1. Determination of sibiromycin and its natural derivatives using new analytical and structural approaches
Miriam Chudomelová, Miroslav Sulc, Markéta Jelínková, Iva Fadrhoncová, Jana Olšovská, Jürgen Felsberg J Chromatogr A . 2011 Jan 7;1218(1):83-91. doi: 10.1016/j.chroma.2010.10.110.
A new separation and quantification method using ultra high-performance liquid chromatography (UHPLC) with UV detection was developed for the detection of sibiromycin in fermentation broth of Streptosporangium sibiricum. The solid phase extraction method based on cation-exchange was employed to pre-concentrate and purify fermentation broth containing sibiromycin prior to UHPLC analysis. The whole assay was validated and showed a linear range of detector response for the quantification of sibiromycin in a concentration from 3.9 to 250.0 μg mL⁻¹, with correlation coefficient of 0.999 and recoveries ranging from 71.66±3.55% to 74.76±5.18%. Method limit of quantification of the assay was determined as 0.18 μg mL⁻¹ and was verified with resulting RSD of 9.6% and accuracy of 97.6%. The developed assay was used to determine the sibiromycin production in 12 different fermentation broths. Moreover, several natural sibiromycin analogues/derivatives were described with pilot characterization using off-line mass spectrometry: the previously described dihydro-sibiromycin (DH-sibiromycin) and tentative bis-glycosyl forms of sibiromycin and its dihydro-analogue.
2. Mutasynthesis of a potent anticancer sibiromycin analogue
Isaac T Yonemoto, Natàlia Reixach, Barbara Gerratana, Ankush Khullar, Wei Li ACS Chem Biol . 2012 Jun 15;7(6):973-7. doi: 10.1021/cb200544u.
Pursuit of the actinomycete pyrrolobenzodiazepine natural product sibiromycin as a chemotherapeutic agent has been limited by its cardiotoxicity. Among pyrrolobenzodiazepines, cardiotoxicity is associated with hydroxylation at position 9. Deletion of the methyltransferase gene sibL abolishes the production of sibiromycin. Supplementation of growth media with 4-methylanthranilic acid can substitute for its native 3-hydroxy congener. Cultures grown in this fashion yielded 9-deoxysibiromycin. In this study, we characterize the structure and biological activity of sibiromycin and 9-deoxysibiromycin methyl carbinolamines. Preliminary in vitro evidence suggests that 9-deoxysibiromycin exhibits reduced cardiotoxicity while gaining antitumor activity. These results strongly support further exploration of the production and evaluation of monomeric and dimeric glycosylated pyrrolobenzodiazepine analogues of sibiromycin.
3. A four-enzyme pathway for 3,5-dihydroxy-4-methylanthranilic acid formation and incorporation into the antitumor antibiotic sibiromycin
Femke I Kraas, Mohamed A Marahiel, Tobias W Giessen Biochemistry . 2011 Jun 28;50(25):5680-92. doi: 10.1021/bi2006114.
The antitumor antibiotic sibiromycin belongs to the class of pyrrolo[1,4]benzodiazepines (PBDs) that are produced by a variety of actinomycetes. PBDs are sequence-specific DNA-alkylating agents and possess significant antitumor properties. Among them, sibiromycin, one of two identified glycosylated PBDs, displays the highest DNA binding affinity and the most potent antitumor activity. In this study, we report the elucidation of the precise reaction sequence leading to the formation and activation of the 3,5-dihydroxy-4-methylanthranilic acid building block found in sibiromycin, starting from the known metabolite 3-hydroxykynurenine (3HK). The investigated pathway consists of four enzymes, which were biochemically characterized in vitro. Starting from 3HK, the SAM-dependent methyltransferase SibL converts the substrate to its 4-methyl derivative, followed by hydrolysis through the action of the PLP-dependent kynureninase SibQ, leading to 3-hydroxy-4-methylanthranilic acid (3H4MAA) formation. Subsequently the NRPS didomain SibE activates 3H4MAA and tethers it to its thiolation domain, where it is hydroxylated at the C5 position by the FAD/NADH-dependent hydroxylase SibG yielding the fully substituted anthranilate moiety found in sibiromycin. These insights about sibiromycin biosynthesis and the substrate specificities of the biosynthetic enzymes involved may guide future attempts to engineer the PBD biosynthetic machinery and help in the production of PBD derivatives.