Engineered gene over-expression as a method of drug target identification.
The proposed target of aminobisphosphonate (aBP) bone resorption inhibitors, both in mammalian osteoclasts and in Dictyostelium, is the enzyme farnesyl diphosphate synthase (FDP synthase). The genetic evidence, obtained with Dictyostelium, derives from variant strains that over-express FDP synthase and that are relatively resistant to aBPs. We show that forced FDP synthase over-expression also leads to aBP resistance; by placing FDP synthase under control of a semi-constitutive promoter, transforming it into Dictyostelium cells and selecting with the aBP alendronate. This combination of drug and dominant selectable marker provides a novel selection system for transformation. We further show that, when a population of Dictyostelium cells expressing an entire growth stage cDNA library is placed under alendronate selection, FDP synthase is the only cDNA insert that confers drug resistance. This confirms FDP synthase as the primary target of aBPs and suggests a general method of drug target identification based upon engineered gene over-expression.
Sugden
Christopher J
CJ
School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dow Street, Dundee DD1 5EH, UK.
Roper
Janine R
JR
Williams
Jeffrey G
JG
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004023:Dictyostelium / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D004164:Diphosphonates / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000379:methods*; D015195:Drug Design; D004351:Drug Resistance / Q000502:physiology*; D051228:Geranyltranstransferase; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
D004164:Diphosphonates; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase