Adam Jarmula

Nencki Institute of Experimental Biology | NENCKI · Neurobiology Center

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...

Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCM...

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, ia...

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, histi...

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 in 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 s...

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...

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 sampl...

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 N5,10-methylenetetrahydrofolate (meTHF) have been determined and deposited in Protein Data Bank under the accession codes 3IHI, 4E5O and 5FCT, respectively. The structures show a s...

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 c...

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 dependences of the inactivation rate on time and accompanied by differing inhibition parameters. Considering importance for chemother...

Predicting FRET pathways in proteins using computer simulation techniques is very important for reliable interpretation of experimental data. A novel and relatively simple methodology has been developed and applied to purine nucleoside phosphorylase (PNP) complexed with a fluorescent ligand — formycin A (FA). FRET occurs between an excited Tyr resi...

Thymidylate synthase ThyA (EC 2.1.1.45; encoded by the Tyms gene), having been for 60 years a molecular target in chemotherapy, catalyses the dUMP pyrimidine ring C(5) methylation reaction, encompassing a transfer of one-carbon group (the methylene one, thus at the formaldehyde oxidation level) from 6R-N5,10-methylenetetrahydrofolate, coupled with...

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. T...

Legend to Supplementary Figure 1S: Two antiparallel arrays of N-terminal non-polar residues stabilizing the molecular crystal packing of the mTS-dUMP-Raltitrexed complex structure.

Purine nucleoside phosphorylase (PNP) is the enzyme which uses orthophosphate to cleave the N-glycosidic bond of β- (deoxy)ribonucleosides. Formycin A (FA) is its aromatic, com- petitive inhibitor with strong fluorescence capabilities. Absorp- tion and emission spectra of PNP result from the presence of ty- rosine residues and are characterized wit...

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. As 31P NMR studies of both the phosphorylated protein and of potassium phosphoramida...

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 . The mechanism by which peptide inhibition takes place remains...

To solve the inhibition mechanism of thymidylate synthase (TS) by N4-hydroxy-dCMP (N4-OH-dCMP), crystallographic studies were undertaken. Structures of three mouse TS (mTS) complexes with the inhibitor were solved, based on crystals formed by the enzyme protein in the presence of either only N4-OH-dCMP [crystal A, belonging to the space group C 1 2...

Crystal structures were solved of the binary complexes Trichinella spiralis and Caenorhabditis elegans thymidylate synthases with deoxyuridine monophosphate (dUMP), with crystals obtained by the vapor diffusion method in hanging drops. For the T. spiralis thymidylate synthase-dUMP complex, the diffraction data were collected at the BESSY Synchrotro...

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 t...

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 elega...

Quantum mechanical, molecular mechanics and molecular dynamics (MD) methods were used to investigate initial steps of 2′-deoxyuridine-5′-monophosphate (dUMP) methylation catalysed by the thymidylate synthase (TS) enzyme. The amino acid residues surrounding the active site within a 10 Å radius sphere were modelled with the combined quantum mechanica...

Inhibitors of thymidylate synthase (TS) play an essential role in the pharmacological management of several tumors. Two antifolates, Raltitrexed and Pemetrexed, are licensed anticancer drugs, with Pemetrexed, unlike Raltitrexed, undergoing further intense clinical development. Other antifolate TS inhibitors, recently/currently tested in clinical st...

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...

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 dynamic...

Crystal structures of mouse thymidylate synthase (mTS), liganded with either 2'-deoxyuridine 5'-monophosphate (dUMP) or the sulfate anion, have been determined and deposited in Protein Data Bank under the accession codes 3IHH and 3IHI, respectively. The structures show a strong overall similarity to the corresponding structures of rat (rTS) and hum...

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 t...

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 dynam...

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 = 21.72 Å, b = 8....

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...

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 bindi...

In order to explain different activities shown by 5-hydroxy-dUMP (substrate) and its close analogue 5-hydroxymethyl-dUMP (slow-binding inhibitor) in the reaction catalyzed by thymidylate synthase, studies have been undertaken involving (i) ab initio RHF simulations, (ii) comparative analysis of crystallographic structures available from CSD, and (i...

To explain the mechanism of the influence of fluorine substituent on the FdUMP activity in thymidylate synthase reaction, the aromaticity based on X-ray determined structures, factor analysis applied to structural data from CSD and ab initio RHF calculations were employed. It was found that fluorine substitution dearomatizes the pyrimidine ring, st...

In order to understand the influence on thymidylate synthase interactions with dUMP analogues of the pyrimidine ring 2- and/or 4-thio, and 5-fluoro substitutions, X-ray diffractions by crystals of 5-fluoro-dUrd and its 2- and 4-thio, and 2,4-dithio analogues were measured, the four structures solved and refined. The following conclusions were sugge...

In order to understand the influence on thymidylate synthase interactions with dUMP analogues of the pyrimidine ring 2- and/or 4-thio, and 5-fluoro substitutions, X-ray diffractions by crystals of 5-fluoro-dUrd and its 2- and 4-thio, and 2,4-dithio analogues were measured, the four structures solved and refined. The following conclusions were sugge...

The 2,4-dithio analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine have been synthesized by thiation of the previously described 2-thio analogues, and then phosphorylated enzymatically or chemically to yield 2,4-dithio-dUMP and 2,4-dithio-5-fluoro-dUMP. In striking contrast to the 2-thio and 4-thio analogues of dUMP, which are good substrates...

The 2,4-dithio analogues of 2′-deoxyuridine and 2′-deoxy-5-fluorouridine have been synthesized by thiation of the previously described 2-thio analogues, and then phosphorylated enzymatically or chemically to yield 2,4-dithio-dUMP and 2,4-dithio-5-fluoro-dUMP. In striking contrast to the 2-thio and 4-thio analogues of dUMP, which are good substrates...

Crystal and molecular structures of 4-(4-methoxyphenyl)-2,6-diphenylpyridine (I) and 1-methyl-4-(4-methoxyphenyl)-2,6-diphenyl pyridinium perchlorate (II) are repoded, with R=0.052 and 0.070, respectively. The lower precision of II is due to disorder of the perchlorate anion. A geometrical analysis of 8 p-substituted derivatives of anisole shows th...