Purification of human malaria parasite hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) using immobilized Reactive Red 120

Malaria is caused by Plasmodium parasite infection. The human malarial parasite does not have a de novo pathway for synthesis of nucleotides and the purine salvage pathway enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) is critical for survival. In our efforts to find inhibitors of the malarial parasite HGXPRT, we have developed a simple but effective purification protocol for this protein expressed in Escherichia coli without an affinity tag. The protocol consists of tandem columns of anion exchange and immobilized Reactive Red 120 resins. The enzyme is inactive as isolated but can be activated by incubation with substrate(s).     

High-throughput screening for potent and selective inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase

Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and urea-based compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC(50) values below 600 nm were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC(50) against malarial DHODH of 16 nm, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.     

Purines and pyrimidines in malarial parasites

In order for the plasmodium malarial parasite to replicate in the human erythrocyte it requires metabolic pathways which are not operative in the host erythrocyte. Thus, the malarial parasite not only synthesizes enzymes for purine salvage and interconversion, for the pyrimidine biosynthetic pathway de novo, and for the folate cycle, but it also alters the host erythrocyte membrane in respect to the transport of purines. Several of the plasmodium enzymes from these pathways have been cloned and these appear to be highly homologous to the corresponding human enzymes. However, enzymes which have been purified from Plasmodium, have demonstrated physicochemical and kinetic differences and may be potential targets for chemotherapy. Inhibition of individual enzymes, such as the dihydroorotate dehydrogenase (DHO-DHase), and inhibition of the inserted pathway from IMP to AMP and IMP to GMP hold considerable promise as chemotherapeutic targets. An entirely new approach in inhibiting malarial growth involves the altered nucleoside transporter in the infected cell membrane through which cytotoxic compounds may be selectively targeted into only the infected cell.     

Targeting a novel Plasmodium falciparum purine recycling pathway with specific immucillins

Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine synthesis are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5'-Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.     

Synthesis of purine N9-[2-hydroxy-3-O-(phosphonomethoxy)propyl] derivatives and their side-chain modified analogs as potential antimalarial agents

6-Oxopurine acyclic nucleoside phosphonates (ANPs) have been shown to be potent inhibitors of hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), a key enzyme of the purine salvage pathway in human malarial parasites. These compounds also exhibit antimalarial activity against parasites grown in culture. Here, a new series of ANPs, hypoxanthine and guanine 9-[2-hydroxy-3-(phosphonomethoxy)propyl] derivatives with different chemical substitutions in the 2'-position of the aliphatic chain were prepared and tested as inhibitors of Plasmodium falciparum (Pf) HGXPRT, Plasmodium vivax (Pv) HGPRT and human HGPRT. The attachment of an hydroxyl group to this position and the movement of the oxygen by one atom distal from N(9) in the purine ring compared with 2-(phosphonoethoxy)ethyl hypoxanthine (PEEHx) and 2-(phosphonoethoxy)ethyl guanine (PEEG) changes the affinity and selectivity for human HGPRT, PfHGXPRT and PvHGPRT. This is attributed to the differences in the three-dimensional structure of these inhibitors which affects their mode of binding. A novel observation is that these molecules are not always strictly competitive with 5-phospho-α-d-ribosyl-1-pyrophosphate. 9-[2-Hydroxy-3-(phosphonomethoxy)propyl]hypoxanthine (iso-HPMP-Hx) is a very weak inhibitor of human HGPRT but remains a good inhibitor of both the parasite enzymes with K(i) values of 2μM and 5μM for PfHGXPRT and PvHGPRT, respectively. The addition of pyrophosphate to the assay decreased the K(i) values for the parasite enzymes by sixfold. This suggests that the covalent attachment of a second group to the ANPs mimicking pyrophosphate and occupying its binding pocket could increase the affinity for these enzymes.     

Development of FRET-based high-throughput screening for viral RNase III inhibitors

The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose–response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.     

A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransferase

Nicotinamide adenine dinucleotide (NAD) plays a crucial role in many cellular processes. As the rate-limiting enzyme of the predominant NAD biosynthesis pathway in mammals, nicotinamide phosphoribosyltransferase (Nampt) regulates the cellular NAD level. Tumor cells are more sensitive to the NAD levels, making them more susceptible to Nampt inhibition than their nontumorigenic counterparts. Experimental evidence has indicated that Nampt might have proangiogenic activity and supports the growth of some tumors, so Nampt inhibitors may be promising as antitumor agents. However, only four Nampt inhibitors have been reported, and no high-throughput screening (HTS) strategy for Nampt has been proposed to date, largely limiting the drug discovery targeting Nampt. Therefore, the development of a robust HTS strategy for Nampt is both imperative and significant. Here we developed a fluorometric method for a Nampt activity assay by measuring the fluorescence of nicotinamide mononucleotide (NMN) derivative resulting from the enzymatic product NMN through simple chemical reactions. Then we set up an HTS system after thorough optimizations of this method and validated that it is feasible and effective through a pilot screening on a small library. This HTS system should expedite the discovery of Nampt inhibitors as antitumor drug candidates.     

A colorimetric assay optimization for high-throughput screening of dihydroorotase by detecting ureido groups

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine biosynthesis pathway and is a potential new antibacterial drug target. No target-based high-throughput screening (HTS) assay for this enzyme has been reported to date. Here, we optimized two colorimetric-based enzymatic assays that detect the ureido moiety of the DHOase substrate, carbamyl-aspartate (Ca-asp). Each assay was developed in a 40-μl assay volume using 384-well plates with a different color mix, diacetylmonoxime (DAMO)–thiosemicarbazide (TSC) or DAMO–antipyrine. The sensitivity and color interference of both color mixes were compared in the presence of common HTS buffer additives, including dimethyl sulfoxide, reducing agents, detergents, and bovine serum albumin. DAMO–TSC (Z′-factors 0.7–0.8) was determined to be superior to DAMO–antipyrine (Z′-factors 0.5–0.6) with significantly less variability within replicates. An HTS pilot screening with 29,552 compounds from four structurally diverse libraries confirmed the quality of our newly optimized colorimetric assay with DAMO–TSC. This robust method has no heating requirement, which was the main obstacle to applying previous assays to HTS. More important, this well-optimized HTS assay for DHOase, the first of its kind, should make it possible to screen large-scale compound libraries to develop new inhibitors against any enzymes that produce ureido functional groups.     

An alternative model for heme biosynthesis in the malarial parasite

The malarial parasite imports an infected host's red blood cell enzymes for heme biosynthesis during the intraerythrocytic stage. This is despite all the genes of the heme-biosynthetic pathway having been identified on the parasite genome. On the basis of predictions of parasite genome-coded enzyme localization, functionality of some of these enzymes and shuttling of intermediates between different parasite compartments, a hybrid model for parasite heme biosynthesis has been proposed. However, this model does not take into account the possible role of imported host enzymes in parasite heme biosynthesis. We propose an alternative model with an extrinsic heme-biosynthetic pathway in the parasite cytosol that uses imported host enzymes, and an intrinsic pathway confined to the organellar fractions that uses the parasite-genome-encoded enzymes.     

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors

A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli (E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ∼27,000 compounds and proved to be highly reliable (average Z′ factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors.     

Multiple e-pharmacophore modelling pooled with high-throughput virtual screening,docking and molecular dynamics simulations to discover potential inhibitors of Plasmodium falciparum lactate dehydrogenase (PfLDH)

Development of new antimalarial drugs continues to be of huge importance because of the resistance of malarial parasite towards currently used drugs. Due to the reliance of parasite on glycolysis for energy generation, glycolytic enzymes have played important role as potential targets for the development of new drugs. Plasmodium falciparum lactate dehydrogenase (PfLDH) is a key enzyme for energy generation of malarial parasites and is considered to be a potential antimalarial target. Presently, there are nearly 15 crystal structures bound with inhibitors and substrate that are available in the protein data bank (PDB). In the present work, we attempted to consider multiple crystal structures with bound inhibitors showing affinity in the range of 1.4 × 10²–1.3 × 10⁶ nM efficacy and optimized the pharmacophore based on the energy involved in binding termed as e-pharmacophore mapping. A high throughput virtual screening (HTVS) combined with molecular docking, ADME predictions and molecular dynamics simulation led to the identification of 20 potential compounds which could be further developed as novel inhibitors for PfLDH.     

A cell-based beta-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replication

This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication. The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells. The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate. Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window. Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors. These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window. HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.     

Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II

Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000-compound library. The novel assay presented here is robust, highly reproducible (Z' = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.     

Characterization of the glyoxalases of the malarial parasite Plasmodium falciparum and comparison with their human counterparts

The glyoxalase system consisting of glyoxalase I (GloI) and glyoxalase II (GloII) constitutes a glutathione-dependent intracellular pathway converting toxic 2-oxoaldehydes, such as methylglyoxal, to the corresponding 2-hydroxyacids. Here we describe a complete glyoxalase system in the malarial parasite Plasmodium falciparum. The biochemical, kinetic and structural properties of cytosolic GloI (cGloI) and two GloIIs (cytosolic GloII named cGloII, and tGloII preceded by a targeting sequence) were directly compared with the respective isofunctional host enzymes. cGloI and cGloII exhibit lower K(m) values and higher catalytic efficiencies (k(cat)/K(m) ) than the human counterparts, pointing to the importance of the system in malarial parasites. A Tyr185Phe mutant of cGloII shows a 2.5-fold increase in K(m) , proving the contribution of Tyr185 to substrate binding. Molecular models suggest very similar active sites/metal binding sites of parasite and host cell enzymes. However, a fourth protein, which has highest similarities to GloI, was found to be unique for malarial parasites; it is likely to act in the apicoplast, and has as yet undefined substrate specificity. Various S-(N-hydroxy-N-arylcarbamoyl)glutathiones tested as P. falciparum Glo inhibitors were active in the lower nanomolar range. The Glo system of Plasmodium will be further evaluated as a target for the development of antimalarial drugs.     

Plasmodium falciparum invasion and intraerythrocytic development are impaired by 2′, 3′-dialdehyde adenosine

Purine nucleotide synthesis in protozoa takes place exclusively via the purine salvage pathway and S-adenosyl-l-homocysteine hydrolase (SAHH) is an important enzyme in the Plasmodium salvage pathway which is not present in erythrocytes. Here, we describe the antimalarial effect of 2′3′-dialdehyde adenosine or oxidized adenosine (oADO), inhibitor of SAHH, on in vitro infection of human erythrocytes by P. falciparum. Treatment of infected erythrocytes with oADO inhibits parasite development and reinvasion of new cells. Erythrocytes pre-treated with oADO have a reduced susceptibility to invasion. Our results suggest that oADO interferes with one or more parasitic enzymes of the purine salvage pathway.     

High-throughput assays for promiscuous inhibitors

High-throughput screening (HTS) searches large libraries of chemical compounds for those that can modulate the activity of a particular biological target; it is the dominant technique used in early-stage drug discovery. A key problem in HTS is the prevalence of nonspecific or 'promiscuous' inhibitors. These molecules have peculiar properties, act on unrelated targets and can dominate the results from screening campaigns. Several explanations have been proposed to account for promiscuous inhibitors, including chemical reactivity, interference in assay read-out, high molecular flexibility and hydrophobicity. The diversity of these models reflects the apparently unrelated molecules whose behaviors they seek to explain. However, a single mechanism may explain the effects of many promiscuous inhibitors: some organic molecules form large colloid-like aggregates that sequester and thereby inhibit enzymes. Hits from HTS, leads for drug discovery and even several drugs appear to act through this mechanism at micromolar concentrations. Here, we report two rapid assays for detecting promiscuous aggregates that we tested against 1,030 'drug-like' molecules. The results from these assays were used to test two preliminary computational models of this phenomenon and as benchmarks to develop new models.     

Determining appropriate substrate conversion for enzymatic assays in high-throughput screening

It is generally accepted that the conversion of substrate should be kept at less than 10% of the total substrate used when studying enzyme kinetics. However, 10% or less substrate conversion often will not produce sufficient signal changes required for robust high-throughput screening (HTS). To increase the signal-to-background ratio, HTS is often performed at higher than 10% substrate conversion. Because the consequences of high substrate conversion are poorly understood, the screening results are sometimes questioned by enzymologists. The quality of an assay is judged by the ability to detect an inhibitor under HTS conditions, which depends on the robustness of the primary detection signal (Z factor) and the sensitivity to an inhibitor. The assay sensitivity to an inhibitor is reflected in the observed IC(50) value or percent inhibition at a fixed compound concentration when single-point data are collected. The major concern for an enzymatic assay under high substrate conversion is that the sensitivity of the screen may be compromised. Here we derive the relationship between the IC(50) value for a given inhibitor and the percentage of substrate conversion using a first-order kinetic model under conditions that obey Henri-Michaelis-Menten kinetics. The derived theory was further verified experimentally with a cAMP-dependent protein kinase. This model provides guidance for assay developers to choose an appropriate substrate conversion in designing an enzymatic assay, balancing the needs for robust signal and sensitivity to inhibitors.     

Purine salvage pathways in the apicomplexan parasite Toxoplasma gondii

We have exploited a variety of molecular genetic, biochemical, and genomic techniques to investigate the roles of purine salvage enzymes in the protozoan parasite Toxoplasma gondii. The ability to generate defined genetic knockouts and target transgenes to specific loci demonstrates that T. gondii uses two (and only two) pathways for purine salvage, defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK). Both HXGPRT and AK are single-copy genes, and either one can be deleted, indicating that either one of these pathways is sufficient to meet parasite purine requirements. Fitness defects suggest both pathways are important for the parasite, however, and that the salvage of adenosine is more important than salvage of hypoxanthine and other purine nucleobases. HXGPRT and AK cannot be deleted simultaneously unless one of these enzymes is provided in trans, indicating that alternative routes of functionally significant purine salvage are lacking. Despite previous reports to the contrary, we found no evidence of adenine phosphoribosyltransferase (APRT) activity when parasites were propagated in APRT-deficient host cells, and no APRT ortholog is evident in the T. gondii genome. Expression of Leishmania donovani APRT in transgenic T. gondii parasites yielded low levels of activity but did not permit genetic deletion of both HXGPRT and AK. A detailed comparative genomic study of the purine salvage pathway in various apicomplexan species highlights important differences among these parasites.     

Development of a microplate-based scintillation proximity assay for MraY using a modified substrate

MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.     

Development and validation of a generic fluorescent methyltransferase activity assay based on the transcreener AMP/GMP assay

Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids, and lipids. Histone methyltransferases (HMTs) are currently of high interest as drug targets because of their role in epigenetic regulation; however, most HMT assay methods are either not amenable to a high-throughput screening (HTS) environment or are applicable to a limited number of enzymes. The authors developed a generic methyltransferase assay method using fluorescent immunodetection of adenosine monophosphate (AMP), which is formed from the MT reaction product S-adenosylhomocysteine in a dual-enzyme coupling step. The detection range of the assay; its suitability for HTS, including stability of reagents following dispensing and after addition to reactions; and the potential for interference from drug-like molecules was investigated. In addition, the use of the assay for measuring inhibitor potencies with peptide or intact protein substrates was examined through pilot screening with selected reference enzymes including HMT G9a. By combining a novel enzymatic coupling step with the well-characterized Transcreener AMP/GMP assay, the authors have developed a robust HTS assay for HMTs that should be broadly applicable to other types of methyltransferases as well.