[Syntheses and Structure-Activity Relationship Studies of Antitumor Bicyclic Hexapeptide RA-VII Analogues].

A series of antitumor bicyclic hexapeptide RA-VII analogues modified at residue 2, 3, or 6 were prepared by the chemical transformation of the hydroxy, methoxy, or carboxy groups or the aromatic rings of natural peptides RA-II, III, V, VII, and X. Analogues with modified side chains or peptide backbones, which cannot be prepared by the chemical transformation of their natural peptides, and newly isolated peptides from Rubia cordifolia roots were synthesized by using protected cycloisodityrosines prepared by the degradation of bis(thioamide) obtained from RA-VII or the diphenyl ether formation of boronodipeptide under the modified Chan-Lam coupling reaction conditions. Studies of the conformational features of the analogues and the newly isolated peptides and their relationships with cytotoxic activities against the HCT-116, HL-60, KATO-III, KB, L1210, MCF-7, and P-388 cell lines revealed the following: the methoxy group at residue 3 is essential for the potent cytotoxic activity; the methyl group at Ala-2 and Ala-4 but not at D-Ala-1 is required to establish the bioactive conformation; the N-methyl group at Tyr-5 is necessary for the peptides to adopt the active conformation preferentially; and the orientation of Tyr-5 and/or Tyr-6 phenyl rings has a significant effect on the cytotoxic activity.

Proline based rationally designed peptide esters against dipeptidyl peptidase-4: Highly potent anti-diabetic agents.

With the target to develop small molecules based anti-diabetic agents, we, herein, report the design, synthesis and biological studies on Lys-Pro and Gly-Pro esters, and a Phe-Pro-Phe tripeptide inhibiting the activity of glycoprotein dipeptidyl peptidase-4 (DPP-4). Since DPP-4 cleaves the glucagon like peptide (GLP-1) and glucose dependent insulinotropic polypeptide (GIP) hormones which are responsible for inducing insulin secretion, the results of present studies could be significant in making control over glycemia. The structural analysis of DPP-4 and its binding mode with the substrate as well as the reported inhibitors provided the background for the design of new molecules. Among the 17 compounds screened against DPP-4, 14 compounds displayed IC50 better than the known drug Sitagliptin. Collectively, a highly encouraging set of molecules was identified that may prove as the clinical candidates for the treatment of diabetes.

Cathepsin D inhibitors based on tasiamide B derivatives with cell membrane permeability.

Cathepsin D (Cath D) has been evidenced as a potential target for cancer therapy. Our previous studies revealed that TB-9, a tasiamide B derivative, exhibited highly potent inhibition against Cath D with satisfactory selectivity over Cath E and BACE1. But this compound was inactive on cell level possibly due to poor membrane permeability. Herein, we report the design, synthesis, and evaluation of two novel Cath D inhibitors (2 and 3) which combining tasiamide B scaffold with a cell penetrating peptide (CPP) specifically targeting the endolysosomal compartment. The results revealed that 2 and 3 not only retained highly potent inhibition against Cath D, but also were active against MDA-MB-231 cell lines.

C-terminal modified Enkephalin-like tetrapeptides with enhanced affinities at the kappa opioid receptor and monoamine transporters.

A new series of enkephalin-like tetrapeptide analogs modified at the C-terminus by an N-(3,4-dichlorophenyl)-N-(piperidin-4-yl)propionamide (DPP) moiety were designed, synthesized, and tested for their binding affinities at opioid receptors and monoamine transporters to evaluate their potential multifunctional activity for the treatment of chronic pain. Most ligands exhibited high binding affinities in the nanomolar range at the opioid receptors with a slight delta-opioid receptor (DOR) selectivity over mu-opioid receptor (MOR) and kappa-opioid receptor (KOR) and low binding affinities in the micromolar range at the monoamine transporters, SERT and NET. Ligands of which the positions 1 and 4 were substituted by Dmt and Phe(4-X) residues, respectively, showed the excellent binding affinities at three opioid receptors. Among them, Dmt-d-Tic-Gly-Phe(4-F)-DPP was the most promising considering its excellent opioid affinities, particularly unexpected high binding affinity (Ki = 0.13 nM) at the KOR, and moderate interactions with serotonin/norepinephrine reuptake inhibitors (SNRIs). Docking studies revealed that the ligand was a good fit for the KOR binding pocket (binding score = 8,750).

Synthetic Oligopeptides Mimicking γ-Core Regions of Cysteine-Rich Peptides of Solanum lycopersicum Possess Antimicrobial Activity against Human and Plant Pathogens.

Plant cysteine-rich peptides (CRPs) represent a diverse group of molecules involved in different aspects of plant physiology. Antimicrobial peptides, which directly suppress the growth of pathogens, are regarded as promising templates for the development of next-generation pharmaceuticals and ecologically friendly plant disease control agents. Their oligopeptide fragments are even more promising because of their low production costs. The goal of this work was to explore the antimicrobial activity of nine short peptides derived from the γ-core-containing regions of tomato CRPs against important plant and human pathogens. We discovered antimicrobial activity in peptides derived from the defensin-like peptides, snakins, and MEG, which demonstrates the direct involvement of these CRPs in defense reactions in tomato. The CRP-derived short peptides appeared particularly active against the gram-positive bacterium Clavibacter michiganensis, which causes bacterial wilt-opening up new possibilities for their use in agriculture to control this dangerous disease. Furthermore, high inhibitory potency of short oligopeptides was demonstrated against the yeast Cryptococcus neoformans, which causes serious diseases in humans, making these peptide molecules promising candidates for the development of next-generation pharmaceuticals. Studies of the mode of action of the two most active peptides indicate fungal membrane permeabilization as a mechanism of antimicrobial action.

Proliferating coacervate droplets as the missing link between chemistry and biology in the origins of life.

The hypothesis that prebiotic molecules were transformed into polymers that evolved into proliferating molecular assemblages and eventually a primitive cell was first proposed about 100 years ago. To the best of our knowledge, however, no model of a proliferating prebiotic system has yet been realised because different conditions are required for polymer generation and self-assembly. In this study, we identify conditions suitable for concurrent peptide generation and self-assembly, and we show how a proliferating peptide-based droplet could be created by using synthesised amino acid thioesters as prebiotic monomers. Oligopeptides generated from the monomers spontaneously formed droplets through liquid-liquid phase separation in water. The droplets underwent a steady growth-division cycle by periodic addition of monomers through autocatalytic self-reproduction. Heterogeneous enrichment of RNA and lipids within droplets enabled RNA to protect the droplet from dissolution by lipids. These results provide experimental constructs for origins-of-life research and open up directions in the development of peptide-based materials.

Antibacterial sulfonimidamide-based oligopeptides as type I signal peptidase inhibitors: Synthesis and biological evaluation.

Oligopeptide boronates with a lipophilic tail are known to inhibit the type I signal peptidase in E. coli, which is a promising drug target for developing novel antibiotics. Antibacterial activity depends on these oligopeptides having a cationic modification to increase their permeation. Unfortunately, this modification is associated with cytotoxicity, motivating the need for novel approaches. The sulfonimidamide functionality has recently gained much interest in drug design and discovery, as a means of introducing chirality and an imine-handle, thus allowing for the incorporation of additional substituents. This in turn can tune the chemical and biological properties, which are here explored. We show that introducing the sulfonimidamide between the lipophilic tail and the peptide in a series of signal peptidase inhibitors resulted in antibacterial activity, while the sulfonamide isostere and previously known non-cationic analogs were inactive. Additionally, we show that replacing the sulfonamide with a sulfonimidamide resulted in decreased cytotoxicity, and similar results were seen by adding a cationic sidechain to the sulfonimidamide motif. This is the first report of incorporation of the sulfonimidamide functional group into bioactive peptides, more specifically into antibacterial oligopeptides, and evaluation of its biological effects.

Solid-phase synthesis and evaluation of linear and cyclic ferrocenoyl/ruthenocenoyl water-soluble hexapeptides as potential antibacterial compounds.

A series of novel water-soluble short peptide-bioconjugates containing a ferrocenoyl (Fc) or ruthenocenoyl (Rc) unit was synthesized and characterized to combine the unique activity of ferrocene and the isoelectronic ruthenocene with precisely designed peptide structures. We aim at evaluating these bioconjugates as a new class of OrganoMetallic Short AntiMicrobial Peptides (OM-SAMPs). The series of OM-SAMPs was designed with a set of linear and "head-to-tail" cyclic metallocene-based hexapeptides derived from the homo-sequence H-KKKKKK-NH2 by substitution of lysine (K) by tryptophan (W) and by orthogonal derivatization of the ε-N-amine group of lysine by a metallocene moiety. Peptide conjugates were characterized by RP-HPLC, mass spectrometry (ESI and MALDI-TOF) and circular dichroism (CD) spectroscopy. Gram-positive and Gram-negative antibacterial activity testings were carried out to explore the role of insertion of the metallocene fragment into the peptide, and the effect of the modification of the cationic charge and aromatic residues on the physiochemical properties of these OM-SAMPs. These results show that the insertion of two tryptophan residues and ferrocenoyl/ruthenocenoyl moieties into a linear homo-sequence peptides increase significantly their antibacterial activity with minimum inhibitory concentration values as low as 5 µM for the most active compounds. However, "head-to-tail" cyclic metallocene-based hexapeptides were not active against Gram-negative bacteria up to concentrations of 50 µM. These studies provide a better understanding of the role of structural modifications to enhance antibacterial peptide activity, which is promising for their therapeutic application.

Chemoproteomic Study Uncovers HemK2/KMT9 As a New Target for NTMT1 Bisubstrate Inhibitors.

Understanding the selectivity of methyltransferase inhibitors is important to dissecting the functions of each methyltransferase target. From this perspective, we report a chemoproteomic study to profile the selectivity of a potent protein N-terminal methyltransferase 1 (NTMT1) bisubstrate inhibitor NAH-C3-GPKK (Ki, app = 7 ± 1 nM) in endogenous proteomes. First, we describe the rational design, synthesis, and biochemical characterization of a new chemical probe 6, a biotinylated analogue of NAH-C3-GPKK. Next, we systematically analyze protein networks that may selectively interact with the biotinylated probe 6 in concert with the competitor NAH-C3-GPKK. Besides NTMT1, the designated NTMT1 bisubstrate inhibitor NAH-C3-GPKK was found to also potently inhibit a methyltransferase complex HemK2-Trm112 (also known as KMT9-Trm112), highlighting the importance of systematic selectivity profiling. Furthermore, this is the first potent inhibitor for HemK2/KMT9 reported until now. Thus, our studies lay the foundation for future efforts to develop selective inhibitors for either methyltransferase.

Single nucleotide translation without ribosomes.

The translation of messenger RNA sequences into polypeptide sequences according to the genetic code is central to life. How this process, which relies on the ribosomal machinery, arose from much simpler precursors is unclear. Here, we demonstrate that single nucleotides charged with an amino acid couple with amino acids linked to the 5'-terminus of an RNA primer in reactions directed by the nucleotides of an RNA template in dilute aqueous solution at 0 °C. When a mixture of U-Val, A-Gly and G-Leu competed for coupling to Gly-RNA, base pairing dictated which dipeptide sequence formed preferentially. The resulting doubly anchored dipeptides can retain their link to the primer for further extension or can be fully released under mild acidic conditions. These results show that a single-nucleotide-based form of translation exists that requires no more than oligoribonucleotides and anchored amino acids.

Bio-Functionalized Chitosan for Bone Tissue Engineering.

Hybrid biomaterials allow for the improvement of the biological properties of materials and have been successfully used for implantology in medical applications. The covalent and selective functionalization of materials with bioactive peptides provides favorable results in tissue engineering by supporting cell attachment to the biomaterial through biochemical cues and interaction with membrane receptors. Since the functionalization with bioactive peptides may alter the chemical and physical properties of the biomaterials, in this study we characterized the biological responses of differently functionalized chitosan analogs. Chitosan analogs were produced through the reaction of GRGDSPK (RGD) or FRHRNRKGY (HVP) sequences, both carrying an aldehyde-terminal group, to chitosan. The bio-functionalized polysaccharides, pure or "diluted" with chitosan, were chemically characterized in depth and evaluated for their antimicrobial activities and biocompatibility toward human primary osteoblast cells. The results obtained indicate that the bio-functionalization of chitosan increases human-osteoblast adhesion (p < 0.005) and proliferation (p < 0.005) as compared with chitosan. Overall, the 1:1 mixture of HVP functionalized-chitosan:chitosan is the best compromise between preserving the antibacterial properties of the material and supporting osteoblast differentiation and calcium deposition (p < 0.005 vs. RGD). In conclusion, our results reported that a selected concentration of HVP supported the biomimetic potential of functionalized chitosan better than RGD and preserved the antibacterial properties of chitosan.

Modified histidine containing amphipathic ultrashort antifungal peptide, His[2-p-(n-butyl)phenyl]-Trp-Arg-OMe exhibits potent anticryptococcal activity.

In pursuit of ultrashort peptide-based antifungals, a new structural class, His(2-aryl)-Trp-Arg is reported. Structural changes were investigated on His-Trp-Arg scaffold to demonstrate the impact of charge and lipophilic character on the biological activity. The presence and size of the aryl moiety on imidazole of histidine modulated overall amphiphilic character, and biological activity. Peptides exhibited IC50 of 0.37-9.66 µg/mL against C. neoformans. Peptide 14f [His(2-p-(n-butyl)phenyl)-Trp-Arg-OMe] exhibited two-fold potency (IC50 = 0.37 µg/mL, MIC = 0.63 µg/mL) related to amphotericin B, without any cytotoxic effects up to 10 µg/mL. Peptide 14f act by nuclear fragmentation, membranes permeabilization, disruption and pore formations in the microbial cells as determined by the mechanistic studies employing Trp-quenching, CLSM, SEM, and HR-TEM. The amalgamation of short sequence, presence of appropriate aryl group on l-histidine, potent anticryptococcal activity, no cytotoxicity, and detailed mechanistic studies directed to the identification of 14f as a new antifungal structural lead.

Allenone-Mediated Racemization/Epimerization-Free Peptide Bond Formation and Its Application in Peptide Synthesis.

Allenone has been identified as a highly effective peptide coupling reagent for the first time. The peptide bond was formed with an α-carbonyl vinyl ester as the key intermediate, the formation and subsequent aminolysis of which proceed spontaneously in a racemization-/epimerization-free manner. The allenone coupling reagent not only is effective for the synthesis of simple amides and dipeptides but is also amenable to peptide fragment condensation and solid-phase peptide synthesis (SPPS). The robustness of the allenone-mediated peptide bond formation was showcased incisively by the synthesis of carfilzomib, which involved a rare racemization-/epimerization-free N to C peptide elongation strategy. Furthermore, the successful synthesis of the model difficult peptide ACP (65-74) on a solid support suggested that this method was compatible with SPPS. This method combines the advantages of conventional active esters and coupling reagents, while overcoming the disadvantages of both strategies. Thus, this allenone-mediated peptide bond formation strategy represents a disruptive innovation in peptide synthesis.

RGD-Peptide Functionalization Affects the In Vivo Diffusion of a Responsive Trimeric MRI Contrast Agent through Interactions with Integrins.

The relevance of MRI as a diagnostic methodology has been expanding significantly with the development of molecular imaging. Partially, the credit for this advancement is due to the increasing potential and performance of targeted MRI contrast agents, which are able to specifically bind distinct receptors or biomarkers. Consequently, these allow for the identification of tissues undergoing a disease, resulting in the over- or underexpression of the particular molecular targets. Here we report a multimeric molecular probe, which combines the established targeting properties of the Arg-Gly-Asp (RGD) peptide sequence toward the integrins with three calcium-responsive, Gd-based paramagnetic moieties. The bifunctional probe showed excellent 1H MRI contrast enhancement upon Ca2+ coordination and demonstrated a longer retention time in the tissue due to the presence of the RGD moiety. The obtained results testify to the potential of combining bioresponsive contrast agents with targeting vectors to develop novel functional molecular imaging methods.

Solvent-tailored ordered self-assembly of oligopeptide amphiphiles to create an anisotropic meso-matrix.

Herein, we have developed a solvent-tailored ordered self-assembly strategy to create anisotropic nanomaterials. A trace amount of water has been found to be a predominant factor to direct peptide self-assembly into an anisotropic meso-matrix in DMSO. The obtained meso-matrix was applied to measure the anisotropic RDC parameter of organic molecules for structural elucidation.

Discovery of novel tripeptide propylene oxide proteasome inhibitors for the treatment of multiple myeloma.

The ubiquitin proteasome pathway (UPP) plays a critical role in the maintenance of cell homeostasis and the development of diseases, such as cancer and neurodegenerative disease. A series of novel tripeptide propylene oxide compounds as proteasome inhibitors were designed, synthesized and biologically investigated in this manuscript. The enzymatic activities of final compounds against 20S human proteasome were investigated and structure-activity relationship (SAR) was summarized. Some potent compounds were further evaluated to inhibit the proliferation of multiple myeloma (MM) cancer cell lines RPMI8226 and U266B. The results showed that some compounds were active against MM cancer cell lines with IC50 values of less than 50 nM. The microsomal metabolic stabilities in human, rat and mice species were carried out and the results showed that compounds 30 and 31 were stable enough to be in vivo investigated. The in vivo pharmacokinetic results showed that compounds 30 and 31 had acceptable biological parameters for both ig and iv administrations. In vivo antitumor activities of compounds 30 and 31 with the doses of 100 mg/kg and 50 mg/kg BIW were performed by using RPMI8226 xenograft nude mouse model. Toxicities of compounds 30 and 31 were not observed during the experiment and dose dependent effect was obvious and the tumor volume was greatly inhibited.

A comprehensive review of chemistry and pharmacological aspects of natural cyanobacterial azoline-based circular and linear oligopeptides.

The cyanobacterial oligopeptides are recognized for being highly selective, efficacious and relatively safer compounds with diverse bioactivities. Azoline-based natural compounds consist of heterocycles which are reduced analogues of five-membered heterocyclic azoles. Among other varieties of azoline-based natural compounds, the heteropeptides bearing oxazoline or thiazoline heterocycles possess intrinsic structural properties with captivating pharmacological profiles, representing excellent templates for the design of novel therapeutics. The specificity of heteropeptides has been translated into prominent safety, tolerability, and efficacy profiles in humans. These peptidic congeners serve as ideal intermediary between small molecules and biopharmaceuticals based on their typically low production complexity compared to the protein-based biopharmaceuticals. The distinct bioproperties and unique structures render these heteropeptides one of the most promising lead compounds for drug discovery. The high degree of chemical diversity in cyanobacterial secondary metabolites may constitute a prolific source of new entities leading to the development of new pharmaceuticals. This review focuses on the azoline-based natural oligopeptides with emphasis on distinctive structural features, stereochemical aspects, biological activities, structure activity relationship, synthetic and biosynthetic aspects as well as mode of action of cyanobacteria-derived peptides.

Investigating the phosphinic acid tripeptide mimetic DG013A as a tool compound inhibitor of the M1-aminopeptidase ERAP1.

ERAP1 is a zinc-dependent M1-aminopeptidase that trims lipophilic amino acids from the N-terminus of peptides. Owing to its importance in the processing of antigens and regulation of the adaptive immune response, dysregulation of the highly polymorphic ERAP1 has been implicated in autoimmune disease and cancer. To test this hypothesis and establish the role of ERAP1 in these disease areas, high affinity, cell permeable and selective chemical probes are essential. DG013A 1, is a phosphinic acid tripeptide mimetic inhibitor with reported low nanomolar affinity for ERAP1. However, this chemotype is a privileged structure for binding to various metal-dependent peptidases and contains a highly charged phosphinic acid moiety, so it was unclear whether it would display the high selectivity and passive permeability required for a chemical probe. Therefore, we designed a new stereoselective route to synthesize a library of DG013A 1 analogues to determine the suitability of this compound as a cellular chemical probe to validate ERAP1 as a drug discovery target.

Naphthalimide-Containing BP100 Leads to Higher Model Membranes Interactions and Antimicrobial Activity.

In a large variety of organisms, antimicrobial peptides (AMPs) are primary defenses against pathogens. BP100 (KKLFKKILKYL-NH2), a short, synthetic, cationic AMP, is active against bacteria and displays low toxicity towards eukaryotic cells. BP100 acquires a α-helical conformation upon interaction with membranes and increases membrane permeability. Despite the volume of information available, the action mechanism of BP100, the selectivity of its biological effects, and possible applications are far from consensual. Our group synthesized a fluorescent BP100 analogue containing naphthalimide linked to its N-terminal end, NAPHT-BP100 (Naphthalimide-AAKKLFKKILKYL-NH2). The fluorescence properties of naphthalimides, especially their spectral sensitivity to microenvironment changes, are well established, and their biological activities against transformed cells and bacteria are known. Naphthalimide derived compounds are known to interact with DNA disturbing related processes as replication and transcription, and used as anticancer agents due to this property. A wide variety of techniques were used to demonstrate that NAPHT-BP100 bound to and permeabilized zwitterionic POPC and negatively charged POPC:POPG liposomes and, upon interaction, acquired a α-helical structure. Membrane surface high peptide/lipid ratios triggered complete permeabilization of the liposomes in a detergent-like manner. Membrane disruption was driven by charge neutralization, lipid aggregation, and bilayer destabilization. NAPHT-BP100 also interacted with double-stranded DNA, indicating that this peptide could also affect other cellular processes besides causing membrane destabilization. NAPHT-BP100 showed increased antibacterial and hemolytic activities, compared to BP100, and may constitute an efficient antimicrobial agent for dermatological use. By conjugating BP100 and naphthalimide DNA binding properties, NAPHT-BP100 bound to a large extent to the bacterial membrane and could more efficiently destabilize it. We also speculate that peptide could enter the bacteria cell and interact with its DNA in the cytoplasm.

Synthesis and Chemical and Biological Evaluation of a Glycine Tripeptide Chelate of Magnesium.

Magnesium (Mg2+) plays a crucial role in over 80% of all metabolic functions. It is becoming increasingly apparent that magnesium deficiency (hypomagnesemia) may play an important role in chronic disease. To counteract magnesium deficiency, there is an unmet clinical need to develop new fully characterized, highly bioavailable, and substantially water-soluble magnesium supplements. To this end, triglycine (HG3), a tripeptide of the amino acid glycine, was chosen as a chelating ligand for magnesium, given its natural occurrence and water solubility, and entropically-driven metal binding. Herein, we discuss the synthesis, chemical and physical characterization, and cellular uptake of a magnesium triglycine chelate (MgG3), an octahedral complex with extraordinary water solubility and improved cellular uptake in CaCo-2 cells than select commonly used magnesium supplements.