Project Description

Description

1. Summary

The COVID pandemic has very serious consequences, killing more than 7 million people worldwide since its initiation in 2019 . Despite the worldwide efforts leading to effective control of the pandemic, the continuous emergence of viral variants has necessitated the ongoing development of SARS-CoV-2 inhibitors. The main protease nsp5 plays a major role in the virus’s life cycle, and also serves as an important drug target for the virus. Our project primarily aims to develop an efficient and reliable in vivo nsp5 inhibitor screening system based on FlipGFP. Additionally, we desire to investigate the possibility of using nsp5, a sequence-specific protease, as a tool enzyme for removing recombinant tags during protein purification.

2. Inspirations and significance

Currently, the screening of nsp5 inhibitors primarily relies on FRET-based high-throughput screening following protein purification, a process that typically takes over a week[1,2,3]. This inspired us to design an inhibitor screening system that does not require protein purification. We introduce engineered FlipGFP[4] in E. coli that responds to nsp5 enzymatic activity, establishing an in vivo inhibitor screening platform without protein purification.

Positive-strand RNA viruses affect a wide range of biological classifications and exhibit a high transmission rate and mutation rate[5], necessitating the continuous development of new inhibitors. Due to the importance of proteases in all positive-sense RNA viruses and the detection flexibility of FlipGFP, our engineered platform has the potential to be extended to the screening of protease inhibitors for all positive-strand RNA viruses. Additionally, as a sequence-specific protease, we also aim to rationally design the nsp5 to enhance its activity and use it as a tool enzyme for the removal of recombinant tags during protein purification.

3. Background

3.1 SARS-CoV-2 and its lifecycle

SARS-CoV-2 is an enveloped, single-stranded, positive-sense RNA virus belonging to the Coronaviridae family and the Betacoronavirus genus. It has a diameter of approximately 80-120 nm, with an average of 24-40 spike proteins on its surface. The internal RNA genome is about 30 kb in length, encoding three structural proteins and 16 non-structural proteins[6](Figure 1).

The life cycle of SARS-CoV-2 consists of the following five steps.

  1. First, the viral particle recognizes and binds to the host cell receptor, angiotensin-converting enzyme 2 (ACE2), via its spike protein. It then attaches to the host cell surface.
  2. The virus then invades the cell through host-mediated endocytosis, releasing its genomic RNA into the host cell cytosol.
  3. Using the host's ribosomes, the virus translates polyprotein 1a (pp1a) and polyprotein 1ab (pp1ab) from its genomic RNA, which are subsequently cleaved by viral proteases into 16 non-structural proteins.
  4. These non-structural proteins assemble into a series of replication-transcription complexes (RTCs), responsible for mRNA transcription and genome replication.
  5. Newly synthesized structural proteins encapsulate the newly replicated genomic RNA, forming new viral particles that are ultimately released from the cell via host-mediated exocytosis.
Figure 1. SARS-Cov-2 genome and life cycle<sup>[6]</sup>.

3.2 The main protease nsp5

Non-structural protein 5 (nsp5) is the most highly conserved non-structural protein across the Coronaviridae family, with a molecular weight of approximately 33 kDa[6]. It is referred to as the main protease due to its pivotal role in the cleavage of polyproteins. Nsp5 has up to 11 conserved cleavage sites on the polyprotein and first undergoes autolytic cleavage to release itself, after which it cleaves at these sites to release other non-structural proteins.

Nsp5 consists of three structural domains: two N-terminal domains with protease activity and a C-terminal domain composed of α-helices[7]. The active form of SARS-CoV-2 nsp5 is a homodimer that recognizes substrates approximately 10 amino acid residues in length, but exhibits selectivity at only four specific positions(Figure 2)[8]. In addition to cleaving viral proteins, nsp5 also suppresses the host innate immune response by degrading host protein factors[9].

Figure 2. Cleavage site of nsp5 and its structure[8].

3.3 Inhibitors discovery of nsp5

As a key drug target for SARS-CoV-2, the development and optimization of nsp5 inhibitors have been ongoing since 2020. nsp5 inhibitors are primarily categorized into peptidomimetic inhibitors and small molecule inhibitors.Among peptidomimetic inhibitors, Nirmatrelvir stands out as one of the most promising. It binds to nsp5 in a manner similar to its natural substrate, and since it binds in a non-covalent form, it exhibits lower cytotoxicity[10](Figure 3). Nirmatrelvir has already been approved by the FDA for clinical use in COVID-19 treatment.In the small molecule inhibitors category, Ensitrelvir is a compound developed through computational virtual screening and structure-based optimization. Like Nirmatrelvir, it also binds to nsp5 non-covalently[11] (Figure 3) and has been approved for clinical use in Japan for the treatment of COVID-19.

While significant success has been achieved in inhibitor development, the potential for nsp5 to evolve drug-resistant mutations[12] necessitates ongoing efforts in the discovery of new inhibitors to address these emerging resistant variants.

Figure 3. Structure and mechanism of inhibition of nirmatrelvir(left panel) and ensitrelvir (right panel)[13].

3.3 FlipGFP

GFP contains 11 β-strands and a central α-helix and can be divided into two parts: (1) β-strands 1-9 (β1-9) and an α-helix, and (2) β-strands 10 (β10) and 11 (β11). Each part contains the components and residues necessary for fluorescence. When these parts are separated from each other, the protein does not fluoresce. However, when β10 and β11 are connected but separated from β1-9, they will rapidly reassemble with the nearby β1-9 strands and emit fluorescence.

FlipGFP is a fluorescence protein dependent on protease activation[4]. In FlipGFP, a dimerization linker (E5 and K5) is inserted between β10 and β11, causing them to align parallel to each other and thus preventing fluorescence by failing to self-assemble with β1-9. By inserting a specific protease recognition sequence between β11 and K5, the protease can recognize and cleave this sequence(Figure 3). Subsequently, β10 and β11 can rearrange into their normal antiparallel positions, restoring fluorescence. This design provides FlipGFP with high specificity and flexibility for detection. By simply replacing the linker between β11 and K5, FlipGFP can be tailored to respond to different proteases.

Figure 4. Activation mechanism of FlipGFP[4].

Project Goals

  • We aim to express and purify the SARS-Cov-2 nsp5 in E. Coli and validate its enzymatic activity
  • We aim to engineer FlipGFP to respond to nsp5 activity, thereby developing an in vivo screening platform for nsp5 inhibitors.
  • We aim to enhance the enzymatic activity of nsp5 through rational design, enabling its application in the removal of recombinant tags during protein purification.

References

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