The melanocortin-3 receptor (MC3R) is a G-protein coupled receptor that regulates appetite and is a potential therapeutic target for anorexia and weight loss treatments. The report of a highly selective synthetic antagonist of MC3R, Cpd11, represents a major advance towards therapeutic targeting of MC3R. However, Cpd11 is challenging to access synthetically, severely limiting its use and additional structural optimization. Here, we outline an improved synthesis of Cpd11 that addresses three major synthetic challenges, including the formation of Cpd11's structurally unique bicyclic core. With these changes, Cpd11 was readily produced (2.3 mg from a .05 mmol scale versus << 0.1 mg using the original synthetic methodology) and utilized in MC3R studies in C57BL/6J male mice. Thus, this new approach will increase the accessibility of Cpd11 and is translatable to related bicyclic agonists and antagonists for other melanocortin receptors and may have general applicability toward the synthesis of other multicyclic poly-cysteine peptides.

The COVID-19 pandemic drove a uniquely fervent pursuit to explore the potential of peptide, antibody, protein, and small-molecule based antiviral agents against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). The interaction between the SARS-CoV2 spike protein with the angiotensin-converting enzyme 2 (ACE2) receptor that mediates viral cell entry was a particularly interesting target given its well described protein-protein interaction (PPI). This PPI is mediated by an α-helical portion of ACE2 binding to the receptor binding domain (RBD) of the spike protein and thought to be susceptible to blockade through molecular mimicry. Small numbers of hydrocarbon-stapled synthetic peptides designed to disrupt or block this interaction were tested individually and were found to have variable efficacy despite having related or overlapping sequences and similarly increased α-helicity. Reasons for these differences are unclear and reported pre-clinical successes have been limited. The current study sought to better understand reasons for these differences through evaluation of a comprehensive collection of hydrocarbon stapled peptides, designed based on four distinct principles: stapling position, number of staples, amino acid sequence, and primary sequence length. Surprisingly, we observed that the helicity and amino acid sequence iterations of hydrocarbon-stapled peptides did not correlate with their bioactivity. Our results highlight the importance of iterative and combinatorial testing of these compounds to determine a configuration that best mimics natural binding and allows for chain flexibility while sacrificing structural helicity.

Monoclonal antibodies (mAbs) that target the P-amyloid peptide (Aβ) are important Alzheimer's disease research tools and are now being used as Alzheimer's disease therapies. Conformation-specific mAbs that target oligomeric and fibrillar Aβ assemblies are of particular interest, as these assemblies are associated with Alzheimer's disease pathogenesis and progression. This paper reports the generation of rabbit mAbs against two different triangular trimers derived from Aβ. These antibodies are the first mAbs generated against Aβ oligomer mimics in which the high-resolution structures of the oligomers are known. We describe the isolation of the mAbs using single B-cell sorting of peripheral blood mononuclear cells (PBMCs) from immunized rabbits, the selectivity of the mAbs for the triangular trimers, the immunoreactivity of the mAbs with aggregated Aβ, and the immunoreactivity of the mAbs in brain tissue from the 5xFAD Alzheimer's disease mouse model. The characterization of these mAbs against structurally defined trimers derived from Aβ enhances understanding of antibody-amyloid recognition and may benefit the development of diagnostics and immunotherapies in Alzheimer's disease.

Coiled coils are one of most common protein quaternary structures and represent the best understood relationship between amino acid sequence and protein conformation. Whereas the roles of residues at the canonical heptad positions the , , , and are understood in precise detail, conventional approaches often assume that the solvent-exposed -, -, and -positions can be varied broadly for application-specific purposes with minimal consequences. However, a growing body of evidence suggests that interactions among these , , and residues can contribute substantially to coiled-coil conformational stability. In the trimeric coiled coil described here, we find that -position Glu10 engages in a stabilizing long-range synergistic interaction with -position Lys18 (ΔΔΔG = -0.65 ± 0.02 kcal/mol). This favorable interaction depends strongly on the presence of two nearby -position residues: Lys 7 and Tyr14. Extensive mutational analysis of these residues in the presence of added salt vs. denaturant suggests that this long-range synergistic interaction is primarily electrostatic in origin, but also depends on the precise location and acidity of a side-chain hydrogen-bond donor within -position Tyr14.

Sunflower trypsin inhibitor-1 (SFTI-1) structure is used for designing grafted peptides as a possible therapeutic agent. The grafted peptide exhibits multiple conformations in solution due to the presence of proline in the structure of the peptide. To lock the grafted peptide into a major conformation in solution, a dibenzofuran moiety (DBF) was incorporated in the peptide backbone structure, replacing the Pro-Pro sequence. NMR studies indicated a major conformation of the grafted peptide in solution. Detailed structural studies suggested that SFTI-DBF adopts a twisted beta-strand structure in solution. The surface plasmon resonance analysis showed that SFTI-DBF binds to CD58 protein. A model for the protein-SFTI-DBF complex was proposed based on docking studies. These studies suggested that SFTI-1 grafted peptide can be used to design stable peptides for therapeutic purposes by grafting organic functional groups and amino acids. However, when a similar strategy was used with another grafted peptide, the resulting peptide did not produce a single major conformation, and its biological activity was lost. Thus, conformational constraints depend on the sequence of amino acids used for SFTI-1 grafting.

Simple and efficient total synthesis of homogeneous and chemically modified protein samples remains a significant challenge. Here, we report development of a convergent hybrid phase native chemical ligation (CHP-NCL) strategy for facile preparation of proteins. In this strategy, proteins are split into ~100-residue blocks, and each block is assembled on solid support from synthetically accessible peptide fragments before ligated together into full-length protein in solution. With the new method, we increase the yield of CENP-A synthesis by 2.5-fold compared to the previous hybrid phase ligation approach. We further extend the new strategy to the total chemical synthesis of 212-residue linker histone H1.2 in unmodified, phosphorylated, and citrullinated forms, each from eight peptide segments with only one single purification. We demonstrate that fully synthetic H1.2 replicates the binding interactions of linker histones to intact mononucleosomes, as a proxy for the essential function of linker histones in the formation and regulation of higher order chromatin structure.

Prion protein misfolding is associated with fatal neurodegenerative disorders such as kuru, Creutzfeldt-Jakob disease, and several animal encephalopathies. While the C-terminal 106-126 peptide has been well studied for its role in prion replication and toxicity, the octapeptide repeat (OPR) sequence found within the N-terminal domain has been relatively under explored. Recent findings that the OPR has both local and long-range effects on prion protein folding and assembly, as well as its ability to bind and regulate transition metal homeostasis, highlights the important role this understudied region may have in prion pathologies. This review attempts to collate this knowledge to advance a deeper understanding on the varied physiologic and pathologic roles the prion OPR plays, and connect these findings to potential therapeutic modalities focused on OPR-metal binding. Continued study of the OPR will not only elucidate a more complete mechanistic model of prion pathology, but may enhance knowledge on other neurodegenerative processes underlying Alzheimer's, Parkinson's, and Huntington's diseases.

The construction of protein-sized synthetic chains that blend natural amino acids with artificial monomers to create so-called heterogeneous-backbones is a powerful approach to generate complex folds and functions from bio-inspired agents. A variety of techniques from structural biology commonly used to study natural proteins have been adapted to investigate folding in these entities. In NMR characterization of proteins, proton chemical shift is a straightforward to acquire, information-rich metric that bears directly on a variety of properties related to folding. Leveraging chemical shift to gain insight into folding requires a set of reference chemical shift values corresponding to each building block type (i.e., the 20 canonical amino acids in the case of natural proteins) in a random coil state and knowledge of systematic changes in chemical shift associated with particular folded conformations. Although well documented for natural proteins, these issues remain unexplored in the context of protein mimetics. Here, we report random coil chemical shift values for a library of artificial amino acid monomers frequently used to construct heterogeneous-backbone protein analogues as well as a spectroscopic signature associated with one monomer class, β-residues bearing proteinogenic side chains, adopting a helical folded conformation. Collectively, these results will facilitate the continued utilization of NMR for the study of structure and dynamics in protein-like artificial backbones.

Bacteria utilize a cell density-dependent communication system called quorum sensing (QS) to coordinate group behaviors. In Gram-positive bacteria, QS involves the production of and response to auto-inducing peptide (AIP) signaling molecules to modulate group phenotypes, including pathogenicity. As such, this bacterial communication system has been identified as a potential therapeutic target against bacterial infections. More specifically, developing synthetic modulators derived from the native peptide signal paves a new way to selectively block the pathogenic behaviors associated with this signaling system. Moreover, rational design and development of potent synthetic peptide modulators allows in depth understanding of the molecular mechanisms that drive QS circuits in diverse bacterial species. Overall, studies aimed at understanding the role of QS in microbial social behavior could result in the accumulation of significant knowledge of microbial interactions, and consequently lead to the development of alternative therapeutic agents to treat bacterial infectivity. In this review, we discuss recent advances in the development of peptide-based modulators to target QS systems in Gram-positive pathogens, with a focus on evaluating the therapeutic potential associated with these bacterial signaling pathways.

Peptide vaccines and immunotherapies against aggregating proteins involved in the pathogenesis and progression of Alzheimer's disease (AD) - the β-amyloid peptide (Aβ) and tau - are promising therapeutic avenues against AD. Two decades of effort has led to the controversial FDA approval of the monoclonal antibody Aducanumab (Aduhelm), which has subsequentially sparked the revival and expedited review of promising monoclonal antibody immunotherapies that target Aβ. In this review, we explore the development of Aβ and tau peptide vaccines and immunotherapies with monoclonal antibodies in clinical trials against AD.

is a highly adaptable pathogen that can rapidly develop resistance to conventional antibiotics such as penicillin. Recently, teixobactin was discovered from uncultivated soil bacteria by using the i-chip technology. This depsipeptide forms an ester bond between the backbone C-terminal isoleucine carboxylic acid and the hydroxyl group of threonine at position 8. Also, it contains multiple nonstandard amino acids, making it costly to synthesize. This study reports new peptides designed by linearizing teixobactin. After linearization and conversion to normal amino acids, teixobactin lost its antibacterial activity. Using this inactive template, a series of peptides were designed via hydrophobic patching and residue replacements. Three out of the five peptides were active. YZ105, only active against Gram-positive bacteria, however, showed the highest cell selectivity index. Different from teixobactin, which inhibits cell wall synthesis, YZ105 targeted the membranes of methicillin-resistant (MRSA) based on kinetic killing, membrane permeation, depolarization, and scanning electron microscopy studies. Moreover, YZ105 could kill nafcillin-resistant MRSA, Staphylococcal clinical strains, and disrupted preformed biofilms. Taken together, YZ105, with a simpler sequence, is a promising lead for developing novel anti-MRSA agents.

C-terminal hydrazides are an important class of synthetic peptides with an ever expanding scope of applications, but their widespread application for chemical protein synthesis has been hampered due to the lack of stable resin linkers for synthesis of longer and more challenging peptide hydrazide fragments. We present a practical method for the regeneration, loading, and storage of trityl-chloride resins for the production of hydrazide containing peptides, leveraging 9-fluorenylmethyl carbazate. We show that these resins are extremely stable under several common resin storage conditions. The application of these resins to solid phase peptide synthesis (SPPS) is demonstrated through the synthesis of the 40-mer GLP-1R agonist peptide "P5". These studies support the broad utility of Fmoc-NHNH-Trt resins for SPPS of C-terminal hydrazide peptides.

Since its first detection in 2019, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been the cause of millions of deaths worldwide. Despite the development and administration of different vaccines, the situation is still worrisome as the virus is constantly mutating to produce newer variants some of which are highly infectious. This raises an urgent requirement to understand the infection mechanism and thereby design therapeutic-based treatment for COVID-19. The gateway of the virus to the host cell is mediated by the binding of the receptor binding domain (RBD) of the virus spike protein to the angiotensin-converting enzyme 2 (ACE2) of the human cell. Therefore, the RBD of SARS-CoV-2 can be used as a target to design therapeutics. The α1 helix of ACE2, which forms direct contact with the RBD surface, has been used as a template in the current study to design stapled peptide therapeutics. Using computer simulation, the mechanism and thermodynamics of the binding of six stapled peptides with RBD have been estimated. Among these, the one with two lactam stapling agents has shown binding affinity, sufficient to overcome RBD-ACE2 binding. Analyses of the mechanistic detail reveal that a reorganization of amino acids at the RBD-ACE2 interface produces favorable enthalpy of binding whereas conformational restriction of the free peptide reduces the loss in entropy to result higher binding affinity. The understanding of the relation of the nature of the stapling agent with their binding affinity opens up the avenue to explore stapled peptides as therapeutic against SARS-CoV-2.

Genetically-encoded cyclic peptide libraries allow rapid screens for inhibitors of any target protein of interest. In particular, the Split Intein Circular Ligation of Protein and Peptides (SICLOPPS) system exploits spontaneous protein splicing of inteins to produce intracellular cyclic peptides. A previous SICLOPPS screen against Aurora B kinase, which plays a critical role during chromosome segregation, identified several candidate inhibitors that we sought to recapitulate by chemical synthesis. We describe the syntheses of cyclic peptide hits and analogs via solution-phase macrocyclization of side chain-protected linear peptides obtained from standard solid-phase peptide synthesis. Cyclic peptide targets, including cyclo-[CTWAR], were designed to match both the variable portions and conserved cysteine residue of their genetically-encoded counterparts. Synthetic products were characterized by tandem high-resolution mass spectrometry to analyze a combination of exact mass, isotopic pattern, and collisional dissociation-induced fragmentation pattern. The latter analyses facilitated the distinction between targets and oligomeric side products, and served to confirm peptidic sequences in a manner that can be readily extended to analyses of complex biological samples. This alternative chemical synthesis approach for cyclic peptides allows cost-effective validation and facile chemical elaboration of hit candidates from SICLOPPS screens.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Covid-19 pandemic has caused high morbidity and mortality rates worldwide. Virus entry into cells can be blocked using several strategies, including inhibition of protein-protein interactions (PPIs) between the viral spike glycoprotein and cellular receptors, as well as blocking of spike protein conformational changes that are required for cleavage/activation and fusogenicity. The spike-mediated viral attachment and entry into cells via fusion of the viral envelope with cellular membranes involve PPIs mediated by short peptide fragments exhibiting particular secondary structures. Thus, peptides that can inhibit these PPIs may be used as potential antiviral agents preventing virus entry and spread. This review is focused on peptides and peptidomimetics as PPI modulators and protease inhibitors against SARS-CoV-2.

is an opportunistic respiratory human pathogen that poses a continuing threat to human health. Natural competence for genetic transformation in plays an important role in aiding pathogenicity and it is the best-characterized feature to acquire antimicrobial resistance genes by a frequent process of recombination. In , competence, along with virulence factor production, is controlled by a cell-density communication mechanism termed the competence regulon. In this review, we present the recent advances in the development of alternative methods to attenuate the pathogenicity of S. by targeting the various stages of the non-essential competence regulon communication system. We mainly focus on new developments related to competitively intercepting the competence regulon signaling through the introduction of promising dominant-negative Competence Stimulating Peptide (dnCSP) scaffolds. We also discuss recent reports on antibiotics that can block CSP export by disturbing the proton motive force (PMF) across the membrane and various ways to control the pneumococcal pathogenicity by activating the counter signaling circuit and targeting the pneumococcal proteome.

HYD1 is an all D-amino acid linear 10-mer peptide that was discovered by one-bead-one-compound screening. HYD1 has five hydrophobic amino acids flanked by polar amino acids. Alanine scanning studies showed that alternating hydrophobic amino acid residues and N- and C-terminal lysine side chains were contributors to the biological activity of the linear 10-mer analogs. This observation led us to hypothesize that display of the hydrophobic pentapeptide sequence of HYD1 in a cyclic beta-hairpin-like scaffold could lead to better bioavailability and biological activity. An amphipathic pentapeptide sequence was used to form an antiparallel strand and those strands were linked via dipeptide-like sequences selected to promote β-turns. Early cyclic analogs were more active but otherwise mimicked the biological activity of the linear HYD1 peptide. The cyclic peptidomimetics were synthesized using standard Fmoc solid phase synthesis to form linear peptides, followed by solution phase or on-resin cyclization. SAR studies were carried out with an aim to increase the potency of these drug candidates for the killing of multiple myeloma cells . The solution structures of , , and were elucidated using NMR spectroscopy. H NMR and 2D TOCSY studies of these peptides revealed a downfield H proton chemical shift and 2D NOE spectral analysis consistent with a β-hairpin-like structure.

Fluorinated compounds, while rarely used by nature, are emerging as fundamental ingredients in biomedical research, with applications in drug discovery, metabolomics, biospectroscopy, and, as the focus of this review, peptide/protein engineering. Leveraging the fluorous effect to direct peptide assembly has evolved an entirely new class of organofluorine building blocks from which unique and bioactive materials can be constructed. Here, we discuss three distinct peptide fluorination strategies used to design and induce peptide assembly into nano-, micro-, and macrosupramolecular states that potentiate high-ordered organization into material scaffolds. These fluorine-tailored peptide assemblies employ the unique fluorous environment to boost biofunctionality for a broad range of applications, from drug delivery to antibacterial coatings. This review provides foundational tactics for peptide fluorination and discusses the utility of these fluorous-directed hierarchical structures as material platforms in diverse biomedical applications.

Here we report a new type of tryptophan-rich short peptides, which act as hydrogelators, form supramolecular assemblies via enzymatic dephosphorylation, and exhibit cell compatibility. The facile synthesis of the peptides starts with the production of phosphotyrosine, then uses solid phase peptide synthesis (SPPS) to build the phosphopeptides that contain multiple tryptophan residues. Besides exhibiting excellent solubility, these phosphopeptides, unlike the previously reported cytotoxic phenylalanine-rich phosphopeptides, are largely compatible toward mammalian cells. Our preliminary mechanistic study suggests that the tryptophan-rich peptides, instead of forming pericellular assemblies, largely accumulate in lysosomes. Such lysosomal localization may account for their cell compatibility. Moreover, these tryptophan-rich peptides are able to transiently reduce the cytotoxicity of phenylalanine-rich peptide assemblies. This rather unexpected result implies that tryptophan may act as a useful aromatic building block for developing cell compatible supramolecular assemblies for soft materials and find applications for protecting cells from cytotoxic peptide assemblies.

Methods to constrain peptides in a bioactive α-helical conformation for inhibition of protein-protein interactions represent an ongoing area of investigation in chemical biology. Recently, the first example of a reversible "stapling" methodology was described which exploits native cysteine or homocysteine residues spaced at the and  + 4 positions in a peptide sequence together with the thiol selective reactivity of dibromomaleimides (a previous study). This manuscript reports on the optimization of the maleimide based constraint, focusing on the kinetics of macrocyclization and the extent to which helicity is promoted with different thiol containing amino acids. The study identified an optimal stapling combination of = L-Cys and = L-Cys in the context of the model peptide Ac-XAAAX-NH, which should prove useful in implementing the dibromomaleimide stapling strategy in peptidomimetic ligand discovery programmes.

Naturally derived antimicrobial peptides have been an area of great interest because of high selectivity against bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. There are also a significant number of venom-derived peptides that exhibit antimicrobial activity in addition to activity against mammals or other organisms. Many venom peptides share the same net cationic, amphiphilic nature as host-defense peptides, making them an attractive target for development as potential antibacterial agents. The peptide ponericin L1 derived from was used as a model to investigate the role of cationic residues and net charge on peptide activity. Using a combination of spectroscopic and microbiological approaches, the role of cationic residues and net charge on antibacterial activity, lipid bilayer interactions, and bilayer and membrane permeabilization were investigated. The L1 peptide and derivatives all showed enhanced binding to lipid vesicles containing anionic lipids, but still bound to zwitterionic vesicles. None of the derivatives were especially effective at permeabilizing lipid bilayers in model vesicles, in-tact , or human red blood cells. Taken together the results indicate that the lack of facial amphiphilicity regarding charge segregation may impact the ability of the L1 peptides to effectively permeabilize bilayers despite effective binding. Additionally, increasing the net charge of the peptide by replacing the lone anionic residue with either Gln or Lys dramatically improved efficacy against several bacterial strains without increasing hemolytic activity.