No abstract

The development of drugs for neglected infectious diseases often uses parasite-specific enzymes as targets. We here demonstrate that parasite enzymes with highly conserved human homologs may represent a promising reservoir of new potential drug targets. The cyclic nucleotide-specific phosphodiesterases (PDEs) of Trypanosoma brucei, causative agent of the fatal human sleeping sickness, are essential for the parasite. The highly conserved human homologs are well-established drug targets. We here describe what is to our knowledge the first pharmacological validation of trypanosomal PDEs as drug targets. High-throughput screening of a proprietary compound library identified a number of potent hits. One compound, the tetrahydrophthalazinone compound A (Cpd A), was further characterized. It causes a dramatic increase of intracellular cyclic adenosine monophosphate (cAMP). Short-term cell viability is not affected, but cell proliferation is inhibited immediately, and cell death occurs within 3 days. Cpd A prevents cytokinesis, resulting in multinucleated, multiflagellated cells that eventually lyse. These observations pharmacologically validate the highly conserved trypanosomal PDEs as potential drug targets.

The effects of the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the selective PDE inhibitors motapizone (type III), rolipram (type IV), zardaverine (type III/IV), and zaprinast (type V and I) on prostaglandin F2 alpha (PFG2 alpha)-induced tone in human pulmonary arteries was investigated. Relaxation was achieved by IBMX [concentration eliciting 50% of maximum response (EC50): 11.3 microM, n = 10], motapizone (EC50:3.0 microM, n = 7), zardaverine (EC50: 3.2 microM, n = 9), and zaprinast (EC50: 31.8 microM, n = 6), whereas rolipram was almost ineffective. The combination of motapizone and zaprinast (10 microM) was the most effective relaxant with supra-additive relaxation and a motapizone EC50 of 575 nM. Biochemical studies revealed the presence of the PDE isozymes I, III, IV and V in the cytosolic and particulate phases of arterial homogenates; PDE II was not detectable. Partial inhibition of adenosine 3',5'-cyclic monophosphate (cAMP)-hydrolyzing PDE activity was achieved with rolipram (26 +/- 2.2%) or motapizone (60 +/- 5.4%), whereas there was almost complete inhibition of total PDE activity with zardaverine (81 +/- 2.0%) or the combination of motapizone and rolipram (82 +/- 2.3%). Inhibition of guanosine 3',5'-cyclic monophosphate (cGMP)-hydrolyzing PDE activity was achieved with zaprinast (62 +/- 2.6%) and motapizone (13 +/- 2.3%), indicating the cGMP-hydrolyzing activity of PDE III. We conclude that four out of the five recognized PDE isozyme families are present in human pulmonary artery. PGF2 alpha-induced tone in this tissue is effectively relaxed through PDE inhibitors with selectivity for type III, III/IV, and type V PDE.

The pleiotropic cytokine tumor necrosis factor-␣ (TNF-␣) and thrombin lead to increased endothelial permeability in sepsis. Numerous studies demonstrated the significance of intracellular cyclic nucleotides for the maintenance of endothelial barrier function. Actions of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are terminated by distinct cyclic nucleotide phosphodiesterases (PDEs). We hypothesized that TNF-␣ could regulate PDE activity in endothelial cells, thereby impairing endothelial barrier function. In cultured human umbilical vein endothelial cells (HUVECs), we found a dramatic increase of PDE2 activity following TNF-␣ stimulation, while PDE3 and PDE4 activities remained unchanged. Significant PDE activities other than PDE2, PDE3, and PDE4 were not detected. TNF-␣ increased PDE2 expression in a p38 mitogen-activated protein kinase (MAPK)-dependent manner. Endothelial barrier function was investigated in HUVECs and in isolated mice lungs. Selective PDE2 up-regulation sensitized HUVECs toward the permeability-increasing agent thrombin. In isolated mice lungs, we demonstrated that PDE2 inhibition was effective in preventing thrombin-induced lung edema, as shown with a reduction in both lung wet-to-dry ratio and albumin flux from the vascular to bronchoalveolar compartment. Our findings suggest that TNF-␣-mediated upregulation of PDE2 may destabilize endothelial barrier function in sepsis. Inhibition of PDE2 is therefore of potential therapeutic interest in sepsis and acute respiratory distress syndrome (ARDS). IntroductionSepsis-the systemic inflammatory response to infection-is the most common cause of death among patients in noncoronary intensive care units. 1 Laboratory markers of inflammation include high circulating levels of tumor necrosis factor ␣ (TNF-␣) and other cytokines, as well as activation of the coagulation cascade. 1 Endothelial hyperpermeability is a hallmark of sepsis. TNF-␣ increases endothelial cell permeability 2 with subsequent vascular leakage contributing to severe organ dysfunction such as adult respiratory distress syndrome (ARDS). 3 Half of the sepsis patients develop disseminated intravascular coagulation 2,4 due to a shift toward a procoagulant state with excessive thrombin and fibrin generation. 1 On a cellular level, thrombin changes the shape of endothelial cells and increases endothelial permeability, 5 thereby impairing endothelial barrier function synergistically with TNF-␣. 1,6 Thrombin, the main effector protease of the coagulation cascade, activates endothelial cells directly via protease-activated receptor 1 (PAR1) and PAR4, 7 thereby inducing hyperpermeability and promoting adhesion of platelets and leukocytes, as well as secretion of plateletactivating factor (PAF) and inflammatory cytokines. 8 After binding to one of the various receptors, TNF-␣ mediates activation of diverse signaling pathways, such as the p38 mitogenactivated protein kinase (p38 MAPK), JUN N-terminal kinase (JNK), and nuclear factor kappa B (NF-B). TNF recepto...