Jutta Eichler

The starting point of this work was a SARS-CoV-2 neutralizing peptide (LW25.13), which binds to the receptor-binding domain of the viral spike protein and inhibits the attachment of the virus to its cellular receptor ACE2. As LW25.13 is unable to neutralize later SARS-CoV-2 variants, such as omicron, we have extended the neutralization breadth of LW25.13 through structural and bioinformatic analysis. This involved the systematic variation of a range of positions and yielded peptides neutralizing SARS-CoV-2 beta and omicron at low nanomolar concentrations, while preserving the strong neutralizing capacity against earlier virus variants (wild-type, alpha, delta), as well as the proteolytic stability and α-helical conformation of the peptide. This gain in neutralizing breadth illustrates the utility of the peptide as a scaffold that can be adapted to different virus variants, which may prove useful for the development of peptides against new coronavirus variants of concern in the future.

Electrostatic interactions between charged amino acid side chains play an important role for the stability of α-helices. The strength of these interactions depends on the protonation state of the residues, which is determined by the pH of the environment. Force fields used for molecular dynamics (MD) simulations of such systems should therefore accurately model charged interactions and their dependence on the protonation state. We performed MD simulations for two distinct protonation states on a set of helical peptides known to exhibit various degrees of pH-dependence in experiments and compared two recommended combinations of AMBER protein force field and water model: ff14SB + TIP3P and ff19SB + OPC. While both combinations model similar side chain interactions for the helical states of the peptides, they showed some differences in the overall helical content and the stability of the hydrogen bonds. Nevertheless, both combinations were capable of detecting protonation-dependent structural changes in the peptides. This could be useful for identifying pH-sensitive sites in helical peptides or for designing pH-dependent switches.

Antibody mimetic peptides have evolved as versatile tools for biomedical applications, based on their ability to interfere with protein–protein interactions. We had previously designed a functional mimic of the broadly neutralizing HIV‐1 antibody b12 that recognizes the CD4 binding site of the HIV‐1 envelope glycoprotein gp120. The molecular details of the interaction of a linear variant of this peptide (H1H3s) with gp120 have now been characterized through cross‐linking mass spectrometry, confirming the proposed involvement of the CD4 binding site of gp120 in the interaction. In addition, a variant of the b12 mimetic peptide composed mostly of D‐amino acids was shown to be stable towards proteolytic degradation, while the binding and HIV‐1 neutralizing properties were largely preserved. Furthermore, a peptide variant in which aspartate residues were replaced with lysine was shown to strongly enhance infection of cells with HIV‐1 and GALV glycoprotein pseudotyped viral vectors, respectively, introducing this peptide as a tool to facilitate retroviral gene transfer. Collectively, the presented results highlight the versatile potential therapeutic and gene transfer applications of H1H3s and its variants in particular, as well as antibody mimetic peptides in general.

A pair of heterodimeric coiled-coil peptides was modified to enable covalent stabilization of the dimer without using enzymes or cysteine. Fusion of the peptides to a protein and a chemical moiety, respectively, facilitates site-selective protein modification.

PG16 is a broadly neutralizing antibody that binds to the gp120 subunit of the HIV-1 Env protein. The major interaction site is formed by the unusually long complementarity determining region (CDR) H3. The CDRH3 residue Tyr100H is known to represent a tyrosine sulfation site; however, this modification is not present in the experimental complex structure of PG16 with full-length HIV-1 Env. To investigate the role of sulfation for this complex, we modeled the sulfation of Tyr100H and compared the dynamics and energetics of the modified and unmodified complex by atomistic molecular dynamics simulations. Our results show that sulfation does not affect the overall conformation of CDRH3, but still enhances gp120 interactions both at the site of mutation and for the neighboring residues. This stabilization affects not only protein-protein contacts, but also the interactions between PG16 and the gp120 glycan shield. Further, we also investigated whether PG16-CDRH3 is a suitable template for the development of peptide mimetics. For a peptide spanning residues 93-105 of PG16 we obtained an experimental EC50 value of 3nM for the binding of gp120 to the peptide. This affinity can be enhanced by almost one order of magnitude by artificial disulfide bonding between residues 99 and 100F. In contrast, any truncation results in significantly lower affinity, suggesting that the entire peptide segment is involved in gp120 recognition. Their high affinity makes PG16-derived peptides useful building blocks for further optimization to obtain a potent inhibitor that efficiently blocks HIV-1 infection.

The replication of human cytomegalovirus (HCMV) involves a process termed nuclear egress, which enables translocation of newly formed viral capsids from the nucleus into the cytoplasm. The HCMV core nuclear egress complex (core NEC), a heterodimer of viral proteins pUL50 and pUL53, is therefore considered a promising target for new antiviral drugs. We have recently shown that a 29-mer peptide presenting an N-terminal alpha-helical hook-like segment of pUL53, through which pUL53 interacts with pUL50, binds to pUL50 with high affinity, and inhibits the pUL50–pUL53 interaction in vitro. Here, we show that this peptide is also able to interfere with HCMV infection of cells, as well as with core NEC formation in HCMV-infected cells. As the target of the peptide, i.e., the pUL50–pUL53 interaction, is localized at the inner nuclear membrane of the cell, the peptide had to be equipped with translocation moieties that facilitate peptide uptake into the cell and the nucleus, respectively. For the resulting fusion peptide (NLS-CPP-Hook), specific cellular and nuclear uptake into HFF cells, as well as inhibition of infection with HCMV, could be demonstrated, further substantiating the HCMV core NEC as a potential antiviral target.

Based on the structure of a de novo designed miniprotein (LCB1) in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, we have generated and characterized truncated peptide variants of LCB1, which present only two of the three LCB1 helices, and which fully retained the virus neutralizing potency against different SARS-CoV-2 variants of concern (VOC). This antiviral activity was even 10-fold stronger for a cyclic variant of the two-helix peptides, as compared to the full-length peptide. Furthermore, the proteolytic stability of the cyclic peptide was substantially improved, rendering it a better potential candidate for SARS-CoV-2 therapy. In a more mechanistic approach, the peptides also served as tools to dissect the role of individual mutations in the RBD for the susceptibility of the resulting virus variants to neutralization by the peptides. As the peptides reported here were generated through chemical synthesis, rather than recombinant protein expression, they are amenable to further chemical modification, including the incorporation of a wide range of non-proteinogenic amino acids, with the aim to further stabilize the peptides against proteolytic degradation, as well as to improve the strength, as well the breadth, of their virus neutralizing capacity.

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV‐1) infection is six‐helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non‐natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV‐1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics.

Varicella zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. Here, the crystal structure of the VZV Orf24-Orf27 complex is described, representing the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled capsids from the nucleus. While previous studies have primarily emphasized the finding that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focusses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. CoIP and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of the NEC formation thermodynamic parameters of three prototypical α-, β- and γ herpesviruses, i.e. VZV, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) reveals highly similar binding affinities for the autologous interaction with some specific differences in the enthalpy and entropy terms. Computational alanine scanning and structural comparisons highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses. Combined, these data allow to explain the distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings might prove highly valuable when attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.