The Function of the Active Site Glutamate in Human Dihydrofolate Reductase

Dihydrofolate reductase (DHFR) plays a vital role in the DNA synthesis by reducing dihydrofolic acid to tetrahydrofolic acid which is an essential component. Synthetic ligands like methotrexate (MTX), aminopterin (AMP) and their analogues act as potential anti metabolites by mimicking the coenzyme dihydrofolic acid (DHFA) they inhibit the activity of DHFR antagonistically. Several ligands which are similar to MTX analogues and 6, 8 substituted 2 – naphthyls (NAP) which can mimic the pteridyl group of DHFA have been computationally designed. These ligands were proposed to hinder the formation N5, N10 methylene tetrahydrofolic acid (THFA) coenzyme, which is essential for the DNA synthesis. The docking studies were done using grid, generated with the 0.9 Vander Waals scaling for non polar bonds in the active site of the receptor. These newly designed ligands such as 14 -21 ,23 and 28 have shown good docking scores and predicted activities when compared to already existing ligands MTX and its analogues.

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2-Tier Bacterial and In Vitro Selection of Active and Methotrexate-Resistant Variants of Human Dihydrofolate Reductase

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108318783 ◽

2008 ◽

Vol 13 (6) ◽

pp. 504-514 ◽

Cited By ~ 10

Author(s):

Elena Fossati ◽

Jordan P. Volpato ◽

Lucie Poulin ◽

Vanessa Guerrero ◽

David-Antoine Dugas ◽

...

Keyword(s):

Dihydrofolate Reductase ◽

Active Site ◽

Drug Target ◽

In Vitro Selection ◽

Fold Increase ◽

Active Site Residues ◽

20 Nm ◽

Human Dihydrofolate Reductase ◽

Selection Of

We report a rapid and reliable 2-tier selection and screen for detection of activity as well as drug-resistance in mutated variants of a clinically-relevant drug-target enzyme. Human dihydrofolate reductase point-mutant libraries were subjected to a 1st-tier bacterial complementation assay, such that bacterial propagation served as an indicator of enzyme activity. Alternatively, when selection was performed in the presence of the inhibitor methotrexate (MTX), propagation indicated MTX resistance. The selected variants were then subjected to a 2nd-tier in vitro screen in 96-well plate format using crude bacterial lysate. Conditions were defined to establish a threshold for activity or for MTX resistance. The 2nd-tier assay allowed rapid detection of the best variants among the leads and provided reliable estimates of relative reactivity, ( kcat) and IC50MTX. Screening saturation libraries of active-site positions 7, 15, 24, 70, and 115 revealed a variety of novel mutations compatible with reactivity as well as 2 novel MTX-resistant variants: V115A and V115C. Both variants displayed KiMTX = 20 nM, a 600-fold increase relative to the wild-type. We also present preliminary results from screening against further antifolates following simple modifications of the protocol. The flexibility and robustness of this method will provide new insights into interactions between ligands and active-site residues of this clinically relevant human enzyme. ( Journal of Biomolecular Screening 2008:504-514)

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Multiple Conformers in Active Site of Human Dihydrofolate Reductase F31R/Q35E Double Mutant Suggest Structural Basis for Methotrexate Resistance

Journal of Biological Chemistry ◽

10.1074/jbc.m109.018010 ◽

2009 ◽

Vol 284 (30) ◽

pp. 20079-20089 ◽

Cited By ~ 25

Author(s):

Jordan P. Volpato ◽

Brahm J. Yachnin ◽

Jonathan Blanchet ◽

Vanessa Guerrero ◽

Lucie Poulin ◽

...

Keyword(s):

Dihydrofolate Reductase ◽

Active Site ◽

Double Mutant ◽

Structural Basis ◽

Methotrexate Resistance ◽

Human Dihydrofolate Reductase ◽

Multiple Conformers

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Computer modeling studies of the structural role of NADPH binding to active site mutants of human dihydrofolate reductase in complex with piritrexim.

Acta Biochimica Polonica ◽

10.18388/abp.2001_3856 ◽

2001 ◽

Vol 48 (4) ◽

pp. 903-916 ◽

Cited By ~ 3

Author(s):

W Nowak ◽

V Cody ◽

A Wojtczak

Keyword(s):

Dihydrofolate Reductase ◽

Active Site ◽

Structural Changes ◽

Energy Substitution ◽

Energy Simulations ◽

Detailed Computer ◽

Human Dihydrofolate Reductase ◽

Folate Cycle ◽

Active Site Mutants

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is one of the enzymes active in the folate cycle which plays an important role in DNA synthesis. Inhibition of DHFR is a key element in the treatment of many diseases, including cancer and AIDS related infections. A search for new selective inhibitors is motivated by the resistance to common drugs observed in the course of treatment. In this paper, results of a detailed computer analysis of human DHFR interactions with the lipophilic inhibitor piritrexim (PTX) are presented. It was found that the NADPH cofactor contributes 30% of the total PTX-enzyme interaction energy. Substitution of the highly conserved Glu30 with alanine does not lead to the release of the inhibitor from the hDHFR pocket. The important L22F point mutation does affect PTX orientation but does not changethe binding energy. Simulations of the dynamics of binary hDHFR-PTX complexes were performed with the use of Extensible Systematic Force Field (ESFF) and the results indicate structural changes in the enzyme induced by NADPH binding.

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