Characterization of a deazaflavin analog as a potent inhibitor of multidrug resistance-associated protein 1.

Selective inhibition of overexpressed ATP binding cassette (ABC) transporters is an attractive approach to enhancing the efficacy of chemotherapeutics in multidrug resistant cancers. Previously, we reported that the cancer sensitizing effect of deazaflavin analogs, an important chemotype for developing combination treatments with topoisomerase II (TOP2) poisons, is associated with increased intracellular drug accumulation. Here we report the characterization of ZW-1226, a deazaflavin analog, as a potent inhibitor of multidrug resistance-associated protein 1 (MRP1). Specifically, ZW-1226 inhibited MRP1 with a 16-fold higher potency than the most widely used positive control MK-571 in vesicular transport assay and displayed excellent selectivity indices exceeding 100 over other major ABC transporters, including P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), MRP2 and MRP3. Mechanistically, we revealed that its MRP1 inhibitory action requires the participation of GSH. In chemo-sensitization test, ZW-1226 fully reversed the MRP1-mediated drug resistance to TOP2 poisons etoposide (ETP) and doxorubicin (DOX) in H69AR cells and conferred CC50s comparable to those in the sensitive parental NCI-H69 cells. The sensitization was associated with boosted intracellular accumulation of ETP and DOX and elevated endogenous GSH. Moreover, ZW-1226 showed potential to occupy the leukotriene C4 binding site in molecular docking with bovine MRP1, presumably with the help of GSH. Lastly, ZW-1226 exhibited high tissue to plasma partitions in mice but did not alter ETP distribution to normal tissues, suggesting it could be a viable lead with desirable pharmacokinetic properties to warrant further investigation.

Interaction between diterpene icetexanes and old yellow enzymes of Leishmania braziliensis and Trypanosoma cruzi.

Old Yellow Enzymes (OYEs) are flavin-dependent redox enzymes that promote the asymmetric reduction of activated alkenes. Due to the high importance of flavoenzymes in the metabolism of organisms, the interaction between OYEs from the parasites Trypanosoma cruzi and Leishmania braziliensis and three diterpene icetexanes (brussonol and two analogs), were evaluated in the present study, and differences in the binding mechanism and inhibition capacity of these molecules were examined. Although the aforementioned compounds showed poor and negligible activities against T. cruzi and L. braziliensis cells, respectively, the experiments with the purified enzymes indicated that the interaction occurs by divergent mechanisms. Overall, the ligands' inhibitory effect depends on their accessibility to the N5 position of the flavin's isoalloxazine ring. The results also indicated that the OYEs found in both parasites share structural similarities and showed affinities for the diterpene icetexanes in the same range. Nevertheless, the interaction between OYEs and ligands is directed by enthalpy and/or entropy in distinct ways. In conclusion, the binding site of both OYEs exhibits remarkable plasticity, and a large range of different molecules, including that can be substrates and inhibitors, can bind this site. This plasticity should be considered in drug design using OYE as a target.

Flavin containing monooxygenase 2 regulates renal tubular cell fibrosis and paracrine secretion via SMURF2 in AKI­CKD transformation.

In the follow­up of hospitalized patients with acute kidney injury (AKI), it has been observed that 15­30% of these patients progress to develop chronic kidney disease (CKD). Impaired adaptive repair of the kidneys following AKI is a fundamental pathophysiological mechanism underlying renal fibrosis and the progression to CKD. Deficient repair of proximal tubular epithelial cells is a key factor in the progression from AKI to CKD. However, the molecular mechanisms involved in the regulation of fibrotic factor paracrine secretion by injured tubular cells remain incompletely understood. Transcriptome analysis and an ischemia­reperfusion injury (IRI) model were used to identify the contribution of flavin­containing monooxygenase 2 (FMO2) in AKI­CKD. Lentivirus­mediated overexpression of FMO2 was performed in mice. Functional experiments were conducted using TGF­ß­induced tubular cell fibrogenesis and paracrine pro­fibrotic factor secretion. Expression of FMO2 attenuated kidney injury induced by renal IRI, renal fibrosis, and immune cell infiltration into the kidneys. Overexpression of FMO2 not only effectively blocked TGF secretion in tubular cell fibrogenesis but also inhibited aberrant paracrine activation of pro­fibrotic factors present in fibroblasts. FMO2 negatively regulated TGF­ß­mediated SMAD2/3 activation by promoting the expression of SMAD ubiquitination regulatory factor 2 (SMURF2) and its nuclear translocation. During the transition from AKI to CKD, FMO2 modulated tubular cell fibrogenesis and paracrine secretion through SMURF2, thereby affecting the outcome of the disease.

The protein disulfide isomerase inhibitor 3-methyltoxoflavin inhibits Chikungunya virus.

Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever, a (re)emerging arbovirus infection, that causes severe and often persistent arthritis, as well as representing a serious health concern worldwide for which no antivirals are currently available. Despite efforts over the last decade to identify and optimize new inhibitors or to reposition existing drugs, no compound has progressed to clinical trials for CHIKV and current prophylaxis is based on vector control, which has shown limited success in containing the virus. Our efforts to rectify this situation were initiated by screening 36 compounds using a replicon system and ultimately identified the natural product derivative 3-methyltoxoflavin with activity against CHIKV using a cell-based assay (EC50 200 nM, SI = 17 in Huh-7 cells). We have additionally screened 3-methyltoxoflavin against a panel of 17 viruses and showed that it only additionally demonstrated inhibition of the yellow fever virus (EC50 370 nM, SI = 3.2 in Huh-7 cells). We have also showed that 3-methyltoxoflavin has excellent in vitro human and mouse microsomal metabolic stability, good solubility and high Caco-2 permeability and it is not likely to be a P-glycoprotein substrate. In summary, we demonstrate that 3-methyltoxoflavin has activity against CHIKV, good in vitro absorption, distribution, metabolism and excretion (ADME) properties as well as good calculated physicochemical properties and may represent a valuable starting point for future optimization to develop inhibitors for this and other related viruses.

Monascin and ankaflavin-Biosynthesis from Monascus purpureus, production methods, pharmacological properties: A review.

Monascus purpureus copiously yields beneficial secondary metabolites , including Monascus pigments, which are broadly used as food additives, as a nitrite substitute in meat products, and as a colorant in the food industry. Monascus yellow pigments (monascin and ankaflavin) have shown potential antidiabetic, antibacterial, anti-inflammatory, antidepressant, antibiotic, anticancer, and antiobesity activities. Cosmetic and textile industries are other areas where it has established its potential as a dye. This paper reviews the production methods of Monascus yellow pigments, biosynthesis of Monascus pigments from M. purpureus, factors affecting yellow pigment production during fermentation, and the pharmacological properties of monascin and ankaflavin.

Synthesis and Structure-Activity Relationship Studies of Pyrido [1,2-e]Purine-2,4(1H,3H)-Dione Derivatives Targeting Flavin-Dependent Thymidylate Synthase in Mycobacterium tuberculosis.

In 2002, a new class of thymidylate synthase (TS) involved in the de novo synthesis of dTMP named Flavin-Dependent Thymidylate Synthase (FDTS) encoded by the thyX gene was discovered; FDTS is present only in 30% of prokaryote pathogens and not in human pathogens, which makes it an attractive target for the development of new antibacterial agents, especially against multi-resistant pathogens. We report herein the synthesis and structure-activity relationship of a novel series of hitherto unknown pyrido[1,2-e]purine-2,4(1H,3H)-dione analogues. Several synthetics efforts were done to optimize regioselective N1-alkylation through organopalladium cross-coupling. Modelling of potential hits were performed to generate a model of interaction into the active pocket of FDTS to understand and guide further synthetic modification. All those compounds were evaluated on an in-house in vitro NADPH oxidase assays screening as well as against Mycobacterium tuberculosis ThyX. The highest inhibition was obtained for compound 23a with 84.3% at 200 µM without significant cytotoxicity (CC50 > 100 µM) on PBM cells.

Monascin and Ankaflavin of Monascus purpureus Prevent Alcoholic Liver Disease through Regulating AMPK-Mediated Lipid Metabolism and Enhancing Both Anti-Inflammatory and Anti-Oxidative Systems.

Alcohol metabolism causes an excessive accumulation of liver lipids and inflammation, resulting in liver damage. The yellow pigments monascin (MS) and ankaflavin (AK) of Monascus purpureus-fermented rice were proven to regulate ethanol-induced damage in HepG2 cells, but the complete anti-inflammatory and anti-fatty liver mechanisms in the animal model are still unclear. This study explored the roles of MS and AK in improving alcoholic liver injury. MS and AK were simultaneously fed to evaluate their effects and mechanisms in C57BL/6J mice fed the Lieber-DeCarli liquid alcohol diet for 6 weeks. The results indicated that MS and AK significantly reduced the serum aspartate aminotransferase and alanine aminotransferase activity, as well as the total liver cholesterol and triglyceride levels. The histopathological results indicated that MS and AK prevented lipid accumulation in the liver. MS and AK effectively enhanced the activity of antioxidant enzymes and reduced the degree of lipid peroxidation; AK was particularly effective and exhibited a superior preventive effect against alcoholic liver injury and fatty liver. In addition to inhibiting the phosphorylation of the MAPK family, MS and AK directly reduced TNF-α, IL-6, and IL-1ß levels, thereby reducing NF-κB and its downstream iNOS and COX-2 expressions, as well as increasing PPAR-γ, Nrf-2, and HO-1 expressions to prevent liver damage. MS and AK also directly reduced TNF-α, IL-6, and IL-1ß expression, thereby reducing the production of NF-κB and its downstream iNOS and COX-2, and increasing PPAR-γ, Nrf-2, and HO-1 expressions, preventing alcohol damage to the liver.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease.

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

The noncovalent conjugations of human serum albumin (HSA) with MS/AK and the effect on anti-oxidant capacity as well as anti-glycation activity of Monascus yellow pigments.

Monascin (MS) and ankaflavin (AK), as typical yellow lipid-soluble pigments identified from Monascus-fermented products, have been confirmed to possess diverse biological activities such as anti-oxidation, reversing diabetes, and anti-atherosclerosis, and have received increasing attention in recent years. Certainly Monascus-fermented product with a high content of MS/AK is also a concern. The current work explored interactions between MS/AK and human serum albumin (HSA) as well as their influence on the anti-oxidant properties of MS/AK. Moreover, the anti-glycation potential of Monascus-fermented products rich in MS and AK (denoted as Mps) was assessed. The results showed that the fluorescence emission of HSA was quenched by MS/AK through a static quenching mechanism, and MS-HSA and AK-HSA complexes were mainly formed by van der Waals forces and hydrophobic interactions, but AK showed a higher binding affinity than MS. Although the DPPH radical-scavenging abilities of MS-HSA and AK-HSA complexes declined, Mps significantly reduced the formation of fructosamine, α-dicarbonyl compounds and advanced glycation end products (AGEs) in the in vitro glycation model (HSA-glucose). Notably, approximately 80% of fluorescent-AGEs were suppressed by Mps at a concentration of 0.95 mg mL-1, while aminoguanidine (AG, a reference standard) caused only 65% decrease at the same concentration. Although radical scavenging and metal chelating activities could justify the observed anti-glycation activity of Mps, in-depth research on the structures of other functional compounds present in Mps except MS/AK and reaction mechanisms should be performed. Overall, the present study proved that Mps would be promising sources of food-based anti-glycation agents because of their superior inhibitory effect on AGEs.

Lumiflavin Enhances the Effects of Ionising Radiation on Ovarian Cancer Stem-Like Cells by Inhibiting Autophagy.

BACKGROUND: The development of Cancer Stem-like Cells (CSCs) is one of the main causes of ovarian cancer tolerance to radiotherapy. Autophagy is an adaptive process by which cells damage due to radiation. As a metabolite of riboflavin, lumiflavin can enhance the chemotherapeutic effects of cisplatin on ovarian cancer CSCs. OBJECTIVE: This study aimed to investigate the synergistic effects of lumiflavin and ionising radiation on ovarian cancer CSCs and explore the association of this metabolite with autophagy. METHODS: CSCs of human ovarian cancer cell lines HO8910 were treated with lumiflavin and rapamycin and then subjected to irradiation at a cumulative dose of 8 Gy. Cell proliferation ability, clonal formation ability, apoptosis rate, autophagy changes and autophagy-related protein changes were detected. RESULTS: Lumiflavin and ionising radiation synergistically reduced cell vitality and clone formation and increased the apoptosis of CSCs compared with irradiation alone. In addition, ionising radiation increased autophagy and the expression of associated proteins, whereas lumiflavin reduced those changes in autophagy progression. Moreover, rapamycin, an autophagy inhibitor, was observed to block the synergistic effects of lumiflavin and ionising radiation on CSC apoptosis. CONCLUSION: Lumiflavin can enhance the effects of ionising radiation on ovarian cancer CSCs. The mechanism by which these effects are exerted is related to blocking the autophagy pathway.

Syn-2, 3-diols and anti-inflammatory indole derivatives from Streptomyces sp. CB09001.

Two new natural diols, (2S, 3S, 4S)-4-methyl-1-phenylhexane-2,3-diol (1) and (2S, 3S)-4-methyl-1-phenylpentane-2,3-diol (2), together with five known compounds, xenocyloins B-D (3-5), lumichrome (6) and thymidine (7) were isolated from Streptomyces sp. CB09001. The absolute configurations of 1 and 2 were established by crystallographic structure analysis. The anti-inflammatory effects of 1-7 were also investigated in RAW246.7 murine macrophage cells stimulated by lipopolysaccharide. The indole derivative xenocyloin B (3) significantly inhibited inducible nitric oxide synthase expression in RAW264.7 cells and could be a potential anti-inflammatory drug lead.

Disruption of hepatic one-carbon metabolism impairs mitochondrial function and enhances macrophage activity in methionine-choline-deficient mice.

One-carbon metabolism is a collection of metabolic cycles that supports methylation and provides one-carbon bound folates for the de novo synthesis of purine and thymidine nucleotides. The methylation of phosphatidylethanolamine to form choline has been extensively studied in the context of fatty liver disease. However, the role of one-carbon metabolism in supporting nucleotide synthesis during liver damage has not been addressed. The objective of this study is to determine how the disruption of one-carbon metabolism influences nucleotide metabolism in the liver after dietary methionine and choline restriction. Mice (n=8) were fed a methionine-choline-deficient or control diet for 3 weeks. We treated mice with the compound alloxazine (0.5 mg/kg), a known adenosine receptor antagonist, every second day during the final week of feeding to probe the function of adenosine signaling during liver damage. We found that concentrations of several hepatic nucleotides were significantly lower in methionine- and choline-deficient mice vs. controls (adenine: 13.9±0.7 vs. 10.1±0.6, guanine: 1.8±0.1 vs. 1.4±0.1, thymidine: 0.0122±0.0027 vs. 0.0059±0.0027 nmol/mg dry tissue). Treatment of alloxazine caused a specific decrease in thymidine nucleotides, decrease in mitochondrial content in the liver and exacerbation of steatohepatitis as shown by the increased hepatic lipid content and altered macrophage morphology. This study demonstrates a role for one-carbon metabolism in supporting de novo nucleotide synthesis and mitochondrial function during liver damage.

Characterization of ankaflavin from Penicillium aculeatum and its cytotoxic properties.

The pigment was extracted from Penicillium aculeatum, purified and characterized as Ankaflavin by spectroscopic analysis. The stability of the pigment was determined under various conditions and was found to possess high stability. The cytotoxicity property of the purified pigment was determined by MTT assay in MCF-7, HCT116 and PC-3 and the studies were compared with its activity in CHOK1 cells. In MCF-7 and in CHOK 1 cells, the pigment exhibited very less toxicity. However, significant cytotoxicity was observed in HCT116 and PC-3 cells with IC50 of 162 µg mL-1 and 85 µg mL-1 for HCT116 and PC-3 cells respectively. In vitro toxicity was tested by haemolysis assay and MTT assay in HEK 293 cells. The pigment showed least cytotoxicity (<5%) at 160 and 320 µg mL-1 concentrations HEK 293 cells and negligible (<5%) toxicity on human erythrocytes at 160 and 320 µg mL-1, the highest concentrations tested.

Novel deazaflavin tyrosyl-DNA phosphodiesterase 2 (TDP2) inhibitors.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a DNA repair enzyme that removes 5'-phosphotyrosyl blockages resulting from topoisomerase II (TOP2)-DNA cleavage complexes trapped by TOP2 inhibitors. TDP2 is a logical target for the development of therapeutics to complement existing treatments based on inhibition of TOP2. There is, however, no TDP2 inhibitor in clinical development at present. Of the reported TDP2 inhibitors, the deazaflavins are the most promising chemical class centered around the lead compound SV-5-153. Recently we reported new subtypes derived within the deazaflavin family with improved membrane permeability properties. In this work we characterize two representative analogues from two new deazaflavin subtypes based on their biochemical TDP2 inhibitory potency and drug-likeness. We demonstrate that the ZW-1288 derivative represents a promising direction for the development of deazaflavins as therapeutic agents. ZW-1288 exhibits potent inhibitory activity at low nanomolar concentrations against recombinant and cellular human TDP2 with profile similar to that of the parent analog SV-5-153 based on high resistance against murine TDP2 and human TDP2 mutated at residue L313H. While expressing weak cytotoxicity on its own, ZW-1288 potentiates the clinical TOP2 inhibitors etoposide (ETP) and mitoxantrone in human prostate DU145 and CCRF-CEM leukemia and chicken lymphoma DT40 cells while not impacting the activity of the topoisomerase I (TOP1) inhibitor camptothecin or the PARP inhibitor olaparib. ZW-1288 increases the uptake of ETP to a lesser extent than SV-5-153 and remained active in TDP2 knockout cells indicating that the deazaflavin TDP2 inhibitors have additional cellular effects that will have to be taken into account for their further development as TDP2 inhibitors.

Inhibitory Effects of Streptomyces sp. MBTH32 Metabolites on Sortase A and Sortase A-Mediated Cell Clumping of Staphylococcus aureus to Fibrinogen.

Sortase A (SrtA), a type of transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall, is important in the virulence of gram-positive bacteria. Three compounds were isolated from marine-derived Streptomyces sp. MBTH32 using various chromatography techniques. The structures of these compounds were determined based on spectroscopic data and comparisons with previously reported data. Among the metabolites tested, lumichrome showed strong inhibitory activity against Staphylococcus aureus SrtA without affecting cell viability. The results of cell clumping activity assessment suggest the potential for using this compound to treat S. aureus infection by inhibiting SrtA activity.

Lumiflavin increases the sensitivity of ovarian cancer stem-like cells to cisplatin by interfering with riboflavin.

Here, we used lumiflavin, an inhibitor of riboflavin, as a new potential therapeutic chemosensitizer to ovarian cancer stem-like cells (CSCs). This study demonstrates that the enrichment of riboflavin in CSCs is an important cause of its resistance to chemotherapy. Lumiflavin can effectively reduce the riboflavin enrichment in CSCs and sensitize the effect of cisplatin Diamminedichloroplatinum (DDP) on CSCs. In this study, CSCs of human ovarian cancer cell lines HO8910 were separated using a magnetic bead (CD133+). We also show the overexpression of the mRNA and protein of riboflavin transporter 2 and the high content of riboflavin in CSCs compared to non-CSCs (NON-CSCs). Moreover, CSCs were less sensitive to DDP than NON-CSCs, whereas, the synergistic effect of lumiflavin and DDP on CSCs was more sensitive than NON-CSCs. Further research showed that lumiflavin had synergistic effects with DDP on CSCs in increasing mitochondrial function damage and apoptosis rates and decreasing clonic function. In addition, we found that the combination of DDP and lumiflavin therapy in vivo has a synergistic cytotoxic effect on an ovarian cancer nude mice model by enhancing the DNA-damage response and increasing the apoptotic protein expression. Notably, the effect of lumiflavin is associated with reduced riboflavin concentration, and riboflavin could reverse the effect of DDP in vitro and in vivo. Accordingly, we conclude that lumiflavin interfered with the riboflavin metabolic pathways, resulting in a significant increase in tumour sensitivity to DDP therapy. Our study suggests that lumiflavin may be a novel treatment alternative for ovarian cancer and its recurrence.

Deazaflavin Inhibitors of TDP2 with Cellular Activity Can Affect Etoposide Influx and/or Efflux.

Tyrosyl DNA phosphodiesterase 2 (TDP2) facilitates the repair of topoisomerase II (TOP2)-linked DNA double-strand breaks and, as a consequence, is required for cellular resistance to TOP2 "poisons". Recently, a deazaflavin series of compounds were identified as potent inhibitors of TDP2, in vitro. Here, however, we show that while some deazaflavins can induce cellular sensitivity to the TOP2 poison etoposide, they do so independently of TDP2 status. Consistent with this, both the cellular level of etoposide-induced TOP2 cleavage complexes and the intracellular concentration of etoposide was increased by incubation with deazaflavin, suggesting an impact of these compounds on etoposide uptake/efflux. In addition, deazaflavin failed to increase the level of TOP2 cleavage complexes or sensitivity induced by m-AMSA, which is a different class of TOP2 poison to which TDP2-defective cells are also sensitive. In conclusion, while deazaflavins are potent inhibitors of TDP2 in vitro, their limited cell permeability and likely interference with etoposide influx/efflux limits their utility in cells.

Lumichrome Inhibits Human Lung Cancer Cell Growth and Induces Apoptosis via a p53-Dependent Mechanism.

Lumichrome, a major derivative of riboflavin, may exhibit pharmacological activity against cancer cells. Riboflavin is a vitamin found in food, however, certain evidence has suggested its possible potentiating effects on cancer progression. Here, we have shown for the first time that unlike riboflavin, lumichrome can suppress lung cancer cell growth and reduce survival in both normal and anchorage-independent conditions. In addition, lumichrome induced apoptosis in lung cancer cells via a p53-dependent mitochondrial mechanism with substantial selectivity, shown by its lesser toxicity to the normal primary dermal papilla cells. The potency of lumichrome in killing lung cancer cells was found to be comparable to that of cisplatin, a standard chemotherapeutic drug for lung cancer treatment. With regard to the mechanism, lumichrome significantly upregulated p53 and decreased its downstream target BCL-2. Such a shift of BCL-2 family protein balance further activated caspase-9 and -3 and finally executed apoptosis. Furthermore, lumichrome potentially suppressed cancer stem cells (CSCs) in lung cancer by dramatically suppressing CSC markers together with the CSC-maintaining cell signaling namely protein kinase B (AKT) and ß-catenin. To conclude, the present study has unraveled a novel role and mechanism of lumichrome against lung cancer that may benefit the development of the compound for management of the disease.

Novel Deazaflavin Analogues Potently Inhibited Tyrosyl DNA Phosphodiesterase 2 (TDP2) and Strongly Sensitized Cancer Cells toward Treatment with Topoisomerase II (TOP2) Poison Etoposide.

Topoisomerase II (TOP2) poisons as anticancer drugs work by trapping TOP2 cleavage complexes (TOP2cc) to generate DNA damage. Repair of such damage by tyrosyl DNA phosphodiesterase 2 (TDP2) could render cancer cells resistant to TOP2 poisons. Inhibiting TDP2, thus, represents an attractive mechanism-based chemosensitization approach. Currently known TDP2 inhibitors lack cellular potency and/or permeability. We report herein two novel subtypes of the deazaflavin TDP2 inhibitor core. By introducing an additional phenyl ring to the N-10 phenyl ring (subtype 11) or to the N-3 site of the deazaflavin scaffold (subtype 12), we have generated novel analogues with considerably improved biochemical potency and/or permeability. Importantly, many analogues of both subtypes, particularly compounds 11a, 11e, 12a, 12b, and 12h, exhibited much stronger cancer cell sensitizing effect than the best previous analogue 4a toward the treatment with etoposide, suggesting that these analogues could serve as effective cellular probes.

Binding of methimazole and NADP(H) to human FMO3: In vitro and in silico studies.

Human flavin-containing monooxygenase isoform 3 (hFMO3) is an important hepatic drug-metabolizing enzyme, catalyzing the monooxygenation of nucleophilic heteroatom-containing xenobiotics. Based on the structure of bacterial FMO, it is proposed that a conserved asparagine is involved in both NADP(H) and substrate binding. In order to explore the role of this amino acid in hFMO3, two mutants were constructed. In the case of N61Q, increasing the steric hindrance above the flavin N5-C4a causes poor NADP(H) binding, destabilizing the catalytic FAD intermediate, whereas the introduction of a negatively charged residue, N61D, interferes mainly with catalytic intermediate formation and its stability. To better understand the substrate-enzyme interaction, in vitro as well as in silico experiments were carried out with methimazole as substrate. Methimazole is a high-affinity substrate of hFMO3 and can competitively suppress the metabolism of other compounds. Our results demonstrate that methimazole Pi-stacks above the isoalloxazine ring of FAD in hFMO3, in a similar way to indole binding to the bacterial FMO. However, for hFMO3 indole is found to act as a non-substrate competitive inhibitor. Finally, understanding the binding mode of methimazole and indole could be advantageous for development of hFMO3 inhibitors, currently investigated as a possible treatment strategy for atherosclerosis.