title: Identifying New Drug Targets for Potent Phospholipase D Inhibitors: Combining Sequence Alignment, Molecular Docking, and Enzyme Activity/Binding Assays.
abstract: Phospholipase D enzymes cleave phospholipid substrates generating choline and phosphatidic acid. Phospholipase D from Streptomyces chromofuscus is a non-HKD (histidine, lysine, and aspartic acid) phospholipase D as the enzyme is more similar to members of the diverse family of metallo-phosphodiesterase/phosphatase enzymes than phospholipase D enzymes with active site HKD repeats. A highly efficient library of phospholipase D inhibitors based on 1,3-disubstituted-4-amino-pyrazolopyrimidine core structure was utilized to evaluate the inhibition of purified S. chromofuscus phospholipase D. The molecules exhibited inhibition of phospholipase D activity (IC50 ) in the nanomolar range with monomeric substrate diC4 PC and micromolar range with phospholipid micelles and vesicles. Binding studies with vesicle substrate and phospholipase D strongly indicate that these inhibitors directly block enzyme vesicle binding. Following these compelling results as a starting point, sequence searches and alignments with S. chromofuscus phospholipase D have identified potential new drug targets. Using AutoDock, inhibitors were docked into the enzymes selected from sequence searches and alignments (when 3D co-ordinates were available) and results analyzed to develop next-generation inhibitors for new targets. In vitro enzyme activity assays with several human phosphatases demonstrated that the predictive protocol was accurate. The strategy of combining sequence comparison, docking, and high-throughput screening assays has helped to identify new drug targets and provided some insight into how to make potential inhibitors more specific to desired targets.
authors.affiliation: STEM Biotechnology Division, Roxbury Community College, Roxbury, MA, USA.
authors.forename: Helene
authors.initials: H
authors.lastname: Djakpa
authors.affiliation: Department of Chemistry, University of Massachusetts Boston, 100 Morrissey Blvd, Boston, MA, 02125, USA.
authors.forename: Aditya
authors.initials: A
authors.lastname: Kulkarni
authors.affiliation: STEM Biotechnology Division, Roxbury Community College, Roxbury, MA, USA.
authors.forename: Scheneque
authors.initials: S
authors.lastname: Barrows-Murphy
authors.affiliation: STEM Biotechnology Division, Roxbury Community College, Roxbury, MA, USA.
authors.forename: Greg
authors.initials: G
authors.lastname: Miller
authors.affiliation: Department of Chemistry, University of Massachusetts Boston, 100 Morrissey Blvd, Boston, MA, 02125, USA.
authors.forename: Weihong
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Department of Chemistry, University of Massachusetts Boston, 100 Morrissey Blvd, Boston, MA, 02125, USA.
authors.forename: Hyejin
authors.initials: H
authors.lastname: Cho
authors.affiliation: Department of Chemistry, University of Massachusetts Boston, 100 Morrissey Blvd, Boston, MA, 02125, USA.
authors.forename: Béla
authors.initials: B
authors.lastname: Török
authors.affiliation: STEM Biotechnology Division, Roxbury Community College, Roxbury, MA, USA.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Stieglitz
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology*; D062105:Molecular Docking Simulation; D010739:Phospholipase D / Q000037:antagonists & inhibitors*; D016415:Sequence Alignment
chemical_list: D004791:Enzyme Inhibitors; D010739:Phospholipase D
keywords: bioinformatics; biological screening; chemical biology; drug design; structural biology