Ulrich Pleiss

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle

Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (α/ß) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. Through a high‐throughput screen we identified BAY 58‐2667, an amino dicarboxylic acid which potently activates sGC in an NO‐independent manner. In contrast to NO, YC‐1 and BAY 41‐2272, the sGC stimulators described recently, BAY 58‐2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem deficient. Binding studies with radiolabelled BAY 58‐2667 show a high affinity site on the enzyme. Using photoaffinity labelling studies we identified the amino acids 371 (α‐subunit) and 231 – 310 (ß‐subunit) as target regions for BAY 58‐2667. sGC activation by BAY 58‐2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not influenced by nitrate tolerance. BAY 58‐2667 shows a potent antihypertensive effect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic effects of BAY 58‐2667 and GTN are very similar on the arterial and venous system. This novel type of sGC activator is a valuable research tool and may offer a new approach for treating cardiovascular diseases.

Synthesis of a radiolabeled enniatin cyclodepsipeptide [3H‐methyl]JES 1798

For receptor binding studies and the elucidation of the mode of action of the potent anthelmintic compound JES 1798 a tritium labeled compound at very high specific activity was necessary. Tritium was introduced by methylation of the N-demethyl precursor JES 2314. The identity of [N-methyl- 3 H]JES 1798 was determined by mass spectrometry. After synthesis and purification, 535 μg [N-methyl- 3 H]JES 1798 were available at a specific activity of 84 Ci/mmol (3.11 TBq/mmol) as determined by mass spectrometry. The total activity was 80 mCi (2.96 GBq). Radiolabeled JES 1798 showed an efficient and specific binding to a membrane fraction from Ascaris suum. Displacement by unlabeled JES 1798 was half-maximal at about 0.72 ± 0.06 μM. Different known enniatins also competed for the [ 3 H]JES 1798-binding in the Ascaris suum membrane preparation. In vitro comparison of JES 1798 with enniatin A, A 1 , B and B 1 or beauvericin revealed that enniatin A showed an anthelmintic activity against Nippostrongylus brasiliensis, Trichinella spiralis and Heterakis spurnosa at a concentration of 5 μg/ml, whereas enniatins A 1 , B and B 1 had an activity at concentrations between 1 and 100 μg/ml. On the other hand beauvericin and JES 1798 exerted their anthelmintic activities at 100 μg/ml and therefore possess minor anthelmintic potency in vitro as compared to the natural occurring enniatins

Synthesis of a radiolabeled cyclodepsipeptide [3H-methyl]PF1022A

For receptor binding studies and the elucidation of the mode of action of the potent anthelmintic compound PF1022A a tritium labeled compound with very high specific activity was necessary. Tritium was introduced into the compound by methylation of the [bis-N-demethyl]precursor of PF1022A (PF1022-219). The identity of [bis-N-methyl- 3 H]PF1022A was determined by LC/MS. After synthesis and purification, 88.9 μg [bis-N-methyl- 3 H]PF1022A were available showing a specific activity of 162 Ci/mmol (5,99 TBq/mmol) determined by mass spectrometry. The total activity was 15 mCi (555 MBq). Radiolabeled PF1022A showed an efficient and specific binding to a membrane fraction from Ascaris suum. Displacement by unlabeled PF1022A was half-maximal at about 40 nM. At 100-fold higher concentrations the biologically much less effective optical antipodean (PF1022-001) competed for maximal 40% of the [ 3 H]PF1022A-binding in the Ascaris suum membrane preparation. In-vitro comparison of PF1022A with its optical antipodean revealed a more than 100-fold higher anthelmintic activity of PF1022A against Heterakis spumova, Nippostrongylus brasiliensis and Trichinella spiralis.

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

SummaryWhole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.