The Antimicrobial Peptide Capitellacin: Chemical Synthesis of Analogues to Probe the Role of Disulphide Bridges and Their Replacement with Vinyl Sulphides.

Capitellacin (1) is a 20-residue antimicrobial ß-hairpin, produced by the marine polychaeta (segmented worms) Capitella teletai. Since its discovery in 2020, only very limited studies have been undertaken to understand capitellacin's structure-activity relationship (SAR). Using fast-flow Fmoc-SPPS, a focused library of capitellacin analogues was prepared to systematically study the influence of the two disulphide bridges on its structure and activity, and their replacement with a vinyl sulphide as a potential bioisostere. Upon studying the resulting peptides' antimicrobial activity and secondary structure, the most terminal disulphide emerged as the most critical element for maintaining both bioactivity and the secondary structure, properties which were demonstrated to be closely interlinked. The removal of the innermost disulphide bridge or disulphide replacement with a vinyl sulphide emerged as strategies with which to tune the activity spectrum, producing selectivity towards E. coli. Additionally, an enantiomeric d-capitellacin analogue revealed mechanistic insights, suggesting that chirality may be an inherent property of capitellacin's bacterial membrane target, or that a hitherto unknown secondary mechanism of action may exist. Additionally, we propose the Alloc protecting group as a more appropriate alternative to the common Dde group during fast-flow Fmoc-SPPS, in particular for short-chain diamino acids.

A turn for the worse: Aß ß-hairpins in Alzheimer's disease.

Amyloid-ß (Aß) oligomers are a cause of neurodegeneration in Alzheimer's disease (AD). These soluble aggregates of the Aß peptide have proven difficult to study due to their inherent metastability and heterogeneity. Strategies to isolate and stabilize homogenous Aß oligomer populations have emerged such as mutations, covalent cross-linking, and protein fusions. These strategies along with molecular dynamics simulations have provided a variety of proposed structures of Aß oligomers, many of which consist of molecules of Aß in ß-hairpin conformations. ß-Hairpins are intramolecular antiparallel ß-sheets composed of two ß-strands connected by a loop or turn. Three decades of research suggests that Aß peptides form several different ß-hairpin conformations, some of which are building blocks of toxic Aß oligomers. The insights from these studies are currently being used to design anti-Aß antibodies and vaccines to treat AD. Research suggests that antibody therapies designed to target oligomeric Aß may be more successful at treating AD than antibodies designed to target linear epitopes of Aß or fibrillar Aß. Aß ß-hairpins are good epitopes to use in antibody development to selectively target oligomeric Aß. This review summarizes the research on ß-hairpins in Aß peptides and discusses the relevance of this conformation in AD pathogenesis and drug development.

First vertebrate BRICHOS antimicrobial peptides: ß-hairpin host defense peptides in limbless amphibia lung resemble those of marine worms.

Innate immunity of invertebrates offers potent antimicrobial peptides (AMPs) against drug-resistant infections. To identify new worm ß-hairpin AMPs, we explored the sequence diversity of proteins with a BRICHOS domain, which comprises worm AMP precursors. Strikingly, we discovered new BRICHOS AMPs not in worms, but in caecilians, the least studied clade of vertebrates. Two precursor proteins from Microcaecilia unicolor and Rhinatrema bivittatum resemble SP-C lung surfactants and bear worm AMP-like peptides at C-termini. The analysis of M. unicolor tissue transcriptomes shows that the AMP precursor is highly expressed in the lung along with regular SP-C, suggesting a different, protective function. The peptides form right-twisted ß-hairpins, change conformation upon lipid binding, and rapidly disrupt bacterial membranes. Both peptides exhibit broad-spectrum activity against multidrug-resistant ESKAPE pathogens with 1-4 µM MICs and remarkably low toxicity, giving 40-70-fold selectivity towards bacteria. These BRICHOS AMPs, previously unseen in vertebrates, reveal a novel lung innate immunity mechanism and offer a promising antibiotics template.

Dimerization of the ß-Hairpin Membrane-Active Cationic Antimicrobial Peptide Capitellacin from Marine Polychaeta: An NMR Structural and Thermodynamic Study.

Capitellacin is the ß-hairpin membrane-active cationic antimicrobial peptide from the marine polychaeta Capitella teleta. Capitellacin exhibits antibacterial activity, including against drug-resistant strains. To gain insight into the mechanism of capitellacin action, we investigated the structure of the peptide in the membrane-mimicking environment of dodecylphosphocholine (DPC) micelles using high-resolution NMR spectroscopy. In DPC solution, two structural forms of capitellacin were observed: a monomeric ß-hairpin was in equilibrium with a dimer formed by the antiparallel association of the N-terminal ß-strands and stabilized by intermonomer hydrogen bonds and Van der Waals interactions. The thermodynamics of the enthalpy-driven dimerization process was studied by varying the temperature and molar ratios of the peptide to detergent. Cooling the peptide/detergent system promoted capitellacin dimerization. Paramagnetic relaxation enhancement induced by lipid-soluble 12-doxylstearate showed that monomeric and dimeric capitellacin interacted with the surface of the micelle and did not penetrate into the micelle interior, which is consistent with the "carpet" mode of membrane activity. An analysis of the known structures of ß-hairpin AMP dimers showed that their dimerization in a membrane-like environment occurs through the association of polar or weakly hydrophobic surfaces. A comparative analysis of the physicochemical properties of ß-hairpin AMPs revealed that dimer stability and hemolytic activity are positively correlated with surface hydrophobicity. An additional positive correlation was observed between hemolytic activity and AMP charge. The data obtained allowed for the provision of a more accurate description of the mechanism of the oligomerization of ß-structural peptides in biological membranes.

Insights into the Adsorption Mechanisms of the Antimicrobial Peptide CIDEM-501 on Membrane Models.

CIDEM-501 is a hybrid antimicrobial peptide rationally designed based on the structure of panusin and panulirin template peptides. The new peptide exhibits significant antibacterial activity against multidrug-resistant pathogens (MIC = 2-4 µM) while conserving no toxicity in human cell lines. We conducted molecular dynamics (MD) simulations using the CHARMM-36 force field to explore the CIDEM-501 adsorption mechanism with different membrane compositions. Several parameters that characterize these interactions were analyzed to elucidate individual residues' structural and thermodynamic contributions. The membrane models were constructed using CHARMM-GUI, mimicking the bacterial and eukaryotic phospholipid compositions. Molecular dynamics simulations were conducted over 500 ns, showing rapid and highly stable peptide adsorption to bacterial lipids components rather than the zwitterionic eucaryotic model membrane. A predominant peptide orientation was observed in all models dominated by an electric dipole. The peptide remained parallel to the membrane surface with the center loop oriented to the lipids. Our findings shed light on the antibacterial activity of CIDEM-501 on bacterial membranes and yield insights valuable for designing potent antimicrobial peptides targeting multi- and extreme drug-resistant bacteria.

Design of Functional Globular ß-Sheet Miniproteins.

The design of discrete ß-sheet peptides is far less advanced than e. g. the design of α-helical peptides. The reputation of ß-sheet peptides as being poorly soluble and aggregation-prone often hinders active design efforts. Here, we show that this reputation is unfounded. We demonstrate this by looking at the ß-hairpin and WW domain. Their structure and folding have been extensively studied and they have long served as model systems to investigate protein folding and folding kinetics. The resulting fundamental understanding has led to the development of hyperstable ß-sheet scaffolds that fold at temperatures of 100 °C or high concentrations of denaturants. These have been used to design functional miniproteins with protein or nucleic acid binding properties, in some cases with such success that medical applications are conceivable. The ß-sheet scaffolds are not always completely rigid, but can be specifically designed to respond to changes in pH, redox potential or presence of metal ions. Some engineered ß-sheet peptides also exhibit catalytic properties, although not comparable to those of natural proteins. Previous reviews have focused on the design of stably folded and non-aggregating ß-sheet sequences. In our review, we now also address design strategies to obtain functional miniproteins from ß-sheet folding motifs.

Sequence-based identification of amyloidogenic ß-hairpins reveals a prostatic acid phosphatase fragment promoting semen amyloid formation.

ß-Structure-rich amyloid fibrils are hallmarks of several diseases, including Alzheimer's (AD), Parkinson's (PD), and type 2 diabetes (T2D). While amyloid fibrils typically consist of parallel ß-sheets, the anti-parallel ß-hairpin is a structural motif accessible to amyloidogenic proteins in their monomeric and oligomeric states. Here, to investigate implications of ß-hairpins in amyloid formation, potential ß-hairpin-forming amyloidogenic segments in the human proteome were predicted based on sequence similarity with ß-hairpins previously observed in Aß, α-synuclein, and islet amyloid polypeptide, amyloidogenic proteins associated with AD, PD, and T2D, respectively. These three ß-hairpins, established upon binding to the engineered binding protein ß-wrapin AS10, are characterized by proximity of two sequence segments rich in hydrophobic and aromatic amino acids, with high ß-aggregation scores according to the TANGO algorithm. Using these criteria, 2505 potential ß-hairpin-forming amyloidogenic segments in 2098 human proteins were identified. Characterization of a test set of eight protein segments showed that seven assembled into Thioflavin T-positive aggregates and four formed ß-hairpins in complex with AS10 according to NMR. One of those is a segment of prostatic acid phosphatase (PAP) comprising amino acids 185-208. PAP is naturally cleaved into fragments, including PAP(248-286) which forms functional amyloid in semen. We find that PAP(185-208) strongly decreases the protein concentrations required for fibril formation of PAP(248-286) and of another semen amyloid peptide, SEM1(86-107), indicating that it promotes nucleation of semen amyloids. In conclusion, ß-hairpin-forming amyloidogenic protein segments could be identified in the human proteome with potential roles in functional or disease-related amyloid formation.

Conformational features and interaction mechanisms of VH H antibodies with ß-hairpin CDR3: A case of Nb8-HigB2 interaction.

The ß-hairpin conformation is regarded as an important basic motif to form and regulate protein-protein interactions. Single-domain VH H antibodies are potential therapeutic and diagnostic tools, and the third complementarity-determining regions of the heavy chains (CDR3s) of these antibodies are critical for antigen recognition. Although the sequences and conformations of the CDR3s are diverse, CDR3s sometimes adopt ß-hairpin conformations. However, characteristic features and interaction mechanisms of ß-hairpin CDR3s remain to be fully elucidated. In this study, we investigated the molecular recognition of the anti-HigB2 VH H antibody Nb8, which has a CDR3 that forms a ß-hairpin conformation. The interaction was analyzed by evaluation of alanine-scanning mutants, molecular dynamics simulations, and hydrogen/deuterium exchange mass spectrometry. These experiments demonstrated that positions 93 and 94 (Chothia numbering) in framework region 3, which is right outside CDR3 by definition, play pivotal roles in maintaining structural stability and binding properties of Nb8. These findings will facilitate the design and optimization of single-domain antibodies.

Nucleation of α-Synuclein Amyloid Fibrils Induced by Cross-Interaction with ß-Hairpin Peptides Derived from Immunoglobulin Light Chains.

Heterologous interactions between different amyloid-forming proteins, also called cross-interactions, may have a critical impact on disease-related amyloid formation. ß-hairpin conformers of amyloid-forming proteins have been shown to affect homologous interactions in the amyloid self-assembly process. Here, we applied two ß-hairpin-forming peptides derived from immunoglobulin light chains as models to test how heterologous ß-hairpins modulate the fibril formation of Parkinson's disease-associated protein α-synuclein (αSyn). The peptides SMAhp and LENhp comprise ß-strands C and C' of the κ4 antibodies SMA and LEN, which are associated with light chain amyloidosis and multiple myeloma, respectively. SMAhp and LENhp bind with high affinity to the ß-hairpin-binding protein ß-wrapin AS10 according to isothermal titration calorimetry and NMR spectroscopy. The addition of SMAhp and LENhp affects the kinetics of αSyn aggregation monitored by Thioflavin T (ThT) fluorescence, with the effect depending on assay conditions, salt concentration, and the applied ß-hairpin peptide. In the absence of agitation, substoichiometric concentrations of the hairpin peptides strongly reduce the lag time of αSyn aggregation, suggesting that they support the nucleation of αSyn amyloid fibrils. The effect is also observed for the aggregation of αSyn fragments lacking the N-terminus or the C-terminus, indicating that the promotion of nucleation involves the interaction of hairpin peptides with the hydrophobic non-amyloid-ß component (NAC) region.

Incorporating a ß-hairpin sequence motif to increase intracellular stability of a peptide-based PROTAC.

Proteolysis targeting chimeras (PROTACs) have emerged as a new class of therapeutics that utilize the ubiquitin-proteasome system (UPS) to facilitate proteasomal degradation of "undruggable" targets. Peptide-based PROTACs contain three essential components: a binding motif for the target protein, a short amino acid sequence recognized by an E3 ligase called a degron, and a cell penetrating peptide to facilitate uptake into intact cells. While peptide-based PROTACs have been shown to successfully degrade numerous targets, they have often been found to exhibit low cell permeability and high protease susceptibility. Prior work identified peptides containing a ß-hairpin sequence motif that function not only as protecting elements, but also as CPPs and degrons. The goal of this study was to investigate if a ß-hairpin sequence could replace commonly used unstructured peptides sequences as the degron and the CPP needed for PROTAC uptake and function. The degradation of the protein Tau was selected as a model system as several published works have identified a Tau binding element that could easily be conjugated to the ß-hairpin sequence. A series of time- and concentration-dependent studies confirmed that the ßhairpin sequence was an adequate alternative CPP and degron to facilitate the proteasomemediated degradation of Tau. Microscopy studies confirmed the time-dependent uptake of the PROTAC and a degradation assay confirmed that the ß-hairpin conjugated PROTAC had a greater lifetime in cells.

The Amphiphilic Property and Structure of ß-Amyloid Peptide Contribute to Its Impacts on the Activities of Horseradish Peroxidase and Alkaline Phosphatase.

Recent studies have found that ß-amyloid (Aß) oligomers may play much more important roles than amyloid plaques in the pathogenesis of Alzheimer's disease (AD). However, due to the complexity of Aß, studying the structural basis of Aß oligomer toxicity is challenging. Here, we assessed the amphiphilic property and ß-hairpin structure of Aß monomer. The potential impacts of Aß oligomers and three sequence-modifying peptides on the enzyme activities of horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were further evaluated. We demonstrated that Aß oligomer possesses the ability to alter the activity of two enzymes. Moreover, modifications on the hydrophobic region and ß-turn structure of Aß monomer significantly alter its impacts on the enzyme activities. In addition, these modifications also change the bonding modes of Aß monomers or oligomers binding to HRP, as assessed by molecular docking. All of these findings provide direct experimental evidence to reveal the critical roles of the amphiphilic property and ß-sheet structure of Aß monomer in its impacts on protein activity.

Solution structure of the N-terminal extension domain of a Schistosoma japonicum asparaginyl-tRNA synthetase.

Several secreted proteins from helminths (parasitic worms) have been shown to have immunomodulatory activities. Asparaginyl-tRNA synthetases are abundantly secreted in the filarial nematode Brugia malayi (BmAsnRS) and the parasitic flatworm Schistosoma japonicum (SjAsnRS), indicating a possible immune function. The suggestion is supported by BmAsnRS alleviating disease symptoms in a T-cell transfer mouse model of colitis. This immunomodulatory function is potentially related to an N-terminal extension domain present in eukaryotic AsnRS proteins but few structure/function studies have been done on this domain. Here we have determined the three-dimensional solution structure of the N-terminal extension domain of SjAsnRS. A protein containing the 114 N-terminal amino acids of SjAsnRS was recombinantly expressed with isotopic labelling to allow structure determination using 3D NMR spectroscopy, and analysis of dynamics using NMR relaxation experiments. Structural comparisons of the N-terminal extension domain of SjAsnRS with filarial and human homologues highlight a high degree of variability in the ß-hairpin region of these eukaryotic N-AsnRS proteins, but similarities in the disorder of the C-terminal regions. Limitations in PrDOS-based intrinsically disordered region (IDR) model predictions were also evident in this comparison. Empirical structural data such as that presented in our study for N-SjAsnRS will enhance the prediction of sequence-homology based structure modelling and prediction of IDRs in the future.Communicated by Ramaswamy H. Sarma.

Two Novel Mosquitocidal Peptides Isolated from the Venom of the Bahia Scarlet Tarantula (Lasiodora klugi).

Effective control of diseases transmitted by Aedes aegypti is primarily achieved through vector control by chemical insecticides. However, the emergence of insecticide resistance in A. aegypti undermines current control efforts. Arachnid venoms are rich in toxins with activity against dipteran insects and we therefore employed a panel of 41 spider and 9 scorpion venoms to screen for mosquitocidal toxins. Using an assay-guided fractionation approach, we isolated two peptides from the venom of the tarantula Lasiodora klugi with activity against adult A. aegypti. The isolated peptides were named U-TRTX-Lk1a and U-TRTX-Lk2a and comprised 41 and 49 residues with monoisotopic masses of 4687.02 Da and 5718.88 Da, respectively. U-TRTX-Lk1a exhibited an LD50 of 38.3 pmol/g when injected into A. aegypti and its modeled structure conformed to the inhibitor cystine knot motif. U-TRTX-Lk2a has an LD50 of 45.4 pmol/g against adult A. aegypti and its predicted structure conforms to the disulfide-directed ß-hairpin motif. These spider-venom peptides represent potential leads for the development of novel control agents for A. aegypti.

Diagonal Interactions between Glutamate and Arginine Analogs with Varying Side-Chain Lengths in a ß-Hairpin.

Cross-strand interactions are important for the stability of ß-sheet structures. Accordingly, cross-strand diagonal interactions between glutamate and arginine analogs with varying side-chain lengths were studied in a series of ß-hairpin peptides. The peptides were analyzed by homonuclear two-dimensional nuclear magnetic resonance methods. The fraction folded population and folding free energy of the peptides were derived from the chemical shift data. The fraction folded population trends could be rationalized using the strand propensity of the constituting residues, which was not the case for the peptides with lysine analogs, highlighting the difference between the arginine analogs and lysine analogs. Double-mutant cycle analysis was used to derive the diagonal ion-pairing interaction energetics. The most stabilizing diagonal cross-strand interaction was between the shortest residues (i.e., Asp2-Agp9), most likely due to the least side-chain conformational penalty for ion-pair formation. The diagonal interaction energetics in this study involving the arginine analogs appears to be consistent with and extend beyond our understanding of diagonal ion-pairing interactions involving lysine analogs. The results should be useful for designing ß-strand-containing molecules to affect biological processes such as amyloid formation and protein-protein interactions.

Unlocking the Potential of the Antimicrobial Peptide Gomesin: From Discovery and Structure-Activity Relationships to Therapeutic Applications.

Gomesin is a cationic antimicrobial peptide which is isolated from the haemocytes of the Brazilian tarantula Acanthoscurria gomesiana and can be produced chemically by Fmoc solid-phase peptide synthesis. Gomesin exhibits a range of biological activities, as demonstrated by its toxicity against therapeutically relevant pathogens such as Gram-positive or Gram-negative bacteria, fungi, cancer cells, and parasites. In recent years, a cyclic version of gomesin has been used for drug design and development as it is more stable than native gomesin in human serum and can penetrate and enter cancer cells. It can therefore interact with intracellular targets and has the potential to be developed as a drug lead for to treat cancer, infectious diseases, and other human diseases. This review provides a perspective on the discovery, structure-activity relationships, mechanism of action, biological activity, and potential clinical applications of gomesin.

Functional Importance of the Hydrophobic Residue 362 in Influenza A PB1 Subunit.

PB1, acting as the catalytic subunit of the influenza polymerase, has numerous sequentially and structurally conserved regions. It has been observed that the slight modification of residues in PB1 would greatly affect the polymerase activity and even host adaptation ability. Here, we identified a critical residue, 362M, on the polymerase activity and virus replication. By means of the minireplicon assay, we assured the importance of the hydrophobicity of PB1 362, and the possibility that the size and charge of the side chain might directly interfere with the polymerase function. We also proposed a hydrophobic core between the PA-arch and the PB1 ß-hairpin motifs and showed the importance of the core to the polymerase function.

Mechanism of ß-hairpin formation in AzoChignolin and Chignolin.

AzoChignolin is a photoswitchable variant of the mini-protein Chignolin with an azobenzene (AMPP) replacing the central loop. AzoChignolin is unfolded with AMPP in the trans-isomer. Transition to the cis-isomer causes ß-hairpin folding similar to Chignolin. The AzoChignolin system is excellently suited for comprehensive analysis of folding nucleation kinetics. Utilizing multiple long-time MD simulations of AzoChignolin and Chignolin in MeOH and water, we estimated Markov models to examine folding kinetics of both peptides. We show that while AzoChignolin mimics Chignolin's structure well, the folding kinetics are quite different. Not only folding times but also intermediate states differ, particularly Chignolin is able to fold in MeOH into an α-helical intermediate which is impossible to form in AzoChignolin. The Markov models demonstrate that AzoChignolin's kinetics are generally faster, specifically when comparing the two main microfolding processes of hydrophobic collapse and turn formation. Photoswitchable loops are used frequently to understand the kinetics of elementary protein folding nucleation. However, our results indicate that intermediates and folding kinetics may differ between natural loops and photoswitchable variants.

Coupled folding-upon-binding of human tumor suppressor MIG6 to lung cancer EGFR kinase domain and molecular trimming/stapling of MIG6-derived ß-hairpins to target the coupling event.

Human epidermal growth factor receptor (EGFR) is involved in strong association with malignant proliferation, which has been shown to play a central role in the development and progression of non-small cell lung cancer and other solid tumors. The tumor-suppressor protein MIG6 is a negative regulator of EGFR kinase activity by binding at the activation interface of asymmetric dimer of EGFR kinase domain to disrupt EGFR dimerization and then inactivate the kinase. The protein adopts two discrete fragments 1 and 2 to directly interact with EGFR. It is revealed that the MIG6 fragment 2 is intrinsically disordered in free unbound state, but would fold into a well-structured ß-hairpin when binding to EGFR, thus characterized by a so-called coupled folding-upon-binding process, which can be regarded as a compromise between favorable direct readout and unfavorable indirect readout. Here, a 23-mer F2P peptide was derived from MIG6 fragment 2, trimmed into a 17-mer tF2P peptide that contains the binding hotspot region of the fragment 2, and then constrained with an ordered hairpin conformation in free unbound state by disulfide stapling, finally resulting in a rationally stapled/trimmed stF2P peptide that largely minimizes the unfavorable indirect readout effect upon its binding to EGFR kinase domain, with affinity improved considerably upon the trimming and stapling/trimming. These rationally designed ß-hairpin peptides may be further exploited as potent anti-lung cancer agents to target the activation event of EGFR dimerization.

Designed symmetrical ß-hairpin peptides for treating multidrug-resistant salmonella typhimurium infections.

The rapid emergence and prevalence of multidrug-resistant salmonellosis lack effective therapies, which causes epidemic health problems and stimulates the development of antimicrobials with novel modes of action. In this research, 10 short symmetrical ß-hairpin peptides are synthesized by combining the ß-turn of Leucocin-A with recurring hydrophobic and cationic amino acid sequences. Those designed peptides exhibited potent antibacterial activities against drug-susceptible and drug-resistant Salmonella. One of the 10 peptides, WK2 ((WK)2CTKSGC(KW)2), displayed best cell selectivity towards Salmonella cells over macrophages and erythrocytes in a co-culture model. Fluorescent measurements and microscopic observations reflected that WK2 exerted its antimicrobial activity through a membrane-lytic mechanism. Moreover, the ß-hairpin peptides can bind to endotoxin (LPS) and suppress the production of LPS-induced proinflammatory cytokines in RAW264.7 cells, indicating as a potent anti-inflammatory activity. The preliminary in vivo studies can also demonstrate that WK2 decreased loads of Salmonella in the liver and spleen, mitigated Salmonella-caused inflammation and maintained the integrity of intestinal mucosal surfaces. Ultimately, the results highlight that WK2 is a promising therapeutic agent to prevent multidrug-resistant S. Typhimurium infections in humans and animals.

Exploiting the Innate Plasticity of the Programmed Cell Death-1 (PD1) Receptor to Design Pembrolizumab H3 Loop Mimics.

Checkpoint blockade of the immunoreceptor programmed cell death-1 (PD1) with its ligand-1 (PDL1) by monoclonal antibodies such as pembrolizumab provided compelling clinical results in various cancer types, yet the molecular mechanism by which this drug blocks the PD1/PDL1 interface remains unclear. To address this question, we examined the conformational motion of PD1 associated with the binding of pembrolizumab. Our results revealed that the innate plasticity of both C'D and FG loops is crucial to form a deep binding groove (371 Å3 ) across several distant epitopes of PD1. This analysis ultimately provided a rational-design to create pembrolizumab H3 loop mimics [RDYRFDMGFD] into ß-hairpin scaffolds. As a result, a 20-residue long ß-hairpin peptide 1 e was identified as a first-in-class potent PD1-inhibitor (EC50 of 0.29 µM; Ki of 41 nM).