title: Identification and optimization of PDE10A inhibitors using fragment-based screening by nanocalorimetry and X-ray crystallography.
abstract: Fragment-based lead discovery (FBLD) is a technique in which small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high-resolution diffraction, with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an X-ray crystallography secondary screen.
authors.affiliation: 1Palo Alto Research Center, Palo Alto, CA, USA.
authors.forename: Michael I
authors.initials: MI
authors.lastname: Recht
authors.forename: Vandana
authors.initials: V
authors.lastname: Sridhar
authors.forename: John
authors.initials: J
authors.lastname: Badger
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Bounaud
authors.forename: Cheyenne
authors.initials: C
authors.lastname: Logan
authors.forename: Barbara
authors.initials: B
authors.lastname: Chie-Leon
authors.forename: Vicki
authors.initials: V
authors.lastname: Nienaber
authors.forename: Francisco E
authors.initials: FE
authors.lastname: Torres
mesh_terms: D000818:Animals; D002151:Calorimetry; D018360:Crystallography, X-Ray; D055808:Drug Discovery / Q000379:methods; D004353:Drug Evaluation, Preclinical; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D036103:Nanotechnology; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D054852:Small Molecule Libraries
chemical_list: D008024:Ligands; D010726:Phosphodiesterase Inhibitors; D054852:Small Molecule Libraries; D010727:Phosphoric Diester Hydrolases
keywords: X-ray crystallography; enzyme assay; fragment-based lead discovery; label-free assay; nanocalorimetry