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  • br Discussion br Drug repurposing screens have several benef

    2020-08-07


    4. Discussion
    Drug repurposing screens have several benefits in drug discovery including the ability to rapidly identify new indications, the relative safety of the drug candidates, and the ability to develop new ther-apeutic targets [36]. From this study, Benp was identified as a potential candidate for anti-metastatic drugs. While our understanding of the cough reflex and hypertussive states has increased, cough treatment remains a significant unmet medical need [37]. Benp is clinically used as an antitussive drug; however, the molecular target and mode of ac-tion of Benp remain unknown. In this study, we identified ARPC2, which directly interacted with Benp in Mocetinostat (MGCD0103, MG0103) (Fig. 5). These results may explain the antitussive effects of Benp in patients and suggest that novel ARPC2 inhibitors may be useful antitussive drugs. Additionally, the information may help elucidate the factors related to hypertussive states, which will improves the options in terms of new medicines for treating cough.
    Despite the significant progress made in the development of effi-cient therapeutics for cancer treatment, cancer still remains the leading cause of death worldwide. Metastasis is the main cause of cancer mortality and is a complex and multistep process; cancer cell migration  Biochemical Pharmacology 163 (2019) 46–59
    is a pivotal step in the metastatic process [2,3]. Therefore, targeting cancer cell migration may be an important approach for discovery of anti-metastatic drugs [7,38]. Benp will be a good candidate for devel-opment of anti-metastatic drugs because it specifically and selectively blocks the migration and invasion of some of cancer cells (Fig. 1C and F). For small molecule drugs, target engagement is crucial to identi-fying the molecular targets underlying the therapeutic effects [39]. For these purposes, non-affinity-based approaches have been developed, such as DARTS, CETSA, stability of proteins from rates of oxidation, and bioinformatics-based analysis of connectivity [40]. Integrating diverse techniques has been proposed for validating target engagement in an-imal tissues and human biopsy samples [41]. Here, we confirmed the direct binding of Benp to ARPC2 by DARTS and CETSA (Fig. 5D and E). In the future, these tools will be useful for pharmacodynamic studies of drugs and thus facilitate clinical application.
    Functional studies of the Arp2/3 complex for migration have been examined by inhibiting or depleting Arp2/3 complex subunits in dif-ferent cell types such as fibroblasts, haematopoietic cells, epithelial cells, and cancer cells [23]. In a variety of cell types, the results of Arp2/3 depletion and chemical inhibition of Arp2/3 activity are con-flicting due to its genetic diversity and heterogeneity in the cells [23]. In MCF-10A cells, treatment with the Arp2/3 inhibitors CK666 and CK869 disrupted lamellipodia formation and directional migration [21]; however, Benp did not inhibit the migration and invasion of MCF-10A cells (Fig. 8B and C) and showed no effect on cell morphology (Fig. 8D). Thus, our data show that cell migration is differentially modulated in different cell types.
    The composition of the Arp2/3 complex subunits represents a cell line-specific difference, giving rise to the functional divergence in dif-ferent cell types [20]. For example, alternative Arp2/3 complexes with Arp2, Arp3, and ARPC2, with or without ARPC1B, were detected and were then coupled with vinculin/α-actinin [42]. In cell cycle progres-sion, Aurora A interacts with ARPC1B, which exists as a stand-alone protein and a part of the Arp2/3 complex [43]. ARPC2 depletion by siRNA-mediated knockdown suppresses cell migration in pancreatic and gastric cancer cells [22,44]. In addition to the role of ARPC2 in actin polymerization, it upregulates cancer-promoting genes and downregulates tumor suppressor genes in gastric cancer cells [22]. Several studies of ARPC2 depletion show the concomitant loss of other subunits of the Arp2/3 complex [24,35]. However, Benp disrupted the function of ARPC2 without affecting the expression levels of Arp2/3 complex subunits, resulting in inhibition of cancer cell migration and metastasis. Therefore, small molecule inhibitors targeting Arp2/3 complex subunits and impairing its function may be a new approach for cancer therapy.
    The expression levels of actin binding proteins have been shown to be correlated with the invasiveness and metastatic potential of cancer cells [10,11]. Since Arp2/3 is activated by positive regulators such as WAVE and N-WASP, the expression and activation state of the reg-ulators should be elucidated in various types of cancer cells [16]. Moreover, negative regulators of the Arp2/3 complex, such as Arpin and Gadkin, are highly expressed in normal cells compared with cancer cells [45,46]. These regulators suppress actin polymerization by binding to the Arp2/3 complex and competing with positive regulators [16]. In the future, additional studies are needed to evaluate the ex-pression levels and binding sites of the negative regulators of the Arp2/ 3 complex in Benp-treated cells.