Gamendazole was recently identified as an orally active antispermatogenic compound with antifertility effects. The cellular mechanism(s) through which these effects occur and the molecular target(s) of
gamendazole action are currently unknown.
Gamendazole was recently designed as a potent orally active antispermatogenic
male contraceptive agent. Here, we report the identification of binding targets and propose a testable mechanism of action for this antispermatogenic agent. Both HSP90AB1 (previously known as HSP90beta [heat shock 90-kDa
protein 1, beta]) and EEF1A1 (previously known as eEF1A [eukaryotic translation
elongation factor 1 alpha 1]) were identified as binding targets by biotinylated
gamendazole (BT-GMZ) affinity purification from testis, Sertoli cells, and ID8
ovarian cancer cells; identification was confirmed by matrix-assisted
laser desorption/ionization-time of flight mass spectrometry and Western blot analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not to TEF3 or HBS1, and was competed by unlabeled
gamendazole. However,
gamendazole did not inhibit
nucleotide binding by EEF1A1.
Gamendazole binding to purified Saccharomyces cerevisiae HSP82 inhibited
luciferase refolding and was not competed by the HSP90 drugs
geldanamycin or
novobiocin analogue, KU-1.
Gamendazole elicited degradation of the HSP90-dependent client
proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggest that
gamendazole may represent a new class of selective HSP90AB1 and EEF1A1 inhibitors. Testis gene microarray analysis from
gamendazole-treated rats showed a marked, rapid increase in three
interleukin 1 genes and Nfkbia (
NF-kappaB inhibitor alpha) 4 h after
oral administration. A spike in II1a transcription was confirmed by RT-PCR in primary Sertoli cells 60 min after exposure to 100 nM
gamendazole, demonstrating that Sertoli cells are a target. AKT1, NFKB, and
interleukin 1 are known regulators of the Sertoli cell-spermatid junctional complexes. A current model for
gamendazole action posits that this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting
infertility.