Investigation of ACE2 N-terminal fragments binding to SARS-CoV-2 Spike RBD

Abstract

28Coronavirus disease 19 is an emerging global health crisis. With over 200,000 29 confirmed cases to date, this pandemic continues to expand, spurring research to discover 30 vaccines and therapies. SARS-CoV-2 is the novel coronavirus responsible for this disease. It 31 initiates entry into human cells by binding to angiotensin-converting enzyme 2 (ACE2) via the 32 receptor binding domain (RBD) of its spike protein (S). Disrupting the SARS-CoV-2-RBD binding 33 to ACE2 with designer drugs has the potential to inhibit the virus from entering human cells, 34 presenting a new modality for therapeutic intervention. Peptide-based binders are an attractive 35 solution to inhibit the RBD-ACE2 interaction by adequately covering the extended protein contact 36interface. Using molecular dynamics simulations based on the recently solved ACE2 and SARS-37CoV-2-RBD co-crystal structure, we observed that the ACE2 peptidase domain (PD) α1 helix is 38 important for binding SARS-CoV-2-RBD. Using automated fast-flow peptide synthesis, we 39 chemically synthesized a 23-mer peptide fragment of the ACE2 PD α1 helix composed entirely of 40 proteinogenic amino acids. Chemical synthesis of this human derived sequence was complete in 41 1.5 hours and after work up and isolation >20 milligrams of pure material was obtained. Bio-layer 42 interferometry revealed that this peptide specifically associates with the SARS-CoV-2-RBD with 43 low nanomolar affinity. This peptide binder to SARS-CoV-2-RBD provides new avenues for 44 COVID-19 treatment and diagnostic modalities by blocking the SARS-CoV-2 spike protein 45 interaction with ACE2 and thus precluding virus entry into human cells. 46 47 Key words ： SARS-CoV-2, peptide binder, protein-protein interaction inhibitor, coronavirus, 48 COVID-19, rapid response, peptide therapeutic, MD simulation, automated flow peptide synthesis 49 50 51 3

The Distal Polybasic Cleavage Sites of SARS-CoV-2 Spike Protein Enhance Spike Protein-ACE2 Binding

“…[5][6][7] Owing to the crucial role of the SARS-CoV-2 RBD, numerous vaccine and drug candidates have been proposed to target the RBD to inhibit the RBD-ACE2 binding. [8][9][10] However, more research is beginning to show that mutations distal from the RBD (non-RBD mutations) of SARS-CoV-2 also influence its transmissibility. For instance, Walls et al 2 found that by mutating the T678NSPRRAR685 residues, which are distributed around 10 nm from the SARS-CoV-2 RBD, to a variant with T678--IL--R685, the transduction efficiency decreased in human ACE2-expressing Baby Hamster Kidney cells.…”

Computational Design of Peptides to Block Binding of the SARS-CoV-2 Spike Protein to Human ACE2

“…They also generated a hybrid peptide by linking two discontinuous fragments from hACE2 (a.a. 22-44 and 351-357) with a glycine, and this 31-mer exhibited a potent antiviral activity with an IC50 of about 0.1 μM, indicating that this artificial peptide had much stronger binding affinity for SARS-CoV RBD than the peptides composed of residues 22-44 or 22-57. Due to the high similarity of the binding interfaces between SARS-CoV RBD/hACE2 and SARS-CoV-2 RBD/hACE2, we think that this artificial peptide may also bind to SARS-CoV-2 more strongly than the peptide 21-43 tested by Zhang et al 14 , which is similar to the peptide 22-44 from Han et al 16 . Although the natural peptides are promising, it has been argued that the sequence of hACE2 is suboptimal for binding the S protein of SARS-CoV-2 17 .…”

“…These positive features make peptides great candidates to serve as therapeutics 12,13 . Recently, Zhang et al 14 reported that the natural 23-mer peptide (a.a. cut from the human ACE2 (hACE2) α1 helix can strongly bind to SARS-CoV-2 RBD with a disassociation constant (Kd) of 47 nM, which was comparable to that of the full-length hACE2 binding to SARS-CoV-2 RBD 15 ; they also showed that a shorter 12-mer peptide (a.a. [27][28][29][30][31][32][33][34][35][36][37][38] from the same helix was not able to bind the virus RBD at all. In an earlier report, Han et al 16 performed a study to identify the critical determinants on hACE2 for SARS-CoV entry, and they found that two natural peptides from hACE2 (a.a. 22-44 and 22-57) exhibited a modest antiviral activity to inhibit the binding of SARS-CoV RBD to hACE2 with IC50 values of about 50 μM and 6 μM, respectively, implying that the peptide composed of residues 22-57 had stronger binding affinity for SARS-CoV RBD.…”

Lectin-like Intestinal Defensin Inhibits 2019-nCoV Spike binding to ACE2

“…Nevertheless, HD5 had less of an effect on ACE2-S1 interaction ( Figure 2B), possibly due to a binding site distant from the RBD. Recent studies have discovered some antibodies and peptides with high affinities binding to the spike 32, 35 . These candidates are placed hopes on suppressing 2019-nCoV.…”