Diverse Proteomimetic Frameworks via Rational Design of π-Stacking Peptide Tectons.

Peptide-based frameworks aim to integrate protein architecture into solid-state materials using simpler building blocks. Despite the growing number of peptide frameworks, there are few strategies to rationally engineer essential properties like pore size and shape. Designing peptide assemblies is generally hindered by the difficulty of predicting complex networks of weak intermolecular interactions. Peptides conjugated to polyaromatic groups are a unique case where assembly appears to be strongly driven by π-π interactions, suggesting that rationally adjusting the geometry of the π-stackers could create novel structures. Here, we report peptide elongation as a simple mechanism to predictably tune the angle between the π-stacking groups to produce a remarkable diversity of pore shapes and sizes, including some that are mesoporous. Notably, rapid jumps in pore size and shape can occur with just a single amino acid insertion. The geometry of the π-stacking residues also significantly influences framework structure, representing an additional dimension for tuning. Lastly, sequence identity can also indirectly modulate the π-π interactions. By correlating each of these factors with detailed crystallographic data, we find that, despite the complexity of peptide structure, the shape and polarity of the tectons are straightforward predictors of framework structure. These guidelines are expected to accelerate the development of advanced porous materials with protein-like capabilities.

Mystery of the Passerini Reaction for the Synthesis of the Antimicrobial Peptidomimetics against Nosocomial Pathogenic Bacteria.

The first example of applying salicylaldehyde derivatives, as well as coumarin with the formyl group at the C8 position in its structure, as carbonyl partners in a three-component Passerini reaction, is presented. As a result of research on the conditions of the Passerini reaction, the important role of the hydroxyl group in the salicylaldehyde used in the course of the multicomponent reaction was revealed. When an aldehyde with an unprotected hydroxyl group is used, only two-component α-hydroxy amide products are obtained. In contrast, the use of acylated aldehyde results in three-component α-acyloxy amide products with high efficiency. The developed protocol gives access to structurally diversified peptidomimetics with good yield. The compounds were also evaluated as antimicrobial agents against selected strains of nosocomial pathogenic bacteria. The structure-activity relationship revealed that inhibitory activity is strongly related to the presence of the trifluoromethyl group (CF3) or the methyl group at the C4 position in an unsaturated lactone ring of the coumarin scaffold. MIC and MBC studies were carried out on eight selected pathogenic bacteria strains (Gram-positive pathogenic Staphylococcus aureus strain (ATCC 23235), as well as on Gram-negative E. coli (K12 (ATCC 25404), R2 (ATCC 39544), R3 (ATCC 11775), and R4 (ATCC 39543)), Acinetobacter baumannii (ATCC 17978), Pseudomonas aeruginosa (ATCC 15442), and Enterobacter cloacae (ATCC 49141) have shown that the tested compounds show a strong bactericidal effect at low concentrations. Among all agents investigated, five exhibit higher antimicrobial activity than those observed for commonly used antibiotics. It should be noted that all the compounds tested showed very high activity against S. aureus, which is the main source of nosocomial infections that cause numerous fatalities. Additionally, the cytotoxicity of sixteen derivatives was measured with the use of the MTT test on BALB/c3T3 mouse fibroblast cell lines. The cytotoxicity studies revealed that the tested substances exert a similar or lower effect on cell proliferation than that observed for commonly used antibiotics within the range of therapeutic doses. A parallel MTT assay using ciprofloxacin, bleomycin, and cloxacillin showed that these antibiotics are more cytotoxic when tested in mammalian cells, and cell viability is in the range of 85.0-89.9%. Furthermore, we have shown that the studied coumarin-based peptidomimetics, depending on their structural characteristics, are nonselective and act efficiently against various Gram-positive and Gram-negative pathogens, which is of great importance for hospitalised patients.

Discovery of macrocyclic covalent inhibitors for severe acute respiratory syndrome coronavirus 2 3CL protease.

The coronavirus disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spread worldwide for more than 3 years. Although the hospitalization rate and mortality have decreased dramatically due to wide vaccination effort and improved treatment options, the disease is still a global health issue due to constant viral mutations, causing negative impact on social and economic activities. In addition, long COVID and complications arising from COVID-19 weeks after infection have become a concern for public health experts. Therefore, better treatments for COVID-19 are still needed. Herein, we describe a class of macrocyclic peptidomimetic compounds that are potent inhibitors of SARS-Cov-2 3CL protease (3CLpro). Significantly, some of the compounds showed a higher stability against human liver microsomes (HLM t1/2 > 180 min) and may be suitable for oral administration without the need for a pharmacokinetic (PK) boosting agent such as ritonavir.

Rational In Silico Design of Selective TMPRSS6 Peptidomimetic Inhibitors via Exploitation of the S2 Subpocket.

TMPRSS6 is a potential therapeutic target for the treatment of iron overload due to its role in regulating levels of hepcidin. Although potent TMPRSS6 inhibitors have been previously developed, their lack of specificity requires optimization to avoid potential side effects before pursuing preclinical development with in vivo models. Here, using computer-aided drug design based on a TMPRSS6 homology model, we reveal that the S2 position of TMPRSS6 offers a potential avenue to achieve selectivity against other members of the TTSP family. Accordingly, we synthesized novel peptidomimetic molecules containing lipophilic amino acids at the P2 position to exploit this unexplored pocket. This enabled us to identify TMPRSS6-selective small molecules with low nanomolar affinity. Finally, pharmacokinetic parameters were determined, and a compound was found to be potent in cellulo toward its primary target while retaining TTSP-subtype selectivity and showing no signs of alteration in in vitro TEER experiments.

Novel selective proline-based peptidomimetics for human cathepsin K inhibition.

Human cathepsin K (CatK) stands out as a promising target for the treatment of osteoporosis, considering its role in degrading the bone matrix. Given the small and shallow S2 subsite of CatK and considering its preference for proline or hydroxyproline, we now propose the rigidification of the leucine fragment found at the P2 position in a dipeptidyl-based inhibitor, generating rigid proline-based analogs. Accordingly, with these new proline-based peptidomimetics inhibitors, we selectively inhibited CatK against other human cathepsins (B, L and S). Among these new ligands, the most active one exhibited a high affinity (pKi = 7.3 - 50.1 nM) for CatK and no inhibition over the other cathepsins. This specific inhibitor harbors two novel substituents never employed in other CatK inhibitors: the trifluoromethylpyrazole and the 4-methylproline at P3 and P2 positions. These results broaden and advance the path toward new potent and selective inhibitors for CatK.

Design, synthesis and evaluation of bioactivity of peptidomimetics based on chloroalkene dipeptide isosteres.

Ample biologically active peptides have been found, identified and modified for use in drug discovery to date. However, several factors, such as low metabolic stability due to proteolysis and non-specific interactions with multiple off-target molecules, might limit the therapeutic use of peptides. To enhance the stability and/or bioactivity of peptides, the development of "peptidomimetics," which mimick peptide molecules, is considered to be idealistic. Hence, chloroalkene dipeptide isosteres (CADIs) was designed, and their synthetic methods have been developed by us. Briefly, in a CADI an amide bond in peptides is replaced with a chloroalkene structure. CADIs might be superior mimetics of amide bonds because the Van der Waals radii (VDR) and the electronegativity value of a chlorine atom are close to those of the replaced oxygen atom. By a developed method of the "liner synthesis", N-tert-butylsulfonyl protected CADIs can be synthesized via a key reaction involving diastereoselective allylic alkylation using organocopper reagents. On the other hand, by a developed method of the "convergent synthesis", N-fluorenylmethoxycarbonyl (Fmoc)-protected carboxylic acids can be also constructed based on N- and C-terminal analogues from corresponding amino acid starting materials via an Evans syn aldol reaction and the Ichikawa allylcyanate rearrangement reaction involving a [3.3] sigmatropic rearrangement. Notably, CADIs can also be applied for Fmoc-based solid-phase peptide synthesis and therefore introduced into bioactive peptides including as the Arg-Gly-Asp (RGD) peptide and the amyloid ß fragment Lys-Leu-Val-Phe-Phe (KLVFF) peptide, which are correlated with cell attachment and Alzheimer's disease (AD), respectively. These CADI-containing peptidomimetics stabilized the conformation and enhanced the potency of the cyclic RGD peptide and the cyclic KLVFF peptide.

Recent Advances in Amphipathic Peptidomimetics as Antimicrobial Agents to Combat Drug Resistance.

The development of antimicrobial drugs with novel structures and clear mechanisms of action that are active against drug-resistant bacteria has become an urgent need of safeguarding human health due to the rise of bacterial drug resistance. The discovery of AMPs and the development of amphipathic peptidomimetics have lay the foundation for novel antimicrobial agents to combat drug resistance due to their overall strong antimicrobial activities and unique membrane-active mechanisms. To break the limitation of AMPs, researchers have invested in great endeavors through various approaches in the past years. This review summarized the recent advances including the development of antibacterial small molecule peptidomimetics and peptide-mimic cationic oligomers/polymers, as well as mechanism-of-action studies. As this exciting interdisciplinary field is continuously expanding and growing, we hope this review will benefit researchers in the rational design of novel antimicrobial peptidomimetics in the future.

Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV-2 Main Protease Inhibitors as Antiviral Drugs.

Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M-1s-1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents.

A Novel Peptidomimetic Insecticide: Dippu-AstR-Based Rational Design and Biological Activity of Allatostatin Analogs.

Insect neuropeptides play an essential role in regulating growth, development, reproduction, nerve conduction, metabolism, and behavior in insects; therefore, G protein-coupled receptors of neuropeptides are considered important targets for designing green insecticides. Cockroach-type allatostatins (ASTs) (FGLamides allatostatins) are important insect neuropeptides in Diploptera punctata that inhibit juvenile hormone (JH) synthesis in the corpora allata and affect growth, development, and reproduction of insects. Therefore, the pursuit of novel insecticides targeting the allatostatin receptor (AstR) holds significant importance. Previously, we identified an AST analogue, H17, as a promising candidate for pest control. Herein, we first modeled the 3D structure of AstR in D. punctata (Dippu-AstR) and predicted the binding mode of H17 with Dippu-AstR to study the critical interactions and residues favorable to its bioactivity. Based on this binding mode, we designed and synthesized a series of H17 derivatives and assessed their insecticidal activity against D. punctata. Among them, compound Q6 showed higher insecticidal activity than H17 against D. punctata by inhibiting JH biosynthesis, indicating that Q6 is a potential candidate for a novel insect growth regulator (IGR)-based insecticide. Moreover, Q6 exhibited insecticidal activity against Plutella xylostella, indicating that these AST analogs may have a wider insecticidal spectrum. The underlying mechanisms and molecular conformations mediating the interactions of Q6 with Dippu-AstR were explored to understand its effects on the bioactivity. The present work clarifies how a target-based strategy facilitates the discovery of new peptide mimics with better bioactivity, enabling improved IGR-based insecticide potency in sustainable agriculture.

Design of Triazolium-Grafted Peptidomimetic Macrocycles with Facial Amphipathicity to Target Pathogenic Bacteria.

Access to 1,2,3-triazolium-grafted peptoid macrocycles was developed by macrocyclization and multivalent postmodification of linear peptoid oligomers carrying an alternance of benzylic and propargyl groups as side chains. X-ray analysis and NMR studies revealed a conformational preference for constrained hairpin-shaped structures leading to the facial amphipathic character of these macrocycles. A preliminary evaluation showed the antimicrobial activities of these new cationic amphipathic architectures.

Targeted genetic and small molecule disruption of N-Ras CaaX cleavage alters its localization and oncogenic potential.

Ras GTPases and other CaaX proteins undergo multiple post-translational modifications at their carboxyl-terminus. These events initiate with prenylation of a cysteine and are followed by endoproteolytic removal of the 'aaX' tripeptide and carboxylmethylation. Some CaaX proteins are only subject to prenylation, however, due to the presence of an uncleavable sequence. In this study, uncleavable sequences were used to stage Ras isoforms in a farnesylated and uncleaved state to address the impact of CaaX proteolysis on protein localization and function. This targeted strategy is more specific than those that chemically inhibit the Rce1 CaaX protease or delete the RCE1 gene because global abrogation of CaaX proteolysis impacts the entire CaaX protein proteome and effects cannot be attributed to any specific CaaX protein of the many concurrently affected. With this targeted strategy, clear mislocalization and reduced activity of farnesylated and uncleaved Ras isoforms was observed. In addition, new peptidomimetics based on cleavable Ras CaaX sequences and the uncleavable CAHQ sequence were synthesized and tested as Rce1 inhibitors using in vitro and cell-based assays. Consistently, these non-hydrolyzable peptidomimetic Rce1 inhibitors recapitulate Ras mislocalization effects when modeled on cleavable but not uncleavable CaaX sequences. These findings indicate that a prenylated and uncleavable CaaX sequence, which can be easily applied to a wide range of mammalian CaaX proteins, can be used to probe the specific impact of CaaX proteolysis on CaaX protein properties under conditions of an otherwise normally processed CaaX protein proteome.

Recent developments in the utility of saturated azaheterocycles in peptidomimetics.

To a large extent, the physical and chemical properties of peptidomimetic molecules are dictated by the integrated heterocyclic scaffolds they contain. Heterocyclic moieties are introduced into a majority of peptide-mimicking molecules to modulate conformational flexibility, improve bioavailability, and fine-tune electronics, and in order to achieve potency similar to or better than that of the natural peptide ligand. This mini-review delineates recent developments, limited to the past five years, in the utility of selected saturated 3- to 6-membered heterocyclic moieties in peptidomimetic design. Also discussed is the chemistry involved in the synthesis of the azaheterocyclic scaffolds and the structural implications of the introduction of these azaheterocycles in peptide backbones as well as side chains of the peptide mimics.

Neutralizing SARS-CoV-2 by dimeric side chain-to-side chain cross-linked ACE2 peptide mimetics.

We present the finding of a dimeric ACE2 peptide mimetic designed through side chain cross-linking and covalent dimerization. It has a binding affinity of 16 nM for the SARS-CoV-2 spike RBD, and effectively inhibits the SARS-CoV-2 pseudovirus in Huh7-hACE2 cells with an IC50 of 190 nM and neutralizes the authentic SARS-CoV-2 in Caco2 cells with an IC50 of 2.4 µM. Our study should provide a new insight for the optimization of peptide-based anti-SARS-CoV-2 inhibitors.

Novel Macrocyclic Peptidomimetics Targeting the Polo-Box Domain of Polo-Like Kinase 1.

The polo-box domain (PBD) of Plk1 is a promising target for cancer therapeutics. We designed and synthesized novel phosphorylated macrocyclic peptidomimetics targeting PBD based on acyclic phosphopeptide PMQSpTPL. The inhibitory activities of 16e on Plk1-PBD is >30-fold higher than those of PMQSpTPL. Both 16a and 16e possess excellent selectivity for Plk1-PBD over Plk2/3-PBD. Analysis of the cocrystal structure of Plk1-PBD in complex with 16a reveals that the 3-(trifluoromethyl)benzoyl group in 16a interacts with Arg516 through a π-stacking interaction. This π-stacking interaction, which has not been reported previously, provides insight into the design of novel and potent Plk1-PBD inhibitors. Furthermore, 16h, a PEGlyated macrocyclic phosphopeptide derivative, induces Plk1 delocalization and mitotic failure in HeLa cells. Also, the number of phospho-H3-positive cells in a zebrafish embryo increases in proportion to the amount of 16a. Collectively, the novel macrocyclic peptidomimetics should serve as valuable templates for the design of potent and novel Plk1-PBD inhibitors.

Development of anti-inflammatory peptidomimetics based on the structure of human alpha1-antitrypsin.

Human α1-antitrypsin (hAAT) has two distinguishing functions: anti-protease activity and regulation of the immune system. In the present study we hypothesized that those two protein functions are mediated by different structural domains on the hAAT surface. Indeed, such biologically active immunoregulatory sites (not associated with canonical anti-protease activity) on the surface of hAAT were identified by in silico methods. Several peptides were derived from those immunoregulatory sites. Four peptides exhibited impressive biological effects in pharmacological concentration ranges. Peptidomimetic (14) was developed, based on the structure of the most druggable and active peptide. The compound exhibited a potent anti-inflammatory activity in vitro and in vivo. Such a compound could be used as a basis for developing novel anti-inflammatory drug candidates and as a research tool for better understanding hAAT functions.

Design, Synthesis, and Biological Evaluation of Peptidomimetic Aldehydes as Broad-Spectrum Inhibitors against Enterovirus and SARS-CoV-2.

A novel series of peptidomimetic aldehydes was designed and synthesized to target 3C protease (3Cpro) of enterovirus 71 (EV71). Most of the compounds exhibited high antiviral activity, and among them, compound 18p demonstrated potent enzyme inhibitory activity and broad-spectrum antiviral activity on a panel of enteroviruses and rhinoviruses. The crystal structure of EV71 3Cpro in complex with 18p determined at a resolution of 1.2 Å revealed that 18p covalently linked to the catalytic Cys147 with an aldehyde group. In addition, these compounds also exhibited good inhibitory activity against the 3CLpro and the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially compound 18p (IC50 = 0.034 µM, EC50 = 0.29 µM). According to our previous work, these compounds have no reasons for concern regarding acute toxicity. Compared with AG7088, compound 18p also exhibited good pharmacokinetic properties and more potent anticoronavirus activity, making it an excellent lead for further development.

3CL Protease Inhibitors with an Electrophilic Arylketone Moiety as Anti-SARS-CoV-2 Agents.

The novel coronavirus, SARS-CoV-2, has been identified as the causative agent for the current coronavirus disease (COVID-19) pandemic. 3CL protease (3CLpro) plays a pivotal role in the processing of viral polyproteins. We report peptidomimetic compounds with a unique benzothiazolyl ketone as a warhead group, which display potent activity against SARS-CoV-2 3CLpro. The most potent inhibitor YH-53 can strongly block the SARS-CoV-2 replication. X-ray structural analysis revealed that YH-53 establishes multiple hydrogen bond interactions with backbone amino acids and a covalent bond with the active site of 3CLpro. Further results from computational and experimental studies, including an in vitro absorption, distribution, metabolism, and excretion profile, in vivo pharmacokinetics, and metabolic analysis of YH-53 suggest that it has a high potential as a lead candidate to compete with COVID-19.

In Search of Small Molecules That Selectively Inhibit MBOAT4.

Ghrelin is a 28-residue peptide hormone produced by stomach P/D1 cells located in oxyntic glands of the fundus mucosa. Post-translational octanoylation of its Ser-3 residue, catalyzed by MBOAT4 (aka ghrelin O-acyl transferase (GOAT)), is essential for the binding of the hormone to its receptor in target tissues. Physiological roles of acyl ghrelin include the regulation of food intake, growth hormone secretion from the pituitary, and inhibition of insulin secretion from the pancreas. Here, we describe a medicinal chemistry campaign that led to the identification of small lipopeptidomimetics that inhibit GOAT in vitro. These molecules compete directly for substrate binding. We further describe the synthesis of heterocyclic inhibitors that compete at the acyl coenzyme A binding site.

Peptidomimetic analogues of an Arg-Trp-x-x-Trp motif responsible for interaction of translocase MraY with bacteriophage ϕX174 lysis protein E.

Translocase MraY is the target for bacteriophage ϕX174 lysis protein E, which interacts via a protein-protein interaction mediated by Phe-288 and Glu-287 of E. coli MraY, and an Arg-Trp-x-x-Trp motif on protein E, also found in several cationic antimicrobial peptides. Analogues of Arg-Trp-octyl ester, found previously to show antimicrobial activity, were tested for antimicrobial activity, with Lys-Trp-oct (MIC50P. fluorescens 5 µg/mL) and Arg-Trp-decyl ester (MIC50P. fluorescens 3 µg/mL) showing enhanced antimicrobial activity. Synthesis and testing of α-helix peptidomimetic analogues for this motif revealed improved antibacterial activity (MIC50E. coli 4-7 µg/mL) for analogues containing two aromatic substituents, mimicking the Arg-Trp-x-x-Trp motif, and MraY inhibition (IC50 140 µM) by one such peptidomimetic. Investigation of mechanism of action using the Alamar Blue membrane permeabilisation assay revealed bacteriostatic and bacteriocidal mechanisms in different members of this set of compounds, raising the possibility of more than one biological target. The observed antimicrobial activity and MraY inhibition shown by peptidomimetic compounds confirms that this site could be targeted by drug-like molecules.

Histamine H3 receptor antagonists with peptidomimetic (keto)piperazine structures to inhibit Aß oligomerisation.

Alzheimers disease (AD) is the most prominent neurodegenerative disorder with high medical need. Protein-protein-interactions (PPI) interactions have a critical role in AD where ß-amyloid structures (Aß) build toxic oligomers. Design of disease modifying multi target directed ligand (MTDL) has been performed, which disable PPI on the one hand and on the other hand, act as procognitive antagonists at the histamine H3 receptor (H3R). The synthetized compounds are structurally based on peptidomimetic amino acid-like structures mainly as keto, diketo-, or acyl variations of a piperazine moiety connected to an H3R pharmacophore. Most of them showed low nanomolar affinities at H3R and some with promising affinity to Aß-monomers. The structure-activity relationships (SAR) described offer new possibilities for MTDL with an optimized profile combining symptomatic and potential causal therapeutic approaches in AD.