Seven 3-alkyl-4-aryl-1,5-dihydro-2H-pyrrol-2-ones were prepared as potential inhibitors of cardiac cAMP phosphodiesterase (PDE). The design of these compounds made use of rolipram, a known inhibitor of the brain cAMP PDE isozyme, as a lead structure and was guided by a model which describes the features required for potent inhibition of the cardiac isozyme. Syntheses for the new compounds are described, together with the results of theoretical and crystallographic studies aimed toward ascertaining their three-dimensional structures. The activities of these compounds as inhibitors of the cardiac and brain cAMP PDE isozymes and their positive inotropic activity in ferret papillary muscle are also reported. Selected compounds were further examined in an in vivo hemodynamic model. One compound 1,5-dihydro-4-[4-(1H-imidazol-1- yl)phenyl]-3-methyl-2H-pyrrol-2-one, was identified as a potent and selective positive inotropic agent and inhibitor of cardiac cAMP PDE.

Methionine synthases are essential enzymes for amino acid and methyl group metabolism in all domains of life. Here, we describe a putatively anciently derived type of methionine synthase yet unknown in bacteria, here referred to as core-MetE. The enzyme appears to represent a minimal MetE form and transfers methyl groups from methylcobalamin instead of methyl-tetrahydrofolate to homocysteine. Accordingly, it does not possess the tetrahydrofolate binding domain described for canonical bacterial MetE proteins. In Dehalococcoides mccartyi strain CBDB1, an obligate anaerobic, mesophilic, slowly growing organohalide-respiring bacterium, it is encoded by the locus cbdbA481. In line with the observation to not accept methyl groups from methyl-tetrahydrofolate, all known genomes of bacteria of the class Dehalococcoidia lack metF encoding for methylene-tetrahydrofolate reductase synthesizing methyl-tetrahydrofolate, but all contain a core-metE gene. We heterologously expressed core-MetE CBDB in E. coli and purified the 38 kDa protein. Core-MetE CBDB exhibited Michaelis-Menten kinetics with respect to methylcob(III)alamin (K M ≈ 240 µM) and L-homocysteine (K M ≈ 50 µM). Only methylcob(III)alamin was found to be active as methyl donor with a k cat ≈ 60 s −1. Core-MetE CBDB did not functionally complement metE-deficient E. coli strain DH5α (ΔmetE::kan) suggesting that core-MetE CBDB and the canonical MetE enzyme from E. coli have different enzymatic specificities also in vivo. Core-MetE appears to be similar to a MetE-ancestor evolved before LUCA (last universal common ancestor) using methylated cobalamins as methyl donor whereas the canonical MetE consists of a tandem repeat and might have evolved by duplication of the core-MetE and diversification of the N-terminal part to a tetrahydrofolate-binding domain. Methionine plays an essential role as proteinogenic amino acid in all domains of life, as an initiation amino acid in protein translation 1 and as a precursor in the formation of cysteine, carnitine, taurine and lecithin 2,3. Moreover, methionine can be converted to S-adenosyl-L-methionine (SAM) 4 , which represents an activated methyl group donor for many fundamental cellular processes 5,6. The final step in methionine de novo synthesis, the methylation of homocysteine to methionine, is catalyzed by different types of methionine synthases including cobalamin-dependent (MetH) and cobalamin-independent methionine synthase (MetE). Some bacteria, e.g. Escherichia coli, possess genes for both enzymes 7 and repress the expression of metE in the presence of vitamin B 12 8. Homocysteine methylation in mammals is catalyzed by mammalian methionine synthases (mMS) similar to bacterial MetH 9 , betaine-L-homocysteine-S-methyltransferase (BHMT) or S-methyl-L-methionine-L-homocysteine-S-methyltransferase (also known as BHMT-2) 10. Fungi and plants encode exclusively MetE 11 or BHMT-2 12. All known methionine synthase types contain a zinc ion in the active site that is essential for homocysteine binding and methyl group transfer 13. ...