Bioorganic Chemistry 105 (2020) 104337 Available online 6 October 2020 0045-2068/© 2020 Elsevier Inc. All rights reserved. Identifcation of the hydantoin alkaloids parazoanthines as novel CXCR4 antagonists by computational and in vitro functional characterization Rosa Maria Vitale a, 1 , Stefano Thellung b, 1 , Francesco Tinto a, 1 , Agnese Solari b , Monica Gatti b , Genoveffa Nuzzo a , Efstathia Ioannou c , Vassilios Roussis c , Maria Letizia Ciavatta a , Emiliano Manzo a, * , Tullio Florio b, d, * , Pietro Amodeo a, * a Institute of Biomolecular Chemistry, National Research Council (ICB-CNR), Via Campi Flegrei 34, 80078, Pozzuoli, (NA), Italy b Section of Pharmacology, Department of Internal Medicine, University of Genova, 16132 Genova, Italy c Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece d IRCCS Policlinico San Martino, 16132 Genova, Italy A R T I C L E INFO Keywords: Hydantoin alkaloids Parazoanthines Natural products CXCR4 antagonists Molecular docking Pharmacophoric model ABSTRACT CXCR4 chemokine receptor represents an attractive pharmacological target due to its key role in cancer metastasis and infammatory diseases. Starting from our previously-developed pharmacophoric model, we applied a combined computational and experimental approach that led to the identifcation of the hydantoin alkaloids parazoanthines, isolated from the Mediterranean Sea anemone Parazoanthus axinellae, as novel CXCR4 antagonists. Parazoanthine analogues were then synthesized to evaluate the contribution of functional groups to the overall activity. Within the panel of synthesized natural and non-natural parazoanthines, parazoanthine-B was identifed as the most potent CXCR4 antagonist with an IC 50 value of 9.3 nM, even though all the investi- gated compounds were able to antagonize in vitro the down-stream effects of CXC12, albeit with variable potency and effcacy. The results of our study strongly support this class of small molecules as potent CXCR4 antagonists in tumoral pathologies characterized by an overexpression of this receptor. Furthermore, their structure–activity relationships allowed the optimization of our pharmacophoric model, useful for large-scale in silico screening. 1. Introduction Chemokines comprise a family of chemotactic cytokines, classifed according to the position of the conserved N-terminal cysteine residues into four main subfamilies: CXC, CC, CX3C and XC [1]. These small proteins exert their homeostatic and/or infammatory functions by binding and activation of cognate receptors, belonging to the G-protein coupled receptors (GPCRs) family. CXCR4, the cognate receptor of the C- X-C chemokine CXCL12, is by far the most largely studied chemokine receptor, due to its relevance in many pathologic processes, including HIV infection [2], infammatory diseases [3–5], cancer [6–9], and WHIM syndrome [10]. In particular, CXCR4 plays a pivotal role in cancer cell survival, proliferation, migration, invasiveness and metas- tasis, representing a relevant therapeutic target in those diseases char- acterized by up-regulation of CXCL12/CXCR4 signaling [11]. Currently, the CXCR4 antagonist AMD3100, initially proposed as an anti-HIV agent, is a FDA-approved drug in patients with multiple myeloma and non-Hodgkin’s lymphoma who have undergone stem cell trans- plantation, for its mobilizing effect on hematopoietic cells [12]. Starting from AMD3100, other molecules have been developed in an attempt to reduce the size and improve the pharmacokinetic profle, including AMD070 [13], GSK81239 [14], TG-0054 [15], KRH-3955 [16], and HF5073 [17]. In a previous study [18], we developed a minimalistic pharmaco- phoric model for CXCR4 ligands in which the essential anchoring points on CXCR4 consisted of an aromatic and a basic functional groups, separated by a properly-sized spacer, resulting in an upper limit of 18 Å for the distance between the centers of mass of the two groups. This model led to the identifcation of phidianidine A (Fig. 1), an alkaloid compound of marine origin, as CXCR4 inhibitor exhibiting low micro- molar activity. Natural compounds, representing an invaluable source of novel chemical scaffolds endowed with a high chemical diversity, are * Corresponding authors. E-mail addresses: emanzo@icb.cnr.it (E. Manzo), tullio.forio@unige.it (T. Florio), pamodeo@icb.cnr.it (P. Amodeo). 1 These authors share co-frst authoriship. Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg https://doi.org/10.1016/j.bioorg.2020.104337 Received 12 August 2020; Received in revised form 19 September 2020; Accepted 30 September 2020