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. 2023 Mar 20;13(13):8779-8793.
doi: 10.1039/d2ra07356k. eCollection 2023 Mar 14.

Dimerized fusion inhibitor peptides targeting the HR1-HR2 interaction of SARS-CoV-2

Kohei Tsuji  1 , Kofi Baffour-Awuah Owusu  1 , Yutaro Miura  1 , Takahiro Ishii  1 , Kouki Shinohara  1 , Takuya Kobayakawa  1 , Akino Emi  2 , Takashi Nakano  2 , Youichi Suzuki  2 , Hirokazu Tamamura  1
Affiliations

Dimerized fusion inhibitor peptides targeting the HR1-HR2 interaction of SARS-CoV-2

Kohei Tsuji et al. RSC Adv. .

Abstract

Membrane fusion is a critical and indispensable step in the replication cycles of viruses such as SARS-CoV-2 and human immunodeficiency virus type-1 (HIV-1). In this step, a trimer of the heptad repeat 1 (HR1) region interacts with the three HR2 regions and forms a 6-helix bundle (6-HB) structure to proceed with membrane fusion of the virus envelope and host cells. Recently, several researchers have developed potent peptidic SARS-CoV-2 fusion inhibitors based on the HR2 sequence and including some modifications. We have developed highly potent HIV-1 fusion inhibitors by dimerization of its HR2 peptides. Here, we report the development of dimerized HR2 peptides of SARS-CoV-2, which showed significantly higher antiviral activity than the corresponding monomers, suggesting that the dimerization strategy can facilitate the design of potent inhibitors of SARS-CoV-2.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1. Schematic representations of entry and fusion mechanisms of SARS-CoV-2. (a) Dual entry pathway of SARS-CoV-2 infection: (1) binding of spike proteins on the virus surface to the receptor, ACE2, on the host cell surface; (2a) processing of the S1 units by exogeneous and membrane bound proteases; (3a) membrane fusion and releasing viral RNA; (2b) internalization via endocytosis; (3b) endosomal acidification and processing of the S1 units by lysosomal proteases; (4b) membrane fusion and releasing virus RNA. (b) Membrane fusion mechanism of SARS-CoV-2: (1) receptor binding and processing of the S1 units by host proteases; (2) insertion of the exposed fusion peptides into the host cell membrane; (3) comformational changes of the S2 units with viral and host cell membrane approach; (4) formig 6-helix bundles and membrane fusion.
Fig. 2
Fig. 2. Representation of the 6-helix bundle (6-HB) structures of SARS-CoV-2 and HIV-1 and the dimerized fusion inhibitors of HIV-1. (a) The SARS-CoV-2 HR1–HR2 crystal structure forming 6-HB (left, PDB code: 6LXT, white: HR1, purple: HR2) and the crystal structure of HIV-1 gp41 HR1 fragment N36 and HR2-derived fusion inhibitor SC34EK forming 6-HB (right, PDB code: 2Z2T, dark gray: HR1, cyan: HR2). (b) Our previous findings in the HIV-1 research; dimerization of HR2-derived peptides resulted highly potent fusion inhibitors. The dimerization at the C-terminus is preferable to that at the N-terminus.
Fig. 3
Fig. 3. (a) The SARS-CoV-2 HR1–HR2 crystal structure forming 6-HB (PDB code: 6LXT). (b) A focused structure around 1185Arg and 1189Val of the HR2 region. A hydrogen bond between 1185Arg (in HR2) and 936Asp (in HR1) is observed. HR1 region: white ribbon with grey surface; HR2 region: green, pink (Glu), red (mutated to Glu in SR9EK13 (2)), light blue (Lys), and blue (mutated to Lys in SR9EK13 (2)). (c) The sequences of SARS-CoV-2 HR2 (1169I–1202E) (1), SR9EK13, described with the corresponding position alignment (2) and its modified sequence (3).
Fig. 4
Fig. 4. Structures of designed monomeric peptides. E (red), K (blue): mutation introduction; E (pink), K (light blue): natural sequence; placement of E (i position) and K (i + 3 or i + 4 position) or placement of K (i position) and E (i + 3 or i + 4 position) leads to a salt bridge formation between their sidechains.
Fig. 5
Fig. 5. Structures of octa-arginine (R8)-conjugated peptides.
Fig. 6
Fig. 6. Structures of C-terminal dimers.
Fig. 7
Fig. 7. Structures of N-terminal dimers.
Scheme 1
Scheme 1. Synthesis of monomer peptides shown in Fig. 4. (a) Preparation of 1a and 2a–7a; (b) preparation of 1b; (c) preparation of 8a.
Scheme 2
Scheme 2. Synthesis of octa-arginine-conjugated peptides shown in Fig. 5. (a) Preparation of 1c, 2b, 3b and 6b; (b) preparation of 1e; (c) preparation of 4b, 5b and 7b. Peptide 1d was synthesized as shown in Scheme 1a.
Scheme 3
Scheme 3. Synthesis of the C-terminal dimers shown in Fig. 6. (a) Preparation of 1Ca, 1Cb, 1Cc, 4C, 5C and 7C; (b) preparation of 2Ca, 2Cb, 2Cc, 3C and 6C; (c) preparation of 8C.
Scheme 4
Scheme 4. Synthesis of the N-terminal dimers shown in Fig. 7. (a) Preparation of 1N–7N; (b) preparation of 8N.
Fig. 8
Fig. 8. CD spectra of the HR2 peptides. CD spectra were measured at 25 °C in PBS (50 mM Na phosphate buffer, 150 mM NaCl, pH 7.2).
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