Different Reaction Specificities of F420H2-Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets.

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated l-proline-like precursors and likely in the microbial central metabolism.

Evolution-guided adaptation of an adenylation domain substrate specificity to an unusual amino acid.

Adenylation domains CcbC and LmbC control the specific incorporation of amino acid precursors in the biosynthesis of lincosamide antibiotics celesticetin and lincomycin. Both proteins originate from a common L-proline-specific ancestor, but LmbC was evolutionary adapted to use an unusual substrate, (2S,4R)-4-propyl-proline (PPL). Using site-directed mutagenesis of the LmbC substrate binding pocket and an ATP-[32P]PPi exchange assay, three residues, G308, A207 and L246, were identified as crucial for the PPL activation, presumably forming together a channel of a proper size, shape and hydrophobicity to accommodate the propyl side chain of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket to the PPL-specific LmbC. The mere change of three amino acid residues in originally strictly L-proline-specific CcbC switched its substrate specificity to prefer PPL and even synthetic alkyl-L-proline derivatives with prolonged side chain. This is the first time that such a comparative study provided an evidence of the evolutionary relevant adaptation of the adenylation domain substrate binding pocket to a new sterically different substrate by a few point mutations. The herein experimentally simulated rearrangement of the substrate binding pocket seems to be the general principle of the de novo genesis of adenylation domains' unusual substrate specificities. However, to keep the overall natural catalytic efficiency of the enzyme, a more comprehensive rearrangement of the whole protein would probably be employed within natural evolution process.

Diversity of Alkylproline Moieties in Pyrrolobenzodiazepines Arises from Postcondensation Modifications of a Unified Building Block.

Anticancer pyrrolobenzodiazepines (PBDs) are one of several groups of natural products that contain unusual 4-alkyl-l-proline derivatives (APDs) in their structure. APD moieties of PBDs are characterized by high structural diversity achieved through unknown biosynthetic machinery. Based on LC-MS analysis of culture broths, feeding experiments, and protein assays, we show that APDs are not incorporated into PBDs in their final form as was previously hypothesized. Instead, a uniform building block, 4-propylidene-l-proline or 4-ethylidene-l-proline, enters the condensation reaction. The subsequent postcondensation steps are initiated by the introduction of an additional double bond catalyzed by a FAD-dependent oxidoreductase, which we demonstrated with Orf7 from anthramycin biosynthesis. The resulting double bond arrangement presumably represents a prerequisite for further modifications of the APD moieties. Our study gives general insight into the diversification of APD moieties of natural PBDs and provides proof-of-principle for precursor directed and combinatorial biosynthesis of new PBD-based antitumor compounds.

New Concept of the Biosynthesis of 4-Alkyl-L-Proline Precursors of Lincomycin, Hormaomycin, and Pyrrolobenzodiazepines: Could a γ-Glutamyltransferase Cleave the C-C Bond?

Structurally different and functionally diverse natural compounds - antitumour agents pyrrolo[1,4]benzodiazepines, bacterial hormone hormaomycin, and lincosamide antibiotic lincomycin - share a common building unit, 4-alkyl-L-proline derivative (APD). APDs arise from L-tyrosine through a special biosynthetic pathway. Its generally accepted scheme, however, did not comply with current state of knowledge. Based on gene inactivation experiments and in vitro functional tests with recombinant enzymes, we designed a new APD biosynthetic scheme for the model of lincomycin biosynthesis. In the new scheme at least one characteristic in each of five final biosynthetic steps has been changed: the order of reactions, assignment of enzymes and/or reaction mechanisms. First, we demonstrate that LmbW methylates a different substrate than previously assumed. Second, we propose a unique reaction mechanism for the next step, in which a putative γ-glutamyltransferase LmbA indirectly cleaves off the oxalyl residue by transient attachment of glutamate to LmbW product. This unprecedented mechanism would represent the first example of the C-C bond cleavage catalyzed by a γ-glutamyltransferase, i.e., an enzyme that appears unsuitable for such activity. Finally, the inactivation experiments show that LmbX is an isomerase indicating that it transforms its substrate into a compound suitable for reduction by LmbY, thereby facilitating its subsequent complete conversion to APD 4-propyl-L-proline. Elucidation of the APD biosynthesis has long time resisted mainly due to the apparent absence of relevant C-C bond cleaving enzymatic activity. Our proposal aims to unblock this situation not only for lincomycin biosynthesis, but generally for all above mentioned groups of bioactive natural products with biotechnological potential.

Effects of 2,3-Dehydrosilybin and Its Galloyl Ester and Methyl Ether Derivatives on Human Umbilical Vein Endothelial Cells.

The effects in vitro of 2,3-dehydrosilybin and several galloyl esters and methyl ethers on the viability, proliferation, and migration of human umbilical vein endothelial cells (HUVECs) were evaluated. The monogalloyl esters were synthesized by a chemoselective esterification method or by Steglich esterification of suitably protected 2,3-dehydrosilybin (1) with protected gallic acid. 2,3-Dehydrosilybin (1) displayed more potent cytotoxic, antiproliferative, and antimigratory activities (IC50 12.0, 5.4, and 12.2 µM, respectively) than silybin. The methylated derivatives were less active, with the least potent being 3,7-di-O-methyl-2,3-dehydrosilybin (6). On the other hand, galloylation at C-7 OH and C-23 OH markedly increased the cytotoxicity and the effects on the proliferation and migration of HUVECs. The most active derivative was 7-O-galloyl-2,3-dehydrosilybin (13; IC50 value of 3.4, 1.6, and 4.7 µM in the cytotoxicity, inhibition of proliferation, and antimigratory assays, respectively). Overall, this preliminary structure-activity relationship study demonstrated the importance of a 2,3-double bond, a C-7 OH group, and a galloyl moiety in enhancing the activity of flavonolignans toward HUVECs.

Deacetylation of mycothiol-derived 'waste product' triggers the last biosynthetic steps of lincosamide antibiotics.

The immediate post-condensation steps in lincomycin biosynthesis are reminiscent of the mycothiol-dependent detoxification system of actinomycetes. This machinery provides the last proven lincomycin intermediate, a mercapturic acid derivative, which formally represents the 'waste product' of the detoxification process. We identified and purified new lincomycin intermediates from the culture broth of deletion mutant strains of Streptomyces lincolnensis and tested these compounds as substrates for proteins putatively involved in lincomycin biosynthesis. The results, based on LC-MS, in-source collision-induced dissociation mass spectrometry and NMR analysis, revealed the final steps of lincomycin biosynthesis, i.e. conversion of the mercapturic acid derivative to lincomycin. Most importantly, we show that deacetylation of the N'-acetyl-S-cysteine residue of the mercapturic acid derivative is required to 'escape' the detoxification-like system and proceed towards completion of the biosynthetic pathway. Additionally, our results, supported by l-cysteine-13C3, 15N incorporation experiments, give evidence that a different type of reaction catalysed by the homologous pair of pyridoxal-5'-phosphate-dependent enzymes, LmbF and CcbF, forms the branch point in the biosynthesis of lincomycin and celesticetin, two related lincosamides.

Lincosamide synthetase--a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism.

In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.

Access to bifunctionalized biomolecular platforms using oxime ligation.

This paper describes an efficient oxime ligation strategy to prepare multivalent conjugates wherein peptides alone or in combination with carbohydrate or oxime groups were coupled to a cyclopeptide scaffold. To demonstrate the versatility of this approach, two classes of conjugates have been prepared. In one class, we attached two or four peptide sequences to the cyclopeptide core together with free oxime groups, while the second class contains an additional substitution with four or two monosaccharides. The well-defined structure of these conjugates was confirmed by high-resolution mass spectrometry.

Anti-cancer efficacy of silybin derivatives -- a structure-activity relationship.

Silybin or silibinin, a flavonolignan isolated from Milk thistle seeds, is one of the popular dietary supplements and has been extensively studied for its antioxidant, hepatoprotective and anti-cancer properties. We have envisioned that potency of silybin could be further enhanced through suitable modification/s in its chemical structure. Accordingly, here, we synthesized and characterized a series of silybin derivatives namely 2,3-dehydrosilybin (DHS), 7-O-methylsilybin (7OM), 7-O-galloylsilybin (7OG), 7,23-disulphatesilybin (DSS), 7-O-palmitoylsilybin (7OP), and 23-O-palmitoylsilybin (23OP); and compared their anti-cancer efficacy using human bladder cancer HTB9, colon cancer HCT116 and prostate carcinoma PC3 cells. In all the 3 cell lines, DHS, 7OM and 7OG demonstrated better growth inhibitory effects and compared to silybin, while other silybin derivatives showed lesser or no efficacy. Next, we prepared the optical isomers (A and B) of silybin, DHS, 7OM and 7OG, and compared their anti-cancer efficacy. Isomers of these three silybin derivatives also showed better efficacy compared with respective silybin isomers, but in each, there was no clear cut silybin A versus B isomer activity preference. Further studies in HTB cells found that DHS, 7OM and 7OG exert better apoptotic activity than silibinin. Clonogenic assays in HTB9 cells further confirmed that both the racemic mixtures as well as pure optical isomers of DHS, 7OM and 7OG were more effective than silybin. Overall, these results clearly suggest that the anti-cancer efficacy of silybin could be significantly enhanced through structural modifications, and identify strong anti-cancer efficacy of silybin derivatives, namely DHS, 7OM, and 7OG, signifying that their efficacy and toxicity should be evaluated in relevant pre-clinical cancer models in rodents.

A novel semisynthetic flavonoid 7-O-galloyltaxifolin upregulates heme oxygenase-1 in RAW264.7 cells via MAPK/Nrf2 pathway.

Quercetin and gallic acid are natural activators of the transcription factor Nrf2, which regulates the expression of many cytoprotective enzymes including heme oxygenase-1 (HO-1). We developed procedures for the synthesis of monogalloyl esters of quercetin and taxifolin (dihydroquercetin), namely, 3-O-galloylquercetin and 7-O-galloyltaxifolin, and examined their effect on the Nrf2 pathway in RAW264.7 cells. Unlike quercetin and free gallic acid, 3-O-galloylquercetin and natural quercetin derivatives isoquercitrin (quercetin-3-O-ß-d-glucoside) and taxifolin had no effect on the expression of HO-1. In contrast, 7-O-galloyltaxifolin increased both mRNA and protein levels of HO-1 at concentrations of 25 µM and above. The induction of HO-1 by 7-O-galloyltaxifolin was primarily associated with the production of reactive oxygen species and phosphorylation of mitogen-activated protein kinases (MAPKs), including p38 MAPKs and ERKs, followed by nuclear accumulation of Nrf2 and downregulation of Keap1, a negative regulator of Nrf2. We conclude that 7-O-galloyltaxifolin upregulates HO-1 via activation of the MAPK/Nrf2 signaling pathway.

Synthesis and antiangiogenic activity of new silybin galloyl esters.

The synthesis of various silybin monogalloyl esters was developed, and their antiangiogenic activities were evaluated in a variety of in vitro tests with human umbilical vein endothelial cells (HUVECs). A structure-activity relationship (SAR) study found the regioselectivity of the silybin galloylation to be highly significant. Silybin (as an equimolar mixture of two diastereomers A and B) exhibited quite poor antiangiogenic activities, whereas its B stereoisomer is more active than silybin A. The galloylation of phenolic OH groups of natural silybin (a mixture of both isomers) leads to increases in their antiangiogenic activities, which is more apparent with the 7-OH than the 20-OH. In contrast, gallates at aliphatic OH groups either had a comparable activity to the parent compound or are even worse than silybin, which was observed in the case of 3-O-galloylsilybin. The most effective compound from this series (7-O-galloylsilybin) has also been prepared from stereochemically pure silybins A and B to evaluate the effect of stereochemistry on the activity. As with silybin itself, the B isomer of 7-O-galloylsilybin was more active than the A isomer.

Dimerisation process of silybin-type flavonolignans: insights from theory.

Natural polyphenols are known to be oxidized by free radicals, which partially explains the antioxidant properties of a number of these compounds. This oxidation may also be used to synthesise new compounds of biological interest, for example, dimers. The present theoretical study describes the existing experimental evidence showing that silybin and dehydrosilybin [natural polyphenols isolated from milk thistle (Silybum marianum)] form dimers regioselectively. Based on DFT calculations, thermodynamic and kinetic values were computed in order to better understand the physicochemical behaviour of these dimerisation processes. Calculations showed that after H-atom transfer (from polyphenol to radical), dimerisation could proceed in two steps: 1) bond formation and, when possible, 2) tautomerisation reorganisation. The former step is the limiting step, while the latter is crucial for the process to be thermodynamically favourable (ΔG<0). If this rearrangement is impossible then dimerisation is not feasible, or at least becomes a minor process (e.g., dehydrosilybin dimerisation after H-atom abstraction from the 3-OH group). This explains the regioselectivity of polyphenol dimerisation.

Dehydrosilybin attenuates the production of ROS in rat cardiomyocyte mitochondria with an uncoupler-like mechanism.

Reactive oxygen species (ROS) originating from mitochondria are perceived as a factor contributing to cell aging and means have been sought to attenuate ROS formation with the aim of extending the cell lifespan. Silybin and dehydrosilybin, two polyphenolic compounds, display a plethora of biological effects generally ascribed to their known antioxidant capacity. When investigating the cytoprotective effects of these two compounds in the primary cell cultures of neonatal rat cardiomyocytes, we noted the ability of dehydrosilybin to de-energize the cells by monitoring JC-1 fluorescence. Experiments evaluating oxygen consumption and membrane potential revealed that dehydrosilybin uncouples the respiration of isolated rat heart mitochondria albeit with a much lower potency than synthetic uncouplers. Furthermore, dehydrosilybin revealed a very high potency in suppressing ROS formation in isolated rat heart mitochondria with IC(50) = 0.15 µM. It is far more effective than its effect in a purely chemical system generating superoxide or in cells capable of oxidative burst, where the IC(50) for dehydrosilybin exceeds 50 µM. Dehydrosilybin also attenuated ROS formation caused by rotenone in the primary cultures of neonatal rat cardiomyocytes. We infer that the apparent uncoupler-like activity of dehydrosilybin is the basis of its ROS modulation effect in neonatal rat cardiomyocytes and leads us to propose a hypothesis on natural ischemia preconditioning by dietary polyphenols.

Synthesis of multivalent glycoconjugates containing the immunoactive LELTE peptide: effect of glycosylation on cellular activation and natural killing by human peripheral blood mononuclear cells.

Pentapeptide diacidic sequence LELTE, derived from the mycobacterial heat shock protein hsp65, has been recently identified as a "danger" signal of the immune system effective via specific binding to the universal leukocyte triggering receptor CD69. This sequence is not active per se, only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using a standard bifunctional reagents. Here we describe an entirely new way of presenting of this peptide based on its attachment to a cyclopeptide RAFT scaffold (K-K-K-P-G)(2) through the epsilon-amino group of lysine residues, alone or in combination with the carbohydrate epitope alphaGalNAc. The ability of such RAFT scaffolds to precipitate the target CD69 receptor or to activate CD69-positive cells is enhanced in compounds 2 and 4 possessing combined peptide/carbohydrate expression. Compounds 2 and 4 are highly efficient activators of natural killer lymphocytes, but they are completely inactive from the point of view of activation-induced apoptosis of lymphocytes by the target cells. These unique properties make the combined peptide/carbohydrate RAFTs highly suitable for future evaluation in animal tumor therapies in vivo and predict them to be readily available and efficient immunoactivators.

Silybin and silymarin--new and emerging applications in medicine.

This review critically surveys the literature published mainly within this millennium on the new and emerging applications of silybin (pure, chemically defined substance) and silymarin (flavonoid complex from Silybum marianum - milk thistle seeds). These compounds used so far mostly as hepatoprotectants were shown to have other interesting activities, e.g. anticancer and canceroprotective and also hypocholesterolemic activity. These effects were demonstrated in a large variety of illnesses of different organs, e.g. prostate, lungs, CNS, kidneys, pancreas and also in the skin protection. Besides the cytoprotective activity of silybin mediated by its antioxidative and radical-scavenging properties also new functions based on the specific receptor interaction were discovered. These were studied on the molecular level and modulation of various cell-signaling pathways with silybin was disclosed--e.g. NF-kappaB, inhibition of EGFR-MAPK/ERK1/2 signaling, activity upon Rb and E2F proteins, IGF-receptor signaling. Proapoptotic activity of silybin in pre- and/or cancerogenic cells and anti-angiogenic activity of silybin are other important findings that bring silymarin preparations closer to respective application in the cancer treatment. Discovery of the inhibition and modulation of drug transporters, P-glycoproteins, estrogenic receptors, nuclear receptors by silybin and some of its new derivatives contribute further to the better understanding of silybin activity on the molecular level. Silymarin application in veterinary medicine is reviewed as well. Recent works using optically pure silybin diastereomers clearly indicate extreme importance of the use of optically active silybin namely in the receptor studies. Significance of silymarin and its components in the medicine is clearly indicated by an exponential growth of publications on this topic--over 800 papers in the last 5 years.

New derivatives of silybin and 2,3-dehydrosilybin and their cytotoxic and P-glycoprotein modulatory activity.

Large series of O-alkyl derivatives (methyl and benzyl) of silybin and 2,3-dehydrosilybin was prepared. Selective alkylation of the silybin molecule was systematically investigated. For the first time we present here, for example, preparation of 19-nor-2,3-dehydrosilybin. All prepared silybin/2,3-dehydrosilybin derivatives were tested for cytotoxicity on a panel of drugs sensitive against multidrug resistant cell lines and the ability to inhibit P-glycoprotein mediated efflux activity. We have identified effective and relatively non-cytotoxic inhibitors of P-gp derived from 2,3-dehydrosilybin. Some of them were more effective inhibitors at concentrations lower than a standard P-gp efflux inhibitor cyclosporin A. Another group of 2,3-dehydrosilybin derivatives also had better inhibitory effects on P-gp efflux but a cytotoxicity comparable with that of parent 2,3-dehydrosilybin. Structural requirements for improving inhibitory activity and reducing toxicity of 2,3-dehydrosilybin were established. Effect of E-ring substitution as well as an influence of the substituent size at the C-7-OH position of A-ring on P-gp-inhibitory activity was evaluated for the first time in this study.

Effects of silymarin flavonolignans and synthetic silybin derivatives on estrogen and aryl hydrocarbon receptor activation.

Silymarin, a standardized mixture of flavonolignans, or its major constituents could be effective for prevention and treatment of hepatic damage or skin cancer. However, their potential side effects, such as modulation of endocrine functions via the disruption of estrogen receptor (ER) and/or aryl hydrocarbon receptor (AhR) activation, are largely unknown. In the present study, we investigated impact of silymarin, its constituents and a series of their synthetic derivatives on ER- and AhR-mediated activities using in vitro reporter gene assays. We found that none of the compounds under study affected the AhR-mediated activity in rat hepatoma cells. Contrary to that, several compounds behaved as either partial or full ER agonists. Silymarin elicited partial ER activation, with silybin B being probably responsible for a majority of the weak ER-mediated activity of silymarin; silybin A and other flavonolignans were found to be inactive and potent ER agonist taxifolin is only a minor constituent of silymarin. To our knowledge, this is probably the first time, when receptor-specific in vitro effects of separated diastereomers have been demonstrated. In contrast to silymarin constituents, the synthetic silybin derivatives, potentially useful as chemoprotective agents, did not modulate the ER-mediated activity, with exception of 23-O-pivaloylsilybin. Interestingly, 7-O-benzylsilybin potentiated ER-mediated activity of 17beta-estradiol despite possessing no estrogenic activity. In conclusion, our data suggest that estrogenicity of some silymarin constituents should be taken in account as their potential side effect when considered as chemopreventive compounds. These results also stress the need to study biological activities of purified or synthesized diastereomers of silybin derivatives.