Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells.

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.

C-terminal binding protein 2 is a novel tumor suppressor targeting the MYC-IRF4 axis in multiple myeloma.

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.

Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs.

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.

Primary saturation of α, ß-unsaturated carbonyl containing fatty acids does not abolish electrophilicity.

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.

TRPS1 drives heterochromatic origin refiring and cancer genome evolution.

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.

Inhibition of hexose oxidase on the dark spots in fresh wet noodle sheets: A feasible prevention of dark spots.

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.

Design, synthesis and evaluation of covalent inhibitors of DprE1 as antitubercular agents.

Decaprenylphosphoryl-ß-d-ribose 2'-oxidoreductase (DprE1) is a promising drug target for the development of novel anti-tubercular agents, and inhibitors of DprE1 are being investigated extensively. Among them, the 1,3-benzothiazinone compounds such as BTZ043, and its closer congener, PBTZ169, are undergoing clinical studies. It has been shown that both BTZ compounds are prodrugs, the nitro group is reduced to nitroso first, to which an adjacent Cys387 in the DprE1 binding pocket is covalently bound and results in suicide enzyme inhibition. We figured that replacement of the nitro with an electrophilic warhead would still achieve covalent interaction with nucleophilic Cys387, while the required reductive activation could be circumvented. To test this hypothesis, a number of covalent inhibitors of DprE1 were designed and prepared. The compounds inhibitory potency against DprE1 and anti-tubercular activity were investigated, their chemical reactivity, formation of covalent adduct between the warhead and the enzyme was demonstrated by mass spectrometry.

Exploring the Potential Inhibition of Candidate Drug Molecules for Clinical Investigation Based on their Docking or Crystallographic Analyses against M. tuberculosis Enzyme Targets.

Tuberculosis (TB) is a devastating disease responsible for millions of humans' deaths worldwide. It is caused by a mycobacterial organism, the tubercle bacillus or Mycobacterium tuberculosis. Although TB can be treated, cured and can be prevented if patients take prescribed medicines, scientists have never come close to wiping it out due to a sharp rise in the incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) mycobacterium strains. Due to long regimen treatment and emergence of MDR and XDR-TB, it is urgent to re-engineer and reposition old drugs for developing new antimycobacterial entities with novel mechanisms of action to achieve effective TB control even against the resistant forms of TB. To combat the dreadful MDR and XDR-TB, potential targets are being extensively searched for the last couple of years for the design and discovery of active potential antitubercular chemotherapeutics. To explore the disease virulence, potential new tubercular target enzymes such as InhA, MmpL3, ATP synthase, DprE1, QcrB and MenA have been taken into consideration in the present study and the structure-based design of the corresponding target inhibitors which are under clinical investigation has been attempted to identify structural features for the discovery of new chemical entities (NCEs) having specificity towards MDR and XDR Mycobacterium tuberculosis (M. tuberculosis).

The epigallocatechin gallate derivative Y6 reduces the cardiotoxicity and enhances the efficacy of daunorubicin against human hepatocellular carcinoma by inhibiting carbonyl reductase 1 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Green tea is the most ancient and popular beverage worldwide and its main constituent epigallocatechin-3-gallate (EGCG) has a potential role in the management of cancer through the modulation of cell signaling pathways. However, EGCG is frangible to oxidation and exhibits low lipid solubility and bioavailability, and we synthesized a derivative of EGCG in an attempt to overcome these limitations. AIM OF THE STUDY: The anthracycline antibiotic daunorubicin (DNR) is a potent anticancer agent. However, its severe cardiotoxic limits its clinical efficacy. Human carbonyl reductase 1 (CBR1) is one of the most effective human reductases for producing hydroxyl metabolites and thus may be involved in increasing the cardiotoxicity and decreasing the antineoplastic effect of anthracycline antibiotics. Accordingly, in this study, we investigated the co-therapeutic effect of Y6, a novel and potent adjuvant obtained by optimization of the structure of EGCG. MATERIAL AND METHODS: The cellular concentrations of DNR and its metabolite DNRol were measured by HPLC to determine the effects of EGCG and Y6 on the inhibition of DNRol formation. The cytotoxic effects of EGCG and Y6 were tested by MTT assay in order to identify non-toxic concentrations of them. To understand their antitumor and cardioprotective mechanisms, hypoxia-inducible factor-1α (HIF-1α) and CBR1 protein expression was measured via Western blotting and immunohistochemical staining while gene expression was analyzed using RT-PCR. Moreover, PI3K/AKT and MEK/ERK signaling pathways were analyzed via Western blotting. HepG2 xenograft model was used to detect the effects of EGCG and Y6 on the antitumor activity and cardiotoxicity of DNR in vivo. Finally, to obtain further insight into the interactions of Y6 and EGCG with HIF-1α and CBR1, we performed a molecular modeling. RESULTS: Y6(10 µg/ml or 55 mg/kg) decreased the expression of HIF-1α and CBR1 at both the mRNA and protein levels during combined drug therapy in vitro as well as in vivo, thereby inhibiting formation of the metabolite DNRol from DNR, with the mechanisms being related to PI3K/AKT and MEK/ERK signaling inhibition. In a human carcinoma xenograft model established with subcutaneous HepG2 cells, Y6(55 mg/kg) enhanced the antitumor effect and reduced the cardiotoxicity of DNR more effectively than EGCG(40 mg/kg). CONCLUSIONS: Y6 has the ability to inhibit CBR1 expression through the coordinate inhibition of PI3K/AKT and MEK/ERK signaling, then synergistically enhances the antitumor effect and reduces the cardiotoxicity of DNR.

Optimization of Hydantoins as Potent Antimycobacterial Decaprenylphosphoryl-ß-d-Ribose Oxidase (DprE1) Inhibitors.

In search of novel drugs against tuberculosis, we previously discovered and profiled a novel hydantoin-based family that demonstrated highly promising in vitro potency against Mycobacterium. tuberculosis. The compounds were found to be noncovalent inhibitors of DprE1, a subunit of decaprenylphosphoryl-ß-d-ribose-2'-epimerase. This protein, localized in the periplasmic space of the mycobacterial cell wall, was shown to be an essential and vulnerable antimycobacterial drug target. Here, we report the further SAR exploration of this chemical family through more than 80 new analogues. Among these, the most active representatives combined submicromolar cellular potency and nanomolar target affinity with balanced physicochemical properties and low human cytotoxicity. Moreover, we demonstrate in vivo activity in an acute Mtb infection model and provide further proof of DprE1 being the target of the hydantoins. Overall, the hydantoin family of DprE1 inhibitors represents a promising noncovalent lead series for the discovery of novel antituberculosis agents.

C-terminal binding proteins 1 and 2 in traumatic brain injury-induced inflammation and their inhibition as an approach for anti-inflammatory treatment.

Traumatic brain injury (TBI) induces an acute inflammatory response in the central nervous system that involves both resident and peripheral immune cells. The ensuing chronic neuroinflammation causes cell death and tissue damage and may contribute to neurodegeneration. The molecular mechanisms involved in the maintenance of this chronic inflammation state remain underexplored. C-terminal binding protein (CtBP) 1 and 2 are transcriptional coregulators that repress diverse cellular processes. Unexpectedly, we find that the CtBPs can transactivate a common set of proinflammatory genes both in lipopolysaccharide-activated microglia, astrocytes and macrophages, and in a mouse model of the mild form of TBI. We also find that the expression of these genes is markedly enhanced by a single mild injury in both brain and peripheral blood leukocytes in a severity- and time-dependent manner. Moreover, we were able to demonstrate that specific inhibitors of the CtBPs effectively suppress the expression of the CtBP target genes and thus improve neurological outcome in mice receiving single and repeated mild TBIs. This discovery suggests new avenues for therapeutic modulation of the inflammatory response to brain injury.

Pentagamavunon-1 (PGV-1) inhibits ROS metabolic enzymes and suppresses tumor cell growth by inducing M phase (prometaphase) arrest and cell senescence.

We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible growth arrest, and causes apoptosis. In this study, we tested Pentagamavunon-1 (PGV-1), a molecule related to curcumin, for its inhibitory activity on tumor cells in vitro and in vivo. PGV-1 exhibited 60 times lower GI50 compared to that of curcumin in K562 cells, and inhibited the proliferation of cell lines derived from leukemia, breast adenocarcinoma, cervical cancer, uterine cancer, and pancreatic cancer. The inhibition of growth by PGV-1 remained after its removal from the medium, which suggests that PGV-1 irreversibly prevents proliferation. PGV-1 specifically induced prometaphase arrest in the M phase of the cell cycle, and efficiently induced cell senescence and cell death by increasing intracellular ROS levels through inhibition of ROS-metabolic enzymes. In a xenograft mouse model, PGV-1 had marked anti-tumor activity with little side effects by oral administration, whereas curcumin rarely inhibited tumor formation by this administration. Therefore, PGV-1 is a potential therapeutic to induce tumor cell apoptosis with few side effects and low risk of relapse.

Active-Site Tryptophan, the Target of Antineoplastic C-Terminal Binding Protein Inhibitors, Mediates Inhibitor Disruption of CtBP Oligomerization and Transcription Coregulatory Activities.

C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g., PUMA, BIK) and metastasis-associated epithelial-mesenchymal transition (e.g., CDH1), and by activating expression of genes that promote migratory and invasive properties of cancer cells (e.g., TIAM1) and genes responsible for enhanced drug resistance (e.g., MDR1). CtBP's transcriptional functions are also critically dependent on oligomerization and nucleation of transcriptional complexes. Recently, we have developed a family of CtBP dehydrogenase inhibitors, based on the parent 2-hydroxyimino-3-phenylpropanoic acid (HIPP), that specifically disrupt cancer cell viability, abrogate CtBP's transcriptional function, and block polyp formation in a mouse model of intestinal polyposis that depends on CtBP's oncogenic functions. Crystallographic analysis revealed that HIPP interacts with CtBP1/2 at a conserved active site tryptophan (W318/324; CtBP1/2) that is unique among eukaryotic D2-dehydrogenases. To better understand the mechanism of action of HIPP-class inhibitors, we investigated the contribution of W324 to CtBP2's biochemical and physiologic activities utilizing mutational analysis. Indeed, W324 was necessary for CtBP2 self-association, as shown by analytical ultracentrifugation and in vivo cross-linking. Additionally, W324 supported CtBP's association with the transcriptional corepressor CoREST, and was critical for CtBP2 induction of cell motility. Notably, the HIPP derivative 4-chloro-HIPP biochemically and biologically phenocopied mutational inactivation of CtBP2 W324. Our data support further optimization of W318/W324-interacting CtBP dehydrogenase inhibitors that are emerging as a novel class of cancer cell-specific therapeutic.

Potent inhibition of human carbonyl reductase 1 (CBR1) by the prenylated chalconoid xanthohumol and its related prenylflavonoids isoxanthohumol and 8-prenylnaringenin.

In terms of drug disposal and metabolism SDR21C1 (carbonyl reductase 1; CBR1) exerts an assorted substrate spectrum among a large variety of clinically relevant substances. Additionally, this short-chain dehydrogenase/reductase is extensively expressed in most tissues of the human body, thus underpinning its role in xenobiotic metabolism. Reduction of the chemotherapeutic daunorubicin (DAUN) to daunorubicinol (DAUNol) is a prominent example of its metabolic properties in terms of chemoresistance and cardiotoxicity. The hop-derived prenylated chalcone xanthohumol (XN) and its physiological metabolites isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) have previously been reported to inhibit other DAUN reducing reductases and dehydrogenases including AKR1B1 and AKR1B10. Also with regard to their effects by means of interacting with cancer-related molecular pathways, XN and related prenylated flavonoids in particular have been in the focus of recent studies. In this study, inhibitory properties of these substances were examined with CBR1-mediated 2,3-hexanedione and DAUN reduction. All substances tested in this study turned out to efficiently inhibit recombinant human CBR1 within a low micromolar to submicromolar range. Among the substances tested, 8-PN turned out to be the most effective inhibitor when using 2,3-hexanedione as a substrate (Ki(app) = 180 ±â€¯20 nM). Inhibition rates of recombinant CBR1-mediated DAUN reduction were somewhat weaker with IC50-values ranging from 11 to 20 µM. XN, IX and 8-PN also efficiently inhibited DAUN reduction by SW480 colon adenocarcinoma cytosol (IC50 = 3.71 ±â€¯0.26 µM with 8-PN as inhibitor). This study identifies prenylated inhibitors, which might potentially interact with endogenous CBR1-driven (de-)toxication systems.

Stem cell-like breast cancer cells with acquired resistance to metformin are sensitive to inhibitors of NADH-dependent CtBP dimerization.

Altered flux through major metabolic pathways is a hallmark of cancer cells and provides opportunities for therapy. Stem cell-like cancer (SCLC) cells can cause metastasis and therapy resistance. They possess metabolic plasticity, theoretically enabling resistance to therapies targeting a specific metabolic state. The C-terminal binding protein (CtBP) transcriptional regulators are potential therapeutic targets in highly glycolytic cancer cells, as they are activated by the glycolytic coenzyme nicotinamide adenine dinucleotide (NADH). However, SCLC cells commonly exist in an oxidative state with low rates of glycolysis. Metformin inhibits complex I of the mitochondrial electron transport chain; it can kill oxidative SCLC cells and has anti-cancer activity in patients. SCLC cells can acquire resistance to metformin through increased glycolysis. Given the potential for long-term metformin therapy, we have studied acquired metformin resistance in cells from the claudin-low subtype of breast cancer. Cells cultured for 8 weeks in sub-IC50 metformin concentration proliferated comparably to untreated cells and exhibited higher rates of glucose uptake. SCLC cells were enriched in metformin-adapted cultures. These SCLC cells acquired sensitivity to multiple methods of inhibition of CtBP function, including a cyclic peptide inhibitor of NADH-induced CtBP dimerization. Single-cell mRNA sequencing identified a reprogramming of epithelial-mesenchymal and stem cell gene expression in the metformin-adapted SCLC cells. These SCLC cells demonstrated an acquired dependency on one of these genes, Tenascin C. Thus, in addition to acquisition of sensitivity to glycolysis-targeting therapeutic strategies, the reprograming of gene expression in the metformin-adapted SCLC cells renders them sensitive to potential therapeutic approaches not directly linked to cell metabolism.

Gossypol inhibits 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase: Its possible use for the treatment of prostate cancer.

Gossypol is a yellow polyphenol isolated from cotton seeds. It has the antitumor activity and it is being tested to treat prostate cancer. However, its underlying mechanisms are still not well understood. The present study investigated the inhibitory effects of gossypol acetate on rat 5α-reductase 1, 3α-hydroxysteroid dehydrogenase, and retinol dehydrogenase 2 for androgen metabolism. Rat 5α-reductase 1, 3α-hydroxysteroid dehydrogenase, and retinol dehydrogenase 2 were expressed in COS-1 cells. Immature Leydig cells that contain these enzymes were isolated from 35-day-old male Sprague Dawley rats. The potency and mode of action of gossypol acetate to inhibit these enzymes in both enzyme-expressed preparations and immature Leydig cells were examined. Molecular docking study of gossypol on the crystal structure of 3α-hydroxysteroid dehydrogenase was performed. Gossypol acetate inhibited 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase with IC50 values of 3.33 ±â€¯0.07 and 0.52 ±â€¯0.06 × 10-6 M in the expressed enzymes as well as 8.512 ±â€¯0.079 and 1.032 ±â€¯0.068 × 10-6 M in intact rat immature Leydig cells, respectively. Gossypol acetate inhibited rat 5α-reductase 1 in a noncompetitive mode and 3α-hydroxysteroid dehydrogenase in a mixed mode when steroid substrates were supplied. Gossypol acetate weakly inhibited retinol dehydrogenase 2 with IC50 value over 1 × 10-4 M. Molecular docking analysis showed that gossypol partially bound to the steroid-binding site of the crystal structure of rat 3α-hydroxysteroid dehydrogenase. Gossypol acetate is a potent inhibitor of rat 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase, possibly inhibiting the formation of androgen in the prostate cancer cells.

Anti-tumor effect of sulfasalazine in neuroblastoma.

Neuroblastoma (NB) is a tumor arising from the sympathetic nervous system during infancy and early childhood. High-risk patients who relapse often fail to respond to further therapy, which results in 5-year survival rate for this patient group below 5%. Therefore, there continues to be an urgent need for innovative treatments. Recently, we found that sulfasalazine (SSZ), an FDA-approved drug for the treatment of rheumatoid arthritis and ulcerative colitis induces anti-proliferative effects in NB tumor cells. SSZ was recently shown to inhibit sepiapterin reductase (SPR), a key enzyme that produces tetrahydrobiopterin (BH4) in the nitric oxide (NO) pathway. Here we tested SSZ against purified SPR in vitro, measured the anti-proliferative effect of SSZ on a panel of MYCN amplified and MYCN non-amplified NB cell lines, and assessed the anti-tumor effect of SSZ in NB tumor-xenografted mice. We found that the expression of both SPR mRNA and SPR protein was significantly higher in cell lines without MYCN amplification. SSZ inhibited SPR enzyme activity in vitro and exhibits anti-proliferative activity in a large number of NB cell lines derived from high-risk tumors. Importantly, oral/intraperitoneal (i.p.) SSZ co-administration resulted in measureable anti-tumor effects in vivo. The FDA-approved drug SSZ, a well-tolerated drug in clinical use, could be repositioned to inhibit tumor growth in NB.

Identification and Profiling of Hydantoins-A Novel Class of Potent Antimycobacterial DprE1 Inhibitors.

Tuberculosis is the leading cause of death worldwide from infectious diseases. With the development of drug-resistant strains of Mycobacterium tuberculosis, there is an acute need for new medicines with novel modes of action. Herein, we report the discovery and profiling of a novel hydantoin-based family of antimycobacterial inhibitors of the decaprenylphospho-ß-d-ribofuranose 2-oxidase (DprE1). In this study, we have prepared a library of more than a 100 compounds and evaluated them for their biological and physicochemical properties. The series is characterized by high enzymatic and whole-cell activity, low cytotoxicity, and a good overall physicochemical profile. In addition, we show that the series acts via reversible inhibition of the DprE1 enzyme. Overall, the novel compound family forms an attractive base for progression to further stages of optimization and may provide a promising drug candidate in the future.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

Fluorescent Benzothiazinone Analogues Efficiently and Selectively Label Dpre1 in Mycobacteria and Actinobacteria.

Benzothiazinones (BTZ) are highly potent bactericidal inhibitors of mycobacteria and the lead compound, BTZ043, and the optimized drug candidate, PBTZ169, have potential for the treatment of tuberculosis. Here, we exploited the tractability of the BTZ scaffold by attaching a range of fluorophores to the 2-substituent of the BTZ ring via short linkers. We show by means of fluorescence imaging that the most advanced derivative, JN108, is capable of efficiently labeling its target, the essential flavoenzyme DprE1, both in cell-free extracts and after purification as well as in growing cells of different actinobacterial species. DprE1 displays a polar localization in Mycobacterium tuberculosis, M. marinum, M. smegmatis, and Nocardia farcinica but not in Corynebacterium glutamicum. Finally, mutation of the cysteine residue in DprE1 in these species, to which BTZ covalently binds, abolishes completely the interaction with JN108, thereby highlighting the specificity of this fluorescent probe.