Cyclic Tetrapeptide Research Articles

Design and Synthesis of Simplified Largazole Analogues as Isoform-Selective Human Lysine Deacetylase Inhibitors.

Selective inhibition of KDAC isoforms while maintaining potency remains a challenge. Using the largazole macrocyclic depsipeptide structure as a starting point for developing new KDACIs with increased selectivity, a combination of four different simplified largazole analogue (SLA) scaffolds with diverse zinc-binding groups (for a total of 60 compounds) were designed, synthesized, and evaluated against class I KDACs 1, 3, and 8, and class II KDAC6. Experimental evidence as well as molecular docking poses converged to establish the cyclic tetrapeptides (CTPs) as the primary determinant of both potency and selectivity by influencing the correct alignment of the zinc-binding group in the KDAC active site, providing a further basis for developing new KDACIs of higher isoform selectivity and potency.

A modular approach to cyclic tetrapeptides related to histone deacetylase inhibition: synthesis of epi-microsporin A

A short new route to the unusual amino acid component (S)-2-amino-8-oxodecanoic acid (Aoda) of some important HDAC inhibitors has been developed using cross metathesis reaction as the key step. An extension of the study employing grafting of a macrocyclic scaffold through cross metathesis has proved to be highly efficient for the synthesis of an isomer of the cyclic tetrapeptide HDAC inhibitor microsporin.

The reactivity and conformational control of cyclic tetrapeptides derived from aziridine-containing amino acids.

Among the smallest of the macrocyclic peptides, 12- and 13-membered cyclic tetrapeptides are particularly noteworthy because they exhibit a broad spectrum of biological activities due to their innate capacity to mimic β-turns in proteins. In this report, we demonstrate that aziridine-containing cyclic tetrapeptides offer a platform to interrogate the conformational properties of tetrapeptides. We show that aziridine ring-opening of 12-membered cyclic tetrapeptides yields exclusively 13-membered α3β macrocycles, regardless of peptide sequence, nucleophile, aziridine β-carbon substitution, or stereochemistry. NMR and computational studies on two related aziridine-containing cyclic tetrapeptides revealed that the amide conformations of their N-acyl aziridines are similar, and are likely the determinant of the observed ring-opening regioselectivity. Interestingly, some of the resulting 13-membered α3β macrocycles were found to be conformationally heterogeneous. This study on the reactivity and conformational control of aziridine-containing cyclic tetrapeptides provides useful insight on the design and development of macrocyclic therapeutics.

ChemInform Abstract: Diversity‐Oriented Synthesis of Analogues of the Novel Macrocyclic Peptide FR‐225497 Through Late Stage Functionalization.

AbstractA concise synthetic approach to a class of biologically interesting cyclic tetrapeptides is reported which involves a late‐stage functionalization of a macrocyclic scaffold through cross metathesis in an attempt to create diversity.

Anthranilic acid-containing cyclic tetrapeptides: at the crossroads of conformational rigidity and synthetic accessibility.

Each amino acid in a peptide contributes three atom units to main-chains, hence natural cyclic peptides can be 9, 12, 15, …. i.e. 3n membered-rings, where n is the number of amino acids. Cyclic peptides that are 9 or 12-membered ring compounds tend to be hard to prepare because of strain, while their one amino acid homologs (15-membered cyclic pentapeptides) are not conformationally homogeneous unless constrained by strategically placed proline or d-amino acid residues. We hypothesized that replacing one genetically encoded amino acid in a cyclic tetrapeptide with a rigid β-amino acid would render peptidomimetic designs that rest at a useful crossroads between synthetic accessibility and conformational rigidity. Thus this research explored non-proline containing 13-membered ring peptides 1 featuring one anthranilic acid (Anth) residue. Twelve cyclic peptides of this type were prepared, and in doing so the viability of both solution- and solid-phase methods was demonstrated. The library produced contained a complete set of four diastereoisomers of the sequence 1aaf (i.e. cyclo-AlaAlaPheAnth). Without exception, these four diastereoisomers each adopted one predominant conformation in solution; basically these conformations feature amide N-H vectors puckering above and below the equatorial plane, and approximately oriented their N-H[combining low line] atoms towards the polar axis. Moreover, the shapes of these conformers varied in a logical and predictable way (NOE, temperature coefficient, D/H exchange, circular dichroism). Comparisons were made of the side-chain orientations presented by compounds 1aaa in solution with ideal secondary structures and protein-protein interaction interfaces. Various 1aaa stereoisomers in solution present side-chains in similar orientations to regular and inverse γ-turns, and to the most common β-turns (types I and II). Consistent with this, compounds 1aaa have a tendency to mimic various turns and bends at protein-protein interfaces. Finally, proteolytic- and hydrolytic stabilities of the compounds at different pHs indicate they are robust relative to related linear peptides, and rates of permeability through an artificial membrane indicate their structures are conducive to cell permeability.

Molecular tectonics: homochiral coordination polymers based on pyridyl-substituted cyclic tetrapeptides

Upon combining enantiopure bis-4-pyridylphenyl-substituted cyclotetrapeptides with HgCl2 and CdCl2, homochiral 1D zigzag coordination polymers were obtained, while the use of a bis-4-pyridyl-substituted cyclotetrapeptide with HgCl2 led to the formation of a different type of 1D coordination network.

Diversity-oriented synthesis of analogues of the novel macrocyclic peptide FR-225497 through late stage functionalization.

A concise synthetic approach to a class of biologically interesting cyclic tetrapeptides is reported which involves a late-stage functionalization of a macrocyclic scaffold through cross metathesis in an attempt to create diversity. The utility of this protocol is demonstrated through the preparation of three structural analogues of the important naturally occurring histone deacetylase inhibitor FR-225497.

Synthetic strategy for bicyclic tetrapeptides HDAC inhibitors using ring closing metathesis

Cyclic peptides show diverse biological activities and are considered as good therapeutic agents due to structural rigidity, receptor selectivity and biochemical stability. We have developed bicyclic tetrapeptide HDAC inhibitors based on different cyclic tetrapeptide scaffolds. For the synthesis of these bicyclic tetrapeptides, two cyclization steps, namely, peptide cyclization and fusion of aliphatic side chains by ring closing metathesis (RCM) were involved. In the course of these syntheses, we have established two facts: a lower limit of aliphatic loop length and better synthetic route for bicyclic tetrapeptide synthesis. It was found that nine methylene loop length is the lower limit for aliphatic loop and the synthetic route selection depended on the configuration of amino acids in the cyclic tetrapeptide scaffold. RCM followed by peptide cyclization was the proper route for LDLD configuration and the reverse route was suitable for LLLD configuration. Nine methylene units is the lower limit for aliphatic loop length and the synthetic route selection depends on the configuration of amino acids in the cyclic tetrapeptide scaffold. RCM followed by peptide cyclization is the proper route for LDLD configuration and reverse route for LLLD configuration.

New cyclic tetrapeptide from the coral-derived endophytic bacteria Brevibacterium sp. L-4 collected from the South China Sea

One new cyclic tetrapeptide cyclic-(Tyr-Ala-Leu-Ser) (1) along with four natural compounds firstly obtained 3H-imidazole-4-carboxylic acid (2), 2-methyl-3H-imidazole-4-carboxylic acid (3), 3-ethylidene-6-isopropyl-piperazine-2,5-dione (4), and 3-isobutylidene-6-methyl piperazine-2,5-dione (5) have been isolated from the coral derived endophytic bacteria Brevibacterium sp. L-4 collected from the South China Sea. Their structures were elucidated through spectroscopic techniques including NMR (1D and 2D), MS, and EA, and their relative configurations were also assigned by NMR analysis.

Combining Stable Isotope Labeling and Molecular Networking for Biosynthetic Pathway Characterization.

Filamentous fungi are a rich source of bioactive compounds, ranging from statins over immunosuppressants to antibiotics. The coupling of genes to metabolites is of large commercial interest for production of the bioactives of the future. To this end, we have investigated the use of stable isotope labeled amino acids (SILAAs). SILAAs were added to the cultivation media of the filamentous fungus Aspergillus nidulans for the study of the cyclic tetrapeptide nidulanin A. Analysis by UHPLC-TOFMS confirmed that the SILAAs were incorporated into produced nidulanin A, and the change in observed m/z could be used to determine whether a compound (known or unknown) incorporated any of the added amino acids. Samples were then analyzed using MS/MS and the data used to perform molecular networking. The molecular network revealed several known and unknown compounds that were also labeled. Assisted by the isotope labeling, it was possible to determine the sequence of several of the compounds, one of which was the known metabolite fungisporin, not previously described in A. nidulans. Several novel analogues of nidulanin A and fungisporin were detected and tentatively identified, and it was determined that these metabolites were all produced by the same nonribosomal peptide synthase. The combination of stable isotope labeling and molecular network generation was shown to very effective for the automated detection of structurally related nonribosomal peptides, while the labeling was effective for determination of the peptide sequence, which could be used to provide information on biosynthesis of bioactive compounds.

Antifouling Compounds from the Marine-Derived Fungus Aspergillus terreus SCSGAF0162

A new cyclic tetrapeptide, asperterrestide B (1), and 11 known compounds (2-12) were isolated from a marine-derived fungus Aspergillus terreus SCSGAF0162. The structure of 1 was elucidated by spectroscopic analysis, and the absolute configuration of 1 was determined by Mosher ester and Marfey's methods. Compounds 4, 6, and 8 had potent antifouling activity against larvae of the barnacle Balanus amphitrite, with EC50 values of 17.1 ± 1.2, 11.6 ± 0.6, and 17.1 ± 0.8 μg x mL(-1), respectively.

Staphylopeptide A, a new cyclic tetrapeptide from culture broth of Staphylococcus sp.

Re-investigation of the culture broth of the marine bacterium Staphylococcus sp. (no. P-100826-4-6) afforded a new cyclic tetrapeptide namely staphylopeptide A (1), along with five known compounds, cyclo (l-prolyl-l-valine) (2), cyclo (l-prolyl-l-tyrosine), (3), cyclo (l-prolyl-l-alanine) (4), l-phenylalanine (5), and l-tryptophan (6). The structures of the isolated compounds were determined by extensive IR, 1D (1H and 13C) and 2D (1H-1H COSY, HSQC, HMBC, and NOESY) NMR and HRFABMS spectral measurements. The antimicrobial activity of the isolated compounds (1–6) was evaluated.

Antitumor and antimicrobial activity of some cyclic tetrapeptides and tripeptides derived from marine bacteria.

Marine derived cyclo(Gly-l-Ser-l-Pro-l-Glu) was selected as a lead to evaluate antitumor-antibiotic activity. Histidine was chosen to replace the serine residue to form cyclo(Gly-l-His-l-Pro-l-Glu). Cyclic tetrapeptides (CtetPs) were then synthesized using a solution phase method, and subjected to antitumor and antibiotic assays. The benzyl group protected CtetPs derivatives, showed better activity against antibiotic-resistant Staphylococcus aureus in the range of 60–120 μM. Benzyl group protected CtetPs 3 and 4, exhibited antitumor activity against several cell lines at a concentration of 80–108 μM. However, shortening the size of the ring to the cyclic tripeptide (CtriP) scaffold, cyclo(Gly-l-Ser-l-Pro), cyclo(Ser-l-Pro-l-Glu) and their analogues showed no antibiotic or antitumor activity. This phenomenon can be explained from their backbone structures.

Autophagy inhibitor sensitizes MCF-7 breast cancer cells to novel cyclic tetrapeptide CTS203-induced caspase-9-dependent apoptotic cell death.

Histone deacetylase (HDAC) inhibitors have been demonstrated to be effective anti-cancer candidates against aggressive malignancies. In previous study, anovel hydroxamic acid derivate, CTS203 (cyclo(-l-Asu(NHOH)-l-A3mc6c-l-Phe-d-Pro-)), demonstrated promising HDAC inhibitory activity. Herein, more biological evaluations including cell viability, cell cycle distribution, cellular morphology, expression quantification as well as protein-protein interactions were measured to investigate its cytotoxic mechanism. Corresponding with its significant HDAC inhibitory activity, CTS203 led to increased acetylation of H3K14, cell cycle arrest as well as consequent apoptotic cell death, with bearable influence on the viability of normal cells. However, schedule-dependent cytotoxicity against MCF-7 breast cancer cells revealed adelayed cellular response to chemo-stimuli. Within this corresponding period, autophagy was rapidly triggered once exposure started, whereas autophagy inhibitor sensitized MCF-7 cells to CTS203, exhibiting synergistically anti-proliferative effects. The expression variation in MCF-7 cells revealed that the cleavage of Beclin 1 mediated by caspase-8 resulted in disabled autophagy, thus ultimately facilitated and fastened caspase-9-dependent apoptotic cell death. Taken together, these findings elucidated the mechanism of CTS203-induced cytotoxicity as well as suggested that appropriate manipulation of autophagy would be an adjunctive strategy to enhance HDAC inhibitor-induced cell death. cyclic tetrapeptide, apoptosis, autophagy, HDAC inhibitor, Beclin 1 cleavage.

Ustiloxins, fungal cyclic peptides, are ribosomally synthesized in Ustilaginoidea virens.

Ustiloxins A and B are toxic cyclic tetrapeptides, Tyr-Val/Ala-Ile-Gly (Y-V/A-I-G), that were originally identified from Ustilaginoidea virens, a pathogenic fungus affecting rice plants. Contrary to our report that ustiloxin B is ribosomally synthesized in Aspergillus flavus, a recent report suggested that ustiloxins are synthesized by a non-ribosomal peptide synthetase in U.virens. Thus, we analyzed the U.virens genome, to identify the responsible gene cluster. The biosynthetic gene cluster was identified from the genome of U.virens based on homologies to the ribosomal peptide biosynthetic gene cluster for ustiloxin B identified from A.flavus. It contains a gene encoding precursor protein having five Tyr-Val-Ile-Gly and three Tyr-Ala-Ile-Gly motifs for ustiloxins A and B, respectively, strongly indicating that ustiloxins A and B from U.virens are ribosomally synthesized. Accession codes of the U.virens and A.flavus gene clusters in NCBI are BR001221 and BR001206, respectively. Supplementary data are available at Bioinformatics online.

A New Cyclic Peptide and a New Steroid from the Fungus Penicillium sp. GD6 Isolated from the Mangrove Bruguiera gymnorrhiza

AbstractA rare new cyclic tetrapeptide, 5,5′‐epoxy‐MKN‐349A (1), featured by a MKN‐349A (5) skeleton and containing an uncommon ether bridge between C(5) and C(5′), and a new steroid, named 11‐O‐acetyl‐NGA0187 (2), together with two known steroids, 3 and 4, were isolated from an endophytic fungus Penicillium sp. GD6 associated with the Chinese mangrove Bruguiera gymnorrhiza. The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and by comparison with the data of related compounds reported in literature. Neither of the compounds 3 and 4, isolated in this study, showed obvious bioactivities in the antibacterial bioassay experiments.

ChemInform Abstract: Synthesis and Conformational Analysis of Cyclic Homooligomers from Pyranoid ε‐Sugar Amino Acids.

New pyranoid e-sugar amino acids were designed as building blocks, in which the carboxylic acid and the amine groups were placed in positions C2 and C3 with respect to the tetrahydropyran oxygen atom. By using standard solution-phase coupling procedures, cyclic homooligomers containing pyranoid e-sugar amino acids were synthesized. Conformation analysis was performed by using NMR spectroscopic experiments, FTIR spectroscopic studies, X-ray analysis, and a theoretical conformation search. These studies reveal that the presence of a methoxy group in the position C4 of the pyran ring produces an important structural change in the cyclodipeptides. When the methoxy groups are present, the structure collapses through interresidue hydrogen bonds between the oxygen atoms of the pyran ring and the amide protons. However, when the cyclodipeptide lacks the methoxy groups, a U-shape structure is adopted, in which there is a hydrophilic concave face with four oxygen atoms and two amide protons directed toward the center of the cavity. Additionally, we found important evidence of the key role played by weak electrostatic interactions, such as the five-membered hydrogen-bonded pseudocycles (C5) between the amide protons and the ether oxygen atoms, in the conformation equilibrium of the macrocycles and in the cyclization step of the cyclic tetrapeptides.

Apicidin F: characterization and genetic manipulation of a new secondary metabolite gene cluster in the rice pathogen Fusarium fujikuroi.

The fungus F. fujikuroi is well known for its production of gibberellins causing the ‘bakanae’ disease of rice. Besides these plant hormones, it is able to produce other secondary metabolites (SMs), such as pigments and mycotoxins. Genome sequencing revealed altogether 45 potential SM gene clusters, most of which are cryptic and silent. In this study we characterize a new non-ribosomal peptide synthetase (NRPS) gene cluster that is responsible for the production of the cyclic tetrapeptide apicidin F (APF). This new SM has structural similarities to the known histone deacetylase inhibitor apicidin. To gain insight into the biosynthetic pathway, most of the 11 cluster genes were deleted, and the mutants were analyzed by HPLC-DAD and HPLC-HRMS for their ability to produce APF or new derivatives. Structure elucidation was carried out be HPLC-HRMS and NMR analysis. We identified two new derivatives of APF named apicidin J and K. Furthermore, we studied the regulation of APF biosynthesis and showed that the cluster genes are expressed under conditions of high nitrogen and acidic pH in a manner dependent on the nitrogen regulator AreB, and the pH regulator PacC. In addition, over-expression of the atypical pathway-specific transcription factor (TF)-encoding gene APF2 led to elevated expression of the cluster genes under inducing and even repressing conditions and to significantly increased product yields. Bioinformatic analyses allowed the identification of a putative Apf2 DNA-binding (“Api-box”) motif in the promoters of the APF genes. Point mutations in this sequence motif caused a drastic decrease of APF production indicating that this motif is essential for activating the cluster genes. Finally, we provide a model of the APF biosynthetic pathway based on chemical identification of derivatives in the cultures of deletion mutants.

A potent HDAC inhibitor, 1-alaninechlamydocin, from a Tolypocladium sp. induces G2/M cell cycle arrest and apoptosis in MIA PaCa-2 cells.

The cyclic tetrapeptide 1-alaninechlamydocin was purified from a Great Lakes-derived fungal isolate identified as a Tolypocladium sp. Although the planar structure was previously described, a detailed analysis of its spectroscopic data and biological activity are reported here for the first time. Its absolute configuration was determined using a combination of spectroscopic (1H–1H ROESY, ECD, and X-ray diffraction) and chemical (Marfey’s analysis) methods. 1-Alaninechlamydocin showed potent antiproliferative/cytotoxic activities in a human pancreatic cancer cell line (MIA PaCa-2) at low-nanomolar concentrations (GI50 5.3 nM, TGI 8.8 nM, LC50 22 nM). Further analysis revealed that 1-alaninechlamydocin induced G2/M cell cycle arrest and apoptosis. Similar to other cyclic epoxytetrapeptides, the inhibitory effects of 1-alaninechlamydocin are proposed to be produced primarily via inhibition of histone deacetylase (HDAC) activity.

βγ-fused turn structures in sugar amino acid (SAA) containing cyclic tetrapeptides with α3δ architecture

The current manuscript describes conformational analysis of 15-membered cyclic tetrapeptides (CTPs), with α3δ architecture, containing sugar amino acids (SAA) having variation in the stereocenter at C5 carbon. Conformational analyses of both the series, in protected and deprotected forms, were carried out in DMSO-d6 using various NMR techniques, supported by restrained MD calculations. It was intriguing to notice that the α3δ macrocycles got stabilized by both 10-membered β-turn as well as a seven-membered γ-turn, fused within the same macrocycle. The presence of fused sub-structures within a 15-membered macrocycle is rare to see. Also, the stereocenter variation at C5 did not affect the fused turn structures and exhibited similar conformations in both the series. The design becomes highly advantageous as fused reverse turn structures are occurring in the cyclic structure with minimalistic size macrocycle and this can be applied to develop suitable pharmacophores in the drug development process.