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

Département de Biochimie, Université de Montréal, Montréal, Québec, Canada

Jordan P. Volpato

Département de Biochimie, Université de Montréal, Montréal, Québec, Canada

Lucie Poulin

Département de Chimie, Université de Montréal, Montréal, Québec, Canada

Vanessa Guerrero

Département de Biochimie, Université de Montréal, Montréal, Québec, Canada

David-Antoine Dugas

Département de Biochimie, Université de Montréal, Montréal, Québec, Canada

Joelle N. Pelletier

Département de Biochimie, Université de Montréal, Montréal, Québec, Canada, joelle.pelletier{at}umontreal.ca, Département de Chimie, Université de Montréal, Montréal, Québec, Canada

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, (k_cat) and IC₅₀^MTX. 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 K_i^MTX = 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)

Key Words: human dihydrofolate reductase • methotrexate • drug resistance • saturation mutagenesis • high-throughput screening

This version was published on July 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 6, 504-514 (2008)
DOI: 10.1177/1087057108318783


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