Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis.

MtmOIV and MtmW catalyze the final two reactions in the mithramycin (MTM) biosynthetic pathway, the Baeyer-Villiger opening of the fourth ring of premithramycin B (PMB), creating the C3 pentyl side chain, strictly followed by reduction of the distal keto group on the new side chain. Unexpectedly this results in a C2 stereoisomer of mithramycin, iso-mithramycin (iso-MTM). Iso-MTM undergoes a non-enzymatic isomerization to MTM catalyzed by Mg2+ ions. Crystal structures of MtmW and its complexes with co-substrate NADPH and PEG, suggest a catalytic mechanism of MtmW. The structures also show that a tetrameric assembly of this enzyme strikingly resembles the ring-shaped ß subunit of a vertebrate ion channel. We show that MtmW and MtmOIV form a complex in the presence of PMB and NADPH, presumably to hand over the unstable MtmOIV product to MtmW, yielding iso-MTM, as a potential self-resistance mechanism against MTM toxicity.

Bioanalytical method for quantitative determination of mithramycin analogs in mouse plasma by HPLC-QTOF.

Mithramycin (MTM) has potent anticancer activity, but severe toxicities restrict its clinical use. Semi-synthetic approaches have yielded novel MTM analogs with potentially lower toxicity and similar efficacy. In an effort to transition these analogs into in vivo models, a bioanalytical method was developed for their quantification in mouse plasma. Here we present the validation of the method for the quantitation of mithramycin SA-tryptophan (MTMSA-Trp) as well as the applicability of the methodology for assaying additional analogs, including MTM, mithramycin SK (MTMSK) and mithramycin SA-phenylalanine (MTMSA-Phe) with run times of 6 min. Assay linearity ranged from 5 to 100 ng/mL. Accuracies of calibration standards and quality control samples were within 15% of nominal with precision variability of <20%. MTMSA-Trp was stable for 30 days at -80°C and for at least three freeze-thaw cycles. Methanol (-80°C) extraction afforded 92% of MTMSA-Trp from plasma. Calibration curves for MTM and analogs were also linear from ≤5 to 100 ng/mL. This versatile method was used to quantitate MTM analogs in plasma samples collected during preclinical pharmacokinetic studies.

Structures of mithramycin analogues bound to DNA and implications for targeting transcription factor FLI1.

Transcription factors have been considered undruggable, but this paradigm has been recently challenged. DNA binding natural product mithramycin (MTM) is a potent antagonist of oncogenic transcription factor EWS-FLI1. Structural details of MTM recognition of DNA, including the FLI1 binding sequence GGA(A/T), are needed to understand how MTM interferes with EWS-FLI1. We report a crystal structure of an MTM analogue MTM SA-Trp bound to a DNA oligomer containing a site GGCC, and two structures of a novel analogue MTM SA-Phe in complex with DNA. MTM SA-Phe is bound to sites AGGG and GGGT on one DNA, and to AGGG and GGGA(T) (a FLI1 binding site) on the other, revealing how MTM recognizes different DNA sequences. Unexpectedly, at sub-micromolar concentrations MTMs stabilize FLI1-DNA complex on GGAA repeats, which are critical for the oncogenic function of EWS-FLI1. We also directly demonstrate by nuclear magnetic resonance formation of a ternary FLI1-DNA-MTM complex on a single GGAA FLI1/MTM binding site. These biochemical and structural data and a new FLI1-DNA structure suggest that MTM binds the minor groove and perturbs FLI1 bound nearby in the major groove. This ternary complex model may lead to development of novel MTM analogues that selectively target EWS-FLI1 or other oncogenic transcription factors, as anti-cancer therapeutics.

Distilbene derivative as a new environment-sensitive bifunctional ligand for the possible induction of serum protein aggregation: a spectroscopic investigation and potential consequences.

The photophysical properties of a new distilbene fluorophore, DPDB, belonging to the conjugated polyene family is found to be well modulated with the variation of the microenvironment. Compared to the ground state, the excited-state photophysical properties of the fluorophore have been altered to larger extents with the variation of polarity and the hydrogen-bonding nature of solvents. The change in the fluorescence intensity of DPDB shows a nice correlation with the aggregation behavior of different surfactants which have been utilized for the determination of the CMC of surfactants. The distribution of DPDB is found to be higher in nonionic micelles. On the other hand, DPDB specifically binds the subdomain IB cavity of serum albumin with a stronger binding ability with HSA compared to BSA. DPDB behaves like a bivalent (bifunctional) ligand and forms a complex of 2:1 stoichiometry with serum albumins. Dynamic light scattering and circular dichroism measurements indicate that DPDB favors the association of serum albumin molecules, promoting their preaggregation state. Aggregation is an important phenomenon and is known to be initiated by heat, extreme pH conditions, very high ionic strength, surfactants, metal ions, and so forth. This study explores a new avenue in bringing about association phenomena of serum albumins and points out that the binding of such a bifunctional ligand may also become an important factor in inducing the protein association.

Angucycline C5 glycosides: regio- and stereocontrolled synthesis and cytotoxicity.

This study discloses a general and convergent route for the regio- and stereospecific construction of the C5 glycosyl angucycline framework of mayamycin. C-Glycosidation, dearomatization, and Hauser annulation are the key steps. The synthetic analogues show cytotoxicity against different human cancer cell lines with IC50 values between 16.4 and 1.2 µM.

Stereoselective synthesis of hydroxy stilbenoids and styrenes by atom-efficient olefination with thiophthalides.

The synthesis of stilbenoids and styryl carboxylic acids is accomplished with high E-stereoselectivity by olefination of aldehydes with thiophthalides under basic conditions. The olefination is highly atom-efficient as it only loses elemental sulfur during the reaction. This olefination, in conjunction with retro Kolbe-Schmitt reaction, allows facile synthesis of E-hydroxystilbenoids with minimal employment of protecting groups. This study also discloses two important findings: formation of i) 4-methylsulfanyl isocoumarins and ii) an 2-arylindenone.

Benzannulation for the regiodefined synthesis of 2-alkyl/aryl-1-naphthols: total synthesis of arnottin I.

The annulation of phthalides with α-alkyl/arylacrylates in the presence of LDA/LHMDS is shown to directly give alkyl/aryl-1-naphthols. The method involving a novel dealkoxycarbonylation obviates the regiochemical issues in the synthesis of polysubstituted naphthalenes, and it forms the key step in a three-step total synthesis of arnottin I, a naphthobenzopyranone natural product.

Secondary metabolites produced by the citrus phytopathogen Phyllosticta citricarpa.

The isolation and structure elucidation of one new fungal metabolite, phenguignardic acid butyl ester (1a), and four previously reported metabolites (1b, 2a, 3-4) from the citrus phytopathogen Phyllosticta citricarpa LGMF06 are described. The new dioxolanone phenguignardic acid butyl ester (1a) had low phytotoxic activity in citrus leaves and fruits (at dose of 100 µg), and its importance as virulence factor in citrus black spot disease needs to be further addressed. Beside the phytotoxic analysis, we also evaluated the antibacterial (against methicillin sensitive and resistant Staphylococcus aureus) and cytotoxic (A549 non-small cell lung cancer, PC3 prostate cancer and HEL 299 normal epithelial lung) activities of the isolated compounds, which revealed that compounds 1a, 1b and 2a were responsible for the antibacterial activity of this strain.

Development of Mithramycin Analogues with Increased Selectivity toward ETS Transcription Factor Expressing Cancers.

Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is <100 nM in ETS cells. Molecular assays demonstrated the inhibitory capacity of these analogues against EWS-FLI1 mediated transcription in Ewing sarcoma. Structural analysis shows that positioning the tryptophan residue in a distal position improves selectivity, presumably via interaction with the ETS transcription factor. Thus, these analogues may present new ways to target transcription factors for clinical use.

Bioprospecting of Diaporthe terebinthifolii LGMF907 for antimicrobial compounds.

Antibiotic-resistant bacteria have been observed with increasing frequency over the past decades, driving the search for new drugs and stimulating the interest in natural products sources. Endophytic fungi from medicinal plants represent a great source of novel bioactive compounds useful to pharmaceutical and agronomical purposes. Diaporthe terebinthifolii is an endophytic species isolated from Schinus terebinthifolius, a plant used in popular medicine for several health problems. The strain D. terebinthifolii LGMF907 was previously reported by our group to produce secondary metabolites with biological activity against phytopathogens. Based on these data, strain LGMF907 was chosen for bioprospecting against microorganisms of clinical importance and for characterization of major secondary metabolites. In this study, different culture conditions were evaluated and the biological activity of this strain was expanded. The crude extracts demonstrated high antibacterial activity against Escherichia coli, Micrococcus luteus, Saccharomyces cerevisiae, methicillin-sensitive Staphylococcus aureus, and methicillin-resistant S. aureus. The compounds diaporthin and orthosporin were characterized and also showed activity against the clinical microorganisms evaluated. This study discloses the first isolation of diaporthin and orthosporin from D. terebinthifolii, and revealed the potential of this endophytic fungus to produce secondary metabolites with antimicrobial activity.

Structural Basis for EarP-Mediated Arginine Glycosylation of Translation Elongation Factor EF-P.

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-ß-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design.IMPORTANCE The structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP not only provides the first molecular insights into arginine glycosylation but also lays the basis for targeted-inhibitor design against Pseudomonas aeruginosa infection.