Alternative boronic acids in the detection of Mycolactone A/B using the thin layer chromatography (f-TLC) method for diagnosis of Buruli ulcer.

BACKGROUND: Mycobacterium ulcerans is the causative agent of Buruli ulcer. The pathology of M. ulcerans disease has been attributed to the secretion of a potent macrolide cytotoxin known as mycolactone which plays an important role in the virulence of the disease. Mycolactone is a biomarker for the diagnosis of BU that can be detected using the fluorescent-thin layer chromatography (f-TLC) technique. The technique relies on the chemical derivatization of mycolactone A/B with 2-naphthylboronic acid (BA) which acts as a fluorogenic chemosensor. However, background interferences due to co-extracted human tissue lipids, especially with clinical samples coupled with the subjectivity of the method call for an investigation to find an alternative to BA. METHODS: Twenty-six commercially available arylboronic acids were initially screened as alternatives to BA using the f-TLC experiment. UV-vis measurements were also conducted to determine the absorption maximum spectra of mycolactone A/B and myco-boronic acid adducts followed by an investigation of the fluorescence-enhancing ability of the boronate ester formation between mycolactone A/B and our three most promising boronic acids (BA15, BA18, and BA21). LC-MS technique was employed to confirm the adduct formation between mycolactone and boronic acids. Furthermore, a comparative study was conducted between BA18 and BA using 6 Polymerase Chain Reaction (PCR) confirmed BU patient samples. RESULTS: Three of the boronic acids (BA15, BA18, and BA21) produced fluorescent band intensities superior to BA. Complexation studies conducted on thin layer chromatography (TLC) using 0.1 M solution of the three boronic acids and various volumes of 10 ng/µL of synthetic mycolactone ranging from 1 µL - 9 µL corresponding to 10 ng - 90 ng gave similar results with myco-BA18 adduct emerging with the most visibly intense fluorescence bands. UV-vis absorption maxima (λmax) for the free mycolactone A/B was observed at 362 nm, and the values for the adducts myco-BA15, myco-BA18, and myco-BA21 were at 272 nm, 270 nm, and 286 nm respectively. The comparable experimental λmax of 362 nm for mycolactone A/B to the calculated Woodward-Fieser value of 367 nm for the fatty acid side chain of mycolactone A/B demonstrate that even though 2 cyclic boronates were formed, only the boronate of the southern side chain with the chromophore was excited by irradiation at 365 nm. Fluorescence experiments have demonstrated that coupling BA18 to mycolactone A/B along the 1,3-diols remarkably enhanced the fluorescence intensity at 537 nm. High-Resolution Mass Spectrometer (HR-MS) was used to confirm the formation of the myco-BA15 adduct. Finally, f-TLC analysis of patient samples with BA18 gave improved BA18-adduct intensities compared to the original BA-adduct. CONCLUSION: Twenty-six commercially available boronic acids were investigated as alternatives to BA, used in the f-TLC analysis for the diagnosis of BU. Three (3) of them BA15, BA18, and BA21 gave superior fluorescence band intensity profiles. They gave profiles that were easier to interpret after the myco-boronic acid adduct formation and in experiments with clinical samples from patients with BA18 the best. BA18, therefore, has been identified as a potential alternative to BA and could provide a solution to the challenge of background interference of co-extracted human tissue lipids from clinical samples currently associated with the use of BA.

Inhibiting Leishmania donovani Sterol Methyltransferase to Identify Lead Compounds Using Molecular Modelling.

The recent outlook of leishmaniasis as a global public health concern coupled with the reportage of resistance and lack of efficacy of most antileishmanial drugs calls for a concerted effort to find new leads. The study combined In silico and in vitro approaches to identify novel potential synthetic small-molecule inhibitors targeting the Leishmania donovani sterol methyltransferase (LdSMT). The LdSMT enzyme in the ergosterol biosynthetic pathway is required for the parasite's membrane fluidity, distribution of membrane proteins, and control of the cell cycle. The lack of LdSMT homologue in the human host and its conserved nature among all Leishmania parasites makes it a viable target for future antileishmanial drugs. Initially, six known inhibitors of LdSMT with IC50 < 10 µM were used to generate a pharmacophore model with a score of 0.9144 using LigandScout. The validated model was used to screen a synthetic library of 95,630 compounds obtained from InterBioScreen limited. Twenty compounds with pharmacophore fit scores above 50 were docked against the modelled three-dimensional structure of LdSMT using AutoDock Vina. Consequently, nine compounds with binding energies ranging from -7.5 to -8.7 kcal/mol were identified as potential hit molecules. Three compounds comprising STOCK6S-06707, STOCK6S-84928, and STOCK6S-65920 with respective binding energies of -8.7, -8.2, and -8.0 kcal/mol, lower than 22,26-azasterol (-7.6 kcal/mol), a known LdSMT inhibitor, were selected as plausible lead molecules. Molecular dynamics simulation studies and molecular mechanics Poisson-Boltzmann surface area calculations showed that the residues Asp25 and Trp208 were critical for ligand binding. The compounds were also predicted to have antileishmanial activity with reasonable pharmacological and toxicity profiles. When the antileishmanial activity of the three hits was evaluated in vitro against the promastigotes of L. donovani, mean half-maximal inhibitory concentrations (IC50) of 21.9 ± 1.5 µM (STOCK6S-06707), 23.5 ± 1.1 µM (STOCK6S-84928), and 118.3 ± 5.8 µM (STOCK6S-65920) were obtained. Furthermore, STOCK6S-84928 and STOCK6S-65920 inhibited the growth of Trypanosoma brucei, with IC50 of 14.3 ± 2.0 µM and 18.1 ± 1.4 µM, respectively. The identified compounds could be optimised to develop potent antileishmanial therapeutic agents.

Correction: Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents.

[This corrects the article DOI: 10.1371/journal.pntd.0008919.].

Evaluation of the fluorescent-thin layer chromatography (f-TLC) for the diagnosis of Buruli ulcer disease in Ghana.

BACKGROUND: Buruli ulcer is a tissue necrosis infection caused by an environmental mycobacterium called Mycobacterium ulcerans (MU). The disease is most prevalent in rural areas with the highest rates in West and Central African countries. The bacterium produces a toxin called mycolactone which can lead to the destruction of the skin, resulting in incapacitating deformities with an enormous economic and social burden on patients and their caregivers. Even though there is an effective antibiotic treatment for BU, the control and management rely on early case detection and rapid diagnosis to avert morbidities. The diagnosis of Mycobacterium ulcerans relies on smear microscopy, culture histopathology, and PCR. Unfortunately, all the current laboratory diagnostics have various limitations and are not available in endemic communities. Consequently, there is a need for a rapid diagnostic tool for use at the community health centre level to enable diagnosis and confirmation of suspected cases for early treatment. The present study corroborated the diagnostic performance and utility of fluorescent-thin layer chromatography (f-TLC) for the diagnosis of Buruli ulcer. METHODOLOGY/PRINCIPAL FINDINGS: The f-TLC method was evaluated for the diagnosis of Buruli ulcer in larger clinical samples than previously reported in an earlier preliminary study Wadagni et al. (2015). A total of 449 patients suspected of BU were included in the final data analysis out of which 122 (27.2%) were positive by f-TLC and 128 (28.5%) by PCR. Using a composite reference method generated from the two diagnostic methods, 85 (18.9%) patients were found to be truly infected with M. ulcerans, 284 (63.3%) were uninfected, while 80 (17.8%) were misidentified as infected or noninfected by the two methods. The data obtained was used to determine the discriminatory accuracy of the f-TLC against the gold standard IS2404 PCR through the analysis of its sensitivity, specificity, positive (+LR), and negative (-LR) likelihood ratio. The positive (PPV) and negative (NPV) predictive values, area under the receiver operating characteristic curve Azevedo et al. (2014), and diagnostic odds ratio were used to assess the predictive accuracy of the f-TLC method. The sensitivity of f-TLC was 66.4% (85/128), specificity was 88.5% (284/321), while the diagnostic accuracy was 82.2% (369/449). The AUC stood at 0.774 while the PPV, NPV, +LR, and-LR were 69.7% (85/122), 86.9% (284/327), 5.76, and 0.38, respectively. The use of the rule-of-thumb interpretation of diagnostic tests suggests that the method is good for use as a diagnostic tool. CONCLUSIONS/SIGNIFICANCE: Larger clinical samples than previously reported had been used to evaluate the f-TLC method for the diagnosis of Buruli ulcer. A sensitivity of 66.4%, a specificity of 88.5%, and diagnostic accuracy of 82.2% were obtained. The method is good for diagnosis and will help in making early clinical decisions about the patients as well as patient management and facilitating treatment decisions. However, it requires a slight modification to address the challenge of background interference and lack of automatic readout to become an excellent diagnostic tool.

Targeting the Ubiquinol-Reduction (Qi) Site of the Mitochondrial Cytochrome bc1 Complex for the Development of Next Generation Quinolone Antimalarials.

Antimalarials targeting the ubiquinol-oxidation (Qo) site of the Plasmodium falciparum bc1 complex, such as atovaquone, have become less effective due to the rapid emergence of resistance linked to point mutations in the Qo site. Recent findings showed a series of 2-aryl quinolones mediate inhibitions of this complex by binding to the ubiquinone-reduction (Qi) site, which offers a potential advantage in circumventing drug resistance. Since it is essential to understand how 2-aryl quinolone lead compounds bind within the Qi site, here we describe the co-crystallization and structure elucidation of the bovine cytochrome bc1 complex with three different antimalarial 4(1H)-quinolone sub-types, including two 2-aryl quinolone derivatives and a 3-aryl quinolone analogue for comparison. Currently, no structural information is available for Plasmodial cytochrome bc1. Our crystallographic studies have enabled comparison of an in-silico homology docking model of P. falciparum with the mammalian's equivalent, enabling an examination of how binding compares for the 2- versus 3-aryl analogues. Based on crystallographic and computational modeling, key differences in human and P. falciparum Qi sites have been mapped that provide new insights that can be exploited for the development of next-generation antimalarials with greater selective inhibitory activity against the parasite bc1 with improved antimalarial properties.

Homology Modeling, de Novo Design of Ligands, and Molecular Docking Identify Potential Inhibitors of Leishmania donovani 24-Sterol Methyltransferase.

The therapeutic challenges pertaining to leishmaniasis due to reported chemoresistance and toxicity necessitate the need to explore novel pathways to identify plausible inhibitory molecules. Leishmania donovani 24-sterol methyltransferase (LdSMT) is vital for the synthesis of ergosterols, the main constituents of Leishmania cellular membranes. So far, mammals have not been shown to possess SMT or ergosterols, making the pathway a prime candidate for drug discovery. The structural model of LdSMT was elucidated using homology modeling to identify potential novel 24-SMT inhibitors via virtual screening, scaffold hopping, and de-novo fragment-based design. Altogether, six potential novel inhibitors were identified with binding energies ranging from -7.0 to -8.4 kcal/mol with e-LEA3D using 22,26-azasterol and S1-S4 obtained from scaffold hopping via the ChEMBL, DrugBank, PubChem, ChemSpider, and ZINC15 databases. These ligands showed comparable binding energy to 22,26-azasterol (-7.6 kcal/mol), the main inhibitor of LdSMT. Moreover, all the compounds had plausible ligand efficiency-dependent lipophilicity (LELP) scores above 3. The binding mechanism identified Tyr92 to be critical for binding, and this was corroborated via molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations. The ligand A1 was predicted to possess antileishmanial properties with a probability of activity (Pa) of 0.362 and a probability of inactivity (Pi) of 0.066, while A5 and A6 possessed dermatological properties with Pa values of 0.205 and 0.249 and Pi values of 0.162 and 0.120, respectively. Structural similarity search via DrugBank identified vabicaserin, daledalin, zanapezil, imipramine, and cefradine with antileishmanial properties suggesting that the de-novo compounds could be explored as potential antileishmanial agents.

Unravelling the myth surrounding sterol biosynthesis as plausible target for drug design against leishmaniasis.

The mortality rate of leishmaniasis is increasing at an alarming rate and is currently second to malaria amongst the other neglected tropical diseases. Unfortunately, many governments and key stakeholders are not investing enough in the development of new therapeutic interventions. The available treatment options targeting different pathways of the parasite have seen inefficiencies, drug resistance, and toxic side effects coupled with longer treatment durations. Numerous studies to understand the biochemistry of leishmaniasis and its pathogenesis have identified druggable targets including ornithine decarboxylase, trypanothione reductase, and pteridine reductase, which are relevant for the survival and growth of the parasites. Another plausible target is the sterol biosynthetic pathway; however, this has not been fully investigated. Sterol biosynthesis is essential for the survival of the Leishmania species because its inhibition could lead to the death of the parasites. This review seeks to evaluate how critical the enzymes involved in sterol biosynthetic pathway are to the survival of the leishmania parasite. The review also highlights both synthetic and natural product compounds with their IC50 values against selected enzymes. Finally, recent advancements in drug design strategies targeting the sterol biosynthesis pathway of Leishmania are discussed.

The Search for Putative Hits in Combating Leishmaniasis: The Contributions of Natural Products Over the Last Decade.

Despite advancements in the areas of omics and chemoinformatics, potent novel biotherapeutic molecules with new modes of actions are needed for leishmaniasis. The socioeconomic burden of leishmaniasis remains alarming in endemic regions. Currently, reports from existing endemic areas such as Nepal, Iran, Brazil, India, Sudan and Afghanistan, as well as newly affected countries such as Peru, Bolivia and Somalia indicate concerns of chemoresistance to the classical antimonial treatment. As a result, effective antileishmanial agents which are safe and affordable are urgently needed. Natural products from both flora and fauna have contributed immensely to chemotherapeutics and serve as vital sources of new chemical agents. This review focuses on a systematic cross-sectional view of all characterized anti-leishmanial compounds from natural sources over the last decade. Furthermore, IC50/EC50, cytotoxicity and suggested mechanisms of action of some of these natural products are provided. The natural product classification includes alkaloids, terpenes, terpenoids, and phenolics. The plethora of reported mechanisms involve calcium channel inhibition, immunomodulation and apoptosis. Making available enriched data pertaining to bioactivity and mechanisms of natural products complement current efforts geared towards unraveling potent leishmanicides of therapeutic relevance.

Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents.

BACKGROUND: Ghana is endemic for some neglected tropical diseases (NTDs) including schistosomiasis, onchocerciasis and lymphatic filariasis. The major intervention for these diseases is mass drug administration of a few repeatedly recycled drugs which is a cause for major concern due to reduced efficacy of the drugs and the emergence of drug resistance. Evidently, new treatments are needed urgently. Medicinal plants, on the other hand, have a reputable history as important sources of potent therapeutic agents in the treatment of various diseases among African populations, Ghana inclusively, and provide very useful starting points for the discovery of much-needed new or alternative drugs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, extracts of fifteen traditional medicines used for treating various NTDs in local communities were screened in vitro for efficacy against schistosomiasis, onchocerciasis and African trypanosomiasis. Two extracts, NTD-B4-DCM and NTD-B7-DCM, prepared from traditional medicines used to treat schistosomiasis, displayed the highest activity (IC50 = 30.5 µg/mL and 30.8 µg/mL, respectively) against Schistosoma mansoni adult worms. NTD-B2-DCM, also obtained from an antischistosomal remedy, was the most active against female and male adult Onchocera ochengi worms (IC50 = 76.2 µg/mL and 76.7 µg/mL, respectively). Antitrypanosomal assay of the extracts against Trypanosoma brucei brucei gave the most promising results (IC50 = 5.63 µg/mL to 18.71 µg/mL). Incidentally, NTD-B4-DCM and NTD-B2-DCM, also exhibited the greatest antitrypanosomal activities (IC50 = 5.63 µg/mL and 7.12 µg/mL, respectively). Following the favourable outcome of the antitrypanosomal screening, this assay was selected for bioactivity-guided fractionation. NTD-B4-DCM, the most active extract, was fractionated and subsequent isolation of bioactive constituents led to an eupatoriochromene-rich oil (42.6%) which was 1.3-fold (IC50 <0.0977 µg/mL) more active than the standard antitrypanosomal drug, diminazene aceturate (IC50 = 0.13 µg/mL). CONCLUSION/SIGNIFICANCE: These findings justify the use of traditional medicines and demonstrate their prospects towards NTDs drug discovery.

Setting Our Sights on Infectious Diseases.

In May 2019, the Wellcome Centre for Anti-Infectives Research (WCAIR) at the University of Dundee, UK, held an international conference with the aim of discussing some key questions around discovering new medicines for infectious diseases and a particular focus on diseases affecting Low and Middle Income Countries. There is an urgent need for new drugs to treat most infectious diseases. We were keen to see if there were lessons that we could learn across different disease areas and between the preclinical and clinical phases with the aim of exploring how we can improve and speed up the drug discovery, translational, and clinical development processes. We started with an introductory session on the current situation and then worked backward from clinical development to combination therapy, pharmacokinetic/pharmacodynamic (PK/PD) studies, drug discovery pathways, and new starting points and targets. This Viewpoint aims to capture some of the learnings.

Synthesis and profiling of benzylmorpholine 1,2,4,5-tetraoxane analogue N205: Towards tetraoxane scaffolds with potential for single dose cure of malaria.

A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84 nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies.

A tetraoxane-based antimalarial drug candidate that overcomes PfK13-C580Y dependent artemisinin resistance.

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P. falciparum and P. vivax in vitro, is efficacious against P. falciparum in in vivo rodent models, produces parasite reduction ratios equivalent to dihydroartemisinin and has pharmacokinetic and pharmacodynamic characteristics compatible with a single-dose cure. In vitro studies with transgenic parasites expressing variant forms of K13 show no cross-resistance with the C580Y mutation, the primary variant observed in Southeast Asia. E209 is a superior next generation endoperoxide with combined pharmacokinetic and pharmacodynamic features that overcome the liabilities of artemisinin derivatives.

Synthesis and evaluation of the antimalarial, anticancer, and caspase 3 activities of tetraoxane dimers.

The synthesis of a range of mono spiro and dispiro 1,2,4,5-tetraoxane dimers is described. Selected molecules were examined in in vitro assays to determine their antimalarial and anticancer potential. Our studies reveal that several molecules possess potent nanomolar antimalarial and single digit micromolar antiproliferative IC(50)s versus colon (HT29-AK and leukemia (HL60) cell lines.

An endoperoxide-based hybrid approach to deliver falcipain inhibitors inside malaria parasites.

The emergence of artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia has reinforced the urgent need to discover novel chemotherapeutic strategies to treat and control malaria. To address this problem, we prepared a set of dual-acting tetraoxane-based hybrid molecules designed to deliver a falcipain-2 (FP-2) inhibitor upon activation by iron(II) in the parasite digestive vacuole. These hybrids are active in the low nanomolar range against chloroquine-sensitive and chloroquine-resistant P. falciparum strains. We also demonstrate that in the presence of FeBr2 or within infected red blood cells, these molecules fragment to release falcipain inhibitors with nanomolar protease inhibitory activity. Molecular docking studies were performed to better understand the molecular interactions established between the tetraoxane-based hybrids and the cysteine protease binding pocket residues. Our results further indicate that the intrinsic activity of the tetraoxane partner compound can be masked, suggesting that a tetraoxane-based delivery system offers the potential to attenuate the off-target effects of known drugs.

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria.

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc(1). Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Identification of novel antimalarial chemotypes via chemoinformatic compound selection methods for a high-throughput screening program against the novel malarial target, PfNDH2: increasing hit rate via virtual screening methods.

Malaria is responsible for approximately 1 million deaths annually; thus, continued efforts to discover new antimalarials are required. A HTS screen was established to identify novel inhibitors of the parasite's mitochondrial enzyme NADH:quinone oxidoreductase (PfNDH2). On the basis of only one known inhibitor of this enzyme, the challenge was to discover novel inhibitors of PfNDH2 with diverse chemical scaffolds. To this end, using a range of ligand-based chemoinformatics methods, ~17000 compounds were selected from a commercial library of ~750000 compounds. Forty-eight compounds were identified with PfNDH2 enzyme inhibition IC(50) values ranging from 100 nM to 40 µM and also displayed exciting whole cell antimalarial activity. These novel inhibitors were identified through sampling 16% of the available chemical space, while only screening 2% of the library. This study confirms the added value of using multiple ligand-based chemoinformatic approaches and has successfully identified novel distinct chemotypes primed for development as new agents against malaria.

Comparison of the reactivity of antimalarial 1,2,4,5-tetraoxanes with 1,2,4-trioxolanes in the presence of ferrous iron salts, heme, and ferrous iron salts/phosphatidylcholine.

Dispiro-1,2,4,5-tetraoxanes and 1,2,4-trioxolanes represent attractive classes of synthetic antimalarial peroxides due to their structural simplicity, good stability, and impressive antimalarial activity. We investigated the reactivity of a series of potent amide functionalized tetraoxanes with Fe(II)gluconate, FeSO(4), FeSO(4)/TEMPO, FeSO(4)/phosphatidylcholine, and heme to gain knowledge of their potential mechanism of bioactivation and to compare the results with the corresponding 1,2,4-trioxolanes. Spin-trapping experiments demonstrate that Fe(II)-mediated peroxide activation of tetraoxanes produces primary and secondary C-radical intermediates. Reaction of tetraoxanes and trioxolanes with phosphatidylcholine, a predominant unsaturated lipid present in the parasite digestive vacuole membrane, under Fenton reaction conditions showed that both endoperoxides share a common reactivity in terms of phospholipid oxidation that differs with that of artemisinin. Significantly, when tetraoxanes undergo bioactivation in the presence of heme, only the secondary C-centered radical is observed, which smoothly produces regioisomeric drug derived-heme adducts. The ability of these tetraoxanes to alkylate the porphyrin ring was also confirmed with Fe(II)TPP and Mn(II)TPP, and docking studies were performed to rationalize the regioselectivity observed in the alkylation process. The efficient process of heme alkylation and extensive lipid peroxidation observed here may play a role in the mechanism of action of these two important classes of synthetic endoperoxide antimalarial.

Endoperoxide carbonyl falcipain 2/3 inhibitor hybrids: toward combination chemotherapy of malaria through a single chemical entity.

We extend our approach of combination chemotherapy through a single prodrug entity (O'Neill et al. Angew. Chem., Int. Ed. 2004, 43, 4193) by using a 1,2,4-trioxolane as a protease inhibitor carbonyl-masking group. These molecules are designed to target the malaria parasite through two independent mechanisms of action: iron(II) decomposition releases the carbonyl protease inhibitor and potentially cytotoxic C-radical species in tandem. Using a proposed target "heme", we also demonstrate heme alkylation/carbonyl inhibitor release and quantitatively measure endoperoxide turnover in parasitized red blood cells.