1. Introduction

The effect of conformational entropy on (macro)molecular interactions should not be underestimated, but at the same time structure rigidification should be meticulously optimized to allow taking on (or at least sampling of) the target-bound conformation ^[1][2]. Achieving the proper pre-organized fold might also lead to improved specificity because off-target binding (associated with a different conformation of the same peptide) is less likely to occur ^[3]. A further benefit of restricting peptide’s flexibility is to enhance its proteolytic stability, as the substrate polypeptide chain typically needs to adopt an extended conformation to be loaded in the protease active site ^[4].

The simplest way of constraining a peptide is to form a cyclized structure, for example, through paired cysteines, head-to-tail peptide bond, or grafting a peptide loop on a short antiparallel β-sheet motif. Although a looped peptide is typically still fairly flexible (depending on the ring size), it can only conform to a subset of structures that are accessible to its linear counterpart. ‘CLIPSing’ of linear peptides harboring cysteine residues with synthetic thiol-reactive scaffolds enables the construction of bicyclic or tricyclic structures capable of complex folds ^[5], functionally reminiscent of antibody paratopes. Such structures were shown to effectively bind to sites considered undruggable with conventional low-molecular-weight compounds, such as flat surfaces engaged in protein–protein interactions ^[6][7] and components or precursors of the bacterial cell wall ^[8][9]. Alternatively, the bicyclic form allows for combining two distinct functionalities in a single molecule, such as the ability to inhibit two non-homologous enzyme targets ^[10], or cell penetration and intracellular target binding ^[11][12]. Enhanced lipid membrane permeability has been reported for cationic amphipathic peptides that assume helical conformation upon interaction with cell membranes ^[13], and stapled and stitched (i.e., containing tandem crosslinks) peptide helices recapitulate this feature ^[14][15]. Cell internalization was shown to be highly dependent on the overall peptide charge, being most efficient for net charges of +3 to +5 ^[14]. Additionally, the stitched peptides displayed better permeability compared to the less stabilized stapled ones. The overall hydrophobic nature of the construct and the positioning of staples are also important for cell penetration (most penetrable helices contained staples at the amphipathic boundary ^[15]). Cell penetration seems to occur via a clathrin- and caveolin-independent, energy-dependent endocytic pathway, and likely proceeds through interaction with the negatively-charged sulfated cell surface proteoglycans ^[14]. Beta hairpin peptides are attractive because of their compatibility with biological library formats, and due to the vast functional diversity of this structural motif. For example, there are reports of β-hairpin peptides mimicking helical protein epitopes ^[16] or forming highly selective complexes with distinct precursor microRNAs ^[17][18].

2. Applications of Constrained Peptides

Antimicrobial agents are traditionally associated with small-molecule chemistry. The development of novel peptidomimetic technologies, however, has steered the field towards macrocycles. ‘t Hart et al. ^[9] reported the use of a 1,3,5-tris(bromomethyl)benzene-CLIPSed bicyclic peptide phage display screen as an avenue to generating novel antimicrobial lipopeptides. Specifically, they have identified unique lipid II-binding peptides that are active against Gram-positive bacteria, including clinically relevant vancomycin-resistant strains. Another similar example came from Adaligil and coworkers ^[8]; using ’mirror image’ phage display (screening against enantiomers of D-alanyl-D-alanine and D-alanyl-D-lactate containing fragments of bacterial cell wall precursors and their structural mimetic cephalosporin, and subsequent D-amino acid peptide synthesis) has led them to bicyclic peptides composed entirely of D-amino acids. Two of the hits displayed relatively high antibacterial (including against methicillin-resistant S. aureus and vancomycin-resistant Enterococci), but no hemolytic activity. Related to antimicrobials, neutralization of bacterial endotoxins is a warranted medicinal application but has hampered small molecule use due to the large contact area of the interacting molecules in the septic shock cascade. By interrogating a synthetic β-hairpin peptide library, Gonzalez-Navarro et al. ^[19] identified novel peptide binders with LPS neutralizing activity. Furthermore, Srinivas et al. ^[20] have identified D-Pro-L-Pro template-stabilized β hairpin mimetics of the antimicrobial peptide protegrin I, highly active against Pseudomonas aeruginosa. Using an elegant approach in which forward genetic screening was combined with functional and biochemical assays they have identified the outer membrane protein LptD as the molecular target for the peptidomimetic antimicrobials. It was concluded that perturbation of the critical LPS transport function of LptD is responsible for bacterial cell death, paving the way to antimicrobials with novel mode of action.

Novel peptidic technologies also support efficient protein–protein interaction probing. In their pioneering work, Timmerman et al. ^[5] have demonstrated the value of chemically crosslinked peptides in discontinuous epitope mapping. They have constructed a synthetic library of dodecamer peptides tiled along the sequence of follicle-stimulating hormone (FSH) β chain, both linear and cyclized, and analyzed binding to a monoclonal antibody using ELISA assay. Interaction was only detected with a specific looped peptide, and systematic substitutions allowed the identification of residues constituting the antigenic determinant (the β3-loop). In a follow-on paper ^[21], immunological properties of cyclized peptide mimetics of the β3-loop were studied. When rats were immunized with the 1,3-bis(bromomethyl)benzene-looped peptide (as opposed to the disulfide-stabilized counterpart), antibodies selectively recognizing the β subunit of FSH were induced. Double stabilization (involving an additional disulfide bond at the loop termini) further augmented the FSH-targeted immunogenicity, clearly demonstrating the importance of subtle conformational differences for inducing cross-reactive antibodies. The successful mimicry of a complex protein surface by a small synthetic peptide attained by CLIPS technology indicates its enormous potential for generating antibodies against difficult protein targets such as integral membrane proteins, where immunization with conventional linear peptides does not deliver results. Iqbal et al. ^[22] devised a high-throughput strategy for hydrocarbon-stapled peptide identification using mRNA display. They reprogrammed the genetic code to incorporate α-methyl cysteine at positions i and i + 4 of short randomized peptides using the PURE system, and stapled them with m-dibromoxylene. Although only a proof of principle screen was conducted with the library, this approach has the potential of evolving into a powerful tool for PPI inhibitor discovery.

Constrained peptides are being increasingly applied in oncology. For example, by screening a phage display library, Anananuchatkul et al. ^[23] identified [i, i + 7]-stapled helical peptides that showed a disruptive ability for the hDM2 oncogene-p53 tumor suppressor interaction. A similar approach has yielded binders for galectin-3 (a cancer-related galactose-binding protein) from a stapled α-helix phage-displayed peptide library ^[24]. Bertoldo et al. ^[6] reported a novel binder, identified by a screening bicyclic phage display peptide library, to the interaction region of the translational Wnt inhibitor ICAT (inhibitor of b-catenin and Tcf), which is a prime target site on β-catenin for therapeutic intervention in oncology. By screening libraries of CLIPSed bicyclic peptides designed on the basis of anti-gastrin 17 antibody complementarity determining loops, Timmerman et al. ^[25] have developed gastrin 17-neutralizing peptidomimetics. As gastrin 17 is a trophic factor in several gastrointestinal tumors, its neutralization presents a warranted anticancer therapeutic strategy. Indeed, the constrained peptides effectively reduced proliferation of two cancer cell lines in vitro, comparably with the parent neutralizing antibodies. The company Bicycle Therapeutics has a large portfolio of conformationally constrained macrocyclic peptides developed through phage display and CLIPS technology, mainly for treatment of cancers. Several of their drug candidates rely on such peptide moieties for selective cytotoxic payload targeting ^[26][27][28] and some have already entered clinical evaluation (clinical study identification codes NCT03486730, NCT04180371, NCT04561362). Others are structured as multiple bicyclic peptides connected by linkers via a central hinge and target and activate CD137 (a co-stimulatory immune checkpoint molecule) on NK and T cells or (combining two different bicycles) simultaneously activate CD137 and target tumor-associated antigens ^[28]. An elegant approach utilizing bicyclic peptides for targeting intracellular PPIs was devised by Trinh et al. ^[11], where one of the rings was an invariant cell-penetrating peptide and the other contained a randomized peptide sequence. The library was screened against the oncoprotein K-Ras G12V and yielded a K-Ras inhibitor of moderate potency that disrupted signaling events downstream of Ras, and induced apoptosis of cancer cells. An identical approach was employed in the discovery of a macrocyclic peptide disruptor of NEMO-IKKβ interaction, inhibiting proliferation of cisplatin-resistant ovarian cancer cells ^[12]. A related principle was adopted by Bernhagen et al. ^[29][30]; they have screened bicyclic libraries with built-in universal integrin-binding sequence Arg-Gly-Asp in the first loop and a randomized tripeptide sequence in the second loop. With the ‘guided’ library, they have probed adjacent binding sites on α₅β₁, and identified high-affinity integrin binders with potential as therapeutics or delivery vehicles in diagnostics and treatment of breast cancer. Furthermore, it has been demonstrated that it is possible to mimic RNA-recognition motifs of natural proteins ^[17]. Encouraged by this finding, Shortridge et al. ^[18] have screened a cyclic β-hairpin peptide library designed to mimic bovine immunodeficiency virus trans-activator of transcription (Tat) RNA-binding domain against the primary miRNA-21 transcript’s stem loop using the electrophoretic mobility shift assay. A structured peptide was identified that bound to an oncogenic microRNA-21 precursor with decent affinity and specificity, and suppressed Dicer processing, preventing downregulation of key tumor-suppressing and proapoptotic factors.

Constrained peptides with PPI-disruptive activities are also being developed for combating inflammatory disorders. Tamada et al. ^[31] have designed a cyclic β-hairpin peptide mimicking the predicted receptor for advanced glycation end-products (RAGE)-binding domain of high mobility group box 1 (HMGB1) protein using in silico methods. As HMGB1, which is secreted from immune and dying cells during cellular infection and injury, is a major promoter of inflammation, the peptide blocking HMGB1/RAGE interaction could become a useful therapeutic against HMGB1/RAGE-mediated sepsis and other inflammatory diseases. Another study identified disulfide-cyclized peptide binders of TNF-α via phage display and utilized them as imaging contrast agents to inflammatory areas by covalently linking them to iron oxide nanoparticles ^[32].

Another exciting research area involving structured peptides is the development of minimalist peptide catalysts in which the residues are spatially arranged to mimic enzymes’ active sites in terms of functional group cooperativity in catalysis. Because de novo design of catalytic peptides would be highly challenging, screening libraries of partially randomized peptides with the propensity to fold into defined structures is a viable alternative. For example, both helical ^[33] and β-hairpin loop scaffolds ^[34][35] have been successfully exploited to nucleate active site-like residue organization with the imidazole ring of a central histidine serving as a nucleophile in acyl-transfer catalysis. Bezer et al. ^[33] designed polyalanine/aminoisobutyric acid-based helical peptide libraries with His at position i, and randomized residues at positions in the immediate vicinity (i.e., [i − 4], [i − 3], [i + 3], or [i + 4]). Similarly, Matsumoto et al. ^[34][35] designed synthetic peptide libraries by randomizing the non-hydrogen-bonding positions of a histidine-harboring β-hairpin loop peptide. Here, the effects of various aromatic and hydrogen-bonding residues in close contact with the histidine (its ±2 neighbors in the linear sequence and those located cross-strand) were probed. Dye-labeled substrates (active esters) were incubated with one-bead-one-compound peptide libraries in dichloromethane. In this reactive tagging assay, acyl groups were transferred on the imidazole group, forming colored N-acyl histidine intermediates stable under nucleophile-free conditions (whereas in presence of competing alcoholic nucleophiles the peptides were deacylated, thus closing the catalytic transesterification cycle). The intensely stained beads were then separated from the non-stained ones, and the peptide catalysts were identified by mass spectrometry. The authors note that both, the type of cooperating residues (enhancing imidazole’s nucleophilic character) and the structural determinants of the peptide (e.g., helix length and type (α vs. 3₁₀), β-hairpin flexibility, and placement of functionalities within the structure), govern catalytic activity in a way that would be difficult to predict. These observations argue in favor of interrogating combinatorial peptide libraries for functional properties other than binding affinity.