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Fragment-based screening for inhibitors of PDE4A using enthalpy arrays and X-ray crystallography
Conferences & Talks

Fragment-based Lead Discovery Conference 2010

12 October 2010



Phosphodiesterase 4 (PDE4) is a family of four enzymes (PDE4A-D) with cAMP-specific activity. PDE4 is expressed in many cell types and tissues, and PDE4 selective inhibitors have been studied as anti-inflammatory therapeutics for treatment of asthma and COPD. PDE4 has also been implicated in psychiatric diseases such as depression and schizophrenia. This study demonstrates the combined use of enthalpy arrays and x-ray crystallography in fragment-based screening of PDE4A, showing the complementarity of these two techniques. Enthalpy arrays are arrays of nanocalorimeters that enable label-free detection of molecular interactions using small sample volumes (~250nL) and short measurement times (3-4 minutes), and in this work we applied them to activity-based screening of fragments, using the measured generation of heat to monitor rates of substrate turnover. We screened a 150 compound library for inhibitors of PDE4A, and we found several competitive inhibitors with a ligand efficiency >0.35 kcal/mol/HA, based on the measured KI. In parallel, we conducted an x-ray crystallographic screen and will discuss how the hits compare to those identified using enthalpy arrays. Whereas typical phosphodiesterase functional assays use radioactive substrates, multiple coupled enzymes, or fluorescence-based detection, none of which are ideal for screening fragment libraries, enthalpy arrays provide a much simpler and more sensitive alternative. Screening fragment libraries at high concentration is possible because calorimetry-based assays do not suffer from issues of compound interference that occur with optical detection techniques, since calorimetry does not require labeled compounds. We will show enthalpy array measurements of competitive inhibition constants between 130 nM and ~2mM, as well as discuss the identification of compounds that cause a significant reduction in kcat. While conventional isothermal titration calorimeters can in principle perform similar measurements, the level of use in screening applications is severely hampered by the need for large samples (˜ 0.2 to 1.5 mL) and long measurement times. Pairing kinetic data from enthalpy array measurements with direct binding and high resolution structural data provides the opportunity for a more complete description of fragment hits.