Statine-based peptidomimetic compounds as inhibitors for SARS-CoV-2 main protease (SARS-CoV­2 Mpro).

COVID-19 is a multisystemic disease caused by the SARS-CoV-2 airborne virus, a member of the Coronaviridae family. It has a positive sense single-stranded RNA genome and encodes two non-structural proteins through viral cysteine-proteases processing. Blocking this step is crucial to control virus replication. In this work, we reported the synthesis of 23 statine-based peptidomimetics to determine their ability to inhibit the main protease (Mpro) activity of SARS-CoV-2. Among the 23 peptidomimetics, 15 compounds effectively inhibited Mpro activity by 50% or more, while three compounds (7d, 8e, and 9g) exhibited maximum inhibition above 70% and IC50 < 1 µM. Compounds 7d, 8e, and 9g inhibited roughly 80% of SARS-CoV-2 replication and proved no cytotoxicity. Molecular docking simulations show putative hydrogen bond and hydrophobic interactions between specific amino acids and these inhibitors. Molecular dynamics simulations further confirmed the stability and persisting interactions in Mpro's subsites, exhibiting favorable free energy binding (ΔGbind) values. These findings suggest the statine-based peptidomimetics as potential therapeutic agents against SARS-CoV-2 by targeting Mpro.

Synthesis and biological evaluation of novel peptidomimetic inhibitors of the coronavirus 3C-like protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and related variants, are responsible for the devastating coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 main protease (Mpro) plays a central role in the replication of the virus and represents an attractive drug target. Herein, we report the discovery of novel SARS-CoV-2 Mpro covalent inhibitors, including highly effective compound NIP-22c which displays high potency against several key variants and clinically relevant nirmatrelvir Mpro E166V mutants.

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

Peptidomimetics as potent dual SARS-CoV-2 cathepsin-L and main protease inhibitors: In silico design, synthesis and pharmacological characterization.

In this paper we present the design, synthesis, and biological evaluation of a new series of peptidomimetics acting as potent anti-SARS-CoV-2 agents. Starting from our previously described Main Protease (MPro) and Papain Like Protease (PLPro) dual inhibitor, CV11, here we disclose its high inhibitory activity against cathepsin L (CTSL) (IC50 = 19.80 ± 4.44 nM), an emerging target in SARS-CoV-2 infection machinery. An in silico design, inspired by the structure of CV11, led to the development of a library of peptidomimetics showing interesting activities against CTSL and Mpro, allowing us to trace the chemical requirements for the binding to both enzymes. The screening in Vero cells infected with 5 different SARS-CoV-2 variants of concerns, highlighted sub-micromolar activities for most of the synthesized compounds (13, 15, 16, 17 and 31) in agreement with the enzymatic inhibition assays results. The compounds showed lack of activity against several different RNA viruses except for the 229E and OC43 human coronavirus strains, also characterized by a cathepsin-L dependent release into the host cells. The most promising derivatives were also evaluated for their chemical and metabolic in-vitro stability, with derivatives 15 and 17 showing a suitable profile for further preclinical characterization.

Molecular insights into the dynamic modulation of bacterial ClpP function and oligomerization by peptidomimetic boronate compounds.

Bacterial caseinolytic protease P subunit (ClpP) is important and vital for cell survival and infectivity. Recent publications describe and discuss the complex structure-function relationship of ClpP and its processive activity mediated by 14 catalytic sites. Even so, there are several aspects yet to be further elucidated, such as the paradoxical allosteric modulation of ClpP by peptidomimetic boronates. These compounds bind to all catalytic sites, and in specific conditions, they stimulate a dysregulated degradation of peptides and globular proteins, instead of inhibiting the enzymatic activity, as expected for serine proteases in general. Aiming to explore and explain this paradoxical effect, we solved and refined the crystal structure of native ClpP from Staphylococcus epidermidis (Se), an opportunistic pathogen involved in nosocomial infections, as well as ClpP in complex with ixazomib at 1.90 Å and 2.33 Å resolution, respectively. The interpretation of the crystal structures, in combination with complementary biochemical and biophysical data, shed light on how ixazomib affects the ClpP conformational state and activity. Moreover, SEC-SAXS and DLS measurements show, for the first time, that a peptidomimetic boronate compound also induces the assembly of the tetradecameric structure from isolated homomeric heptameric rings of a gram-positive organism.

Targeted therapy, immunotherapy, and small molecules and peptidomimetics as emerging immunoregulatory agents for melanoma.

Primary cutaneous melanoma is the most lethal of all skin neoplasms and its incidence is increasing. Clinical management of advanced melanoma in the last decade has been revolutionised by the availability of immunotherapies and targeted therapies, used alone and in combination. This article summarizes advances in the treatment of late-stage melanoma including use of protein kinase inhibitors, antibody-based immune checkpoint inhibitors, adoptive immunotherapy, vaccines and more recently, small molecules and peptidomimetics as emerging immunoregulatory agents.

Hepatitis C: The Story of a Long Journey through First, Second, and Third Generation NS3/4A Peptidomimetic Inhibitors. What Did We Learn?

Hepatitis C viral (HCV) infection is the leading cause of liver failure and still represents a global health burden. Over the past decade, great advancements made HCV curable, and sustained viral remission significantly improved to more than 98%. Historical treatment with pegylated interferon alpha and ribavirin has been displaced by combinations of direct-acting antivirals. These regimens include drugs targeting different stages of the HCV life cycle. However, the emergence of viral resistance remains a big concern. The design of peptidomimetic inhibitors (PIs) able to fit and fill the conserved substrate envelope region within the active site helped avoid contact with the vulnerable sites of the most common resistance-associated substitutions Arg155, Ala156, and Asp168. Herein, we give an overview of HCV NS3 PIs discovered during the past decade, and we deeply discuss the rationale behind the structural optimization efforts essential to achieve pangenotypic activity.

Synthetic Peptides and Peptidomimetics: From Basic Science to Biomedical Applications-Second Edition.

Peptides are increasingly emerging as a drug class for a wide range of human diseases due to their intrinsic properties, such as excellent recognition abilities and biocompatibility [...].

Biaryl carboxamide-based peptidomimetics analogs as potential pancreatic lipase inhibitors for treating obesity.

A series of 1,1'-biphenyl-3-carboxamide and furan-phenyl-carboxamide analogs were synthesized using an optimized scheme and confirmed by 1H and 13C nuclear magnetic resonance and high-resolution mass spectrometry techniques. The synthesized peptidomimetics analogs were screened in vitro to understand the inhibitory potential of pancreatic lipase (PL). Analogs were assessed for the PL inhibitory activity based on interactions, geometric complementarity, and docking score. Among the synthesized analogs, 9, 29, and 24 were found to have the most potent PL inhibitory activity with IC50 values of 3.87, 4.95, and 5.34 µM, respectively, compared to that of the standard drug, that is, orlistat, which inhibits PL with an IC50 value of 0.99 µM. The most potent analog, 9, exhibited a competitive-type inhibition with an inhibition constant (Ki) of 2.72 µM. In silico molecular docking of analog 9 with the PL (PDB ID:1LPB) showed a docking score of -11.00 kcal/mol. Analog 9 formed crucial hydrogen bond interaction with Ser152, His263, π-cation interaction with Asp79, Arg256, and π-π stacking with Phe77, Tyr114 at the protein's active site. The molecular dynamic simulation confirmed that analog 9 forms stable interactions with PL at the end of 200 ns with root mean square deviation values of 2.5 and 6 Å. No toxicity was observed for analog 9 (concentration range of 1-20 µM) when tested by MTT assay in RAW 264.7 cells.

Structure-based design of peptidomimetic inhibitors of PSD-95 with improved affinity for the PDZ3 domain.

Aberrant brain-derived neurotrophic factor (BDNF) signaling has been proposed to contribute to the pathophysiology of depression and other neurological disorders such as Angelman syndrome. We have previously shown that targeting the tropomyosin receptor kinase B/postsynaptic density protein-95 (PSD-95) nexus in the BDNF signaling pathway by peptidomimetic inhibitors is a promising approach for therapeutic intervention. Here, we used structure-based knowledge to develop a new Syn3 peptidomimetic compound series that fuses peptides derived from the PSD-95-binding protein SynGAP to our prototype compound CN2097. The new compounds target the PSD-95 PDZ3 domain and adjoining αC helix to achieve bivalent binding that results in up to 7-fold stronger affinity compared to CN2097. These compounds were designed to improve CN2097 specificity for the PSD-95 PDZ3 domain, and structure-activity relationship studies were performed to improve their resistance to proteolysis.

Inhibition of Aurora-A/N-Myc Protein-Protein Interaction Using Peptidomimetics: Understanding the Role of Peptide Cyclization.

Using N-Myc61-89 as a starting template we showcase the systematic use of truncation and maleimide constraining to develop peptidomimetic inhibitors of the N-Myc/Aurora-A protein-protein interaction (PPI); a potential anticancer drug discovery target. The most promising of these - N-Myc73-94-N85C/G89C-mal - is shown to favour a more Aurora-A compliant binding ensemble in comparison to the linear wild-type sequence as observed through fluorescence anisotropy competition assays, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Further in silico investigation of this peptide in its Aurora-A bound state, by molecular dynamics (MD) simulations, imply (i) the bound conformation is more stable as a consequence of the constraint, which likely suppresses dissociation and (ii) the constraint may make further stabilizing interactions with the Aurora-A surface. Taken together this work unveils the first orthosteric N-Myc/Aurora-A inhibitor and provides useful insights on the biophysical properties and thus design of constrained peptides, an attractive therapeutic modality.

Antidiabetic Effect of a New Original NT-3 Dipeptide Mimetic.

It was previously established that the original dipeptide mimetic of the 4th loop of NT-3, hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301), has a pronounced neuroprotective effect in vitro at concentrations of 10-5-10-12 М. In the present study, experiments on the streptozotocin-induced diabetes model in C57Bl/6 mice showed that GTS-301, when administered intraperitoneally for 32 days at doses of 0.1 and 0.5 mg/kg, has antidiabetic activity manifested in a reduction of hyperglycemia and polydipsia and in an increase in animal survival. The results obtained confirm the concept of the similarity of neurochemical mechanisms underlying the regulation of functions of neurons and ß-cells.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Pyrazolidinone-based peptidomimetic SARS-CoV-2 Mpro inhibitors.

The main protease (Mpro) of SARS-CoV-2 is an attractive drug target for COVID-19 treatment as it plays an integral role in the proliferation of coronavirus. Herein, we describe the investigation of ß- and γ-lactams as electrophilic "warheads" for covalent binding to Cys145 of the Mpro active site. The highest inhibitory activity (IC50 = 45 ± 3 µM) was achieved using a pyrazolidinone warhead attached to the targeting dipeptide. Importantly, the synergy of the warhead and the targeting dipeptide is crucial for the successful inhibition of Mpro.

Peptidomimetics based on ammonium decasubstituted pillar[5]arenes: Influence of the alpha-amino acid residue nature on cholinesterase inhibition.

Cholinesterase inhibitors are a group of medicines that are widely used for the treatment of cognitive impairments accompanying Alzheimer's disease as well as for the treatment of pathological muscle weaknesses syndromes such as myasthenia gravis. The search for novel non-toxic and effective cholinesterase inhibitors for creating neuroprotective and neurotransmitter agents is an urgent interdisciplinary problem. For the first time, the application of water-soluble pillar[5]arenes containing amino acid residues as effective cholinesterase inhibitors was shown. The influence of the nature of aliphatic and aromatic alpha-amino acid residues (glycine, l-alanine, l-phenylalanine and l-tryptophan) on self-assembly, aggregate's stability, cytotoxicity on A549 and LEK cells and cholinesterase inhibition was studied. It was found that the studied compounds with aliphatic amino acid residues showed a low inhibitory ability against cholinesterases. It was established that the pillar[5]arene containing fragments of l-phenylalanine is the most promising inhibitor of butyrylcholinesterase (IC50 = 0.32 ± 0.01 µM), the pillar[5]arene with l-tryptophan residues is the most promising inhibitor of acetylcholinesterase (IC50 = 0.32 ± 0.01 µM). This study has shown a possible application of peptidomimetics based on pillar[5]arenes to inhibit cholinesterase, as well as control the binding affinity to a particular enzyme in a structure-dependent manner.

Antimicrobial Peptidomimetics Prevent the Development of Resistance against Gentamicin and Ciprofloxacin in Staphylococcus and Pseudomonas Bacteria.

Bacteria readily acquire resistance to traditional antibiotics, resulting in pan-resistant strains with no available treatment. Antimicrobial resistance is a global challenge and without the development of effective antimicrobials, the foundation of modern medicine is at risk. Combination therapies such as antibiotic-antibiotic and antibiotic-adjuvant combinations are strategies used to combat antibiotic resistance. Current research focuses on antimicrobial peptidomimetics as adjuvant compounds, due to their promising activity against antibiotic-resistant bacteria. Here, for the first time we demonstrate that antibiotic-peptidomimetic combinations mitigate the development of antibiotic resistance in Staphylococcus aureus and Pseudomonas aeruginosa. When ciprofloxacin and gentamicin were passaged individually at sub-inhibitory concentrations for 10 days, the minimum inhibitory concentrations (MICs) increased up to 32-fold and 128-fold for S. aureus and P. aeruginosa, respectively. In contrast, when antibiotics were passaged in combination with peptidomimetics (Melimine, Mel4, RK758), the MICs of both antibiotics and peptidomimetics remained constant, indicating these combinations were able to mitigate the development of antibiotic-resistance. Furthermore, antibiotic-peptidomimetic combinations demonstrated synergistic activity against both Gram-positive and Gram-negative bacteria, reducing the concentration needed for bactericidal activity. This has significant potential clinical applications-including preventing the spread of antibiotic-resistant strains in hospitals and communities, reviving ineffective antibiotics, and lowering the toxicity of antimicrobial chemotherapy.

Synthesis of peptidomimetics as antibiotic adjuvants for combination with aztreonam to combat MDR Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the multipledrug-resistant (MDR) Gram-negative pathogens with few drugs available for treatment. Antibiotic adjuvant approach provides an alternative and complementary strategy. In this study, the stereo-structure-activity relationship of monobactams against MDR Gram-negative organisms was extended. Meanwhile, a series of novel peptidemimetic derivatives as antibiotic adjuvants was synthesized and evaluated for their synergistic effects with aztreonam (AZT) against P. aeruginosa, using dipeptide PAßN as the lead. Among the analogues, compound 22j showed a significant synergistic effect against MDR P. aeruginosa in vitro and in vivo, presumably through the mechanism of affecting the permeability of outer membrane. Thus, we identified 22j as a novel peptidemimetic lead compound to potentiate the activity of AZT against MDR P. aeruginosa, which is worthy of further development as antibiotic adjuvant candidates.

Advancement from Small Peptide Pharmaceuticals to Orally Active Piperazine-2,5-dion-Based Cyclopeptides.

The oral delivery of peptide pharmaceuticals has long been a fundamental challenge in drug development. A new chemical platform was designed based on branched piperazine-2,5-diones for creating orally available biologically active peptidomimetics. The platform includes a bio-carrier with "built-in" functionally active peptide fragments or bioactive molecules that are covalently attached via linkers. The developed platform allows for a small peptide to be taken with a particular biological activity and to be transformed into an orally stable compound displaying the same activity. Based on this approach, various peptidomimetics exhibiting hemostimulating, hemosuppressing, and adjuvant activity were prepared. In addition, new examples of a rare phenomenon when enantiomeric molecules demonstrate reciprocal biological activity are presented. Finally, the review summarizes the evolutionary approach of the short peptide pharmaceutical development from the immunocompetent organ separation to orally active cyclopeptides and peptidomimetics.

Chromone-embedded peptidomimetics and furopyrimidines as highly potent SARS-CoV-2 infection inhibitors: docking and MD simulation study.

BACKGROUND: COVID-19 is a respiratory illness caused by SARS-CoV-2. Pharmaceutical companies aim to control virus spread through effective drugs. This study investigates chromone compound derivatives' ability to inhibit viral entry and prevent replication. METHOD: This study investigated the inhibitory effect of chromone-embedded peptidomimetics and furopyrimidines on 7BZ5 from Severe Acute Respiratory Syndrome CoV-2, Homo sapiens, and 6LU7 from Bat SARS-like CoV using molecular docking. The crystal structure of these proteins was obtained from the Protein Data Bank, and the inhibition site was determined using ligand binding interaction options. The 3D structure was protonated and energetically minimised using MOE software. Chromone derivatives were designed in three dimensions, and their energy was minimised using MOE 2019. The molecular drug-likeness was calculated using SwissADME, Lipinski and Benigni-Bossa's rule, and toxicity was calculated using Toxtree v3.1.0 software. Compounds with pharmacological properties were selected for molecular docking, and interactions were assessed using MOE 2019. MD simulations of Mpro-ch-p complexes were performed to evaluate root mean square fluctuations (RMSF) and measure protein stability. RESULT: The pharmacokinetic tests revealed that chromone derivatives of the peptidomimetic family have acceptable pharmacokinetic activity in the human body. Some compounds, such as Ch-p1, Ch-p2, Ch-p6, Ch-p7, Ch-p12, and Ch-p13, have pronounced medicinal properties. Molecular docking revealed high affinity for binding to SARS-CoV-2 protease. Ch-p7 had the highest binding energy, likely due to its inhibitory property. A 10 ns molecular dynamics study confirmed the stability of the protein-ligand complex, resulting in minimal fluctuations in the system's backbone. The MM-GBSA analysis revealed free energies of binding of - 19.54 kcal/mol. CONCLUSIONS: The study investigated the inhibition of viral replication using chromone derivatives, finding high inhibitory effects in the peptidomimetic family compared to other studies.

Rational Design of Novel Peptidomimetics against Influenza A Virus: Biological and Computational Studies.

Effective therapy against the influenza virus is still an unmet goal. Drugs with antiviral effects exist, but the appearance of resistant viruses pushes towards the discovery of drugs with different mechanisms of action. New anti-influenza molecules should target a good candidate, as a new anti-influenza molecule could be an inhibitor of the influenza A virus hemagglutinin (HA), which plays a key role during the early phases of infection. In previous work, we identified two tetrapeptide sequences, SLDC (1) and SKHS (2), derived from bovine lactoferrin (bLf) C-lobe fragment 418-429, which were able to bind HA and inhibit cell infection at picomolar concentration. Considering the above, the aim of this study was to synthesize a new library of peptidomimetics active against the influenza virus. In order to test their ability to bind HA, we carried out a preliminary screening using biophysical assays such as surface plasmon resonance (SPR) and orthogonal immobilization-free microscale thermophoresis (MST). Biological and computational studies on the most interesting compounds were carried out. The methods applied allowed for the identification of a N-methyl peptide, S(N-Me)LDC, which, through high affinity binding of influenza virus hemagglutinin, was able to inhibit virus-induced hemagglutination and cell infection at picomolar concentration. This small sequence, with high activity, represents a good starting point for the design of new peptidomimetics and small molecules.