Adjudin disrupts spermatogenesis by targeting drug transporters: Lesson from the breast cancer resistance protein (BCRP)

Abstract

For non-hormonal male contraceptives that exert their effects in the testis locally instead of via the hypothalamic-pituitary-testicular axis, such as adjudin that disrupts germ cell adhesion, a major hurdle in their development is to improve their bioavailability so that they can be efficiently delivered to the seminiferous epithelium by transporting across the blood-testis barrier (BTB). If this can be done, it would widen the gap between their efficacy and general toxicity. However, Sertoli cells that constitute the BTB, peritubular myoid cells in the tunica propria, germ cells at different stages of their development, as well as endothelial cells that constitute the microvessels in the interstitium are all equipped with multiple drug transporters, most notably efflux drug transporters, such as P-glycoprotein, multidrug resistance-related protein 1 (MRP1) and breast cancer resistance protein (BCRP) that can actively prevent drugs (e.g., adjudin) from entering the seminiferous epithelium to exert their effects. Recent studies have shown that BCRP is highly expressed by endothelial cells of the microvessels in the interstitium in the testis and also peritubular myoid cells in tunica propria even though it is absent from Sertoli cells at the site of the BTB. Furthermore, BCRP is also expressed spatiotemporally by Sertoli cells and step 19 spermatids in the rat testis and stage-specifically, limiting to stage VII‒VIII of the epithelial cycle, and restricted to the apical ectoplasmic specialization [apical ES, a testis-specific F-actin-rich adherens junction (AJ)]. Interestingly, adjudin was recently shown to be capable of downregulating BCRP expression at the apical ES. In this Opinion article, we critically discuss the latest findings on BCRP; in particular, we provide some findings utilizing molecular modeling to define the interacting domains of BCRP with adjudin. Based on this information, it is hoped that the next generation of adjudin analogs to be synthesized can improve their efficacy in downregulating BCRP and perhaps other drug efflux transporters in the testis to improve their efficacy to traverse the BTB by modifying their interacting domains.

Keywords: Bcrp; adjudin; ectoplasmic specialization; male contraception; spermatogenesis; testis.

Figure 1. Stage-specific expression of BCRP at the apical ES during the epithelial cycle of spermatogenesis in the rat testis. BCRP (red fluorescence) was highly expressed at the apical ES, co-localized with a putative apical ES protein Eps8 (green fluorescence; epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein; it also displays stage-specific and spatiotemporal expression at the ES in the rat testis²³) in stage VII tubules, but this expression rapidly subsided and diminished to a level almost undetectable at stage VIII when the release of sperm takes place at spermiation. Cell nuclei were visualized by DAPI (4′,6-diamidino-2-phenylindole) staining. Detailed of the information on antibodies and procedures used to obtain results on this study can be found in reference . Scale bar = 10 μm, which applies to all other micrographs.

Figure 2. Adjudin effectively downregulates the expression of Bcrp at the apical ES. Rats (n = 3 for each time point) were treated with a single oral dose of adjudin at 50 mg/kg b.w. at time 0 h (hour) (control). Thereafter, rats were terminated at 12-, 48- and 96 h. BCRP (red fluorescence) was found to co-localize with F-actin (green fluorescence, visualized using FITC-phalloidin) in control testes and expressed prominently in this stage VII tubule. However, a rapid decline in BCRP expression at the apical ES was detected as soon as 12 h after adjudin treatment, and by 48 and 96 h, BCRP expression was completely abolished. Cell nuclei stained by DAPI. Scale bar = 10 μm, which applies to all other micrographs. Detailed of the information on antibodies and procedures used to obtain results on this study can be found in reference .

Figure 3. Ramachandran plot for the modeled human (A) and rat (B) BCRP. The amino acid sequences of breast cancer resistance protein (BCRP/ABCG2) of Homo sapiens (UniProtKB ID: Q9UNQ0) and of Rattus rattus (UniProtKB ID:Q80W57) were retrieved from UniProt (www.uniprot.org). A BLASTp search was performed to find appropriate proteins with significant amino acid sequence and structural similarity to BCRP by searching the Protein Data Bank (PDB) database (PDB, www.rscb.org/pdb/). The search was refined to find a suitable structural homolog for the modeling of BCRP in human and also in rat (seeFigs. 4 and 5). The amino acid sequence of these two proteins and their template sequences were aligned using the web based interface MultAlin. Based on the alignment generated, the tertiary structure of BCRP of human and rat were predicted using Modeler v9.11. Discrete Optimized Protein Energy (DOPE) and Modeler Objective Function (MOF) scores of the resulting models were used to select the most reliable model. The predicted structures were energy minimized by Smart Minimizer algorithm in Discovery Studio 3.1. The minimization was performed in 500 steps by applying CHARMM (Chemistry at HARvard Macromolecular Mechanics) force field and then subjected to validation. Backbone conformation was evaluated by examining the Psi/Phi interactions in Ramachandran Plot, obtained from PROCHECK, for human (A) and rat (B) BCRP and shown herein. Based on the plot, residues in the disallowed regions were refined using Loop Refinement (MODELER) module, from DS3.1. The final refined model was tested for their stability and reliability using ERRAT.

Figure 4. Docked complex of human BCRP with adjudin. (A) Entire modeled human BCRP in ribbon format, colored as per its secondary structure and the docked adjudin in CPK model. White for H; gray for C; blue for N; red for O; purple for P; green for CI. (B) Enlarged view of the docking site in (A). Adjudin is depicted in scaled ball and stick model, interacting residues are in stick model and their interactions in its 3D conformation. (C) Two dimension representations of molecular interactions between adjudin and the human BCRP. Green circles represent residues involved in van der Waal’s interactions; pink circles represent residues involved in hydrogen bond, polar or charge interactions; blue halo around residues represent solvent accessible surface of an interacting residue. Orange lines represent Pi-Pi and Pi-cation interactions between Phe470 and adjudin. Blue dotted line represents hydrogen bond formation with side chain of Glu451.

Figure 5. Docked complex of rat BCRP with adjudin. (A) This figure depicts the entire modeled protein docked with adjudin. (B) An enlarged view of the interacting residues in 3D conformation. (C) Orange line represents Pi-Pi interaction between Arg465 and adjudin. Blue dotted line represents hydrogen bond formation between side chain of Glu451 and donor oxygen atom of Adjudin. See Figure 4C for residue color code.