Discovery of the First Human Arylsulfatase A Reversible Inhibitor Impairing Mouse Oocyte Fertilization.,ACS Chemical Biology

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Discovery of the First Human Arylsulfatase A Reversible Inhibitor Impairing Mouse Oocyte Fertilization.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-04-02 , DOI: 10.1021/acschembio.9b00999
Silvia Caroselli ₁ , Clemens Zwergel _{2,

3} , Adele Pirolli _{2,

4} , Manuela Sabatino ₂ , Zhanjie Xu ₅ , Gilbert Kirsch ₅ , Antonello Mai _{2,

6} , Gianni Colotti ₇ , Fabio Altieri ₈ , Rita Canipari ₁ , Sergio Valente ₂ , Rino Ragno _{2,

9}

Affiliation

Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Section of Histology and Medical Embryology, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy.
Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy.
Department of Precision Medicine, Luigi Vanvitelli, University of Campania, Via L. De Crecchio 7, 80138 Naples, Italy.
Department of Information Technology, IRBM Science Park, Via Pontina km 30, 600, 00071 Pomezia, Rome, Italy.
Université de Lorraine, UMR CNRS 7565, Structure et Réactivité des Systèmes Moléculaires Complexes, Equipe 3 (HECRIN), 1 Boulevard Arago, 57078, Metz Technopôle, France.
Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Viale Regina Elena 291, 00161 Rome, Italy.
Institute of Molecular Biology and Pathology, Italian National Research Council, c/o Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy.
Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy.
Rome Center for Molecular Design, Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy.

Arylsulfatase A (ARSA) plays a crucial role in the reproduction of mammals due to its involvement in the specific gamete interaction preceding sperm and egg fusion leading to fertilization. Recently, it has been shown that zona pellucida (ZP) sperm binding and in vivo fertilization in mice are markedly hampered by using a specific anti-ARSA antibody. Herein, the design and discovery of the first ARSA small molecule inhibitor based on a coumarin-containing polycycle are presented. Through a structure-based approach applied on our in-house library, compound 1r was identified as an ARSA reversible inhibitor (ARSAi); then its activity was validated through both surface plasmon resonance and biochemical inhibition experiments, the first providing a K_D value of 21 μM and the latter an IC₅₀ value of 13.2 μM. Further investigations highlighted that compound 1r induced 20% sperm death at 25 μM and also impaired sperm motility; nevertheless both the effects were mediated by ROS production, since they were rescued by the cotreatment of 1r and N-acetyl cysteine (NAC). Interestingly, while 1r was not able to hamper the ZP/sperm binding, it markedly decreased the in vitro oocyte fertilization by mouse sperm up to 60%. Notably, this effect was not hampered by 1r/NAC coadministration, hence allowing the ruling out of an ROS-dependent mechanism. In conclusion, herein is reported the first ever hit of ARSAi as a chemical tool that will enable better exploration of ARSA’s biological role in fertilization as well as provide a starting point for developing 1r structure optimization aimed at increasing enzyme inhibition potency but also providing a deeper understanding of the involvement of ARSA in the fertilization pathway mechanism.

中文翻译：

第一个人类芳基硫酸酯酶的可逆抑制剂的发现损害小鼠卵母细胞受精。

芳基硫酸酯酶A（ARSA）在哺乳动物的繁殖中起着至关重要的作用，因为它参与了精子和卵融合导致受精之前的特定配子相互作用。最近，已经显示，通过使用特异性抗ARSA抗体显着阻碍了小鼠透明带（ZP）精子的结合和体内受精。在本文中，提出了基于含香豆素的多环化合物的第一个ARSA小分子抑制剂的设计和发现。通过在我们的内部图书馆中使用的基于结构的方法，化合物1r被鉴定为ARSA可逆抑制剂（ARSAi）；然后通过表面等离振子共振和生化抑制实验验证了其活性，首先提供了K _D值为21μM，后者的IC ₅₀值为13.2μM。进一步的研究表明，化合物1r在25μM时可导致20％的精子死亡，并且还会损害精子的运动能力。但是，这两种作用均由ROS产生介导，因为它们是通过1r和N-乙酰半胱氨酸（NAC）的共同治疗而挽救的。有趣的是，虽然1r不能阻止ZP /精子结合，但它显着降低了小鼠精子的体外卵母细胞受精率，最高可达60％。值得注意的是，此效果并未受到1r的影响/ NAC共同管理，因此可以排除依赖ROS的机制。总而言之，本文报道了ARSAi作为化学工具的首次成功应用，它将能够更好地探索ARSA在受精过程中的生物学作用，并为开发旨在提高酶抑制潜能的1r结构优化提供起点，同时也提供了更深的了解ARSA在受精途径机制中的参与。

更新日期：2020-04-02

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