S-Adenosylhomocysteine Analogue of a Fairy Chemical, Imidazole-4-carboxamide, as its Metabolite in Rice and Yeast and Synthetic Investigations of Related Compounds.

During the course of our investigations of fairy chemicals (FCs), we found S-ICAr-H (8a), as a metabolite of imidazole-4-carboxamide (ICA) in rice and yeast (Saccharomyces cerevisiae). In order to determine its absolute configuration, an efficient synthetic method of 8a was developed. This synthetic strategy was applicable to the preparation of analogues of 8a that might be biologically very important, such as S-ICAr-M (9), S-AICAr-H (10), and S-AICAr-M (11).

Novel parameter describing restriction endonucleases: Secondary-Cognate-Specificity and chemical stimulation of TsoI leading to substrate specificity change.

Over 470 prototype Type II restriction endonucleases (REases) are currently known. Most recognise specific DNA sequences 4-8 bp long, with very few exceptions cleaving DNA more frequently. TsoI is a thermostable Type IIC enzyme that recognises the DNA sequence TARCCA (R = A or G) and cleaves downstream at N11/N9. The enzyme exhibits extensive top-strand nicking of the supercoiled single-site DNA substrate. The second DNA strand of such substrate is specifically cleaved only in the presence of duplex oligonucleotides containing a cognate site. We have previously shown that some Type IIC/IIG/IIS enzymes from the Thermus-family exhibit 'affinity star' activity, which can be induced by the S-adenosyl-L-methionine (SAM) cofactor analogue-sinefungin (SIN). Here, we define a novel type of inherently built-in 'star' activity, exemplified by TsoI. The TsoI 'star' activity cannot be described under the definition of the classic 'star' activity as it is independent of the reaction conditions used and cannot be separated from the cognate specificity. Therefore, we define this phenomenon as Secondary-Cognate-Specificity (SCS). The TsoI SCS comprises several degenerated variants of the cognate site. Although the efficiency of TsoI SCS cleavage is lower in comparison to the cognate TsoI recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The TsoI/SAC REase may serve as a novel tool for DNA manipulation.

Identifying Unknown Enzyme-Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry.

The enzyme-substrate complex is inherently transient, rendering its detection difficult. In our framework designed for bisubstrate systems-isotope-labeled, activity-based identification and tracking (IsoLAIT)-the common substrate, such as S-adenosyl-l-methionine (AdoMet) for methyltransferases, is replaced by an analogue (e.g., S-adenosyl-l-vinthionine) that, as a probe, creates a tightly bound [enzyme⋅substrate⋅probe] complex upon catalysis by thiopurine-S-methyltransferase (TPMT, EC 2.1.1.67). This persistent complex is then identified by native mass spectrometry from the cellular milieu without separation. Furthermore, the probe's isotope pattern flags even unknown substrates and enzymes. IsoLAIT is broadly applicable for other enzyme systems, particularly those catalyzing group transfer and with multiple substrates, such as glycosyltransferases and kinases.

SAM/SAH Analogs as Versatile Tools for SAM-Dependent Methyltransferases.

S-Adenosyl-L-methionine (SAM) is a sulfonium molecule with a structural hybrid of methionine and adenosine. As the second largest cofactor in the human body, its major function is to serve as methyl donor for SAM-dependent methyltransferases (MTases). The resultant transmethylation of biomolecules constitutes a significant biochemical mechanism in epigenetic regulation, cellular signaling, and metabolite degradation. Recently, numerous SAM analogs have been developed as synthetic cofactors to transfer the activated groups on MTase substrates for downstream ligation and identification. Meanwhile, new compounds built upon or derived from the SAM scaffold have been designed and tested as selective inhibitors for important MTase targets. Here, we summarized the recent development and application of SAM analogs as chemical biology tools for MTases.

Synthesis and antiviral activities of 3-deaza-3-fluoroaristeromycin and its 5' analogues.

The naturally occurring adenine based carbocyclic nucleosides aristeromycin and neplanocin A and their 3-deaza analogues have found a prominent place in the search for diverse antiviral activity agent scaffolds because of their ability to inhibit S-adenosylhomocysteine (AdoHcy) hydrolase. Following the lead of these compounds, their 3-deaza-3-fluoroaristeromycin analogues have been synthesized and their effect on S-adenosylhomocysteine hydrolase and RNA and DNA viruses determined.

Bromo-deaza-SAH: a potent and selective DOT1L inhibitor.

Chemical inhibition of proteins involved in chromatin-mediated signaling is an emerging strategy to control chromatin compaction with the aim to reprogram expression networks to alter disease states. Protein methyltransferases constitute one of the protein families that participate in epigenetic control of gene expression, and represent a novel therapeutic target class. Recruitment of the protein lysine methyltransferase DOT1L at aberrant loci is a frequent mechanism driving acute lymphoid and myeloid leukemias, particularly in infants, and pharmacological inhibition of DOT1L extends survival in a mouse model of mixed lineage leukemia. A better understanding of the structural chemistry of DOT1L inhibition would accelerate the development of improved compounds. Here, we report that the addition of a single halogen atom at a critical position in the cofactor product S-adenosylhomocysteine (SAH, an inhibitor of SAM-dependent methyltransferases) results in an 8-fold increase in potency against DOT1L, and reduced activities against other protein and non-protein methyltransferases. We solved the crystal structure of DOT1L in complex with Bromo-deaza-SAH and rationalized the observed effects. This discovery reveals a simple strategy to engineer selectivity and potency towards DOT1L into the adenosine scaffold of the cofactor shared by all methyltransferases, and can be exploited towards the development of clinical candidates against mixed lineage leukemia.

Structure-guided design of a methyl donor cofactor that controls a viral histone H3 lysine 27 methyltransferase activity.

vSET (a viral SET domain protein) is an attractive polycomb repressive complex 2 (PRC2) surrogate to study the effect of histone H3 lysine 27 (H3K27) methylation on gene transcription, as both catalyze histone H3K27 trimethylation. To control the enzymatic activity of vSET in vivo with an engineered S-adenosyl-l-methionine (SAM) analogue as methyl donor cofactor, we have carried out structure-guided design, synthesis, and characterization of orthogonal vSET methyltransferase mutant/SAM analogue pairs using a "bump-and-hole" strategy.

Binding and inhibition of human spermidine synthase by decarboxylated S-adenosylhomocysteine.

Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well-studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S-adenosylmethionine and a short-chain polyamine (putrescine) to make a medium-chain polyamine (spermidine) and 5'-deoxy-5'-methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S-adenosylhomocysteine (dcSAH). The inhibitor was synthesized, and dose-dependent inhibition of human, Thermatoga maritima, and Plasmodium falciparum spermidine synthases, as well as functionally homologous human spermine synthase, was determined. The human SpdS/dcSAH complex structure was determined by X-ray crystallography at 2.0 Å resolution and showed consistent active site positioning and coordination with previously known structures. Isothermal calorimetry binding assays confirmed inhibitor binding to human SpdS with K(d) of 1.1 ± 0.3 µM in the absence of putrescine and 3.2 ± 0.1 µM in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold.

Synthesis of S-adenosyl-L-homocysteine capture compounds for selective photoinduced isolation of methyltransferases.

Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S-adenosyl-L-homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase-cofactor complexes.

Activation and inhibition of DNA methyltransferases by S-adenosyl-L-homocysteine analogues.

The inhibition of methyltransferases is currently of high interest, particularly in the areas of microbial infection and cell proliferation, as there have been serious attempts to develop novel anti-microbial agents. In the present investigation, a series of 11 S-adenosyl-l-homocysteine analogues have been synthesized and effect of these analogues on DNA methylation catalyzed by DNA methyltransferases was studied. It was found that, while 5'-S-(propionic acid)5'-deoxy-9-(1'-beta-d-ribofuranosyl)1,3-dideazaadenine was an activator of EcoP15I and HhaI DNA methyltransferases, 5'-S-(propionic acid)5'-deoxy-9-(1'-beta-dribofuranosyl)adenine inhibited the methyltransferases in a non-competitive manner. An understanding of the binding of analogues to DNA methyltransferases will greatly assist the design of novel anti-microbial compounds.

Cofactor dependence of reduction potentials for [4Fe-4S]2+/1+ in lysine 2,3-aminomutase.

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of l-lysine and l-beta-lysine by a free radical mechanism. The 5'-deoxyadenosyl radical derived from the reductive cleavage of S-adenosyl-l-methionine (SAM) initiates substrate-radical formation. The [4Fe-4S](1+) cluster in LAM is the one-electron source in the reductive cleavage of SAM, which is directly ligated to the unique iron site in the cluster. We here report the midpoint reduction potentials of the [4Fe-4S](2+/1+) couple in the presence of SAM, S-adenosyl-l-homocysteine (SAH), or 5'-{N-[(3S)-3-aminocarboxypropyl]-N-methylamino}-5'-deoxyadenosine (azaSAM) as measured by spectroelectrochemistry. The reduction potentials are -430 +/- 2 mV in the presence of SAM, -460 +/- 3 mV in the presence of SAH, and -497 +/- 10 mV in the presence of azaSAM. In the absence of SAM or an analogue and the presence of dithiothreitol, dihydrolipoate, or cysteine as ligands to the unique iron, the midpoint potentials are -479 +/- 5, -516 +/- 5, and -484 +/- 3 mV, respectively. LAM is a member of the radical SAM superfamily of enzymes, in which the CxxxCxxC motif donates three thiolate ligands to iron in the [4Fe-4S] cluster and SAM donates the alpha-amino and alpha-carboxylate groups of the methionyl moiety as ligands to the fourth iron. The results show the reduction potentials in the midrange for ferredoxin-like [4Fe-4S] clusters. They show that SAM elevates the reduction potential by 86 mV relative to that of dihydrolipoate as the cluster ligand. This difference accounts for the SAM-dependent reduction of the [4Fe-4S](2+) cluster by dithionite reported earlier. Analogues of SAM have a weakened capacity to raise the potential. We conclude that the midpoint reduction potential of the cluster ligated to SAM is 1.2 V less negative than the half-wave potential for the one-electron reductive cleavage of simple alkylsulfonium ions in aqueous solution. The energetic barrier in the reductive cleavage of SAM may be overcome through the use of binding energy.

Determinants of cofactor binding to DNA methyltransferases: insights from a systematic series of structural variants of S-adenosylhomocysteine.

S-Adenosylmethionine (AdoMet) is a commonly used cofactor, second only to ATP in the variety of reactions in which it participates. It is the methyl donor in the majority of methyl transfer reactions, including methylation of DNA, RNA, proteins and small molecules. Almost all structurally characterised methyltransferases share a conserved AdoMet-dependent methyltransferase fold, in which AdoMet is bound in the same orientation. Although potential interactions between the cofactor and methyltransferases have been inferred from crystal structures, there has not been a systematic study of the contributions of each functional group to binding. To explore the binding interaction we synthesised a series of seven analogues of the methyltransferase inhibitor S-adenosylhomocysteine (AdoHcy), each containing a single modification, and tested them for the ability to inhibit methylation by HhaI and HaeIII DNA methyltransferase. Comparison of the Ki values highlights the structural determinants for cofactor binding, and indicates which nucleoside and amino acid functional groups contribute significantly to AdoMet binding. An understanding of the binding of AdoHyc to methyltransferases will greatly assist the design of AdoMet inhibitors.

Nucleosides XI. Synthesis and antiviral evaluation of 5'-alkylthio-5'-deoxy quinazolinone nucleoside derivatives as S-adenosyl-L-homocysteine analogs.

4-amino-1-(beta-D-ribofuranosyl)quinazolin-2-one (3) was prepared by a direct glycosylation of 4-aminoquinazolin-2-one (7) using the Vorbruggen's silylation method and provided exclusively the beta-anomer. This quinazoline nucleoside and its 2',3'-O-isopropylidene derivative (9) did not undergo the coupling reaction with dialkyl disulfides in the presence of tri-n-butylphosphine unless their 4-amino groups were protected by N,N-dimethylaminomethylidene. This approach provides a viable alternative synthetic route to 5'-alkylthio-5'-deoxy nucleosides.

Synthesis of an alpha-aminophosphonate nucleoside as an inhibitor of S-adenosyl-L-homocysteine hydrolase.

A phosphonic acid analogue of S-adenosyl-L-homocysteine was prepared by a novel method and the epimeric mixture separated. Preliminary studies indicate that each epimer causes time-dependent inactivation of S-adenosyl-L-homocysteine hydrolase, however each presented distinct kinetic characteristics.

Design and synthesis of mimics of S-adenosyl-L-homocysteine as potential inhibitors of erythromycin methyltransferases.

A series of indanotriazine C-ribosides were prepared as SAH mimics, and tested for their ability to inhibit erythromycin resistance methylases Erm AM and Erm C'. A carbocyclic analogue derived from quinic acid was also synthesized and tested.

Acyl homoserine-lactone quorum-sensing signal generation.

Acyl homoserine lactones (acyl-HSLs) are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression. These signals are synthesized by members of the LuxI family of proteins. To understand the mechanism of acyl-HSL synthesis we have purified the Pseudomonas aeruginosa RhlI protein and analyzed the kinetics of acyl-HSL synthesis by this enzyme. Purified RhlI catalyzes the synthesis of acyl-HSLs from acyl-acyl carrier proteins and S-adenosylmethionine. An analysis of the patterns of product inhibition indicated that RhlI catalyzes signal synthesis by a sequential, ordered reaction mechanism in which S-adenosylmethionine binds to RhlI as the initial step in the enzymatic mechanism. Because pathogenic bacteria such as P. aeruginosa use acyl-HSL signals to regulate virulence genes, an understanding of the mechanism of signal synthesis and identification of inhibitors of signal synthesis has implications for development of quorum sensing-targeted antivirulence molecules.

Measurement of plasma S-adenosylmethionine and S-adenosylhomocysteine as their fluorescent isoindoles.

The low levels of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) in plasma can be measured by formation of the fluorescent isoindole derivatives of these compounds. The procedure involves an initial separation of AdoMet and AdoHcy in deproteinized plasma by HPLC on a C-8 column followed by derivatization with naphthalenedialdehyde and cyanide for 10 min at pH 9.0. The fluorescent derivatives of AdoMet and AdoHcy are then chromatographed by HPLC on a C-18 column monitored with a fluorescence monitor. The formation of the isoindole goes to 85% completion and the overall recovery of standards added to plasma is about 65%. Correction for recovery is made by addition of known quantities of AdoMet and AdoHcy to plasma. Normal values (+/- SE) for AdoMet were 102.7 nM +/- 9.9 and for AdoHcy were 22.7 +/- 3.1.

Enzyme-mediated cytosine deamination by the bacterial methyltransferase M.MspI.

Most prokaryotic (cytosine-5)-DNA methyltransferases increase the frequency of deamination at the cytosine targeted for methylation in vitro in the absence of the cofactor S-adenosylmethionine (AdoMet) or the reaction product S-adenosylhomocysteine (AdoHcy). We show here that, under the same in vitro conditions, the prokaryotic methyltransferase, M.MspI (from Moraxella sp.), causes very few cytosine deaminations, suggesting a mechanism in which M.MspI may avoid enzyme-mediated cytosine deamination. Two analogues of AdoMet, sinefungin and 5'-amino-5'-deoxyadenosine, greatly increased the frequency of cytosine deamination mediated by M.MspI presumably by introducing a proton-donating amino group into the catalytic centre, thus facilitating the formation of an unstable enzyme-dihydrocytosine intermediate and hydrolytic deamination. Interestingly, two naturally occurring analogues, adenosine and 5'-methylthio-5'-deoxyadenosine, which do not contain a proton-donating amino group, also weakly increased the deamination frequency by M.MspI, even in the presence of AdoMet or AdoHcy. These analogues may trigger a conformational change in the enzyme without completely inhibiting the access of solvent water to the catalytic centre, thus allowing hydrolytic deamination of the enzyme-dihydrocytosine intermediate. Under normal physiological conditions the enzymes M.HpaII (from Haemophilus parainfluenzae), M. HhaI (from Haemophilus hemolytica) and M.MspI all increased the in vivo deamination frequency at the target cytosines with comparable efficiency.

Methylation inhibitors can increase the rate of cytosine deamination by (cytosine-5)-DNA methyltransferase.

The target cytosines of (cytosine-5)-DNA methyltransferases in prokaryotic and eukaryotic DNA show increased rates of C-->T transition mutations compared to non-target cytosines. These mutations are induced either by the spontaneous deamination of 5-mC-->T generating inefficiently repaired G:T rather than G:U mismatches, or by the enzyme-induced C-->U deamination which occurs under conditions of reduced levels of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy). We tested whether various inhibitors of (cytosine-5)-DNA methyltransferases analogous to AdoMet and AdoHcy would affect the rate of enzyme-induced deamination of the target cytosine by M.HpaII and M.SssI. Interestingly, we found two compounds, sinefungin and 5'-amino-5'-deoxyadenosine, that increased the rate of deamination 10(3)-fold in the presence and 10(4)-fold in the absence of AdoMet and AdoHcy. We have therefore identified the first mutagenic compounds specific for the target sites of (cytosine-5)-DNA methyltransferases. A number of analogs of AdoMet and AdoHcy have been considered as possible antiviral, anticancer, antifungal and antiparasitic agents. Our findings show that chemotherapeutic agents with affinities to the cofactor binding pocket of (cytosine-5)-DNA methyltransferase should be tested for their potential mutagenic effects.

N6-methyladenosine inhibits murine erythroleukemia cell maturation by blocking methylation of RNA and memory via conversion to S-(N6-methyl)-adenosylhomocysteine.

We have shown earlier that N6-methyladenosine (N6mAdo) and other methylated derivatives block commitment of murine erythroleukemia (MEL) cells to terminal erythroid maturation. In this study, we further investigated the mechanism of this blockade. Treatment of MEL cells with N6mAdo inhibited cell growth, prevented accumulation of committed cells, suppressed methylation of total cytoplasmic RNA, and erased the expression of "memory" response, an event that precedes initiation of commitment. Furthermore, N6mAdo increased the level of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) and altered the SAH/SAM ratio that influences methylation of ribonucleic acid (RNA). Moreover, analysis of the intracellular extracts revealed that N6-mAdo is converted into S-(N6-methyl)-adenosylhomocysteine (N6-SAH) in MEL cells, an active intermediate that affects methylation of RNA. Therefore, we conclude that N6-mAdo prevents induction of MEL cell differentiation by affecting methylation of critical RNA transcripts involved in expression of "memory" and initiation of commitment. It is likely that this inhibition occurs via conversion of N6mAdo into N6-SAH.