Conformational Changes of Anoplin,W-MreB1–9, and (KFF)3K Peptides near the Membranes

Many peptides interact with biological membranes, but elucidating these interactions is challenging because cellular membranes are complex and peptides are structurally flexible. To contribute to understanding how the membrane-active peptides behave near the membranes, we investigated peptide structural changes in different lipid surroundings. We focused on two antimicrobial peptides, anoplin and W-MreB_1–9, and one cell-penetrating peptide, (KFF)₃K. Firstly, by using circular dichroism spectroscopy, we determined the secondary structures of these peptides when interacting with micelles, liposomes, E. coli lipopolysaccharides, and live E. coli bacteria. The peptides were disordered in the buffer, but anoplin and W-MreB_1–9 displayed lipid-induced helicity. Yet, structural changes of the peptide depended on the composition and concentration of the membranes. Secondly, we quantified the destructive activity of peptides against liposomes by monitoring the release of a fluorescent dye (calcein) from the liposomes treated with peptides. We observed that only for anoplin and W-MreB_1–9 calcein leakage from liposomes depended on the peptide concentration. Thirdly, bacterial growth inhibition assays showed that peptide conformational changes, evoked by the lipid environments, do not directly correlate with the antimicrobial activity of the peptides. However, understanding the relation between peptide structural properties, mechanisms of membrane disruption, and their biological activities can guide the design of membrane-active peptides.     

Structure–Activity Relationship of Synthetic Ginkgolic Acid Analogs for Treating Type 2 Diabetes by PTPN9 Inhibition

Ginkgolic acid (C13:0) (GA), isolated from Ginkgo biloba, is a potential therapeutic agent for type 2 diabetes. A series of GA analogs were designed and synthesized for the evaluation of their structure–activity relationship with respect to their antidiabetic effects. Unlike GA, the synthetic analog 1e exhibited improved inhibitory activity against PTPN9 and significantly stimulated glucose uptake via AMPK phosphorylation in differentiated 3T3-L1 adipocytes and C2C12 myotubes; it also induced insulin-dependent AKT activation in C2C12 myotubes in a concentration-dependent manner. Docking simulation results showed that 1e had a better binding affinity through a unique hydrophobic interaction with a PTPN9 hydrophobic groove. Moreover, 1e ameliorated palmitate-induced insulin resistance in C2C12 cells. This study showed that 1e increases glucose uptake and suppresses palmitate-induced insulin resistance in C2C12 myotubes via PTPN9 inhibition; thus, it is a promising therapeutic candidate for treating type 2 diabetes.     

Antimicrobial and Anticancer Properties of Synthetic Peptides Derived from the Wasp Parachartergus fraternus

Agelaia-MPI and protonectin are antimicrobial peptides isolated from the wasp Parachartergus fraternus that show antimicrobial and neuroactive activities. Previously, two analogues of these peptides, neuroVAL and protonectin-F, were designed to reduce nonspecific toxicity and improve potency. Here, the three-dimensional structures of neuroVAL, protonectin and protonectin-F were determined by using circular dichroism and NMR spectroscopy. Antibacterial, antifungal, cytotoxic and hemolytic activities were tested for the parent peptides and analogues. All peptides showed moderate antimicrobial activity against Gram-positive bacteria, with agelaia-MPI being the most active. Protonectin and protonectin-F were found to be toxic to cancerous and noncancerous cell lines. Internalization experiments revealed that these peptides accumulate inside both cell types. By contrast, neuroVAL was nontoxic to all tested cells and was able to enter cells without accumulating. In summary, neuroVAL has potential as a nontoxic cell-penetrating peptide, while protonectin-F needs further modification to realize its potential as an antitumor peptide.     

Manipulating Active Structure and Function of Cationic Antimicrobial Peptide CM15 with the Polysulfonated Drug Suramin: A Step Closer to in Vivo Complexity

Antimicrobial peptides (AMPs) kill bacteria by targeting their membranes through various mechanisms involving peptide assembly, often coupled with disorder-to-order structural transition. However, for several AMPs, similar conformational changes in cases in which small organic compounds of both endogenous and exogenous origin have induced folded peptide conformations have recently been reported. Thus, the function of AMPs and of natural host defence peptides can be significantly affected by the local complex molecular environment in vivo; nonetheless, this area is hardly explored. To address the relevance of such interactions with regard to structure and function, we have tested the effects of the therapeutic drug suramin on the membrane activity and antibacterial efficiency of CM15, a potent hybrid AMP. The results provided insight into a dynamic system in which peptide interaction with lipid bilayers is interfered with by the competitive binding of CM15 to suramin, resulting in an equilibrium dependent on peptide-to-drug ratio and vesicle surface charge. In vitro bacterial tests showed that when CM15 ⋅ suramin complex formation dominates over membrane binding, antimicrobial activity is abolished. On the basis of this case study, it is proposed that small-molecule secondary structure regulators can modify AMP function and that this should be considered and could potentially be exploited in future development of AMP-based antimicrobial agents.     

Broad-Spectrum Antiviral Activity of the Amphibian Antimicrobial Peptide Temporin L and Its Analogs

The COVID-19 pandemic has evidenced the urgent need for the discovery of broad-spectrum antiviral therapies that could be deployed in the case of future emergence of novel viral threats, as well as to back up current therapeutic options in the case of drug resistance development. Most current antivirals are directed to inhibit specific viruses since these therapeutic molecules are designed to act on a specific viral target with the objective of interfering with a precise step in the replication cycle. Therefore, antimicrobial peptides (AMPs) have been identified as promising antiviral agents that could help to overcome this limitation and provide compounds able to act on more than a single viral family. We evaluated the antiviral activity of an amphibian peptide known for its strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, namely Temporin L (TL). Previous studies have revealed that TL is endowed with widespread antimicrobial activity and possesses marked haemolytic activity. Therefore, we analyzed TL and a previously identified TL derivative (Pro³, DLeu⁹ TL, where glutamine at position 3 is replaced with proline, and the D-Leucine enantiomer is present at position 9) as well as its analogs, for their activity against a wide panel of viruses comprising enveloped, naked, DNA and RNA viruses. We report significant inhibition activity against herpesviruses, paramyxoviruses, influenza virus and coronaviruses, including SARS-CoV-2. Moreover, we further modified our best candidate by lipidation and demonstrated a highly reduced cytotoxicity with improved antiviral effect. Our results show a potent and selective antiviral activity of TL peptides, indicating that the novel lipidated temporin-based antiviral agents could prove to be useful additions to current drugs in combatting rising drug resistance and epidemic/pandemic emergencies.     

Structure–Activity Relationship Studies of Amino Acid Substitutions in Radiolabeled Neurotensin Conjugates

Radiolabeled derivatives of the peptide neurotensin (NT) and its binding sequence NT(8–13) have been studied as potential imaging probes and therapeutics for NT‐1‐receptor‐positive cancer. However, a direct comparison of reported NT analogues, even if radiolabeled with the same radionuclide, is difficult because different techniques and models have been used for preclinical evaluations. In an effort to identify a suitable derivative of NT(8–13) for radiotracer development, we herein report a side‐by‐side in vitro comparison of radiometallated NT derivatives bearing some of the most commonly reported amino acid substitutions in their sequence. Performed investigations include cell internalization experiments, determinations of receptor affinity, measurements of the distribution coefficient, and blood serum stability studies. Of the [¹⁷⁷Lu]‐1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA)‐labeled examples studied, analogues of NT(8–13) containing a short hydrophilic tetraethylene glycol (PEG₄) spacer between the peptide and the radiometal complex, and a minimum number of substitutions of amino acid residues, exhibited the most promising properties in vitro.     

Synthesis and Structure–Activity Relationship Study of Organometallic Bioconjugates of the Cyclic Octapeptide Octreotate

The chemically stabilized somatostatin‐derived cyclic octapeptide octreotate has a number of interesting applications in medicinal chemistry. Here, a number of different organometallic derivatives of octreotate were prepared, and their properties were investigated. Specifically, we report the synthesis and characterization of ruthenocene, ferrocene, and cobaltocenium octreotate derivatives and their fluorophore‐labeled conjugates as well as a dicobalt hexacarbonyl alkyne functionalized octreotate. To provide further insights into their characteristics, the log P values and electrochemical properties of the novel metal conjugates were compared. For biological activity, we determined their toxicity in three different cell lines. Cellular uptake and colocalization of selected compounds were studied by fluorescence microscopy with particular focus on efficiency and specificity of their uptake through the somatostatin receptor SSTR to elucidate the value of the metallocene head group for its potential use as a nontoxic and universal peptide label.     

A Parallel Semisynthetic Approach for Structure–Activity Relationship Studies of Peptide YY

The gut hormone peptide YY (PYY) is postprandially secreted from enteroendocrine L cells and is involved in the regulation of energy homeostasis. The N‐terminal truncated version PYY(3–36) decreases food intake and has potential as an anti‐obesity agent. The anorectic effect of PYY(3–36) is mediated through Y₂ receptors in the hypothalamus, vagus, and brainstem regions, and it is well known that the C‐terminal tetrapeptide sequence of PYY(3–36) is crucial for Y₂ receptor activation. The aim of this work was to develop a semisynthetic methodology for the generation of a library of C‐terminally modified PYY(3–36) analogues. By using an intein‐based expression system, PYY(3–29) was generated as a C‐terminal peptide α‐thioester. Heptapeptides bearing an N‐terminal cysteine and modifications at one of the four C‐terminal positions were synthesized in a 96‐well plate by parallel solid‐phase synthesis. In the plate format, an array of [Ala30]PYY(3–36) analogues were generated by ligation, desulfurization, and subsequent solid‐phase extraction. The generated analogues, in which either Arg33, Gln34, Arg35, or Tyr36 had been substituted with proteinogenic or non‐proteinogenic amino acids, were tested in a functional Y₂ receptor assay. Generally, substitutions of Tyr36 were better tolerated than modifications of Arg33, Gln34, and Arg35. Two analogues showed significantly improved Y₂ receptor selectivity; therefore, these results could be used to design new drug candidates for the treatment of obesity.     

Membrane Interactions of Latarcins: Antimicrobial Peptides from Spider Venom

A group of seven peptides from spider venom with diverse sequences constitute the latarcin family. They have been described as membrane-active antibiotics, but their lipid interactions have not yet been addressed. Using circular dichroism and solid-state ¹⁵N-NMR, we systematically characterized and compared the conformation and helix alignment of all seven peptides in their membrane-bound state. These structural results could be correlated with activity assays (antimicrobial, hemolysis, fluorescence vesicle leakage). Functional synergy was not observed amongst any of the latarcins. In the presence of lipids, all peptides fold into amphiphilic α-helices as expected, the helices being either surface-bound or tilted in the bilayer. The most tilted peptide, Ltc2a, possesses a novel kind of amphiphilic profile with a coiled-coil-like hydrophobic strip and is the most aggressive of all. It indiscriminately permeabilizes natural membranes (antimicrobial, hemolysis) as well as artificial lipid bilayers through the segregation of anionic lipids and possibly enhanced motional averaging. Ltc1, Ltc3a, Ltc4a, and Ltc5a are efficient and selective in killing bacteria but without causing significant bilayer disturbance. They act rather slowly or may even translocate towards intracellular targets, suggesting more subtle lipid interactions. Ltc6a and Ltc7, finally, do not show much antimicrobial action but can nonetheless perturb model bilayers.     

Asymmetric Contribution of Aromatic Interactions Stems from Spatial Positioning of the Interacting Aryl Pairs in β‐Hairpins

Isolated aromatic interactions in designed octapeptide β‐hairpin scaffolds display a near‐universal T‐shaped face‐to‐edge geometry in all positional permutations, with the exception of aryl‐Trp interactions. The heterogeneous asymmetric indole ring of Trp competes for a “shielding” face geometry, which lowers the scaffold stability in FtE aryl‐Trp pairs. Assessment of the contributions of aryl pairs (in the absence of solvent‐driven interactions) to the overall β‐hairpin structure reveals the superiority of Trp‐Phe and Trp‐Tyr contributions over the well‐established scaffold stabilization by Trp‐Trp.     

Analogs of a Natural Peptaibol Exert Anticancer Activity in Both Cisplatin- and Doxorubicin-Resistant Cells and in Multicellular Tumor Spheroids

Peptaibols, by disturbing the permeability of phospholipid membranes, can overcome anticancer drug resistance, but their natural hydrophobicity hampers their administration. By a green peptide synthesis protocol, we produced two water-soluble analogs of the peptaibol trichogin GA IV, termed K6-Lol and K6-NH2. To reduce production costs, we successfully explored the possibility of changing the naturally occurring 1,2-aminoalcohol leucinol to a C-terminal amide. Peptaibol activity was evaluated in ovarian cancer (OvCa) and Hodgkin lymphoma (HL) cell lines. Peptaibols exerted comparable cytotoxic effects in cancer cell lines that were sensitive—and had acquired resistance—to cisplatin and doxorubicin, as well as in the extrinsic-drug-resistant OvCa 3-dimensional spheroids. Peptaibols, rapidly taken up by tumor cells, deeply penetrated and killed OvCa-spheroids. They led to cell membrane permeabilization and phosphatidylserine exposure and were taken up faster by cancer cells than normal cells. They were resistant to proteolysis and maintained a stable helical structure in the presence of cancer cells. In conclusion, these promising results strongly point out the need for further preclinical evaluation of our peptaibols as new anticancer agents.     

Systematic Extraction of Structure–Activity Relationship Information from Biological Screening Data

A data mining approach is introduced that automatically extracts SAR information from high‐throughput screening data sets and that helps to select active compounds for chemical exploration and hit‐to‐lead projects. SAR pathways are systematically identified consisting of sequences of similar active compounds with gradual increases in potency. Fully enumerated SAR pathway sets are subjected to pathway scoring, filtering, and mining, and pathways with the most significant SAR information content are prioritized. High‐scoring SAR pathways often reveal activity cliffs contained in screening data. Subsets of SAR pathways are analyzed in SAR trees that make it possible to identify microenvironments of significant SAR discontinuity from which hits are preferentially selected. SAR trees of alternative pathways leading to activity cliffs identify key compounds and help to develop chemically intuitive SAR hypotheses.     

Metal Binding Properties of Fluorescent Analogues of Trichogin GA IV: A Conformational Study by Time‐Resolved Spectroscopy and Molecular Mechanics Investigations

The metal ion binding properties of two fluorescent analogues of trichogin GA IV, which is a natural undecapeptide showing significant antimicrobial activity, were studied by circular dichroism, time‐resolved optical spectroscopy, and molecular mechanics calculations. Binding of Ca^II and Gd^III to the peptides investigated was shown to promote a structural transition from highly helical conformations to folded structures characterized by formation of a loop that embedded the metal ion. Time‐resolved spectroscopy revealed that peptide dynamics is also remarkably affected by ion binding: peptide‐backbone motions slowed down to the microsecond time scale. Finally, molecular mechanics calculations emphasized the role of the central Gly5‐Gly6 motif, which allowed for the twisting of the peptide segment that gave rise to the formation of the binding cavity.     

Anticoagulant Oligonucleotide–Peptide Conjugates: Identification of Thrombin Aptamer Conjugates with Improved Characteristics

Oligonucleotide–peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3–Ser–Phe–Asn (SFN) or T3–Tyr–Trp–Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.     

Discovery of Flavonoids as Novel Inhibitors of ATP Citrate Lyase: Structure–Activity Relationship and Inhibition Profiles

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism and its aberrantly high expression is closely associated with various cancers, hyperlipemia and atherosclerotic cardiovascular diseases. Prospects of ACLY inhibitors as treatments of these diseases are excellent. To date, flavonoids have not been extensively reported as ACLY inhibitors. In our study, 138 flavonoids were screened and 21 of them were subjected to concentration–response curves. A remarkable structure–activity relationship (SAR) trend was found: ortho-dihydroxyphenyl and a conjugated system maintained by a pyrone ring were critical for inhibitory activity. Among these flavonoids, herbacetin had a typical structure and showed a non–aggregated state in solution and a high inhibition potency (IC₅₀ = 0.50 ± 0.08 μM), and therefore was selected as a representative for the ligand–protein interaction study. In thermal shift assays, herbacetin improved the thermal stability of ACLY, suggesting a direct interaction with ACLY. Kinetic studies determined that herbacetin was a noncompetitive inhibitor of ACLY, as illustrated by molecular docking and dynamics simulation. Together, this work demonstrated flavonoids as novel and potent ACLY inhibitors with a remarkable SAR trend, which may help design high–potency ACLY inhibitors. In–depth studies of herbacetin deepened our understanding of the interactions between flavonoids and ACLY.     

Structure–Activity Relationship (SAR) Study of Spautin-1 to Entail the Discovery of Novel NEK4 Inhibitors

Lung cancer is one of the most frequently diagnosed cancers accounting for the highest number of cancer-related deaths in the world. Despite significant progress including targeted therapies and immunotherapy, the treatment of advanced lung cancer remains challenging. Targeted therapies are highly efficacious at prolonging life, but not curative. In prior work we have identified Ubiquitin Specific Protease 13 (USP13) as a potential target to significantly enhance the efficacy of mutant EGFR inhibition. The current study aimed to develop lead molecules for the treatment of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) by developing potent USP13 inhibitors initially starting from Spautin-1, the only available USP13 inhibitor. A SAR study was performed which revealed that increasing the chain length between the secondary amine and phenyl group and introducing a halogen capable of inducing a halogen bond at position 4’ of the phenyl group, dramatically increased the activity. However, we could not confirm the binding between Spautin-1 (or its analogues) and USP13 using isothermal titration calorimetry (ITC) or thermal shift assay (TSA) but do not exclude binding under physiological conditions. Nevertheless, we found that the anti-proliferative activity displayed by Spautin-1 towards EGFR-mutant NSCLC cells in vitro was at least partially associated with kinase inhibition. In this work, we present N-[2-(substituted-phenyl)ethyl]-6-fluoro-4-quinazolinamines as promising lead compounds for the treatment of NSCLC. These analogues are significantly more effective towards EGFR-mutant NSCLC cells than Spautin-1 and act as potent never in mitosis A related kinase 4 (NEK4) inhibitors (IC₅₀~1 µM) with moderate selectivity over other kinases.     

A Novel Antimicrobial Peptide Spampcin56–86 from Scylla paramamosain Exerting Rapid Bactericidal and Anti-Biofilm Activity In Vitro and Anti-Infection In Vivo

The abuse of antibiotics leads to the increase of bacterial resistance, which seriously threatens human health. Therefore, there is an urgent need to find effective alternatives to antibiotics, and antimicrobial peptides (AMPs) are the most promising antibacterial agents and have received extensive attention. In this study, a novel potential AMP was identified from the marine invertebrate Scylla paramamosain and named Spampcin. After bioinformatics analysis and AMP database prediction, four truncated peptides (Spa31, Spa22, Spa20 and Spa14) derived from Spampcin were screened, all of which showed potent antimicrobial activity with different antibacterial spectrum. Among them, Spampcin_56–86 (Spa31 for short) exhibited strong bactericidal activity against a variety of clinical pathogens and could rapidly kill the tested bacteria within minutes. Further analysis of the antibacterial mechanism revealed that Spa31 disrupted the integrity of the bacterial membrane (as confirmed by scanning electron microscopy observation, NPN, and PI staining assays), leading to bacterial rupture, leakage of cellular contents (such as elevated extracellular ATP), increased ROS production, and ultimately cell death. Furthermore, Spa31 was found to interact with LPS and effectively inhibit bacterial biofilms. The antibacterial activity of Spa31 had good thermal stability, certain ion tolerance, and no obvious cytotoxicity. It is worth noting that Spa31 could significantly improve the survival rate of zebrafish Danio rerio infected with Pseudomonas aeruginosa, indicating that Spa31 played an important role in anti-infection in vivo. This study will enrich the database of marine animal AMPs and provide theoretical reference and scientific basis for the application of marine AMPs in medical fields.     

Insights into the Action Mechanism of the Antimicrobial Peptide Lasioglossin III

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee Lasioglossum laticeps. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-rac-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.     

A Deep Learning-Based Quantitative Structure–Activity Relationship System Construct Prediction Model of Agonist and Antagonist with High Performance

Molecular design and evaluation for drug development and chemical safety assessment have been advanced by quantitative structure–activity relationship (QSAR) using artificial intelligence techniques, such as deep learning (DL). Previously, we have reported the high performance of prediction models molecular initiation events (MIEs) on the adverse toxicological outcome using a DL-based QSAR method, called DeepSnap-DL. This method can extract feature values from images generated on a three-dimensional (3D)-chemical structure as a novel QSAR analytical system. However, there is room for improvement of this system’s time-consumption. Therefore, in this study, we constructed an improved DeepSnap-DL system by combining the processes of generating an image from a 3D-chemical structure, DL using the image as input data, and statistical calculation of prediction-performance. Consequently, we obtained that the three prediction models of agonists or antagonists of MIEs achieved high prediction-performance by optimizing the parameters of DeepSnap, such as the angle used in the depiction of the image of a 3D-chemical structure, data-split, and hyperparameters in DL. The improved DeepSnap-DL system will be a powerful tool for computer-aided molecular design as a novel QSAR system.