Stapled Peptides Based on Human Angiotensin-Converting Enzyme 2 (ACE2) Potently Inhibit SARS-CoV-2 Infection In Vitro.

SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as the primary receptor to enter host cells and initiate the infection. The critical binding region of ACE2 is an ∼30-amino-acid (aa)-long helix. Here, we report the design of four stapled peptides based on the ACE2 helix, which is expected to bind to SARS-CoV-2 and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50 to 94% helicity). In contrast, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus-based single-cycle assay in HT1080/ACE2 cells and human lung cell line A549/ACE2, overexpressing ACE2. Three of the four stapled peptides showed potent antiviral activity in HT1080/ACE2 (50% inhibitory concentration [IC50]: 1.9 to 4.1 µM) and A549/ACE2 (IC50: 2.2 to 2.8 µM) cells. The linear peptide NYBSP-C and the double-stapled peptide StRIP16, used as controls, showed no antiviral activity. Most significantly, none of the stapled peptides show any cytotoxicity at the highest dose tested. We also evaluated the antiviral activity of the peptides by infecting Vero E6 cells with the replication-competent authentic SARS-CoV-2 (US_WA-1/2020). NYBSP-1 was the most efficient, preventing the complete formation of cytopathic effects (CPEs) at an IC100 of 17.2 µM. NYBSP-2 and NYBSP-4 also prevented the formation of the virus-induced CPE with an IC100 of about 33 µM. We determined the proteolytic stability of one of the most active stapled peptides, NYBSP-4, in human plasma, which showed a half-life (T1/2) of >289 min.IMPORTANCE SARS-CoV-2 is a novel virus with many unknowns. No vaccine or specific therapy is available yet to prevent and treat this deadly virus. Therefore, there is an urgent need to develop novel therapeutics. Structural studies revealed critical interactions between the binding site helix of the ACE2 receptor and SARS-CoV-2 receptor-binding domain (RBD). Therefore, targeting the entry pathway of SARS-CoV-2 is ideal for both prevention and treatment as it blocks the first step of the viral life cycle. We report the design of four double-stapled peptides, three of which showed potent antiviral activity in HT1080/ACE2 cells and human lung carcinoma cells, A549/ACE2. Most significantly, the active stapled peptides with antiviral activity against SARS-CoV-2 showed high α-helicity (60 to 94%). The most active stapled peptide, NYBSP-4, showed substantial resistance to degradation by proteolytic enzymes in human plasma. The lead stapled peptides are expected to pave the way for further optimization of a clinical candidate.

Synthesis, antiviral activity and resistance of a novel small molecule HIV-1 entry inhibitor.

One of the most critical requirements of the infection of the human immunodeficiency virus type 1 (HIV-1) is the interaction of its surface envelope glycoprotein gp120 with the cellular receptor CD4, which initiates virus entry to cells. Therefore, envelope glycoprotein gp120 has been validated as a potential target to develop HIV-1 entry inhibitors. Here we report the evaluation of a novel non-natural amino acid, termed 882376, reported earlier as a precursor of a CD4-mimetic miniprotein, as HIV-1 entry inhibitor. 882376 showed HIV-1 inhibitory activity against a large panel of primary isolates of different subtype. Moreover, genotyping of 882376 resistant HIV-1 virus revealed three amino acid substitutions in the gp120 including one in the CD4 binding site suggesting that this molecule may bind to gp120 and prevent its binding to CD4. Additional neutralization experiments indicate that 882376 is not active against mutant pseudoviruses carrying the amino acid substitutions S375H and S375Y located in the 'Phe43 cavity' which is the major site of CD4 binding, suggesting that this compound may interfere with the interaction between gp120 and CD4. The unnatural amino acid, 882376, is expected to serve as a lead for further optimization to more potent HIV-1 entry inhibitors.

Dual-acting stapled peptides target both HIV-1 entry and assembly.

BACKGROUND: Previously, we reported the conversion of the 12-mer linear and cell-impermeable peptide CAI to a cell-penetrating peptide NYAD-1 by using an i,i + 4 hydrocarbon stapling technique and confirmed its binding to the C-terminal domain (CTD) of the HIV-1 capsid (CA) protein with an improved affinity (K(d) ~ 1 µM) compared to CAI (K(d) ~ 15 µM). NYAD-1 disrupts the formation of both immature- and mature-like virus particles in in vitro and cell-based assembly assays. In addition, it displays potent anti-HIV-1 activity in cell culture against a range of laboratory-adapted and primary HIV-1 isolates. RESULTS: In this report, we expanded the study to i,i + 7 hydrocarbon-stapled peptides to delineate their mechanism of action and antiviral activity. We identified three potent inhibitors, NYAD-36, -66 and -67, which showed strong binding to CA in NMR and isothermal titration calorimetry (ITC) studies and disrupted the formation of mature-like particles. They showed typical α-helical structures and penetrated cells; however, the cell penetration was not as efficient as observed with the i,i + 4 peptides. Unlike NYAD-1, the i,i + 7 peptides did not have any effect on virus release; however, they impaired Gag precursor processing. HIV-1 particles produced in the presence of these peptides displayed impaired infectivity. Consistent with an effect on virus entry, selection for viral resistance led to the emergence of two mutations in the gp120 subunit of the viral envelope (Env) glycoprotein, V120Q and A327P, located in the conserved region 1 (C1) and the base of the V3 loop, respectively. CONCLUSION: The i,i + 7 stapled peptides derived from CAI unexpectedly target both CA and the V3 loop of gp120. This dual-targeted activity is dependent on their ability to penetrate cells as well as their net charge. This mechanistic revelation will be useful in further modifying these peptides as potent anti-HIV-1 agents.

Flexible antibodies with nonprotein hinges.

There is a significant need for antibodies that can bind targets with greater affinity. Here we describe a novel strategy employing chemical semisynthesis to produce symmetroadhesins: antibody-like molecules having nonprotein hinge regions that are more flexible and extendible and are capable of two-handed binding. Native chemical ligation was carried out under mild, non-denaturing conditions to join a ligand binding domain (Aß peptide) to an IgG1 Fc dimer via discrete oxyethylene oligomers of various lengths. Two-handed Aß-Fc fusion proteins were obtained in quantitative yield and shown by surface plasmon resonance to bind an anti-Aß antibody with a K(D) at least two orders of magnitude greater than the cognate Aß peptide. MALDI-TOF MS analysis confirmed the protein/nonprotein/protein structure of the two-handed molecules, demonstrating its power to characterize complex protein-nonprotein hybrids by virtue of desorption/ionization mediated by peptide sequences contained therein. We anticipate many applications for symmetroadhesins that combine the target specificity of antibodies with the novel physical, chemical and biological properties of nonprotein hinges.

Measurement of plasma renin concentration in cats by use of a fluorescence resonance energy transfer peptide substrate of renin.

OBJECTIVE: To evaluate the use of a commercially available 5-carboxyfluorescein-based, intramolecularly quenched, fluorescence resonance energy transfer (FRET) peptide substrate of renin for measurement of plasma renin concentration in cats. SAMPLE POPULATION: Plasma samples obtained during a previous study of renal autograft ischemia-reperfusion injury in 10 cats and samples of fetal bovine serum containing recombinant human renin (rh-renin). PROCEDURES: Experiments involving samples of fetal bovine serum containing rh-renin were conducted to identify a suitable control vehicle, optimal substrate concentration, and appropriate duration of incubation. With the use of the identified assay conditions, a standard curve was constructed to allow conversion of relative fluorescent units into values of renin concentration (ng/mL). Subsequently, plasma samples obtained from cats before and after renal autograft ischemia-reperfusion injury were assayed to determine endogenous renin concentration. RESULTS: Under conditions of a 1:50 substrate dilution and 4-hour incubation period, the assay detected small amounts of rh-renin in fetal bovine serum. A linear relationship (R(2) = 0.996) between the relative fluorescent units generated and exogenous rh-renin concentration was evident. The assay detected renin in plasma samples obtained from cats after renal autograft ischemia-reperfusion, and renin concentrations on days 1 and 2 after transplant differed significantly. CONCLUSIONS AND CLINICAL RELEVANCE: The study data indicated that the assay involving the FRET peptide substrate of renin is potentially a rapid and specific method for measurement of plasma renin concentration in cats.

A cell-penetrating helical peptide as a potential HIV-1 inhibitor.

The capsid domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is a critical determinant of virus assembly, and is therefore a potential target for developing drugs for AIDS therapy. Recently, a 12-mer alpha-helical peptide (CAI) was reported to disrupt immature- and mature-like capsid particle assembly in vitro; however, it failed to inhibit HIV-1 in cell culture due to its inability to penetrate cells. The same group reported the X-ray crystal structure of CAI in complex with the C-terminal domain of capsid (C-CA) at a resolution of 1.7 A. Using this structural information, we have utilized a structure-based rational design approach to stabilize the alpha-helical structure of CAI and convert it to a cell-penetrating peptide (CPP). The modified peptide (NYAD-1) showed enhanced alpha-helicity. Experiments with laser scanning confocal microscopy indicated that NYAD-1 penetrated cells and colocalized with the Gag polyprotein during its trafficking to the plasma membrane where virus assembly takes place. NYAD-1 disrupted the assembly of both immature- and mature-like virus particles in cell-free and cell-based in vitro systems. NMR chemical shift perturbation analysis mapped the binding site of NYAD-1 to residues 169-191 of the C-terminal domain of HIV-1 capsid encompassing the hydrophobic cavity and the critical dimerization domain with an improved binding affinity over CAI. Furthermore, experimental data indicate that NYAD-1 most likely targets capsid at a post-entry stage. Most significantly, NYAD-1 inhibited a large panel of HIV-1 isolates in cell culture at low micromolar potency. Our study demonstrates how a structure-based rational design strategy can be used to convert a cell-impermeable peptide to a cell-permeable peptide that displays activity in cell-based assays without compromising its mechanism of action. This proof-of-concept cell-penetrating peptide may aid validation of capsid as an anti-HIV-1 drug target and may help in designing peptidomimetics and small molecule drugs targeted to this protein.