Structure–activity relationships in ultrashort cationic lipopeptides: the effects of amino acid ring constraint on antibacterial activity

Taking a minimalistic approach in efforts to lower the cost for the development of new synthetic antimicrobial peptides, ultrashort cationic lipopeptides were designed to mimic the amphiphilic nature crucial for their activity but with only a very short peptide sequence ligated to a lipidic acid. Nine ultrashort cationic lipopeptides were prepared to study the effects of ring constraint in the amino acid side chain of the peptide component. USCL-P_Cat1, consisting of only four l-4R-aminoproline residues and acylated with palmitic acid at the N-terminus, was found to populate a polyproline II helical secondary conformation that is stable to different pHs and temperatures using circular dichroism. The synthesized lipopeptides were found to have a micellar structure in water using negative staining transmission electron microscopy. We found that constraining the side chain of the amino acid component is not beneficial to the antimicrobial activity. USCL-Dab1, USCL-Dab3 and USCL-K1 showed promising activity against a panel of laboratory reference and clinically isolated Gram-positive and Gram-negative bacterial strains, some of which are multidrug resistant. No appreciable cytotoxicity against human monocytic THP-1 cells was observed up to concentrations of 20–40 µM for all synthesized compounds. Moreover, all USCLs did not induce the production of either pro-inflammatory cytokines or chemokines up to 40 µM.     

Novel Nociceptin Analogues: Synthesis and Biological Activity

Syntheses are described of the nociceptin (1–13) amide [NC(1–13)-NH₂] and of several analogues in which either one or both the phenylalanine residues (positions 1 and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and β-alanine, respectively. The preparation is also described of NC(1–13)-NH₂ analogues in which either galactose or N-acetyl-galactosamine are β-O-glycosidically linked to Thr⁵ and/or to Ser¹⁰. Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe⁴, Thr⁵, Ala⁷ and Arg⁸ are crucial residues for OP₄ receptor activation. Manipulation of Phe¹ yielded peptides endowed with antagonist activity but [Nphe¹] NC(1–13)-NH₂ acted as an antagonist still possessing weak agonist activity. Introduction of the βAla residue either in position 7 or 11 of the [Nphe¹] NC(1–13)-NH₂ sequence, abolished any residual agonist activity and [Nphe¹, βAla⁷] NC(1–13)-NH₂ and [Nphe¹, βAla¹¹] NC(1–13)-NH₂ acted as competitive antagonists only. Modification of both Ala⁷ and Ala¹¹ abolished the antagonist activity of [Nphe¹]NC(1–13)-NH₂ probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(βGal)¹⁰] NC(1–13)-NH₂ displayed an activity comparable with that of NC(1–13)-NH₂, [Ser(βGalNAc)¹⁰] NC(1–13)-NH₂ and [βAla¹¹] NC(1–13)-NH₂ were five and 10 times less active, respectively.The α-amino acid residues are of the l-configuration. Standard abbreviations for amino acid derivatives and peptides are according to the suggestions of the IUPAC-IUB Commission on Biochemical Nomeclature (1984), Eur. J. Biochem. 138, 9–37. Abbreviations listed in the guide published in (2003), J. Peptide Sci. 9, 1–8 are used without explanation.     

Studies on the Formation and the Degradation of Glucose 1,6-Bisphosphate in the Beef Liver Homogenate

Four kinds of the enzyme reactions have been reported for the synthesis of Glc-1,6-P₂. However, any activity of Glc-1-P dismutase and phosphoglucokinase was not observed in the beef liver homogenate. When the liver homogenate was incubated with Glc-1-P and Fru-1,6-P₂, a significant amount of Glc-1,6-P₂ was formed. The Glc-1,6-P₂ synthesis activity from Glc-1-P and Fru-1,6-P₂ was caused by the action of phosphoglucomutase present in the liver homogenate. The most remarkable activity for Glc-1,6-P₂ synthesis was observed when the homogenate was incubated with Glc-1-P and glycerate-1,3-P₂. The Glc-1,6-P₂ synthesis activity from Glc-1-P and glycerate-1,3-P₂ was separated from the major peak of phosphoglucomutase activity by DEAE-Sephadex chromatography. The peak of Glc-1,6-P₂ synthesis activity, however, still retained phosphoglucomutase activity.

Glc-1,6-P₂ phosphatase activity was mainly observed in the mitochondria and microsome fraction. The properties of Glc-1,6-P₂ phosphatase were differentiated from those of acid phosphatase and Glc-6-P phosphatase.     

Substrate Specificity of Barley Cysteine Endoproteases EP-A and EP-B

The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgare L.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P₂-P₁-P₁′-P₂′-tyrosine(NO₂)-aspartic acid, in which cleavage occurs between P₁ and P₁′, showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P₂. Arginine was preferred to glutamine at P₁, whereas proline at P₂, P₁, or P₁′ greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower K_m values.     

Effect of self-assembly on antimicrobial activity of double-chain short cationic lipopeptides

Short cationic antimicrobial lipopeptides with surfactant-like structure are promising antibiotic candidates that preferentially target microbial membranes. Therefore, we focused our study on double-chain lipopeptides, (C_10-16)₂Dab-KKK-NH₂ and (C_10-16)₂Dap-KKK-NH₂, where Dab and Dap are 2,4-diaminobutyric and 2,3-diaminopropionic acids, respectively. We tried to answer a question how the self-assembly behaviour affects biological activities of the tested compounds. The subject compounds were synthesized by solid-phase method and screened for their antimicrobial and haemolytic activities. Cytotoxicity tests on human keratinocytes were carried out for the most promising lipopeptides. Self-assembly properties were evaluated by both experimental and theoretical methods. Interactions with membrane models were examined using the ITC and FTIR techniques. All the lipopeptides studied showed the tendency to self-assembly in solution, and this behaviour was affected by the length of the hydrocarbon chains. Acyl chain elongation supported the formation of the bilayer structure and deprived the lipopeptides of antimicrobial activity. A multi-step mechanism of interaction with a negatively charged membrane was observed for the short-chain lipopeptides, indicating other processes accompanying the binding process. Short-chain lipopeptides were able to penetrate into the liposome’s interior and/or cause the rupture of the liposome, this being compatible with their high antimicrobial activity.     

Short arginine-rich lipopeptides: From self-assembly to antimicrobial activity

In this paper, we examine antimicrobial and cytotoxic activities, self-assembly and interactions with anionic and zwitterionic membranes of short arginine-rich lipopeptides: C₁₆-RRRR-NH₂, C₁₄-RRRR-NH₂, C₁₂-RRRR-NH₂, and C₁₆-PRRR-NH₂. They show a tendency to self-assembly into micelles, but it is not required for antimicrobial activity. The membrane binding of the lipopeptides can be accompanied by other factors such as: peptide aggregation, pore formation or micellization of phospholipid bilayer. The shortening of the acyl chain results in compounds with a lower haemolytic activity and a slightly improved antimicrobial activity against Gram-positive bacteria, what indicates enhanced cell specificity. Results of coarse-grained molecular dynamics simulations indicate different organization of membrane lipids upon binding of arginine-based lipopeptides and the previously studied lysine-based ones.     

The purification and kinetic properties of biophosphoglycerate synthase from horse red blood cells.

Bisphosphoglycerate synthase from horse red cells has been purified to apparent homogeneity by a simple and efficient new procedure incorporating chromatography on a column of Sepharose 4B derivatized with blue dextran. The enzyme is similar to the human red cell synthase in subunit size. It is phosphorylated by either glycerate-1,3-P₂ or glycerate-2,3-P₂ to form a phosphoenzyme with the acid-lability of a histidyl phosphate. In addition to the synthase activity (glycerate-1,3-P₂ → glycerate-2,3-P₂), k_cat 12.5 s^?1, the enzyme has bisphosphoglycerate phosphatase activity in the presence of glycolate-2-P (glycerate-2,3-P₂ → glycerate-P + P_i), k_cat 2.6 s^?1 and phosphoglycerate mutase activity (3-PGA ? 2-PGA), k_cat 1.7 s^?1. The energy of activation for the synthase reaction is 9.38 kcal/mol. Lineweaver-Burk plots of the kinetic data are parallel lines. In contrast intersecting patterns were obtained from similar experiments done with the human red cell enzyme. Further investigation is required to explain these differences. This enzyme may function as both synthase and phosphatase for bisphosphoglycerate in the red blood cell.     

Multifunctional Properties of Beef Liver Phosphoglucomutase

Liver phosphoglucomutase was found to catalyze also the reaction of Glc-1,6-P₂ formation from Glc-1-P and Fru-1,6-P_z or Glc-1-P and glycerate-1,3-P₂. The specific activity of Glc-1,6-P₂ formation from Glc-1-P and Fru-1,6-P₂ was 1/9200 of that of the mutase activity. The activity of Glc-1,6-P₂ formation from Glc-1-P and glycerate-1,3-P₂ was 1/122,000 of the mutase activity. From the results of the kinetics and the thermal inactivation experiments, the reaction of the mutase and Glc-1,6-P₂ synthesis were strongly suggested to occur at the same active site of liver phosphoglucomutase.

Liver phosphoglucomutase exhibited the Glc-1,6-P₂ phosphatase activity only in the presence of xylose 1-phosphate. The specific activity of phosphatase was only 1/154,000 of that of the mutase activity.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

Comparative in vivo evaluation of polyalkoxy substituted 4H-chromenes and oxa-podophyllotoxins as microtubule destabilizing agents in the phenotypic sea urchin embryo assay

A series of polyalkoxy substituted 7-hydroxy- and 7-methoxy-4-aryl-4H-chromenes were evaluated using the sea urchin embryo model to yield several compounds exhibiting potent antimitotic microtubule destabilizing activity. Data obtained by the assay were further confirmed in the NCI60 human cancer cell screen. The replacement of methylenedioxy ring A and lactone ring D in podophyllotoxin analogues by 7-methoxy, 2-NH₂, and 3-CN groups in 4-aryl-4H-chromenes resulted in potent antimitotic microtubule destabilizing agents. Feasible synthesis and high yields render 7-methoxy-4H-chromenes to be a promising series for further anticancer drug development.     

Ultrasensitive internally quenched substrates of human cathepsin L

Internally quenched cathepsin L (Cat L) substrate ABZ-Bip-Arg-Ala-Gln-Tyr(3-NO₂)-NH₂ with high specificity constant (k_cat/K_M = 2.6 × 10⁷ M⁻¹ s⁻¹) was synthesized. The resultant compound displayed high selectivity over other members of the cathepsin family (B, S, X, V, C, K, H, F, D, and A). Activity of Cat L at picomolar (pM) concentrations was found using this substrate. Moreover, it was established that the presence of the selective Cat L inhibitor suppressed the proteolysis of the substrate to a non-detectable level. Incubation of the synthesized compound with a cell lysate of healthy and cancer cell lines indicated significant differences in Cat L activity. Based on the obtained results, it is proposed that this substrate could be used for selective monitoring of Cat L activity in biological systems.     

Novel diphenylthiazole derivatives with multi-target mechanism: Synthesis,docking study,anticancer and anti-inflammatory activities

Over the last few decades, a growing body of studies addressed the anticancer activity of NSAIDs, particularly selective COX-2 inhibitors. However, their exact molecular mechanism is still unclear and is not fully investigated. In this regard, a novel series of compounds bearing a COXs privilege scaffold, diphenyl thiazole, was synthesized and evaluated for their anticancer activity against a panel of cancer cell lines. The most active compounds 10b, 14a,b, 16a, 17a,b and 18b were evaluated in vitro for COX-1/COX-2 inhibitory activity. These compounds were suggested to exert their anticancer activity through a multi-target mechanism based on their structural features. Thus, compounds 10b and 17b with the least IC₅₀ values in MTT assay were tested against three known anticancer targets; EGFR, BRAF and tubulin. Compounds 10b and 17b showed remarkable activity against EGFR with IC₅₀ values of 0.4 and 0.2 μM, respectively and good activity against BRAF with IC₅₀ values of 1.3 and 1.7 μM, respectively. In contrast, they showed weak activity in tubulin polymerization assay. The in vivo anti-inflammatory potential was assessed and interestingly, compound 17b was the most potent compound. Together, this study offers some important insights into the correlation between COXs inhibition and cancer treatment. Additionally, the results demonstrated the promising activity of these compounds with a multi-target mechanism as good candidates for further development into potential anticancer agents.     

Octapeptide Analogs of Somatostatin Containing α,α-Dialkylated Amino Acids with Potent Anticancer Activity

We describe the synthesis and anticancer activities of octapeptide analogs of somatostatin incorporating α,α-dialkylated amino acids. The designed analogs of somatostatin are: d-Phe¹-Cys²-Tyr³-d-Trp⁴-Orn⁵-Xxx⁶-Pen⁷-Thr⁸-NH₂ where Xxx=α-Aminoisobutyric acid (Aib), Diethyl glycine (Deg), 1-Aminocyclopentane carboxylic acid (Ac5c), and, d-Phe¹-Cys²-Tyr³-d-Trp⁴-Lys⁵-Ac5c⁶-Pen⁷-Thr⁸-NH₂ (disulphide bond between Cys² and Pen⁷ in all analogs). The conformational studies two of the designed analogs were carried out by NMR techniques and the experimental results suggest a β-turn structure for one of the designed analog. In vivo tumor regression study of two designed analogs on human primary colon tumor xenografts in nude mice demonstrates the anticancer potential of the synthesized analogs.     

Functionalization of peptide nucleolipid bioconjugates and their structure anti-cancer activity relationship studies

In the search for more potent peptide-based anti-cancer conjugates the generation of new, functionally diverse nucleolipid derived D-(KLAKLAK)₂-AK sequences has enabled a structure and anti-cancer activity relationship study. A reductive amination approach was key for the synthesis of alkylamine, diamine and polyamine derived nucleolipids as well as those incorporating heterocyclic functionality. The carboxy-derived nucleolipids were then coupled to the C-terminus of the D-(KLAKLAK)₂-AK killer peptide sequence and produced with and without the FITC fluorophore for investigating biological activity in cancer cells. The amphiphilic, α-helical peptide-nucleolipid bioconjugates were found to exhibit variable effects on the viability of MM.1S cells, with the histamine derived nucleolipid peptide bioconjugate displaying the most significant anti-cancer effects. Thus, functionally diverse nucleolipids have been developed to fine-tune the structure and anti-cancer properties of killer peptide sequences, such as D-(KLAKLAK)₂-AK.     

A potent antibacterial activity of new short d-enantiomeric lipopeptide against multi drug resistant bacteria

The emergence of drug-resistant pathogenic bacteria threatens human health. Resistance to existing antibiotics is increasing, while the emergence of new antibiotics is slowing. Cationic antimicrobial peptides (CAMPs) are fascinating alternative antibiotics because they possess a broad spectrum of activity, being active against both Gram-positive and Gram-negative bacteria including those resistant to traditional antibiotics. However, low bioavailability resulting from enzymatic degradation and attenuation by divalent cations like Mg²⁺ and Ca²⁺ limits their use as antibiotic agents. Here, we report the design of new CAMPs showing both high antibacterial activity and serum stability under physiological ion concentrations. The peptides were designed by applying two approaches, the use of d-enantiomer and lipidation. Based on the sequence of the CopW (LLWIALRKK-NH₂), a nonapeptide derived from coprisin, a series of novel d-form CopW lipopeptides with different acyl chain lengths (C6, C8, C10, C12, C14, and C16) were synthesized and evaluated with respect to their activity and salt sensitivity. Among the analogs, the d-form lipopeptide dCopW3 exhibited MIC values ranging from 1.25 to 5?μM against multidrug-resistant bacteria. Significantly, this compound did not induce bacterial resistance and was highly stable in human serum proteases. The results emphasize the potential of cationic d-form lipopeptide as therapeutically valuable antibiotics for treating drug-resistant bacterial infections.     

Discovery of 1,2,4-triazine-based derivatives as novel neddylation inhibitors and anticancer activity studies against gastric cancer MGC-803 cells

Neddylation modification is often over-expressed in a variety of human tumor cells. Therefore, targeting neddylation pathway may represent a potential approach to the treatment of human tumors. Herein, we describe the discovery of a hit scaffold from our in-house library and further structure-based optimizations. In this work, compound V11 could block the neddylation and inhibit the activity of NAE (with an EC₅₀ value of 3.56 µM), and a dose-dependent reduction of the Ubc12-NEDD8 conjugations was also observed. Molecular docking results suggest compound V11 could bind tightly to NAE via hydrogen bonds and hydrophobic interactions. Compound V11 showed the best antiproliferative ability with an IC₅₀ value of 8.22 μM against gastric cancer MGC-803 cells. Further anticancer activity studies suggested that compound V11 inhibited MGC-803 cell growth, caused a cell cycle arrestment at G2/M phase and induced apoptosis via extrinsic and intrinsic apoptosis pathways. All the findings suggest that 1,2,4-triazine scaffold might provide a novel scaffold for the further development of neddylation inhibitors and compound V11 might be a potential neddylation inhibitor with anticancer activity.     

Carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing elevated fructose 2,6-bisphosphate levels

The aim of this work was to investigate the role of fructose 2,6-bisphosphate (Fru 2,6-P₂) during photosynthesis. The level of Fru 2,6-P₂ in tobacco plants was elevated by the introduction of a modified mammalian gene encoding 6-phosphofructo-2-kinase (6-PF-2-K). Estimates of the metabolite control coefficient (C) for Fru 2,6-P₂ levels in response to increased 6-PF-2-K activity, suggest that small increases in 6-PF-2-K activity have little effect upon steady-state Fru 2,6-P₂ levels (_C = +0.08 for a 0–58% increase in 6-PF-2-K activity). However, larger changes resulted in dramatic rises in Fru 2,6-P₂ levels (C = +3.35 for 206–268% increase in 6-PF-2-K activity). Transgenic plants contained Fru 2,6-P₂ levels in the dark that ranged from 104 to 230% of the level in wild-type tobacco. Plants with altered levels of Fru 2,6-P₂ were used to determine the effects of this signal metabolite upon carbohydrate metabolism during the initial phase of the light period. Here we provide direct evidence that Fru 2,6-P₂ contributes to the regulation of carbon partitioning in tobacco leaves by inhibiting sucrose synthesis.     

Synthesis and characterization of novel 1,2,4-triazine derivatives with antiproliferative activity

A series of novel small molecules with a 1,2,4-triazine scaffold was obtained according to a recently published and highly efficient synthetic route. Screening for antiproliferative and cytotoxic activity revealed distinct anticancer effects against the human leukemia cell line K-562 combined with a remarkable low cytotoxicity. All compounds were in agreement with the ‘rule-of-five’ claims by Lipinski and calculated log P_calc values were experimentally confirmed (log P_exp). For the most active compounds, in vitro serum albumin binding was investigated and structure–activity relationships were established.     

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P₂) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P₂ were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P₂ had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-¹³C]glucose, in the lines possessing the highest amounts of Fru-2,6-P₂, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of ¹⁴C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P₂ can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates. Received: 29 March 2000 / Accepted: 13 June 2000