Thesis (M.Sc.)--York University, 2006. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references (leaves 123-137). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR19750

<p> Dihydrofolate reductase from a subline of the L1210 lymphoma was purified by affinity chromatography using substituted Sepharose -4B to which was coupled methotrexate, a specific, tight binding inhibitor of the enzyme. The purified enzyme was subjected to disc gel electrophoresis at pH 8.5. At least two bands of activity were detected on the gel by the formation of a reduced formazan. Their ratios were dependent on enzyme concentration. Similar bands were found in the presence of EDTA (10^-6M), 4M and SM urea. When a substrate, NADPH (5xl0^-5M), was added to the buffers used in electrophoresis, three bands of enzyme activity were present in a fixed ratio which was independent of enzyme concentration. Protein bands showed a different but constant ratio. When folate replaced dihydrofolate as substrate in the assay mixture, the bands of activity corresponded at high concentrations of the enzyme. When activity was detected in the presence of an increasing concentration of methotrexate, different inhibition of the bands resulted. Preliminary experiments with crude extracts of the same subline gave activity profiles with multiple peaks.</p> / Thesis / Master of Science (MSc)

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The main objective of the research described in this thesis has been the design and synthesis of inhibitors of the enzyme dihydrofolate reductase (DHFR) intended for local administration and devoid of systemic side-effects. The blocking of the enzymatic activity of DHFR is a key element in the treatment of many diseases, including cancer, bacterial and protozoal infections, and also opportunistic infections associated with AIDS (Pneumocystis carinii pneumonia, PCP). Recent research indicates that the enzyme also is involved in various autoimmune diseases, e.g., rheumatoid arthritis, inflammatory bowel diseases and psoriasis. Many useful antifolates have been developed to date although problems remain with toxicity and selectivity, e.g., the well-established, classical antifolate methotrexate exerts a high activity but also high toxicity. The new antifolates described herein were designed to retain the pharmacophore of methotrexate, but encompassing an ester group, so that they also would serve as substrates for the endogenous hydrolytic enzymes, e.g., esterases. Such antifolates would optimally comprise good examples of soft drugs because they in a controlled fashion would be rapidly and predictably metabolized to non-toxic metabolites after having exerted their biological effect at the site of administration. A preliminary screening of a large series of simpler aromatic esters as model compounds in a biological assay consisting of esterases from different sources was performed. The structural features of the least reactive ester were substituted for the methyleneamino bridge in methotrexate to produce analogues that were chemically stable but potential substrates for DHFR as well as for the esterases. The new inhibitor showed desirable activity towards rat liver DHFR, being only eight times less potent then methotrexate. Furthermore, the derived metabolites were found to be poor substrates for the same enzyme. The new compound showed good activity in a mice colitis model in vivo, but a pharmacokinetic study revealed that the half-life of the new compound was similar to methotrexate. A series of compounds characterized by a high lipophilicity and thus expected to provide better esterase substrates were designed and synthesized. One of these analogues in which three methoxy groups were substituted for the glutamic residue of methotrexate exhibited favorable pharmacokinetics. This compound is structurally similar to another potent DHFR inhibitor, trimetrexate, used in the therapy of PCP (vide supra). The new inhibitor that undergoes a fast metabolism in vivo is suitable as a model to further investigate the soft drug concept.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Šedivá Supervisor: Mgr. Eva Novotná, Ph.D. Title of diploma thesis: Cloning, expression and purification of mycobacterial dihydrofolate reductase Dihydrofolate reductase is an enzyme essential for the metabolism of folic acid - it catalyzes the reduction of dihydrofolate to tetrahydrofolate. Tetrahydrofolate is an important cofactor involved in one-cabron transfer reactions. Dihydrofolate reductase plays a key role in the synthesis of DNA, RNA and proteins. Dihydrofolate reductase was also found in M. tuberculosis. This bacterium is the most common causative agent of tuberculosis in humans. Thus dihydrofolate reductase could be a potential target for the design of new antituberculotics. The recombinant protein dihydrofolate reductase was prepared in several steps. The coding sequence of the protein was first amplified by polymerase chain reaction. A recombinant plasmid, obtained by the ligation of an amplified segment of DNA with plasmid pET-28b(+), was transformed into competent cells E. coli strain BH101 by the heat shock method. Cells E. coli strain BL21(DE3) were used for the protein expression. The expression was induced by the addition of isopropyl-β-D-...

Protein motions are complex, including occurring at different time scales, and their roles in enzyme-catalyzed reactions have always been of great interest among enzymologists. In order to characterize the potential factors that play a role on the chemical step of enzymatic reactions, variants of dihydrofolate reductase have been used as a benchmark system to study the motions of proteins correlated with the chemical step. A “global dynamic network” of coupled residues in Escherichia coli dihydrofolate reductase (ecDHFR), which assists in catalyzing the chemical step, has been demonstrated through quantum mechanical/molecular mechanical and molecular dynamic (QM/MM/MD) simulations, as well as bioinformatic analyses. A few specific residues — M42, G121, and I14 — were shown to function synergistically with measurements of single turnover rates and the temperature dependence of intrinsic kinetic isotope effects (KIEsint) of site-directed mutants. Although similar networks have been found in other enzymes, the general features of these networks are still unclear. This project focuses on exploring homologous residues of the proposed global network in human DHFR through computer simulations and measurements of the temperature dependence of KIEsint. The mutants M53W and S145V, both remote residues, showed significant decreases in catalytic efficiency. Non-additive isotope effects on activation energy were observed between M53 and S145, indicating their synergistic effect on hydride transfer in human DHFR. Apart from the effects of the conserved residues, we also extend our studies to exploring three potential phylogenetic events that account for the discrepancies between E. coli and human DHFR. They are L28, PP insertion and PEKN insertions by phylogenetic sequence analysis. Two of them (N23PP and G51PEKN E. coli DHFR) have been proved to be important both by MD simulation and experimental probe of KIEs measurement. The experiments have found that PP insertion itself rigidified the M20 loop and motions coupled to hydride transfer were impaired, however, loop rigidification was improved after incorporating PEKN. Furthermore, deletion of PP and PEKN of the engineered human enzyme also show a similar outcome. However, the effect of the key residue of L28 is not clear. In this project, we have step-wise engineered the human DHFR to be like hagfish (F31M) and E. coli (F32L). And it is found out that there is an increase in the temperature dependence of KIEs when the enzyme was bacterilized into a more primitive variant. This indicates that not only is residue F32 important and correlated with the chemical step as indicated by bioinformatic studies, but it is possible to trace the evolutionary trajectory. A triple mutation F32L-PP26N-PEKN62G on the human DHFR was also conducted, and it is not surprising to find out that the temperature dependence of KIEs has retained its behavior like wild-type human DHFR. These results suggest that the three predicted phylogenetically coherent events coevolved together to maintain the evolutionary preservation of the protein dynamics to enable H-tunneling from well-reorganized active sites. As has been indicated by the previous project, as the enzyme evolves, the active site of the enzyme will “reorganize” to form the optimal transition state for chemical step (from F32L-F32M-wild type DHFR). Here in this project, we aimed to systematically address this point of view through a series of cyclic permutation DHFR from directed evolutions. As this primitive enzyme is 7 orders of magnitude less efficient than the well-evolved human DHFR, together with four generations of evolved variants (cp, cp’ and cp”), this provides a good model system for explorations of the molecular basis of enzyme evolution. It is found that the organizations of transition state are improved before the catalytic efficiency is enhanced as the enzyme evolves.

<p>The main objective of the research described in this thesis has been the design and synthesis of inhibitors of the enzyme dihydrofolate reductase (DHFR) intended for local administration and devoid of systemic side-effects. The blocking of the enzymatic activity of DHFR is a key element in the treatment of many diseases, including cancer, bacterial and protozoal infections, and also opportunistic infections associated with AIDS (<i>Pneumocystis carinii</i> pneumonia, PCP). Recent research indicates that the enzyme also is involved in various autoimmune diseases, e.g., rheumatoid arthritis, inflammatory bowel diseases and psoriasis. Many useful antifolates have been developed to date although problems remain with toxicity and selectivity, e.g., the well-established, classical antifolate methotrexate exerts a high activity but also high toxicity. The new antifolates described herein were designed to retain the pharmacophore of methotrexate, but encompassing an ester group, so that they also would serve as substrates for the endogenous hydrolytic enzymes, e.g., esterases. Such antifolates would optimally comprise good examples of <i>soft drugs</i> because they in a controlled fashion would be rapidly and predictably metabolized to non-toxic metabolites after having exerted their biological effect at the site of administration.</p><p>A preliminary screening of a large series of simpler aromatic esters as model compounds in a biological assay consisting of esterases from different sources was performed. The structural features of the least reactive ester were substituted for the methyleneamino bridge in methotrexate to produce analogues that were chemically stable but potential substrates for DHFR as well as for the esterases.</p><p>The new inhibitor showed desirable activity towards rat liver DHFR, being only eight times less potent then methotrexate. Furthermore, the derived metabolites were found to be poor substrates for the same enzyme. The new compound showed good activity in a mice colitis model <i>in vivo</i>, but a pharmacokinetic study revealed that the half-life of the new compound was similar to methotrexate. A series of compounds characterized by a high lipophilicity and thus expected to provide better esterase substrates were designed and synthesized. One of these analogues in which three methoxy groups were substituted for the glutamic residue of methotrexate exhibited favorable pharmacokinetics. This compound is structurally similar to another potent DHFR inhibitor, trimetrexate, used in the therapy of PCP (<i>vide supra</i>). The new inhibitor that undergoes a fast metabolism <i>in vivo</i> is suitable as a model to further investigate the soft drug concept.</p>