Biosynthesis of Anti-Tumour Molecules PDF Print E-mail

Unit Heads

Jose Antonio Salas Fernández

Mª Carmen Méndez Fernández

Location

Department of Functional Biology

Microbiology Area

Faculty of Medicine


 

Research Lines

Techniques

Biography

Publications

Members

 

 


Research Lines

 

The main objective of the research lines of the Biosynthesis of Anti tumour Molecules group is the generation of new derivatives of anti tumour compounds. This is done through the application of genetic engineering techniques to microorganisms in order to isolate and characterise the genetic groupings implicated in the biosynthesis of anti tumour compounds and their subsequent manipulation. In general terms this manipulation consists of genetic inactivation and in the expression of genes with two or more distinct routes of biosynthesis in the microorganism which produces the anti-tumour compound. The ultimate aim of this technology, known as "Combinatorial Biosynthesis" is the generation of new molecules, most of which are hybrid molecules, with different structural characteristics to those of naturally occurring products. Currently we are working on the identification of new antitumour compounds produced by actinomyces isolated from Peruvian leafcutter ants. These ants are also the object of study in relation to the metabolic routes and systems of regulation involved in the biosynthesis of these antitumour compounds with the aim of improving their production.

At the present the generation of new derivatives by Combinatorial Biosynthesis is focussing on the following groups of compounds:

 

DNA replication and transcription inhibitors: mithramycin and cromomycin.

 

The majority of antitumour compounds currently used in cancer chemotherapy act via their interaction with cellular DNA by inhibiting its replication and transcription processes. In recent years our group has isolated and characterized the routes of biosynthesis of antitumours of the aureolic acid group; mithramycin and cromomycin, produced by the organisms Streptomyces argillaceus and Streptomyces griseus ssp. griseus, respectively.Over 70 derivatives have been developed from these compounds, using modifications to the lateral chain as well as glycosylation and acetylation patterns. One of these compounds, mithramycin SK, exhibits approximately 80 times more antitumour activity than mithramycin. In addition, work is being carried out to develop new derivatives of cromomycin, mithramycin and mithramycin SK with further modifications which will facilitate increased antitumour activity.

 

Indolocarbozole DNA topoisomerase inhibitors (rebeccamycin) and protein kinases (staurosporine)

 

The indolocarbozole family includes compounds which inhibit the activity of DNA tooisomerases such as rebeccamycin, or which inhibit protein kinases such as staurosporine. Derivatives of these compounds are currently being clinically trialled to determine their application against different types of cancer. The routes of biosynthesis of rebeccamycin and of staurosporine, produced by Lechevaleria aerocolonigenes and Streptomyces longisporoflavus, respectively, have been isolated and characterized in our laboratories in recent years. The manipulation of these routes and their expression in non producing hosts has enabled the generation of over 20 new carbozoles which are currently being analysed in order to determine their protein kinase inhibition activity and possible future use as antitumour agents.

 

Oviedomycin, an apoptosis inducer.

 

This compound, from the angucycline family of antitumour compounds, was discovered by our group from cultures of Streptomyces antibioticus. Oviedomycin demonstrates antitumour activity and has also been shown to induce programmememed cell death ("apoptosis") in HeLa cells from uterus neck carcinoma. The isolation and characterization of its rbiosynthetic route and its expression in non producing hosts has facilitated the generation of 8 new derivatives with different patterns of hydroxylation, three of which exhibit better antitumour activity than oviedomycin itself.

 

Colimycin, a 2,2'-bipyridine compound with antitumour properties

 

Colimycin is a molecule of the 2,2'-bipyridine family of compounds with a chemical structure derived from picolinic acid. The biosynthetic route of this compound, produced by Streptomyces sp. CS40, a strain isolated from leafcutter ants (Acromytmex octospinosus) is in the process of being characterized. Colimycin exhibits cytotoxic activity against mouse P388 leukemia cells and has been approved as a carcinostatic agent for oral and perenteral administartaion, per se or in combination with other antitumour agents. Colimycin is, in addition, an inhibitor of the union of dexamethasone and glucorticoids and may posess the ability to inhibit oxidative stress in cells.

 

Streptolydigin, an RNA polymerase and terminal deoxynucleotidyltransferase inhibitor.

 

Currently work is underway to isolate and characterize the biosynthetic route of the antibiotic, acyltetramic acid, an inhibitor of the RNA polymerase streptolydigin produced by Streptomyces lydicus. The compounds of the tetramic acid family such as streptolydigin and tyrandamycin demonstrate potent inhibitory activity in relation to the enzyme terminal deoxynucleotidyltransferase (TDT), which is found in great quantities in the leucocytes of patients with acute lymphoblastic leukemia and chronic myelocitic leukemia. This activity has resulted in these compounds being considered as potential treatments for TDT+ leukemias. We have characterized the biosynthetic route of streptolydigin and are currently conducting experiments directed towards the generation of new derivatives of both streptolydgin and tyrandamycin.


 

 

Techniques

 

 

The main aim of the Biosynthesis of Antitumour Molecules Group is the generation of new compounds with potential antitumour activity through the genetic or metabolic manipulation antitumourcompound producing microorganisms. The Unit has a great deal of experience in Molecular Biology and Genetic Engineering techniques among which it is worth highlighting the Unit's expertise in:

 

-Genetic manipulation of microorganisms

-Setting up of gene libraries

-DNA sequencing

-Analysis of DNA sequences

-PCR and RT-PCR

-Microarrays in Streptomyces

-Purification of antitumour compounds

-HPLC and MS analysis

-Expession and purification of proteins

-Enzymatic reaction trials

 

 

Group Head Biography

 

 

José Antonio Salas completed his degree in Biological Sciences and PhD at the University of Oviedo. After his doctoral thesis he spent several periods as a postdoctoral fellow at Cambridge and Leicester Universities (UK) in the laboratories of Professor David J. Ellar and Professor Eric Cundliffe.  In 1984 he was appointed Senior Lecturer in Microbiology at the University of Oviedo and has been Professor of Microbiology at the same university since 1999. He has published more than 160 scientific articles and is the inventor of 14 patents. He has supervised 26 doctoral theses and has been the head researcher in 20 research projects funded by Spanish institutions and 10 European Union projects. He has also carried out 5 projects with private companies. His current line of research is focused on the application of combinatorial biosynthesis in the generation of new derivatives of anti-tumour compounds produced by microorganisms with better anti-tumour activity or improved pharmacological properties.

 

Carmen Méndez Fernández completed her degree in Biological Sciences at the University of Oviedo, graduating with distinction in 1979. She then joined the Interfaculty Department of Microbiology at the University of Oviedo, where she completed her doctoral thesis, a study of the Streptomyces cell cycle and the organisation and development of its colonies, and received her PhD in 1984. After spending two years as a postdoctoral fellow in the Streptomyces genetics group at the John Innes Institute in Norwich (UK), she re-joined the Microbiology Area at the University of Oviedo and in 1988 was appointed Senior Lecturer in this area. Her work as a researcher has primarily focused on the characterization of groups of genes which biosynthesise bioactive compounds, such as anti-tumour compounds, and the use of these genes to create new compounds which may have higher anti-tumour activity or improved pharmacological properties.


 

Selected Publications

 

  • 1. Remsing,L.L., J.Garcia-Bernardo, A.Gonzalez, E.Künzel, U.Rix, A.F.Braña, D.W.Bearden, C.Méndez, J.A.Salas and J.Rohr (2002). Ketopremithramycins and Ketomithramycins, four new aureolic acid-type compounds obtained upon inactivation of two genes involved in the biosynthesis of the deoxysugar moieties of the antitumor drug mithramycin by Streptomyces argillaceus, reveal novel insights into post-PKS tailoring steps of the mithramycin biosynthetic pathway. Journal of the American Chemical Society 124: 1606-1614.

 

  • 2. Méndez,C., E.Künzel, F.Lombó, W.Cotham, M.Walla, D.W.Bearden, A.F. Braña, J.A.Salas and J.Rohr (2002). Oviedomycin, a novel unusual angucyclinone encoded by genes of the oleandomycin-producer Streptomyces antibioticus ATCC11891 identified by expressing its biosynthetic gene cluster into Streptomyces albus R-?M?-. Journal of Natural Products 65: 779-782.

 

  • 3. Sánchez, C., I.A.Butovich, A.F.Braña, J.Rohr, C.Méndez and J.A.Salas (2002). The biosynthetic gene cluster for the antitumor rebeccamycin: characterization and generation of indolocarbazole derivatives. Chemistry & Biology 9: 519-531.

 

  • 4. Trefzer,A., G.Blanco, L.Remsing, E.Künzel, U.Rix, F.Lipata, A.F.Braña, C.Méndez, J.Rohr, A.Bechthold and J.A.Salas (2002). Rationally designed glycosylated premithramycins: hybrid aromatic polyketides using genes from three different biosynthetic pathways. Journal of the American Chemical Society 124: 6056-6062.

 

  • 5. Rodríguez,L., I.Aguirrezabalaga, N.Allende, A.F.Braña, C.Méndez and J.A.Salas (2002). Engineering deoxysugar biosynthetic pathways from antibiotic-producing microorganisms: a tool to produce novel glycosylated bioactive compounds. Chemistry & Biology 9: 721-729.

 

  • 6. Remsing,L., González,A.M., M.Nur-e-Alam, Mª J.Fernández-Lozano, A.F.Braña, U.Rix, M.A.Oliveira, C.Méndez, J.A.Salas and J.Rohr (2003). Mithramycin SK, a novel antitumor drug with improved therapeutic index generated by combinatorial biosynthesis in the mithramycin producer Streptomyces argillaceus. Journal of the American Chemical Society 125: 5745-5753

 

  • 7. Méndez,C. and J.A.Salas (2003). On the generation of novel anticancer drugs by recombinant DNA technology: the use of combinatorial biosynthesis to produce novel drugs. Comb. Chem. High Throughput. Screen. 6: 513-526.

 

  • 8. Menéndez,N., N. Mohammad, A.F.Braña, J.Rohr, J.A.Salas and C.Méndez (2004). Biosynthesis of the antitumor chromomycin A3 in Streptomyces griseus: analysis of the gene cluster and rational design of novel chromomycin analogues. Chem. Biol. 11: 21-32

 

  • 9. Olano,C., B.Wilkinson, C.Sánchez, S.J.Moss, R.Sheridan, V.Math, A.J.Weston, A.F.Braña, C.J.Martin, M.Oliynyk, C.Méndez, P.F.Leadlay and J.A.Salas (2004). Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: cluster analysis and assignment of functions. Chem. Biol 11: 87-97.

 

  • 10. Rodriguez, D., L.M.Quirós and J.A. Salas (2004). MtmMII-mediated C-methylation during biosynthesis of the antitumor drug mithramycin is essential for biological activity and DNA-drug interaction. J. Biol. Chem. 279: 8149-8158.

 

  • 11. Olano,C, S.J.Moss, A.F.Braña, R.M.Sheridan, V.Math, A.J.Weston, C.Méndez, P.F.Leadlay, B.Wilkinson and J.A.Salas (2004). Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tu4055: insights into nitrile formation. Mol Microbiol 52: 1745-1756.

 

  • 12. Lombó, F., A.F. Braña, J.A.Salas and C. Méndez (2004). Genetic organization of the biosynthetic gene cluster for the antitumor angucycline oviedomycin in Streptomyces antibioticus ATCC 11891. Chembiochem 9: 1181-1187.

 

  • 13. Lombó, F., M.Gibson, L.Greenwell, A.F.Braña, J.Rohr, J.A.Salas and C.Méndez (2004). Engineering biosynthetic pathway for deoxysugars: branched-chain sugar pathways and novel derivatives from the antitumor tetracenomycin. Chem. Biol. 11: 1709-1718.

 

  • 14. Sánchez, C., L. Zhu, A.F.Braña, A.P.Salas, J.Rohr, C.Méndez and J.A.Salas (2005). Combinatorial biosynthesis of antitumor indolocarbazole compounds. Proc. Natl. Acad. Sci. USA 102: 461-466.

 

  • 15. Salas, A.P., Zhu, L., Sánchez, C., Brana, A.F., Rohr, J., Méndez, C. and Salas, J.A. (2005). Deciphering the late steps in the biosynthesis of the anti-tumour indolocarbazole staurosporine: sugar donor substrate flexibility of the StaG glycosyltransferase. Mol. Microbiol. 58: 17-27.

 

  • 16. Lombo, F., Velasco, A., Castro, A., de la Calle, F., Brana, A.F., Sanchez-Puelles, J.M., Mendez, C. and Salas, J.A. (2006). Deciphering the biosynthesis pathway of the antitumor thiocoraline from a marine actinomycete and its expression in two streptomyces species. Chembiochem 7: 366-376.

 

  • 17. Sánchez, C., Méndez, C. and Salas, J.A. (2006). Indolocarbazole natural products: occurrence, biosynthesis, and biological activity. Nat. Prod. Rep. 23:1007-1045.

 

  • 18. Salas, J.A. and Méndez, C. (2007). Engineering the glycosylation of natural products in actinomycetes. Trends Microbiol. 15: 219-232.

 

  • 19. Pérez, M., Baig, I., Braña, A.F., Salas, J.A., Rohr, J. and Méndez, C. (2008). Generation of novel derivatives of the antitumor mithramycin by altering the glycosylation pattern through combinatorial biosynthesis. Chembiochem 9: 2295-2304.

 

  • 20. Lombó, F., Abdelfattah, M.S., Braña, A.F., Salas, J.A., Rohr, J. and Méndez, C. (2009). Elucidation of oxygenation steps during oviedomycin biosynthesis and generation of derivatives with increased antitumor activity. Chembiochem 10:296-303.

 

 

Members

biosintesis_de_moleculas_antitumorales_m

 

Name

Position

e-mail

Phone number

Jose Antonio Salas Fernández

Group leader

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985 103 652

Carmen Méndez Fernández

Group leader

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985 103 558

Gloria Blanco Blanco

Investigator

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985 103 205

Alfredo Braña Fernández

Investigator

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985 102 738

Carlos Olano Álvarez

Investigator

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985 105 288

Ignacio García Llorente

Postdoc

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985 105 288

Marina Sánchez Hidalgo

Postdoc

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985 105 287

Ana Belén Flórez García

Postdoc

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985 105 287

Beatriz García Fernández

Predoc

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985 105 286

María Pardo Solís

Predoc

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985 105 286

Carlos Alberto Sialer Guerrero

Predoc

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985 105 287

Cristina Gómez Suárez

Predoc

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985 105 288

Dina Heidi Horna Inga

Predoc

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985 105 288

Natalia Miguel Vior

Predoc

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985 105 288

Daniel Zabala

Predoc

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985 105 287

Leire Peña Noval

Lab technician

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985 105 287

Patricia Oro Carbajosa

Lab technician

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985 105 288