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.

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.

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.

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.

Synthesis and biological evaluation of indole-based peptidomimetics as antibacterial agents against Gram-positive bacteria.

The emergence of bacterial multidrug resistance and the lack of new antimicrobial agents urgently demand the discovery and development of novel antibacterials that avoid bacterial resistance. Antimicrobial peptidomimetics represent a promising approach for overcoming antibiotic resistance. Herein we report the synthesis and evaluation of indole-based amphiphilic antimicrobial peptidomimetics, bearing hydrophobic side chains and hydrophilic cationic moieties. Among these derivatives, compound 28 demonstrated potent antimicrobial activity against Gram-positive bacteria, low hemolytic activity and low toxicity towards mammalian cells, as well as good stability in salt conditions. Moreover, compound 28 showed the rapid killing of bacteria via membrane-targeting action without developing bacterial resistance. More importantly, compound 28 displayed high antimicrobial potency against Gram-positive bacteria in a murine model of bacterial keratitis, and was found to be more efficient than vancomycin. Thus, compound 28 had great potential as a promising lead compound for the treatment of Gram-positive bacterial infection.

A novel peptidomimetic therapeutic for selective suppression of lung cancer stem cells over non-stem cancer cells.

Cancers are highly heterogeneous and typically contain a small subset of drug-resisting cells called tumor initiating cells or cancer stem cells (CSCs). CSCs can self-renew, divide asymmetrically, and often cause tumor invasion and metastasis. Therefore, treatments specifically targeting CSCs are critical to improve patient survival. Recently, we identified a highly specific peptidomimetic (peptoid - PCS2) that selectively binds to the CSC subpopulation of lung cancer over the remaining cancer cells (non-CSCs). Subsequently, we identified plectin as the target of PCS2. Plectin is an intracellular structural protein, which is involved in tumor invasion and metastasis when it appears on cell surface. While PCS2 monomer did not display any anti-cancer activity, we designed a series of homo-dimeric versions of PCS2, and identified PCS2D1.2 optimized homo-dimer that displayed highly specific cytotoxicity towards CSCs over non-CSCs. PCS2D1.2 effectively blocked the in vitro colony formation and cell migration, hallmarks of CSCs. Furthermore, PCS2D1.2 reduced the in vivo tumor formation. In both in vitro and in vivo studies, PCS2D1.2 effectively reduced plectin expression and/or plectin-rich CSCs, but had no effect on non-CSCs. Therefore, PCS2D1.2 has the potential to be developed as a highly CSC specific drug candidate, which can be used in combination with current anti-cancer drugs.

Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors.

The dynamic interplay of post-translational modifications (PTMs) in chromatin provides a communication system for the regulation of gene expression. An increasing number of studies have highlighted the role that such crosstalk between PTMs plays in chromatin recognition. In this study, (bio)chemical and structural approaches were applied to specifically probe the impact of acetylation of Lys18 in the histone H3 tail peptide on peptide recognition by the protein methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). Peptidomimetics that recapitulate the transition state of protein arginine N-methyltransferases, were designed based on the H3 peptide wherein the target Arg17 was flanked by either a free or an acetylated lysine. Structural studies with these peptidomimetics and the catalytic domain of CARM1 provide new insights into the binding of the H3 peptide within the enzyme active site. While the co-crystal structures reveal that lysine acetylation results in minor conformational differences for both CARM1 and the H3 peptide, acetylation of Lys18 does lead to additional interactions (Van der Waals and hydrogen bonding) and likely reduces the cost of desolvation upon binding, resulting in increased affinity. Informed by these findings a series of smaller peptidomimetics were also prepared and found to maintain potent and selective CARM1 inhibition. These findings provide new insights both into the mechanism of crosstalk between arginine methylation and lysine acetylation as well as towards the development of peptidomimetic CARM1 inhibitors.

Coupling Interrupted Fischer and Multicomponent Joullié-Ugi to Chase Chemical Diversity: from Batch to Sustainable Flow Synthesis of Peptidomimetics.

The generation of peptidomimetic substructures for medicinal chemistry purposes requires effective and divergent synthetic methods. We present in this work an efficient flow process that allows quick modulation of reagents for Joullié-Ugi multicomponent reaction, using spiroindolenines as core motifs. This sterically hindered imine equivalent could successfully be diversified using various isocyanides and amino acids in generally good space-time yields. A telescoped flow process combining interrupted Fischer reaction for spiroindolenine synthesis and subsequent Joullié-Ugi-type modification resulted in product formation in very good overall yield in less than 2 hours compared to 48 hours required in batch mode. The developed protocol can be seen as a general tool for rapid and facile generation of peptidomimetic compounds. We also showcase preliminary biological assessments for the prepared compounds.

Synthetic and computational efforts towards the development of peptidomimetics and small-molecule SARS-CoV 3CLpro inhibitors.

Severe Acute Respiratory Syndrome (SARS) is a severe febrile respiratory disease caused by the beta genus of human coronavirus, known as SARS-CoV. Last year, 2019-n-CoV (COVID-19) was a global threat for everyone caused by the outbreak of SARS-CoV-2. 3CLpro, chymotrypsin-like protease, is a major cysteine protease that substantially contributes throughout the viral life cycle of SARS-CoV and SARS-CoV-2. It is a prospective target for the development of SARS-CoV inhibitors by applying a repurposing strategy. This review focuses on a detailed overview of the chemical synthesis and computational chemistry perspectives of peptidomimetic inhibitors (PIs) and small-molecule inhibitors (SMIs) targeting viral proteinase discovered from 2004 to 2020. The PIs and SMIs are one of the primary therapeutic inventions for SARS-CoV. The journey of different analogues towards the evolution of SARS-CoV 3CLpro inhibitors and complete synthetic preparation of nineteen derivatives of PIs and ten derivatives of SMIs and their computational chemistry perspectives were reviewed. From each class of derivatives, we have identified and highlighted the most compelling PIs and SMIs for SARS-CoV 3CLpro. The protein-ligand interaction of 29 inhibitors were also studied that involved with the 3CLpro inhibition, and the frequent amino acid residues of the protease were also analyzed that are responsible for the interactions with the inhibitors. This work will provide an initiative to encourage further research for the development of effective and drug-like 3CLpro inhibitors against coronaviruses in the near future.

Discovery of First-in-Class Peptidomimetic Neurolysin Activators Possessing Enhanced Brain Penetration and Stability.

Peptidase neurolysin (Nln) is an enzyme that functions to cleave various neuropeptides. Upregulation of Nln after stroke has identified the enzyme as a critical endogenous cerebroprotective mechanism and validated target for the treatment of ischemic stroke. Overexpression of Nln in a mouse model of stroke results in dramatic improvement of stroke outcomes, while pharmacological inhibition aggravates them. Activation of Nln has therefore emerged as an intriguing target for drug discovery efforts for ischemic stroke. Herein, we report the discovery and hit-to-lead optimization of first-in-class Nln activators based on histidine-containing dipeptide hits identified from a virtual screen. Adopting a peptidomimetic approach provided lead compounds that retain the pharmacophoric histidine moiety and possess single-digit micromolar potency over 40-fold greater than the hit scaffolds. These compounds exhibit 5-fold increased brain penetration, significant selectivity over highly homologous peptidases, greater than 65-fold increase in mouse brain stability, and 'drug-like' fraction unbound in the brain.

Rhodaelectro-catalyzed access to chromones via formyl C-H activation towards peptide electro-labeling.

Chromones represent a privileged scaffold in medicinal chemistry and are an omnipresent structural motif in natural products. Chemically encoded non-natural peptidomimetics feature improved stability towards enzymatic degradation, cell permeability and binding affinity, translating into a considerable impact on pharmaceutical industry. Herein, a strategy for the sustainable assembly of chromones via electro-formyl C-H activation is presented. The rational design of the rhodaelectro-catalysis is guided by detailed mechanistic insights and provides versatile access to tyrosine-based fluorogenic peptidomimetics.

Macrocyclic Tetramers-Structural Investigation of Peptide-Peptoid Hybrids.

Outstanding affinity and specificity are the main characteristics of peptides, rendering them interesting compounds for basic and medicinal research. However, their biological applicability is limited due to fast proteolytic degradation. The use of mimetic peptoids overcomes this disadvantage, though they lack stereochemical information at the α-carbon. Hybrids composed of amino acids and peptoid monomers combine the unique properties of both parent classes. Rigidification of the backbone increases the affinity towards various targets. However, only little is known about the spatial structure of such constrained hybrids. The determination of the three-dimensional structure is a key step for the identification of new targets as well as the rational design of bioactive compounds. Herein, we report the synthesis and the structural elucidation of novel tetrameric macrocycles. Measurements were taken in solid and solution states with the help of X-ray scattering and NMR spectroscopy. The investigations made will help to find diverse applications for this new, promising compound class.

Peptide/ß-Peptoid Hybrids with Ultrashort PEG-Like Moieties: Effects on Hydrophobicity, Antibacterial Activity and Hemolytic Properties.

PEGylation of antimicrobial peptides as a shielding tool that increases stability toward proteolytic degradation typically leads to concomitant loss of activity, whereas incorporation of ultrashort PEG-like amino acids (sPEGs) remains essentially unexplored. Here, modification of a peptide/ß-peptoid hybrid with sPEGs was examined with respect to influence on hydrophobicity, antibacterial activity and effect on viability of mammalian cells for a set of 18 oligomers. Intriguingly, the degree of sPEG modification did not significantly affect hydrophobicity as measured by retention in reverse-phase HPLC. Antibacterial activity against both wild-type and drug-resistant strains of Escherichia coli and Acinetobacter baumannii (both Gram-negative pathogens) was retained or slightly improved (MICs in the range 2-16 µg/mL equal to 0.7-5.2 µM). All compounds in the series exhibited less than 10% hemolysis at 400 µg/mL. While the number of sPEG moieties appeared not to be clearly correlated with hemolytic activity, a trend toward slightly increased hemolytic activity was observed for analogues displaying the longest sPEGs. In contrast, within a subseries the viability of HepG2 liver cells was least affected by analogues displaying the longer sPEGs (with IC50 values of ~1280 µg/mL) as compared to most other analogues and the parent peptidomimetic (IC50 values in the range 330-800 µg/mL).

Design, synthesis and pharmacological screening of novel renoprotective methionine-based peptidomimetics: Amelioration of cisplatin-induced nephrotoxicity.

Cisplatin (CP) is an effective chemotherapeutic agent for treatment of various types of cancer, however efforts are needed to reduce its toxic side effect. Previous studies revealed promising effect of peptides in decreasing CP induced nephrotoxicity. Herein, novel Met-based peptidomimetics were synthesized using N-acylbenzotriazole as acylating agent in high yield. Evaluation of renoprotective effect of the synthesized targets on CP treated kidney cell line (LLC-PK1) revealed that pretreatment with 1/3 IC50 of targets II, IIIa-g attenuated CP induced cell death where the IC50 of CP was raised from 3.28 µM to 9.25-41.1 µM. The most potent compounds IIIg, II and IIIb exhibited antioxidative stress in CP-treated LLC-PK1 cells as confirmed by raising GSH/GSSG ratio and SOD concentration as well as decreasing ROS and MDA. Additionally, in vivo experiments using Sprague Dawley rats showed renoprotective effect of IIIg against CP-induced nephrotoxicity as evidenced by improved results of renal function tests and attenuated CP-induced renal structural injury. Moreover, antioxidant activity of IIIg was demonstrated via its ability to reduce renal MDA level and up-regulate renal antioxidant element GSH level. Further, immunohistochemistry of renal specimens showed the ability of IIIg to restore CP-induced suppression of Nrf2. Interestingly, in vivo and in vitro studies demonstrated that IIIg had no effect on CP antiproliferative activity. An assessment of the ADMET properties revealed that targets IIIg, II and IIIb showed good drug-likeness in terms of their physicochemical, pharmacokinetic properties. The findings presented here showcase that IIIg is a promising renoprotective candidate with antioxidative stress potential.

γ-AApeptides as a New Class of Peptidomimetics: Design, Synthesis, and Applications.

Peptidomimetics are studied for medicinal application because of their ability to mimic hierarchical structures of peptides and proteins. To break the limitation and expand the peptidomimetics family, a new class of peptidomimetics based on peptide nucleic acids (PNAs) backbone - "γ-AApeptides" was developed. Compared with previous peptidomimetics, γ-AApeptides possess prominent advantages such as resistance to proteolytic degradation, enhanced chemodiversity, good selectivity and outstanding bioactivity. The synthesis of γ-AApeptides is carried out using a ''monomer building block'' strategy which is facile and efficient. γ-AApeptides are able to mimic primary and secondary structures of therapeutic peptides, which make them promising candidates for molecular probes and potential drug leads. In the past decade, several interesting structures and applications of γ-AApeptides have been developed by different approaches such as structure-based design, combinatorial library screening, and peptides selfassembly and folding. By following the mechanism of host-defense peptides (HDPs), antibiotic γ- AApeptides showed broad-spectrum activity. At the same time, γ-AApeptides can be used for combinatorial library screening because of their structural stability and their chemodiversity. Anticancer agents, anti-T2DM (Type 2 diabetes mellitus) agents, anti-HIV (human immuno-deficiency virus) agents and anti-Alzheimer's disease agents were developed by combinatorial screening and rational design. Furthermore, γ-AApeptides as biopolymers, nanomaterials, supramolecular structures and self-assembly architectures were studied due to their unique backbone structures. Therefore, γ-AApeptides may play an important role in the development of peptidomimetics.