Fosmidomycin uptake into Plasmodium and Babesia-infected erythrocytes is facilitated by parasite-induced new permeability pathways

Background

Highly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative {"type":"entrez-nucleotide","attrs":{"text":"FR900098","term_id":"525219861","term_text":"FR900098"}}FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii.

Methodology/Principal Findings

Since non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled {"type":"entrez-nucleotide","attrs":{"text":"FR900098","term_id":"525219861","term_text":"FR900098"}}FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite.

Conclusions/Significance

Our findings provide an explanation for the observed differences in activity of fosmidomycin and {"type":"entrez-nucleotide","attrs":{"text":"FR900098","term_id":"525219861","term_text":"FR900098"}}FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery.     

Acyloxyalkyl ester prodrugs of FR900098 with improved in vivo anti-malarial activity

FR900098 represents an improved derivative of the new antimalarial drug fosmidomycin and acts through inhibition of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, an essential enzyme of the mevalonate independent pathway of isoprenoid biosynthesis. Prodrugs with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield acyloxyalkyl esters. The most successful compound demonstrated 2-fold increased activity in mice infected with the rodent malaria parasite Plasmodium vinckei.     

Synthesis of beta- and gamma-oxa isosteres of fosmidomycin and FR900098 as antimalarial candidates

To expand the structure-activity relationships of fosmidomycin and FR900098, two potent antimalarials interfering with the MEP-pathway, we decided to replace a methylene group in beta-position of the phosphonate moiety of these leads by an oxygen atom. beta-oxa-FR900098 (11) proved equally active as the parent compound. When applied to 4-[hydroxyl(methyl)amino]-4-oxobutyl phosphonic acid, featuring a hydroxamate instead of the retrohydroxamate moiety, a beta-oxa modification yielded a derivative (13) with superior activity against the Plasmodium falciparum 3D7 strain than fosmidomycin, while a gamma-oxa modification resulted in less active derivatives. A bis(pivaloyloxymethyl)ester of phosphonate 13 proved twice as active in inhibiting cultured parasites as a similar prodrug of FR900098.     

Synthesis of alpha-substituted fosmidomycin analogues as highly potent Plasmodium falciparum growth inhibitors

In view of the promising antimalarial activity of fosmidomycin or its N-acetyl homologue FR900098, the objective of this work was to investigate the influence of aromatic substituents in the alpha-position of the phosphonate moiety. The envisaged analogues were prepared using a linear route involving a 3-aryl-3-phosphoryl propanal intermediate. The activities of all compounds were evaluated on Eschericia coli 1-deoxy-d-xylulose 5-phosphate reductoisomerase and against two Plasmodium falciparum strains. Compared with fosmidomycin, several analogues displayed enhanced activity towards the P. falciparum strains. Compound 1e with a 3,4-dichlorophenyl substitution in the alpha-position of fosmidomycin emerged as the most potent analogue of this series. It is approximately three times more potent in inhibiting the growth of P. falciparum than FR900098, the most potent representative of this class reported so far.     

Synthesis and antimalarial activity of chain substituted pivaloyloxymethyl ester analogues of Fosmidomycin and FR900098

Fosmidomycin is a promising antimalarial drug candidate with a unique chemical structure and a novel mode of action. Chain substituted pivaloyloxymethyl ester derivatives of Fosmidomycin and its acetyl analogue FR900098 have been synthesized and their in vitro antimalarial activity versus the Chloroquine sensitive strain 3D7 of Plasmodium falciparum has been determined.     

Substitution of the phosphonic acid and hydroxamic acid functionalities of the DXR inhibitor FR900098: an attempt to improve the activity against Mycobacterium tuberculosis

Two series of FR900098/fosmidomycin analogs were synthesized and evaluated for MtDXR inhibition and Mycobacterium tuberculosis whole-cell activity. The design rationale of these compounds involved the exchange of either the phosphonic acid or the hydroxamic acid part for alternative acidic and metal-coordinating functionalities. The best inhibitors provided IC(50) values in the micromolar range, with a best value of 41 μM.     

Synthesis and evaluation of alpha,beta-unsaturated alpha-aryl-substituted fosmidomycin analogues as DXR inhibitors

Fosmidomycin, which acts through inhibition of 1-deoxy-D-xylulose phosphate reductoisomerase (DXR) in the non-mevalonate pathway, represents a valuable recent addition to the armamentarium against uncomplicated malaria. In this paper, we describe the synthesis and biological evaluation of E- and Z-alpha,beta-unsaturated alpha-aryl-substituted analogues of FR900098, a fosmidomycin congener, utilizing a Stille or a Suzuki coupling to introduce the aryl group. In contrast with our expectations based on the promising activity earlier observed for several alpha-substituted fosmidomycin analogues, all synthesized analogues exhibited much lower binding affinity for DXR than fosmidomycin.     

Antibacterial and antitubercular activity of fosmidomycin, FR900098, and their lipophilic analogs

The nonmevalonate pathway (NMP) of isoprene biosynthesis is an exciting new route toward novel antibiotic development. Inhibitors against several enzymes in this pathway are currently under examination. A significant liability of many of these agents is poor cell penetration. To overcome and improve our understanding of this problem, we have synthesized a series of lipophilic, prodrug analogs of fosmidomycin and FR900098, inhibitors of the NMP enzyme Dxr. Several of these compounds show improved antibacterial activity against a panel of organisms relative to the parent compound, including activity against Mycobacterium tuberculosis (Mtb). Our results show that this strategy can be an effective way for improving whole cell activity of NMP inhibitors.     

Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of K_M ^DXP = 252 µM and K_M ^NADPH = 13 µM, IC₅₀ values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and K_i values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development.     

FR207944, an antifungal antibiotic from Chaetomium sp. no. 217 I. Taxonomy, fermentation, and biological properties

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.     

New N-acetyltransferase fold in the structure and mechanism of the phosphonate biosynthetic enzyme FrbF

The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 ? resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.     

Differences in hypothalamic monoaminergic mechanisms in heterogeneous conditioned reflexes in rats

Monoamines and aminoacids were microinjected in the middle medial hypothalamus of rats following the elaboration of conditioned food-procuring or avoidance reflexes. The glutamine acid had no effect on the avoidance reflex (AR), but stimulated the motor activity and the food-procuring reflex (FR), shortening its latencies, increasing the number of the conditioned food-procuring movements and the reflex size. Dopamine decreased the motor activity, but did not influence the AR latencies, whereas norepinephrine prolonged the AR latencies without changing the motor activity. Both catecholamines, while having no effect on the muscle tone, suppressed FR, prolonging the latencies and decreasing the reflex and the number of conditioned food-procuring movements. Local administration into the hypothalamus of GABA prolonged the latencies of heteromodal conditioned reflexes, whereas the serotonin had no effect on the recorded FR parameters, but facilitated AR significantly reducing its latencies.     

Effect of Endothelin on Vasomotor and Respiratory Neurons in the Rostral Ventrolateral Medulla in Rats

1. We have previously shown that intracisternal administration of endothelin-1 (ET-1) elicited cardiorespiratory responses acting on the ventral surface of the medulla oblongata (VSM) subjacent to the rostral ventrolateral medulla (RVLM). In this study, we examined whether vasomotor and respiratory neurons in RVLM participate in above-mentioned responses and whether those neurons respond to direct iontophoretic application of ET-1 and/or an ET-A receptor antagonist, FR139317.2. Unit activity of vasomotor, respiratory, or nociceptive neurons in RVLM was recorded together with arterial blood pressure (AP) and heart rate (HR) in urethane-anesthetized Sprague-Dawley rats.3. Intracisternal administration or topical application of ET-1 (0.1–1 pmol) to VSM caused excitation of the majority of vasomotor neurons (15/18) and respiratory neurons (10/11) but not in nociceptive neurons (0/7). Changes in neuronal activity were in similar time course with corresponding changes in AP and HR. Iontophoretic application of ET-1 to the vicinity of recording neuron caused excitation in 19 of 21 vasomotor neurons without affecting AP nor HR. Remaining two neurons were insensitive to ET-1. FR139317 did not affect basal activity of the vasomotor neurons but inhibited ET-1-evoked excitation. Twenty-four of 40 respiratory neurons were excited and 13 were inhibited by iontophoretic application of ET-1. Five of ET-1-excited respiratory neurons were inhibited by FR139317 alone while six of ET-1-inhibited neurons were not affected by FR139317 alone. In both cases, FR139317 inhibited the effect of simultaneously applied ET-1. Iontophoretic application of ET-1 excited only one out of 10 nociceptive neurons so far tested.4. These results support the view that intracisternally administered ET-1 alters activity of vasomotor and respiratory neurons in the RVLM, at least in part by acting directly on neurons themselves and hence causes systemic cardiorespiratory changes. Majority of vasomotor and respiratory neurons should express ET-A receptors and some respiratory neurons are under tonic excitatory control by ET-1.     

Ascorbic acid blockade of muscle contractions by neurotransmitters and 2-aminoethanol.

Rats given intracisternal administration of 5–6 dihydroxytryptamine (5,6 DHT) 5 days after birth were tested at two subsequent periods for alterations in locomotor activity and responsiveness to dl-amphetamine and scopolamine. Later in life they were given amphetamine while performing on an FR-6 operant schedule for food reward. Biochemical analyses revealed that the 5,6 DHT treatment produced reductions in forebrain serotonin content. There was no alteration in the treated animals in locomotor activity or in responsiveness to amphetamine in the open field. However, animals given the highest dose (75 μg) of 5,6 DHT neonatally showed open field activity increases at high doses of scopolamine significantly more frequently than the other groups of animals. Although treatment with 5,6 DHT did not affect response rates on the FR task under no-drug conditions, the rate-suppressive effects of amphetamine on the FR task were greatly enhanced in the treated animals.     

The effect of chain length and unsaturation on Mtb Dxr inhibition and antitubercular killing activity of FR900098 analogs

Inhibition of the nonmevalonate pathway (NMP) of isoprene biosynthesis has been examined as a source of new antibiotics with novel mechanisms of action. Dxr is the best studied of the NMP enzymes and several reports have described potent Dxr inhibitors. Many of these compounds are structurally related to natural products fosmidomycin and FR900098, each bearing retrohydroxamate and phosphonate groups. We synthesized a series of compounds with two to five methylene units separating these groups to examine what linker length was optimal and tested for inhibition against Mtb Dxr. We synthesized ethyl and pivaloyl esters of these compounds to increase lipophilicity and improve inhibition of Mtb growth. Our results show that propyl or propenyl linker chains are optimal. Propenyl analog 22 has an IC₅₀ of 1.07 μM against Mtb Dxr. The pivaloyl ester of 22, compound 26, has an MIC of 9.4 μg/mL, representing a significant improvement in antitubercular potency in this class of compounds.     

Novel adenosine A1 receptor antagonists. Synthesis and structure-activity relationships of a novel series of 3-(2-cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5 -a]pyridines

A novel series of 3-(2-cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazol o[1,5-a]pyridines was synthesized and evaluated for in vitro adenosine A1 and A2A receptor binding activities. Most of the cyclohexenyl derivatives (7a-e, 8a-s) were found to be potent adenosine A1 receptor antagonists. In a series of analogues of FR166124 (3a), alcohol 7c, nitrile 7e and amide derivatives (7d, 8c, 8r) were found to be more potent A1 antagonists with higher A2A/A1 selectivity than FR166124. Amongst them, 8r showed considerable water solubility (33.3 mg/mL), but lower than that of the sodium salt of FR166124 (> 200 mg/mL). Additionally, FR166124 had strong diuretic activity by both p.o. and iv administration in rats (minimum effective dose=0.1 and 0.032 mg/kg, respectively).     

Amelioration of Cold Injury-Induced Cortical Brain Edema Formation by Selective Endothelin ETB Receptor Antagonists in Mice

Brain edema is a potentially fatal pathological condition that often occurs in stroke and head trauma. Following brain insults, endothelins (ETs) are increased and promote several pathophysiological responses. This study examined the effects of ET_B antagonists on brain edema formation and disruption of the blood-brain barrier in a mouse cold injury model (Five- to six-week-old male ddY mice). Cold injury increased the water content of the injured cerebrum, and promoted extravasation of both Evans blue and endogenous albumin. In the injury area, expression of prepro-ET-1 mRNA and ET-1 peptide increased. Intracerebroventricular (ICV) administration of BQ788 (ET_B antagonist), IRL-2500 (ET_B antagonist), or {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (ET_A antagonist) prior to cold injury significantly attenuated the increase in brain water content. Bolus administration of BQ788, IRL-2500, or {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 also inhibited the cold injury-induced extravasation of Evans blue and albumin. Repeated administration of BQ788 and IRL-2500 beginning at 24 h after cold injury attenuated both the increase in brain water content and extravasation of markers. In contrast, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 had no effect on edema formation when administrated after cold injury. Cold injury stimulated induction of glial fibrillary acidic protein-positive reactive astrocytes in the injured cerebrum. Induction of reactive astrocytes after cold injury was attenuated by ICV administration of BQ788 or IRL-2500. These results suggest that ET_B receptor antagonists may be an effective approach to ameliorate brain edema formation following brain insults.     

Influence on adipocyte plasma membrane bound protein kinase by feedback regulator.

Protein kinase, phosphodiesterase and adenylate cyclase of plasma membrane of adipocytes and the effect of the feedback regulator (FR) on these three enzymes was measured and compared. The basal level ratio of adenylate cyclase to phosphodiesterase to protein kinase was 1:1.9:3.0. Epinephrine and/or FR alters this ratio. FR stimulated protein kinase activity up to 3 fold in the presence of a wide range of enzyme concentrations, 5-50 mug membrane protein/tube. The concentration of FR effective for stimulation of membrane protein kinase was much greater than that needed for inhibition of adenylate cyclase and phosphodiesterases. The inhibition by FR on adenylate cyclase was the most potent effect among the 3 enzymes. 1 U (or 2 U/ml) of FR inhibited 50% of the adenylate cyclase activity in a defined system. The maximum effective concentration of FR for stimulation of membrane protein kinase was greater than 10 U/ml. Histone type 11A was the best substrate for protein phosphorylation so far observed. The FR stimulatory effect was observed at all substrate concentrations used ranging from 1-5 mg/ml. A NaF concentration curve shows that 15 mM NaF gave maximum phosphorylation. The stimulatory effect of FR was observed both in the presence and absence of NaF. Protein kinase of adipocyte plasma membrane was mainly cAMP-independent. The effect of FR (20 U/ml) in stimulation of protein phosphorylation was much greater than that of cAMP (1 X 10(-6) M). The cAMP and FR effects seemed to be additive. Preincubation of plasma membrane with FR in the absence of ATP resulted in no decrease but slight increase in protein kinase activity. A shift in protein kinase, phosphodiesterase and adenylate cyclase ratios by FR suggests the regulatory role of FR in cAMP metabolism in adipocytes.     

Interactions among unit-price, fixed-ratio value, and dosing regimen in determining effects of repeated cocaine administration

Previous research has shown tolerance to cocaine that was dependent on fixed-ratio (FR) parameter size in the context of a multiple FR schedule of food reinforcement. Completion of the FR requirement in these studies resulted in the same magnitude of reinforcement, regardless of ratio size. The cost-to-benefits ratio (unit-price) was therefore not equated across the different FR components. The current study examined the effects of repeated administration of cocaine to pigeons when unit-price under FR schedules was either the same or different. Additionally, the role of a chronic variable-dosing versus chronic fixed-dosing procedure was examined when unit-price was equated across different ratio values. Pigeons were trained to key peck in daily sessions under a three-component multiple FR schedule of food presentation, according to which either 10, 30, or 100 pecks were required for each delivery of food. In Experiment 1, completion of the FR 10, FR 30, and FR 100 resulted in 1.5, 4.5, and 15.0 s access to food, respectively. That is, the response requirement was correlated with access to food time so that unit-price (pecks per second of access to food) was equated across components. After assessing acute effects of a range of doses of cocaine, drug administration occurred daily before each session, with the dose varying from day to day. Tolerance, the magnitude of which was unrelated to the peck requirement, developed under the repeated-dosing regimen. In order to assess whether having equal unit-price was responsible for producing similar levels of tolerance across components, daily drug administration continued in Experiment 2 using the variable-dose regimen, but the amount of food presented each time was fixed at 4.5 s access to food, yielding different unit-prices under the three pecking requirements. Subsequently, the conditions of Experiment 1 were reinstated, i.e., unit-price was equated. Making unit-price different or the same had little influence on effects of cocaine. In Experiment 3, a fixed dose of cocaine was administered before each session while programmed unit-price remained the same across components. Under these conditions, tolerance became peck-requirement related. Specifically, tolerance was most prevalent under the smaller requirements and less robust or absent when the largest requirement was in effect. Differences in unit-price, therefore, were not related to degree of tolerance, but work requirement was. Differences in effects of cocaine across responses requirements, however, were observed only when each session was preceded by the same dose, not when dose varied from session to session.