Consecutive Aromatic Residues Are Required for Improved Efficacy of β-Sheet Breakers.

Alzheimer's disease is a fatal neurodegenerative malady which up to very recently did not have approved therapy modifying its course. After controversial approval of aducanumab (monoclonal antibody clearing β-amyloid plaques) by FDA for use in very early stages of disease, possibly new avenue opened for the treatment of patients. In line with this approach is search for compounds blocking aggregation into amyloid oligomers subsequently forming fibrils or compounds helping in getting rid of plaques formed by β-amyloid fibrils.

Molecular Mechanism of Thymidylate Synthase Inhibition by N-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies.

Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N-hydroxy-dCMP (N-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N-OH-dCMP and N-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf.

Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues.

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively.

β-sheet breakers with consecutive phenylalanines: Insights into mechanism of dissolution of β-amyloid fibrils.

β-sheet breakers (BSB) constitute a class of peptide inhibitors of amyloidogenesis, a process which is a hallmark of many diseases called amyloidoses, including Alzheimer's disease (AD); however, the molecular details of their action are still not fully understood. Here we describe the results of the computational investigation of the three BSBs, iaβ6 (LPFFFD), iaβ5 (LPFFD), and iaβ6_Gly (LPFGFD), in complex with the fibril model of Aβ42 and propose the kinetically probable mechanism of their action. The mechanism involves the binding of BSB to the central hydrophobic core (CHC) region (LVFFA) of Aβ fibril and the π-stacking of its Phe rings both internally and with the Aβ fibril.

Interactions between motor domains in kinesin-14 Ncd - a molecular dynamics study.

Minus-end directed, non-processive kinesin-14 Ncd is a dimeric protein with C-terminally located motor domains (heads). Generation of the power-stroke by Ncd consists of a lever-like rotation of a long superhelical 'stalk' segment while one of the kinesin's heads is bound to the microtubule. The last ∼30 amino acids of Ncd head play a crucial but still poorly understood role in this process.

ANO5 mutations in the Polish limb girdle muscular dystrophy patients: Effects on the protein structure.

LGMD2L is a subtype of limb-girdle muscular dystrophy (LGMD), caused by recessive mutations in ANO5, encoding anoctamin-5 (ANO5). We present the analysis of five patients with skeletal muscle weakness for whom heterozygous mutations within ANO5 were identified by whole exome sequencing (WES). Patients varied in the age of the disease onset (from 22 to 38 years) and severity of the morphological and clinical phenotypes.

Combining Rosetta with molecular dynamics (MD): A benchmark of the MD-based ensemble protein design.

Computational protein design is a set of procedures for computing amino acid sequences that will fold into a specified structure. Rosetta Design, a commonly used software for protein design, allows for the effective identification of sequences compatible with a given backbone structure, while molecular dynamics (MD) simulations can thoroughly sample near-native conformations. We benchmarked a procedure in which Rosetta design is started on MD-derived structural ensembles and showed that such a combined approach generates 20-30% more diverse sequences than currently available methods with only a slight increase in computation time.

Crystal structures of nematode (parasitic T. spiralis and free living C. elegans), compared to mammalian, thymidylate synthases (TS). Molecular docking and molecular dynamics simulations in search for nematode-specific inhibitors of TS.

Three crystal structures are presented of nematode thymidylate synthases (TS), including Caenorhabditis elegans (Ce) enzyme without ligands and its ternary complex with dUMP and Raltitrexed, and binary complex of Trichinella spiralis (Ts) enzyme with dUMP. In search of differences potentially relevant for the development of species-specific inhibitors of the nematode enzyme, a comparison was made of the present Ce and Ts enzyme structures, as well as binary complex of Ce enzyme with dUMP, with the corresponding mammalian (human, mouse and rat) enzyme crystal structures. To complement the comparison, tCONCOORD computations were performed to evaluate dynamic behaviors of mammalian and nematode TS structures.

Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells.

Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex in vitro and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells.

Phosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N(4)-hydroxy-dCMP.

Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependence of the inactivation rate on time and accompanied by differing inhibition parameters. Considering its importance for chemotherapeutic drug resistance, the possible effect of thymidylate synthase inhibition by post-translational modification was tested, e.

Properties of phosphorylated thymidylate synthase.

Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results.

Crystal structure of mouse thymidylate synthase in tertiary complex with dUMP and raltitrexed reveals N-terminus architecture and two different active site conformations.

The crystal structure of mouse thymidylate synthase (mTS) in complex with substrate dUMP and antifolate inhibitor Raltitrexed is reported. The structure reveals, for the first time in the group of mammalian TS structures, a well-ordered segment of 13 N-terminal amino acids, whose ordered conformation is stabilized due to specific crystal packing. The structure consists of two homodimers, differing in conformation, one being more closed (dimer AB) and thus supporting tighter binding of ligands, and the other being more open (dimer CD) and thus allowing weaker binding of ligands.

Crystal structure of phosphoramide-phosphorylated thymidylate synthase reveals pSer127, reflecting probably pHis to pSer phosphotransfer.

Crystal structure is presented of the binary complex between potassium phosphoramidate-phosphorylated recombinant C. elegans thymidylate synthase and dUMP. On each monomer a single phosphoserine residue (Ser127) was identified, instead of expected phosphohistidine.

The β-sheet breakers and π-stacking.

Fibrillation of β-amyloid is recognized as a key process leading to the development of Alzheimer's disease. Small peptides called β-sheet breakers were found to inhibit the process of β-amyloid fibrillation and to dissolve amyloid fibrils in vitro, in vivo, and in cell culture studies [1,2]. The mechanism by which peptide inhibition takes place remains elusive and a detailed model needs to be established.

Computational study of the effects of protein tyrosine nitrations on the catalytic activity of human thymidylate synthase.

Tyrosine nitration is a widespread post-translational modification capable of affecting both the function and structure of the host protein molecule. Enzyme thymidylate synthase (TS), a homodimer, is a molecular target for anticancer therapy. Recently purified TS preparations, isolated from mammalian tissues, were found to be nitrated, suggesting this modification to appear endogenously in normal and tumor tissues.

Tyrosine nitration affects thymidylate synthase properties.

Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C.

The aromaticity of 5,6-dihydroborauracil, borauracil and benzoborauracil systems.

The nucleus-independent chemical shift (NICS) indices of aromaticity, calculated for four boron compounds, 4-hydroxy-5,6-dihydroborauracil, 4-hydroxyborauracil, borazine and 4-hydroxybenzoborauracil, and parent uracil, were analyzed in parallel with the NMR properties, in order to learn more about the aromaticity of those heterocyclic systems. The existence of a unique solvent-dependent aromaticity of 4-hydroxyborauracil is indicated.

Mechanism of influence of phosphorylation on serine 124 on a decrease of catalytic activity of human thymidylate synthase.

Regulation by phosphorylation is a well-established mechanism for controlling biological activity of proteins. Recently, phosphorylation of serine 124 in human thymidylate synthase (hTS) has been shown to lower the catalytic activity of the enzyme. To clarify a possible mechanism of the observed influence, molecular dynamics (MD), essential dynamics (ED) and MM-GBSA studies were undertaken.

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.

The effect of 5-substitution in the pyrimidine ring of dUMP on the interaction with thymidylate synthase: molecular modeling and QSAR.

Thymidylate synthase (TS) is a target enzyme for a number of anticancer agents including the 5-fluorouracil metabolite, FdUMP. The present paper reports on molecular modeling studies of the effect of substitution at C(5) position in the pyrimidine ring of the TS substrate, dUMP, on the binding affinity for the enzyme. The results of molecular dynamics simulations show that the binding of C(5) analogues of dUMP to TS in the binary complexes does not undergo changes, unless a substituent with a large steric effect, such as the propyl group, is involved.

5,10-Methylene-5,6,7,8-tetrahydrofolate conformational transitions upon binding to thymidylate synthase: molecular mechanics and continuum solvent studies.

We applied the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach to evaluate relative stability of the extended (flat) and C-shaped (bent) solution conformational forms of the 5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) molecule in aqueous solution. Calculations indicated that both forms have similar free energies in aqueous solution but detailed energy components are different. The bent solution form has lower intramolecular electrostatic and van der Waals interaction energies.

Relative free energies of binding to thymidylate synthase of 2- and/or 4-thio and/or 5-fluoro analogues of dUMP.

Free energy perturbation calculations have been applied to evaluate the relative free energies of binding of 2'-deoxyuridine-5'-monophosphate (dUMP) and its 2- and/or 4-thio and/or 5-fluoro analogues to the wild-type E. coli thymidylate synthase (ecTS). The results accurately reproduce experimentally measured differences in the free energy of binding of dUMP versus 5-fluoro-dUMP to thymidylate synthase.