The ratio of M1 to M2 microglia in the striatum determines the severity of L-Dopa-induced dyskinesias.

L-Dopa, while treating motor symptoms of Parkinson's disease, can lead to debilitating L-Dopa-induced dyskinesias, limiting its use. To investigate the causative relationship between neuro-inflammation and dyskinesias, we assessed if striatal M1 and M2 microglia numbers correlated with dyskinesia severity and whether the anti-inflammatories, minocycline and indomethacin, reverse these numbers and mitigate against dyskinesia. In 6-OHDA lesioned mice, we used stereology to assess numbers of striatal M1 and M2 microglia populations in non-lesioned (naïve) and lesioned mice that either received no L-Dopa (PD), remained non-dyskinetic even after L-Dopa (non-LID) or became dyskinetic after L-Dopa treatment (LID). We also assessed the effect of minocycline/indomethacin treatment on striatal M1 and M2 microglia and its anti-dyskinetic potential via AIMs scoring. We report that L-Dopa treatment leading to LIDs exacerbates activated microglia numbers beyond that associated with the PD state; the severity of LIDs is strongly correlated to the ratio of the striatal M1 to M2 microglial numbers; in non-dyskinetic mice, there is no M1/M2 microglia ratio increase above that seen in PD mice; and reducing M1/M2 microglia ratio using anti-inflammatories is anti-dyskinetic. Parkinson's disease is associated with increased inflammation, but this is insufficient to underpin dyskinesia. Given that L-Dopa-treated non-LID mice show the same ratio of M1/M2 microglia as PD mice that received no L-Dopa, and, given minocycline/indomethacin reduces both the ratio of M1/M2 microglia and dyskinesia severity, our data suggest the increased microglial M1/M2 ratio that occurs following L-Dopa treatment is a contributing cause of dyskinesias.

Heme Detoxification in the Malaria Parasite: A Target for Antimalarial Drug Development.

Over the last century, malaria deaths have decreased by more than 85%. Nonetheless, there were 405 000 deaths in 2018, mostly resulting from Plasmodium falciparum infection. In the 21st century, much of the advance has arisen from the deployment of insecticide-treated bed nets and artemisinin combination therapy. However, over the past few decades parasites with a delayed artemisinin clearance phenotype have appeared in Southeast Asia, threatening further gains. The effort to find new drugs is thus urgent. A prominent process in blood stage malaria parasites, which we contend remains a viable drug target, is hemozoin formation. This crystalline material consisting of heme can be readily seen when parasites are viewed microscopically. The process of its formation in the parasite, however, is still not fully understood.In early work, we recognized hemozoin formation as a biomineralization process. We have subsequently investigated the kinetics of synthetic hemozoin (ß-hematin) crystallization catalyzed at lipid-aqueous interfaces under biomimetic conditions. This led us to the use of neutral detergent-based high-throughput screening (HTS) for inhibitors of ß-hematin formation. A good hit rate against malaria parasites was obtained. Simultaneously, we developed a pyridine-based assay which proved successful in measuring the concentrations of hematin not converted to ß-hematin.The pyridine assay was adapted to determine the effects of chloroquine and other clinical antimalarials on hemozoin formation in the cell. This permitted the determination of the dose-dependent amounts of exchangeable heme and hemozoin in P. falciparum for the first time. These studies have shown that hemozoin inhibitors cause a dose-dependent increase in exchangeable heme, correlated with decreased parasite survival. Electron spectroscopic imaging (ESI) showed a relocation of heme iron into the parasite cytoplasm, while electron microscopy provided evidence of the disruption of hemozoin crystals. This cellular assay was subsequently extended to top-ranked hits from a wide range of scaffolds found by HTS. Intriguingly, the amounts of exchangeable heme at the parasite growth IC50 values of these scaffolds showed substantial variation. The amount of exchangeable heme was found to be correlated with the amount of inhibitor accumulated in the parasitized red blood cell. This suggests that heme-inhibitor complexes, rather than free heme, lead to parasite death. This was supported by ESI using a Br-containing compound which showed the colocalization of Fe and Br as well as by confocal Raman microscopy which confirmed the presence of a complex in the parasite. Current evidence indicates that inhibitors block hemozoin formation by surface adsorption. Indeed, we have successfully introduced molecular docking with hemozoin to find new inhibitors. It follows that the resulting increase in free heme leads to the formation of the parasiticidal heme-inhibitor complex. We have reported crystal structures of heme-drug complexes for several aryl methanol antimalarials in nonaqueous media. These form coordination complexes but most other inhibitors interact noncovalently, and the determination of their structures remains a major challenge.It is our view that key future developments will include improved assays to measure cellular heme levels, better in silico approaches for predicting ß-hematin inhibition, and a concerted effort to determine the structure and properties of heme-inhibitor complexes.

Adapting a Dialysis Service for Delivery of Percutaneous Arteriovenous Fistulas.

The percutaneous arteriovenous fistula (pAVF) is an exciting and novel addition to the vascular access options available to patients with end-stage kidney disease who require dialysis. Early clinical results have been promising, with high rates of maturation and low rates of reintervention. To successfully adapt an existing hemodialysis service to include the provision of pAVF formation, it is essential to identify and align the interests of key clinical and nonclinical stakeholders. Only through strong collaboration can the service be supported. The authors provide a comprehensive overview of the planning fundamentals required, including the referral pathway, screening and clinical assessment, and practical procedural elements and considerations, as well as follow-up requirements such as cannulation, fistula surveillance, and maintenance. Key staffing requirements are highlighted, including those pertaining to vascular US screening and dialysis nurse training. A broad and structured planning approach ensures that the entire network of key stakeholder interests is included and provides a strong foundation for a compelling business plan to attract the necessary funding and managerial support for the service. The authors present a systematic framework of the essential considerations necessary to facilitate the planning, funding, and ultimately delivery of a successful pAVF service. Online supplemental material is available for this article. ©RSNA, 2022.

THC shows activity against cultured Plasmodium falciparum.

The FDA approved drug Dronabinol was identified in a previous study applying virtual screening using the haemozoin crystal as a target against malaria parasites. The active ingredient of dronabinol is synthetic tetrahydrocannabinol (THC), which is one of the major cannabinoids from Cannabis sativa. Traditional use of cannabis for malaria fever was reported in the world's oldest pharmacopoeia, dating to around 5000 years ago. In this research we report that THC inhibits ß-haematin (synthetic haemozoin) and malaria parasite growth. Due the psychoactivity of THC, CBD, the other major naturally occurring cannabinoid that lacks the off-target psychoactive effects of THC, was also tested and inhibited ß-haematin but showed only a mild antimalarial activity. To evaluate whether THC inhibit haemozoin formation, we performed a cellular haem fractionation assay that indicated that is not the likely mechanism of action. For the first time, the cannabinoid chemical structure is raised as a new chemical class to be further studied for malaria treatment, aiming to overcome the undesirable psychoactive effects of THC and optimize the antimalarial effects.

Targeting the cannabinoid receptor CB2 in a mouse model of l-dopa induced dyskinesia.

L-dopa induced dyskinesia (LID) is a debilitating side-effect of the primary treatment used in Parkinson's disease (PD), l-dopa. Here we investigate the effect of HU-308, a cannabinoid CB2 receptor agonist, on LIDs. Utilizing a mouse model of PD and LIDs, induced by 6-OHDA and subsequent l-dopa treatment, we show that HU-308 reduced LIDs as effectively as amantadine, the current frontline treatment. Furthermore, treatment with HU-308 plus amantadine resulted in a greater anti-dyskinetic effect than maximally achieved with HU-308 alone, potentially suggesting a synergistic effect of these two treatments. Lastly, we demonstrated that treatment with HU-308 and amantadine either alone, or in combination, decreased striatal neuroinflammation, a mechanism which has been suggested to contribute to LIDs. Taken together, our results suggest pharmacological treatments with CB2 agonists merit further investigation as therapies for LIDs in PD patients. Furthermore, since CB2 receptors are thought to be primarily expressed on, and signal through, glia, our data provide weight to suggestion that neuroinflammation, or more specifically, altered glial function, plays a role in development of LIDs.

Intrinsic fluorescence properties of antimalarial pyrido[1,2-a]benzimidazoles facilitate subcellular accumulation and mechanistic studies in the human malaria parasite Plasmodium falciparum.

The intrinsic fluorescence properties of two related pyrido[1,2-a]benzimidazole antimalarial compounds suitable for the cellular imaging of the human malaria parasite Plasmodium falciparum without the need to attach extrinsic fluorophores are described. Although these compounds are structurally related, they have been shown by confocal microscopy to not only accumulate selectively within P. falciparum but to also accumulate differently in the organelles investigated. Localization to the digestive vacuole and nearby neutral lipids was observed for compound 2 which was shown to inhibit hemozoin formation using a cellular fractionation assay indicating that this is a contributing mechanism of action. By contrast, compound 1, which differs from compound 2 by the replacement of the imidazole[1,2-a:4,5-b']dipyridine core with the benzimidazole core as well as the presence of Cl substituents, shows very different localisation patterns and shows no evidence of hemozoin inhibition, suggesting a different mechanism of antimalarial action. Docking profiles of both compounds on the hemozoin surface further provided insight into their mechanisms of action.

Lapatinib, Nilotinib and Lomitapide Inhibit Haemozoin Formation in Malaria Parasites.

With the continued loss of antimalarials to resistance, drug repositioning may have a role in maximising efficiency and accelerating the discovery of new antimalarial drugs. Bayesian statistics was previously used as a tool to virtually screen USFDA approved drugs for predicted ß-haematin (synthetic haemozoin) inhibition and in vitro antimalarial activity. Here, we report the experimental evaluation of nine of the highest ranked drugs, confirming the accuracy of the model by showing an overall 93% hit rate. Lapatinib, nilotinib, and lomitapide showed the best activity for inhibition of ß-haematin formation and parasite growth and were found to inhibit haemozoin formation in the parasite, providing mechanistic insights into their mode of antimalarial action. We then screened the USFDA approved drugs for binding to the ß-haematin crystal, applying a docking method in order to evaluate its performance. The docking method correctly identified imatinib, lapatinib, nilotinib, and lomitapide. Experimental evaluation of 22 of the highest ranked purchasable drugs showed a 24% hit rate. Lapatinib and nilotinib were chosen as templates for shape and electrostatic similarity screening for lead hopping using the in-stock ChemDiv compound catalogue. The actives were novel structures worthy of future investigation. This study presents a comparison of different in silico methods to identify new haemozoin-inhibiting chemotherapeutic alternatives for malaria that proved to be useful in different ways when taking into consideration their strengths and limitations.

Prediction Model for Antimalarial Activities of Hemozoin Inhibitors by Using Physicochemical Properties.

The rapid spread of strains of malaria parasites that are resistant to several drugs has threatened global malaria control. Hence, the aim of this study was to predict the antimalarial activity of chemical compounds that possess anti-hemozoin-formation activity as a new means of antimalarial drug discovery. After the initial in vitro anti-hemozoin-formation high-throughput screening (HTS) of 9,600 compounds, a total of 224 hit compounds were identified as hemozoin inhibitors. These 224 compounds were tested for in vitro erythrocytic antimalarial activity at 10 µM by using chloroquine-mefloquine-sensitive Plasmodium falciparum strain 3D7A. Two independent experiments were conducted. The physicochemical properties of the active compounds were extracted from the ChemSpider and SciFinder databases. We analyzed the extracted data by using Bayesian model averaging (BMA). Our findings revealed that lower numbers of S atoms; lower distribution coefficient (log D) values at pH 3, 4, and 5; and higher predicted distribution coefficient (ACD log D) values at pH 7.4 had significant associations with antimalarial activity among compounds that possess anti-hemozoin-formation activity. The BMA model revealed an accuracy of 91.23%. We report new prediction models containing physicochemical properties that shed light on effective chemical groups for synthetic antimalarial compounds and help with in silico screening for novel antimalarial drugs.

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology.

Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize novel pfcrt alleles that can undermine the efficacy of first-line antimalarial drug regimens.

Interplay between Plasmodium falciparum haemozoin and L-arginine: implication for nitric oxide production.

BACKGROUND: Plasmodium falciparum haemozoin, a detoxification product of digested haemoglobin from infected erythrocytes, is released into the bloodstream upon schizont rupture and accumulates in leukocytes. High levels of haemozoin correlate with disease severity. Some studies have shown that concentrations of the substrate of inducible nitric oxide synthase (iNOS), L-arginine, as well as nitric oxide are low in patients infected with P. falciparum malaria. The present study investigates, in vitro, the role of P. falciparum haemozoin on nitric oxide production, iNOS expression in macrophages, and the possible interaction between L-arginine and haemozoin. METHODS: Plasmodium falciparum haemozoin was obtained from in vitro cultures through magnetic isolation. Phagocytosis of haemozoin by immortalized bone marrow derived macrophages was detected by confocal reflection combined with fluorescence microscopy. Nitrite concentrations in the supernatants was evaluated by Griess assay as a standard indication of nitric oxide production, while iNOS expression was detected on cell extracts by western blotting. Detection of L-arginine in haemozoin-treated or untreated media was achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Haemozoin synergizes in vitro with interferon-gamma to produce nitric oxide. However, when mouse macrophages were stimulated with haemozoin, a proportional increase of nitric oxide was observed up to 25 µM of haemozoin, followed by a decrease with doses up to 100 µM, when nitric oxide release was completely abrogated. This was not due to reactive oxygen species production, nor to an effect on iNOS activity. Interestingly, when at 24 h, haemozoin-treated macrophages were washed and incubated in fresh medium for further 24 h, the nitric oxide production was restored in a dose-response manner. Similar results were seen when L-arginine-enriched media was used in the stimulation. Moreover, muramyldipeptide, a strong nitric oxide inducer, was unable to activate macrophages to release nitric oxide in the presence of haemozoin-treated medium. By LC-MS/MS a complete depletion of L-arginine was observed in this haemozoin-treated, conditioned medium. CONCLUSIONS: It is proposed that haemozoin interacts with L-arginine reducing its availability for iNOS, and thus decreasing nitric oxide production. The clinical (or pathological) implications of these results are discussed.

Fe(III) Protoporphyrin IX Encapsulated in a Zinc Metal-Organic Framework Shows Dramatically Enhanced Peroxidatic Activity.

Two MOFs, [H2N(CH3)2][Zn3(TATB)2(HCOO)]·HN(CH3)2·DMF·6H2O (1) and Zn-HKUST-1 (2), were investigated as potential hosts to encapsulate Fe(III) heme (Fe(III) protoporphyrin IX = Fe(III)PPIX). Methyl orange (MO) adsorption was used as an initial model for substrate uptake. MOF 1 showed good adsorption of MO (10.3 ± 0.8 mg g-1) which could undergo in situ protonation upon exposure to aqueous HCl vapor. By contrast, MO uptake by 2 was much lower (2 ± 1 mg g-1), and PXRD indicated that structural instability on exposure to water was the likely cause. Two methods for Fe(III)PPIX-1 preparation were investigated: soaking and encapsulation. Encapsulation was verified by SEM-EDS and showed comparable concentrations of Fe(III)PPIX on exposed interior surfaces and on the original surface of fractured crystals. SEM-EDS results were consistent with ICP-OES data on bulk material (1.2 ± 0.1 mass % Fe). PXRD data showed that the framework in 1 was unchanged after encapsulation of Fe(III)PPIX. MO adsorption (5.8 ± 1.2 mg g-1) by Fe(III)PPIX-1 confirmed there is space for substrate diffusion into the framework, while the UV-vis spectrum of solubilized crystals confirmed that Fe(III)PPIX retained its integrity. A solid-state UV-vis spectrum of Fe(III)PPIX-1 indicated that Fe(III)PPIX was not in a µ-oxo dimeric form. Although single-crystal XRD data did not allow for full refinement of the encapsulated Fe(III)PPIX molecule owing to disorder of the metalloporphyrin, the Fe atom and pyrrole N atoms were located, enabling rigid-body modeling of the porphine core. Reaction of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) with H2O2, catalyzed by Fe(III)PPIX-1 and -2, showed that Fe(III)PPIX-1 is significantly more efficient than Fe(III)PPIX-2 and is superior to solid Fe(III)PPIX-Cl. Fe(III)PPIX-1 was used to catalyze the oxidation of hydroquinone, thymol, benzyl alcohol, and phenyl ethanol by tert-butyl-hydroperoxide with t1/2 values that increase with increasing substrate molecular volume.

Shining new light on ancient drugs: preparation and subcellular localisation of novel fluorescent analogues of Cinchona alkaloids in intraerythrocytic Plasmodium falciparum.

Fluorescent derivatives of the archetypal antimalarial quinine and its diastereomer, quinidine, suitable for cellular imaging have been synthesised by attaching the small extrinsic fluorophore, NBD. Interactions of these derivatives with ferriprotoporphyrin IX were evaluated to verify that insights generated by live-cell imaging were relevant to the parent molecules. These analogues are shown by confocal and super-resolution microscopy to accumulate selectively in Plasmodium falciparum. Localisation to the region corresponding to the digestive vacuole supports the putative primary role of these alkaloids as haemozoin inhibitors. Quantitative analysis revealed minimal accumulation within the nucleus, rejecting the disruption of DNA replication as a possible mode of action. While extensive localisation to phospholipid structures and associated organelles was observed, the analogues did not show evidence of association with neutral lipid bodies.

Identification and deconvolution of cross-resistance signals from antimalarial compounds using multidrug-resistant Plasmodium falciparum strains.

Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs.

Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials.

BACKGROUND: The activity of several well-known anti-malarials, including chloroquine (CQ), is attributed to their ability to inhibit the formation of haemozoin (Hz) in the malaria parasite. The formation of inert Hz, or malaria pigment, from toxic haem acquired from the host red blood cell of the parasite during haemoglobin digestion represents a pathway essential for parasite survival. Inhibition of this critical pathway therefore remains a desirable target for novel anti-malarials. A recent publication described the results of a haem fractionation assay used to directly determine haemoglobin, free haem and Hz in Plasmodium falciparum inoculated with CQ. CQ was shown to cause a dose-dependent increase in cellular-free haem that was correlated with decreased parasite survival. The method provided valuable information but was limited due to its low throughput and high demand on parasite starting material. Here, this haem fractionation assay has been successfully adapted to a higher throughput method in 24-well plates, significantly reducing lead times and starting material volumes. METHODS: All major haem species in P. falciparum trophozoites, isolated through a series of cellular fractionation steps were determined spectrophotometrically in aqueous pyridine (5 % v/v, pH 7.5) as a low spin complex with haematin. Cell counts were determined using a haemocytometer and a rapid novel fluorescent flow cytometry method. RESULTS: A higher throughput haem fractionation assay in 24-well plates, containing at most ten million trophozoites was validated against the original published method using CQ and its robustness was confirmed. It provided a minimum six-fold improvement in productivity and 24-fold reduction in starting material volume. The assay was successfully applied to amodiaquine (AQ), which was shown to inhibit Hz formation, while the antifolate pyrimethamine (PYR) and the mitochondrial electron transporter inhibitor atovaquone (Atov) demonstrated no increase in toxic cellular free haem. CONCLUSIONS: This higher throughput cellular haem fractionation assay can easily be applied to novel anti-malarials with a significantly decreased lead time, providing a valuable tool with which to probe the mechanisms of action of both new and established anti-malarials.

Antiplasmodial activity, in vivo pharmacokinetics and anti-malarial efficacy evaluation of hydroxypyridinone hybrids in a mouse model.

BACKGROUND: During the erythrocytic stage in humans, malaria parasites digest haemoglobin of the host cell, and the toxic haem moiety crystallizes into haemozoin. Chloroquine acts by forming toxic complexes with haem molecules and interfering with their crystallization. In chloroquine-resistant strains, the drug is excluded from the site of action, which causes the parasites to accumulate less chloroquine in their acid food vacuoles than chloroquine-sensitive parasites. 3-Hydroxylpyridin-4-ones are known to chelate iron; hydroxypyridone-chloroquine (HPO-CQ) hybrids were synthesized in order to determine whether they can inhibit parasites proliferation in the parasitic digestive vacuole by withholding iron from plasmodial parasite metabolic pathway. METHODS: Two HPO-CQ hybrids were tested against Plasmodium falciparum chloroquine-sensitive (D10 and 3D7) and -resistant strains (Dd2 and K1). The pharmacokinetic properties of active compounds were determined using a mouse model and blood samples were collected at different time intervals and analysed using LC-MS/MS. For in vivo efficacy the mice were infected with Plasmodium berghei in a 4-day Peters' test. The parasitaemia was determined from day 3 and the course of the infection was followed by microscopic examination of stained blood films every 2-3 days until a rise in parasitaemia was observed in all test subjects. RESULTS: IC50 values of the two compounds for sensitive and resistant strains were 0.064 and 0.047 µM (compound 1), 0.041 and 0.122 µM (compound 2) and 0.505 and 0.463 µM (compound 1), 0.089 and 0.076 µM (compound 2), respectively. Pharmacokinetic evaluation of these compounds showed low oral bioavailability and this affected in vivo efficacy when compounds were dosed orally. However, when dosed intravenously compound 1 showed a clearance rate of 28 ml/min/kg, an apparent volume of distribution of 20 l/kg and a half-life of 4.3 h. A reduction in parasitaemia was observed when compound 1 was dosed intravenously for four consecutive days in P. berghei-infected mice. However, a rise in parasitaemia levels was observed on day 6 and on day 9 for chloroquine-treated mice. CONCLUSION: The hybrid compounds that were tested were able to reduce parasitaemia levels in P. berghei-infected mice when dosed intravenously, but parasites recrudesced 24 h after the administration of the least dose. Despite low oral bioavailability, the IV data obtained suggests that further structural modifications may lead to the identification of more HPO-CQ hybrids with improved pharmacokinetic properties and in vivo efficacy.

Bayesian models trained with HTS data for predicting ß-haematin inhibition and in vitro antimalarial activity.

A large quantity of high throughput screening (HTS) data for antimalarial activity has become available in recent years. This includes both phenotypic and target-based activity. Realising the maximum value of these data remains a challenge. In this respect, methods that allow such data to be used for virtual screening maximise efficiency and reduce costs. In this study both in vitro antimalarial activity and inhibitory data for ß-haematin formation, largely obtained from publically available sources, has been used to develop Bayesian models for inhibitors of ß-haematin formation and in vitro antimalarial activity. These models were used to screen two in silico compound libraries. In the first, the 1510 U.S. Food and Drug Administration approved drugs available on PubChem were ranked from highest to lowest Bayesian score based on a training set of ß-haematin inhibiting compounds active against Plasmodium falciparum that did not include any of the clinical antimalarials or close analogues. The six known clinical antimalarials that inhibit ß-haematin formation were ranked in the top 2.1% of compounds. Furthermore, the in vitro antimalarial hit-rate for this prioritised set of compounds was found to be 81% in the case of the subset where activity data are available in PubChem. In the second, a library of about 5000 commercially available compounds (Aldrich(CPR)) was virtually screened for ability to inhibit ß-haematin formation and then for in vitro antimalarial activity. A selection of 34 compounds was purchased and tested, of which 24 were predicted to be ß-haematin inhibitors. The hit rate for inhibition of ß-haematin formation was found to be 25% and a third of these were active against P. falciparum, corresponding to enrichments estimated at about 25- and 140-fold relative to random screening, respectively.

Antimalarial benzoheterocyclic 4-aminoquinolines: Structure-activity relationship, in vivo evaluation, mechanistic and bioactivation studies.

A novel class of benzoheterocyclic analogues of amodiaquine designed to avoid toxic reactive metabolite formation was synthesized and evaluated for antiplasmodial activity against K1 (multidrug resistant) and NF54 (sensitive) strains of the malaria parasite Plasmodium falciparum. Structure-activity relationship studies led to the identification of highly promising analogues, the most potent of which had IC50s in the nanomolar range against both strains. The compounds further demonstrated good in vitro microsomal metabolic stability while those subjected to in vivo pharmacokinetic studies had desirable pharmacokinetic profiles. In vivo antimalarial efficacy in Plasmodium berghei infected mice was evaluated for four compounds, all of which showed good activity following oral administration. In particular, compound 19 completely cured treated mice at a low multiple dose of 4×10mg/kg. Mechanistic and bioactivation studies suggest hemozoin formation inhibition and a low likelihood of forming quinone-imine reactive metabolites, respectively.

N10,N11-di-alkylamine indolo[3,2-b]quinolines as hemozoin inhibitors: design, synthesis and antiplasmodial activity.

We recently reported that potent N10,O11-bis-alkylamine indolo[3,2-b]quinoline antimalarials act as hemozoin (Hz) growth inhibitors. To improve access and binding to the target we have now designed novel N10,N11-di-alkylamine bioisosteres. 3-Chloro derivatives (10a-f) showed selectivity for malaria parasite compared to human cells, high activity against Plasmodium falciparum chloroquine (CQ)-resistant strain W2 (IC50s between 20 and 158nM), good correlation with ß-hematin inhibition and improved vacuolar accumulation ratios, thus suggesting inhibition of Hz growth as one possible mechanism of action for these compounds. Moreover, our studies show that Hz is a valid target for the development of new antimalarials able to overcome CQ resistance.

Interrogating alkyl and arylalkylpolyamino (bis)urea and (bis)thiourea isosteres as potent antimalarial chemotypes against multiple lifecycle forms of Plasmodium falciparum parasites.

A new series of potent potent aryl/alkylated (bis)urea- and (bis)thiourea polyamine analogues were synthesized and evaluated in vitro for their antiplasmodial activity. Altering the carbon backbone and terminal substituents increased the potency of analogues in the compound library 3-fold, with the most active compounds, 15 and 16, showing half-maximal inhibitory concentrations (IC50 values) of 28 and 30 nM, respectively, against various Plasmodium falciparum parasite strains without any cross-resistance. In vitro evaluation of the cytotoxicity of these analogues revealed marked selectivity towards targeting malaria parasites compared to mammalian HepG2 cells (>5000-fold lower IC50 against the parasite). Preliminary biological evaluation of the polyamine analogue antiplasmodial phenotype revealed that (bis)urea compounds target parasite asexual proliferation, whereas (bis)thiourea compounds of the same series have the unique ability to block transmissible gametocyte forms of the parasite, indicating pluripharmacology against proliferative and non-proliferative forms of the parasite. In this manuscript, we describe these results and postulate a refined structure-activity relationship (SAR) model for antiplasmodial polyamine analogues. The terminally aryl/alkylated (bis)urea- and (bis)thiourea-polyamine analogues featuring a 3-5-3 or 3-6-3 carbon backbone represent a structurally novel and distinct class of potential antiplasmodials with activities in the low nanomolar range, and high selectivity against various lifecycle forms of P. falciparum parasites.

Kojic acid derived hydroxypyridinone-chloroquine hybrids: synthesis, crystal structure, antiplasmodial activity and ß-haematin inhibition.

Aminochloroquinoline-kojic acid hybrids were synthesized and evaluated for ß-haematin inhibition and antiplasmodial activity against drug resistant (K1) and sensitive (3D7) strains of Plasmodium falciparum. Compound 7j was the most potent compound in both strains (IC50(3D7)=0.004 µM; IC50(K1)=0.03 µM) and had the best ß-haematin inhibition activity (0.07 IC50 equiv vs 1.91 IC50 equiv for chloroquine). One compound 8c was found to be equipotent in both strains (IC50=0.04 µM).