Tumor suppressive activities of solvatochromic 3,3'-azadimethylene dinaphthospiropyran in colon cancer model.

Spiropyrans have been extensively investigated because of their thermo- and photochromic characteristics, but their biotherapeutic properties have not been explored much. We report anti-proliferative properties of a novel 3,3'-azadimethylene dinaphthospiropyran 11. Dibenzospiropyrans and dinaphthospiropyrans were synthesized by a simple and expedient method using acid-catalyzed aldol condensation of salicylaldehyde and 2-hydroxy-1-naphthaldehyde, respectively, with cyclic ketones. Together with structural elucidation by 2D NMR and X-ray crystallography studies, we provide a putative mechanism for their formation. Compound 11 showed solvatochromism and exhibited altered spectral characteristics depending on the pH. In acidic conditions, 11 remains in open form, whereas upon alkalinization it reverts back to closed form. Based on the in vitro anti-proliferative activity in H441, HCT-116, MiaPaCa-2, and Panc-1 cancer cell lines, 11 was submitted to further investigation. It reduced HCT116 colonosphere formation and demonstrated induction of caspase cascade, suggesting apoptosis. In vitro proliferation assays also suggested that HCl and trifluoroacetate salts of 11 are more effective. Treatment of mice carrying HCT-116 xenografts with 11 (5 µg/day, intraperitoneal for 3 weeks) suppressed tumor growth by 62%. Overall, the results reveal a new series of structurally complex, but relatively easy to synthesize molecules of which compound 11 represents a lead for anticancer development.

Acceptor substrate determines donor specificity of an aromatic prenyltransferase: expanding the biocatalytic potential of NphB.

Aromatic prenyltransferases are known for their extensive promiscuity toward aromatic acceptor substrates and their ability to form various carbon-carbon and carbon-heteroatom bonds. Of particular interest among the prenyltransferases is NphB, whose ability to geranylate cannabinoid precursors has been utilized in several in vivo and in vitro systems. It has therefore been established that prenyltransferases can be utilized as biocatalysts for the generation of useful compounds. However, recent observations of non-native alkyl-donor promiscuity among prenyltransferases indicate the role of NphB in biocatalysis could be expanded beyond geranylation reactions. Therefore, the goal of this study was to elucidate the donor promiscuity of NphB using different acceptor substrates. Herein, we report distinct donor profiles between NphB-catalyzed reactions involving the known substrate 1,6-dihydroxynaphthalene and an FDA-approved drug molecule sulfabenzamide. Furthermore, we report the first instance of regiospecific, NphB-catalyzed N-alkylation of sulfabenzamide using a library of non-native alkyl-donors, indicating the biocatalytic potential of NphB as a late-stage diversification tool. KEY POINTS: • NphB can utilize the antibacterial drug sulfabenzamide as an acceptor. • The donor profile of NphB changes dramatically with the choice of acceptor. • NphB performs a previously unknown regiospecific N-alkylation on sulfabenzamide. • Prenyltransferases like NphB can be utilized as drug-alkylating biocatalysts.

FgaPT2, a biocatalytic tool for alkyl-diversification of indole natural products.

Aromatic prenyltransferases from natural product biosynthetic pathways display relaxed specificity for their aromatic substrates. While a growing body of evidence suggests aromatic prenyltransferases to be more tolerant towards their alkyl-donor substrates, most studies aimed at probing their donor-substrate specificity are limited to only a small set of alkyl pyrophosphate donors, restricting their broader utility as biocatalysts for synthetic applications. Here, we assess the donor substrate specificity of an l-tryptophan C4-prenyltransferase, also known as C4-dimethylallyltryptophan synthase, FgaPT2 from Aspergillus fumigatus, using an array of 34 synthetic unnatural alkyl-pyrophosphate analogues, and demonstrate FgaPT2 can catalyze the transfer of 25 of the 34 non-native alkyl groups from their corresponding synthetic alkyl-pyrophosphate analogues at N1, C3, C4 and C5 position of tryptophan in a normal and reverse manner. The kinetic studies and regio-chemical analysis of the alkyl-l-tryptophan products suggest that the alkyl-donor transfer by FgaPT2 is a function of the stability of the carbocation and the steric factors in the active site of the enzyme. Further, to demonstrate the biocatalytic utility of FgaPT2, this study also highlights the FgaPT2-catalyzed synthesis of a small set of alkyl-diversified indolocarbazole analogues. These results reveal FgaPT2 to be more tolerant to diverse non-native alkyl-donor substrates beyond their known acceptor substrate promiscuity and set the stage for its development as a novel biocatalytic tool for the differential alkylation of natural products for drug discovery and other synthetic applications.

Development and quantitative characterization of the precursor rheology of hyaluronic acid hydrogels for bioprinting.

Bioprinting technologies have tremendous potential for advancing regenerative medicine due to the precise spatial control over depositing a printable biomaterial, or bioink. Despite the growing interest in bioprinting, the field is challenged with developing biomaterials for extrusion-based bioprinting. The paradigm of contemporary bioink studies relies on trial-and-error methods for discovering printable biomaterials, which has little practical use for others who endeavor to develop bioinks. There is pressing need to follow the precedent set by a few pioneering studies that have attempted to standardize bioink characterizations for determining the properties that define printability. Here, we developed a pentenoate-functionalized hyaluronic acid hydrogel (PHA) into a printable bioink and used three recommended, quantitative rheological assessments to characterize the printability: 1) yield stress, 2) viscosity, and 3) storage modulus recovery. The most important characteristic is the yield stress; we found a yield stress upper limit of ∼1000 Pa for PHA. Measuring the viscosity was advantageous for determining shear-thinning behavior, which aided in extruding highly viscous PHA through a nozzle. Post-printing recovery is required to maintain shape fidelity and we found storage modulus recoveries above ∼85% were sufficient for PHA. Two formulations had superior printability (i.e., 1.5 MDa PHA - 4 wt%, and 1 MDa PHA - 8 wt%), and increasing cell concentrations in PHA up to 9 × 106 cells/mL had minimal effects on the printability. Even so, other factors such as sterilization and peptide modifications to enhance bioactivity may influence printability, highlighting the need for investigators to consider such factors when developing new bioinks. STATEMENT OF SIGNIFICANCE: Bioprinting has potential for regenerating damaged tissues; however, there are a limited number of printable biomaterials, and developing new bioinks is challenging because the required material physical properties for extrusion-based printing are not yet known. Most new bioinks are developed by trial-and-error, which is neither efficient nor comparable across materials. There is a need for the field to begin utilizing standard methods proposed by a few pioneering studies to characterize new bioinks. Therefore, we have developed the printability of a hyaluronic acid based-hydrogel and characterized the material with three quantitative rheological tests. The current work impacts the bioprinting field by demonstrating and encouraging the use of universal bioink characterizations and by providing printability windows to advance new bioink development.

Consequences of Depsipeptide Substitution on the ClpP Activation Activity of Antibacterial Acyldepsipeptides.

The acyldepsipeptide (ADEP) antibiotics operate through a clinically unexploited mechanism of action and thus have attracted attention from several antibacterial development groups. The ADEP scaffold is synthetically tractable, and deep-seated modifications have produced extremely potent antibacterial leads against Gram-positive pathogens. Although newly identified ADEP analogs demonstrate remarkable antibacterial activity against bacterial isolates and in mouse models of bacterial infections, stability issues pertaining to the depsipeptide core remain. To date, no study has been reported on the natural ADEP scaffold that evaluates the sole importance of the macrocyclic linkage on target engagement, molecular conformation, and bioactivity. To address this gap in ADEP structure-activity relationships, we synthesized three ADEP analogs that only differ in the linkage motif (i.e., ester, amide, and N-methyl amide) and provide a side-by-side comparison of conformational behavior and biological activity. We demonstrate that while replacement of the naturally occurring ester linkage with a secondary amide maintains in vitro biochemical activity, this simple substitution results in a significant drop in whole-cell activity. This study provides direct evidence that ester to amide linkage substitution is unlikely to provide a reasonable solution for ADEP instability.

Efficacy of ampicillin against methicillin-resistant Staphylococcus aureus restored through synergy with branched poly(ethylenimine).

ß-Lactam antibiotics kill Staphylococcus aureus bacteria by inhibiting the function of cell wall penicillin-binding proteins (PBPs) 1 and 3. However, ß-lactams are ineffective against PBP2a, used by methicillin-resistant S. aureus (MRSA) to perform essential cell wall crosslinking functions. PBP2a requires teichoic acid to properly locate and orient the enzyme, and thus MRSA is susceptible to antibiotics that prevent teichoic acid synthesis in the bacterial cytoplasm. As an alternative, we have used branched poly(ethylenimine), BPEI, to target teichoic acid in the bacterial cell wall. The result is restoration of MRSA susceptibility to the ß-lactam antibiotic ampicillin with a MIC of 1 µg ml-1, superior to that of vancomycin (MIC=3.7 µg ml-1). A checkerboard assay shows synergy of BPEI and ampicillin. NMR data show that BPEI alters the teichoic acid chemical environment. Laser scanning confocal microscopy images show BPEI residing on the bacterial cell wall, where teichoic acids and PBPs are located.

Transferring Fungi to a Deuterium-Enriched Medium Results in Assorted, Conditional Changes in Secondary Metabolite Production.

Deuterium is one of the few stable isotopes that have the capacity to significantly alter a compound's chemical and biological properties. The addition of a single neutron to a protium atom results in the near doubling of its mass, which gives rise to deuterium's characteristic isotope effects. Since the incorporation of deuterium into organic substrates is known to alter enzyme/protein-substrate interactions, we tested the extent to which deuterium enrichment would modify fungal secondary metabolite production. Several fungal cultures were tested, and in all cases their secondary metabolomes were marked by changes in natural product production. Workup of one Aspergillus sp. grown under deuterium-enrichment conditions resulted in the production of several secondary metabolites not previously detected from the fungus. Bioassay testing revealed that in comparison to the inactive crude fungal extract derived from growing the fungus under non-deuterium-enriched conditions, an extract derived from the same isolate cultured in a deuterium-enriched medium inhibited methicillin-resistant Staphylococcus aureus. Using an assortment of NMR and mass spectrometry experiments, we were able to identify the bacterial inhibitor as an isotope-labeled version of pigmentosin A (6). Five additional isotopically labeled metabolites were also obtained from the fungus including brevianamide F (1), stephacidin A (2), notoamide D (3), notoamide L (4), and notoamide C (5). Given the assorted changes observed in the secondary metabolite profiles of this and other fungi grown in deuterium-enriched environments, as well as the fact that 1 and 3-6 had not been previously observed from the Aspergillus sp. isolate used in this study, we propose that deuterium enrichment might offer an effective method for further expanding a fungus's chemical diversity potential.

Virus-based photo-responsive nanowires formed by linking site-directed mutagenesis and chemical reaction.

Owing to the genetic flexibility and error-free bulk production, bio-nanostructures such as filamentous phage showed great potential in materials synthesis, however, their photo-responsive behaviour is neither explored nor unveiled. Here we show M13 phage genetically engineered with tyrosine residues precisely fused to the major coat protein is converted into a photo-responsive organic nanowire by a site-specific chemical reaction with an aromatic amine to form an azo dye structure on the surface. The resulting azo-M13-phage nanowire exhibits reversible photo-responsive properties due to the photo-switchable cis-trans isomerisation of the azo unit formed on the phage. This result shows that site-specific display of a peptide on bio-nanostructures through site-directed genetic mutagenesis can be translated into site-directed chemical reaction for developing advanced materials. The photo-responsive properties of the azo-M13-phage nanowires may open the door for the development of light controllable smart devices for use in non-linear optics, holography data storage, molecular antenna, and actuators.

Oxide Formation on Biological Nanostructures via a Structure-Directing Agent: Towards an Understanding of Precise Structural Transcription.

Biomimetic silica formation is strongly dependent on the presence of cationic amine groups which hydrolyze organosilicate precursors and bind to silicate oligomers. Since most biological species possess anionic surfaces, the dependence on amine groups limits utilization of biotemplates for fabricating materials with specific morphologies and pore structures. Here, we report a general aminopropyltriethoxysilane (APTES) directed method for preparing hollow silica with well-defined morphologies using varying biotemplates (proteins, viruses, flagella, bacteria and fungi). Control experiments, pH evolution measurements and 29Si NMR spectroscopic studies have revealed a mechanism of the assembly of APTES on bio-surfaces with subsequent nucleation and growth of silica. The APTES assembly and nuclei formation on bio-surfaces ensured precise transcription of the morphologies of biotemplates to the resulting silica. This method could be extended to the preparation of other oxides.

4-hydroxy-2-nonenal mediates genotoxicity and bystander effects caused by Enterococcus faecalis-infected macrophages.

BACKGROUND & AIMS: Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide and promotes chromosome instability via macrophage-induced bystander effects. We investigated the ability of 4-hydroxy-2-nonenal (4-HNE), a diffusible breakdown product of ω-6 polyunsaturated fatty acids, to mediate these effects. METHODS: 4-HNE was purified from E faecalis-infected macrophages; its genotoxicity was assessed in human colon cancer (HCT116) and primary murine colon epithelial (YAMC) cell lines. RESULTS: 4-HNE induced G(2)-M cell cycle arrest, led to formation γH2AX foci, and disrupted the mitotic spindle in both cell lines. Binucleate tetraploid cells that formed after incubation with 4-HNE were associated with the activation of stathmin and microtubule catastrophe. Silencing glutathione S-transferase α4, a scavenger of 4-HNE, increased the susceptibility of epithelial cells to 4-HNE-induced genotoxicity. Interleukin-10 knockout mice colonized with superoxide-producing E faecalis developed inflammation and colorectal cancer, whereas colonization with a superoxide-deficient strain resulted in inflammation but not cancer. 4-HNE-protein adducts were found in the lamina propria and macrophages in areas of colorectal inflammation. CONCLUSIONS: 4-HNE can act as an autochthonous mitotic spindle poison in normal colonic epithelial and colon cancer cells. This finding links the macrophage-induced bystander effects to colorectal carcinogenesis.

Phenylethanoid and lignan glycosides from polar extracts of Lantana, a genus of verbenaceous plants widely used in traditional herbal therapies.

Many references to the use of Lantana spp. can be found in the ethnopharmacological literature from locations around the globe. This study was focused on examining constituents from the polar extracts of Lantana radula Sw. and Lantana canescens Kunth, for which no prior chemical investigations had been reported. A new phenylethanoid glycoside, raduloside, and lignan glycoside, radulignan, were identified along with the known compounds alyssonoside, arenarioside, calceolarioside E, isonuomioside, samioside, and verbascoside.

Molecular structure and dynamic properties of a sulfonamide derivative of glutathione that is produced under conditions of oxidative stress by hypochlorous acid.

Reduced glutathione (GSH) is a cornerstone of the antioxidant stratagem for eukaryotes and some prokaryotes. Hypochlorous acid (HOCl), which is produced by neutrophilic myeloperoxidase, reacts rapidly with excess GSH to yield mainly oxidized glutathione (GSSG). GSSG can be further oxidized to give first N-chloro derivatives and, later, higher oxidation states at the S centers. Under certain conditions, another major species that is observed during the oxidation of GSH by HOCl (and a minor species for other oxidants) exhibits a molecular mass that is 30 mass units heavier than GSH. This GSH+2O-2H species, which has been employed as a biomarker for oxidative stress, has been previously proposed to be a sulfonamide. Employing NMR spectroscopy and mass spectrometry, we demonstrate that the GSH+2O-2H species is indeed a nine-membered cyclic sulfonamide. Alternative formulations, including six-membered 1,2,5-oxathiazine heterocycles, have been ruled out. Remarkably, the sulfonamide exists as a 2:1 equilibrium mixture of two diastereomers. Isotope tracer studies have demonstrated that it is the Glu C alpha center that has undergone racemization. It is proposed that the racemization takes place via an acyclic imine-sulfinic acid intermediate. The glutathione sulfonamides are stable products of GSH that have been detected in physiological systems. Elucidation of the structures of the glutathione sulfonamides provides further impetus to explore their potential as biomarkers of hypochlorous acid formation.

Chemical mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae.

Homoisocitrate dehydrogenase (HIcDH, 3-carboxy-2-hydroxyadipate dehydrogenase) catalyzes the fourth reaction of the alpha-aminoadipate pathway for lysine biosynthesis, the conversion of homoisocitrate to alpha-ketoadipate using NAD as an oxidizing agent. A chemical mechanism for HIcDH is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. According to the pH-rate profiles, two enzyme groups act as acid-base catalysts in the reaction. A group with a p K a of approximately 6.5-7 acts as a general base accepting a proton as the beta-hydroxy acid is oxidized to the beta-keto acid, and this residue participates in all three of the chemical steps, acting to shuttle a proton between the C2 hydroxyl and itself. The second group acts as a general acid with a p K a of 9.5 and likely catalyzes the tautomerization step by donating a proton to the enol to give the final product. The general acid is observed in only the V pH-rate profile with homoisocitrate as a substrate, but not with isocitrate as a substrate, because the oxidative decarboxylation portion of the isocitrate reaction is limiting overall. With isocitrate as the substrate, the observed primary deuterium and (13)C isotope effects indicate that hydride transfer and decarboxylation steps contribute to rate limitation, and that the decarboxylation step is the more rate-limiting of the two. The multiple-substrate deuterium/ (13)C isotope effects suggest a stepwise mechanism with hydride transfer preceding decarboxylation. With homoisocitrate as the substrate, no primary deuterium isotope effect was observed, and a small (13)C kinetic isotope effect (1.0057) indicates that the decarboxylation step contributes only slightly to rate limitation. Thus, the chemical steps do not contribute significantly to rate limitation with the native substrate. On the basis of data from solvent deuterium kinetic isotope effects, viscosity effects, and multiple-solvent deuterium/ (13)C kinetic isotope effects, the proton transfer step(s) is slow and likely reflects a conformational change prior to catalysis.