Aquaporins mediate transport of water or small, uncharged solutes across cellular membranes according to the prevailing osmotic and chemical gradients. Because of their implication in human diseases and pathophysiological states, aquaporins are considered as potential drug targets. Yet, specific aquaporin inhibitors for in vivo studies are not available. Common functional aquaporin assays that monitor biophysical parameters related to volume changes, such as light scattering or fluorescence quenching, are time consuming and require costly equipment. Hence, they are not well geared for screening large numbers of compounds. In this paper, we describe a less demanding phenotypic yeast-based assay on 96-well microplates. The assay uses a methylamine-sensitive yeast strain in which a methylamine-permeable test aquaporin is expressed to rescue proliferation on selection plates. Specific inhibition of the aquaporin directly correlates to reduced cell proliferation.

Free endogenous methylarginines, N(ω)-monomethyl-L-arginine (L-NMMA) and N(ω),N(ω')-dimethyl-L-arginine (ADMA), inhibit NO synthases (NOSs) and are metabolized by dimethylargininedimethylaminohydrolase (DDAH). A postulated metabolism has been shown several times for DDAH-1, but the involvement of DDAH-2 in the degradation of ADMA and L-NMMA is still a matter of debate. Determination of the isoform-specific DDAH protein expression profiles in various porcine tissue types shows a correlation of DDAH activity only with DDAH-1 levels. DDAH activity (measured as L-citrulline formation from the conversion of methylarginines and alternative DDAH substrates) was detected in DDAH-1-rich porcine tissue types, that is, kidney, liver, and brain, but not in DDAH-2-rich porcine fractions, that is, spleen and thyroid. Furthermore, several ex vivo studies showed DDAH activity to be important for L-citrulline formation in porcine tissue and indicated the absence of an endogenous DDAH inhibitor in porcine tissue. This study provides new insights into tissue distributions as well as substrate selectivity for both DDAH isoforms. Although DDAH-1 is known to metabolize the endogenous NOS inhibitors L-NMMA and ADMA, a physiological function for DDAH-2 has yet to be determined. Hence, determining DDAH activity by methylarginine conversion is not suitable for analyzing isoform selectivity of DDAH-1 inhibitors as postulated.

N-(4-Aminobutyl)-N′-(2-methoxyethyl)­guanidine (8a) is a potent inhibitor targeting the hDDAH-1 active site (K i = 18 μM) and derived from a series of guanidine- and amidine-based inhibitors. Its nonamino acid nature leads to high selectivities toward other enzymes of the nitric oxide-modulating system. Crystallographic data of 8a-bound hDDAH-1 illuminated a unique binding mode. Together with its developed N-hydroxyguanidine prodrug 11, 8a will serve as a most widely applicable, pharmacological tool to target DDAH-1-associated diseases.