Engineered gene over-expression as a method of drug target identification

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Aminobisphosphonates (aBPs) are bone resorption inhibitors.
• Farnesyl diphosphate (FDP) synthase is the proposed target of aBPs in mammalian osteoclasts and Dictyostelium.
• Genetic evidence from Dictyostelium variant strains suggests FDP synthase over-expression confers resistance to aBPs.

Purpose of the Study:

• To confirm FDP synthase as the primary target of aBPs.
• To establish a novel selection system for genetic transformation using aBP resistance.
• To demonstrate a generalizable method for drug target identification.

Main Methods:

• Forced over-expression of FDP synthase in Dictyostelium under a semi-constitutive promoter.
• Genetic transformation of Dictyostelium using alendronate as a selectable marker.
• Screening of a Dictyostelium cDNA library under alendronate selection.

Main Results:

• Forced FDP synthase over-expression conferred significant resistance to alendronate.
• The combination of FDP synthase gene and alendronate provided a novel and effective selection system for transformation.
• Screening the cDNA library identified FDP synthase as the sole gene conferring alendronate resistance.

Conclusions:

• Farnesyl diphosphate synthase is confirmed as the primary molecular target of aminobisphosphonates.
• Engineered gene over-expression coupled with drug selection offers a robust platform for drug target identification.
• This study validates a powerful approach for discovering drug targets in various biological systems.