Virtual screening and in vitro validation of natural compound inhibitors against SARS-CoV-2 spike protein.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to a major public health burden and has resulted in millions of deaths worldwide. As effective treatments are limited, there is a significant requirement for high-throughput, low resource methods for the discovery of novel antivirals. The SARS-CoV-2 spike protein plays a key role in viral entry and has been identified as a therapeutic target. Using the available spike crystal structure, we performed a virtual screen with a library of 527 209 natural compounds against the receptor binding domain of this protein. Top hits from this screen were subjected to a second, more comprehensive molecular docking experiment and filtered for favourable ADMET properties. The in vitro activity of 10 highly ranked compounds was assessed using a virus neutralisation assay designed to facilitate viral entry in a physiologically relevant manner via the plasma membrane route. Subsequently, four compounds ZINC02111387, ZINC02122196, SN00074072 and ZINC04090608 were identified to possess antiviral activity in the µM range. These findings validate the virtual screening method as a tool for identifying novel antivirals and provide a basis for future drug development against SARS-CoV-2.

Synthesis and Characterization of Bone Binding Antibiotic-1 (BBA-1), a Novel Antimicrobial for Orthopedic Applications.

Osteomyelitis and orthopedic infections are major clinical problems, limited by a lack of antibiotics specialized for such applications. In this paper, we describe the design and synthesis of a novel bone-binding antibiotic (BBA-1) and its subsequent structural and functional characterization. The synthesis of BBA-1 was the result of a two-step chemical conjugation of cationic selective antimicrobial-90 (CSA-90) and the bisphosphonate alendronate (ALN) via a heterobifunctional linker. This was analytically confirmed by HPLC, FT-IR, MS and NMR spectroscopy. BBA-1 showed rapid binding and high affinity to bone mineral in an in vitro hydroxyapatite binding assay. Kirby-Baur assays confirmed that BBA-1 shows a potent antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus comparable to CSA-90. Differentiation of cultured osteoblasts in media supplemented with BBA-1 led to increased alkaline phosphatase expression, which is consistent with the pro-osteogenic activity of CSA-90. Bisphosphonates, such as ALN, are inhibitors of protein prenylation, however, the amine conjugation of ALN to CSA-90 disrupted this activity in an in vitro protein prenylation assay. Overall, these findings support the antimicrobial, bone-binding, and pro-osteogenic activities of BBA-1. The compound and related agents have the potential to ensure lasting activity against osteomyelitis after systemic delivery.

A Bioactive Coating Enhances Bone Allografts in Rat Models of Bone Formation and Critical Defect Repair.

Bone allografts are inferior to autografts for the repair of critical-sized defects. Prior studies have suggested that bone morphogenetic protein-2 (BMP-2) can be combined with allografts to produce superior healing. We created a bioactive coating on bone allografts using polycondensed deoxyribose isobutyrate ester (PDIB) polymer to deliver BMP-2 ± the bisphosphonate zoledronic acid (ZA) and tested its ability to enhance the functional utility of allografts in preclinical Wistar rat models. One ex vivo and two in vivo proof-of-concept studies were performed. First, PDIB was shown to be able to coat bone grafts (BGs). Second, PDIB was used to coat structural allogenic corticocancellous BG with BMP-2 ± ZA ± hydroxyapatite (HA) microparticles and compared with PDIB-coated grafts in a rat muscle pouch model. Next, a rat critical defect model was performed with treatment groups including (i) empty defect, (ii) BG, (iii) collagen sponge + BMP-2, (iv) BG + PDIB/BMP-2, and (v) BG + PDIB/BMP-2/ZA. Key outcome measures included detection of fluorescent bone labels, microcomputed tomography (CT) quantification of bone, and radiographic healing. In the muscle pouch study, BMP-2 did not increase net bone volume measured by microCT, however, fluorescent labeling showed large amounts of new bone. Addition of ZA increased BV by sevenfold (p < 0.01). In the critical defect model, allografts were insufficient to promote reliable union, however, union was achieved in collagen/BMP-2 and all BG/BMP-2 groups. Statement of clinical significance: These data support the concept that PDIB is a viable delivery method for BMP-2 and ZA delivery to enhance the bone forming potential of allografts. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2278-2286, 2019.

Effect of citrus peel extracts on the cellular quiescence of prostate cancer cells.

The re-entry of quiescent cancer cells to the cell cycle plays a key role in cancer recurrence, which can pose a high risk after primary treatment. Citrus peel extracts (CPEs) contain compounds that can potentially impair tumour growth; however the mechanism of action and effects on cell cycle regulation remain unclear. In this study, the capacity of an ethyl acetate : hexane extract (CPE/hexane) and water extract (CPE/water) to modulate the cell cycle re-entry of quiescent (PC-3 and LNCaP) prostate cancer cells was tested in an in vitro culture system. Cell cycle analysis showed that the quiescent PC-3 and LNCaP cancer cells in the presence of CPE/water were impaired in their ability to enter the S phase where only 2-3% reduction of G0/G1 cells was noted compared to 12-18% reduction of control cells. In contrast, the CPE/hexane did not show any cell cycle inhibition activity in both cell lines. A low DNA synthesis rate and weak apoptosis were observed in quiescent cancer cells treated with CPEs. Hesperidin and narirutin, the predominant flavonoids found in citrus fruits, were not responsible for the observed biological activity, implicating alternative bioactive compounds. Notably, citric acid was identified as one of the compounds present in CPEs that acts as a cell cycle re-entry inhibitor. Citric acid exhibited a higher cell toxicity effect on PC-3 prostate cancer cells than non-cancerous RWPE-1 prostate cells, suggesting specific benefits for cancer treatment. In conclusion, CPE containing citric acid together with various bioactive compounds may be used as a chemopreventive agent for post-therapy cancer patients.

A Novel Strategy for Softening Gelatin-Bioactive-Glass Hybrids.

The brittle structure of polymer-bioactive-glass hybrids is a hurdle for their biomedical applications. To address this issue here, we developed a novel method to cease the overcondensation of bioactive-glass by polymer cross-linking. Here, an organosilane-functionalized gelatin methacrylate (GelMA) is covalently bonded to a bioactive-glass during the sol-gel process, and the condensation of silica networks is controlled by photo-cross-linking of GelMA. The physicochemical properties and mechanical strength of these hybrids are tunable by the incorporation of secondary cross-linking agents. These hydrogels display elastic properties with ultimate compression strain above 0.2 mm·mm(-1) and tunable compressive modulus in the range of 42-530 kPa. In addition, these hydrogels are bioactive because they promoted the alkaline phosphatase activity of bone progenitor cells. They are also well-tolerated in the mice subcutaneous model. Therefore, our method is efficient for the prevention of overcondensation and allows preparation of soft bioactive hydrogels from organic-inorganic matrices, suitable for soft and hard tissue regeneration.

Vitamin K series: current status and future prospects.

The Vitamin K series, particularly menaquinone, have been attracting research attention, due to the potential in reducing both osteoporosis and cardiovascular diseases. This review provides an overview of the types of vitamin K and their health benefits. This is followed by a critical review of the various biotechnological approaches used in the production of menaquinone, including solid and liquid state fermentations, extraction and recovery. The currently available market information is summarized and future growth prospects are discussed. Recommendations are also given for areas of future research in order to improve the production process for menaquinone and reduce costs.

Designing of an intensification process for biosynthesis and recovery of menaquinone-7.

A nutritional food rich in menaquinone-7 has a potential in preventing osteoporosis and cardiovascular diseases. The static fermentation of Bacillus subtilis natto is widely regarded as an optimum process for menaquinone-7 production. The major issues for the bulk production of menaquinone-7 are the low fermentation yield, biofilm formation and the use of organic solvents for the vitamin extraction. In this study, we demonstrate that the dynamic fermentation involving high stirring and aeration rates enhances the yield of fermentation process significantly compared to static system. The menaquinone-7 concentration of 226 mg/L was produced at 1,000 rpm, 5 vvm, 40 °C after 5 days of fermentation. This concentration is 70-fold higher than commercially available food products such as natto. Additionally, it was found that more than 80% of menaquinone-7 was recovered in situ in the vegetable oil that was gradually added to the system as an anti-foaming agent. The intensification process developed in this study has a capacity to produce an oil rich in menaquinone-7 in one step and eliminate the use of organic solvents for recovery of this compound. This oil can, therefore, be used for the preparation of broad range of supplementary and dietary food products rich in menaquinone-7 to reduce the risk of osteoporotic fractures and cardiovascular diseases.

Sterilization of ginseng using a high pressure CO2 at moderate temperatures.

The aim of this study was to determine the feasibility of using high pressure CO2 for sterilization of Ginseng powder, as an alternative method to conventional techniques such as gamma-irradiation and ethylene oxide. The Ginseng sample used in this study was originally contaminated with fungi and 5 x 10(7) bacteria/g that was not suitable for oral use. This is the first time that high pressure CO2 has been used for the sterilization of herbal medicine to decrease the total aerobic microbial count (TAMC) and fungi. The effect of the process duration, operating pressure, temperature, and amount of additives on the sterilization efficiency of high pressure CO2 were investigated. The process duration was varied over 15 h; the pressure between 100 and 200 bar and the temperature between 25 and 75 degrees C. A 2.67-log reduction of bacteria in the Ginseng sample was achieved after long treatment time of 15 h at 60 degrees C and 100 bar, when using neat carbon dioxide. However, the addition of a small quantity of water/ethanol/H2O2 mixture, as low as 0.02 mL of each additive/g Ginseng powder, was sufficient for complete inactivation of fungi within 6 h at 60 degrees C and 100 bar. At these conditions the bacterial count was decreased from 5 x 10(7) to 2.0 x 10(3) TAMC/g complying with the TGA standard for orally ingested products. A 4.3 log reduction in bacteria was achieved at 150 bar and 30 degrees C, decreasing the TAMC in Ginseng sample to 2,000, below the allowable limit. However, fungi still remained in the sample. The complete inactivation of both bacteria and fungi was achieved within 2 h at 30 degrees C and 170 bar using 0.1 mL of each additive/g Ginseng. Microbial inactivation at this low temperature opens an avenue for the sterilization of many thermally labile pharmaceutical and food products that may involve sensitive compounds to gamma-radiation and chemically reactive antiseptic agents.