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Adenosine Kinase of T. b. Rhodesiense identified as the putative target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine using chemical proteomics.

Kuettel S, Mosimann M, Mäser P, Kaiser M, Brun R, Scapozza L, Perozzo R - PLoS Negl Trop Dis (2009)

Bottom Line: This finding was confirmed by RNA interference experiments showing that down-regulation of adenosine kinase counteracts compound 1 activity.The subsequent kinetic analysis provided strong evidence that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition.The results suggest that TbrAK is the putative target of this compound, and that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.

ABSTRACT

Background: Human African trypanosomiasis (HAT), a major parasitic disease spread in Africa, urgently needs novel targets and new efficacious chemotherapeutic agents. Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) exhibits specific antitrypanosomal activity with an IC(50) of 1.0 microM on Trypanosoma brucei rhodesiense (T. b. rhodesiense), the causative agent of the acute form of HAT.

Methodology/principal findings: In this work we show adenosine kinase of T. b. rhodesiense (TbrAK), a key enzyme of the parasite purine salvage pathway which is vital for parasite survival, to be the putative intracellular target of compound 1 using a chemical proteomics approach. This finding was confirmed by RNA interference experiments showing that down-regulation of adenosine kinase counteracts compound 1 activity. Further chemical validation demonstrated that compound 1 interacts specifically and tightly with TbrAK with nanomolar affinity, and in vitro activity measurements showed that compound 1 is an enhancer of TbrAK activity. The subsequent kinetic analysis provided strong evidence that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition.

Conclusions/significance: The results suggest that TbrAK is the putative target of this compound, and that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides.

No MeSH data available.


Related in: MedlinePlus

Chemical structures of compounds used in this study.The title compound 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (1) and its amino substituted derivatives 4-[4-(5-morpholine-4-yl-1H-pyrazol-3-yl)-phenoxy]phenylamine (2), 4-[5-morpholine-4-yl-3-(4-phenoxyphenyl)-pyrazol-1-yl]phenylamine (3) and 1-[5-(4-phenoxyphenyl-2H-pyrazol-3-yl]piperazine (4) are presented. 3-(4-phenoxyphenyl)-1H-pyrazole (5) showed very low antiparasitic activity (IC50 of >65 µM) as well as general toxicity (IC50 of >136 µM), therefore it was used as negative control for the biochemical, biophysical and compound sensitivity tests.
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pntd-0000506-g001: Chemical structures of compounds used in this study.The title compound 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (1) and its amino substituted derivatives 4-[4-(5-morpholine-4-yl-1H-pyrazol-3-yl)-phenoxy]phenylamine (2), 4-[5-morpholine-4-yl-3-(4-phenoxyphenyl)-pyrazol-1-yl]phenylamine (3) and 1-[5-(4-phenoxyphenyl-2H-pyrazol-3-yl]piperazine (4) are presented. 3-(4-phenoxyphenyl)-1H-pyrazole (5) showed very low antiparasitic activity (IC50 of >65 µM) as well as general toxicity (IC50 of >136 µM), therefore it was used as negative control for the biochemical, biophysical and compound sensitivity tests.

Mentions: Recently we reported the synthesis and evaluation of new 4-[5-(4-phenoxy-phenyl-2H-pyrazol-3-yl]morpholine derivatives against several parasites [7]. One of the compounds, 4-[5-(4-phenoxy-phenyl-2H-pyrazol-3-yl]morpholine (Fig. 1, compound 1), exhibited good activity toward T. b. rhodesiense with an IC50 of 1 µM and low cytotoxicity [7]. This finding prompted us to address the question regarding the cellular target and the molecular mechanism underlying the observed toxicity toward the parasite. To this end, here we report a chemical proteomics approach that led to the identification of adenosine kinase as the putative target. Subsequent biochemical and biophysical characterization with respect to compound 1 binding as well as drug sensitivity tests on the corresponding knock-down strain allowed its validation and suggested hyperactivation of adenosine kinase as the molecular mechanism underlying the biological activity.


Adenosine Kinase of T. b. Rhodesiense identified as the putative target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine using chemical proteomics.

Kuettel S, Mosimann M, Mäser P, Kaiser M, Brun R, Scapozza L, Perozzo R - PLoS Negl Trop Dis (2009)

Chemical structures of compounds used in this study.The title compound 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (1) and its amino substituted derivatives 4-[4-(5-morpholine-4-yl-1H-pyrazol-3-yl)-phenoxy]phenylamine (2), 4-[5-morpholine-4-yl-3-(4-phenoxyphenyl)-pyrazol-1-yl]phenylamine (3) and 1-[5-(4-phenoxyphenyl-2H-pyrazol-3-yl]piperazine (4) are presented. 3-(4-phenoxyphenyl)-1H-pyrazole (5) showed very low antiparasitic activity (IC50 of >65 µM) as well as general toxicity (IC50 of >136 µM), therefore it was used as negative control for the biochemical, biophysical and compound sensitivity tests.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2724708&req=5

pntd-0000506-g001: Chemical structures of compounds used in this study.The title compound 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (1) and its amino substituted derivatives 4-[4-(5-morpholine-4-yl-1H-pyrazol-3-yl)-phenoxy]phenylamine (2), 4-[5-morpholine-4-yl-3-(4-phenoxyphenyl)-pyrazol-1-yl]phenylamine (3) and 1-[5-(4-phenoxyphenyl-2H-pyrazol-3-yl]piperazine (4) are presented. 3-(4-phenoxyphenyl)-1H-pyrazole (5) showed very low antiparasitic activity (IC50 of >65 µM) as well as general toxicity (IC50 of >136 µM), therefore it was used as negative control for the biochemical, biophysical and compound sensitivity tests.
Mentions: Recently we reported the synthesis and evaluation of new 4-[5-(4-phenoxy-phenyl-2H-pyrazol-3-yl]morpholine derivatives against several parasites [7]. One of the compounds, 4-[5-(4-phenoxy-phenyl-2H-pyrazol-3-yl]morpholine (Fig. 1, compound 1), exhibited good activity toward T. b. rhodesiense with an IC50 of 1 µM and low cytotoxicity [7]. This finding prompted us to address the question regarding the cellular target and the molecular mechanism underlying the observed toxicity toward the parasite. To this end, here we report a chemical proteomics approach that led to the identification of adenosine kinase as the putative target. Subsequent biochemical and biophysical characterization with respect to compound 1 binding as well as drug sensitivity tests on the corresponding knock-down strain allowed its validation and suggested hyperactivation of adenosine kinase as the molecular mechanism underlying the biological activity.

Bottom Line: This finding was confirmed by RNA interference experiments showing that down-regulation of adenosine kinase counteracts compound 1 activity.The subsequent kinetic analysis provided strong evidence that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition.The results suggest that TbrAK is the putative target of this compound, and that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.

ABSTRACT

Background: Human African trypanosomiasis (HAT), a major parasitic disease spread in Africa, urgently needs novel targets and new efficacious chemotherapeutic agents. Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) exhibits specific antitrypanosomal activity with an IC(50) of 1.0 microM on Trypanosoma brucei rhodesiense (T. b. rhodesiense), the causative agent of the acute form of HAT.

Methodology/principal findings: In this work we show adenosine kinase of T. b. rhodesiense (TbrAK), a key enzyme of the parasite purine salvage pathway which is vital for parasite survival, to be the putative intracellular target of compound 1 using a chemical proteomics approach. This finding was confirmed by RNA interference experiments showing that down-regulation of adenosine kinase counteracts compound 1 activity. Further chemical validation demonstrated that compound 1 interacts specifically and tightly with TbrAK with nanomolar affinity, and in vitro activity measurements showed that compound 1 is an enhancer of TbrAK activity. The subsequent kinetic analysis provided strong evidence that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition.

Conclusions/significance: The results suggest that TbrAK is the putative target of this compound, and that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides.

No MeSH data available.


Related in: MedlinePlus