The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.

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Osseointegration, the direct functional and structural connection between device and bone is influenced by multiple factors such as implant macrogeometry and surgical technique. This study investigated the effects of osseodensification drilling techniques on implant stability and osseointegration using trabecular metal (TM) and tapered-screw vent (TSV) implants in a low-density bone. Six skeletally mature sheep were used where six osteotomy sites were prepared in each of the ilia, (n = 2/technique: Regular [R] (subtractive), clockwise [CW], and counterclockwise [CCW]). One TM and one TSV implant was subsequently placed with R osteotomy sites prepared using a conventional (subtractive) drilling protocol as recommended by the implant manufacturer for low density bone. CW and CCW drilling sites were subjected to osseodensification (OD) (additive) drilling. Evaluation of insertion torque as a function of drilling technique showed implants subjected to R drilling yielded a significant lower insertion torque relative to samples implanted in OD (CW/CCW) sites (p < 0.05). Histomorphometric analysis shows that the osseodensification demonstrates significantly greater values for bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO). Histological analysis shows the presence of bone remnants, which acted as nucleating surfaces for osteoblastic bone deposition, facilitating the bridging of bone between the surrounding native bone and implant surface, as well as within the open spaces of the trabecular network in the TM implants. Devices that were implanted via OD demonstrated atemporal biomechanical stability and osseointegration. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2516-2523, 2018.