Na,K-ATPase alpha4 Isoform and HSP90 molecular chaperone as molecular targets for drug discovery

Abstract

Na,K-ATPase alpha4 is specifically expressed in male germ cells of the testis and abundant in the sperm flagellum, and crucial for sperm motility. It binds ouabain with a higher affinity than other Na,K-ATPase alpha isoforms, and provides the opportunity to pharmacologically target the Na,K-ATPase alpha4 for male contraception. In search for additional chemical matter a HTVS was performed to identify selective inhibitors of the Na,K-ATPase alpha4 following homology modeling of the Na,K-ATPase alpha4. Based on subsequent docking screening cetirizine was identified as a potential Na,K-ATPase alpha4 binder. In vitro assay results reveal that cetirizine is a selective Na,K-ATPase alpha4 inhibitor that reduces sperm motility and has the potential to be a promising scaffold for male contraceptive development. Ouabain, SS-I-24, SS-I-42, and SS-I-54 are highly selective inhibitors of the rat alpha4 over the rat alpha1 isoform. Computational simulations including homology modeling, molecular docking, MM-GBSA calculations, and MD simulations were employed to investigate the structural origin of their selectivity. His118 and Asn129 of Loop1 of the rat alpha4 isoform stabilize the compound-enzyme complex more dynamically than the corresponding amino acid residues of the rat alpha1 isoform. The computational simulation results provide a possible reason for the higher affinities of ouabain and its analogues for the rat alpha4 compare to the rat alpha1 isoform. Testis-specific HSP90beta inhibition induces apoptosis in the testis. Therefore, it was hypothesized that a highly selective HSP90beta inhibitor should impair spermatogenesis without undesired effects. TF-1 (7a), an HSP90beta inhibitor identified during a high throughput screening campaign, and its analogues were synthesized and evaluated for their antispermatogenic activity in LE rats. No antispermatogenic effects were observed. Subsequent in vitro assays showed that the inhibitors exerted antiproliferative activities at submicromolar to low micromolar IC50s. Compound 7c exhibited antiproliferative activity against various cancer cell lines at submicromolar IC50s. Results from our in vitro assays including cell-based Her2 ELISA, cell-based NF-kappaB functional assay, and Western blot analysis of cellular proteins suggested that they exhibit antiproliferative activities via the inhibition of the AKT/NF-kappaB signaling pathway following the inhibition of the HSP90 molecular chaperone.