Programmed cell death-1 is involved with peripheral blood immune cell profiles in patients with hepatitis C virus antiviral therapy.

Mutations in the non-structural protein regions of hepatitis C virus (HCV) are a cause of a non-sustained virological response (SVR) to treatment with direct-acting antivirals (DAAs) for chronic hepatitis; however, there are non-SVR cases without these mutations. In this study, we examined immune cell profiles in peripheral blood before and after ombitasvir/paritaprevir/ritonavir treatment and screened for genes that could be used to predict the therapeutic effects of DAAs. Fluorescence-activated cell sorting analysis indicated that the median frequencies of programmed cell death-1-positive (PD-1+) effector regulatory T cells (eTregs), PD-1+CD8+ T cells, and PD-1+Helper T cells were decreased significantly in SVR cases, but without significant changes in non-SVR cases. The frequency of PD-1+ naïve Tregs was significantly higher in the SVR group than in the non-SVR group before and after treatment. Similar results were found in patients treated with other DAAs (e.g., daclatasvir plus asunaprevir) and supported an immune response after HCV therapy. RNA-sequencing analysis indicated a significant increase in the expression of genes associated with the immune response in the SVR group, while genes related to intracellular and extracellular signal transduction were highly expressed in the non-SVR group. Therefore, we searched for genes associated with PD-1+ eTregs and CD8+ T cells that were significantly different between the SVR and non-SVR groups and found that T-box transcription factor 21 was associated with the non-SVR state. These results indicate that PD-1-related signaling pathways are associated with a non-SVR mechanism after DAAs treatment separate from mutation-related drug resistance.

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.

Macrocyclization strategy for improving candidate profiles in medicinal chemistry.

Macrocycles are defined as cyclic compounds with 12 or more members. In medicinal chemistry, they are categorized based on their core chemistry into cyclic peptides and macrocycles. Macrocycles are advantageous because of their structural diversity and ability to achieve high affinity and selectivity towards challenging targets that are often not addressable by conventional small molecules. The potential of macrocyclization to optimize drug-like properties while maintaining adequate bioavailability and permeability has been emphasized as a key innovation in medicinal chemistry. This review provides a detailed case study of the application of macrocyclization over the past 5 years, starting from the initial analysis of acyclic active compounds to optimization of the resulting macrocycles for improved efficacy and drug-like properties. Additionally, it illustrates the strategic value of macrocyclization in contemporary drug discovery efforts.

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.

Modular Biomimetic Strategy Enables Discovery and SAR Exploration of Oxime Macrocycles as Influenza A Virus (H1N1) Inhibitors.

Although vaccination remains the prevalent prophylactic means for controlling Influenza A virus (IAV) infections, novel structural antivirus small-molecule drugs with new mechanisms of action for treating IAV are highly desirable. Herein, we describe a modular biomimetic strategy to expeditiously achieve a new class of macrocycles featuring oxime, which might target the hemagglutinin (HA)-mediated IAV entry into the host cells. SAR analysis revealed that the size and linker of the macrocycles play an important role in improving potency. Particularly, as a 14-membered macrocyclic oxime, 37 exhibited potent inhibitory activity against IAV H1N1 with an EC50 value of 23 nM and low cytotoxicity, which alleviated cytopathic effects and protected cell survival obviously after H1N1 infection. Furthermore, 37 showed significant synergistic activity with neuraminidase inhibitor oseltamivir in vitro.

Synthesis, structural, multitargeted molecular docking analysis of anti-cancer, anti-tubercular, DNA interactions of benzotriazole based macrocyclic ligand.

Biologically important macromolecule 1, 1', 3, 3' Bis - [2,3,5,6-Tetramethyl-p-phenylenebis(methylene)] dibenzotriazlinium dibromide hydrate (BTD) was synthesized and characterized using FT-IR, NMR and single-crystal XRD (SCXRD). SCXRD revealed that the compound was crystallized as a monoclinic system and associated through weak intermolecular interactions like H-bonding and π- π stacking interactions. These weak intermolecular interactions in BTD were studied using Crystal Explorer and Gaussian. The calculated energies for the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) showed the stability and reactivity of the title compound. Molecular electrostatic potential (MEP) surface analysis was used to investigate the crystal's nucleophilic and electrophilic reactive sites. The molecular shape and intermolecular interactions in the crystal structure were determined using Hirshfeld surface analysis and fingerprint plots. Anticancer, anti-bacterial and DNA binding ability of BTD were investigated by experimental and theoretical techniques. The obtained results suggest that BTD possesses better anti-cancer, anti-bacterial and DNA binding abilities. The mode of action of antibiotic and anticancer approach was discussed. This provides promising therapeutic advantages for further development.

Brain-Permeable Immunoproteasome-Targeting Macrocyclic Peptide Epoxyketones for Alzheimer's Disease.

Previously, we demonstrated that linear peptide epoxyketones targeting the immunoproteasome (iP) could ameliorate cognitive deficits in mouse models of Alzheimer's disease (AD) independently of amyloid deposition. We also reported the first iP-targeting macrocyclic peptide epoxyketones, which exhibit improved metabolic stability compared with their linear counterparts. Here, we prepared additional macrocyclic peptide epoxyketones and compared them with existing macrocyclic iP inhibitors by assessing Caco2 cell-based permeability and microsomal stability, providing the four best macrocyclic iP inhibitors. We then evaluated the four compounds using the Ames test and the potency assays in BV2 cells, selecting compound 5 as our AD drug lead. When 5 was administered intravenously (40 mg/kg) or orally (150 mg/kg) into healthy BALB/c mice, we observed considerable iP inhibition in the mouse brain, indicating good blood-brain barrier permeability and target engagement. Combined results suggest that 5 is a promising AD drug lead that may need further investigation.

Synthesis of calix (4) resorcinarene based amphiphilic macrocycle as an efficient nanocarrier for Amphotericin-B to enhance its oral bioavailability.

The supramolecular-based macrocyclic amphiphiles have fascinating attention and find extensive utilization in the pharmaceutical industry for efficient drug delivery. In this study, we designed and synthesized a new supramolecular amphiphilic macrocycle to serve as an efficient nanocarrier, achieved by treating 4-hydroxybenzaldehyde with 1-bromotetradecane. The derivatized product was subsequently treated with resorcinol to cyclize, resulting in the formation of a calix(4)-resorcinarene-based supramolecular amphiphilic macrocycle. The synthesized macrocycle and intermediate products were characterized using mass spectrometry, IR, and 1H NMR spectroscopic techniques. The amphotericin-B (Amph-B)-loaded and unloaded amphiphiles were screened for biocompatibility studies, vesicle formation, particle shape, size, surface charge, drug entrapment, in-vitro release profile, and stability through atomic force microscopy (AFM), Zetasizer, HPLC, and FT-IR. Amph-B -loaded macrocycle-based niosomal vesicles were investigated for in-vivo bioavailability in rabbits. The synthesized macrocycle exhibited no cytotoxicity against normal mouse fibroblast cells and was found to be hemocompatible and safe in mice following an acute toxicity study. The drug-loaded macrocycle-based vesicles appeared spherical, nano-sized, and homogeneous in size, with a notable negative surface charge. The vesicles remained stable after 30 days of storage. The results of Amph-B oral bioavailability and pharmacokinetics revealed that the newly tailored niosomal formulation enhanced drug solubility, protected drug degradation at gastric pH, facilitated sustained drug release at the specific target site, and delayed plasma drug clearance. Incorporating such advanced niosomal formulations in the field of drug delivery systems has the potential to revolutionize therapeutic outcomes and improve the quality of patient well-being.

Anti-Neuroinflammatory Effects of a Macrocyclic Peptide-Peptoid Hybrid in Lipopolysaccharide-Stimulated BV2 Microglial Cells.

Inflammation processes of the central nervous system (CNS) play a vital role in the pathogenesis of several neurological and psychiatric disorders like depression. These processes are characterized by the activation of glia cells, such as microglia. Clinical studies showed a decrease in symptoms associated with the mentioned diseases after the treatment with anti-inflammatory drugs. Therefore, the investigation of novel anti-inflammatory drugs could hold substantial potential in the treatment of disorders with a neuroinflammatory background. In this in vitro study, we report the anti-inflammatory effects of a novel hexacyclic peptide-peptoid hybrid in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The macrocyclic compound X15856 significantly suppressed Interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), c-c motif chemokine ligand 2 (CCL2), CCL3, C-X-C motif chemokine ligand 2 (CXCL2), and CXCL10 expression and release in LPS-treated BV2 microglial cells. The anti-inflammatory effects of the compound are partially explained by the modulation of the phosphorylation of p38 mitogen-activated protein kinases (MAPK), p42/44 MAPK (ERK 1/2), protein kinase C (PKC), and the nuclear factor (NF)-κB, respectively. Due to its remarkable anti-inflammatory properties, this compound emerges as an encouraging option for additional research and potential utilization in disorders influenced by inflammation, such as depression.

Design and Synthesis of Novel Macrocyclic Derivatives as Potent and Selective Cyclin-Dependent Kinase 7 Inhibitors.

The duality of function (cell cycle regulation and gene transcription) of cyclin-dependent kinase 7 (CDK7) makes it an attractive oncology target and the discovery of CDK7 inhibitors has been a long-term pursuit by academia and pharmaceutical companies. However, achieving selective leading compounds is still difficult owing to the similarities among the ATP binding pocket. Herein, we detail the design and synthesis of a series of macrocyclic derivatives with pyrazolo[1,5-a]-1,3,5-triazine core structure as potent and selective CDK7 inhibitors. The diverse manners of macrocyclization led to distinguished selectivity profiles of the CDK family. Molecular dynamics (MD) simulation explained the binding difference between 15- and 16-membered macrocyclic compounds. Further optimization generated compound 37 exhibiting good CDK7 inhibitory activity and high selectivity over other CDKs. This work clearly demonstrated macrocyclization is a versatile method to finely tune the selectivity profile of small molecules and MD simulation can be a valuable tool in prioritizing designs of the macrocycle.

The Cyclic Imine Core Common to the Marine Macrocyclic Toxins Is Sufficient to Dictate Nicotinic Acetylcholine Receptor Antagonism.

Macrocyclic imine phycotoxins are an emerging class of chemical compounds associated with harmful algal blooms and shellfish toxicity. Earlier binding and electrophysiology experiments on nAChR subtypes and their soluble AChBP surrogates evidenced common trends for substantial antagonism, binding affinities, and receptor-subtype selectivity. Earlier, complementary crystal structures of AChBP complexes showed that common determinants within the binding nest at each subunit interface confer high-affinity toxin binding, while distinctive determinants from the flexible loop C, and either capping the nest or extending toward peripheral subsites, dictate broad versus narrow receptor subtype selectivity. From these data, small spiroimine enantiomers mimicking the functional core motif of phycotoxins were chemically synthesized and characterized. Voltage-clamp analyses involving three nAChR subtypes revealed preserved antagonism for both enantiomers, despite lower subtype specificity and binding affinities associated with faster reversibility compared with their macrocyclic relatives. Binding and structural analyses involving two AChBPs pointed to modest affinities and positional variability of the spiroimines, along with a range of AChBP loop-C conformations denoting a prevalence of antagonistic properties. These data highlight the major contribution of the spiroimine core to binding within the nAChR nest and confirm the need for an extended interaction network as established by the macrocyclic toxins to define high affinities and marked subtype specificity. This study identifies a minimal set of functional pharmacophores and binding determinants as templates for designing new antagonists targeting disease-associated nAChR subtypes.

Concurrent Optimizations of Efficacy and Blood-Brain Barrier Permeability in New Macrocyclic LRRK2 Inhibitors for Potential Parkinson's Disease Therapeutics.

The elevated activity of leucine-rich repeat kinase 2 (LRRK2) is implicated in the pathogenesis of Parkinson's disease (PD). The quest for effective LRRK2 inhibitors has been impeded by the formidable challenge of crossing the blood-brain barrier (BBB). We leveraged structure-based de novo design and developed robust three-dimensional quantitative structure-activity relationship (3D-QSAR) models to predict BBB permeability, enhancing the likelihood of the inhibitor's brain accessibility. Our strategy involved the synthesis of macrocyclic molecules by linking the two terminal nitrogen atoms of HG-10-102-01 with an alkyl chain ranging from 2 to 4 units, laying the groundwork for innovative LRRK2 inhibitor designs. Through meticulous computational and synthetic optimization of both biochemical efficacy and BBB permeability, 9 out of 14 synthesized candidates demonstrated potent low-nanomolar inhibition and significant BBB penetration. Further assessments of in vitro and in vivo effectiveness, coupled with pharmacological profiling, highlighted 8 as the promising new lead compound for PD therapeutics.

Bioactive peptide relieves glucocorticoid-induced osteoporosis by giant macrocyclic encapsulation.

Bioactive peptides play a crucial role in the field of regenerative medicine and tissue engineering. However, their application in vivo and clinic is hindered by their poor stability, short half-life, and low retention rate. Herein, we propose a novel strategy for encapsulating bioactive peptides using giant macrocycles. Platelet-derived growth factor (PDGF) bioactive mimicking peptide Nap-FFGVRKKP (P) was selected as the representative of a bioactive peptide. Quaterphen[4]arene (4) exhibited extensive host-guest complexation with P, and the binding constant was (1.16 ± 0.10) × 107 M-1. In vitro cell experiments confirmed that P + 4 could promote the proliferation of BMSCs by 2.27 times. Even with the addition of the inhibitor dexamethasone (Dex), P + 4 was still able to save 76.94% of the cells in the control group. Compared to the Dex group, the bone mass of the mice with osteoporosis in the P + 4 group was significantly increased. The mean trabecular thickness (Tb.Th) increased by 17.03%, and the trabecular bone volume fraction (BV/TV) values increased by 40.55%. This supramolecular bioactive peptide delivery strategy provides a general approach for delivering bioactive peptides and opens up new opportunities for the development of peptide-based drugs.

Structural Analysis of the Macrocyclic Inhibitor BI-4020 Binding to EGFR Kinase.

A novel macrocyclic inhibitor of mutant EGFR (BI-4020) has shown promise in pre-clinical studies of T790M and C797S drug-resistant non-small cell lung cancer. To better understand the molecular basis for BI-4020 selectivity and potency, we have carried out biochemical activity assays and structural analysis with X-ray crystallography. Biochemical potencies agree with previous studies indicating that BI-4020 is uniquely potent against drug-resistant L858R/T790M and L858R/T790M/C797S variants. X-ray structures with wild-type (2.4 Å) and T790M/V948R (3.1 Å) EGFR kinase domains show that BI-4020 is likely rendered selective due to interactions with the kinase domain hinge region as well as T790M, akin to Osimertinib. Additionally, BI-4020 is also rendered more potent due to its constrained macrocycle geometry as well as additional H-bonds to conserved K745 and T845 residues in both active and inactive conformations. These findings taken together show how this novel macrocyclic inhibitor is both highly potent and selective for mutant EGFR in a reversible mechanism and motivate structure-inspired approaches to developing targeted therapies in medicinal oncology.

Design, synthesis, characterization, in vitro cytotoxic, antimicrobial, antioxidant studies, DFT, thermal and molecular docking evaluation of biocompatible Co(II) complexes of N4O4-macrocyclic ligands.

Bioactive cobalt (II) macrocyclic complexes [Co(N4O4ML1)Cl2]-[Co(N4O4ML3)Cl2] have been synthesized by using the macrocyclic ligands [N4O4ML1], [N4O4ML2], and [N4O4ML3] that have an N4O4 core. These three macrocyclic ligands were all isolated in pure form, together with their complexes. Microanalytical investigations, FT-IR NMR, Mass, magnetic moments, electronic, PXRD, TGA, and EPR spectrum studies were used to analyse their structures. For these complexes, an octahedral geometry is proposed for the metal ion. By using molecular weights and conductivity measurements the monomeric and non-electrolytic nature has been confirmed. The Coats-Redfern and FWO methods are used to determine the thermodynamic characteristics of the ligands and their Co(II) complexes. The molecular modelling using the DFT technique displays the bond angle, bond lengths and quantum chemical properties. To determine their ability to prevent the growth of harmful fungus and bacteria, the ligands [N4O4ML1]- [N4O4ML3] and their complexes were tested in vitro against A. Niger, C. albicans and B. subtilis, S. aureus, E. coli and S. typhi fungal and bacterial organisms, respectively. By using DPPH free radical scavenger assays, the in vitro antioxidant capabilities of each compound were evaluated. The [Co(N4O4ML3)Cl2] antioxidative capabilities revealed significant radical scavenging power. The MTT assay was used to assess the toxicity of all the synthesised compounds under inquiry on MCF-7, HeLa, and A549 cancer cells. The findings revealed that the ligand and the compounds gave outstanding IC50 values in the range of 9.07-36.25 (uM) at a concentration of 25 ppm. Among all the substances evaluated, [Co(N4O4ML3)Cl2] complex was discovered to be the most active and least cytotoxic. Additionally, docking investigations of the produced compounds were carried out in order to validate the biological outcomes.

Macrocycles and macrocyclization in anticancer drug discovery: Important pieces of the puzzle.

Increasing disease-related proteins have been identified as novel therapeutic targets. Macrocycles are emerging as potential solutions, bridging the gap between conventional small molecules and biomacromolecules in drug discovery. Inspired by successful macrocyclic drugs of natural origins, macrocycles are attracting more attention for enhanced binding affinity and target selectivity. Due to the conformation constraint and structure preorganization, macrocycles can reach bioactive conformations more easily than parent acyclic compounds. Also, rational macrocyclization combined with sequent structural modification will help improve oral bioavailability and combat drug resistance. This review introduces various strategies to enhance membrane permeability in macrocyclization and subsequent modification, such as N-methylation, intramolecular hydrogen bonding modulation, isomerization, and reversible bicyclization. Several case studies highlight macrocyclic inhibitors targeting kinases, HDAC, and protein-protein interactions. Finally, some macrocyclic agents targeting tumor microenvironments are illustrated.

Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED.

Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus (HCV) infection. Its structures have been elusive to the public until recently when one of the crystal forms is solved by microcrystal electron diffraction (MicroED). In this work, the MicroED structures of two distinct polymorphic crystal forms of paritaprevir are reported from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropyl sulfonamide and methyl pyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the HCV non-structural 3/4A (NS3/4A) serine protease target, and can interact with the pocket and catalytic triad via hydrophobic interactions and hydrogen bonds. These results can provide further insight for optimization of the binding of acyl sulfonamide inhibitors to the HCV NS3/4A serine protease. In addition, this also demonstrates the opportunity to derive different polymorphs and distinct macrocycle conformations from the same experiments using MicroED.

The macrocycle inhibitor landscape of SLC-transporter.

In the past years the interest in Solute Carrier Transporters (SLC) has increased due to their potential as drug targets. At the same time, macrocycles demonstrated promising activities as therapeutic agents. However, the overall macrocycle/SLC-transporter interaction landscape has not been fully revealed yet. In this study, we present a statistical analysis of macrocycles with measured activity against SLC-transporter. Using a data mining pipeline based on KNIME retrieved in total 825 bioactivity data points of macrocycles interacting with SLC-transporter. For further analysis of the SLC inhibitor profiles we developed an interactive KNIME workflow as well as an interactive map of the chemical space coverage utilizing parametric t-SNE models. The parametric t-SNE models provide a good discrimination ability among several corresponding SLC subfamilies' targets. The KNIME workflow, the dataset, and the visualization tool are freely available to the community.

Discovery of Selective and Potent Macrocyclic CDK9 Inhibitors for the Treatment of Osimertinib-Resistant Non-Small-Cell Lung Cancer.

Effectiveness of epidermal growth factor receptor (EGFR) inhibitors, including Osimertinib, for treating non-small-cell lung cancer (NSCLC) is limited due to the continuous emergence of drug resistance. Hence, it is urgent to develop new therapeutic approaches. CDK9, a key regulator of RNA transcription, has emerged as a promising target for the development of antitumor drugs due to its crucial role in modulating the levels of antiapoptotic protein Mcl-1. Herein, we present the synthesis, optimization, and evaluation of selective CDK9 inhibitors with a macrocyclic scaffold that effectively suppresses the growth of NSCLC cells. Notably, compound Z11, a potent CDK9 inhibitor (IC50 = 3.20 nM) with good kinase selectivity, significantly inhibits cell proliferation and colony formation and induces apoptosis in Osimertinib-resistant H1975 cells. Furthermore, Z11 demonstrates a significant suppression of tumor growth in six patient-derived organoids, including three organoids resistant to Osimertinib. Overall, Z11 served as a promising macrocycle-based CDK9 inhibitor for treating Osimertinib-resistant NSCLC.