Synthesis and evaluation of bifunctional sGC regulators: optimization of a connecting linker

Hybrid molecules composed of PpIX and cobyrinic acid derivatives conjugated through linkers of varying length and composition were prepared via 1,3-dipolar cycloaddition (CuAAC) or amidation/esteryfication reactions. They were tested for activation of soluble guanylyl cyclase (sGC), a key enzyme in the NO/cGMP signaling pathway, by an in vitro GTP→cGMP conversion assay. Using purified heme-deficient sGC and truncated sGC variants lacking a heme-binding domain, we demonstrated that such hybrid molecules may activate sGC by targeting heme-binding and/or catalytic domain. While all conjugates activated sGC, only selected compounds served as bifunctional regulators and were capable of simultaneous targeting both heme and catalytic domains of sGC. The length and type of a linker connecting both components had a profound effect on the extent of sGC activation, indicating that the linker's type is crucial for their binding affinities with regulatory and catalytic domains. Only hybrids with the conjugated linker of 13-16 atom length synergistically target both domains and displayed the lowest EC50 and highest activating potency. Compounds with shorter connecting linkers were much less potent and were no more active than the cobyrinic acid component alone. The most active conjugate, which showed a 60-fold activation of sGC, was compound 11, in which PpIX and cobyrinic acid components are separated by 11 atoms chain with the triazole moiety in between.