Hydride transfer versus hydrogen radical transfer in thymidylate synthase

The nature of a H-transfer in the thymidylate synthase catalyzed reaction was investigated by comparison of the wild-type enzyme with the W80M mutant. The nature of the H-transfer was not affected, as indicated by intrinsic isotope effects and their temperature dependence. These findings support a single-step hydride transfer instead of a two-step radical transfer.     

Nonclassical 5-substituted tetrahydroquinazolines as potential inhibitors of thymidylate synthase

Classical inhibitors of thymidylate synthase such as N^l0-propargyl-5,8-dideazafolic acid (1), N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid (ZD1694, 2) and N-[2-amino-4-oxo-3,4-dihydro(pyrrolo[2,3-d]pyrintidin-5-yl)ethylbenzoyl]-L-glutamic acid (LY231514, 3) while potent, suffer from a number of potential disadvantages, such as impaired uptake due to an alteration of the active transport system required for their cellular uptake, as well as formation of long acting, non-effluxing polyglutamates via the action of folylpolyglutamate synthetase, which are responsible for toxicity. To overcome some of the disadvantages of classical inhibitors, there has been considerable interest in the synthesis and evaluation of nonclassical thymidylate synthase inhibitors, which could enter cells via passive diffusion. In an attempt to elucidate the role of saturation of the B-ring of non-classical, quinazoline antifolate inhibitors of thymidylate synthase, analogues 7-17 were designed. Analogues 13-17 which contain a methyl group at the 7-position, were synthesized in an attempt to align the methyl group in an orientation which allows interaction with tryptophan-80 in the active site of thymidylate synthase. The synthesis of these analogues was achieved via the reaction of guanidine with the appropriately substituted cyclohexanone-ketoester. These ketoesters were in turn synthesized via a Michael addition of the appropriate thiophenol with 2-carbethoxycyclohexen-1-one or 5-methyl-2-carbethoxycyclo-hexen-1-one to afford a mixture of diastereomers. The most inhibitory compound was the 3,4-dichloro, 7-methyl derivative 17 which inhibited the Escherichia coli and Pneumocystis carinii thymidylate syntheses 50% at 5 × 10⁵ M. Our results confirm the importance of the 7-CH₃ group and electron withdrawing groups on the aromatic side chain for thymidylate synthase inhibition.     

The relationships between oxidase and synthase activities of flavin dependent thymidylate synthase (FDTS)

New findings lead to a revised understanding of the substrates' binding order, the role of the substrate as an activator, and the observed lag phase in the FDTS catalyzed reaction.     

Tyrosine nitration site specificity identified by LC/MS in nitrite-modified collagen type IV

Non-enzymatic nitrite induced collagen cross-linking results in changes reminiscent of age-related damage and parallels the well-known model system, non-enzymatic glycation. We have recently observed that nitrite modification of basement membrane proteins can induce deleterious effects on overlying retinal pigment epithelial cells in studies relevant to age-related macular degeneration. The present work was undertaken in order to confirm 3-nitro-tyrosine (3-NT) as a product of the reaction and to identify the site specificity of nitration in collagen IV, a major component of basement membranes. Human collagen type IV was modified via incubation with 200 mM NaNO(2) (pH=7.38) for one week at 37(o)C. The modified protein was prepared in 2 different ways, including acid hydrolysis and trypsin digestion for site specificity determination. The samples were analyzed by LC/MS using a C(12) RP column. Site specificity was determined from tandem MS/MS data utilizing TurboSEQUEST software and the Swiss-Prot sequence database. 3-NT was detected in protein digests and acid hydrolysates of nitrite modified collagen IV. Positive identification with standard 3-NT was confirmed by identical R(t), lambda(max)=279 nm and 355 nm, and m/z=227. Analyses of tryptic digests identified four sites of tyrosine nitration, alpha1(IV)Y348, alpha1(IV)Y534, alpha2(IV)Y327, and alpha2(IV)Y1081. These sites are located in the triple-helical region of the protein and provide clues regarding potential sites for nitrite modification in collagen type IV.     

Tyrosine nitration of human ERK1 introduces an intra-hydrogen bond by molecular dynamics simulations

Structural Chemistry - ERK1 is an important kinase in Ras–Raf–MEK signaling. We have recently demonstrated by mass spectrometry that Tyr210 of ERK1 can be nitrated, and the nitration...     

Pharmacodynamic assay of thymidylate synthase activity in peripheral blood mononuclear cells

A simple, selective, and sensitive method utilizing tritium (³H) release from ³H-deoxyuridine 5′-monophosphate (dUMP) substrate for accurate and precise determination of the low basal thymidylate synthase activity (TSA) in normal healthy peripheral blood mononuclear cells (PBMCs) was developed and validated. The method is based on the removal of the remaining substrate after the TSA reaction by absorption onto activated carbon and measurement of the supernatant fluid by liquid scintillation counting. The method background was substantially decreased by using lyophilized substrate and optimized binding conditions of remaining substrate onto carbon after TSA reaction. The concentration of cofactor N ₅,N ₁₀ methylene-(6R,S)-tetrahydrofolate was increased to obtain maximal TSA. Method sensitivity was further increased by omission of ethylenediaminetetraacetic acid from the reaction mix and by using longer reaction times. The validation parameters included specificity, linearity, sensitivity, precision, and stability. The lower limit of quantification was 25 μg PBMC cytosolic lysate, which released 1.4 pmol?³H/h. TSA was stable in PBMC pellets stored for 6 months at ?80 °C. The applicability of the method was demonstrated by the successful determination of TSA in PBMC cytosolic lysates from ten healthy volunteers with and without the specific TSA inhibitor FdUMP.

Tyrosine 92 of aristolochene synthase directs cyclisation of farnesyl pyrophosphate

A mutant of Aristolochene Synthase (AS), in which Tyr 92 was replaced by Val, produced the alicyclic beta-(E)-farnesene as the major product, indicating that cyclisation of FPP is controlled by Tyr 92 in AS.     

Trapping of an intermediate in the reaction catalyzed by flavin-dependent thymidylate synthase

Thymidylate is a DNA nucleotide that is essential to all organisms and is synthesized by the enzyme thymidylate synthase (TSase). Several human pathogens rely on an alternative flavin-dependent thymidylate synthase (FDTS), which differs from the human TSase both in structure and molecular mechanism. It has recently been shown that FDTS catalysis does not rely on an enzymatic nucleophile and that the proposed reaction intermediates are not covalently bound to the enzyme during catalysis, an important distinction from the human TSase. Here we report the chemical trapping, isolation, and identification of a derivative of such an intermediate in the FDTS-catalyzed reaction. The chemically modified reaction intermediate is consistent with currently proposed FDTS mechanisms that do not involve an enzymatic nucleophile, and it has never been observed during any other TSase reaction. These findings establish the timing of the methylene transfer during FDTS catalysis. The presented methodology provides an important experimental tool for further studies of FDTS, which may assist efforts directed toward the rational design of inhibitors as leads for future antibiotics.     

Structure-based discovery and in-parallel optimization of novel competitive inhibitors of thymidylate synthase.

BACKGROUND: The substrate sites of enzymes are attractive targets for structure-based inhibitor design. Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites. First, novel inhibitors often bind at nonsubstrate sites. Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging. RESULTS: In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied. The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site. Five high-ranking compounds were selected for testing. Activity and docking studies led to a derivative of one of these, dansyltyrosine (Ki 65 microM). Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dissimilar to the substrate but bind competitively with it. The most active analog had a Ki of 1.3 microM. The tighter binding inhibitors were also the most specific for LcTS versus related enzymes. CONCLUSIONS: TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate. Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can be envisaged.     

Microcalorimetric study of the binding of methotrexate and its metabolites to thymidylate synthase

Direct microcalorimetric measurements allow determination of both the δH and association constant of biological complexes ifK _a value does not exceed 10⁶ M ^?1. For higherK _a values, δH can obviously be determined; this paper describes an original microcalorimetric method that permits determination of such high association constants. This method is based on the analysis of the competitive effect between two ligands having the same binding site in their receptor. As an example, the affinity constant for thymidylate synthase of a novel antifolate, CB 3717. was found to be 1.4 · 10⁷ M ^?1 using methotrexate polyglutamate MTX-G2 (K _a=2.3·10⁵ M ^?1) as competitor.     

Predicting and harnessing protein flexibility in the design of species-specific inhibitors of thymidylate synthase

BACKGROUND: Protein plasticity in response to ligand binding abrogates the notion of a rigid receptor site. Thus, computational docking alone misses important prospective drug design leads. Bacterial-specific inhibitors of an essential enzyme, thymidylate synthase (TS), were developed using a combination of computer-based screening followed by in-parallel synthetic elaboration and enzyme assay [Tondi et al. (1999) Chem. Biol. 6, 319-331]. Specificity was achieved through protein plasticity and despite the very high sequence conservation of the enzyme between species. RESULTS: The most potent of the inhibitors synthesized, N,O-didansyl-L-tyrosine (DDT), binds to Lactobacillus casei TS (LcTS) with 35-fold higher affinity and to Escherichia coli TS (EcTS) with 24-fold higher affinity than to human TS (hTS). To reveal the molecular basis for this specificity, we have determined the crystal structure of EcTS complexed with DDT and 2'-deoxyuridine-5'-monophosphate (dUMP). The 2.0 A structure shows that DDT binds to EcTS in a conformation not predicted by molecular docking studies and substantially differently than other TS inhibitors. Binding of DDT is accompanied by large rearrangements of the protein both near and distal to the enzyme's active site with movement of C alpha carbons up to 6 A relative to other ternary complexes. This protein plasticity results in novel interactions with DDT including the formation of hydrogen bonds and van der Waals interactions to residues conserved in bacterial TS but not hTS and which are hypothesized to account for DDT's specificity. The conformation DDT adopts when bound to EcTS explains the activity of several other LcTS inhibitors synthesized in-parallel with DDT suggesting that DDT binds to the two enzymes in similar orientations. CONCLUSIONS: Dramatic protein rearrangements involving both main and side chain atoms play an important role in the recognition of DDT by EcTS and highlight the importance of incorporating protein plasticity in drug design. The crystal structure of the EcTS/dUMP/DDT complex is a model system to develop more selective TS inhibitors aimed at pathogenic bacterial species. The crystal structure also suggests a general formula for identifying regions of TS and other enzymes that may be treated as flexible to aid in computational methods of drug discovery.     

Temperature dependence of the kinetic isotope effects in thymidylate synthase. A theoretical study

In recent years, the temperature dependence of primary kinetic isotope effects (KIE) has been used as indicator for the physical nature of enzyme-catalyzed H-transfer reactions. An interactive study where experimental data and calculations examine the same chemical transformation is a critical means to interpret more properly temperature dependence of KIEs. Here, the rate-limiting step of the thymidylate synthase-catalyzed reaction has been studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) simulations in the theoretical framework of the ensemble-averaged variational transition-state theory with multidimensional tunneling (EA-VTST/MT) combined with Grote-Hynes theory. The KIEs were calculated across the same temperature range examined experimentally, revealing a temperature independent behavior, in agreement with experimental findings. The calculations show that the H-transfer proceeds with ～91% by tunneling in the case of protium and ～80% when the transferred protium is replaced by tritium. Dynamic recrossing coefficients are almost invariant with temperature and in all cases far from unity, showing significant coupling between protein motions and the reaction coordinate. In particular, the relative movement of a conserved arginine (Arg166 in Escherichia coli ) promotes the departure of a conserved cysteine (Cys146 in E. coli ) from the dUMP by polarizing the thioether bond thus facilitating this bond breaking that takes place concomitantly with the hydride transfer. These promoting vibrations of the enzyme, which represent some of the dimensions of the real reaction coordinate, would limit the search through configurational space to efficiently find those decreasing both barrier height and width, thereby enhancing the probability of H-transfer by either tunneling (through barrier) or classical (over-the-barrier) mechanisms. In other words, the thermal fluctuations that are coupled to the reaction coordinate, together with transition-state geometries and tunneling, are the same in different bath temperatures (within the limited experimental range examined). All these terms contribute to the observed temperature independent KIEs in thymidylate synthase.     

Direct observation of the participation of flavin in product formation by thyX-encoded thymidylate synthase

The synthesis of thymine for DNA is catalyzed by the enzyme thymidylate synthase (TS). A family of flavin-dependent TSs encoded by the thyX gene has been discovered recently. These newly discovered TSs require a reducing substrate in addition to 2'-deoxyuridine monophosphate (dUMP) and 5,10-methylenetetrahydrofolate (CH2THF), suggesting that the enzyme-bound flavin is a redox intermediary in catalysis. The oxidation of the reduced flavin of the TS from Campylobacter jejuni has been observed directly upon mixing with dUMP and CH2THF under anaerobic conditions, thus providing the first direct demonstration of its redox role in catalysis. Product analysis showed that the one mole of 2'-deoxythymidine monophosphate is formed along with one mole of tetrahydrofolate for each mole of reduced enzyme-bound flavin. The classic TS inactivator 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) was able to bind to the reduced enzyme but was unable to oxidize the flavin, even in the presence of CH2THF. Furthermore, the nucleotide binding site of the enzyme treated with FdUMP and CH2THF was irreversibly blocked, suggesting the formation of a stable substrate adduct analogous to that formed by the well-studied thyA-encoded TS. The formation of inactivated enzyme without flavin oxidation indicates that methylene transfer from the folate to the nucleotide occurs prior to flavin redox chemistry.     

The synthesis of novel nonclassical reversed bridge quinazoline antifolates as inhibitors of thymidylate synthase

2‐Amino‐6‐methyl‐5‐(pyridin‐4‐ylsulfanyl)‐3H‐quinazolin‐4‐one ( 3 , AG337) a lipophilic thymidylate synthase inhibitor, is currently in clinical trials as an antitumor agent. On the basis of the crystal structure of 3 and the classical inhibitor 10‐propargyl‐5,8‐dideazafolic acid ( 1 , PDDF) with thymidylate synthase, we designed and synthesized a series of nonclassical 2‐amino‐6‐substituted‐3H‐quinazolin‐4‐ones 4–13 , with a variety of electron withdrawing groups in the side chain (with the exception of compound 4 ). Molecular modeling indicates that these reversed bridge (N9–C10) 6‐substituted analogues orient their side chain C10‐substituent such that it lies between that of 1 and 3 . These compounds were obtained by reduc tive amination of 6‐aminoquinazoline 16 and the appropriate aryl aldehyde 17 or aryl ketone 18 . For ana logues 11–13 , the yield depended on the substitutents on the aryl ketone 18 (comparison of 11 and 13 ). With the exception of analogue 13 , all the compounds in the series were poor inhibitors of thymidylate synthase from Lactobacillus casei, Pneumocystis carinii and human sources.     

High-performance liquid chromatographic assay for thymidylate synthase from the human malaria parasite, Plasmodium falciparum

A rapid and highly sensitive high-performance liquid chromatographic assay for thymidylate synthase activity is described. The assay is based on the separation of the substrate, deoxyuridylate (dUMP), and its product, deoxythymidylate (dTMP), on a LiChrosorb RP-8 reversed-phase column with 44 mM triethylammonium phosphate (pH 7.0) as mobile phase and a flow-rate of 1.0 ml/min. In addition, using a mu Bondapak C18 reversed-phase column with 10 mM potassium phosphate (pH 4.0) and a gradient of 0-28% methanol, dUMP, dTMP and deoxythymidine (dTdR) are well separated within 30 min. The latter system is also applied to assay thymidine kinase activity with dTdR and dTMP as substrate and product, respectively. This method is sensitive enough to measure dTMP at concentrations as low as 25 pmol, and it was used to show that crude extracts of the human malaria parasite Plasmodium falciparum contain thymidylate synthase but not thymidine kinase activity.     

A novel rearrangement of fluorescent human thymidylate synthase inhibitor analogues in ESI tandem mass spectrometry

Cu(I) catalyzed alkyne-azide cycloaddition reaction was employed to synthesize a series of anthracene-based human thymidylate synthase (hTS) inhibitor analogues. The triazolo-anthracene derivatives were characterized by ESI-MS/MS and a novel rearrangement reaction in ESI-MS/MS was observed. The mechanism is proposed whereby the protonated triazolo-anthracene derivative forms a carbocation, and then the carbocation electrophilically attacks an anthracene moiety resulting in formation of a rearrangement ion. Moreover, the carbocation prefers to attack the γ position rather than the α or β position of the anthracene moiety by an electrophilic substitution mechanism.     

Mouse thymidylate synthase does not show the inactive conformation,observed for the human enzyme

Crystal structures of mouse thymidylate synthase (mTS) in complexes with (1) sulfate anion, (2) 2′-deoxyuridine 5′-monophosphate (dUMP) and (3) 5-fluoro-dUMP (FdUMP) and N ^5,10-methylenetetrahydrofolate (meTHF) have been determined and deposited in Protein Data Bank under the accession codes 3IHI, 4E5O and 5FCT, respectively. The structures show a strong overall similarity to the corresponding structures of rat and human thymidylate synthases (rTS and hTS, respectively). Unlike with hTS, whose unliganded and liganded forms assume different conformations (“inactive” and “active,” respectively) in the loop 181–197, in each of the three mTS structures, the loop 175–191, homologous to hTS loop 181–197, populates the active conformer, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP/FdUMP, pointing to protein’s inability to adopt the inactive conformation. The binary structures of either dUMP- or sulfate-bound mTS, showing the enzyme with open active site and extended C-terminus, differ from the structure of the mTS–5-FdUMP–meTHF ternary complex, with the active site closed and C-terminus folded inward, thus covering the active site cleft. Another difference pertains to the conformation of the Arg44 side chain in the active site-flanking loop 41–47, forming strong hydrogen bonds with the dUMP/FdUMP phosphate moiety in each of the two liganded mTS structures, but turning away from the active site entrance and loosing the possibility of H-bonding with sulfate in the sulfate-bound mTS structure.     

Identification of the tyrosine nitration sites in human endothelial nitric oxide synthase by liquid chromatography-mass spectrometry

The formation of nitric oxide (NO) in biological systems has led to the discovery of a number of post- translational protein modifications that can affect biological conditions such as vasodilation. Studies both from our laboratory and others have shown that beside its effect on cGMP generation from soluble guanylate cylcase, NO can produce protein modifications through both S-nitrosylation of cysteine residues. Previously, we have identified the potential S-nitrosylation sites on endothelial NO synthase (eNOS). Thus, the goal of this study was to further increase our understanding of reactive nitrogen protein modifications of eNOS by identifing tyrosine residues within eNOS that are susceptible to nitration in vitro. To accomplish this, nitration was carried out using tetranitromethane followed by tryptic digest of the protein. The resulting tryptic peptides were analyzed by liquid chromatography/mass spectrometry (LC/MS) and the position of nitrated tyrosines in eNOS were identified. The eNOS sequence contains 30 tyrosine residues and our data indicate that multiple tyrosine residues are capable of being nitrated. We could identify 25 of the 30 residues in our tryptic digests and 19 of these were susceptible to nitration. Interstingly, our data identified four tyrosine residues that can be modified by nitration that are located in the region of eNOS responsible for the binding to heat shock protein 90 (Hsp90), which is responsible for ensuring efficient coupling of eNOS.