A molecular modeling study of the interaction of 2'-fluoro-substituted analogues of dUMP/FdUMP with thymidylate synthase

Molecular dynamics simulations and free energy calculations are presented, exploring previously described experimentally studied interactions of a series of 2'-fluoro-substituted dUMP/FdUMP analogues with thymidylate synthase (TS). The results show the inhibitory behaviors of 2'-F-ara-UMP, 2',2'-diF-dUMP and 2',5-diF-ara-UMP to be dependent upon the binding positions and orientations adopted by the molecules of these compounds in the active site of TS. The binding mode of 2',5-diF-ara-UMP suggests a novel role of the active site residue Trp 80, stabilizing through hydrophobic stacking the binding position of the pyrimidine ring in 2',5-diF-ara-UMP.     

Electronic structure calculations on the thiazole-containing antibiotic thiostrepton: molecular mechanics, semi-empirical and ab initio analyses

Thiostrepton is a highly complex cyclic thiazoyl peptide antibiotic and is active against Gram-positive bacteria. Molecular mechanics, semi-empirical and ab initio studies were utilized to further understand the structural and electronic properties of this antibiotic.     

Exploring the cause of aggregation and reduced Zn binding affinity by G85R mutation in SOD1 rendering amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disorder is characterized by the degeneration of upper and lower motor neuron. ALS occurs due to various notably prominent missense mutations, in gene encoding Cu‐Zn superoxide dismutase (SOD1) thereby leading to aggregation, dysfunction and reduced Zn binding affinity. In this study, one such mutation, G85R was explored in comparison with wild type SOD1, using discrete molecular dynamics (DMD). Accordingly, the conformational changes were significantly observed in mutant SOD1, through various geometrical parameters, which substantiated the difference in conformational deviation, flexibility and compactness, thus stipulating a root cause for aggregation. Followed by, analysis of essential dynamics further authenticated the cause behind the protein dysfunction. In particular, the high content of beta sheet with structural deviations, down to dysfunction was established in mutant as compared to wild type, while passing through secondary structure analysis. Subsequently, the deviation of distance in Zn binding residues was distinctly portrayed in mutant as compared to wild type, thus confirming the cause of reduced Zn binding affinity. In addition, the steered molecular dynamics analysis also authenticated the above results indicating the reduced Zn binding affinity in the mutant as compared to that of the wild type. Hence, this work revealed the theoretical mechanism to unravel the mutational effects of cofactor dependent protein. Proteins 2017; 85:1276–1286. © 2017 Wiley Periodicals, Inc.     

31P NMR studies on the interaction of deoxyuridylate with thymidylate synthase.

The 31P nuclear magnetic resonance signal of deoxyuridylate was studied in the presence and absence of thymidlate synthase. In the absence of enzyme the chemical shift of deoxyuridylate is pH dependent with a pKa of 6.25. In the presence of enzyme, a peak corresponding to the dianioinc form of deoxyuridylate is observed which is independent of pH between pH 5.7 and pH 7.4. The pKa of the phosphate in the deoxyuridylate-thymidylate synthase complex is therefore less than 5. The release of inorganic phosphate from deoxyuridylate catalyzed by contaminating phosphatase was also observed.     

Structural basis for recognition of polyglutamyl folates by thymidylate synthase.

Thymidylate synthase (TS) catalyzes the final step in the de novo synthesis of thymidine. In vivo TS binds a polyglutamyl cofactor, polyglutamyl methylenetetrahydrofolate (CH2-H4folate), which serves as a carbon donor. Glutamate residues on the cofactor contribute as much as 3.7 kcal to the interaction between the cofactor, substrate, and enzyme. Because many ligand/receptor interactions appear to be driven largely by hydrophobic forces, it is surprising that the addition of hydrophilic, soluble groups such as glutamates increases the affinity of the cofactor for TS. The structure of a polyglutamyl cofactor analog bound in ternary complex with deoxyuridine monophosphate (dUMP) and Escherichia coli TS reveals how the polyglutamyl moiety is positioned in TS and accounts in a qualitative way for the binding contributions of the different individual glutamate residues. The polyglutamyl moiety is not rigidly fixed by its interaction with the protein except for the first glutamate residue nearest the p-aminobenzoic acid ring of folate. Each additional glutamate is progressively more disordered than the previous one in the chain. The position of the second and third glutamate residues on the protein surface suggests that the polyglutamyl binding site could be utilized by a new family of inhibitors that might fill the binding area more effectively than polyglutamate.     

Interaction of pyridoxal phosphate with thymidylate synthase: Spectral and equilibrium dialysis studies

• 1.1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents.
• 2.2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 ± 1.6 μM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 ± 0.4, and the Hill coefficient was 0.97.
• 3.3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylenetetrahydrofoliate.
• 4.4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.

     

Inhibition of thymidylate synthase by pergularinine, tylophorinidine and deoxytubulosine

The activity of thymidylate synthase (TS) purified in our laboratory from Lactobacillus leichmannii was inhibited by pergularinine (PGL) and tylophorinidine (TPD) and deoxytubulosine (DTB) isolated from the Indian medicinal plants Pergularia pallida and Alangium lamarckii respectively. Cytotoxicity studies showed that cell growth of L. leichmannii was inhibited (IC50 = 40-45 microM) by all the three alkaloids, the concentrations > 80-90 microM resulting in complete loss of the enzyme activity. Ki values of the enzyme calculated from Lineweaver-Burk and Dixon plots for PGL, TPD and DTB were 10 x 10(-6) M, 9 x 10(-6) M and 7 x 10(-6) M respectively. These are typed as 'non-competitive' inhibitors of TS. All the three alkaloids inhibited (IC50 = 50 microM) the elevated TS activity of leukocytes in cancer patients with clinically diagnosed chronic myelocytic leukemia (n = 10), acute lymphocytic leukemia (n = 8) and metastatic solid tumours (n = 3).     

Structural determinants for the intracellular degradation of human thymidylate synthase

Thymidylate synthase (EC 2.1.1.45) (TS) catalyzes the conversion of dUMP to dTMP and is therefore indispensable for DNA replication in actively dividing cells. The enzyme is a critical target at which chemotherapeutic agents such as fluoropyrimidines (e.g., 5-fluorouracil and 5-fluoro-2'-deoxyuridine) and folic acid analogues (e.g., raltitrexed, LY231514, ZD9331, and BW1843U89) are directed. These agents exert their effects through the generation of metabolites that bind the active site of TS and inhibit catalytic activity. The binding of ligands to the TS molecule leads to dramatic changes in the conformation of the enzyme, particularly within the C-terminal domain. Stabilization of the enzyme and an increase in its intracellular level are associated with ligand binding and may be important in cellular response to TS-directed drugs. In the present study, we have examined molecular features of the TS molecule that control its degradation. We find that the C-terminal conformational shift is not required for ligand-mediated stabilization of the enzyme. In addition, we demonstrate that the N-terminus of the TS polypeptide, which is extended in the mammalian enzyme and is disordered in crystal structures, is a primary determinant of the enzyme's half-life. Finally, we show that TS turnover is carried out by the 26S proteasome in a ubiquitin-independent manner. These findings provide the basis for a mechanistic understanding of TS degradation and its regulation by antimetabolites.     

Mechanistic and structural basis for inhibition of thymidylate synthase ThyX

Nature has established two mechanistically and structurally unrelated families of thymidylate synthases that produce de novo thymidylate or dTMP, an essential DNA precursor. Representatives of the alternative flavin-dependent thymidylate synthase family, ThyX, are found in a large number of microbial genomes, but are absent in humans. We have exploited the nucleotide binding pocket of ThyX proteins to identify non-substrate-based tight-binding ThyX inhibitors that inhibited growth of genetically modified Escherichia coli cells dependent on thyX in a manner mimicking a genetic knockout of thymidylate synthase. We also solved the crystal structure of a viral ThyX bound to 2-hydroxy-3-(4-methoxybenzyl)-1,4-naphthoquinone at a resolution of 2.6 Å. This inhibitor was found to bind within the conserved active site of the tetrameric ThyX enzyme, at the interface of two monomers, partially overlapping with the dUMP binding pocket. Our studies provide new chemical tools for investigating the ThyX reaction mechanism and establish a novel mechanistic and structural basis for inhibition of thymidylate synthesis. As essential ThyX proteins are found e.g. in Mycobacterium tuberculosis and Helicobacter pylori, our studies have also potential to pave the way towards the development of new anti-microbial compounds.     

Crystal structure of the D94S/G98E variant of rat alpha-parvalbumin. An explanation for the reduced divalent ion affinity

Simultaneous replacement of Asp-94 with serine and Gly-98 with glutamate in rat alpha-parvalbumin creates a CD-site ligand array in the context of the EF-site binding loop. Previous work has shown that, relative to the wild-type CD site, this engineered site has markedly reduced Ca(2+) affinity. Seeking an explanation for this phenomenon, we have obtained the crystal structure of the alpha D94S/G98E variant. The Ca(2+) coordination within the engineered EF site of the 94/98E variant is nearly identical to that within the CD site, suggesting that the attenuated affinity of the EF site in 94/98E is not a consequence of suboptimal coordination geometry. We have also examined the divalent ion binding properties of the alpha 94/98E variant in both Na(+)- and K(+)-containing buffers. Although the Ca(2+) and Mg(2+) affinities are higher in K(+) solution, the increases are comparable to those observed for wild-type alpha. Consistent with that finding, the apparent Na(+) stoichiometry, estimated from stability studies conducted as a function of Na(+) concentration, is 1.0 +/- 0.1, identical to that of wild-type alpha. Thus, the reduced affinity for divalent ions is evidently not the result of heightened monovalent ion competition. The thermodynamic analysis indicates that the less favorable Gibbs free energy of binding reflects a substantial enthalpic penalty. Significantly, the crystal structure reveals a steric clash between Phe-57 and the C(gamma) atom of Glu-98. The consequent displacement of Phe-57 also produces a close contact with Ser-55. Thus, steric interference may be the source of the enthalpic penalty.     

Semi-empirical, ab initio and molecular modeling studies on the DNA binding of a calicheamicinone-polyamide conjugate

AM1 semi-empirical and ab initio calculations were performed on certain synthetic polyamide conjugates of the aglycone of the minor groove binding antibiotic calicheamicin. Geometry optimized conformations and heats of formation were obtained. The binding of the optimized conformations of the drug to both alternating and non-alternating (AT)n and to (G)n x (C)n sequences were studied and the energies of binding were compared to each other. The results can be utilized in the design of novel enediyne-based drugs.     

Analysis of polymorphisms and haplotype structure of the human thymidylate synthase genetic region: a tool for pharmacogenetic studies

5-Fluorouracil (5FU), a widely used chemotherapeutic drug, inhibits the DNA replicative enzyme, thymidylate synthase (Tyms). Prior studies implicated a VNTR (variable numbers of tandem repeats) polymorphism in the 5'-untranslated region (5'-UTR) of the TYMS gene as a determinant of Tyms expression in tumors and normal tissues and proposed that these VNTR genotypes could help decide fluoropyrimidine dosing. Clinical associations between 5FU-related toxicity and the TYMS VNTR were reported, however, results were inconsistent, suggesting that additional genetic variation in the TYMS gene might influence Tyms expression. We thus conducted a detailed genetic analysis of this region, defining new polymorphisms in this gene including mononucleotide (poly A:T) repeats and novel single nucleotide polymorphisms (SNPs) flanking the VNTR in the TYMS genetic region. Our haplotype analysis of this region used data from both established and novel genetic variants and found nine SNP haplotypes accounting for more than 90% of the studied population. We observed non-exclusive relationships between the VNTR and adjacent SNP haplotypes, such that each type of VNTR commonly occurred on several haplotype backgrounds. Our results confirmed the expectation that the VNTR alleles exhibit homoplasy and lack the common ancestry required for a reliable marker of a linked adjacent locus that might govern toxicity. We propose that it may be necessary in a clinical trial to assay multiple types of genetic polymorphisms in the TYMS region to meaningfully model linkage of genetic markers to 5FU-related toxicity. The presence of multiple long (up to 26 nt), polymorphic monothymidine repeats in the promoter region of the sole human thymidylate synthetic enzyme is intriguing.     

Structural basis for reduced activity of 1-aminocyclopropane-1-carboxylate synthase affected by a mutation linked to andromonoecy

1-aminocyclopropane-1-carboxylate synthase (ACS) is a key enzyme in the biosynthesis of the plant hormone ethylene. Recently, a new biological role for ACS has been found in Cucumis melo where a single point mutation (A57V) of one isoform of the enzyme, causing reduced activity, results in andromonoecious plants. We present here a straightforward structural basis for the reduced activity of the A57V mutant, based on our work on Malus domestica ACS, including a new structure of the unliganded apple enzyme at 1.35 Å resolution.     

Complementation of a Dictyostelium discoideum thymidylate synthase mutation with the mouse gene provides a new selectable marker for transformation.

A cDNA encoding mouse thymidylate synthase has been inserted 3' to the Dictyostelium discoideum actin 15 promoter in an E. coli-D.discoideum shuttle vector. When this construct was introduced into a D.discoideum thymidylate synthase mutant strain HPS400, stable transformants were obtained at high frequency. These transformants grew in standard axenic medium without requiring exogenous thymidine. This construct provides a second selectable marker for use in transformation of D.discoideum.     

Polymorphism of the thymidylate synthase gene and thymidylate synthase levels in colon cancer cell lines and different tissues of colorectal cancer patients

In a panel of 18 colon cancer cell lines we found that the thymidylate synthase (TS) genotype was related to TS enzyme activity, but not to TS protein and mRNA levels. In addition, no relation with drug sensitivity was observed. TS genotyping of different tissues from 78 colorectal cancer patients revealed a high level of homology in polymorphic status between normal and malignant tissues and the heterozygous genotype to be the most frequent.     

Density-functional theory and ab initio Hartree-Fork studies on the structural parameters and chemical activity of the free radicals generated by benzoquinone and hydroquinone

Density-functional theory (DFT) calculations were performed for calculation of the theoretical spectra and the chemical activities of free radicals generated by benzoquinone and hydroquinone as well as the transition states, and the calculated spectra were used for the assignment of the frequencies observed in the experimental IR spectra. The calculated geometrical parameters, the predicted IR spectra, and the chemical activities of free radicals and transition states were also compared with those of benzoquinone and hydroquinone. The reactive mechanisms of free radicals generated by benzoquinone and hydroquinone are also discussed using ab initio Hartree-Fork (HF) methods.     

Vibrational spectral assignments of paraldehyde by ab initio and density functional methods

The FTIR and Laser-Raman spectra of paraldehyde have been recorded in the regions 4000–400 cm⁻¹ and 3500–250 cm⁻¹ respectively. Molecular electronic energy, geometrical structure, harmonic vibrational spectra, infrared intensities and Raman scattering activities have been computed at the HF/6-31G(d,p) and B3LYP/6-31G(d,p) levels of theory. The results were compared with experimental values with the help of scaling procedures. The observed wave numbers in FTIR and Laser-Raman spectra were analyzed and assigned to different normal modes of the molecule. Most of the modes have wave numbers in the expected range and are in good agreement with computed values.