Chicken liver DHFR

One possible interpretation of this type of rule is the enzyme ligand binding of trimethoprim with bacterial DHFR and chicken liver DHFR. (17,18)... 

This data shows a noticeable drop in binding affinity for trimethoprim and chicken liver DHFR. Figure 8 illustrates steric interaction between the 5-OMe of trimethoprim (green) with the sidechain of Tyr 31 of native chicken liver DHFR (red). There is no steric interaction seen between the 5-OMe of trimethoprim (green) and the sidechain of Phe 30 of L. casei DHFR (red). (Right view chicken liver DHFR Left View L. casei DHFR) It is known from x-ray crystallographic results that the sidechain of Tyr 31 of chicken liver DHFR rotates to accommodate trimethoprim. ( ... 

Fig. 11 Comparison of experimental STDs (shaded circles) and calculated STD values (solid line) from CORCEMA-ST method for the crystal structure of chicken liver DHFR/TMP complex. The optimized parameters are tl =0.101 ns tp = 20.43 ns t i = 0.81 ps Tm2 = 3.04 ps Tm3 = 3.26 ps leakage factor = 0.065 and NOE R-factor = 0.076. Reprinted with permission from [75] 2005, American Chemical Society...

A quick comparison of QSAR 1.82-1.84 reveals the strong similarity between the avian and mammalian models. In fact because of its increased stability, chicken liver DHFR has often been used as a surrogate for human DHFR in enzyme-inhibition studies. The intercepts, coefficients with Tr g and optimum tt q for avian (6.33, 1.01, 1.9), human (6.07, 1.07, 2.0), and mouse leukemia (6.12, 0.98, 1.76) can be compared to the corresponding values for P. carinii (6.48, 0.73, 3.99) and Leishmania major (5.05, 0.65, 4.54). ( SAR 1.81 and 1.87 are not included in the comparison because crude pigeon enzyme was used in... 

The ability of 7,8-dihydro and 5,6,7,8-tetrahydro derivatives of /,L- and flf,L-(IV.107) to inhibit DHFR was also assayed [135]. Activity was increased in both the di- and tetrahydro series, and, as expected, the activity of reduced mc,L-(IV.108) derivatives was intermediate between that of reduced /,l- and reduced f,L-derivatives. The most potent member of the series, 7,8-dihydro-d,L-(IV.107), had an IC50 value of 25 nM against purified L. casei and chicken liver DHFR (versus 10 and 20 nM, respectively, for MTX) and IC50 values of 0.057 and 2.3 nM against S. faecium and L. casei (versus 0.028 and 0.84 nM, respectively, for MTX). 

To the small extent to which trimethoprim binds to chicken liver DHFR, X-ray crystallography reveals it to be squeezed into a butterfly conformation, less energetically favourable than the position it occupies in the E. coli enzyme where the two rings are nearly perpendicular to one another (Matthews, 1981). 

E. coli enzyme. But, studies on the chicken liver enzyme may not be relevant to the human enzyme. Indeed, crystal studies of Oefner et al. (1988) on complexes of human DHFR with folate, methotrexate and trimethoprim itself do not support the explanation given by Matthews, and so we are still not sure of the basis for the species selectivity exhibited by trimethoprim. 

Fig. 2. Alignment of the amino acid sequence of H. volcanii DHFR with the amino acid sequences of DHFRs of E. coli, L. casei, and chicken liver. From Zusman et al. (1989), with permission.

In this example, the R group on the 2-nitrogen was restricted to an (3-X-phenyl) aromatic ring (205). Accurate Kf values were obtained from highly purified DHFR isolated from chicken liver. In most cases, 77 represented the hydrophobicity of the substituent except in certain instances where X = -OR or -CH2ZCeH4-Y.lt was ascertained that alkoxy substituents were not making direct hydro-... 

Another potent inhibitor of DHFR is trimethoprim. The crystal structures of two enzyme-inhibitor complexes (DHFR from Escherichia coli and chicken liver) have been determined [87]. Surprisingly, the inhibitor adopts different conformations in the two proteins. In the chicken liver enzyme, a butterfly-like conformation is observed (t,/t2 = -85°/102 ) in the bacterial enzyme, the aromatic rings are oriented nearly perpendicular to each other ( twisted conformation, r,/T2 = 177°/76°). The inhibitor shows higher affinity for the bacterial enzyme, and the lower energy of the twisted conformation may be partially responsible for this. 

The binding of the dansyl derivative (VIII.307) to DHFR from L. casei, human lymphoblasts (WI-L2 cells), and chicken liver has been studied [367, 373, 374]. The ratio [I]/[E] for extrapolated 100% inhibition of enzyme activity by spectrophotometric measurement was found to be 1.0-1.4 for (VIII.307), 1.9-3.5 for (VIII.264), and 1.0 for MTX. This demonstrated that the dansyl group was well tolerated by the active site of DHFR, and that the... 

C. Hansch, B. A. Hathaway, Z. R. Guo, C. D. Selassie, S. W. Dietrich, J. M. Blaney, R. Langridge, K. W. Volz, and B. T. Kaufman,/. Med. Chem., 27,129 (1984). Crystallography, QSAR and Molecular Graphics in a Comparative Analysis of the Inhibition of DHFR from Chicken Liver and Lactobacillus casei by 4,6-Diamino-l,2-dihydro-2,2-di-methyl-1 -(substituted-phenyl)-s-triazines. 

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