term medication. Four molecules are in clinical trials, which could become the drugs of the future for TB-therapy.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Tuberculosis/drug therapy , Animals , Drug Design , Drug Development , Drug Resistance, Multiple, Bacterial , Humans , Mycobacterium tuberculosis/drug effects , Patents as Topic , Tuberculosis/microbiologyABSTRACT
Joining the global fight against Tuberculosis, the world's most deadly infectious disease, herein we present the design and synthesis of novel isatin-nicotinohydrazide hybrids (5a-m and 9a-c) as promising anti-tubercular and antibacterial agents. The anti-tubercular activity of the target hybrids was evaluated against drug-susceptible M. tuberculosis strain (ATCC 27294) where hybrids 5d, 5g and 5h were found to be as potent as INH with MIC = 0.24 µg/mL, also the activity was evaluated against Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823) where compounds 5g and 5h showed excellent activity (MIC = 3.9 µg/mL). Moreover, the target hybrids were examined against six bronchitis causing-bacteria. Most derivatives exhibited excellent antibacterial activity. K. pneumonia emerged as the most sensitive strain with MIC range: 0.49-7.81 µg/mL. Furthermore, a molecular docking study has proposed DprE1 as a probable enzymatic target for herein reported isatin-nicotinohydrazide hybrids, and explored the binding interactions within the vicinity of DprE1 active site.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins/antagonists & inhibitors , Drug Resistance, Bacterial/drug effects , Hydrazines/chemistry , Isatin/chemistry , Mycobacterium tuberculosis/enzymology , Alcohol Oxidoreductases/chemistry , Alcohol Oxidoreductases/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Bordetella pertussis/chemistry , Bordetella pertussis/enzymology , Bordetella pertussis/isolation & purification , Bronchitis/drug therapy , Bronchitis/microbiology , Drug Design , Drug Resistance, Bacterial/genetics , Haemophilus influenzae/chemistry , Haemophilus influenzae/enzymology , Haemophilus influenzae/isolation & purification , Isoniazid/pharmacology , Klebsiella pneumoniae/chemistry , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Molecular Docking Simulation , Moraxella catarrhalis/chemistry , Moraxella catarrhalis/enzymology , Moraxella catarrhalis/isolation & purification , Mycobacterium tuberculosis/chemistry , Mycobacterium tuberculosis/isolation & purification , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Streptococcus pneumoniae/chemistry , Streptococcus pneumoniae/enzymology , Streptococcus pneumoniae/isolation & purification , Streptomycin/pharmacology , Structure-Activity Relationship , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
Lumasiran (Oxlumo™) is a subcutaneously administered small interfering RNA (siRNA) targeting the mRNA for hydroxyacid oxidase 1 gene (HAO1; encodes glycolate oxidase) and was developed by Alnylam Pharmaceuticals for the treatment of primary hyperoxaluria type 1 (PH1). By silencing the gene encoding glycolate oxidase, lumasiran depletes glycolate oxidase and thereby inhibits the synthesis of oxalate, which is the toxic metabolite that is directly associated with the clinical manifestations of PH1. On 19 November 2020, lumasiran received its first global approval in the EU for the treatment of PH1 in all age groups. On 23 November 2020, lumasiran was approved in the USA for the treatment of adult and paediatric patients with PH1. This article summarizes the milestones in the development of lumasiran leading to this first approval.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Hyperoxaluria, Primary/drug therapy , RNA, Small Interfering/pharmacology , Adult , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Child , Humans , Hyperoxaluria, Primary/metabolism , Injections, Subcutaneous , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/administration & dosageABSTRACT
The spread of antibiotic resistance within the ESKAPE group of human pathogenic bacteria poses severe challenges in the treatment of infections and maintenance of safe hospital environments. This motivates efforts to validate novel target proteins within these species that could be pursued as potential targets for antibiotic development. Genetic data suggest that the enzyme FabG, which is part of the bacterial fatty acid biosynthetic system FAS-II, is essential in several ESKAPE pathogens. FabG catalyzes the NADPH dependent reduction of 3-keto-acyl-ACP during fatty acid elongation, thus enabling lipid supply for production and maintenance of the cell envelope. Here we report on small-molecule screening on the FabG enzymes from A. baumannii and S. typhimurium to identify a set of µM inhibitors, with the most potent representative (1) demonstrating activity against six FabG-orthologues. A co-crystal structure with FabG from A. baumannii (PDB:6T65) confirms inhibitor binding at an allosteric site located in the subunit interface, as previously demonstrated for other sub-µM inhibitors of FabG from P. aeruginosa. We show that inhibitor binding distorts the oligomerization interface in the FabG tetramer and displaces crucial residues involved in the interaction with the co-substrate NADPH. These observations suggest a conserved allosteric site across the FabG family, which can be potentially targeted for interference with fatty acid biosynthesis in clinically relevant ESKAPE pathogens.
Subject(s)
Acinetobacter baumannii/enzymology , Alcohol Oxidoreductases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Pseudomonas aeruginosa/enzymology , Salmonella typhimurium/enzymology , Alcohol Oxidoreductases/metabolism , Binding Sites/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Molecular Structure , Structure-Activity RelationshipABSTRACT
Hexose oxidase was a feasible prevention for the dark spots in the fresh wet noodle sheets (FWNS). The chemical mechanism that hexose oxidase recucing the melanins of dark spots was discussed basis on the UPLC-TOF-MS analysis of the polyphenol oxidase (PPO)-catechol system. In the process of PPO browning, hexose oxidase catalyzed the oxidation of o-benzoquinone derivatives and their oligomers, hindering the formation of melanins. Hexose oxidase was efficient in FWNS with low ash content when water addition was 24%~44% or pH range was 4 ~ 7.5. Hexose oxidase could inhubit dark spots in the presence of 10 metal ions. The recommended addition amount was 40 ~ 60 ppm, by which the dark spots could be compolitely inhibited. Hexose oxidase was also suitable for wholewheat and oat FWNS, ΔL6d of wholewheat and oat FWNS were reduced by 4 and 7.98, respectively.
Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Food , Biocatalysis , Catechol Oxidase/metabolism , Color , Feasibility Studies , Oxidation-ReductionABSTRACT
We report, for the first time, the three-dimensional structure and biochemical properties of a UDP-galactose 4-epimerase-like l-threonine 3-dehydrogenase (GalE-like L-ThrDH) from Phytophthora infestans, a plant disease-causing fungus. We identified GalE-like L-ThrDH using Kyoto Encyclopedia of Genes and Genomes (KEGG) database as a candidate target for the development of a new fungicide. The GalE-like L-ThrDH gene was expressed in Escherichia coli, and its product was purified and characterized. N-Acetylglycine was found to act as a competitive inhibitor of the enzyme (Ki =0.18 mM). The crystal structure of the unique hexameric GalE-like L-ThrDH was determined using the molecular replacement method at a resolution of 2.3 Å, in the presence of NAD+ and citrate, an analogue of the substrate. Based on the molecular docking simulation, N-acetylglycine molecule was modeled into the active site and the binding mode and inhibition mechanism of N-acetylglycine were elucidated.