Bioorthogonal profiling of protein methylation using azido derivative of S-adenosyl-L-methionine

Protein methyltransferases (PMTs) play critical roles in multiple biological processes. Because PMTs often function in vivo through forming multimeric protein complexes, dissecting their activities in the native contexts is challenging but relevant. To address such a need, we envisioned a Bioorthogonal Profiling of Protein Methylation (BPPM) technology, in which a SAM analogue cofactor can be utilized by multiple rationally engineered PMTs to label substrates of the corresponding native PMTs. Here, 4-azidobut-2-enyl derivative of S-adenosyl-L-methionine (Ab-SAM) was reported as a suitable BPPM cofactor. The resultant cofactor-enzyme pairs were implemented to label specifically the substrates of closely related PMTs (e.g., EuHMT1 and EuHMT2) in a complex cellular mixture. The BPPM approach, coupled with mass spectrometric analysis, enables the identification of the nonhistone targets of EuHMT1/2. Comparison of EuHMT1/2's methylomes indicates that the two human PMTs, although similar in terms of their primary sequences, can act on the distinct sets of nonhistone targets. Given the conserved active sites of PMTs, Ab-SAM and its use in BPPM are expected to be transferable to other PMTs for target identification.     

A high throughput scintillation proximity imaging assay for protein methyltransferases

Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z' values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue, was found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use.     

Establishment of an ectopically expressed and functional PRMT1 for proteomic analysis of arginine-methylated proteins

Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), plays crucial roles in a variety of cellular processes. Mammalian PRMT1 exists in a large protein complex in cells, which has been implied in modulating the regulatory and catalytic properties of this enzyme. Establishment of a mammalian comparative approach will help to identify putative substrates of PRMT1 in an authentic condition. Here, we showed that ectopically expressed PRMT1 in mammalian HEK293 cells not only exhibited catalytic properties comparable to the endogenous enzyme but also existed in a functional complex together with endogenous PRMT1 and thus functioned as an endogenous counterpart. In addition, the measured methylation level of cellular proteins using a tritium-labeled methyl donor was accordingly enhanced upon ectopic expression of PRMT1. Subsequent proteomic analysis with such PRMT1-expressing cells allowed us to identify several known and putative methylated proteins. In vitro methylation of selected proteins, eukaryotic translation initiation factor 4A-I and vimentin, by cellular PRMT1 was shown. Together, we have demonstrated the functional equivalence of ectopically expressed PRMT1 in HEK293 cells and its application to systematically identify the substrate proteins in a mammalian cell context.     

Se-Adenosyl-l-selenomethionine Cofactor Analogue as a Reporter of Protein Methylation

Posttranslational methylation by S-adenosyl-l-methionine(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases. However, broad application of these compounds has been limited by their general incompatibility with native enzymes. Here we report a SAM surrogate, ProSeAM (propargylic Se-adenosyl-l-selenomethionine), as a reporter of methyltransferases. ProSeAM can be processed by multiple protein methyltransferases for substrate labeling. In contrast, sulfur-based propargylic SAM undergoes rapid decomposition at physiological pH, likely via an allene intermediate. In conjunction with fluorescent/affinity-based azide probes, copper-catalyzed azide-alkyne cycloaddition chemistry, in-gel fluorescence visualization and proteomic analysis, we further demonstrated ProSeAM's utility to profile substrates of endogenous methyltransferases in diverse cellular contexts. These results thus feature ProSeAM as a convenient probe to study the activities of endogenous protein methyltransferases.     

Structure-based design, synthesis, and in vitro evaluation of bisubstrate inhibitors for catechol O-methyltransferase (COMT)

The enzyme catechol O-methyltransferase (COMT) catalyzes the Me group transfer from the cofactor S-adenosylmethionine (SAM) to the hydroxy group of catechol substrates. Potential bisubstrate inhibitors of COMT were developed by structure-based design and synthesized. The compounds were tested for in vitro inhibitory activity against COMT obtained from rat liver, and the inhibition kinetics were examined with regard to the binding sites of cofactor and substrate. One of the designed molecules was found to be a bisubstrate inhibitor of COMT with an IC50 = 2 microM. It exhibits competitive kinetics for the SAM and noncompetitive kinetics for the catechol binding site. Useful structure-activity relationships were established which provide important guidelines for the design of future generations of bisubstrate inhibitors of COMT.     

Cfr and RlmN contain a single [4Fe-4S] cluster, which directs two distinct reactivities for S-adenosylmethionine: methyl transfer by SN2 displacement and radical generation

The radical SAM (RS) proteins RlmN and Cfr catalyze methylation of carbons 2 and 8, respectively, of adenosine 2503 in 23S rRNA. Both reactions are similar in scope, entailing the synthesis of a methyl group partially derived from S-adenosylmethionine (SAM) onto electrophilic sp(2)-hybridized carbon atoms via the intermediacy of a protein S-methylcysteinyl (mCys) residue. Both proteins contain five conserved Cys residues, each required for turnover. Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe-4S]-cluster cofactor; the remaining two are at opposite ends of the polypeptide. Here we show that each protein contains only the one "radical SAM" [4Fe-4S] cluster and the two remaining conserved cysteines do not coordinate additional iron-containing species. In addition, we show that, while wild-type RlmN bears the C355 mCys residue in its as-isolated state, RlmN that is either engineered to lack the [4Fe-4S] cluster by substitution of the coordinating cysteines or isolated from Escherichia coli cultured under iron-limiting conditions does not bear a C355 mCys residue. Reconstitution of the [4Fe-4S] cluster on wild-type apo RlmN followed by addition of SAM results in rapid production of S-adenosylhomocysteine (SAH) and the mCys residue, while treatment of apo RlmN with SAM affords no observable reaction. These results indicate that in Cfr and RlmN, SAM bound to the unique iron of the [4Fe-4S] cluster displays two reactivities. It serves to methylate C355 of RlmN (C338 of Cfr), or to generate the 5'-deoxyadenosyl 5'-radical, required for substrate-dependent methyl synthase activity.     

Expanding Radical SAM Chemistry by Using Radical Addition Reactions and SAM Analogues

Radical S‐adenosyl‐l ‐methionine (SAM) enzymes utilize a [4Fe‐4S] cluster to bind SAM and reductively cleave its carbon–sulfur bond to produce a highly reactive 5′‐deoxyadenosyl (dAdo) radical. In almost all cases, the dAdo radical abstracts a hydrogen atom from the substrates or from enzymes, thereby initiating a highly diverse array of reactions. Herein, we report a change of the dAdo radical‐based chemistry from hydrogen abstraction to radical addition in the reaction of the radical SAM enzyme NosL. This change was achieved by using a substrate analogue containing an olefin moiety. We also showed that two SAM analogues containing different nucleoside functionalities initiate the radical‐based reactions with high efficiencies. The radical adduct with the olefin produced in the reaction was found to undergo two divergent reactions, and the mechanistic insights into this process were investigated in detail. Our study demonstrates a promising strategy in expanding radical SAM chemistry, providing an effective way to access nucleoside‐containing compounds by using radical SAM‐dependent reactions.     

Hydrogen Transfer in SAM‐Mediated Enzymatic Radical Reactions

S‐Adenosylmethionine (SAM) plays an essential role in a variety of enzyme‐mediated radical reactions. One‐electron reduction of SAM is currently believed to generate the C5′‐desoxyadenosyl radical, which subsequently abstracts a hydrogen atom from the actual substrate in a catalytic or a non‐catalytic fashion. Using a combination of theoretical and experimental bond dissociation energy (BDE) data, the energetics of these radical processes have now been quantified. SAM‐derived radicals are found to react with their respective substrates in an exothermic fashion in enzymes using SAM in a stoichiometric (non‐catalytic) way. In contrast, the catalytic use of SAM appears to be linked to a sequence of moderately endothermic and exothermic reaction steps. The use of SAM in spore photoproduct lyase (SPL) appears to fit neither of these general categories and appears to constitute the first example of a SAM‐initiated radical reaction propagated independently of the cofactor.     

Engineering Orthogonal Methyltransferases to Create Alternative Bioalkylation Pathways

S‐adenosyl‐l ‐methionine (SAM)‐dependent methyltransferases (MTs) catalyse the methylation of a vast array of small metabolites and biomacromolecules. Recently, rare carboxymethylation pathways have been discovered, including carboxymethyltransferase enzymes that utilise a carboxy‐SAM (cxSAM) cofactor generated from SAM by a cxSAM synthase (CmoA). We show how MT enzymes can utilise cxSAM to catalyse carboxymethylation of tetrahydroisoquinoline (THIQ) and catechol substrates. Site‐directed mutagenesis was used to create orthogonal MTs possessing improved catalytic activity and selectivity for cxSAM, with subsequent coupling to CmoA resulting in more efficient and selective carboxymethylation. An enzymatic approach was also developed to generate a previously undescribed co‐factor, carboxy‐S‐adenosyl‐l ‐ethionine (cxSAE), thereby enabling the stereoselective transfer of a chiral 1‐carboxyethyl group to the substrate.     

Mixed self-assembled monolayers of semirigid tetrahydro-4H-thiopyran end-capped oligo(cyclohexylidenes)

Single-component and mixed self-assembled monolayers (SAMs) of one- and three-ring semirigid tetrahydro-4H-thiopyran end-capped oligo(cyclohexylidenes)-that is, thiopyran (1), 4-(4-cyclohexylidene-cyclohexylidene)tetrahydro-4H-thiopyran (2), and 4-(tetrahydro-4H-thiopyran-4-cyclohexylidene-4'-ylidene)tetrahydro-4H-thiopyran (3)--on Au(111) substrates have been prepared and studied by cyclic voltammetry (CV), atomic force microscopy (AFM), and scanning tunneling microscopy (STM). It was found that the shortest adsorbate 1 more readily forms a SAM than 2 or 3. Notwithstanding, the SAMs of 2 or 3 are thermodynamically more stable due to favorable intermolecular attractions. Holes were made with the AFM tip establishing tilt angles of 30-50 degrees with respect to the surface normal for all SAMs. STM imaging showed well-ordered, line-shaped packing patterns with molecular resolution for the SAM of 2. Similar patterned structures were not observed for 1 and 3. Mixed SAMs were prepared by exposing a SAM of 1 to ethanol solutions of either 2 or 3. STM imaging revealed that domains of molecules of 2 or 3 amidst a monolayer of 1 are formed in both cases. Whereas in the mixed SAM of 1 and 2 the domains are irregularly shaped, circular islands of uniform size are found in the mixed SAM of 1 and 3.     

S‐Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C‐Alkylation

A tandem enzymatic strategy to enhance the scope of C‐alkylation of small molecules via the in situ formation of S‐adenosyl methionine (SAM) cofactor analogues is described. A solvent‐exposed channel present in the SAM‐forming enzyme SalL tolerates 5′‐chloro‐5′‐deoxyadenosine (ClDA) analogues modified at the 2‐position of the adenine nucleobase. Coupling SalL‐catalyzed cofactor production with C‐(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C‐(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants that influence C‐alkylation provides the basis to develop a late‐stage enzymatic platform for the preparation of high value small molecules.     

Substrate Profiling of Glutathione S‐transferase with Engineered Enzymes and Matched Glutathione Analogues

The identification of specific substrates of glutathione S‐transferases (GSTs) is important for understanding drug metabolism. A method termed bioorthogonal identification of GST substrates (BIGS) was developed, in which a reduced glutathione (GSH) analogue was developed for recognition by a rationally engineered GST to label the substrates of the corresponding native GST. A K44G‐W40A‐R41A mutant (GST‐KWR) of the mu‐class glutathione S‐transferases GSTM1 was shown to be active with a clickable GSH analogue (GSH‐R1) as the cosubstrate. The GSH‐R1 conjugation products can react with an azido‐based biotin probe for ready enrichment and MS identification. Proof‐of‐principle studies were carried to detect the products of GSH‐R1 conjugation to 1‐chloro‐2,4‐dinitrobenzene (CDNB) and dopamine quinone. The BIGS technology was then used to identify GSTM1 substrates in the Chinese herbal medicine Ganmaocongji.     

Synthesis, characterization and theoretical interpretation of vibrational spectra of poly(2-methylbut-2-enyl thiophene-3-carboxylate)

Electroactive thiophene substituted polyacetylene derivative, namely poly(2-methylbut-2-enyl thiophene-3-carboxylate) (PMT) was synthesized. First, novel acetylene monomer was synthesized by the reaction of 3-thiophenecarboxylic acid with propargyl bromide and then polymerized with a Rh catalyst to give the corresponding polymer. FTIR and FT-Raman spectra were recorded experimentally and used for complete normal mode analysis using Wilson’s GF matrix method and Phonon dispersion of poly(2-methylbut-2-enyl thiophene-3-carboxylate). The simple non-redundant set of internal coordinates and a simplified Urey-Bradley force-field approximation was employed in normal coordinate analysis and to calculate the potential energy distribution (PED) for each fundamental vibration. The PED contribution corresponding to each of the observed frequencies shows the reliability and accuracy of the spectral analysis. Apart from detailed assignments of modes, various characteristic features of dispersion curves have also been explained as arising due to internal symmetry in energy momentum space. Predicted values of intramolecular contribution to the heat capacity of this polymer calculated by density-of-states are also being reported.     

Copper catalyzed asymmetric synthesis of chiral allylic esters

The complex derived from Taniaphos ligand 4 and CuBr*Me2S catalyzes the asymmetric addition of Grignard reagents to 3-bromopropenyl esters 1 to provide allylic esters 2 in high yields and high chemio-, regio-, and enantioselectivities. The work demonstrates that allylic asymmetric alkylation (AAA) can be done on substrates bearing a heteroatom at the gamma-position. The method is a practical route to chiral, nonracemic allylic alcohols. The use of functionalized substrates 1 or Grignard reagents leads to more complex products 2, which can be further manipulated as demonstrated in conversion to (S)-5-ethyl-2(5H)-furanone 6 and (S)-benzoic acid-cyclopent-2-enyl ester 7.     

3-pyrrolines are mechanism-based inactivators of the quinone-dependent amine oxidases but only substrates of the flavin-dependent amine oxidases

We previously reported that 3-pyrroline and 3-phenyl-3-pyrroline effect a time-dependent inactivation of the copper-containing quinone-dependent amine oxidase from bovine plasma (BPAO) (Lee et al. J. Am. Chem. Soc. 1996, 118, 7241-7242). Quinone cofactor model studies suggested a mechanism involving stoichiometric turnover to a stable pyrrolylated cofactor. Full details of the model studies are now reported along with data on the inhibition of BPAO by a family of 3-aryl-3-pyrrolines (aryl = substituted phenyl, 1-naphthyl, 2-naphthyl), with the 4-methoxy-3-nitrophenyl analogue being the most potent. At the same time, the parent 3-phenyl analogue is a pure substrate for the flavin-dependent mitochondrial monoamine oxidase B from bovine liver. Spectroscopic studies (including resonance Raman) on BPAO inactivated by the 4-methoxy-3-nitrophenyl analogue are consistent with covalent derivatization of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The distinction of a class of compounds acting as an inactivator of one amine oxidase family and a pure substrate of another amine oxidase family represents a unique lead to the development of selective inhibitors of the mammalian copper-containing amine oxidases.     

Tripod self-assembled monolayer on Au(111) prepared by reaction of hydroxyl-terminated alkylthiols with SiCl4

Infrared reflection spectroscopy (IRS), single wavelength ellipsometry, and density functional theory were used to elucidate the structure of a molecular tripod self-assembled monolayer (SAM) on polycrystalline gold{111} substrates. The tripod SAM was formed by the reaction of SiCl4 with a densely packed monolayer of 2-mercaptoethanol, 6-mercaptohexanol, and 16-mercaptohexadecanol under inert atmosphere. After reaction with SiCl4, IRS spectra show an intense absorption at approximately 1112 cm(-1) that is attributed to Si-O-C asymmetric stretching vibration of a molecular tripod structure. Harmonic vibrational frequencies computed at the B3LYP/6-311+g** level of theory for the mercaptoethanol tripod SAM closely match the experimental IRS spectra, giving further support for the tripod structure. When rinsed with methanol or water, the Si-Cl-terminated SAM becomes capped with Si-OMe or Si-OH. The silanol-terminated tripod SAM is expected to find use in the preparation of thin zeolite and silica films on gold substrates.     

Synthesis of a 6-aryloxymethyl-5-hydroxy-2,3,4,5-tetrahydro-[1H]-2-benzazepin-4-one: a muscarinic (M3) antagonist

A synthesis of the racemic 6-aryloxymethyl-5-hydroxy-2,3,4,5-[1H]-2-tetrahydrobenzazepin-4-one , for evaluation as a muscarinic (M(3)) antagonist, is described. 2-[2-tert-Butyldimethylsilyloxymethyl-6-(2,6-dimethoxyphenoxymethyl)phenyl]propan-2-ol was prepared from 2,6-dimethyl-1-bromobenzene and taken through to N-[3-(2,6-dimethoxyphenoxymethyl)-2-(propen-2-yl)phenyl]methyl-N-prop-2-enyl 2-nitrobenzene sulfonamide . However, attempts to cyclise this diene by alkene metathesis were unsuccessful, the open-chain alkene being the only product isolated in yields of up to 70%. In a second approach to the 6-aryloxymethyl-5-hydroxytetrahydrobenzazepin-4-one, methyl (Z)-3-[2-(1-tert-butyldimethylsilyloxymethyl)-6-(1,6-dimethoxyphenoxymethyl)phenyl]but-2-enoate was converted into (Z)-3-[2-hydroxymethyl-6-(2,6-dimethoxyphenoxymethyl)phenyl]but-2-enyl 2-nitrobenzene sulfonamide which was cyclised under Mitsunobu conditions to the corresponding 2,3-dihydro-[1H]-2-benzazepine . The structure of this was confirmed by an X-ray crystal structure of its 2-(4-bromophenylsulfonyl) analogue , and functional group modification including hydroxylation, attachment of the requisite side-chain at C(2) and further oxidation gave the target compound which was assayed for muscarinic (M(3)) activity.     

The Nitrogenase FeMo‐Cofactor Precursor Formed by NifB Protein: A Diamagnetic Cluster Containing Eight Iron Atoms

The biological activation of N₂ occurs at the FeMo‐cofactor, a 7Fe–9S–Mo–C–homocitrate cluster. FeMo‐cofactor formation involves assembly of a Fe_6–8–S_X–C core precursor, NifB‐co, which occurs on the NifB protein. Characterization of NifB‐co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent [4Fe–4S] cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB‐co and deliver it to the NifEN protein, upon which FeMo‐cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB‐co‐loaded form provided an opportunity for Mössbauer analysis of NifB‐co. The results indicate that NifB‐co is a diamagnetic (S=0) 8‐Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the ⁵⁷Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB‐co is either a 4Fe²⁺–4Fe³⁺ or 6Fe²⁺–2Fe³⁺ cluster having valence‐delocalized states.