Phosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N4-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.g. phosphorylation, by comparing sensitivities to inhibition by two slow-binding inhibitors, 5-fluoro-dUMP and N4-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparations, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered both the pattern of dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters, with each inhibitor studied. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of the inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N5,10-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N4-hydroxy-dCMP was found to be strongly dependent on [Mg2+], cations demonstrated previously to also influence the activity of endogenous mouse TS isolated from tumour cells

Limits of duty to help others

Celem pracy jest przedstawienie własnej krytyki stanowiska Petera Singera dotyczącego obowiązku niesienia pomocy innym ludziom. Bo to zrobić, przeprowadzam krytyczną analizę argumentacji Singera oraz zasady moralnej symetrii, której używa do wsparcia swojej tezy. Następnie przyjmuję jego argumentację i używam koncepcji prawa do obrony Judith Thomson, by wykazać, że wszyscy potrzebujący ludzie, przy danych założeniach, mogą zostać potraktowani jako zagrożenie. Ma to wskazać luki zarówno w konecpcji Singera jak i Thomson. Jako aleternatywę dla wyznaczania moralnych obowiązków wobec potrzebujących, proponuję użycie koncepcji Thomson wraz z zasadą moralnej symetrii.The main objective of this paper is to present own critique of Peter Singer's position about the duty to help people in need. To achieve this I carry out a critical analysis of his arguments and moral symmetry principle that he uses to support his claims. Next, I take those arguments and Judith Thomson's concept of the right to killing in self-defense, to show that with Singer's assumption, it's possible to categorize people in need as an innocent treat. It shows the weaknesses of Singer's and Thomson's points. As a reasonable alternative to Singer's position, I propose to use moral symmetry principle and Thomson's right to self-defense as arguments for minimal duty to help others

Acceptability of killing in self-defence

Praca jest próbą krytycznej analizy tekstu J. Thomson "Obrona Własna", w której przedstawione oraz wsparte argumentami są następujące tezy:1.Człowiek nigdy nie traci prawa do życia, dlatego zabicie kogoś może być jedynie usprawiedliwione.2.Dopuszczalność zabicia w obronie własnej nie zależy od intencji atakującego.3.Usprawiedliwione jest zabicie w obronie osób trzecich.4.Zabicie w przypadku niezawinionego zagrożenia nie jest usprawiedliwione.Przedstawiona zostaje także definicja "napastnika", która oparta jest na klasycznej teorii wojny i to ona stanowi klucz do analizy wszelkich przykładów.This thesis is an attempt at critical analysis of J.J. Thomson’s "Self-Defense" in which the following statements are presented and validated:1. A person never loses their right to live, therefore killing is never permissible and must be justified.2. Justification of killing in self-defense does not depend on aggressor’s intentions. 3. Killing in defense of other people can be justified.4. In case of the innocent threat, killing the aggressor is not justified.The paper also states the definition of an aggressor based on classical theory of war which is crucial for understanding and analysis of all cases

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. Here we present in silico work on 627 sixtapeptide β-sheet breakers (BSBs) containing consecutive three aromatic residues. Three of these BSBs caused dissociation of one or two β-amyloid chains from U-shaped β-amyloid protofibril model 2BEG after docking and subsequent molecular dynamics simulations. Thorough analysis of our results let us postulate that the first steps of binding these successful BSBs involve π–π interactions with stacked chains of F19 and later also with F20 (F3 and F4 in 2BEG model of protofibril). The consecutive location of aromatic residues in BSBs makes them more attractive for chains of stacked F3 and F4 within the 2BEG model. Spotted by us, BSBs may be prospective lead compounds for an anti-Alzheimer’s therapy

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. This difference indicates an asymmetrical effect of the binding of Raltitrexed to two independent mTS molecules. Conformational changes leading to a ligand-induced closing of the active site cleft are observed by comparing the crystal structures of mTS in three different states along the catalytic pathway: ligand-free, dUMP-bound, and dUMP- and Raltitrexed-bound. Possible interaction routes between hydrophobic residues of the mTS protein N-terminal segment and the active site are also discussed

X-ray Crystal and Ab Initio Structures of 3 ,5 -di-O-Acetyl-N(4)-Hydroxy-2 -Deoxycytidine and Its 5-Fluoro Analogue: Models of the N(4)-OH-dCMP and N(4)-OH-FdCMP Molecules Interacting with Thymidylate Synthase

The crystal and molecular structures of the 3 ,5 -di-O-acetyl-N (4)-hydroxy-2 -deoxycytidine molecule and its 5-fluoro congener have been determined by X-ray single crystal diffraction. The 3 ,5 -di-O-acetyl-N (4)-hydroxy-5-fluoro-2 -deoxycytidine molecule crystallizes in the space group C2 with the following unit cell parameters: a = 21.72Å, b = 8.72Å, c = 8.61Å, and β = 90.42 • . 3 ,5 -di-O-acetyl-N (4)-hydroxy-2 -deoxycytidine also belongs to the monoclinic space group C2 and the unit cell parameters are: a = 39.54Å, b = 8.72Å, c = 22.89Å, and β = 95.26 • . The nonfluorine analogue demonstrates a rare example of crystal structure with five symmetry-independent molecules in the unit cell. All the molecules in both crystal structures have the sugar residue anti oriented with respect to the base, as well as have the N(4)-OH residue in cis conformation relatively to the N(3)-nitrogen atom. In addition to the molecular geometries from X-ray experiment, the optimized molecular geometries have been obtained with the use of theoretical ab initio calculations at the RHF/6-31G(d) level. The corresponding geometric parameters in the molecules of 3 ,5 -di-O-acetyl-N (4)-hydroxy-2 -deoxycytidine and its 5-fluoro congener have been compared. The differences including the C(5) C(6) bond shortening and C(4) C(5) C(6) angle widening in the fluorine analogue are discussed in this paper in relation to the molecular mechanism of enzyme, thymidylate synthase, inhibition by N (4)-hydroxy-2 -deoxycytidine monophosphate and its 5-fluoro congener. KEY WORDS: Crystal structure; ab initio structure; nucleoside analogues of N (4)-OH-dCMP and N (4)-OHFdCMP; thymidylate synthase

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. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson–Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme–ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the ”abortive reaction” inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form