Rhodamine labeling of 3-hydroxy-4-pyridinone iron chelators is an important contribution to target Mycobacterium avium infection.

J Inorg Biochem. 2013 Apr;121:156-66.

Moniz T, Nunes A, Silva AM, Queirós C, Ivanova G, Gomes MS, Rangel M.

REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, 40169-007 Porto, Portugal.

 

Abstract

We have recently demonstrated that tripodal hexadentate chelators, based on 3-hydroxy-4-pyridinone units, can limit the access of iron to bacteria and have a significant inhibitory effect in the intramacrophagic growth of Mycobacterium avium. The results showed that the chelation of iron is a determinant although not sufficient property for antimicrobial activity. The rhodamine B isothiocyanate labelled chelator (MRH7) exhibited the strongest inhibitory activity and was identified as a lead compound since a dose response effect was observed. Significant inhibition of M. avium growth was achieved at a concentration as low as 1 μM. To identify key molecular features essential for the biological activity we designed parent hexadentate and bidentate chelators, in which different structural groups are introduced in the molecular framework. Herein, we report the work concerning three novel fluorescent chelators: a hexadentate ligand labelled with 5(6)-carboxytetramethylrhodamine (MRH8) and two 3-hydroxy-4-pyridinone fluorescent bidentate ligands labelled with rhodamine B isothiocyanate (MRB7) and 5(6)-carboxytetramethylrhodamine (MRB8). The results show that all fluorescent chelators are capable of restricting the intramacrophagic growth of M. avium and that the inhibitory effect is dependent on the fluorophore. In fact, for compounds bearing the same fluorophore the results obtained with the hexadentate or bidentate chelator (MRH7/MRB7 or MRH8/MRB8) are identical as long as the appropriate stoichiometric amount of chelator is used. The inhibitory effect of the rhodamine B isothiocyanate labelled compounds (MRH7 and MRB7) is significantly greater than that observed for the other two chelators, thus pointing out the significance of the rhodamine B isothiocyanate molecular fragment. Copyright © 2013 Elsevier Inc.

PMID: 23384853

 

Supplement

Iron Chelators to fight Infection

New strategies to fight infection based on the concept of iron deprivation.

To fight infection we are developing chelating units with higher affinity for iron(III) when compared with mycobacteria siderophores. To enhance the activity of the chelators these units are being combined with other molecular frameworks which are known to target bacterial membranes.

Our group has recently demonstrated that tripodal hexadentate chelators can limit the access of iron to bacteria and have a significant inhibitory effect on the intramacrophagic growth of M. avium. The results confirm that chelation of iron is a determinant but not sufficient property for antimicrobial activity. The rhodamine B labelled chelator exhibited the strongest inhibitory activity and partition studies of the fluorescent chelators in liposomes demonstrated that, contrasting with others, the rhodamine B labeled chelators strongly interact with lipid phases. Confocal microscopy studies showed that the distribution of the chelators within macrophages is different and suggested that the rhodamine B labeled chelators remain largely membrane bound when distributing throughout the cell endocytic pathway. The spectroscopic studies were crucial to realize that a co-localization with lipid phases may be determinant to situate the drug. From the biophysical point of view, the above result is an interesting illustration of the circumstance of a dramatic change of biological activity due to a significant alteration of molecular features and consequently permeation properties through biological membranes introduced by the attachment of a spectroscopic active label. To identify the key molecular features essential for biological activity we designed parent hexadentate and bidentate chelators functionalized with other fluorophores. The biological results show that the activity is dependent on the fluorophore and the percentage of inhibitory effect is significantly greater for the rhodamine B labeled compounds.

The permeation process of drugs across a lipid bilayer is crucial to understand the mechanism of drug action and a significant contribution to the development of new bioactive molecules. The research team includes researchers with strong expertise in computational biochemistry and biological spectroscopy that will analyze the new compounds by means of theoretical and experimental methodologies. The group has developed liposome preparation methodologies and has been using steady-state fluorescence, fluorescence anisotropy, EPR and more recently NMR that will be applied.

Related references

“Rhodamine labeling of 3-hydroxy-4-pyridinone iron chelators is an important contribution to target Mycobacterium avium infection”

Tânia Moniz, Ana Nunes, Ana M.G. Silva, Carla Queirós, Galya Ivanova,

M.S. Gomes, Maria Rangel, J. Inorg. Biochem., 2013, 121, 156–166.

“Fluorescent 3-hydroxy-4-pyridinone hexadentate iron chelators: intracellular distribution and the relevance to anti-mycobacterial properties” Ana Nunes, Maria Podinovskaia, Andreia Leite, Paula Gameiro, Tao Zhou, Yongmin Ma, Xiaole Kong, Ulrich E. Schaible, Robert C. Hider and Maria Rangel, J. Biol. Inorg. Chem., 2010, 15, 861-877.

“Identification of a new hexadentate iron chelator capable of restricting the intramacrophagic growth of Mycobacterium avium”, Fernandes, Sofia. S. Fernandes, Ana Nunes, Ana R. Gomes, Baltazar Castro, Robert C. Hider, Maria Rangel, Rui Appelberg, Maria Salomé Gomes, Microbes and Infection, 2010, 12, 287-294.

 

Research interests of Dr. Maria Rangel’s group

The research interests of the group are centred in Bioinorganic Chemistry. At present, the research focus is on the design of molecules that may be of use in novel therapeutic strategies to fight Infection, Iron Overload and Diabetes. The first two are related with a particular interest in Iron Biology and the third with the potential insulin-like effect of Zinc and Vanadium complexes.

The design of metal ion chelators is the common factor: in the first two scenarios the drug is the ligand itself and in the third is a metal ion complex. The labelling of chelators with molecules that allow visualization of their pathways within the cell and the study of their interaction with biological membranes by means of spectroscopic methods is a fascinating and relevant area for the development of new drugs.

 

Photos

Maria Rangel-1

 

Maria Rangel-2

 

Maria Rangel-3

 

Maria Rangel-4

Dr. Maria Rangel

REQUIMTE, Instituto de Ciências Biomédicas de Abel Salazar

Rua Jorge Viterbo Ferreira, 228, 4050-313 PORTO

Portugal

mcrangel@fc.up.pt, mrangel@icbas.up.pt

Maria Rangel-5

 

 

 

 

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