Genetic and Structural Characterization of a Thermo-Tolerant, Cold-Active, and Acidic Endo-β-1,4-glucanase from Antarctic Springtail, <i>Cryptopygus antarcticus</i>

The <i>CaCel</i> gene from Antarctic springtail <i>Cryptopygus antarcticus</i> codes for a cellulase belonging to the glycosyl hydrolase family 45 (GHF45). Phylogenetic, biochemical, and structural analyses revealed that the <i>CaCel</i> gene product (CaCel) is closely related to fungal GHF45 endo-β-1,4-glucanases. The organization of five introns within the open reading frame of the <i>CaCel</i> gene indicates its endogenous origin in the genome of the species, which suggests the horizontal transfer of the gene from fungi to the springtail. CaCel exhibited optimal activity at pH 3.5, retained 80% of its activity at 0–10 °C, and maintained a half-life of 4 h at 70 °C. Based on the structural comparison between CaCel and a fungal homologue, we deduced the structural basis for the unusual characteristics of CaCel. Under acidic conditions at 50 °C, CaCel was effective to digest the green algae (<i>Ulva pertusa</i>), suggesting that it could be exploited for biofuel production from seaweeds

Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception

Na,K-ATPase α4 is a testis-specific plasma membrane Na⁺ and K⁺ transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase α4 is characterized by a high affinity for the cardiac glycoside ouabain. With the goal of discovering selective inhibitors of the Na,K-ATPase α4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue <b>25</b>, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding α4 isoform selectivity profile. Moreover, compound <b>25</b> decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca²⁺, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase α4 and sperm function

Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception

Na,K-ATPase α4 is a testis-specific plasma membrane Na⁺ and K⁺ transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase α4 is characterized by a high affinity for the cardiac glycoside ouabain. With the goal of discovering selective inhibitors of the Na,K-ATPase α4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue <b>25</b>, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding α4 isoform selectivity profile. Moreover, compound <b>25</b> decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca²⁺, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase α4 and sperm function

Structural Basis of ALDH1A2 Inhibition by Irreversible and Reversible Small Molecule Inhibitors

Enzymes of the ALDH1A subfamily of aldehyde dehydrogenases are crucial in regulating retinoic acid (RA) signaling and have received attention as potential drug targets. ALDH1A2 is the primary RA-synthesizing enzyme in mammalian spermatogenesis and is therefore considered a viable drug target for male contraceptive development. However, only a small number of ALDH1A2 inhibitors have been reported, and information on the structure of ALDH1A2 was limited to the NAD-liganded enzyme void of substrate or inhibitors. Herein, we describe the mechanism of action of structurally unrelated reversible and irreversible inhibitors of human ALDH1A2 using direct binding studies and X-ray crystallography. All inhibitors bind to the active sites of tetrameric ALDH1A2. Compound WIN18,446 covalently reacts with the side chain of the catalytic residue Cys320, resulting in a chiral adduct in (<i>R</i>) configuration. The covalent adduct directly affects the neighboring NAD molecule, which assumes a contracted conformation suboptimal for the dehydrogenase reaction. The reversible inhibitors interact predominantly through direct hydrogen bonding interactions with residues in the vicinity of Cys320 without affecting NAD. Upon interaction with inhibitors, a large flexible loop assumes regular structure, thereby shielding the active site from solvent. The precise knowledge of the binding modes provides a new framework for the rational design of novel inhibitors of ALDH1A2 with improved potency and selectivity profiles

Structural Basis of ALDH1A2 Inhibition by Irreversible and Reversible Small Molecule Inhibitors

Enzymes of the ALDH1A subfamily of aldehyde dehydrogenases are crucial in regulating retinoic acid (RA) signaling and have received attention as potential drug targets. ALDH1A2 is the primary RA-synthesizing enzyme in mammalian spermatogenesis and is therefore considered a viable drug target for male contraceptive development. However, only a small number of ALDH1A2 inhibitors have been reported, and information on the structure of ALDH1A2 was limited to the NAD-liganded enzyme void of substrate or inhibitors. Herein, we describe the mechanism of action of structurally unrelated reversible and irreversible inhibitors of human ALDH1A2 using direct binding studies and X-ray crystallography. All inhibitors bind to the active sites of tetrameric ALDH1A2. Compound WIN18,446 covalently reacts with the side chain of the catalytic residue Cys320, resulting in a chiral adduct in (<i>R</i>) configuration. The covalent adduct directly affects the neighboring NAD molecule, which assumes a contracted conformation suboptimal for the dehydrogenase reaction. The reversible inhibitors interact predominantly through direct hydrogen bonding interactions with residues in the vicinity of Cys320 without affecting NAD. Upon interaction with inhibitors, a large flexible loop assumes regular structure, thereby shielding the active site from solvent. The precise knowledge of the binding modes provides a new framework for the rational design of novel inhibitors of ALDH1A2 with improved potency and selectivity profiles