Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

For any detailed NMR conformational study of a protein-ligand complex it is essential to have specific resonance assignments. We have now assigned the pyrophosphate 31P resonances in spectra of NADPH bound to Lactobacillus casei dihydrofolate reductase (DHFR) by using a combination of 1H-31P-heteronuclear shift-correlation (HETCOR), 1H-31P-heteronuclear multiple-quantum-coherence correlation spectroscopy (HMQC-COSY), 1H-1H COSY, homonuclear Hartmann-Hahn (HOHAHA) and NOE spectroscopy (NOESY) experiments. The nicotinamide pyrophosphate phosphorus, P(n), has been unequivocally assigned to a signal (-14.07 ppm) which shows a large 3JP-O-C-H coupling constant. Such a coupling constant when combined with the appropriate Karplus relationship provides conformational information about the P-O-C-H torsion angle. The torsion angle changes by 65 degrees +/- 10 degrees for the binary complex compared with the value in free NADPH. The observed coupling constants for the binary (DHFR--NADPH) and ternary (DHFR--NADPH--methotrexate) complexes (12.3 and 10.5 +/- 0.6 Hz, respectively) indicate that the pyrophosphate group has a similar conformation in the two complexes.
 ...
PMID:31P-NMR assignment and conformational study of NADPH bound to Lactobacillus casei dihydrofolate reductase based on two-dimensional 1H-31P-heteronuclear and 1H-detected 1H-31P-shift-correlation experiments. 174 Jan 27

The 5-deaza and 5,8-dideaza analogues of N alpha-pteroyl-L-ornithine (Pter-Orn), the 5-deaza, 8-deaza, and 5,8-dideaza analogues of N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-Orn), and the N delta-carboxymethyl derivative of N alpha-(4-amino-4-deoxy-N10-methylpteroyl)-L-ornithine (mAPA-Orn) were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of tumor cell growth in culture. Reductive amination of 2-acetamido-6-formylpyrido[2,3-d]pyrimidine-4(3H)-one with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate followed by removal of the blocking groups afforded the 5-deaza analogue of Pter-Orn, whereas N-alkylation of methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate with 2-amino-6-(bromomethyl)quinazolin-4(3H)-one and deprotection gave the corresponding 5,8-dideaza analogue. Reductive coupling of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile and 4-aminobenzoic acid followed by reaction with 95-97% formic acid yielded 4-amino-4-deoxy-5-deaza-N10-formylpteroic acid, which on condensation with methyl N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection gave the 5-deaza analogue of APA-Orn. A similar sequence starting from 2,4-diamino-quinazoline-6-carbonitrile led to the corresponding 5,8-dideaza compound, whereas treatment of 2,4-diamino-pyrido[3,2-d]pyrimidine-6-methanol with phosphorus tribromide followed by condensation with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection afforded the 8-deaza analogue. For the preparation of the N delta-carboxymethyl derivative of mAPA-Orn, N alpha-(benzyloxycarbonyl)-L-ornithine was subjected to N delta-monoalkylation with glyoxylic acid and sodium cyanoborohydride, followed by N delta-acylation with ethyl trifluoroacetate, N alpha-deprotection by hydrogenolysis, condensation with 4-amino-4-deoxy-N10-methylpteroic acid, and N delta-deprotection by gentle treatment with ammonia. The 2,4-diamino derivatives all inhibited the growth of tumor cells in culture, with IC50 values of 0.2-2 microM, and inhibited purified DHFR with IC50 values of 0.02-0.08 microM. Deletion of ring nitrogens and N delta-carboxymethylation both increased potency in the cell growth assay; however, the ornithine derivatives were less potent than aminopterin or methotrexate.
 ...
PMID:Synthesis and in vitro biological activity of new deaza analogues of folic acid, aminopterin, and methotrexate with an L-ornithine side chain. 201 22

31P-NMR spectra of NADPH and NADPH bound to Lactobacillus casei dihydrofolate reductase have been recorded using the techniques of cross-polarization, magic-angle spinning and high-power proton-decoupling on both lyophilized and hydrated samples. Previous studies on the lyophilized complex of L. casei dihydrofolate reductase with NADPH and methotrexate, measuring the isotropic shifts and principal components of the chemical shift tensors, have shown that the 2'-phosphate group of bound NADPH exists as a mixture of the dianionic and monoanionic states [Gerothanassis, I. P, Barrie, P. J., Birdsall, B. & Feeney, J. (1994) Eur J. Biochem. 226, 211-218]. In the present study on hydrated samples, the characterization of the isotropic shift and chemical shift tensors of the 2'-phosphate signal indicates that the 2'-phosphate is almost exclusively in the dianionic state. This is in agreement with earlier 31P-NMR studies in solution [Feeney, J., Birdsall, B., Roberts, G. C. K. & Burgen, A. S. V. (1975) Nature 257, 564-566]. In experiments examining progressively hydrated (6%, 12%, 15%, by mass) samples, the observed signals become increasingly narrower probably because the microenvironments of the 31P nuclei become more homogeneous upon sample hydration. Chemical exchange between mobile water molecules and bound protons close to individual sites on NADPH has been indirectly monitored on a hydrated sample (15% water, by mass) using a pulse sequence proposed by Harbison and coworkers [Harbison, G. S., Roberts, J. E., Herzfeld, J. & Griffin, R. G. (1988) J. Am. Chem. Soc. 110, 7221-7223]. In this experiment, the two diphosphate signals are totally suppressed while the 2'-phosphate phosphorus signal remains: this indicates a significant polarization of the 2'-phosphate nuclei from protons in exchange with those of mobile water molecules.
 ...
PMID:31P solid-state NMR measurements used to detect interactions between NADPH and water and to determine the ionisation state of NADPH in a protein-ligand complex subjected to low-level hydration. 863 40