FAD-binding Pocket Research Articles

LSD1-Based Reversible Inhibitors Virtual Screening and Binding Mechanism Computational Study.

As one of the crucial targets of epigenetics, histone lysine-specific demethylase 1 (LSD1) is significant in the occurrence and development of various tumors. Although several irreversible covalent LSD1 inhibitors have entered clinical trials, the large size and polarity of the FAD-binding pocket and undesired toxicity have focused interest on developing reversible LSD1 inhibitors. In this study, targeting the substrate-binding pocket of LSD1, structure-based and ligand-based virtual screenings were adopted to expand the potential novel structures with molecular docking and pharmacophore model strategies, respectively. Through drug-likeness evaluation, ADMET screening, molecular dynamics simulations, and binding free energy screening, we screened out one and four hit compounds from the databases of 2,029,554 compounds, respectively. Generally, these hit compounds can be divided into two categories, amide (Lig2 and Comp2) and 1,2,4-triazolo-4,3-α-quinazoline (Comp3, Comp4, Comp7). Among them, Comp4 exhibits the strongest binding affinity. Finally, the binding mechanisms of the hit compounds were further calculated in detail by the residue free energy decomposition. It was found that van der Waals interactions contribute most to the binding, and FAD is also helpful in stabilizing the binding and avoiding off-target effects. We believe this work not only provides a solid theoretical foundation for the design of LSD1 substrate reversible inhibitors, but also expands the diversity of parent nucleus, offering new insights for synthetic chemists.

The structural and functional roles of the flavin cofactor FAD in mammalian cryptochromes.

The importance of circadian rhythms in human health and disease calls for a thorough understanding of the underlying molecular machinery, including its key components, the flavin adenine dinucleotide (FAD)-containing flavoproteins cryptochrome 1 and 2. Contrary to their Drosophila counterparts, mammalian cryptochromes are direct suppressors of circadian transcription and act independently of light. Light-independence poses the question regarding the role of the cofactor FAD in mammalian cryptochromes. The weak binding of the cofactor in vitro argues against its relevance and might be a functionless evolutionary remnant. From the other side, the FAD-binding pocket constitutes the part of mammalian cryptochromes directly related to their ubiquitylation by the ubiquitin ligase Fbxl3 and is the target for protein-stabilizing small molecules. Increased supplies of FAD stabilize cryptochromes in cell culture, and the depletion of the FAD precursor riboflavin with simultaneous knock-down of riboflavin kinase affects the expression of circadian genes in mice. This review presents the classical and more recent studies in the field, which help to comprehend the role of FAD for the stability and function of mammalian cryptochromes.

Crystal structure determination of the halogenase CtcP from Streptomyces aureofaciens.

Chlortetracycline (CTC), a derivative of tetracycline (TC), is a broadly used antibiotic that inhibits the synthesis of bacterial proteins by competing with the A-site tRNA on ribosomes. A recent study showed that during the biosynthesis of CTC in Streptomyces aureofaciens, the halogenase CtcP catalyzes the final chlorination reaction and transforms TC into CTC. However, the structure of this fundamental enzyme is still lacking. Here, selenomethionine-derivatized CtcP from S. aureofaciens was overexpressed and purified and its structure was determined at 2.7 Å resolution. The structure of CtcP reveals the conserved monooxygenase domain shared by all flavin-dependent halogenases and a unique C-terminal domain. Although FAD was not observed in the structure, the monooxygenase domain has a conserved FAD-binding pocket and active center. The C-terminal domain displays an α-helical bundle fold, which could contribute to substrate specificity. This work provides a molecular basis for enzyme engineering to improve the industrial production of CTC.

A two-component monooxygenase for continuous denitration and dechlorination of chlorinated 4-nitrophenol in Ensifer sp. strain 22-1

The environmental fates of chlorinated 4-nitrophenols, 2,6-dichloro-4-nitrophenol (2,6-DCNP) and 2-chloro-4-nitrophenol (2C4NP), mediated via microbial catabolism have attracted great attention due to their high toxicity and persistence in the environment. In this study, a strain of Ensifer sp. 22–1 that was capable of degrading both 2,6-DCNP and 2C4NP was isolated from a halogenated aromatic-contaminated soil sample. A gene cluster cnpBADCERM was predicted to be involved in the catabolism of 2,6-DCNP and 2C4NP based on genome sequence analysis. A two-component monooxygenase CnpAB, composed of an oxygenase component (CnpA) and a reductase component (CnpB), was confirmed to catalyze the continuous denitration and dechlorination of 2,6-DCNP and 2C4NP to 6-chlorohydroxyquinol (6-CHQ) and hydroxyquinol (HQ), respectively. Knockout of cnpA resulted in the complete loss of the capacity for strain 22-1 to degrade 2,6-DCNP and 2C4NP. Homologous modeling and docking showed that Val155~Ala159, Phe206~Pro209 and Phe446~Arg461 of CnpA participated in the formation of the FAD-binding pocket, and Arg101, Val155 and Asn447 formed hydrogen bonds with 2,6-DCNP/2C4NP in the substrate-binding pocket. This work characterized a new two-component monooxygenase for 2,6-DCNP and 2C4NP, and enriched our understanding of the degradation mechanism of chlorinated nitrophenols (CNPs) by microorganisms.

Exploration of 5-cyano-6-phenylpyrimidin derivatives containing an 1,2,3-triazole moiety as potent FAD-based LSD1 inhibitors

Histone lysine specific demethylase 1 (LSD1) has become a potential therapeutic target for the treatment of cancer. Discovery and develop novel and potent LSD1 inhibitors is a challenge, although several of them have already entered into clinical trials. Herein, for the first time, we reported the discovery of a series of 5-cyano-6-phenylpyrimidine derivatives as LSD1 inhibitors using flavin adenine dinucleotide (FAD) similarity-based designing strategy, of which compound 14q was finally identified to repress LSD1 with IC50 = 183 nmol/L. Docking analysis suggested that compound 14q fitted well into the FAD-binding pocket. Further mechanism studies showed that compound 14q may inhibit LSD1 activity competitively by occupying the FAD binding sites of LSD1 and inhibit cell migration and invasion by reversing epithelial to mesenchymal transition (EMT). Overall, these findings showed that compound 14q is a suitable candidate for further development of novel FAD similarity-based LSD1 inhibitors.

Anion-specific interaction with human NQO1 inhibits flavin binding

Ion binding to biomacromolecules can modulate their activity and stability in vivo. It is of particular interest to understand the structural and energetic basis of anion binding to functional sites of biomacromolecules. In this work, binding of anions to the FAD binding pocket of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with cancer due to a common polymorphism causing a P187S amino acid substitution, was investigated. It is known that NQO1 stability in vivo is strongly modulated by binding of its flavin cofactor. Herein, binding and protein stability analyses were carried out to show that anion binding to the apo-state of NQO1 P187S inhibits FAD binding with increasing strength following the chaotropic behavior of anions. These inhibitory effects were significant for some anions even at low millimolar concentrations. Additional pH dependent analyses suggested that protonation of histidine residues in the FAD binding pocket was not critical for anion or flavin binding. Overall, this detailed biophysical analysis helps to understanding how anions modulate NQO1 functionality in vitro, thus allowing hypothesize that NQO1 stability in vivo could be modulated by differential anion binding and subsequent inhibition of FAD binding.

Identifying the novel inhibitors of lysine-specific demethylase 1 (LSD1) combining pharmacophore-based and structure-based virtual screening

Lysine-specific demethylase 1 (LSD1) has been reported to connect with a range of solid tumors. Thus, the exploration of LSD1 inhibitors has emerged as an effective strategy for cancer treatment. In this study, we constructed a pharmacophore model based on a series of flavin adenine dinucleotide (FAD)-competing inhibitors bearing triazole − dithiocarbamate scaffold combining docking, structure–activity relationship (SAR) study, and molecular dynamic (MD) simulation. Meanwhile, another pharmacophore model was also constructed manually, relying on several speculated substrate-competing inhibitors and reported putative vital interactions with LSD1. On the basis of the two pharmacophore models, multi-step virtual screenings (VSs) were performed against substrate-binding pocket and FAD-binding pocket, respectively, combining pharmacophore-based and structure-based strategy to exploit novel LSD1 inhibitors. After bioassay evaluation, four compounds among 21 hits with diverse and novel scaffolds exhibited inhibition activity at the range of 3.63–101.43 μM. Furthermore, substructure-based enrichment was performed, and four compounds with a more potent activity were identified. After that, the time-dependent assay proved that the most potent compound with IC50 2.21 μM inhibits LSD1 activity in a manner of time-independent. In addition, the compound exhibited a cellular inhibitory effect against LSD1 in MGC-803 cells and may inhibit cell migration and invasion by reversing EMT in cultured gastric cancer cells. Considering the binding mode and SAR of the series of compounds, we could roughly deem that these compounds containing 3-methylxanthine scaffold act through occupying substrate-binding pocket competitively. This study presented a new starting point to develop novel LSD1 inhibitors.

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.

Enhancement of photophysical and photosensitizing properties of flavin adenine dinucleotide by mutagenesis of the C-terminal extension of a bacterial flavodoxin reductase.

The role of the mobile C-terminal extension present in Rhodobacter capsulatus ferredoxin-NADP(H) reductase (RcFPR) was evaluated using steady-state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C-terminal amino acids beyond Ala266 was combined with the replacement A266Y to emulate the structure of plastidic reductases. Our results show that these modifications of the wild-type RcFPR produce subtle global conformational changes, but strongly reduce the local rigidity of the FAD-binding pocket, exposing the isoalloxazine ring to the solvent. Thus, the ultrafast charge-transfer quenching of (1) FAD* by the conserved Tyr66 residue was absent in the mutant series, producing enhancement of the excited singlet- and triplet-state properties of FAD. This work highlights the delicate balance of the specific interactions between FAD and the surrounding amino acids, and how the functionality and/or photostability of redox flavoproteins can be modified.

Identification of NOX2 regions for normal biosynthesis of cytochrome b558 in phagocytes highlighting essential residues for p22phox binding.

Cytochrome b558, the redox core of the NADPH oxidase (NOX) complex in phagocytes, is composed of NOX2 and p22phox, the synthesis of which is intimately connected but not fully understood. We reproduced 10 rare X-minus chronic granulomatous disease (CGD) mutations of highly conserved residues in NOX1-NOX4, in X0-CGD PLB-985 cells in order to analyse their impact on the synthesis of cytochrome b558. According to the impact of these mutations on the level of expression of NADPH oxidase 2 (NOX2) and its activity, mutants were categorized into group A (W18C, E309K, K315del and I325F), characterized by a linear relationship between NOX2 expression and NOX activity, and group B (H338Y, P339H, G389A and F656-F570del), showing an absence of NOX activity associated with variable levels of NOX2 expression. These last residues belong to the FAD-binding pocket of NOX2, suggesting that this functional domain also plays a role in the structural integrity of NOX2. Finally, we observed an abnormal accumulation of p65 (65-kDa monomer), the NOX2 precursor and p65-p22phox dissociation in the W18C, E309K, I325F and G389A mutants, pointing out a possible role of the first transmembrane domain (Trp18), and the region between the membrane and the dehydrogenase domain of NOX2 (Glu309, Ile325 and Gly389), in the binding with p22phox.

Exploring nicotinamide cofactor promiscuity in NAD(P)H-dependent flavin containing monooxygenases (FMOs) using natural variation within the phosphate binding loop. Structure and activity of FMOs from Cellvibrio sp. BR and Pseudomonas stutzeri NF13

Flavin-containing monooxygenases (FMOs) catalyse asymmetric oxidation reactions that have potential for preparative organic synthesis, but most use the more expensive, phosphorylated nicotinamide cofactor NADPH to reduce FAD to FADH2 prior to formation of the (hydro)peroxy intermediate required for substrate oxygenation. A comparison of the structures of NADPH-dependent FMO from Methylophaga aminisulfidivorans (mFMO) and SMFMO from Stenotrophomonas maltophilia, which is able to use both NADPH and NADH, suggested that the promiscuity of the latter enzyme may be due in part to the substitution of an Arg–Thr couple in the NADPH phosphate recognition site in mFMO, for a Gln–His couple in SMFMO (Jensen et al., 2012, Chembiochem, 13, 872–878). Natural variation within the phosphate binding region, and its influence on nicotinamide cofactor promiscuity, was explored through the cloning, expression, characterisation and structural studies of FMOs from Cellvibrio sp. BR (CFMO) and Pseudomonas stutzeri NF13 (PSFMO), which possess Thr–Ser and Gln–Glu in the putative phosphate recognition positions, respectively. CFMO and PSFMO displayed 5- and 1.5-fold greater activity, respectively, than SMFMO for the reduction of FAD with NADH, and were also cofactor promiscuous, displaying a ratio of activity with NADH:NADPH of 1.7:1 and 1:1.3, respectively. The structures of CFMO and PSFMO revealed the context of the phosphate binding loop in each case, and also clarified the structure of the mobile helix–loop–helix motif that appears to shield the FAD-binding pocket from bulk solvent in this class of FMOs, a feature that was absent from the structure of SMFMO.

Interaction of Circadian Clock Proteins CRY1 and PER2 Is Modulated by Zinc Binding and Disulfide Bond Formation

Period (PER) proteins are essential components of the mammalian circadian clock. They form complexes with cryptochromes (CRY), which negatively regulate CLOCK/BMAL1-dependent transactivation of clock and clock-controlled genes. To define the roles of mammalian CRY/PER complexes in the circadian clock, we have determined the crystal structure of a complex comprising the photolyase homology region of mouse CRY1 (mCRY1) and a C-terminal mouse PER2 (mPER2) fragment. mPER2 winds around the helical mCRY1 domain covering the binding sites of FBXL3 and CLOCK/BMAL1, but not the FAD binding pocket. Our structure revealed an unexpected zinc ion in one interface, which stabilizes mCRY1-mPER2 interactions in vivo. We provide evidence that mCRY1/mPER2 complex formation is modulated by an interplay of zinc binding and mCRY1 disulfide bond formation, which may be influenced by the redox state of the cell. Our studies may allow for the development of circadian and metabolic modulators.

Structural and Functional Insights into the Catalytic Inactivity of the Major Fraction of Buffalo Milk Xanthine Oxidoreductase

BackgroundXanthine oxidoreductase (XOR) existing in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species.Methods and FindingsBuffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (k cat/K m) of 0.11 sec−1 µM−1 of buffalo XO was 8–10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (Δε430 nm) of 46,000 M−1 cm−1 suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ∼30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum) were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD+ entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum.ConclusionA major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way.

SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket.

The flavoprotein cryptochromes (CRYs) act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCFFbxl3 ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitylation and degradation. Yet, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound, and Fbxl3-Skp1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Strikingly, the F-box protein Fbxl3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved C-terminal tail and burying its PER-binding interface. This novel F-box protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitylation.

Pre-Clinical Efficacy of Combined Therapy with LSD1 Antagonist SP-2509 and Pan-Histone Deacetylase Inhibitor Against AML Blast Progenitor Cells

AbstractAbstract 868LSD1 (KDM1A) is an FAD-dependent histone demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K) 4 on histone H3, reducing the permissive H3K4Me3 chromatin mark for gene expression. LSD1 forms a complex with the histone deacetylases (HDAC) 1 and 2 and with the co-repressor CoREST, which stimulates the activity of LSD1 toward nucleosomes. While high LSD1 expression may be an effector of blocked differentiation and confers poor prognosis in AML, LSD1 inhibition induces the expression of myeloid–differentiation associated genes and attenuates growth of AML blast progenitor cells (BPCs). Recently, LSD1 was shown to sustain the in vivo leukemogenic potential of MLL-AF9 expressing leukemia stem cells. Also, co-treatment with the LSD1 inhibitor tranylcypromine (TCP) and all-trans retinoic acid (ATRA) was shown to diminish the engraftment of primary AML BPCs in vivo in NOD-SCID-γIL-2 receptor deficient (NSG) mice. Previous studies have shown that HDAC inhibitors attenuate the levels of LSD1 through Sp1 inhibition. SP-2509 is a potent and selective FAD-binding pocket, non-MAOA and MAOB, inhibitor with an IC50 of 13 nM for LSD1. In the present studies, we determined the chromatin effects and anti-AML efficacy of SP-2509 alone and in combination with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharmaceuticals) in cultured (HL-60, OCI-AML3, MV4-11, MOLM13, THP1 and SKM1 cells) and primary human AML BPCs. Treatment with SP-2509 (250 to 1000 nM) dose-dependently increased the levels of H3K4Me2 & Me3 chromatin mark, and chromatin immunoprecipitation followed by QPCR analyses showed an increase in the H3K4Me3 mark on the gene promoters of KLF4, HMOX1, p57 and p21 in AML BPCs. SP-2509 treatment attenuated the binding of LSD1 with CoREST, accompanied with increased levels of p16, p21 and p27 in AML BPCs. Consistent with this, treatment with SP-2509 inhibited the suspension and colony growth of AML BPCs regardless of whether they expressed MLL fusion oncoproteins. Knockdown of LSD1 by shRNA also inhibited the suspension and colony growth of AML blast progenitor cells. SP-2509 also induced C/EBPα expression and features of morphologic differentiation in the cultured and primary AML BPCs. Following tail vein infusion and establishment of AML by OCI-AML3 or MOLM13 cells in NOD/SCID mice, treatment with SP-2509 (25 mg/kg b.i.w. via IP injection) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). We have previously reported that treatment with PS depleted polycomb repressive complex proteins EZH2, SUZ12 and BMI1 but also reduced LSD1 expression in AML cells. Co-treatment with PS enhanced SP-2509-induced chromatin effects and differentiation of AML cells. Also, PS and SP-2509 synergistically induced apoptosis of the cultured AML OCI-AML3, MOLM13 and MV4-11cells (combination indices, CI <1.0). Additionally, co-treatment with SP-2509 sensitized AML cells to ATRA-induced differentiation. Notably, co-treatment with SP-2509 and PS also induced significantly greater loss of viability of primary AML BPCs but not of normal CD34+ cells. SP-2509 treatment (15 mg/kg b.i.w. IP) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts. Survival was further significantly improved upon co-treatment with SP-2509 and PS (5 mg/kg IP, MWF) (p < 0.001). Mice did not experience any toxicity or weight loss. Taken together, these findings demonstrate promising pre-clinical activity of combined therapy with SP-2509 and PS, warranting further in vivo development and testing of SP-2509 against human AML. Disclosures:Sharma:Salarius Pharmaceuticals: Equity Ownership.

Crystal structure of Bombyx mori nucleopolyhedrovirus ORF75 reveals a pseudo-dimer of thiol oxidase domains with a putative substrate-binding pocket

Bombyx mori nucleopolyhedrovirus (BmNPV) triggers the global shutdown of host silkworm gene expression and protein synthesis approximately 12-18 h post-infection. Genome sequence analysis suggests that BmNPV ORF75 could be a flavin adenine dinucleotide (FAD)-linked thiol oxidase essential for virion assembly and virus propagation. Here, we report the crystal structure of BmNPV ORF75 at 2.1 Å (0.21 nm). The structure of BmNPV ORF75 resembles that of the thiol oxidase domain of human quiescin thiol oxidase (QSOX), displaying a pseudo-dimer of canonical and non-canonical thiol oxidase domains. However, BmNPV ORF75 is further dimerized by its C-terminal canonical thiol oxidase domain. Within the unique quaternary structural arrangement, the FAD-binding pocket and the characteristic CXXC motif from each monomer is 35 Å (3.5 nm) away from that of its corresponding molecule, which suggests that BmNPV ORF75 might adopt a deviant mechanism from that of QSOX to catalyse disulfide bond formation. Our thiol oxidase activity assay on the point mutations of the conserved residues participating in FAD recognition reveals an aromatic cage next to the FAD isoalloxazine moiety for substrate binding. These data suggest that the thiol oxidase activity of BmNPV ORF75 could be critical to catalyse the formation of the disulfide bonds of certain BmNPV proteins essential for BmNPV virion assembly.

Abstract 1043: Combined targeting of chromatin modifying enzymes LSD1, histone deacetylases (HDACs) and EZH2 has superior efficacy against human acute myeloid leukemia cells.

Abstract LSD1 or KDM1A is a FAD-dependent demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K)4 on histone H3, reducing the permissive H3K4Me3 chromatin mark. LSD1 inhibition attenuates growth of pluripotent cancer cells with OCT4 and SOX2 expression. LSD1 complexes with HDAC1/2 and Co-REST, and high LSD1 expression confers poor prognosis in cancers. Previous studies have shown that HDAC inhibitors downregulate LSD1 thru Sp1 inhibition. Inhibition of LSD1 leads to increase in H3K4Me3-a permissive mark for gene expression. HCI2509 is an FAD-binding pocket, non-MAOA and MAOB LSD1 inhibitor. In the present studies, we determined the chromatin-modifying and cytotoxic effects of HCI2509 alone and in combination with the pan-histone deacetylase inhibitor, panobinostat (PS) in cultured (HL-60 and OCI-AML3) and primary human acute myeloid leukemia (AML) cells. Treatment with HCI2509 (100 to 500 nM), dose-dependently increased the levels of H3K4Me2 &amp; Me3, p16 and p27, which was associated with inhibition of cell proliferation as measured by a decrease in Ki-67 expression. Chromatin immunoprecipitation followed by PCR demonstrated that treatment with HCI2509 increased the H3K4Me3 mark on the promoters of KLF4, HMOX1, and CDH1 in AML cells. Treatment with HCI2509 also induced C/EBPα expression and features of morphologic differentiation of cultured and primary AML cells. Treatment with HCI2509 (25 mg/kg B.I.W. via IP injection) significantly improved the survival of NOD/SCID mice bearing OCI-AML3- AML xenografts. We have previously reported that treatment with PS (Novartis Pharma) depleted PRC2 complex proteins EZH2, and SUZ12 but also modestly depleted LSD1 expression in AML cells. Co-treatment with PS enhanced the chromatin modifying effects of HCI2509 on K4 of Histone H3 in AML cells. Co-treatment with HCI2509 and PS synergistically induced apoptosis of the cultured AML cells (combination indices, CI &amp;lt;1.0). This was associated with greater induction of p16, and p27. Co-treatment with HCI2509 and PS also induced significantly greater loss of viability of primary AML cells but not of normal CD34+ cells. We have previously reported that treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, dose-dependently depleted EZH2, and SUZ12 levels in cultured and primary AML cells. Combined treatment with HCI2509 enhanced the apoptosis of AML cells induced by DZNep. Taken together, these findings indicate that combined targeted depletion of the level and activity of LSD1 and HDACs by PS and HCI2509, along with PS-mediated depletion of PRC2 proteins, exerts superior and selective cytotoxic activity against AML cells. These findings also support the in vivo testing of combined epigenetic therapies in the treatment of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1043. doi:1538-7445.AM2012-1043

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening.

Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme inthe absence of the cofactor flavin adenine dinucleotide (FAD) is reported. Theapo structure of GCDH from Burkholderia pseudomallei reveals a loss of secondary structure and increased disorder in the FAD-binding pocket relative to the ternary complex of the highly homologous human GCDH. After conducting a fragment-based screen, four small molecules were identified which bind to GCDH from B. pseudomallei. Complex structures were determined for these fragments, which cause backbone and side-chain perturbations to key active-site residues. Structural insights from this investigation highlight differences from apo GCDH and the utility of small-molecular fragments as chemical probes for capturing alternative conformational states of preformed protein crystals.

A mild phenotype of dihydropyrimidine dehydrogenase deficiency and developmental retardation associated with a missense mutation affecting cofactor binding

Objectives Evaluation of a non-synonymous mutation associated with dihydropyrimidine dehydrogenase (DPD) deficiency. Design and methods DPD enzyme analysis, mutation analysis and molecular dynamics simulations based on the 3D-model of DPD. Results The substitution Lys63Glu is likely to affect the FAD binding pocket within the DPD protein and contributes to a near-complete DPD deficiency in a patient with developmental retardation. Conclusions Like other DPD variants attenuating FAD binding, Lys63Glu should be included in screening for DPD deficiency.

Gain‐of‐function mutations cluster in distinct regions associated with the signalling pathway in the PAS domain of the aerotaxis receptor, Aer

The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD-containing PAS domain. Here, we randomly mutagenized the region encoding residues 14-119 of the PAS domain and found 72 aerotaxis-defective mutants, 24 of which were gain-of-function, signal-on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS-FAD domain in NifL from Azotobacter vinlandii. Signal-on lesions clustered in the FAD binding pocket, the beta-scaffolding and in the N-cap loop. We suggest that the signal-on lesions mimic the 'signal-on' state of the PAS domain, and therefore may be markers for the signal-in and signal-out regions of this domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the beta-scaffolding and the N-cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS beta-scaffold and residue Q248C in the HAMP AS-2 helix.