The relative stereochemistry of both

The relative stereochemistry of both the major and minor diastereoisomer of (±)-2-(hydroxymethyl)blebbistatin (±)- (and thus (±)-2-(allyloxymethyl)blebbistatin (±)-) was determined with 1D-NOESY experiments (), as crystallization efforts failed. These experiments revealed an interaction between H and the hydroxyl hydrogen on C in the major diastereoisomer. This NOE was absent in the minor diastereoisomer. These results suggest a - and -configuration for the major and minor diastereoisomer, respectively. The myosin II ATPase inhibitory potency of the thus obtained blebbistatin derivatives (±)--2-(hydroxymethyl)blebbistatin (±)--, (±)--2-(hydroxymethyl)-blebbistatin (±)-- and (±)-2-(allyloxymethyl)blebbistatin (±)- (the latter containing an 84:16 mixture of the - and -diastereoisomer) was subsequently evaluated. To this extend, our in-house-developed steady-state ATPase assay against rabbit skeletal-muscle myosinII was applied., , Rabbit skeletal muscle myosin II is a suitable model system for the human protein, as crucial residues and Rhapontigenin areas in the binding site have been conserved across species, and experimental responses to ()-blebbistatin and its analogs in the ATPase assay are highly similar for rabbit and human isoforms. ()-Blebbistatin ()-, obtained as earlier described, was used as a benchmark (). Contrary to what was expected based on the observation made in A, (±)--2-(hydroxymethyl)blebbistatin (±)-- displayed no inhibition at a concentration as high as 100 µM, while (±)--2-(hydroxymethyl)blebbistatin (±)-- possessed an IC value of ∼75 µM (vs 0.95 µM for ()-blebbistatin ()-). Further, an 84:16 mixture of (±)-- and (±)--2-(allyloxymethyl)-blebbistatin (±)-- and -- showed no inhibition of the ATPase activity at 100 µM. These data reveal that small modification of ring C can have a large negative impact on the myosin II ATPase inhibitory potency. The results also indicate, as observed previously for A- and D-ring modification,, , that rational modification of the blebbistatin scaffold based on analysis of residues lining the binding pocket (cfr. A) is not straightforward. We recently investigated the inability of this approach to correctly predict ligand discrimination using an array of computational techniques. We concluded that structure-based methods perform poor because these techniques do not account for steric and temporal (due to the kinetics of the chemo-mechanical cycle) restrictions in the path(s) leading toward the binding site, as had also been suggested earlier., In the same study, a ligand-based method using dissimilarity distances between compounds calculated by extended chemical fingerprints proved able to select for actives. In conclusion, the synthesis of a small set of blebbistatin derivatives with C-ring modifications at C was attempted in order to (i) enable an additional hydrogen bonding interaction and (ii) optimize filling of the binding pocket. (±)--2-(hydroxymethyl)blebbistatin (±)--, (±)--2-(hydroxy-methyl)blebbistatin (±)-- and (±)-2-(allyloxymethyl)-blebbistatin (±)- (an 84:16 mixture of the - and -diastereoisomer) were synthesized. The myosin II ATPase inhibitory potency of these analogs with small C-ring modifications was lower than that of parent compound ()-blebbistatin ()-. Potency improvement from C-ring modification thus seems not straightforward, yet additional C-ring-modified analogs must be evaluated in order to test this hypothesis. Such compounds should preferentially be designed using ligand-based methods. Acknowledgments
Introduction Although bradycardia is a wide spread clinical problem, there is no causal curative therapy and pacemaker implantation often remains the only option [1]. Genetic screens in humans have shown that propensity to bradycardia might be associated with mutations in distinct cardiac ion channels and genes that are known to modulate ion channel function [[2], [3], [4], [5]]. However, a thorough dissection of the molecular mechanisms that control the vertebrate heart rate under physiological conditions is limited by the lack of suitable genetic model organisms [6,7].