Testis

Gamendazole, an Orally Active Indazole Carboxylic Acid Male Contraceptive Agent, Targets HSP90AB1 (HSP90BETA) and EEF1A1 (eEF1A), and Stimulates Il1a Transcription in Rat Sertoli Cells

Abstract
Gamendazole was recently identified as an orally active anti-spermatogenic compound with anti-fertility 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 anti-spermatogenic male contraceptive agent. We report here identification of binding targets and propose a testable mechanism of action for this anti-spermatogenic agent. 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, and confirmed by MALDI-TOF MS and western analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not TEF3 nor 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 HSP90 drugs geldanamycin or novobiocin analogue, KU-1. Gamendazole elicited degradation of HSP90-dependent client proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggested 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) 4hrs after oral administration. A spike in IL1a transcription was confirmed by rt-PCR in primary Sertoli cells 60min after exposure to 100nM gamendazole, demonstrating 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 this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting infertility.

Key words: Testis • Fertilization • Sertoli cells • Spermatogenesis • male contraception

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