Donnell Bowen

Ligand Binding and Circular Permutation Modify Residue Interaction Network in DHFR

Residue interaction networks and loop motions are important for catalysis in dihydrofolate reductase (DHFR). Here, we investigate the effects of ligand binding and chain connectivity on network communication in DHFR. We carry out systematic network analysis and molecular dynamics simulations of the native DHFR and 19 of its circularly permuted variants by breaking the chain connections in ten folding element regions and in nine nonfolding element regions as observed by experiment. Our studies suggest that chain cleavage in folding element areas may deactivate DHFR due to large perturbations in the network properties near the active site. The protein active site is near or coincides with residues through which the shortest paths in the residue interaction network tend to go. Further, our network analysis reveals that ligand binding has “network-bridging effects” on the DHFR structure. Our results suggest that ligand binding leads to a modification, with most of the interaction networks now passing through the cofactor, shortening the average shortest path. Ligand binding at the active site has profound effects on the network centrality, especially the closeness.

Modulation of high-dose methotrexate toxicity by a non-toxic level of 5-fluorouracil☆

High-dose methotrexate (MTX) toxicity is reduced by a non-toxic dose of 5-fluorouracil (FU) when these agents are used in combination. Changes in the hematopoietic system (platelets, erythrocytes, leukocytes, hemoglobin, and hematocrit), ileal tissue, body weight, and mean survival were used as parameters to assess toxicity. For all parameters studied, there were no significant differences between the scheduling of MTX (245 mg/kg) after a priming dose of FU (25 mg/kg), simultaneous MTX and FU, FU alone, and control. However, sequential treatment with MTX followed by FU, and MTX alone resulted in: a marked decrease in the hematopoietic parameters; significant morphological changes in ileal tissue; a reduction of body weight; and increased mortality of animals. Hence, this study suggests that FU, a cytotoxic agent, may protect against MTX toxicity and improve its therapeutic index when FU administration precedes MTX or when these agents are given simultaneously.

Rate-limiting steps in the interactions of fluoropyrimidines and methotrexate☆

Rate-limiting steps are defined between methotrexate (MTX) and 5-fluorouracil (FU) or 5-fluorodeoxyuridine (FUdR) and [14C]-formate incorporation into RNA, DNA and protein as a function of the basal rate of dTMP synthesis. When Ehrlich cells are incubated with 0.1 microM FU dR, 1 microM FU and 50 microM MTX for 1-35 min. [3H]-deoxyuridine (UdR) incorporation into DNA is maximally inhibited within 1, 10 and 15 min respectively. The delay in suppression of [3H]-UdR incorporation into MTX-exposed cells compared to cells exposed to FU or FUdR is related to the slow transport of MTX and the increasing free intracellular MTX levels. Influx of MTX is 4 and 10 times slower than FU and FUdR respectively. At 2.5, 5, 10 and 15 min the free intracellular MTX levels (nmol/g dry wt) are 5.8, 7.4, 8.7 and 8.8 respectively. Free intracellular FdUMP is identified 1 min after exposure of cells to FU and FUdR. Antagonism to MTX-suppression of [14C]-formate incorporation into RNA, DNA and protein occurs when cells are simultaneously exposed to MTX and FU or FUdR. However, [14C]-formate incorporation into RNA, DNA and protein is maximally inhibited when Ehrlich tumor cells are incubated with 50 microM MTX for 10 min and then exposed to 1 microM FU for 1 min (a time in which free intracellular MTX is maximal and [3H]-UdR incorporation is maximally suppressed). Hence the sequence and time of administration of FU or FUdR and MTX inhibition of formate incorporation into RNA, DNA and protein is related to the rate of (a) FU, FUdR and MTX transport, (b) FU and FUdR metabolism to FdUMP and (c) generation of maximal free intracellular MTX.

Interaction of 5′-deoxy-5-fluorouridine and methotrexate

Methotrexate (MTX) toxicity is reduced significantly by a non-toxic dose of 5′-deoxy-5-fluorouridine (5′dFUr). Changes in the hematopoietic system (platelets, erythrocytes, leukocytes, and hematocrit), ileal tissue, and body weight were used as parameters to assess toxicity. MTX treatment alone resulted in: (a) a reduction of body weight; (b) significantly morphological changes in ileal tissue; and (c) a marked decrease in the hematopoietic parameters. Sequential treatment with MTX followed by 5′dFUr resulted in reversal of MTX depression of animal body weight and ileal tissue necrosis, and partial reversal in MTX toxicity to the hematopoietic system. Also, for all parameters studied, there were no significant differences between scheduling of MTX after a priming dose of 5′dFUr, 5′dFUr alone, and control. Hence, this study suggests that 5′dFUr is a pharmacological antidote for MTX toxicity, and, therefore, 5′dFUr in combination with MTX may provide a basis whereby more intense and effective MTX therapy may be given.

The interaction between fluoropyrimidines and methotrexate, and [14C]-formate incorporation into nucleic acids and protein

SummaryChanges are reported in [14C]-formate incorporation into nucleic acids and protein of Ehrlich ascites tumor cells during exposure to methotrexate (MTX) and fluoropyrimidines. The rate of [14C]-formate incorporation into RNA, DNA, and protein in the presence of only MTX was inhibited by 82%, 91%, and 75% respectively, when compared with control rates. However, in the presence of 5-fluorodeoxyuridine (FdUrd) plus MTX, formate incorporation into RNA, DNA, and protein was inhibited by 67%, 85%, and 66%. Incubation of cells in vitro with [3H]-dihydrofolate (DHF) results in its rapid conversion to [3H]-tetrahydrofolate (THF). The THF/DHF ratio from the soluble fraction of cells that were incubated with [3H]-DHF was 43% greater in the presence of FdUrd and MTX than in the presence of MTX alone. As the rate of [3H]-dUrd incorporation into DNA was reduced by 88% and 99% by pretreating cells with 0.1 μM and 1 μM FdUrd, respectively, the inhibitory effect of MTX on [14C]-formate incorporation into (a) RNA was decreased by 63% and 46%; (b) DNA was decreased by 74% and 61%; and (c) protein was decreased by 63% and 32%. These data suggest that fluoropyrimidines can antagonize the effects of MTX on purines or nucleic acid synthesis and protein synthesis by preventing the consumption of THF for dTMP synthesis.

Molecular modelling of trimethoprim complexes of human wild-type and mutant dihydrofolate reductases: identification of two subsets of binding residues in the antifolate binding site

Computer‐assisted molecular modelling was used to generate structures for the trimethoprim (TMP):NADPH:dihydrofolate reductase (DHFR) ternary complexes for human wild‐type DHFR and for five DHFR mutants (L22R, L22F, F31S, F31W and Q35P). The mutants correspond to DHFR proteins that have been isolated from tissues exposed to chronic or high dose methotrexate (MTX) and show decreased sensitivity to antifolate inhibition. Analysis of the TMP:DHFR interactions suggest the presence of two subsets of TMP binding residues in the DHFR antifolate binding site. One subset of these residues (GLU30, PHE34, ILE60 and VAL115) are common to each DHFR complex studied and are referred to as core residues. The other TMP binding residues vary among the DHFR complexes studied and are referred to as noncore residues. The core residues exhibit a greater number of TMP contacts/residue and form more stable TMP interactions than noncore residues. Additionally, the core and noncore residues make contact with different regions of the TMP structure. Information presented here provides additional insight into the design of new agents for the improved inhibition of wild‐type DHFR and the simultaneous inhibition of both wild‐type and mutant DHFR molecules. Copyright

Interaction Energy Analysis of Nonclassical Antifolates with Human Dihydrofolate Reductase

Abstract The x-ray structure of the PTX:NADPH:L22F human mutant DHFR ternary complex was used as a structural template to generate structural models for the following wild type DHFR complexes: PTX:DHFR:NADPH, TMP:DHFR:NADPH, EPM:DHFR:NADPH, and TMQ:DHFR:NADPH. Each of these complexes were subsequently modeled in a 60 Å cube of explicit water and minimized to a rms gradient of from 1.0-3.0·10-5 kcal·Å-1. For each complex, interaction energies were calculated for the antifolate interaction with each of the following: the DHFR binding site residues, the entire DHFR protein, the solvated complex (containing DHFR, NADPH, and solvent water), water alone, and NADPH. Additionally, each antifolate was subdivided into distinct substructural regions and interaction energy calculations were performed in order to evaluate their contributions to overall antifolate interaction. Each antifolate showed its most stable interaction with the solvated complex. Substructural regions which consisted of a nitrogen containing aromatic ring system contributed most to the stability of the antifolate interactions, while the hydrocarbon aromatic rings, methoxy, and ethoxy groups showed much less stable interaction energies. Since the different substructural regions of nonclassical antifolates differ in their contributions to overall antifolate binding, those substructural regions which exhibit relatively unfavorable interaction energies may constitute important targets in the design of improved DHFR inhibitors.