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Mounting evidence indicates that the neuropeptide FF (NPFF) system is involved in the side effects of opioid usage, including antinociceptive tolerance, hyperalgesia, abuse, constipation, and respiratory depression. Our group recently discovered that the multitarget opioid/NPFF receptor agonist DN-9 exhibits peripheral antinociceptive activity. To improve its metabolic stability, antinociceptive potency, and duration, in this study, we designed and synthesized a novel cyclic disulfide analogue of DN-9, OFP011, and examined its bioactivity through

Effectively presenting epitopes on immunogens, in order to raise conformationally selective antibodies through active immunization, is a central problem in treating protein misfolding diseases, particularly neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease. We seek to selectively target conformations enriched in toxic, oligomeric propagating species while sparing the healthy forms of the protein that are often more abundant. To this end, we computationally modelled scaffolded epitopes in cyclic peptides by inserting/deleting a variable number of flanking glycines (“glycindels”), to best mimic a misfolding-specific conformation of an epitope of α-synuclein enriched in the oligomer ensemble, as characterized by a region most readily disordered and solvent-exposed in a stressed, partially denatured protofibril. We screen and rank the cyclic peptide scaffolds of α-synuclein in silico based on their ensemble overlap properties with the fibril, oligomer-model, and isolated monomer ensembles. We present experimental data of seeded aggregation that supports nucleation rates consistent with computationally predicted cyclic peptide conformational similarity. We also introduce a method for screening against structured off-pathway targets in the human proteome, by selecting scaffolds with minimal conformational similarity between their epitope and the same solvent-exposed primary sequence in structured human proteins. Different cyclic peptide scaffolds with variable numbers of glycines are predicted computationally to have markedly different conformational ensembles. Ensemble comparison and overlap was quantified by the Jensen-Shannon Divergence, and a new measure introduced here—the embedding depth, which determines the extent to which a given ensemble is subsumed by another ensemble, and which may be a more useful measure in developing immunogens that confer conformational-selectivity to an antibody.Graphical TOC Entry

A cyclic hexapeptide, RA-VII isolated from the Rubiaceae family of plants, has high cytotoxic activity. Although RA-VII has been shown to inhibit protein synthesis in eukaryotic cells, the molecular mode of its action is not clear. Here we investigate the mechanism of the RAVII action on the translation apparatus. Biochemical functional assays showed that RA-VII inhibits poly(U)-dependent polyphenylalanine synthesis in the presence of animal elongation factors eEF1A and eEF2. Furthermore, RAVII prevented eEF2/ribosome-dependent GTPase activity, but not eEF-1A/ribosome-dependent activity. A filter binding assay demonstrated that RA-VII markedly enhances the binding affinity of eEF2 for GTP, but not for GDP, and prevents exchange of GTP in the eEF2-GTP complex, even after addition of a large excess of GTP/GDP. Limited proteolysis experiments indicated that RA-VII prevents the digestion of eEF2 in the presence of either GTP or GMPPCP, but not with GDP. Further footprint analysis and a translocation assay showed that the eEF2•GMPPNP•RA-VII complex binds to the conserved rRNA regions at the factor-binding center of the ribosome and retains the ability to translocate the A site-bound tRNA to the P-site. These results suggest that RA-VII tightly stabilizes the GTP•eEF2 complex structure, which is able to bind to the ribosomal functional site, but seems to suppress normal turnover of eEF2 after translocation. The properties of RA-VII make it a novel ligand for probing the action of eEF2 in the process of translocation on the ribosome.

Macrocyclic peptides are sought-after molecular scaffolds for drug discovery, and new methods to access diverse libraries are of increasing interest. Here, we report the enzymatic synthesis of pyridine-based macrocyclic peptides (pyritides) from linear precursor peptides. Pyritides are a recently described class of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are related to the long-known thiopeptide natural products. RiPP precursors typically contain an N-terminal leader region that is physically engaged by the biosynthetic proteins that catalyze modification of the C-terminal core region of the precursor peptide. We demonstrate that pyritide-forming enzymes recognize both the leader region and a C-terminal tripeptide motif, with each contributing to site-selective substrate modification. Substitutions in the core region were well-tolerated and facilitated the generation of a wide range of pyritide analogues, with variations in macrocycle sequence and size. A combination of the pyritide biosynthetic pathway with azole-forming enzymes was utilized to generate a thiazole-containing pyritide (historically known as a thiopeptide) with no similarity in sequence and macrocycle size to the naturally encoded pyritides. The broad substrate scope of the pyritide biosynthetic enzymes serves as a future platform for macrocyclic peptide lead discovery and optimization.

The emergence of resistance to clinically used antibiotics by bacteria presents a significant problem in public health. Natural antimicrobial peptides (AMPs) are a valuable source of antibiotics that act by a mechanism less prone to the evolutionary development of resistance. In an effort to realize the promise of AMPs while overcoming limitations such as poor biostability, researchers have sought sequence-defined oligomers with artificial amide-based backbones that show membrane-disrupting functions similar to natural agents. Most of this precedent has focused on short peptidomimetic analogues of unstructured chains or secondary folds; however, the natural antimicrobial arsenal includes a number of small- and medium-sized proteins that act via an ordered tertiary structure. Generating proteomimetic analogues of these scaffolds poses a challenge due to the increased complexity of the target for mimicry. Here, we report the development of heterogeneous-backbone variants of lasiocepsin, a 27-residue disulfide-rich AMP found in bee venom that adopts a compact tertiary fold. Iterative cycles of design, synthesis, and biological evaluation yielded analogues of the natural domain with ∼30 to 40% artificial backbone content, comparable antibacterial activity, reduced host cell toxicity, and improved stability to proteolytic degradation. High-resolution structures determined for several variants by NMR provide insights into the interplay among backbone composition, tertiary fold, and biological properties. Collectively, the results reported here broaden the scope of protein functional mimicry by artificial backbone analogues of tertiary folding patterns and suggest protein backbone engineering as a means to tune protein function by exerting site-specific control over protein folded structure.

In recent years, some peptide ligases have been identified, such as bacterial sortases and certain plant asparaginyl or prolyl endopeptidases. Peptide ligases have wide applications in protein labelling and cyclic peptide synthesis. To characterize various known peptide ligases or identify new ones, we propose a general bioluminescent activity assay via the genetic fusion of a recognition motif of peptide ligase(s) to the C-terminus of an inactive large NanoLuc fragment (LgBiT) and the chemical introduction of a nucleophilic motif preferred by the peptide ligase(s) to the N-terminus of the low-affinity SmBiT complementation tag. After the inactive ligation version LgBiT protein was ligated with the low-affinity ligation version SmBiT tag by the expected peptide ligase(s), its luciferase activity would be restored and could be quantified sensitively according to the measured bioluminescence. In the present study, we first validated the bioluminescent activity assay using bacterial sortase A and plant-derived butelase-1. Subsequently, we screened novel peptide ligases from crude extracts of selected plants using two LgBiT-SmBiT ligation pairs. Among 80 common higher plants, we identified that five of them likely express asparaginyl endopeptidase-type peptide ligase and four of them likely express prolyl endopeptidase-type peptide ligase, suggesting that peptide ligases are not so rare in higher plants and more of them await discovery. The present bioluminescent activity assay is ultrasensitive, convenient for use, and resistant to protease interference, and thus would have wide applications for characterizing known peptide ligases or screening new ones from various sources in future studies.

Here, we report a novel rapid arene triazene strategy for the macrocyclization of peptides that generates an inbuilt chromophoric triazene moiety at the site of cyclization within a minute. The rapid arene triazene chemistry is chemoselective for secondary amines and

Aims: The present investigation is targeted towards the synthesis of a novel analogue of a natural peptide of marine origin. Background: Marine sponges are enriched with bioactive secondary metabolites, especially circu-lar peptides. Heterocycles are established organic compounds with potential biological value. Tak-ing into consideration the bio-properties of heterocycles and marine sponge-derived natural pep-tides, an effort was made for the synthesis of a heterocyclic analogue of a natural cyclopeptide. Objective : A heterocyclic analogue of a sponge-derived proline-containing cyclic peptide, rolloam-ide A, was synthesized by interaction of Boc-protected L-histidinyl-L-prolyl-L-valine and L-prolyl-L-leucyl-L-prolyl-L-isoleucine methyl ester and compared with synthetic rolloamide A with bioac-tivity against bacteria, fungi, and earthworms. Methods: The synthesis of cycloheptapeptide was accomplished employing the liquid phase method. The larger peptide segment was prepared by interaction of Boc-protected L-prolyl-L-leu-cine with L-prolyl-L-isoleucine methyl ester. Similarly, the tripeptide unit was synthesized from Boc-protected L-histidinyl-L-proline with L-valine ester. The linear heptapeptide segment (7) was cyclized by utilizing pentafluorophenyl (pfp) ester, and the structure was elucidated by elemental and spectral (IR, 1H/13C NMR, MS) analysis. The peptide was also screened for diverse bioactivities such as antibacterial, antifungal, and potential against earthworms and cytotoxicity. Results: The novel cyclooligopeptide was synthesized with 84% yield by making use of car-bodiimides. The synthesized cyclopeptide exhibited significant cytotoxicity against two cell lines. In addition, promising antifungal and antihelmintic properties were observed for newly synthesized heterocyclic peptide derivative (8) against dermatophytes and three earthworm species at 6 μg/mL and 2 mg/mL, respectively. Conclusion: Solution-phase technique employing carbodiimide chemistry was established to be promising for synthesizing the cycloheptapeptide derivative (8), and C5H5N was proved to be a better base for heptapeptide circling when compared to N-methylmorpholine and triethylamine.

Aromatic cyclic β

A new type of peptide bond formation utilizing silacyclic amino acids or peptides is described. This work has the following advantages: (1) imidazolylsilane is a highly fascinating coupling reagent for dipeptide synthesis from N-,C-terminal unprotected amino acids with amino acid

Posttreatment evaluation of gastric mucosa-associated lymphoid tissue (MALT) lymphoma currently relies on esophagogastroduodenoscopy with histological assessment of biopsies. Overexpression of the G protein–coupled C-X-C chemokine receptor type 4 (CXCR4) has been previously observed in MALT lymphoma. The aim of this prospective study was to evaluate positron emission tomography (PET) with the novel CXCR4 tracer [68Ga]Pentixafor as a potential alternative to follow up biopsies for assessment of residual disease (noncomplete remission [CR]) after first-line Helicobacter pylori eradication. Forty-six post–H pylori eradication [68Ga]Pentixafor–PET/magnetic resonance imaging (MRI) examinations of 26 gastric MALT lymphoma patients, and 20 [68Ga]Pentixafor–PET/MRI examinations of 20 control group patients without lymphoma, were analyzed. In the MALT lymphoma group, time-matched gastric biopsies were used as reference standard and showed CR in 6 cases. Pooled examination-based accuracy, sensitivity, specificity, and positive and negative predictive values of [68Ga]Pentixafor–PET for detection of residual gastric MALT lymphoma at follow-up were 97.0%, 95.0%, 100.0%, 100.0%, and 92.9%, respectively. Maximum and mean PET standardized uptake values showed moderate correlation with immunohistochemistry-based CXCR4+ cell counts, with correlation coefficients of r = 0.51 and r = 0.52 (P = .008 and P = .006). In summary, CXCR4 imaging with [68Ga]Pentixafor–PET may represent a promising test for assessment of residual gastric MALT lymphomas after H pylori eradication.

We report the first total synthesis of micrococcin P2 (MP2, 1) by a diversity-oriented route that incorporates a number of refinements relative to earlier syntheses. Biological data regarding the activity of 1 against a range of human pathogens are also provided. Furthermore, we disclose a chemical property of MP2 that greatly facilitates medicinal chemistry work in the micrococcin area and describe a method to obtain MP2 by fermentation in B. subtilis

Backbone-cyclized peptides, which have applications in the pharmaceutical and agricultural industries, can be made efficiently in plants by co-expressing them with a cyclizing enzyme., Cyclotides are ultra-stable, backbone-cyclized plant defence peptides that have attracted considerable interest in the pharmaceutical industry. This is due to their range of native bioactivities as well as their ability to stabilize other bioactive peptides within their framework. However, a hindrance to their widespread application is the lack of scalable, cost-effective production strategies. Plant-based production is an attractive, benign option since all biosynthetic steps are performed in planta. Nonetheless, cyclization in non-cyclotide-producing plants is poor. Here, we show that cyclic peptides can be produced efficiently in Nicotiana benthamiana, one of the leading plant-based protein production platforms, by co-expressing cyclotide precursors with asparaginyl endopeptidases that catalyse peptide backbone cyclization. This approach was successful in a range of other plants (tobacco, bush bean, lettuce, and canola), either transiently or stably expressed, and was applicable to both native and engineered cyclic peptides. We also describe the use of the transgenic system to rapidly identify new asparaginyl endopeptidase cyclases and interrogate their substrate sequence requirements. Our results pave the way for exploiting cyclotides for pest protection in transgenic crops as well as large-scale production of cyclic peptide pharmaceuticals in plants.

Background: Peptides and peptide-based therapeutics are biomolecules that demarcate a significant chemical space to bridge small molecules with biological therapeutics, such as antibodies, recombinant proteins, and protein domains. Introduction: Cyclooligopeptides and depsipeptides, particularly cyanobacteria-derived thiazoline-based polypeptides (CTBCs), exhibit a wide array of pharmacological activities due to their unique structural features and interesting bioactions, which furnish them as promising leads for drug discovery. Methods: In the present study, we comprehensively review the natural sources, distinguishing chemistries, and pertinent bioprofiles of CTBCs. We analyze their structural peculiarities counting the mode of actions for biological portrayals which render CTBCs as indispensable sources for emergence of prospective peptide-based therapeutics. In this milieu, metal organic frameworks and their biomedical applications are also briefly discussed. To boot, the challenges, approaches, and clinical status of peptide-based therapeutics are conferred. Results: Based on these analyses, CTBCs can be appraised as ideal drug targets that have always remained a challenge for traditional small molecules, like those involved in protein- protein interactions or to be developed as potential cancer-targeting nanomaterials. Cyclization-induced reduced conformational freedom of these cyclooligopeptides contribute to improved metabolic stability and binding affinity to their molecular targets. Clinical success of several cyclic peptides provokes the large library-screening and synthesis of natural product-like cyclic peptides to address the unmet medical needs. Conclusion: CTBCs can be considered as the most promising lead compounds for drug discovery. Adopting the amalgamation of advanced biological and biopharmaceutical strategies might endure these cyclopeptides to be prospective biomolecules for futuristic therapeutic applications in the coming times.

The incorporation of noncanonical amino acids (ncAAs) with different side chains into a peptide is a promising technique for changing the functional properties of that peptide. Of particular interest is the incorporation of ncAAs into peptide-derived natural products to optimize their biophysical properties for medical and industrial applications. Here, we present the first instance of ncAA incorporation into the natural product cinnamycin in streptomycetes using the orthogonal pyrrolysyl-tRNA synthetase/tRNAPyl pair from Methanosarcina barkeri. This approach allows site-specific incorporation of ncAAs via the read-through of a stop codon by the suppressor tRNAPyl, which can carry different pyrrolysine analogues. Five new deoxycinnamycin derivatives were obtained with three distinct pyrrolysine analogues incorporated into diverse positions of the antibiotic. The combination of partial hydrolysis and MS/MS fragmentation analysis was used to verify the exact position of the incorporation events. The introdu...

COVID-19 has shaken all the countries across the globe and researchers are trying to find promising antiviral to cure the patients suffering from infection and can decrease the death. Even, different nations are using repurposing drugs to cure the symptoms and these repurposing drugs are hydroxychloroquine, remdesivir, and lopinavir, and recently, India has recently given the approval for the 2-deoxy-d-glucose for emergency purpose to cure the patients suffering from the COVID-19. Plitidepsin is a popular molecule and can be used in treatment of myeloma. Plitidepsin was explored by scientists experimentally against the COVID-19 and was given to the patient. It is found to be more a promising repurposing drug against the COVID-19 than the remdesivir. Therefore, there is a need to understand the interaction of plitidepsin with the main protease of SARS-CoV-2. Molecular docking of the plitidepsin against Mpro of SARS-CoV-2 was performed and the binding energy was found to be − 137.992 kcal/mol. Furthermore, authors have performed the molecular dynamics simulations of the main protease of SARS-CoV-2 in presence of plitidepsin at 300 and 325 K. It was found that the plitidepsin binds effectively with the main protease of SARS-CoV-2 at 300 K.

Antimicrobial resistance (AMR) has been increasing unrelentingly worldwide, thus negatively impacting human health. The discovery and development of novel antibiotics is an urgent unmet need of the hour. However, it has become more challenging, requiring increasingly time-consuming efforts with increased commercial risks. Hence, alternative strategies are urgently needed to potentiate the existing antibiotics. In this context, short cationic peptides or peptide-based antimicrobials that mimic the activity of naturally occurring antimicrobial peptides (AMPs) could overcome the disadvantages of AMPs having evolved as potent antibacterial agents. Besides their potent antibacterial efficacy, short peptide conjugates have also gained attention as potent adjuvants to conventional antibiotics. Such peptide antibiotic combinations have become an increasingly cost-effective therapeutic option to tackle AMR. This Review summarizes the recent progress for peptide-based small molecules as promising antimicrobials and as adjuvants for conventional antibiotics to counter multidrug resistant (MDR) pathogens.

Cytosolic peptide: N-glycanase (PNGase; NGLY1), an enzyme responsible for de-glycosylation of N-glycans on glycoproteins, is known to play pivotal roles in a variety of biological processes. In 2012, NGLY1 deficiency, a rare genetic disorder, was reported and since then, more than 100 patients have now been identified worldwide. Patients with this disease exhibit several common symptoms that are caused by the dysfunction of NGLY1. However, correlation between the severity of patient symptoms and the extent of the reduction in NGLY1 activity in these patients remains to be clarified, mainly due to the absence of a facile quantitative assay system for this enzyme, especially in a crude extract as an enzyme source. In this study, a quantitative, non-radioisotope (RI)-based assay method for measuring recombinant NGLY1 activity was established using a BODIPY-labeled asialoglycopeptide (BODIPY-ASGP) derived from hen eggs. With this assay, the activities of 27 recombinant NGLY1 mutants that are associated with the deficiency were examined. It was found that the activities of three (R469X, R458fs and H494fs) out of the 27 recombinant mutant proteins were 30–70% of the activities of wild-type NGLY1. We further developed a method for measuring endogenous NGLY1 activity in crude extracts derived from cultured cells, patients’ fibroblasts, iPS cells or peripheral blood mononuclear cells (PBMCs), using a glycosylated cyclopeptide (GCP) that exhibited resistance to the endogenous proteases in the extract. Our methods will not only provide new insights into the molecular mechanism responsible for this disease but also promises to be applicable for its diagnosis.

Microviridins, tricyclic peptide natural products originally isolated from cyanobacteria, function as inhibitors of diverse serine-type proteases. Here we report the structure and biochemical characterization of AMdnB, a unique iterative macrocyclase involved in a microviridin biosynthetic pathway from Anabaena sp. PCC 7120. The ATP-dependent cyclase, along with the homologous AMdnC, introduce up to nine macrocyclizations on three distinct core regions of a precursor peptide, AMdnA. The results presented here provide structural and mechanistic insight into the iterative chemistry of AMdnB. In vitro AMdnB-catalyzed cyclization reactions demonstrate the synthesis of the two predicted tricyclic products from a multi-core precursor peptide substrate, consistent with a distributive mode of catalysis. The X-ray structure of AMdnB shows a structural motif common to ATP-grasp cyclases involved in RiPPs biosynthesis. Additionally, comparison with the non-iterative MdnB allows insight into the structural basis for the iterative chemistry. Overall, the presented results provide insight into the general mechanism of iterative enzymes in ribosomally synthesized and post-translationally modified peptide biosynthetic pathways.

Backbone-dependent rotamer libraries are commonly used to assign the side chain dihedral angles of amino acids when modeling protein structures. Most rotamer libraries are created by curating protein crystal structure data and using various methods to extrapolate the existing data to cover all possible backbone conformations. However, these rotamer libraries may not be suitable for modeling the structures of cyclic peptides and other constrained peptides because these molecules frequently sample backbone conformations rarely seen in the crystal structures of linear proteins. To provide backbone-dependent side chain information beyond the α-helix, β-sheet, and PPII regions, we used explicit-solvent metadynamics simulations of model dipeptides to create a new rotamer library that has high coverage in the (ϕ, ψ) space. Furthermore, this approach can be applied to build high-coverage rotamer libraries for noncanonical amino acids. The resulting Metadynamics of Dipeptides for Rotamer Distribution (MEDFORD) rotamer library predicts the side chain conformations of high-resolution protein crystal structures with similar accuracy (~80%) to a state-of-the-art rotamer library. Our ability to test the accuracy of MEDFORD at predicting the side chain dihedral angles of amino acids in noncanonical backbone conformation is restricted by the limited structural data available for cyclic peptides. For the cyclic peptide data that are currently available, MEDFORD and the state-of-the-art rotamer library perform comparably. However, the two rotamer libraries indeed make different rotamer predictions in noncanonical (ϕ, ψ) regions. For noncanonical amino acids, the MEDFORD rotamer library predicts the χ

A family of novel cyclic lipopeptides named tasikamides A􀀀 H (Tsk A􀀀 H) were discovered recently in Streptomyces tasikensis P46. Aside from the unique cyclic pentapeptide scaffold shared by the tasikamides, Tsk A􀀀 C contain a hydrazone bridge that connects the cyclic pentapeptide to the lipophilic alkyl 5- hydroxylanthranilate (AHA) moiety. Here we report the production of tasikamides I􀀀 K (Tsk I􀀀 K) by a mutant strain of S. tasikensis P46 that overexpresses two pathway-specific transcription regulators. Unlike Tsk A􀀀 C, Tsk I􀀀 K feature a rare enaminone-bridge that links the cyclic peptide scaffold to the AHA moiety. Our experimental data suggest that Tsk I􀀀 K are generated by the coupling of two biosynthetic pathways via a nonenzymatic condensation reaction between an arylamine and a β-keto aldehyde-containing precursor. The results underscore the nucleophilic and electrophilic reactivity of the β-keto aldehyde moiety and its ability to Ministry of Education (MOE) National Research Foundation (NRF) Submitted/Accepted version We acknowledge the generous funding support from the Ministry of Education of Singapore (ARC Tier 2 grant (T2EP30221-0029) awarded to Z.-X.L.) and National Research Foundation of Singapore (SBP01 grant awarded to Z.-X.L.).

Cell-permeable, macrocyclic peptides antagonize either the active or the inactive states of Gα s to fine-tune signaling.

Peptides are ideal candidates for the development of antiviral therapeutics due to their specificity, chemical diversity and potential for highly potent, safe, molecular interventions. By restricting conformational freedom and flexibility, cyclic peptides frequently increase peptide stability. Viral targets are often very challenging as their evasive strategies for infectivity can preclude standard therapies. In recent years, several peptides from natural sources mitigated an array of viral infections. In parallel, short peptides derived from key viral proteins, modified with chemical groups such as lipids and cell-penetrating sequences, led to highly effective antiviral inhibitor designs. These strategies have been further developed during the recent COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2. Several anti-SARS-CoV-2 peptides are gaining ground in pre-clinical development. Overall, peptides are strong contenders for lead compounds against many life-threatening viruses and may prove to be the key to future efforts revealing viral mechanisms of action and alleviating their effects.

Lanthipeptides are a class of cyclic peptides characterized by the presence of one or more lanthionine (Lan) or methyllanthionine (MeLan) thioether rings. These cross-links are produced by α,β-unsaturation of Ser or Thr residues in peptide substrates by dehydration, followed by a Michael-type conjugate addition of Cys residues onto the dehydroamino acids. Lanthipeptides may be broadly classified into at least five different classes, and the biosynthesis of classes I-IV lanthipeptides requires catalysis by LanC cyclases that control both the site-specificity and the stereochemistry of the conjugate addition. In contrast, there are no current examples of LanCs that occur in class V biosynthetic clusters, despite the presence of lanthionine rings in these compounds. In this work, bioinformatics-guided co-occurrence analysis identifies more than 240 putative class V lanthipeptide clusters that contain a LanC cyclase. Reconstitution studies demonstrate that the cyclase-catalyzed product is notably distinct from the product formed spontaneously. Stereochemical analysis shows that the cyclase diverts the final product to a configuration that is distinct from one that is energetically favored. Structural characterization of the final product by multi-dimensional NMR spectroscopy reveals that it forms a helical stapled peptide. Mutational analysis identified a plausible order for cyclization and suggests that enzymatic rerouting to the final structure is largely directed by the construction of the first lanthionine ring. These studies show that lanthipeptide cyclases are needed for the biosynthesis of some constrained peptides, the formations of which would otherwise be energetically unfavored.

Here we explore electrochemical dethiylation in processes relevant to Native Chemical Ligation (NCL). NCL's reliance on the redox active amino acid cysteine and β-mercaptamine derivatives suggests a potential role for electrochemistry. We show that the application of a 1 V potential to platinum electrodes in 0.15 M TCEP solution is sufficient to convert Cys to Ala in cyclic peptides, and to cleave the 2-mercapto-2-phenethyl class of acyl transfer auxiliary.

Cyclic lipopeptides produced by Bacillus species exhibit interesting therapeutic potential. However, their clinical use remains limited due to their low stability, undesirable interactions with host macromolecules, and their potential toxicity to mammalian cells. The present work aims to develop suitable lipopeptide-loaded chitosan nanoparticles with improved biological properties and reduced toxicity. Surfactin and bacillomycin D lipopeptides produced by Bacillus amyloliquefaciens B84 strain were loaded onto chitosan nanoparticles by ionotropic gelation process. Nanoformulated lipopeptides exhibit an average size of 569 nm, a zeta potential range of 38.8 mV, and encapsulation efficiency (EE) of 85.58%. Treatment of Candida (C.) albicans cells with encapsulated lipopeptides induced anti-adhesive activity of 81.17% and decreased cell surface hydrophobicity (CSH) by 25.53% at 2000 µg/mL. Nanoformulated lipopeptides also induced antileishmanial activity against Leishmania (L.) major promastigote and amastigote forms at respective IC50 values of 14.37 µg/mL and 22.45 µg/mL. Nanoencapsulated lipopeptides exerted low cytotoxicity towards human erythrocytes and Raw 264.7 macrophage cell line with respective HC50 and LC50 values of 770 µg/mL and 234.56 µg/mL. Nanoencapsulated lipopeptides could be used as a potential delivery system of lipopeptides to improve their anti-adhesive effect against C. albicans cells colonizing medical devices and their anti-infectious activity against leishmania.

Antimicrobial peptide (AMP) derived from the horseshoe crab, tachyplesin I (KWCFRVCYRGICYRRCR-NH2 ), displayed the apparent antimicrobial activity with low cytotoxicity, suggesting its efficacy as an attractive agent but still lacks the understandings regarding its mechanism(s). Hence, the study focused on investigating the antibacterial mode of action of tachyplesin I against Escherichia coli. Based on the reactive oxygen species generation displayed in several antimicrobial effects, the detection of superoxide anion and nitric oxide were verified after tachyplesin I treatment. Substantial increment of two molecules was followed by the imbalance in intracellular ion concentration, noticeably magnesium and calcium. The series of stages led to hydroxyl radical generation with reduced glutathione, followed by damage in DNA due to oxidative stress. Eventually, the apoptosis-like death in E. coli was monitored in DNA fragmentation-dependent manner due to the tachyplesin I treatment, verified by membrane depolarization, caspase-like protein activation, and phosphatidylserine exposure. Accordingly, tachyplesin I induces apoptosis-like death in E. coli, suggesting the potential of being a candidate for regulating bacterial infection.

β-strands are a fundamental component of protein structure, and these extended peptide regions serve as binding epitopes for numerous protein-protein complexes. However, synthetic mimics that capture the conformation of these epitopes and inhibit selected protein-protein interactions are rare. Here we describe covalent and non-covalent β-hairpin mimics of an extended strand region mediating the Tcf4/β-catenin interaction. Our efforts afford a rationally designed lead for an underexplored region of β-catenin, which has been the subject of numerous ligand discovery campaigns.

Cordyheptapeptide A is a lipophilic cyclic peptide from the prized Cordyceps fungal genus that shows potent cytotoxicity in multiple cancer cell lines. To better understand the bioactivity and physicochemical properties of cordyheptapeptide A with the ultimate goal of identifying its cellular target, we developed a solid-phase synthesis of this multiply N-methylated cyclic heptapeptide which enabled rapid access to both side chain- and backbone-modified derivatives. Removal of one of the backbone amide N-methyl (N-Me) groups maintained bioactivity, while membrane permeability was also preserved due to the formation of a new intramolecular hydrogen bond in a low dielectric solvent. Based on its cytotoxicity profile in the NCI-60 cell line panel, as well as its phenotype in a microscopy-based cytological assay, we hypothesized that cordyheptapeptide was acting on cells as a protein synthesis inhibitor. Further studies revealed the molecular target of cordyheptapeptide A to be the eukaryotic translation elongation factor 1A (eEF1A), a target shared by other lipophilic cyclic peptide natural products. This work offers a strategy to study and improve cyclic peptide natural products while highlighting the ability of these lipophilic compounds to effectively inhibit intracellular disease targets.

Mutanobactin D is a non-ribosomal, cyclic peptide isolated from Streptococcus mutans and shows activity reducing yeast-to-hyphae transition as well as biofilm formation of the pathogenic yeast Candida albicans. We report the first total synthesis of this natural product, which relies on enantioselective, zinc-mediated 1,3-dipolar cycloaddition and a sequence of cascading reactions, providing the key lipidated γ-amino acid found in mutanobactin D. The synthesis enables configurational assignment, determination of the dominant solution-state structure, and studies to assess the stability of the lipopeptide substructure found in the natural product. The information stored in the fingerprint region of the IR spectra in combination with quantum chemical calculations proved key to distinguishing between epimers of the α-substituted β-keto amide. Synthetic mutanobactin D drives discovery and analysis of its effect on growth of other members of the human oral consortium. Our results showcase how total synthesis is central for elucidating the complex network of interspecies communications of human colonizers.

Cyanobacteria produce a variety of chemically diverse cyclic lipopeptides with potent antifungal activities. These cyclic lipopeptides have an amphipathic structure comprised of a polar peptide cycle and hydrophobic fatty acid side chain. Many have antibiotic activity against a range of human and plant fungal pathogens. This review article aims to summarize the present knowledge on the chemical diversity and cellular effects of cyanobacterial cyclic lipopeptides that display antifungal activity. Cyclic antifungal lipopeptides from cyanobacteria commonly fall into four structural classes; hassallidins, puwainaphycins, laxaphycins, and anabaenolysins. Many of these antifungal cyclic lipopeptides act through cholesterol and ergosterol-dependent disruption of membranes. In many cases, the cyclic lipopeptides also exert cytotoxicity in human cells, and a more extensive examination of their biological activity and structure–activity relationship is warranted. The hassallidin, puwainaphycin, laxaphycin, and anabaenolysin structural classes are unified through shared complex biosynthetic pathways that encode a variety of unusual lipoinitiation mechanisms and branched biosynthesis that promote their chemical diversity. However, the biosynthetic origins of some cyanobacterial cyclic lipopeptides and the mechanisms, which drive their structural diversification in general, remain poorly understood. The strong functional convergence of differently organized chemical structures suggests that the production of lipopeptide confers benefits for their producer. Whether these benefits originate from their antifungal activity or some other physiological function remains to be answered in the future. However, it is clear that cyanobacteria encode a wealth of new cyclic lipopeptides with novel biotechnological and therapeutic applications. publishedVersion

CD55 is a major regulator of the complement system, a complex network of proteins that cooperate to clear tissue and blood pathogens from the organism. Indeed, overexpression of CD55 is associated with many diseases and is connected to the resistance mechanisms exhibited by several cancers towards immunotherapy approaches. High level of CD55 expression on tumour cells renders it a good target for both imaging and immunotherapy. Indeed, a conceivable approach to tackle disease is to interfere with CD55-mediated complement regulation with the use of CD55-targeting antibodies. However, the large size and poor tissue penetration together with to the high costs of antibodies often limits their widespread therapeutic use. Here, we employed bioinformatic and chemical approaches to design and synthesize molecules of small dimensions able to mimic a CD55 blocking antibody. As a result, a bicyclic peptide, named as miniAB55, proved to bind CD55 with nanomolar affinity. This molecule represents an attracting chemical scaffold for CD55-directed diagnostic tools in diseases associated with CD55 overproduction. To further support the applicative potential of miniAB55, we prove that the miniAB55 binds CD55 on the same region involved in inactivation of the complement C3 and C5 convertases, thus opening promising scenarios for the development of complement-modulating tools.

The contact system comprises a series of serine proteases that mediate procoagulant and proinflammatory activities via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. Inhibition of Factor XIIa (FXIIa), an initiator of the contact system, has been demonstrated to lead to thrombo-protection and anti-inflammatory effects in animal models and serves as a potentially safer target for the development of antithrombotics. Herein, we describe the use of the Randomised Nonstandard Peptide Integrated Discovery (RaPID) mRNA display technology to identify a series of potent and selective cyclic peptide inhibitors of FXIIa. Cyclic peptides were evaluated in vitro, and three lead compounds exhibited significant prolongation of aPTT, a reduction in thrombin generation, and an inhibition of bradykinin formation. We also describe our efforts to identify the critical residues for binding FXIIa through alanine scanning, analogue generation, and via in silico methods to predict the binding mode of our lead cyclic peptide inhibitors.

Silver nanoparticles (AgNPs) are widely used in wound dressing, but are limited in the application due to its high toxicity at effective concentrations. iturin-AgNPs was previously found to have much higher antibacterial activity at lower AgNPs content than the commercial AgNPs. To verify its potential application in the promotion of wound healing, a chitosan (CS) composite sponge dressing-loaded iturin-AgNPs was developed and evaluated for their antibacterial activity in vitro and used for wound healing in vivo. As results, the synthesized CS dressing had high porosity and water absorption. As expected, the antibacterial activity of CS dressing was significantly promoted by the incorporation of iturin-AgNPs. The CS dressing-loaded iturin-AgNPs showed more effective inhibition of bacterial infection and promotion of wound healing processing and quality than the commercial wound dressing loaded AgNPs in vivo. The mechanisms for the promotion of wound healing by the CS dressing-loaded iturin-AgNPs were found as the enhancement of re-epithelialization and collagen formation, as well as the increased antibacerial activity. No toxicity was found to all organs of mice. The study developed an efficient way to enhance the antibacterial activity of CS dressing loaded AgNPs at low toxicity, which has great potential in wound care application.

Simple and rapid methods are required for screening and analysis of water samples to detect cyanobacterial cyclic peptide hepatotoxins: microcystin/nodularin. Previously, we reported a highly sensitive non-competitive heterogeneous assay for microcystin/nodularin utilizing a generic anti-immunocomplex (anti-IC) single-chain fragment of antibody variable domains (scFv) isolated from a synthetic antibody library together with a generic adda ((2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid)-specific monoclonal antibody (Mab) recognizing the common adda part of the microcystin/nodularin. Using the same antibody pair, here we report a homogeneous non-competitive assay for microcystin/nodularin based on TR-FRET (time-resolved Förster resonance energy transfer) measurement. The anti-IC scFv labeled with Alexa Fluor 680 and the Mab labeled with europium enabled the FRET process to occur in the presence of microcystin/nodularin. The TR-FRET signal is proportional to the toxin concentration in the sample. The rapid (15 min) homogeneous assay without requiring any washing step detected all the tested nine toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and nodularin-R). Very good signal to blank ratio (~13) was achieved using microcystin-LR and the sample detection limit (blank+3SD of blank) for microcystin-LR was ~0.3 μg/L (~0.08 μg/L in 80-μL reaction well). The practical application of the TR-FRET assay was demonstrated with water samples spiked with microcystin-LR as well as with environmental water. The average recoveries of microcystin-LR from spiked water ranged from 65 to 123%. Good correlation (r2 = 0.73 to 0.99) with other methods (liquid chromatography-mass spectrometry and previously reported heterogeneous assay) was found when environmental samples were analyzed. The developed wash-free assay has the potential to play as a quick screening tool to detect microcystin/nodularin from water below the World Health Organization’s guideline limit (1 μg/L of microcystin-LR). Graphical abstract

Fengycins are cyclic lipo-depsipeptides produced by Bacillus spp. that display potent antifungal properties but are chemically unstable. This instability has meant that no total synthesis of any fengycin has been published. Here we report the synthesis of fengycin A analogues that display enhanced antifungal properties and chemical stability under both basic and acidic conditions. The analogues prepared also demonstrate that the fengycin core structure can be modified and simplified without the loss of antifungal activity.

Targeting of extracellular protein–protein interactions (PPI) is emerging as a major application for de novo discovered macrocyclic peptides. Modern discovery platforms can routinely identify macrocyclic peptide ligands capable of highly selective modulation of extracellular signaling pathways; amenability to chemical synthesis and natural modularity of peptides additionally provides an avenue for their further structural elaboration, while the challenge of cell internalization can be minimized. Here, we discuss the recent progress in targeting extracellular PPIs with macrocyclic peptides by focusing on a number of recent case studies. We analyze the scope and potential limitations of the discovery systems in identifying functional macrocyclic ligands. We also highlight the recent technical advancements allowing for a more streamlined discovery pipeline and our brief perspective in this field.

Cyclopeptide alkaloids with different biological activities are present in plants of the family Rhamnaceae. Plants of this family grow in a symbiotic relationship with aerobic Gram-positive actinomycetes belonging to the genus Frankia. This goal of this research was a study of the comparative profile of alkaloids present in Discaria chacaye and to establish a connection between the presence or absence of Frankia sp. and the alkaloids. In addition, insecticidal activities of the alkaloidal extract were examined. A total of 24 alkaloids were identified, of which 12 have a benzylisoquinoline skeleton, 9 were cyclopeptides, 2 isoquinolines, and 1 aporphine. The presence of cyclopeptide alkaloids is associated with Frankia nodules in the plant root. The alkaloid extracts showed insecticidal activity with mortality dose-dependence and LD

Iturin, a metabolite produced by Bacillus subtilis, has a broad-spectrum antibacterial effect, and because they are secreted in the rhizosphere of plants, iturins are often mixed with many organic molecules. In recent years, people have improved their separation and purification methods but still cannot achieve simple and effective procedures, making Iturins an ideal biological control agent for insects and bacteria; commercial value still cannot be realized. With the in-depth studies of Iturins, its anti-cancer, hemolysis and other biological activities have gradually been discovered. This article reviews the branches of the Iturin family, structural features of these metabolite, separation and purification methods used for producing it, culture optimization, and various biological activities of the Iturin family, such as insecticidal, antibacterial, hemolytic and anticarcinogenic properties, among others have been summarized. Furthermore, this review revealed some commercial applications of Iturins and their relevance in research works. For example, in food packaging, clean water has good development potential.This can promote the commercial application of Iturins instead of other chemical and biological control agents that are environmentally friendly, pollution-free and have no side effects on humans. Furthermore, work documented anticancer, hemolytic and other biological activities of Iturin.

The incorporation of non-proteinogenic amino acids (NPAAs) enriches the structural diversity of nonribosomal peptides. Recently, four NPAA-containing cyclic hexapeptides, longicatenamides A-D, were isolated using a combined-culture strategy. Based on in silico analysis, we discovered their putative biosynthetic gene cluster (lon) and proposed a possible biosynthetic mechanism. Surprisingly, the lon22 gene encodes an atypical arginine dihydrolase, which can also catalyze the hydrolysis of citrulline to ornithine. Phylogenetic analysis showed that Lon22-like proteins form a novel clade that is separated from other guanidine-modifying enzymes. After rational design, the catalytic efficiencies of a Lon22 Y80F mutant for arginine and citrulline substrates were 2.31- and 4.70-fold that of the wild-type (WT), respectively. In addition, characterization of the Lon20-A4 adenylation domain suggested that it can incorporate both ornithine and lysine into the final products.

Angiogenesis is important for tumor growth, and accordingly, targeting angiogenesis has become an important pathway for antitumor therapy. A novel proapoptotic peptide, CIGB-300 (P15-Tat), has been shown to be involved in the casein kinase II phosphorylation pathway, conferring it with antiangiogenic activity. Cyclic peptides have been widely used as scaffolds in drug design studies due to their high stability and favorable biopharmaceutical properties. Here, we chose two very stable cyclic trypsin inhibitors, MCoTI-II and SFTI-1, as frameworks to incorporate the bioactive epitope P15 into various backbone loops. NMR studies revealed that all re-engineered analogs had similar secondary structures to their native cyclic frameworks. One key analog, MCoP15, displayed significant improvement for inhibiting human umbilical vein endothelial cell migration, was nontoxic, and had higher stability than the P15 epitope alone. Overall, the results show the value of P15 being engineered into cyclic trypsin inhibitor scaffolds for improving antiangiogenic activity and stability. More broadly, the study highlights the versatility of cyclic peptide frameworks in drug design for antiangiogenic therapies.

We conducted global genome mining of 162,672 bacterial genomes and identified 829 cyclodipeptide (CDP) biosynthesis gene clusters (BGC) containing a cytochrome P450 gene. Heterologous expression of BGC from Saccharopolyspora hirsuta DSM 44795 led to the identification of two novel crownlike CDPs, cyctetryptomycin A (4) and B (5), which possess unprecedented complex macrocycle and show neuroprotective activity. The two cytochrome P450s found in the BGC catalyze sequential reactions leading to the cyclization of diketopiperazine dimers.

d/l-Hybrid peptides are an attractive class of molecular modality because they are able to exhibit high proteolytic stability and unique structural diversity which cannot be accessed by those consisting of only proteinogenic l-amino acids. Despite such an expectation, it has not been possible to devise de novo d/l-hybrid peptides capable of disrupting the function of a protein target(s) due to the lack of an effective method that reliably constructs a highly diverse library and screens active species. Here we report for the first time construction of a library consisting of 1012 members of macrocyclic d/l-hybrid peptides containing five kinds of d-amino acids and performance of the RaPID selection against human EGFR as a showcase to uncover PPI (protein-protein interaction) inhibitors.

Clostridia coordinate many important processes such as toxin production, infection, and survival by density‐dependent communication (quorum sensing) using autoinducing peptides (AIPs). Although clostridial AIPs have been proposed to be (thio)lactone‐containing peptides, their true structures remain elusive. Here, we report the genome‐guided discovery of an AIP that controls endospore formation in Ruminiclostridium cellulolyticum. Through a combination of chemical synthesis and chemical complementation assays with a mutant strain, we reveal that the genuine chemical mediator is a homodetic cyclopeptide (cAIP). Kinetic analyses indicate that the mature cAIP is produced via a cryptic thiolactone intermediate that undergoes a rapid S→N acyl shift, in a manner similar to intramolecular native chemical ligation (NCL). Finally, by implementing a chemical probe in a targeted screen, we show that this novel enzyme‐primed, intramolecular NCL is a widespread feature of clostridial AIP biosynthesis., The genome‐guided discovery of an autoinducing peptide (AIP) that controls endospore formation in Ruminiclostridium cellulolyticum is reported. A combination of chemical synthesis and chemical complementation assays with a mutant strain reveals that the genuine chemical mediator is a homodetic cyclopeptide (cAIP).

Isopedopeptins are antibiotic cyclic lipodepsipeptides containing the subsequence L-Thr—L-2,3-diaminopropanoic acid—D-Phe—L-Val/L-3-hydroxyvaline. Acidic hydrolysis of isopedopeptins in D2O showed the D-Phe residues to racemize extensively in peptides with L-3-hydroxyvaline but not in peptides with L-Val. Similarly, one Leu residue in pedopeptins, which are related peptides containing the subsequence Leu—2,3-diaminopropanoic acid—Leu—L-Val/L-3-hydroxyvaline, was found to racemize in peptides with L-3-hydroxyvaline. Model tetrapeptides, L-Ala—L-Phe—L-Val/3-hydroxyvaline—L-Ala, gave the corresponding results, i.e. racemization of L-Phe only when linked to a L-3-hydroxyvaline. We propose the racemization to proceed via an oxazoline intermediate involving Phe/Leu and the L-3-hydroxyvaline residues. The 3-hydroxyvaline residue may form a stable tertiary carbocation by loss of the sidechain hydroxyl group as water after protonation. Elimination of the Phe/Leu H-2 and ring-closure from the carbonyl oxygen onto the carbocation results in the suggested oxazoline intermediate. The reversed reaction leads to either retained or inversed configuration of Phe/Leu. Such racemization during acidic hydrolysis may occur whenever a 3-hydroxyvaline residue or any amino acid that can form a stable carbocation on the C-3, is present in a peptide. The proposed mechanism for racemization was supported by incorporation of 18O in the 3-hydroxyvaline sidechain when the acidic hydrolysis was performed in H2O/H218O (1:1). The 2,3-diaminopropanoic residues of isopedopeptins and pedopeptins were also found to racemize during acidic hydrolysis, as previously described. Based on the results, the configuration of the Leu and 2,3-diaminopropanoic acid residues of the pedopeptins were reassigned to be L-Leu and D-Leu, and 2 × L-2,3-diaminopropanoic acid.

The quest for mastering the controlled engineering of dynamic molecular assemblies is the basis of molecular architectonics. The rational use of noncovalent interactions to programme the molecular assemblies allow the construction of diverse molecular and material architectures with novel functional properties and applications. Understanding and controlling the assembly of molecular systems are daunting tasks owing to the complex factors that govern at the molecular level. Molecular architectures depend on the design of functional molecular modules through the judicious selection of functional core and auxiliary units to guide the precise molecular assembly and co-assembly patterns. Biomolecules with built-in information for molecular recognition are the ultimate examples of evolutionary guided molecular recognition systems that define the structure and functions of living organisms. Explicit use of biomolecules as auxiliary units to command the molecular assemblies of functional molecules is an intriguing exercise in the scheme of molecular architectonics. In this minireview, we discuss the implementation of the principles of molecular architectonics for the development of novel biomaterials with functional properties and applications ranging from sensing, drug delivery to neurogeneration and tissue engineering. We present the molecular designs pioneered by our group owing to the requirement and scope of the article while acknowledging the designs pursued by several research groups that befit the concept.

Blocking the terminal complement pathway with the C5 inhibitor eculizumab has revolutionized the clinical management of several complement-mediated diseases and has boosted the clinical development of new inhibitors. Data on the C3 inhibitor Compstatin and the C5 inhibitors eculizumab and Coversin reported here demonstrate that C3/C5 convertases function differently from prevailing concepts. Stoichiometric C3 inhibition failed to inhibit C5 activation and lytic activity during strong classical pathway activation, demonstrating a “C3 bypass” activation of C5. We show that, instead of C3b, surface-deposited C4b alone can also recruit and prime C5 for consecutive proteolytic activation. Surface-bound C3b and C4b possess similar affinities for C5. By demonstrating that the fluid phase convertase C3bBb is sufficient to cleave C5 as long as C5 is bound on C3b/C4b-decorated surfaces, we show that surface fixation is necessary only for the C3b/C4b opsonins that prime C5 but not for the catalytic convertase unit C3bBb. Of note, at very high C3b densities, we observed membrane attack complex formation in absence of C5-activating enzymes. This is explained by a conformational activation in which C5 adopts a C5b-like conformation when bound to densely C3b-opsonized surfaces. Stoichiometric C5 inhibitors failed to prevent conformational C5 activation, which explains the clinical phenomenon of residual C5 activity documented for different inhibitors of C5. The new insights into the mechanism of C3/C5 convertases provided here have important implications for the development and therapeutic use of complement inhibitors as well as the interpretation of former clinical and preclinical data.

Calmodulin (CaM), a Ca2+ binding protein, plays a critical role in cancer initiation and progression through binding and activating numerous target proteins, including Ca2+ /calmodulin-dependent protein kinase (CaMK) family proteins. However, the mechanisms underlying the effects of CaM/CaMKs on the survival capability of liver cancer cells is unclear, and this study investigates this mechanism in apicidin-persistent HA22T cells. CaM level was upregulated, especially in the cytosol, in apicidin-persistent HA22T cells than in parental HA22T cells and was positively associated with cell proliferation and migration capacity of apicidin-persistent HA22T cells. Further, the expression of CaM-activated CaMKs-dependent signaling cascades, including CaMKK2, CaMKIV, CaMKII-γ, and p-CaMKII was observed in apicidin-persistent HA22T cells, which were transiently activated by mitogen-activated protein kinase oncogenic signaling, such as CREB, ERK1/2, and c-fos. Furthermore, a specific CaM inhibitor trifluoperazine reduced the levels of p-CREB, p-ERK1/2, and c-fos in apicidin-persistent HA22T cells than in parental HA22T cells. Additionally, inhibition of CaM also suppressed CaM-induced Bcl-XL (an antiapoptotic protein) expression in apicidin-persistent HA22T cells. Our finding emphasizes an essential role of CaM/CaMKs in augmentation of the survival capability of apicidin-persistent liver cancer cells and suggests that CaM inhibition significantly attenuates CaM-induced tumor growth and abrogates antiapoptotic function and also offers a promising therapeutic target for cancer treatment.

Background: Increasing the resistance issue has become the reason for the development of new antibacterial in crucial condition. Many ways are tracked to determine the most effective antibacterial agent. Some proteins that are a key role in bacteria metabolism are targeted, including MurA in cell wall biosynthesis and gelatinase biosynthesis-activating pheromone (GBAP) in Fsr Quorum Sensing (QS) system. Objective: The objective of this research is the analysis of compounds 1-4 from M. pendans as antibacterial and anti-QS activity trough protein inhibition by in silico study; focus on the structure-activity relationships, to appraise their role as an antibacterial and anti-QS agent in the molecular level. Methods: Both activities of M. pendans compounds (1-4) were analyzed by in silico, compared to Fosfomycin, Ambuic acid, Quercetin, and Taxifolin as a standard. Chemical structures of M. pendans compounds were converted using an online program molview. The compounds were docked to MurA, GBAP, gelatinase and serine protease using Autodock Vina in Pyrx 0.8 followed PYMOL to visualization and proteis.plus program to analyze of the complex. Results: All compounds from M. pendans bound on MurA, GBAP, gelatinase and serine protease except compound 2. This biflavonoid did not attach to MurA and serine protease yet is the favorable ligand for GBAP and gelatinase with the binding affinity of -6.9 and -9.4 Kcal/mol respectively. Meanwhile, for MurA and serine protease, compound 4 is the highest of bonding energy with values of -8.7 and -6.4 Kcal/mol before quercetin (MurA, -8.9 Kcal/mol) and taxifolin (serine protease, -6.6 Kcal/mol). Conclusion: Based on the data, biflavonoid acts better as anti-QS than an inhibitor of MurA enzyme while the others can be acted into both of them either the therapeutic agent of anti-QS or antibacterial agent of MurA inhibitor.