Compound which reduces sperm production can lead to male contraceptive

Although ?4% of the mammalian genome encodes genes expressed in male germ cells during spermatogenesis (Schultz et al., 2003), contraceptive drugs for men have remained elusive. To date, the only drugs in clinical trials are testosterone analogs that alter endogenous androgen production, although there is a short list of other possible targets (e.g., GAPDHS) and drugs (e.g., gamendazole) (Aitken et al., 2008). This lack of contraceptive alternatives for men is partially responsible for the high rate of unplanned pregnancies, especially in teenagers, and contributes to the maternal mortality, ethical, social, and financial costs associated with abortions and deliveries to single mothers. To approach this dearth of contraceptive alternatives for men, we have undertaken to develop small molecules that could target spermatogenic-specific proteins that have been shown to be essential for both spermatogenesis and fertility in mammals. One such contraceptive target is the testis-specific and bromodomain-containing protein BRDT.

BRDT is a tissue-restricted, chromatin-associated protein expressed in pachytene spermatocytes, diplotene spermatocytes, and round spermatids (Shang et al., 2007). During postmeiotic maturation, BRDT localizes to the nucleus and reorganizes hyperacetylated histones through twin acetyl-lysine recognition modules, or bromodomains (Berkovits and Wolgemuth, 2011; Morinière et al., 2009; Shang et al., 2007). The essential role of BRDT in spermatogenesis is mediated by the first bromodomain (BRDT(1); Figure 1A), which binds the tetra-acetylated amino-terminal tail of histone 4 (H4Kac4) with moderate potency (20 ìM) (Morinière et al., 2009). Structural studies of murine BRDT have demonstrated that BRDT(1) binds a diacetylated histone 4 peptide (H4K5ac8ac) in part through a conserved asparagine (Morinière et al., 2009), akin to other bromodomain coactivator proteins (Dhalluin et al., 1999). Genetic studies of BRDT have demonstrated that selective deletion of the BRDT(1)-encoding region is sufficient to confer sterility in homozygous hypomorphic male mice (Shang et al., 2007), and a recently published genome-wide association study of idiopathic male infertility identified single-nucleotide polymorphisms of BRDT as significantly associated with oligozoospermia or azoospermia in European men (Aston et al., 2010). These insights establish a compelling rationale to target BRDT for a contraceptive effect.

Recently, we established the feasibility of targeting human bromodomains with acetyl-lysine competitive small molecules (Filippakopoulos et al., 2010). Our index study described a potent thienodiazepine inhibitor ((+)-JQ1; Figure 1B; Kd = 90 nM) of the BET family coactivator protein BRD4, which is implicated in the pathogenesis of cancer via transcriptional control of the MYC oncogene (Delmore et al., 2011; Zuber et al., 2011). Protein sequence alignment of human BRD4(1) to human BRDT(1) reveals 81% identity and 89% similarity, including all surface residues predicted to contact (+)-JQ1 (Figure 1C and Data S1 and S2 available online). Based on these insights and preliminary evidence of binding to BRDT(1) established by differential scanning fluorometry (Filippakopoulos et al., 2010), we endeavored to characterize the biochemical and functional effects of (+)-JQ1 on BRDT(1).

We report here that direct inhibition of BRDT by a small-molecule bromodomain inhibitor exerts a dose- and time-dependent inhibitory effect on spermatogenesis. Structural studies of BRDT(1) bound to JQ1 reveal ligand-receptor shape complementarity and complete occlusion of the acetyl-lysine recognition cavity. These and further biochemical studies establish the molecular mechanism of potent BRDT inhibition observed in vivo, where JQ1 targets meiotic and postmeiotic male germ cells, leading to impaired spermatogenesis and compromised motility. In mating studies, JQ1 accomplishes a complete and reversible contraceptive effect in males without adversely affecting testosterone levels or mating behaviors and without prompting obvious teratogenic effects in offspring. These results indicate that targeting a developmental epigenetic reader protein with an orally bioavailable small molecule can modulate male fertility for a contraceptive effect.

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