Thesis
Reference
Novel GC-rich DNA binding compounds generated by metabolic engineering of the mithramycin biosynthetic pathway in
"Streptomyces argillaceus" : characterization of anti-transcriptional and anti-cancer activity
ALBERTINI, Veronica
Abstract
Les buts généraux étaient d'identifier de nouveaux analogues de la mithramycine, MTM, un acide auréolique déjà connu pour ses activités anti-tumorales, aux propriétés pharmacologiques et toxicologiques améliorées, et d'examiner les effets et le mécanisme d'action de ces composés dans les cellules cancéreuses et normales. L'activité anti-tumorale de la MTM dépend de sa capacité à se lier à des séquences de l'ADN riches en GC. Nous avons créé différents analogues par biosynthèse. Nos études ont montré que l'un deux, la SDK, est un inhibiteur de la transcription dépendant du Sp1 beaucoup plus puissants que la MTM. La capacité de la SDK de moduler rapidement l'expression de plusieurs gènes impliqués dans des fonctions cellulaires typiques du cancer et à des doses faibles est une caractéristique intéressante qui pourrait être exploitée pour des applications thérapeutiques dans le domaine du cancer et d'autres maladies.
ALBERTINI, Veronica. Novel GC-rich DNA binding compounds generated by metabolic engineering of the mithramycin biosynthetic pathway in "Streptomyces argillaceus" : characterization of anti-transcriptional and anti-cancer activity . Thèse de doctorat : Univ. Genève, 2007, no. Sc. 3872
URN : urn:nbn:ch:unige-6139
DOI : 10.13097/archive-ouverte/unige:613
Available at:
http://archive-ouverte.unige.ch/unige:613
Disclaimer: layout of this document may differ from the published version.
UNIVERSITE DE GENEVE FACULTE DES SCIENCES
Section des Sciences Pharmaceutiques Professeur Leonardo Scapozza
ONCOLOGY INSTITUTE OF SOUTHERN SWITZERLAND
Laboratory of experimental oncology Docteur Carlo Catapano, MD
Novel GC-Rich DNA Binding Compounds Generated By Metabolic Engineering Of The Mithramycin
Biosynthetic Pathway In Streptomyces Argillaceus:
Characterization Of Anti-Transcriptional And Anti- Cancer Activity
THÈSE
présentée à la Faculté des sciences de l'Université de Genève pour obtenir le grade de Docteur ès sciences, mention interdisciplinaire
par
Veronica ALBERTINI de
Como (Italie)
Thèse N
o3872
GENEVE2007
To my husband
Facts are the air of scientists. Without them, you can never fly.
Linus C. Pauling
TABLE OF CONTENTS
ACKNOWLEDGEMENTS 5
LIST OF ABBREVIATIONS 6 SUMMARY 7
INTRODUCTION 10
Mithramycin and generation of novel mithramycin analogues
11
Transcription factors as therapeutic targets: the Sp1 transcription factor
20
Involvement of p53 dependent and independent pathways in the cellular responses to MTM
22
Overall hypothesis and experimental plan
27
MATERIALS AND METHODS 28
RESULTS 37
DISCUSSION 70
BIBLIOGRAPHY 79
Résumé de thèse 88
Introduction
89
Résultat et discussion
95
SUPPLEMENTAL DATA 102
PUBBLICATIONS AND POSTERS 115
CURRICULUM VITAE 118
ACKNOWLEDGEMENTS
First of all I want to thank Professor Franco Cavalli, director of Oncology Institute Of Southern Switzerland (IOSI), for allowing me to work in this important institute for the cancer research; Doctor Carlo Catapano, co- director of my thesis, for his support and for allowing me to work at this very interesting project. During my thesis I took advantage of his experience in laboratory and also of his patience!
A particular acknowledgement to Professor Leonardo Scapozza to allow me to do my doctorat in his group at the University of Geneva.
A very big thanks to all my collegues , IOSI group, I spent with you many magic moments and I will never forget you.
Thank you to all the members of the jury who have accepted to read this thesis.
Finally, but the most important, a particular thanks to my family, my mother and my father, to allow me to arrive where I am and to be what I am.
This thesis is dedicated to my husband; he followed me in all my decisions
and over all he had a lot of patience during the moments when I was tired.
LIST OF ABBREVIATIONS
MTM Mithramycin
SDK Mithramycin Short side chain DiKeto
SK Mithramycin Short side chain Keto
NMR Nuclear Magnetic Resonance
MtmW Mithramycin W Ketoreductase
MtmOIV Mithramycin oxigenase IV
M7W1 Mutant strain of Streptomyces Argillaceus
HPLC High Performance Liquid Chromatography
X2 A2780X2-CX2 cells
SUMMARY
Aureolic acid antibiotics, like mithramycin (MTM), are interesting lead compounds for drug development. MTM binds to GC-rich DNA sequences, blocks binding of Sp1-family transcription factors and inhibits preferentially transcription of Sp1-regulated genes.
Deregulation of Sp1 transcription factor activity is recognized as an important event in the pathogenesis of a growing number of human diseases, including cancer, chronic inflammatory, cardiovascular and neurodegenerative disorders. Aberrant activity of Sp1 transcription factors is involved in multiple aspects of cancer pathogenesis and may represent a logical target for development of new anticancer drugs. Compounds able to interfere selectively with Sp1 transcription factor activity and modulate expression of critical target genes could be very promising drug candidates. The overall objectives of this study were to identify new aureolic acid antibiotic derivatives with the same mechanism of action of MTM but improved pharmacological properties and to investigate their effects in normal and cancer cells.
New MTM analogues were generated by combinatorial biosynthesis or metabolic engineering of the MTM biosynthetic pathway in S. argillaceus. Initial testing of these compounds using luciferase reporter assays led to the identification of two MTM derivatives, namely MTM SK (SK) and MTM SDK (SDK), with interesting biological activity. SDK and SK differed with respect to MTM only in the structure and length of the pentyl side chain in C-3. Our studies revealed that SDK was a particularly potent inhibitor of Sp1- dependent reporter activity and interfered minimally with other transcription factors, indicating that it retained a high degree of selectivity toward GC-rich DNA binding transcription factors. Gene expression studies by RT-PCR and microarrays showed that SDK inhibited transcription of multiple genes involved in cancer development and progression, consistent with the pleiotropic role of Sp1 family transcription factors. Moreover, SDK inhibited cell proliferation and was a potent inducer of apoptosis in cancer cells while it had minimal effects on viability of normal fibroblasts.
The response of normal and cancer cells to SDK was influenced by the p53 status of the cells and the drug’s ability to activate different apoptotic pathways. In cells with wild type p53, SDK pro-apoptotic activity depended on p53 with concomitant activation of both the extrinsic and intrinsic apoptotic pathway. This was accomplished via nuclear localization of p53 and transcriptional activation of p53 target genes as well as direct translocation of p53 to the mitochondria. The apoptotic response of p53wt cells was favored by the ability of SDK to prevent p53 dependent induction of p21.This eliminated a potential protective factor favoring apoptosis instead of cell cycle arrest. In p53 deficient cells, SDK ability to induce apoptosis was reduced compared to p53wt cells and depended largely on the activation of the extrinsic pathway. In cells carrying p53 mutations that impaired transcriptional activity (i.e., DNA binding domain mutants), SDK induced apoptosis more effectively than in p53 null cells via translocation of p53 to the mitochondria and direct activation of the intrinsic pathway. Unlike cancer cells, normal cells appeared to be protected from SDK cytotoxic effects because of the p53 dependent induction of p21 leading to cell cycle arrest rather than cell death.
Taken together, these results suggest that the new MTM derivative SDK could be an effective agent for treatment of cancer and other diseases associated with abnormal activity of the Sp1 transcription factor.
INTRODUCTION
In 2004 there were approximately 2.8 millions of new cases of cancer and 1.7 millions of cancer deaths in Europe (1). These numbers clearly highlight the needs of new, more effective agents for prevention and treatment of cancer. Natural products that are secondary metabolites of plants, fungi and other microorganisms provide an immense source of biologically active compounds. Actinomycetes in particular produce over 60% of known biologically active secondary metabolites, with nearly 80% of them produced by the genus Streptomyces. These products include antiparasitic drugs, antibiotics (e.g., chloramfenicol, erythromycin A and neomycin), antifungal agents and antitumor drugs (e.g., daunorubicin and doxorubicin) (2). Many of these compounds are polyketides and are generally produced through linear enzymatic condensation of a starter unit, like acetyl- CoA, with several extender units such as malonyl-CoA. The result is a poli-Ketone, which can be subsequently modified by alkylation, cyclization, glycosilation, oxidation and reduction, to obtain compounds with a wide range of structural diversity and activity.
In this project we focused on the aureolic acid antibiotic mithramycin (MTM, also known as mithracin or plicamycin), which is produced by soil bacteria like Streptomyces argillaceus and plicatus. The overall goals were to identify new analogues with improved pharmacological and toxicological properties and to investigate the effects and mechanism of action of the new compounds in normal and cancer cells.
Mithramycin and generation of novel mithramycin analogues
The aureolic acid group of antibiotics includes MTM, chromomycin A3 (CHR), olivomicin A (OLI), UCH9 and durhamycin A (DUR) (3). All these compounds contain the same tricyclic core moiety with a unique dihydroxy-methoxy-oxo-pentyl side chain attached at C3 and a residue at C7, which is either an H-atom or a small alkyl chain (Fig. 1). The compounds differ in the nature of the oligosaccharide chains, which consist of various 2,6-dideoxysugar
residues (4). These structural variations impart differences in the DNA binding and activity profiles of the members of this group of antibiotics (5,6,7). Many of these compounds have biological and pharmacological activity but MTM is the only aureolic acid antibiotic approved for clinical use. MTM has been used clinically for many years to treat testicular carcinoma as well as hypercalcemia in patients with metastatic bone lesions and Paget’s disease (8,9,10). Its current clinical use is limited by its severe side effects that include gastrointestinal, hepatic, kidney and bone marrow toxicity (11,12). Nevertheless, MTM has attracted renewed attention as an experimental therapeutic agent in cancer and non- cancer related disorders (13-22). Moreover, MTM can be a lead compound to create new analogues with greater biological activity and lower toxicity.
The anticancer activity of MTM is due to its ability to bind DNA preferentially in correspondence of GC-rich sequences. The DNA binding properties of MTM have been extensively investigated by NMR and X-ray crystallography (5,7,23-27). MTM binds DNA as a dimer in a MTM:Mg+2-complex (2:1) with the two MTM molecules disposed in a head-to- tail fashion and Mg+2 interacting with the 1-O carbonyl oxygen and 9-O phenolate anion of each aglycon in the dimer (Fig. 2). DNA sequence specificity is due to hydrogen bonds that form between the 8-O hydroxyl group of the aglycon and the NH2 of the guanine. The oligosaccharide moieties of MTM extend across the entire minor groove, with the A and B sugars crossing the phosphate backbone and lying near the major groove. The C-D units of the thrisaccharide chains stack on the aromatic core of the dimer-partner aglycon (Fig.
2). The C-D-E thrisaccharide unit is essential for dimer formation as well as optimal DNA binding (28). The pentyl side chain seems to play a role in DNA binding as demonstrated by the inactivity of tetracyclic MTM intermediates that do not have it. Knowledge of the role of different elements in the MTM structure has been essential to guide the efforts to modify critical portions of the molecule and generate new derivatives.
Figure 1. Structures of natural aureolic acid compounds
Figure 2. Binding of mithramycin to DNA.
Surface representation of a Mg2+ coordinated mithramycin dimer bound to DNA viewed from the minor groove (left) and backbone (right). Mg2+
and water are depicted in pink and blue, respectively. Various parts of the MTM molecule are indicated. Modified from Ming-hon Hou et al., 2005.
A,BA,B
C,D,E C,D,E pentyl
pentylside side chain chain
A,BA,B
C,D,E C,D,E pentyl
pentylside side chain chain
OH O OH
H3CO
O CH3
O OH OH H
O O O
O HOO C
H3 CHCCO
CH3 CH3 CH3
OH HO O O
O CH3 O CH3 O H3C
O H
7 1
1' 3'
3
Olivomycin A
O H3CCO
O
H3C H3C
OH O OH
H3CO
O CH3
O OH OH H C
H3
O O O
O HOO O
HH3C
CH3 CH3 CH3
OH HO O O
O CH3 O HO CH3 O HHO
7 1
1' 3'
3
Mithramycin OH O OH
H3CO
O CH3
O OH OH H C
H3
O O O
O HOO C
H3 H3CCO
CH3 CH3 CH3
OH HO O O
O CH3
O CH3 O H3C
O H
7 1
1' 3'
3
Chromomycin A3
O H3CCO
O
OH O OH
H3CO
O CH3
O OH OH H3CH2CHCH3C H
O O O
O HOO H3CO
CH3 CH3 CH3
OH HO O
O CH3
7 1
1' 3'
3
UCH9
HO HO
O CH3 O HO
HO
OH O OH
H3CO
O CH3
O OH OH H
O O O
O HOO HO CH3 CH3 CH3
OH
7 1
1' 3'
3
Durhamycin A
O CH3 O HO
HO
O O
O CH3 O H O CH3
O HHO
H3CCO O H3CH2CHCH3C OH O
OH
H3CO
O CH3
O OH OH H
O O O
O HOO C
H3 CHCCO
CH3 CH3 CH3
OH HO O O
O CH3 O CH3 O H3C
O H
7 1
1' 3'
3
Olivomycin A
O H3CCO
O
H3C H3C
OH O OH
H3CO
O CH3
O OH OH H
O O O
O HOO C
H3 CHCCO
CH3 CH3 CH3
OH HO O O
O CH3 O CH3 O H3C
O H
7 1
1' 3'
3
Olivomycin A
O H3CCO
O
H3C H3C
OH O OH
H3CO
O CH3
O OH OH H C
H3
O O O
O HOO O
HH3C
CH3 CH3 CH3
OH HO O O
O CH3 O HO CH3 O HHO
7 1
1' 3'
3
Mithramycin
OH O OH
H3CO
O CH3
O OH OH H C
H3
O O O
O HOO O
HH3C
CH3 CH3 CH3
OH HO O O
O CH3 O HO CH3 O HHO
7 1
1' 3'
3
Mithramycin OH O OH
H3CO
O CH3
O OH OH H C
H3
O O O
O HOO C
H3 H3CCO
CH3 CH3 CH3
OH HO O O
O CH3
O CH3 O H3C
O H
7 1
1' 3'
3
Chromomycin A3
O H3CCO
O
OH O OH
H3CO
O CH3
O OH OH H C
H3
O O O
O HOO C
H3 H3CCO
CH3 CH3 CH3
OH HO O O
O CH3
O CH3 O H3C
O H
7 1
1' 3'
3
Chromomycin A3
O H3CCO
O
OH O OH
H3CO
O CH3
O OH OH H3CH2CHCH3C H
O O O
O HOO H3CO
CH3 CH3 CH3
OH HO O
O CH3
7 1
1' 3'
3
UCH9
HO HO
O CH3 O HO
HO
OH O OH
H3CO
O CH3
O OH OH H3CH2CHCH3C H
O O O
O HOO H3CO
CH3 CH3 CH3
OH HO O
O CH3
7 1
1' 3'
3
UCH9
HO HO
O CH3 O HO
HO CH3 O HO
HO
OH O OH
H3CO
O CH3
O OH OH H
O O O
O HOO HO CH3 CH3 CH3
OH
7 1
1' 3'
3
Durhamycin A
O CH3 O HO
HO
O O
O CH3 O H O CH3
O HHO
H3CCO O H3CH2CHCH3C
OH O OH
H3CO
O CH3
O OH OH H
O O O
O HOO HO CH3 CH3 CH3
OH
7 1
1' 3'
3
Durhamycin A
O CH3 O HO
HO
O O
O CH3 O H O CH3
O HHO
H3CCO O H3CH2CHCH3C
OH O OH
H3CO
O CH3
O OH OH H
O O O
O HOO HO CH3 CH3 CH3
OH
7 1
1' 3'
3
Durhamycin A
O CH3 O HO
HO CH3 O HO
HO
O O
O CH3 O H O CH3
O HHO
O O
O CH3 O H O CH3
O HHO
H3CCO O H3CCO
O H3CH2CHCH3C
Metabolic engineering of MTM biosynthetic pathway
The use of synthetic or semi-synthetic approaches to obtain new analogues has proven to be very difficult in the case of aureolic acid antibiotics because of the large size, diverse chemical functionalities and stereochemical controls necessary to produce this type of compounds. A valid alternative is combinatorial biosynthesis or metabolic engineering, where the genes of the compound’s biosynthetic pathway are altered through gene inactivation or recombination in the producing organisms. This approach requires knowledge of the multiple steps involved in the biosysnthetic pathway of the compounds.
The biosynthetic pathway that produces MTM has been elucidated almost in its entirety in recent years, in large part thanks to the efforts of Dr. J. Rohr’s group (29-35).
Approximately 30 distinct genes involved in the biosynthesis of MTM have bee identified so far (Fig. 3). Genes belonging to this pathway include: polyketide synthase genes (PKS) that catalyze the production of the polyketide backbone; post-PKS genes like ketoreductases, oxygenases, glycosyl-transferases and methyltransferases; and complementary genes that are involved in early sugar biosynthesis and regulatory functions. The function of many of these enzymes has been assigned through data bank comparison and by applying genetic approaches, such as gene inactivation, to the MTM producer S. argillaceus. These experiments yielded various MTM derivatives exhibiting a variety of structural changes in the saccharide chains as well as in the aglycon moiety. Analysis of these MTM intermediates has helped to determine the consecutive steps of the biosynthetic pathway.
Figure 3. Genetic organization of the MTM biosynthetic gene cluster in S. argillaceus.
Biosynthesis of MTM proceeds through the condensation of multiple acyl-coA monomers catalyzed by type II polyketide synthases. The initial condensation phase leads to the formation of the tetracyclic intermediate premithramycinone, which is subsequently modified by the addition of saccharide chains leading to the formation of premithramycin B (Fig. 4). The final steps, which are catalyzed by the oxygenase MtmOIV and ketoreductase MtmW, are the oxidative cleavage of the fourth ring of premithramycin B followed by the decarboxylation and ketoreduction of the pentyl substituent in C-3 (29,33).
Genetic approaches have been applied to MTM producing microorganisms by Dr. J.
Rohr’s group, in the attempt to produce compounds that would share the same basic mechanism of action of MTM, but have increased potency and/or improved therapeutic index. Attempts made over the years have yielded compounds exhibiting distinct structural changes and have provided novel information on the structure-activity relationships of aureolic acid-type compounds (10,27-29). Particularly, while modifications of the carbohydrate and aglycon moieties disrupted almost completely activity, changes in the 3-side chain appeared more promising (32,33,36).
Deoxysugar Biosynthesis/ Dehydratase Acyl Carrier Protein
RRegulatory Gene Oxygenase ß-Ketosynthase Methyltransferase SAM Synthase /
THF Reductase AcylCoA Ligase Acyltransferase Glycosyltransferase dTDP-Glu-Synthase Aromatase Chain Length Factor XResistance Genes
Ketoreductase Cyclase Reductase (Deoxysugar Biosynthesis
P K
X Q OII
TII OIII L S
D E TI
R A F
REG SAM SAM
THF SYN DEH KR OXY OXY LE ARO CYC KS/AT CLF ACP KR
OI Y TIII
OXY CYC KR
C U V W GIV GIII MII MI GII GI
MT R? DEH? R? GT GT MT MT GT GT
X Y A B mtm
OXY
1 kB
RG RG RG RG
OIV
Deoxysugar Biosynthesis/ Dehydratase Acyl Carrier Protein
RRegulatory Gene Oxygenase ß-Ketosynthase Methyltransferase SAM Synthase /
THF Reductase AcylCoA Ligase Acyltransferase Glycosyltransferase dTDP-Glu-Synthase Aromatase Chain Length Factor XResistance Genes
Ketoreductase Cyclase Reductase (Deoxysugar Biosynthesis Deoxysugar Biosynthesis/ Dehydratase Acyl Carrier Protein
Deoxysugar Biosynthesis/ Dehydratase Acyl Carrier Protein
RRegulatory Gene Oxygenase ß-Ketosynthase Methyltransferase RRegulatory Gene Oxygenase ß-Ketosynthase Methyltransferase
SAM Synthase /
THF Reductase AcylCoA Ligase Acyltransferase Glycosyltransferase SAM Synthase /
THF Reductase AcylCoA Ligase Acyltransferase Glycosyltransferase dTDP-Glu-Synthase Aromatase Chain Length Factor XResistance Genes dTDP-Glu-Synthase Aromatase Chain Length Factor XResistance Genes
Ketoreductase Cyclase Reductase (Deoxysugar Biosynthesis Ketoreductase Cyclase Reductase (Deoxysugar Biosynthesis
P K
X Q OII
TII OIII L S
D E TI
R A F
REG SAM SAM
THF SYN DEH KR OXY OXY LE ARO CYC KS/AT CLF ACP KR
OI Y TIII
OXY CYC KR
C U V W GIV GIII MII MI GII GI
MT R? DEH? R? GT GT MT MT GT GT
X Y A B mtm
OXY
1 kB
RG RG RG RG
OIV
P K
X Q OII
TII OIII L S
D E TI
R A F
REG SAM SAM
THF SYN DEH KR OXY OXY LE ARO CYC KS/AT CLF ACP KR
P K
X Q OII
TII OIII L S
D E TI
R A F
REG SAM SAM
THF SYN DEH KR OXY OXY LE ARO CYC KS/AT CLF ACP KR
OI Y TIII
OXY CYC KR
C U V W GIV GIII MII MI GII GI
MT R? DEH? R? GT GT MT MT GT GT
OI Y TIII
OXY CYC KR
C U V W GIV GIII MII MI GII GI
MT R? DEH? R? GT GT MT MT GT GT
X Y A B mtm
OXY
1 kB
RG RG RG RG
OIV X Y A B mtm
OXY
1 kB
RG RG RG RG
OIV
Figure 4. Compounds involved in the late steps of mithramycin biosynthesis.
In the effort to find new analogs with improved properties, two new compounds that differed only in the structure and length of the substituent at C-3 position (named here 3- side chain) with respect to MTM were identified. The new derivatives were isolated from
the S. argillaceus M7W1 mutant strain that carried a targeted inactivation of the ketoreductase MtmW gene. This ketoreductase along with the oxygenase MtmOIV catalyzes the last steps in the MTM biosynthesis leading to the formation of the 3-side chain (29,33). Targeted inactivation of the MtmW gene had led first to the isolation of MTM SK (SK). This compound had a butyl side chain with a keto and a secondary alcohol group (Fig. 5).
Figure 5. Structure of Mithramycin (MTM) and its new analogues Mithramycin SDK (SDK) and mithramycin SK (SK) obtained by combinatorial biosynthesis. The compounds differs on the substituent at position C-3, named 3-side chain.
Modifications of the isolation procedure of the secondary metabolites from the S.
argillaceus M7W1 mutant strain showed that inactivation of the ketoreductase MtmW resulted in the accumulation of a second product, MTM SDK (SDK), in addition to the previously identified SK. Detection of SDK, which is acid sensitive and poorly soluble in ethylacetate, had been missed in earlier work because an ethylacetate extraction step
OH O OH
H3CO
O CH3 O OH
OH
H C
H3
O O O
O HOO O
H C H3
CH3 CH3 CH3
OH HO O O
O CH3
O HO CH3 O HHO
A B
D C E
7 1
1' 3'
3
Mithramycin
C52H76O24 (1085.17 g/Mol)
OH O OH
H3CO
O CH3 O
O H C
H3
O O O
O HOO O
HH3C
CH3 CH3 CH3
OH HO O O
O CH3 O HO CH3 O
HHO B A
D C E
7 1
1' 3'
3
Mithramycin SDK C51H72O23 (1053.15 g/Mol)
OH O OH
H3CO
OH CH3 O
O
H C
H3
O O O
O HOO O
HH3C
CH3 CH3 CH3
OH HO O O
O CH3 O HO CH3 O HHO
A B
D C E
7 1
1' 3'
3
Mithramycin SK C51H74O23 (1055.14 g/Mol) OH O
OH
H3CO
O CH3 O OH
OH
H C
H3
O O O
O HOO O
H C H3
CH3 CH3 CH3
OH HO O O
O CH3
O HO CH3 O HHO
A B
D C E
7 1
1' 3'
3
Mithramycin
C52H76O24 (1085.17 g/Mol)
OH O OH
H3CO
O CH3 O
O H C
H3
O O O
O HOO O
HH3C
CH3 CH3 CH3
OH HO O O
O CH3 O HO CH3 O
HHO B A
D C E
7 1
1' 3'
3
Mithramycin SDK C51H72O23 (1053.15 g/Mol)
OH O OH
H3CO
OH CH3 O
O
H C
H3
O O O
O HOO O
HH3C
CH3 CH3 CH3
OH HO O O
O CH3 O HO CH3 O HHO
A B
D C E
7 1
1' 3'
3
Mithramycin SK C51H74O23 (1055.14 g/Mol)
and HPLC acidic elution solvent were used in the isolation procedure (29). The new compound had also a fully glycosylated tricyclic aglycon and differed with respect to MTM only in the structure and length of the 3-side chain. In particular, the newly discovered analogue SDK (for short side chain, diketo) had 3-butyl side chain with two keto groups instead the keto- and secondary alcohol group present in the SK side chain.
The proposed pathway leading to the formation of SK and SDK via a rearrangement process triggered by the inactivation of MtmW is shown in Figure 6. In the absence of the ketoreductase, the product of the MtmOIV reaction, MTM DK, undergoes a spontaneous rearrangement in its 3-β-diketo side chain, triggered by a β-shift and followed by elimination of either formic acid or formaldehyde to form SK and SDK, respectively. This view has been recently confirmed through studies with the isolated enzyme MtmOIV, showing that SDK and SK are the major end products of the conversion of premithramycin B at pH 8.25 (37). This mechanism is also supported by 13C incorporation studies, which proved that C-3’ is excised during the formation of SK (29). A similar compound with a short α-diketo-butyl side chain was found as a product of a mutant of the chromomycin producer S. griseus (38).
In preliminary studies the two MTM compounds SDK and SK showed interesting properties warranting further investigation of their biological activity. In particular, footprinting experiments performed in Dr. Rohr’s laboratory using a previously isolated 223- bp fragment of the c-src gene promoter (36) had shown similar binding activity of SDK, SK and MTM to GC-rich DNA sequences, although slight differences were demonstrated in binding affinity and site selectivity among the three compounds.
