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 β-coronavirus SARS-CoV-2 has caused a global pandemic. Affinity reagents targeting the SARS-CoV-2 spike protein are of interest for the development of therapeutics and diagnostics. We used affinity selection–mass spectrometry for the rapid discovery of synthetic high-affinity peptide binders for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. From library screening with 800 million synthetic peptides, we identified three sequences with nanomolar affinities (dissociation constants K d = 80–970 nM) for RBD and selectivity over human serum proteins. Nanomolar RBD concentrations in a biological matrix could be detected using the biotinylated lead peptide in ELISA format. These peptides do not compete for ACE2 binding, and their site of interaction on the SARS-CoV-2-spike-RBD might be unrelated to the ACE2 binding site, making them potential orthogonal reagents for sandwich immunoassays. These findings serve as a starting point for the development of SARS-CoV-2 diagnostics or conjugates for virus-directed delivery of therapeutics.
FK506-binding proteins (FKBPs) are evolutionarily conserved proteins that display peptidyl-prolyl isomerase activities and act as coreceptors for immunosuppressants. Microbial macrophage-infectivity-potentiator (Mip)-type FKBPs can enhance infectivity. However, developing druglike ligands for FKBPs or Mips has proven difficult, and many FKBPs and Mips still lack biologically useful ligands. To explore the scope and potential of C-substituted [4.3.1]-aza-bicyclic sulfonamides as a broadly applicable class of FKBP inhibitors, we developed a new synthesis method for the bicyclic core scaffold and used it to prepare an FKBP- and Mip-focused library. This allowed us to perform a systematic structure-activity-relationship analysis across key human FKBPs and microbial Mips, yielding highly improved inhibitors for all the FKBPs studied. A cocrystal structure confirmed the molecular-binding mode of the core structure and explained the affinity gained as a result of the preferred substituents. The best FKBP and Mip ligands showed promising antimalarial, antileginonellal, and antichlamydial properties in cellular models of infectivity, suggesting that substituted [4.3.1]-aza-bicyclic sulfonamides could be a novel class of anti-infectives.
Enhancement by displacement. A single methyl group displaces a water molecule from the binding site of FKBPs, resulting in the most potent binders known, outperforming the natural products FK506 and rapamycin in biochemical and cellular assays.
To create highly efficient inhibitors for FK506-binding proteins, a new asymmetric synthesis for pro-(S)-C(5) -branched [4.3.1] aza-amide bicycles was developed. The key step of the synthesis is an HF-driven N-acyliminium cyclization. Functionalization of the C(5) moiety resulted in novel protein contacts with the psychiatric risk factor FKBP51, which led to a more than 280-fold enhancement in affinity. The most potent ligands facilitated the differentiation of N2a neuroblastoma cells with low nanomolar potency.
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