Optimizing variant-specific therapeutic SARS-CoV-2 decoys using deep-learning-guided molecular dynamics simulations.

Treatment of COVID-19 with a soluble version of ACE2 that binds to SARS-CoV-2 virions before they enter host cells is a promising approach, however it needs to be optimized and adapted to emerging viral variants. The computational workflow presented here consists of molecular dynamics simulations for spike RBD-hACE2 binding affinity assessments of multiple spike RBD/hACE2 variants and a novel convolutional neural network architecture working on pairs of voxelized force-fields for efficient search-space reduction. We identified hACE2-Fc K31W and multi-mutation variants as high-affinity candidates, which we validated in vitro with virus neutralization assays. We evaluated binding affinities of these ACE2 variants with the RBDs of Omicron BA.3, Omicron BA.4/BA.5, and Omicron BA.2.75 in silico. In addition, candidates produced in Nicotiana benthamiana, an expression organism for potential large-scale production, showed a 4.6-fold reduction in half-maximal inhibitory concentration (IC50) compared with the same variant produced in CHO cells and an almost six-fold IC50 reduction compared with wild-type hACE2-Fc.

Design of a bifunctional fusion protein for ovarian cancer drug delivery: single-chain anti-CA125 core-streptavidin fusion protein.

We have developed a universal ovarian cancer cell targeting vehicle that can deliver biotinylated therapeutic drugs. A single-chain antibody variable domain (scFv) that recognizes the CA125 antigen of ovarian cancer cells was fused with a core-streptavidin domain (core-streptavidin-VL-VH and VL-VH-core-streptavidin orientations) using recombinant DNA technology and then expressed in Escherichia coli using the T7 expression system. The bifunctional fusion protein (bfFp) was expressed in a shaker flask culture, extracted from the periplasmic soluble protein, and affinity purified using an IMAC column. The two distinct activities (biotin binding and anti-CA125) of the bfFp were demonstrated using ELISA, Western blot and confocal laser-scanning microscopy (CLSM). The ELISA method utilized human NIH OVCAR-3 cells along with biotinylated bovine serum albumin (B-BSA) or biotinylated liposomes, whereas, the Western blot involved probing with B-BSA. The CLSM study has shown specificity in binding to the OVCAR-3 cell-line. ELISA and Western blot studies have confirmed the bifunctional activity and specificity. In the presence of bfFp, there was enhanced binding of biotinylated antigen and liposome to OVCAR-3 cells. In contrast, the control EMT6 cells, which do not express the CA125 antigen, showed minimal binding of the bfFp. Consequently, bfFp based targeting of biotinylated therapeutic drugs, proteins, liposomes, or nanoparticles could be an alternative, convenient method to deliver effective therapy to ovarian cancer patients. Peritoneal infusion of the bfFp-therapeutic complex could also be effective in locally targeting the most common site of metastatic spread.

Biotin carboxyl carrier protein co-purifies as a contaminant in core-streptavidin preparations.

We have successfully cloned and expressed core-streptavidin in Escherichia coli. Core-streptavidin was expressed in shaker flask culture as a soluble protein, isolated by periplasmic extraction, purified by immobilized metal affinity chromatography column, and analyzed for its size, thermal stability, and biotin-binding activity. In Western blots using streptavidin-horseradish peroxidase (HRP) as a probe, we identified a contaminant that co-purified with core-streptavidin, identified as biotin carboxyl carrier protein (BCCP). Although BCCP cannot be detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it appears as a prominent band in Western blot when probed with streptavidin peroxidase conjugate. Based on the results from in vitro gel digestion, mass spectrometry and Mascot database search results, we confirmed the presence of BCCP. It was found that BCCP can complex with core-streptavidin and can dissociate when heated above 80 degrees C. BCCP could be successfully removed and recovered by using core-streptavidin immobilized magnetic beads under mild conditions. In addition, the enriched fractions of core-streptavidin oligotetramers were separated, which may be the by-products of BCCP binding to core-streptavidin in various ratios. Finally, enzyme linked immunosorbent assay results have shown that the amount of biotin-HRP binding to core-streptavidin was higher compared to commercially available streptavidin.