*P?P?Baloxavir ?(Fig.6A,C).6A,C). Additional analogs, SCYX827830 and SCYX1454139, had significant anti\HBV activities (Fig. ?(Fig.6A,C).6A,C). Alisporivir (Debio 025), an anti\HCV drug candidate,38 also decreased HBV infection to the equivalent level to CsA (Fig. ?(Fig.6B).6B). Figure 6D shows a dose\dependent reduction of HBs secretion by treatment with SCYX618806, SCYX827830, and SCYX1454139, all of which had more potent anti\HBV activities than CsA (compare Fig. ?Fig.6D6D with Fig. ?Fig.2A).2A). These results indicate that anti\HBV activity is not disrupted by at least some changes to the 3\glycine, 4\leucine, and 8\alanine residues of CsA, although additional analogs will need to be evaluated for a full understanding of the structure\activity relationships. Notably, SCYX618806 and alisporivir bear modifications on the 4\leucine residue of the CsA backbone that prevent CN binding and immunosuppressive activity (Table S1), further confirming that anti\HBV activity does not require immunosuppressive activity. Notably, SCYX1454139 showed the strongest anti\HBV entry activity among 50 CsA derivatives examined (data not shown and Fig. ?Fig.6E).6E). The median inhibitory concentrations (IC50s) for anti\HBV activity as well as CC50s determined by an MTT\based cell viability assay are shown in Fig. ?Fig.6E.6E. The IC50 and CC50 of SCYX1454139 were 0.17??0.02 and >10 Baloxavir M, respectively, a profile superior to that of CsA (IC50 and CC50 of 1 1.17??0.22 and >10 M, respectively). Inhibition of HBV infection by treatment with SCYX1454139 was also observed in PHHs, in which also the anti\HBV effect of SCYX1454139 was more remarkable than that of CsA (Fig. ?(Fig.6F).6F). These results clearly indicate that analogs of CsA may include compounds with greater anti\HBV potency than that of CsA itself. Open in a separate window Figure 6 Analysis of CsA analogs. (A,B) Anti\HBV activity of CsA analogs. HepaRG cells were treated with or without dimethyl sulfoxide (DMSO), heparin 10 U/mL, lamivudine 1 M, CsA 4 M, or its analogs, SCYX618806, SCYX1774198, SCYX827830, and SCYX1454139 (A) or alisporivir (B) at 4 M, as shown in Fig. ?Fig.1A1A to measure HBs and HBe secretion level. (C) Chemical structures of CsA and its derivatives. (D) Dose\response curves for CsA analogs. HepaRG cells were treated with or without various concentrations of SCYX618806, SCYX827830, or SCYX1454139 (0.25, 0.5, 1, 2, and 4 M) as shown in Fig. ?Fig.1A.1A. (E) IC50s (M) for Rabbit Polyclonal to HTR4 CsA and its analogs in blocking HBV infection are shown. CC50s (M) determined by the MTT cell viability assay are also shown. (F) PHHs were treated with CsA and its derivatives at 4 M or left untreated according to Baloxavir the protocol in Fig. ?Fig.1A,1A, and HBV infection was monitored by HBs protein secretion. *P?P?