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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

Representative plots for TbrAK kinetics (AMP formation) with respect to adenosine in absence (○) and presence (•) of compound 1 (33 µM).Adenosine strongly inhibits TbrAK at concentrations >2 µM and follows typical substrate-inhibition kinetics. In contrast, compound 1 abolishes substrate-inhibition. The solid lines indicate the fit of the raw data with the substrate-inhibition model (see Table 2 for fitting results).
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pntd-0000506-g005: Representative plots for TbrAK kinetics (AMP formation) with respect to adenosine in absence (○) and presence (•) of compound 1 (33 µM).Adenosine strongly inhibits TbrAK at concentrations >2 µM and follows typical substrate-inhibition kinetics. In contrast, compound 1 abolishes substrate-inhibition. The solid lines indicate the fit of the raw data with the substrate-inhibition model (see Table 2 for fitting results).

Mentions: A common property of adenosine kinases from various organisms is their control via a substrate-inhibition mechanism [15]–[18], and recently it was shown that T. brucei AK was inhibited by high adenosine concentrations to prevent non-physiologically high intracellular purine nucleotide levels [19]. This prompted us to analyze the influence of compound 1 toward TbrAK with respect to substrate transformation by determining the kinetic parameters in absence and presence of the activator. TbrAK activity was measured at increasing adenosine concentrations and a fixed concentration of 167 µM ATP. All data are given in Table 2. Adenosine kinetics in absence of compound 1 displayed non-hyperbolic progress plots (Fig. 5). After increasing activity up to a maximum at 2–3 µM adenosine, the enzyme activity declined at higher substrate concentrations which is a typical finding for substrate-inhibition. Thus the observed kinetic data were fit to the substrate-inhibition model [10]. Indeed, a good fit was obtained for all data points, yielding a Km of 0.99±0.05 µM and a Vmax of 19.60±0.33 nM/min. kcat and catalytic efficiency were found to be 0.37±0.01 s−1 and 0.38±0.01 µM−1 s−1, respectively. TbrAK was inhibited by adenosine with a Ki of 6.1±1.4 µM. In contrast, in presence of compound 1 substrate-inhibition was strongly reduced (Fig. 5), and adenosine inhibited TbrAK with a more than ten-fold increased Ki value (78.4±2.2 µM). While Km (0.65±0.04 µM), Vmax (14.20±0.14 µM) and kcat (0.0.27±0.01 s−1) were slightly reduced in presence of compound 1, the catalytic efficency kcat/Km (0.42±0.01 µM−1 s−1) did not change significantly (Table 2).


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)

Representative plots for TbrAK kinetics (AMP formation) with respect to adenosine in absence (○) and presence (•) of compound 1 (33 µM).Adenosine strongly inhibits TbrAK at concentrations >2 µM and follows typical substrate-inhibition kinetics. In contrast, compound 1 abolishes substrate-inhibition. The solid lines indicate the fit of the raw data with the substrate-inhibition model (see Table 2 for fitting results).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000506-g005: Representative plots for TbrAK kinetics (AMP formation) with respect to adenosine in absence (○) and presence (•) of compound 1 (33 µM).Adenosine strongly inhibits TbrAK at concentrations >2 µM and follows typical substrate-inhibition kinetics. In contrast, compound 1 abolishes substrate-inhibition. The solid lines indicate the fit of the raw data with the substrate-inhibition model (see Table 2 for fitting results).
Mentions: A common property of adenosine kinases from various organisms is their control via a substrate-inhibition mechanism [15]–[18], and recently it was shown that T. brucei AK was inhibited by high adenosine concentrations to prevent non-physiologically high intracellular purine nucleotide levels [19]. This prompted us to analyze the influence of compound 1 toward TbrAK with respect to substrate transformation by determining the kinetic parameters in absence and presence of the activator. TbrAK activity was measured at increasing adenosine concentrations and a fixed concentration of 167 µM ATP. All data are given in Table 2. Adenosine kinetics in absence of compound 1 displayed non-hyperbolic progress plots (Fig. 5). After increasing activity up to a maximum at 2–3 µM adenosine, the enzyme activity declined at higher substrate concentrations which is a typical finding for substrate-inhibition. Thus the observed kinetic data were fit to the substrate-inhibition model [10]. Indeed, a good fit was obtained for all data points, yielding a Km of 0.99±0.05 µM and a Vmax of 19.60±0.33 nM/min. kcat and catalytic efficiency were found to be 0.37±0.01 s−1 and 0.38±0.01 µM−1 s−1, respectively. TbrAK was inhibited by adenosine with a Ki of 6.1±1.4 µM. In contrast, in presence of compound 1 substrate-inhibition was strongly reduced (Fig. 5), and adenosine inhibited TbrAK with a more than ten-fold increased Ki value (78.4±2.2 µM). While Km (0.65±0.04 µM), Vmax (14.20±0.14 µM) and kcat (0.0.27±0.01 s−1) were slightly reduced in presence of compound 1, the catalytic efficency kcat/Km (0.42±0.01 µM−1 s−1) did not change significantly (Table 2).

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