L. casei DHFR

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. ( ... 

In addition to nonlinear lipophilicity relationships for the transport and distribution of drugs, nonlinear relationships on molar refractivity are frequently observed in QSAR studies of enzyme inhibition data (provided that MR values are scaled by a factor of 0.1, as usual) [60,63,64,66-68]. Two such examples are given in Eq. (63) (Escherichia coli DHFR) and Eq. (64) (Lactobacillus casei DHFR) [101]. The differences between both models could be explained after the 3D structure of the enzyme became known. Whereas all substituents of a benzyl ring contribute to biological activities in E. coli DHFR, only the 3- and 4-substituents show up in the QSAR model for L. casei DHFR but not the 5-substituents. This results from a narrower binding pocket in L. casei DHFR a (3-branched leucine hinders the accommodation of 5-substituents, whereas a more flexible methionine in the same position of E. coli DHFR opens a wider binding pocket [101] ... 

In antibacterial assays against L. casei and S. faecium, the 11-thia analogue (IV.77) was not inhibitory at concentrations below 3 /xM, was neither a substrate nor an inhibitor of L. casei DHFR, and was not an inhibitor of L. casei TS [116]. Replacement of CH2CH2O by CH2CH2S in the 2-amino-4(3/f)-oxo series therefore led to severe loss of antibacterial activity. How-... 

The biochemical and biological activities of the 2 - and 3 -fluoro derivatives (V.13) and (V.14) of AMT have been described by Henkin and Washtien [199]. In assays against E. coli and L. casei DHFR, the Kj values of (V.13) were 0.08 and 0.095 nM, respectively, while those of (V.14) were 0.03 and 0.06 nM. The corresponding values for AMT were 0.09 and 0.11 nM. Thus, a 3 -fluoro substituent appeared to increase DHFR affinity some 2- to 3-fold,... 

While the equations look the same at a first glance, some striking differences can be seen on a closer inspection. First, the vertebrate, but not the bacterial DHFR equations contain an electronic parameter in addition to lipophilicity and molar refractivity terms. Second, in the case of L. casei (eq. 137) the 5-position of the benzyl group does not at all contribute to biological activities. An explanation could be derived by a comparison of the 3D structure of L. casei DHFR with the E. coli DHFR structure. The active sites of both enzymes are more or less identical in the geometries of the protein backbone and the amino acid side chains. However, there is one significant difference E. coli DHFR contains a methionine side chain in the area where the 5-substituents bind, while there is a relatively rigid leucine side chain in the L. casei DHFR which obviously interferes with the 5-substituents. Therefore, the active site of L. casei DHFR is sterically more constrained and the positive lipophilicity and polarizability contributions of the 5-substituents are counterbalanced by their steric hindrance [432, 682]. 

Equations (14) and (15) differ in one significant respect there is no positive influence of the 5 -substituents in L. casei-DHFR. After the 3D structures of both enzymes had been determined, this could easily be explained. The binding site for the benzyl group is similar in both enzymes, but there is one striking difference. At the place, where the 5 -substituents bind, there is a flexible methionine side chain in E. co/i-DHFR, whereas there is a much more rigid leucine side chain in the L casei -DHFR. Thus, the binding site is more narrow in L case/-DHFR, as compared with E. cr>//-DHFR. Any positive influence resulting... 

The crystal structures of the E. coli DHFR-methotrexate binary complex (Bolin et al., 1982), of the Lactobacillus casei (DHFR-NADPH-methotrexate ternary complex (Filman et al., 1982), of the human DHFR-folate binary complex (Oefner et al., 1988), and of the mouse (DHFR-NADPH-trimethoprim tertiary complex (Stammers et al., 1987) have been resolved at a resolution of 2 A or better. The crystal structures of the mouse DHFR-NADPH-methotrexate (Stammers et al., 1987) and the avian DHFR—phenyltriazine (Volz et al., 1982) complexes were determined at resolutions of 2.5 and 2.9 A, respectively. Recently, the crystal structure of the E. coli DHFR—NADP + binary and DHFR-NADP+-folate tertiary complexes were resolved at resolutions of 2.4 and 2.5 A, respectively (Bystroff et al., 1990). DHFR is therefore the first dehydrogenase system for which so many structures of different complexes have been resolved. Despite less than 30% homology between the amino acid sequences of the E. coli and the L. casei enzymes, the two backbone structures are similar. When the coordinates of 142 a-carbon atoms (out of 159) of E. coli DHFR are matched to equivalent carbons of the L. casei enzyme, the root-mean-square deviation is only 1.07 A (Bolin et al., 1982). Not only are the three-dimensional structures of DHFRs from different sources similar, but, as we shall see later, the overall kinetic schemes for E. coli (Fierke et al., 1987), L. casei (Andrews et al., 1989), and mouse (Thillet et al., 1990) DHFRs have been determined and are also similar. That the structural properties of DHFRs from different sources are very similar, in spite of the considerable differences in their sequences, suggests that in the absence, so far, of structural information for ADHFR it is possible to assume, at least as a first approximation, that the a-carbon chain of the halophilic enzyme will not deviate considerably from those of the nonhalophilic ones. 

The alignment of the amino acid sequence of ADHFR with those of L. casei and E. coli shows a homology of 23 and 30%, respectively (see Fig. 2). Some amino acid residues are conserved in almost all known DHFRs (Beverley et al., 1986 Volz et al., 1982). Most of these residues are also conserved in ADHFR. The functions of some of the residues that are also conserved in ADHFR were inferred from the crystal structures. For instance, in E. coli DHFR, Ala-7, Ser-49, and Leu-54 are involved in binding of dihydrofolate and its analogous inhibitor methotrexate (Bolin et al., 1982). The bond between Gly-... 

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.

Figure 11. A Gibbs free energy plot of the kinetic profiles for E. coli (solid) and L. casei (dotted) DHFR (adapted from ref. 19).

Table I. Comparison of DHFR Active Site Residues from E. coli and L. casei...

In assays of DHFR and TS inhibition as well as S. faecium and L. casei growth inhibition [32a], (III. 112) and (HI.l 13) proved considerably less active than (III.l 11). In the DHFR assay, potency was decreased 140-fold by 5-Me substitution and 400-fold by 5,10-Me2 substitution. Potency against S.faecium relative to (III.l 11) was decreased 4,200-fold in (III.l 12) and 330-fold in (III.l 13), whereas potency against L. casei was decreased 11-fold in (III.l 12) and 85-fold in (III.l 13). It thus appeared that modification of... 

Bacterial growth inhibition data have been reported for the -octyl and propargyl analogues (IV.3) and (IV.6) in comparison with MTX [63]. The IC50 values (IV.3) against S. faecium and L. casei were 0.72 and 0.030 nM. The corresponding values for (IV.6) were 0.41 and 0.027 nM, respectively, while those for MTX were 0.2 and 0.03 nM. Thus, the antibacterial activities of (IV.3) and (IV.6) were similar to MTX and were consistent with their anti-DHFR activity. Neither compound overcame MTX resistance in L. ca-sei/MTX. However, S. faeciumj Wn X was about 50-fold less resistant to (IV.3) than to MTX. 

In assays against purified DHFR from L. casei, lO-deaza-lO-oxaAMT (IV.52), lO-deaza-lO-thiaAMT (IV.58), and 10-deazaAMT (IV.91 vide infra) all showed an IC50 value of 4 nM, whereas the values observed with 11-deaza-... 

The ability of lO-deaza-lO-thiafolic acid (IV.75) to inhibit purified DHFR (IC50 = 50//M) and TS (IC50 > 30/zM) from L. casei has been found to be... 

The enzyme inhibitory activity of 10-deazaAMT (IV.91) was evaluated initially against purified DHFR and TS from L. casei [118]. An IC50 value of 4.5 nM was obtained against DHFR, as compared with 3.3 nM for MTX. As with MTX, inhibitory activity against TS was very low (IC50 > 100 fiM). Of interest was that the 7,8-dihydro and 5,6,7,8-tetrahydro derivatives of (IV.91), which may be viewed as examples of dual modification of regions B and C (vide infra), showed moderate levels of TS inhibition, with IC50 values of 10 and 5.7 /iM, respectively. 

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). 

Nair et al. [171] confirmed in 1983 that CB3717 was a much more potent inhibitor of TS than of DHFR in assays using bacterial enzymes. Against TS and DHFR from L. casei, the IC50 values were 0.013 and 22 /xM, respectively, while against the same enzymes from S.faecium the corresponding ICso values were 0.01 and 0.3 /xM. Thus, the different effect between TS and DHFR inhibition appeared to be species-related, with the S. faecium enzyme probably being more predictive of the effect on mammalian enzyme. In the same... 

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