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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

Specific binding of compound 1 to TbrAK measured by ITC.The top panel shows heat signals upon 27 injections of compound 1 (trace I) or control compound 5 (trace II) into the sample cell containing 7 µM TbrAK. The binding isotherm obtained by integration and normalization of the raw data and by correction for the heat of ligand dilution is shown on the lower panel. The solid line represents the non-linear least square fit based on a two-sites non-interacting binding model. Compound 1 binds to TbrAK via a high affinity binding site with a KD of 75±20 nM and a ΔHbind of −3.05±0.77 kcal/mol, and a low affinity site exhibiting a KD of 497±34 nM and a ΔHbind of −1.13±0.24 kcal/mol. No specific heat release was shown for the negative control (trace II). The mean of three independent experiments are reported.
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pntd-0000506-g004: Specific binding of compound 1 to TbrAK measured by ITC.The top panel shows heat signals upon 27 injections of compound 1 (trace I) or control compound 5 (trace II) into the sample cell containing 7 µM TbrAK. The binding isotherm obtained by integration and normalization of the raw data and by correction for the heat of ligand dilution is shown on the lower panel. The solid line represents the non-linear least square fit based on a two-sites non-interacting binding model. Compound 1 binds to TbrAK via a high affinity binding site with a KD of 75±20 nM and a ΔHbind of −3.05±0.77 kcal/mol, and a low affinity site exhibiting a KD of 497±34 nM and a ΔHbind of −1.13±0.24 kcal/mol. No specific heat release was shown for the negative control (trace II). The mean of three independent experiments are reported.

Mentions: As an orthogonal method to CD we used isothermal titration calorimetry (ITC) to analyze binding of compound 1 to TbrAK. The titration of TbrAK with compound 1 revealed a complex and enthalpy driven binding mode with two molecules of 1 binding to one molecule of enzyme. Exothermic binding heats were corrected for heats of dilution, integrated and plotted against the molar ratio of 1 and enzyme (trace I in Fig. 4). The binding isotherm was best described using a non-linear least square fit assuming a two-sites non-interacting binding model. Subsequent analysis revealed a high affinity binding site with a KD of 75±20 nM and a ΔHbind of −3.05±0.77 kcal/mol, while the low affinity site exhibited a KD of 497±34 nM and a ΔHbind of −1.13±0.24 kcal/mol. The negative control using compound 5 showed no specific heat release (see trace II in Fig. 4).


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)

Specific binding of compound 1 to TbrAK measured by ITC.The top panel shows heat signals upon 27 injections of compound 1 (trace I) or control compound 5 (trace II) into the sample cell containing 7 µM TbrAK. The binding isotherm obtained by integration and normalization of the raw data and by correction for the heat of ligand dilution is shown on the lower panel. The solid line represents the non-linear least square fit based on a two-sites non-interacting binding model. Compound 1 binds to TbrAK via a high affinity binding site with a KD of 75±20 nM and a ΔHbind of −3.05±0.77 kcal/mol, and a low affinity site exhibiting a KD of 497±34 nM and a ΔHbind of −1.13±0.24 kcal/mol. No specific heat release was shown for the negative control (trace II). The mean of three independent experiments are reported.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000506-g004: Specific binding of compound 1 to TbrAK measured by ITC.The top panel shows heat signals upon 27 injections of compound 1 (trace I) or control compound 5 (trace II) into the sample cell containing 7 µM TbrAK. The binding isotherm obtained by integration and normalization of the raw data and by correction for the heat of ligand dilution is shown on the lower panel. The solid line represents the non-linear least square fit based on a two-sites non-interacting binding model. Compound 1 binds to TbrAK via a high affinity binding site with a KD of 75±20 nM and a ΔHbind of −3.05±0.77 kcal/mol, and a low affinity site exhibiting a KD of 497±34 nM and a ΔHbind of −1.13±0.24 kcal/mol. No specific heat release was shown for the negative control (trace II). The mean of three independent experiments are reported.
Mentions: As an orthogonal method to CD we used isothermal titration calorimetry (ITC) to analyze binding of compound 1 to TbrAK. The titration of TbrAK with compound 1 revealed a complex and enthalpy driven binding mode with two molecules of 1 binding to one molecule of enzyme. Exothermic binding heats were corrected for heats of dilution, integrated and plotted against the molar ratio of 1 and enzyme (trace I in Fig. 4). The binding isotherm was best described using a non-linear least square fit assuming a two-sites non-interacting binding model. Subsequent analysis revealed a high affinity binding site with a KD of 75±20 nM and a ΔHbind of −3.05±0.77 kcal/mol, while the low affinity site exhibited a KD of 497±34 nM and a ΔHbind of −1.13±0.24 kcal/mol. The negative control using compound 5 showed no specific heat release (see trace II in Fig. 4).

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