Flavin-dependent Thymidylate Synthase Research Articles

Biosynthesis of ansamitocin P-3 incurs stress on the producing strain Actinosynnema pretiosum at multiple targets

Microbial bioactive natural products mediate ecologically beneficial functions to the producing strains, and have been widely used in clinic and agriculture with clearly defined targets and underlying mechanisms. However, the physiological effects of their biosynthesis on the producing strains remain largely unknown. The antitumor ansamitocin P-3 (AP-3), produced by Actinosynnema pretiosum ATCC 31280, was found to repress the growth of the producing strain at high concentration and target the FtsZ protein involved in cell division. Previous work suggested the presence of additional cryptic targets of AP-3 in ATCC 31280. Herein we use chemoproteomic approach with an AP-3-derived photoaffinity probe to profile the proteome-wide interactions of AP-3. AP-3 exhibits specific bindings to the seemingly unrelated deoxythymidine diphosphate glucose-4,6-dehydratase, aldehyde dehydrogenase, and flavin-dependent thymidylate synthase, which are involved in cell wall assembly, central carbon metabolism and nucleotide biosynthesis, respectively. AP-3 functions as a non-competitive inhibitor of all three above target proteins, generating physiological stress on the producing strain through interfering diverse metabolic pathways. Overexpression of these target proteins increases strain biomass and markedly boosts AP-3 titers. This finding demonstrates that identification and engineering of cryptic targets of bioactive natural products can lead to in-depth understanding of microbial physiology and improved product titers.

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

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

Attempt of thyX gene silencing and construction of a thyX deleted clone in a Mycobacterium bovis BCG.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possess flavin-dependent thymidylate synthase, ThyX. Since thyX is absent in humans and was shown to be essential for M. tuberculosis normal growth, ThyX is thought to be an attractive novel TB drug target. This study assessed thyX essentiality in Mycobacterium bovis BCG strains using CRISPR interference based gene silencing and found that thyX is not essential in an M. bovis BCG Tokyo derivative strain. A thyX deletion mutant strain was successfully constructed from that strain, which reinforces the non-essentiality of thyX under a certain genetic background.

Synthesis of acyclic nucleoside phosphonates targeting flavin-dependent thymidylate synthase in Mycobacterium tuberculosis

Flavin-Dependent Thymidylate Synthase (FDTS) encoded by ThyX gene was discovered as a new class of thymidylate synthase involved in the de novo synthesis of dTMP named only in 30 % of human pathogenic bacteria. This target was pursed for the development of new antibacterial agents against multiresistant pathogens. We have developed a new class of ANPs based on the mimic of two natural’s cofactors (dUMP and FAD) as inhibitors against Mycobacterium tuberculosis ThyX. Several synthetic efforts were performed to optimize regioselective N1-alkylation, cross-coupling metathesis and Sonogashira cross-coupling. Compound 19c showed a poor 31.8% inhibitory effect on ThyX at 200 μM.

Multifaceted role of drugs: a potential weapon to outsmart Mycobacterium tuberculosis resistance by targeting its essential ThyX

Tuberculosis (TB) is one of the prominent cause of deaths across the world and multidrug-resistant and extensively drug-resistant TB continues to pose challenges for clinicians and public health centers. The risk of death is extremely high in individuals who have compromised immune systems, HIV infection, or diabetes. Research institutes and pharmaceutical companies have been working on repurposing existing drugs as effective therapeutic options against TB. The identification of suitable drugs with multi-target affinity profiles is a widely accepted way to combat the development of resistance. Flavin-dependent thymidylate synthase (FDTS), known as ThyX, is in the class of methyltransferases and is a possible target in the discovery of novel anti-TB drugs. In this study, we aimed to repurpose existing drugs approved by Food and Drug Administration (FDA) that could be used in the treatment of TB. An integrated screening was performed based on computational procedures: high-throughput molecular docking techniques, followed by molecular dynamics simulations of the target enzyme, ThyX. After performing in silico screening using a library of 3,967 FDA-approved drugs, the two highest-scoring drugs, Carglumic acid and Mesalazine, were selected as potential candidates that could be repurposed to treat TB. Communicated by Ramaswamy H. Sarma

Ultrafast dynamics of fully reduced flavin in catalytic structures of thymidylate synthase ThyX.

Thymidylate is a vital DNA precursor synthesized by thymidylate synthases. ThyX is a flavin-dependent thymidylate synthase found in several human pathogens and absent in humans, which makes it a potential target for antimicrobial drugs. This enzyme methylates the 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate (dTMP) using a reduced flavin adenine dinucleotide (FADH-) as prosthetic group and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF) as a methylene donor. Recently, it was shown that ThyX-catalyzed reaction is a complex process wherein FADH- promotes both methylene transfer and reduction of the transferred methylene into a methyl group. Here, we studied the dynamic and photophysics of FADH- bound to ThyX, in several substrate-binding states (no substrate, in the presence of dUMP or folate or both) by femtosecond transient absorption spectroscopy. This methodology provides valuable information about the ground-state configuration of the isoalloxazine moiety of FADH- and the rigidity of its local environment, through spectra shape and excited-state lifetime parameters. In the absence of substrate, the environment of FADH- in ThyX is only mildly more constrained than that of free FADH- in solution. The addition of dUMP however narrows the distribution of ground-state configurations and increases the constraints on the butterfly bending motion in the excited state. Folate binding results in the selection of new ground-state configurations, presumably located at a greater distance from the conical intersection where excited-state decay occurs. When both substrates are present, the ground-state configuration appears on the contrary rather limited to a geometry close to the conical intersection, which explains the relatively fast excited-state decay (100 ps on the average), even if the environment of the isoalloxazine is densely packed. Hence, although the environment of the flavin is dramatically constrained, FADH- retains a dynamic necessary to shuttle carbon from folate to dUMP. Our study demonstrates the high sensitivity of FADH- photophysics to the constraints exerted by its immediate surroundings.

Glycol and Phosphate Depot Forms of 4- and/or 5-Modified Nucleosides Exhibiting Antibacterial Activity

Resistance developed to the majority of drugs used to treat infectious diseases warrants the design of new compounds effective against drug-resistant strains of pathogens. Recently, several groups of modified nucleosides have been synthesized and showed significant antibacterial activity in vitro, but their further studies were difficult to undertake because of their low solubility in aqueous solutions. Nevertheless, new compounds, well soluble in water-organic solutions, were synthesized and found to be more effective in inhibiting the growth of Gram-positive bacteria and mycobacteria. The water-soluble forms of modified nucleosides under study were assumed to be their depot forms. To check the assumption, the compounds were tested for hydrolysis in various media and their molecular docking was performed into the active center of the putative target, Mycobacterium tuberculosis flavin-dependent thymidylate synthase ThyX. Computer modelling showed that the water-soluble analogs do not act as ThyX inhibitors, supporting the assumption of their depot nature. The compounds were resistant to chemical hydrolysis but were hydrolyzed when incubated with porcine liver carboxylesterase, human serum, or Staphylococcus aureus 209P. The results demonstrate that the compounds are most likely depot forms of modified nucleosides.

The Dual-Targeting Activity of the Metabolite Substrate of Para-amino Salicyclic Acid in the Mycobacterial Folate Pathway: Atomistic and Structural Perspectives

Therapeutic targeting of folate biosynthetic pathway has recently been explored as a viable strategy in the treatment of tuberculosis. The bioactive metabolite substrate of Para-amino salicyclic acid (PAS-M) reportedly dual-targets dihydrofolate reductase (DHFR) and flavin-dependent thymidylate synthase (FDTS), two essential enzymes in folate biosynthetic pathway. However, the molecular mechanisms and structural dynamics of this dual inhibitory activity of the PAS-M remain elusive. Molecular dynamics simulations revealed that binding of PAS-M towards DHFR is characterized by a recurrence of strong conventional hydrogen bond interactions between a peculiar DHFR binding site residue (Asp27) and the 2-amino-decahydropteridin-4-ol group of PAS-M. Similarly, the binding of PAS-M towards FDTS also involved consistent strong conventional hydrogen bond interactions between some specific residues (Tyr101, Arg172, Thr4, Gln103, Arg87 and Gln106) and, the 2-amino-decahydropteridin-4-ol group, thus establishing the cruciality of the group. Structural dynamics of the bound complexes of both enzymes revealed that, upon binding, PAS-M is anchored at the entrance of hydrophobic pockets by strong hydrogen bond interactions while the rest of the structure gains access to deeper hydrophobic residues to engage in favorable interactions. Further analysis of atomistic changes of both enzymes showed increased C-α atom deviations as well as an increase C-α atoms radius of gyration consistent with structural disorientations. These conformational changes possibly interfered with the biological functions of the enzymes and hence their inhibition as experimentally reported. Structural Insights provided could open up a novel paradigm of structure-based design of multi-targeting inhibitors of biological targets in the folate biosynthetic pathway toward tuberculosis therapy.

Crystal structure of the flavin-dependent thymidylate synthase Thy1 from Thermus thermophilus with an extra C-terminal domain

The thymidylate synthases ThyA and Thy1 are enzymes that catalyse the formation of thymidine monophosphate from 2'-deoxyuridine monophosphate. Thy1 (or ThyX) requires flavin for catalytic reactions, while ThyA does not. In the present study, the crystal structure of the flavin-dependent thymidylate synthase Thy1 from Thermus thermophilus HB8 (TtThy1, TTHA1096) was determined in complex with FAD and phosphate at 2.5 Å resolution. TtThy1 is a tetrameric molecule like other Thy1 proteins, to which four FAD molecules are bound. In the crystal of TtThy1, two phosphate ions were bound to each dUMP-binding site. The characteristic feature of TtThy1 is the existence of an extra C-terminal domain (CTD) consisting of three α-helices and a β-strand. The function of the CTD is unknown and database analysis showed that this CTD is only shared by part of the Deinococcus-Thermus phylum.

Targeting Methyltransferases in Human Pathogenic Bacteria: Insights into Thymidylate Synthase (TS) and Flavin-Dependent TS (FDTS).

In cells, thymidylate synthases provide the only de novo source of 2′-deoxythymidine-5′-monophosphate (dTMP), required for DNA synthesis. The activity of these enzymes is pivotal for cell survival and proliferation. Two main families of thymidylate synthases have been identified in bacteria, folate-dependent thymidylate synthase (TS) and flavin-dependent TS (FDTS). TS and FDTS are highly divergent enzymes, characterized by exclusive catalytic mechanisms, involving different sets of cofactors. TS and FDTS mechanisms of action have been recently revised, providing new perspectives for the development of antibacterial drugs targeting these enzymes. Nonetheless, some catalytic details still remain elusive. For bacterial TSs, half-site reactivity is still an open debate and the recent evidences are somehow controversial. Furthermore, different behaviors have been identified among bacterial TSs, compromising the definition of common mechanisms. Moreover, the redox reaction responsible for the regeneration of reduced flavin in FDTSs is not completely clarified. This review describes the recent advances in the structural and functional characterization of bacterial TSs and FDTSs and the current understanding of their mechanisms of action. Furthermore, the recent progresses in the development of inhibitors targeting TS and FDTS in human pathogenic bacteria are summarized.

Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents

The folate biosynthetic pathway offers many druggable targets that have yet to be exploited in tuberculosis therapy. Herein, we have identified a series of small molecules that interrupt Mycobacterium tuberculosis (Mtb) folate metabolism by dual targeting ofdihydrofolate reductase (DHFR), a key enzyme inthe folate pathway, and its functional analog, Rv2671. We have also compared the antifolate activity of these compounds with that of para-aminosalicylic acid (PAS). We found that the bioactive metabolite of PAS, in addition to previously reported activity against DHFR, inhibits flavin-dependent thymidylate synthase in Mtb, suggesting a multi-targeted mechanism of action for this drug. Finally, we have shown that antifolate treatment in Mtb decreases the production of mycolic acids, most likely due to perturbation of the activated methyl cycle. We conclude that multi-targeting of the folate pathway in Mtb is associated with highly potent anti-mycobacterial activity.

Novel 5-substituted derivatives of 2'-deoxy-6-azauridine with antibacterial activity.

The emergence of new drug-resistant strains of bacteria necessitates the development of principally new antibacterial agents. One of the novel classes of antibacterial agents is nucleoside analogs. We have developed a fast and simple one-pot method for preparation of α- and β-anomers of 5-modified 6-aza- and 2-thio-6-aza-2'-deoxyuridine derivatives in high yields. 2-Thio derivatives demonstrated moderate activity against Mycobacterium smegmatis (MIC = 0.2-0.8 mM), Staphylococcus aureus (MIC = 0.03-0.9 mM) and some other Gram-positive bacteria. 2'-Deoxy-2-thio-5-phenyl-6-azauridine (2b) effectively suppressed the growth of Gram-negative bacteria Pseudomonas aeruginosa ATCC 27853 (MIC = 0.03 mM)-the one that causes diseases difficult to treat due to high resistance to antibiotics. 5'-Monophosphates of compounds 2a, b and 3a, b were docked into a binding site of Mycobacterium tuberculosis flavin-dependent thymidylate synthase (ThyX) enzyme. The molecular modeling demonstrates the possibility of binding of the 5-modified 2-thio-6-aza-2'-deoxyuridine 5'-monophosphates within the active site of the enzyme and thereby inhibiting the growth of the bacteria.

Synthesis and Structure-Activity Relationship Studies of Benzo[b][1,4]oxazin-3(4H)-one Analogues as Inhibitors of Mycobacterial Thymidylate Synthase X.

Since the discovery of a flavin-dependent thymidylate synthase (ThyX or FDTS) that is absent in humans but crucial for DNA biosynthesis in a diverse group of pathogens, the enzyme has been pursued for the development of new antibacterial agents against Mycobacterium tuberculosis, the causative agent of the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous screening efforts and report herein an optimization campaign of a novel series of inhibitors with a unique inhibition profile. The inhibitors display competitive inhibition toward the methylene tetrahydrofolate cofactor of ThyX, enabling us to generate a model of the compounds bound to their target, thus offering insight into their structure-activity relationships.

Chemical quenching and identification of intermediates in flavoenzyme-catalyzed reactions.

Chemical quenching offers a complementary approach to studying the mechanism of a flavoenzyme, supplementing the information learned from spectroscopic, structural, and computational methods. Generally, in a chemical quench experiment, an enzymatic turnover is quickly stopped at various stages with a chemical agent, and the individual reaction mixtures at each time point are analyzed for the reactants, products and any intermediates. The order by which bonds are made and broken in the reaction is indicated by the identities of the captured intermediates, and the rates of individual steps in the mechanism are determined from the amounts of various chemical species at different time points. This chapter outlines general considerations in selecting a chemical quencher of a particular enzyme-catalyzed reaction and methods for analyzing captured reaction intermediates, with a focus on flavoenzymes. The investigation of flavin-dependent thymidylate synthase is used as a case study to illustrate the concepts and workflow of quenching, isolating, and characterizing quencher-modified reaction intermediates and drawing mechanistic conclusions from the identities of these molecules.

Structural and functional insight into serine hydroxymethyltransferase from Helicobacter pylori.

Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a ubiquitous pyridoxal 5’-phosphate (PLP)-dependent enzyme that catalyzes the formation of glycine from serine. The thereby generated 5,10-methylene tetrahydrofolate (MTHF) is a major source of cellular one-carbon units and a key intermediate in thymidylate biosynthesis. While in virtually all eukaryotic and many bacterial systems thymidylate synthase ThyA, SHMT and dihydrofolate reductase (DHFR) are part of the thymidylate/folate cycle, the situation is different in organisms using flavin-dependent thymidylate synthase ThyX. Here the distinct catalytic reaction directly produces tetrahydrofolate (THF) and consequently in most ThyX-containing organisms, DHFR is absent. While the resulting influence on the folate metabolism of ThyX-containing bacteria is not fully understood, the presence of ThyX may provide growth benefits under conditions where the level of reduced folate derivatives is compromised. Interestingly, the third key enzyme implicated in generation of MTHF, serine hydroxymethyltransferase (SHMT), has a universal phylogenetic distribution, but remains understudied in ThyX-containg bacteria. To obtain functional insight into these ThyX-dependent thymidylate/folate cycles, we characterized the predicted SHMT from the ThyX-containing bacterium Helicobacter pylori. Serine hydroxymethyltransferase activity was confirmed by functional genetic complementation of a glyA-inactivated E. coli strain. A H. pylori ΔglyA strain was obtained, but exhibited markedly slowed growth and had lost the virulence factor CagA. Biochemical and spectroscopic evidence indicated formation of a characteristic enzyme-PLP-glycine-folate complex and revealed unexpectedly weak binding affinity of PLP. The three-dimensional structure of the H. pylori SHMT apoprotein was determined at 2.8Ǻ resolution, suggesting a structural basis for the low affinity of the enzyme for its cofactor. Stabilization of the proposed inactive configuration using small molecules has potential to provide a specific way for inhibiting HpSHMT.

Unique Features and Anti-microbial Targeting of Folate- and Flavin-Dependent Methyltransferases Required for Accurate Maintenance of Genetic Information.

Comparative genome analyses have led to the discovery and characterization of novel flavin- and folate-dependent methyltransferases that mainly function in DNA precursor synthesis and post-transcriptional RNA modification by forming (ribo) thymidylate and its derivatives. Here we discuss the recent literature on the novel mechanistic features of these enzymes sometimes referred to as “uracil methyltransferases,” albeit we prefer to refer to them as (ribo) thymidylate synthases. These enzyme families attest to the convergent evolution of nucleic acid methylation. Special focus is given to describing the unique characteristics of these flavin- and folate-dependent enzymes that have emerged as new models for studying the non-canonical roles of reduced flavin co-factors (FADH2) in relaying carbon atoms between enzyme substrates. This ancient enzymatic methylation mechanism with a very wide phylogenetic distribution may be more commonly used for biological methylation reactions than previously anticipated. This notion is exemplified by the recent discovery of additional substrates for these enzymes. Moreover, similar reaction mechanisms can be reversed by demethylases, which remove methyl groups e.g., from human histones. Future work is now required to address whether the use of different methyl donors facilitates the regulation of distinct methylation reactions in the cell. It will also be of great interest to address whether the low activity flavin-dependent thymidylate synthases ThyX represent ancestral enzymes that were eventually replaced by the more active thymidylate synthases of the ThyA family to facilitate the maintenance of larger genomes in fast-growing microbes. Moreover, we discuss the recent efforts from several laboratories to identify selective anti-microbial compounds that target flavin-dependent thymidylate synthase ThyX. Altogether we underline how the discovery of the alternative flavoproteins required for methylation of DNA and/or RNA nucleotides, in addition to providing novel targets for antibiotics, has provided new insight into microbial physiology and virulence.

Drugging the Folate Pathway in Mycobacterium Tuberculosis: The Role of Multi-Targeting Agents

The folate biosynthetic pathway, responsible for the generation of reduced folate cofactors, offers many druggable targets that have not been exploited in TB therapy. Here, we have identified a series of novel small molecules that interrupt Mycobacterium tuberculosis folate metabolism by dual targeting of dihydrofolate reductase (DHFR), a key enzyme in the folate pathway, and its functional analog, Rv2671. We have also compared the antifolate activity of these compounds to that of para-aminosalicylic acid (PAS). We found the bioactive metabolite of PAS, in addition to previously reported activity against DHFR, inhibits flavin-dependent thymidylate synthase (FDTS) in Mtb, suggesting a multi-targeted mechanism of action for this drug. Finally, we have shown that antifolate treatment in Mtb decreases the production of mycolic acids, most likely due to perturbation of the activated methyl cycle. We conclude that multi-targeting of the folate pathway in Mtb is a promising strategy to develop novel anti-TB agents.

Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase.

Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2'-deoxythymidine 5'-monophosphate (dTMP) from 2'-deoxyuridine 5'-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans and a very different mechanism. Here we report nuclear magnetic resonance evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogues also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.

Flavin-Dependent Thymidylate Synthase as a New Antibiotic Target.

In humans de novo synthesis of 2′-deoxythymidine-5′-monophosphate (dTMP), an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate synthase (TSase) and dihydrofolate reductase (DHFR). The enzyme flavin-dependent thymidylate synthase (FDTS) represents an alternative enzymatic pathway to synthesize dTMP, which is not present in human cells. A number of pathogenic bacteria, however, depend on this enzyme in lieu of or in conjunction with the analogous human pathway. Thus, inhibitors of this enzyme may serve as antibiotics. Here, we review the similarities and differences of FDTS vs. TSase including aspects of their structure and chemical mechanism. In addition, we review current progress in the search for inhibitors of flavin dependent thymidylate synthase as potential novel therapeutics.

Synthesis and evaluation of C-5 modified 2′-deoxyuridine monophosphates as inhibitors of M. tuberculosis thymidylate synthase

A series of 5′-monophosphates of 5-substituted 2′-deoxyuridine analogs, which recently demonstrated in vitro substantial suppression of two strains of Mycobacterium tuberculosis growth (virulent laboratory H37Rv and multiple resistant MS-115), has been synthesized and evaluated as potential inhibitors of M. tuberculosis thymidylate synthases: classical (ThyA) and flavin dependent thymidylate synthase (ThyX). A systematic SAR study and docking revealed 5-undecyloxymethyl-2′-deoxyuridine 5′-monophosphate 3b, displaying an IC50 value against ThyX of 8.32μM. All derivatives lack activity against the ThyA. It can be assumed that the mechanism of action of 3b may be partially associated with the inhibition of the ThyX.