De novo-designed mini-protein against SARS-CoV-2

By Tarun Sai LomteReviewed by Danielle Ellis, B.Sc.Jun 7 2022

In a recent study published in the International Journal of Molecular Sciences, researchers characterized truncated forms of LCB1, a de novo-designed mini-protein, against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Background

During the early coronavirus disease 2019 (COVID-19) pandemic, therapeutic options were limited, but many treatment strategies have been developed, including drugs targeting SARS-CoV-2. Monoclonal antibodies (mAbs) against SARS-CoV-2 spike (S) protein are the first approved therapeutics. mAbs bind to S protein and prevent viral attachment to host cells. Lately, two oral drugs, molnupiravir and nirmatrelvir have been introduced.

The therapeutic strategy of preventing viral attachment to host cells is most effective when drugs are administered early in the clinical course. Given that therapeutic mAbs are administered in a hospital setting through intravenous infusions, they are less likely to be used in the initial phases of the disease.

Moreover, the large size of antibodies might also hamper tissue penetration. Therefore, research is focused on exploring alternatives like small molecules capable of inhibiting viral interactions with host cells. Over the last two years, smaller proteins/peptides have been designed computationally either through de novo design based on receptor-binding domain structure or based on the structure of N-terminal α-helices of angiotensin-converting enzyme 2 (ACE2), the host cell receptor.

These mini-proteins or peptides could bind to RBD similarly to antibodies, preventing interactions with ACE2. LCB1 is one such mini-protein, which is 55 amino acids long and folds into three α-helices. Produced recombinantly, LCB1 has been shown to bind to RBD and efficiently inhibit infections of cells with SARS-CoV-2.

The study and results

In the present study, researchers truncated LCB1 harboring only one or two α-helices and evaluated their antiviral potency. The authors generated smaller versions of LCB1 harboring individual helix 1 (peptide LW25.5) and helix 2 (LW26.5), and both helices 1 and 2 (LW25.1). Besides, the full-length LCB1 (LW25.3) with acetylated N-terminus and amidated C-terminus was also tested.

These LCB1 variants were assessed for their ability to inhibit the interaction of soluble recombinant ACE2 with RBD. They found that inhibitory activity of LW25.3 was preserved in the truncated LW25.1 variant, confirming that helix 3 was not essential for inhibitory activity. Moreover, both the single-helix variants (LW25.5 and LW26.5.) were nonfunctional, implying that two helices were required for interactions with RBD.

A 35-mer peptide (LW32.4) was identified upon further truncation of helix 2 C-terminus in the two-helix variant (LW25.1), which also retained the inhibitory potential. In subsequent investigations, the team noted that LW32.4, LW25.1, and LW25.3 efficiently inhibited infection of A549 cells expressing human ACE2 and transmembrane protease, serine 2 (TMPRSS2) in a pseudovirus neutralization assay.

The three helices of LCB1 are interconnected by short loops composed of a single amino acid between helices 1 and 2 and four amino acids between helices 2 and 3. The helices are aligned anti-parallel, such that the helix 1 N-terminus lies adjacent to the C-terminal end of helix 2. This proximity is most prominent between K2 and G39 residues 8.7 Å apart. These residues in the LW25.1 variant were replaced with cysteine (LW25.13), introducing the disulfide bridge.

Intriguingly, the team noted a 20- and nine-fold stronger inhibitory activity than LW25.3 and LW32.4, respectively. This indicated that a covalent bridge between the C- and N-termini of peptide stabilized the bioactive conformation. The RBD-ACE2 binding inhibition and neutralization of pseudovirus of wildtype SARS-CoV-2, Alpha, Beta, Delta, and Omicron variants by LW25.3, LW25.13, and LW32.4 were tested.

Delta variant was susceptible to inhibition by the three peptides, whereas Alpha and Omicron variants were resistant. The susceptibility of LW32.4 and LW25.13 to proteolytic cleavage by pepsin was examined. While LW32.4 was hydrolyzed at the L31 site, LW25.13 was resistant to or stable for 60 minutes at least. This was also consistent with another protease, neutrophil elastase, expressed in inflamed lungs. LW32.4 was rapidly hydrolyzed, whereas LW25.13 was intact.

Conclusions

To summarize, the researchers demonstrated that truncated forms of LCB1 containing two of its three helices inhibited RBD-ACE2 interactions and retained SARS-CoV-2 neutralizing activity. Further, the cyclic variant, LW25.13, exhibited approximately 10-fold greater neutralizing potential than LW25.3. The peptide, LW25.13, was also relatively more stable toward the action of pepsin and neutrophil elastase.

Since the peptides were products of chemical synthesis rather than recombinant protein expression, they could be further chemically modified, such as the inclusion of non-proteinogenic amino acids. These modifications could further stabilize the peptide and improve the strength and breadth of antiviral potency. Overall, the findings indicated that LW25.13 might represent a promising candidate for therapeutic intervention against SARS-CoV-2.