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Bisphosphonate inhibition of a Plasmodium farnesyl diphosphate synthase and a general method for predicting cell-based activity from enzyme data.

ABSTRACT: We screened 26 bisphosphonates against a farnesyl diphosphate synthase from Plasmodium vivax, finding a poor correlation between enzyme and cell growth inhibition (R(2) = 0.06). To better predict cell activity data, we then used a combinatorial descriptor search in which pIC(50)(cell) = a pIC(50)(enzyme) + bB + cC + d, where B and C are descriptors (such as SlogP), and a-d are coefficients. R(2) increased from 0.01 to 0.74 (for a leave-two-out test set of 26 predictions). The method was then further validated using data for nine other systems, including bacterial, viral, and mammalian cell systems. On average, experimental/predicted cell pIC(50) correlations increased from R(2) = 0.28 (for an enzyme-only test set) to 0.70 (for enzyme plus two descriptor test set predictions), while predictions based on scrambled cell activity had no predictive value (R(2) = 0.13). These results are of interest since they represent a general way to predict cell from enzyme inhibition data, with in three cases, R(2) values increasing from approximately 0.02 to 0.72.

SUBMITTER: Mukkamala D

PROVIDER: S-EPMC2765246 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Bisphosphonate inhibition of a Plasmodium farnesyl diphosphate synthase and a general method for predicting cell-based activity from enzyme data.

Mukkamala Dushyant D No Joo Hwan JH Cass Lauren M LM Chang Ting-Kai TK Oldfield Eric E

Journal of medicinal chemistry 20081201 24

We screened 26 bisphosphonates against a farnesyl diphosphate synthase from Plasmodium vivax, finding a poor correlation between enzyme and cell growth inhibition (R(2) = 0.06). To better predict cell activity data, we then used a combinatorial descriptor search in which pIC(50)(cell) = a pIC(50)(enzyme) + bB + cC + d, where B and C are descriptors (such as SlogP), and a-d are coefficients. R(2) increased from 0.01 to 0.74 (for a leave-two-out test set of 26 predictions). The method was then fur ...[more]

PMID: 19053772

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Project description:BackgroundIsoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites.MethodsThe isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate.ResultsThe expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68?±?5 ?M, 7.8?±?1.3 ?M and 2.06?±?0.4 ?M. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate.ConclusionsThe rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Dataset Information

Bisphosphonate inhibition of a Plasmodium farnesyl diphosphate synthase and a general method for predicting cell-based activity from enzyme data.

Publications

Bisphosphonate inhibition of a Plasmodium farnesyl diphosphate synthase and a general method for predicting cell-based activity from enzyme data.

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