Inhibition of dihydrofolate reductase: effect of NADPH on the selectivity and affinity of diaminobenzylpyrimidines

Baccanari, David P.; Daluge, Susan M.; King, Roy W.

doi:10.1021/bi00263a034

Cited by 104 publications

(91 citation statements)

References 32 publications

(50 reference statements)

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“…Pyrimethamine binds tightly to the drugsensitive T. gondii enzyme, but not by a slow binding mechanism (51): there is no apparent barrier to enzyme-inhibitor association when cofactor is abundant and no suggestion of inhibitor isomerization. Pyrimethamine may achieve its tight binding characteristics via binding synergism with the NADPH cofactor as shown for trimethoprim interaction with bacterial DHFR (57,61) and suggested by structural predictions (see below). If so, changes in the enzyme ternary complex might be expected to influence inhibitor potency.…”

Section: Parasite Drug Resistance Phenotypes In Toxoplasma Andmentioning

confidence: 90%

A Biochemical and Genetic Model for Parasite Resistance to Antifolates

Reynolds

Roos

1998

Journal of Biological Chemistry

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We have exploited the experimental accessibility of the protozoan parasite Toxoplasma gondii and its similarity to Plasmodium falciparum to investigate the influence of specific dihydrofolate reductase polymorphisms known from field isolates of drug-resistant malaria. By engineering appropriate recombinant shuttle vectors, it is feasible to examine mutations by transient or stable transformation of T. gondii parasites, in bacterial and yeast complementation assays, and through biochemical analysis of purified enzyme. A series of mutant alleles that mirror P. falciparum variants reveals that the key mutation Asn-108 (Asn-83 in T. gondii) probably confers resistance to pyrimethamine by affecting critical interactions in the ternary complex. Mutations such as Arg-59 (T. gondii 36) have limited effect in isolation, but in combination with other mutations they enhance the competitive ability of folate by increasing the speed of product turnover. Val-16 (T. gondii 10) confers low level resistance to cycloguanil but hypersensitivity to pyrimethamine. This mutation precludes Asn-108, probably because compression of the folate binding pocket introduced by this combination is incompatible with enzyme function. These studies permit detailed biochemical, kinetic, and structural analysis of drug resistance mutations and reconstruction of the probable phylogeny of antifolate resistance in malaria.

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Section: Parasite Drug Resistance Phenotypes In Toxoplasma Andmentioning

confidence: 90%

A Biochemical and Genetic Model for Parasite Resistance to Antifolates

Reynolds

Roos

1998

Journal of Biological Chemistry

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“…11 The bacterial binding site of Ec dhfr is significantly different than the mammalian binding site, resulting in greater than three to four orders of magnitude difference in TMP binding affinity between the two proteins. 11,12 Thus, we suspected that TMP and radiochemical derivatives of TMP would be relatively orthogonal (biologically inert) in mammalian tissues, allowing accumulation in Ec dhfr engineered cells. As a transgene, Ec dhfr may also have potential for immunogenicity, however, several features of the protein may make that less likely, including that the protein size is far smaller (18 kDa) than HSV1-tk, leading to fewer possible immunogenic epitopes (see the Discussion).…”

Section: Introductionmentioning

confidence: 99%

Quantitative PET Reporter Gene Imaging with [11C]Trimethoprim

et al. 2017

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There is a need for improved methods to image genetically engineered cells, including immune cells used for cell-based therapy. Given the genetic manipulation inherent to gene therapy, the use of a reporter protein is a logical solution and positron emission tomography (PET) can provide the desired sensitivity and spatial localization. We developed a broadly applicable PET imaging strategy based on the small bacterial protein E. coli dihydrofolate reductase (Ec dhfr) and its highly specific small molecule inhibitor, trimethoprim (TMP). The difference in TMP affinity for bacterial compared to mammalian DHFR suggests that a TMP radioligand would have a low background in unmodified mammalian tissues and high retention in Ec dhfr engineered cells, providing high contrast imaging. Here, we describe the in vitro properties of [

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“…(27,34 Table 3. Comparison with the measured dissociation rate constants (32,33) shows that the rate offlipping is >100 times faster, so that this motion of the trimethoxybenzyl ring must take place in the bound state.…”

mentioning

confidence: 94%

“…Asp-26 hydrogen bond. The interaction between the pyrimidine ring and Asp-26 is thus breaking and re-forming at a rate more than an order of magnitude faster than that at which the whole drug molecule dissociates from the protein (32,33). This must in turn involve movements of the pyrimidine ring and/or (10).…”

mentioning

confidence: 99%

Dynamics of trimethoprim bound to dihydrofolate reductase.

Searle¹,

Forster²,

Birdsall³

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, we report the use of 'H and 13C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its "target," dihydrofolate reductase.`3C relaxation measurements reveal dihedral angle changes of ± 25°to ± 350 on the subnanosecond time scale, while 13C line-shape analysis demonstrates dihedral angle changes of at least ± 65°o n the millisecond time scale. 'H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.A knowledge of the internal motions of proteins is of considerable importance for understanding their structurefunction relationships (1)(2)(3)(4). In recent years, a variety of theoretical (4-6) and experimental (3, 7-14) methods have been brought to bear on this problem, and a picture of the kinds of motion that take place is beginning to emerge. Hitherto, most attention has been focused on the atoms of the protein itself, although similar dynamic behavior would be expected for small molecules bound to proteins, and this has indeed been observed in a few cases (10, 15-18). We have been studying the binding of the antibacterial drug trimethoprim ( Fig. 1 (26,27), with the spectral density function J(w) = 5 'TR_ + (1 -S2)T )[2]The internal motion is characterized by two parameters: Tint, an effective correlation time, and S2, the square of the order parameter for the motion.

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Inhibition of dihydrofolate reductase: effect of NADPH on the selectivity and affinity of diaminobenzylpyrimidines

Cited by 104 publications

References 32 publications

A Biochemical and Genetic Model for Parasite Resistance to Antifolates

A Biochemical and Genetic Model for Parasite Resistance to Antifolates

Quantitative PET Reporter Gene Imaging with [11C]Trimethoprim

Dynamics of trimethoprim bound to dihydrofolate reductase.

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