Determining the amino acids involved in inhibition of PDE4B by structurally-diverse compounds

The enzyme phosphodiesterase (PDE) catalyze the degradation of the second messenger cAMP, an important regulator of many cellular transduction pathways, including the production of cortisol and breakdown of lipids. The 11 human PDE families have different tissue distribution and control different pathways. PDE4B, the focus of this study, controls the production of TNF-α in response to bacterial liposaccharide stimulation. Selective inhibition of PDE4B could treat a range of autoimmune diseases. This study identified amino acids involved in PDE4B inhibition and a possible allosteric regulatory site, informing rational drug design of a selective PDE4B inhibitor. We transformed a PDE-deficient strain of S. pombe with randomly mutated PDE4B genes generated via PCR. Screening of candidates identified inhibitor-resistant mutant PDEs, which were subsequently sequenced. Comparison of the DNA sequences of these mutant PDE4B with wild-type PDE4B identified amino acid substitutions conferring inhibitor resistance. An aspartic acid to glycine substitution occurred at position 468 in the primary sequence. Structural analysis of the mutant PDE4B reveals a likely allosteric regulatory site close to Gly-468, which is a possible target site for drug inhibitors.