Recombinant Protein Micelles to Block Transduction by SARS-CoV-2 Pseudovirus

Abstract

The continuing emergence of variants of the SARS-CoV-2 virus requires the development of modular molecular therapies. Here, we engineered a recombinant amphiphilic protein, oleosin, to spontaneously self-assemble into multivalent micellar nanostructures which can block the Spike S1 protein of SARS-CoV-2 pseudoviruses (PVs). Short recombinant proteins like oleosin can be formulated more easily than antibodies and can be functionalized with precision through genetic engineering. We cloned S1-binding mini-protein genes called LCB x, previously designed by David Baker’s laboratory (UW Seattle), to the N-terminus of oleosin, expressing Oleo-LCB x proteins in E. coli . These proteins largely formed 10–100 nm micelles as verified by dynamic light scattering. Two proteins, Oleo-LCB1 and Oleo-LCB3, were seen to completely and irreversibly block transduction by both wild-type and delta variant PVs into 293T-hsACE2 cells at 10 μM. Presented in multivalent micelles, these proteins reduced transduction by PVs down to a functional protein concentration of 5 nM. Additionally, Oleo-LCB1 micelles outperformed corresponding synthetic LCB1 mini-proteins in reducing transduction by PVs. Tunable aqueous solubility of recombinant oleosin allowed incorporation of peptides/mini-proteins at high concentrations within micelles, thus enhancing drug loading. To validate the potential multifunctionality of the micelles, we showed that certain combinations of Oleo-LCB1 and Oleo-LCB3 performed much better than the individual proteins at the same concentration. These micelles, which we showed to be non-toxic to human cells, are thus a promising step toward the design of modular, multifunctional therapeutics that could bind to and inactivate multiple receptors and proteins necessary for the infection of the SARS-CoV-2 virus.

Susceptibility and Resistance of SARS-CoV-2 Variants to LCB1 and Its Multivalent Derivatives

“…By using a truncation and lipid modification strategy, we further generated several lipopeptide-based LCB1 inhibitors that were highly effective in inhibiting the LCB1-resistant variants [ 17 ]. To overcome the LCB1 resistance problem, other strategies including cyclization and multimerization have also been used to improve the antiviral activity of LCB1-derived inhibitors against divergent emerging SARS-CoV-2 variants [ 15 , 31 , 32 , 33 ]. Regrettably, the emerging virus variants still possessed mild resistance to the modified LCB1 inhibitors’ inhibition.…”

Lipidation Alters the Structure and Hydration of Myristoylated Intrinsically Disordered Proteins

“…The development of new peptides/proteins has traditionally focused on engineering their amino acid sequence to regulate the structure–function for desired applications, including materials, − vaccines and biopharmaceuticals, − sensors, , and others. − In contrast, nature leverages posttranslational modifications (PTMs)the decoration of proteins with motifs such as phosphate, carbohydrates, and lipids, among othersto modulate protein structure, function, and location with exquisite spatiotemporal control . The chemical diversity of PTMs far surpasses the canonical design space of the 20–22 naturally occurring amino acids, exponentially increasing the diversity of proteinaceous molecules available to regulate the spatiotemporal flow of life-sustaining matter, energy, and information.…”