There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
Methotrexate-resistant forms of human dihydrofolate reductase have the potential to
protect healthy cells from the toxicity of methotrexate (MTX), to improve prognosis
during cancer therapy. It has been shown that synergistic MTX-resistance can be obtained
by combining two active-site mutations that independently confer weak MTX-resistance.
In order to obtain more highly MTX-resistant human dihydrofolate reductase (hDHFR)
variants for this application, we used a semi-rational approach to obtain combinatorial
active-site mutants of hDHFR that are highly resistant towards MTX. We created a combinatorial
mutant library encoding various amino acids at residues Phe31, Phe34 and Gln35. In
vivo library selection was achieved in a bacterial system on medium containing high
concentrations of MTX. We characterized ten novel MTX-resistant mutants with different
amino acid combinations at residues 31, 34 and 35. Kinetic and inhibition parameters
of the purified mutants revealed that higher MTX-resistance roughly correlated with
a greater number of mutations, the most highly-resistant mutants containing three
active site mutations (Ki(MTX)=59-180 nM; wild-type Ki(MTX)<0.03 nM). An inverse correlation
was observed between resistance and catalytic efficiency, which decreased mostly as
a result of increased KM toward the substrate dihydrofolate. We verified that the
MTX-resistant hDHFRs can protect eukaryotic cells from MTX toxicity by transfecting
the most resistant mutants into DHFR-knock-out CHO cells. The transfected variants
conferred survival at concentrations of MTX between 100-fold and >4000-fold higher
than the wild-type enzyme, the most resistant triple mutant offering protection beyond
the maximal concentration of MTX that could be included in the medium. These highly
resistant variants of hDHFR offer potential for myeloprotection during administration
of MTX in cancer treatment.