Cyclic nucleotide phosphodiesterase (PDE) catalyzes the hydrolysis of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) to produce, respectively, 5'-AMP and 5'-GMP. PDE is a key enzyme to control cellular concentrations of cAMP that is known as """"""""second messenger"""""""" and mediates the response of cells to a wide variety of hormones and neurotransmitters. Ten families and twenty two subtypes of human PDE have been identified. The mRNAs of the 22 subtype PDEs are further spliced to generate over 60 isoforms of PDE. The distinct isoforms of PDE are located in different cellular compartments and possess different specificity of substrate. These two features of PDE have attracted great attention from pharmaceutical companies in the past decade. Many selective PDE inhibitors have been studied as therapeutic agents such as cardiotonic agents, vasodilators, antiasthma, atithrombic compounds, smooth muscle relaxants and antidepressants. For example, VIAGRA, an inhibitor of PDE5, is a prescription drug for erectile dysfunction of male patients. This proposal aims at characterization of substrate specificity and inhibitor selectivity by the approach of crystallography.
The specific aims are to determine crystal structures of PDEs and their complexes with inhibitors including (1) the catalytic domain of PDE4B, (2) the catalytic domain of PDE4B complexed with the inhibitors rolipram and iodonated cAMP, (3) full length PDE4D and its complexes with the inhibitors rolipram and denbufylline and (4) the catalytic domain of PDE3 and its complex with cilostamide. The structures in this proposal will reveal the details of inhibitor binding at the active site and provide insight into catalytic mechanism. Docking cGMP into the active site of PDE4B, together with the structure of PDE4B-cAMP analog, will shed light on the substrate specificity. Comparison of the structures of PDE-inhibited complexes will shed light on the selectivity of inhibition of different families of PDE, and thus provide a structural basis for design of selective drugs. The structures will be determined by multiple isomorphous replacement, multiwavelength anomalous diffraction, or molecular replacement. The structural models will be built with the program O and refined by the program CNS.
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