Identification and Profiling of a Novel Diazaspiro[3.4]octane Chemical Series Active against Multiple Stages of the Human Malaria Parasite Plasmodium falciparum and Optimization Efforts.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

Structure-Activity Relationship Studies and Plasmodium Life Cycle Profiling Identifies Pan-Active N-Aryl-3-trifluoromethyl Pyrido[1,2- a]benzimidazoles Which Are Efficacious in an in Vivo Mouse Model of Malaria.

Structure-activity relationship studies involving N-aryl-3-trifluoromethyl pyrido[1,2- a]benzimidazoles (PBI) identified several compounds possessing potent in vitro activities against the asexual blood, liver, and gametocyte stages of the Plasmodium parasite with no cross-resistance to chloroquine. Frontrunner lead compounds with good in vitro absorption, distribution, metabolism, and excretion (ADME) profiles were subjected to in vivo proof-of-concept studies in NMRI mice harboring the rodent P. berghei infection. This led to the identification of compounds 10 and 49, effecting 98% and 99.93% reduction in parasitemia with mean survival days of 12 and 14, respectively, at an oral dose of 4 × 50 mg/kg. In vivo pharmacokinetics studies on 10 revealed slow absorption, low volume of distribution, and low clearance profiles. Furthermore, this series displayed a low propensity to inhibit the human ether-a-go-go-related gene (hERG) potassium ion channel whose inhibition is associated with cardiotoxicity.

Multistage Antiplasmodium Activity of Astemizole Analogues and Inhibition of Hemozoin Formation as a Contributor to Their Mode of Action.

A drug repositioning approach was leveraged to derivatize astemizole (AST), an antihistamine drug whose antimalarial activity was previously identified in a high-throughput screen. The multistage activity potential against the Plasmodium parasite's life cycle of the subsequent analogues was examined by evaluating against the parasite asexual blood, liver, and sexual gametocytic stages. In addition, the previously reported contribution of heme detoxification to the compound's mode of action was interrogated. Ten of the 17 derivatives showed half-maximal inhibitory concentrations (IC50s) of <0.1 µM against the chloroquine (CQ)-sensitive Plasmodium falciparum NF54 ( PfNF54) strain while maintaining submicromolar potency against the multidrug-resistant strain, PfK1, with most showing low likelihood of cross-resistance with CQ. Selected analogues ( PfNF54-IC50 < 0.1 µM) were tested for cytotoxicity on Chinese hamster ovarian (CHO) cells and found to be highly selective (selectivity index > 100). Screening of AST and its analogues against gametocytes revealed their moderate activity (IC50: 1-5 µM) against late stage P. falciparum gametocytes, while the evaluation of activity against P. berghei liver stages identified one compound (3) with 3-fold greater activity than the parent AST compound. Mechanistic studies showed a strong correlation between in vitro inhibition of ß-hematin formation by the AST derivatives and their antiplasmodium IC50s. Analyses of intracellular inhibition of hemozoin formation within the parasite further yielded signatures attributable to a possible perturbation of the heme detoxification machinery.

Potent Plasmodium falciparum gametocytocidal compounds identified by exploring the kinase inhibitor chemical space for dual active antimalarials.

Objectives: Novel chemical tools to eliminate malaria should ideally target both the asexual parasites and transmissible gametocytes. Several imidazopyridazines (IMPs) and 2-aminopyridines (2-APs) have been described as potent antimalarial candidates targeting lipid kinases. However, these have not been extensively explored for stage-specific inhibition of gametocytes in Plasmodium falciparum parasites. Here we provide an in-depth evaluation of the gametocytocidal activity of compounds from these chemotypes and identify novel starting points for dual-acting antimalarials. Methods: We evaluated compounds against P. falciparum gametocytes using several assay platforms for cross-validation and stringently identified hits that were further profiled for stage specificity, speed of action and ex vivo efficacy. Physicochemical feature extraction and chemogenomic fingerprinting were applied to explore the kinase inhibition susceptibility profile. Results: We identified 34 compounds with submicromolar activity against late stage gametocytes, validated across several assay platforms. Of these, 12 were potent at <100 nM (8 were IMPs and 4 were 2-APs) and were also active against early stage gametocytes and asexual parasites, with >1000-fold selectivity towards the parasite over mammalian cells. Front-runner compounds targeted mature gametocytes within 48 h and blocked transmission to mosquitoes. The resultant chemogenomic fingerprint of parasites treated with the lead compounds revealed the importance of targeting kinases in asexual parasites and gametocytes. Conclusions: This study encompasses an in-depth evaluation of the kinase inhibitor space for gametocytocidal activity. Potent lead compounds have enticing dual activities and highlight the importance of targeting the kinase superfamily in malaria elimination strategies.

Antimalarial Pyrido[1,2-a]benzimidazoles: Lead Optimization, Parasite Life Cycle Stage Profile, Mechanistic Evaluation, Killing Kinetics, and in Vivo Oral Efficacy in a Mouse Model.

Further structure-activity relationship (SAR) studies on the recently identified pyrido[1,2-a]benzimidazole (PBI) antimalarials have led to the identification of potent, metabolically stable compounds with improved in vivo oral efficacy in the P. berghei mouse model and additional activity against parasite liver and gametocyte stages, making them potential candidates for preclinical development. Inhibition of hemozoin formation possibly contributes to the mechanism of action.

A Novel Pyrazolopyridine with in Vivo Activity in Plasmodium berghei- and Plasmodium falciparum-Infected Mouse Models from Structure-Activity Relationship Studies around the Core of Recently Identified Antimalarial Imidazopyridazines.

Toward improving pharmacokinetics, in vivo efficacy, and selectivity over hERG, structure-activity relationship studies around the central core of antimalarial imidazopyridazines were conducted. This study led to the identification of potent pyrazolopyridines, which showed good in vivo efficacy and pharmacokinetics profiles. The lead compounds also proved to be very potent in the parasite liver and gametocyte stages, which makes them of high interest.

Nowhere to hide: interrogating different metabolic parameters of Plasmodium falciparum gametocytes in a transmission blocking drug discovery pipeline towards malaria elimination.

BACKGROUND: The discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions. METHODS: Gametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable. RESULTS: A highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4%. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident. CONCLUSIONS: With this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broad-diversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.

Receptor-based identification of an inhibitory peptide against blood stage malaria.

Plasmodium falciparum uses multiple host receptors to attach and invade human erythrocytes. Glycophorins have been implicated as receptors for parasite invasion in human erythrocytes. Here, we screened a phage display cDNA library of P. falciparum (FCR3, a sialic acid-dependent strain) using purified glycophorins and erythrocytes as bait. Several phage clones were identified that bound to immobilized glycophorins and contained the same 74 bp insert encoding the 7-amino acids sequence ETTLKSF. A similar screen using intact human erythrocytes in solution identified additional phage clones containing the same 7-amino acids sequence. Using ELISA and immunofluorescence, direct binding of ETTLKSF peptide to glycophorins and erythrocytes was confirmed. Pull-down and protease treatment assays suggest that ETTLKSF peptide specifically interacts with glycophorin C. The synthetic ETTLKSF peptide partially blocks merozoite invasion in human erythrocytes. Further characterization of ETTLKSF peptide could lead to the development of a novel class of inhibitors against the blood stage malaria.

The M18 aspartyl aminopeptidase of Plasmodium falciparum binds to human erythrocyte spectrin in vitro.

BACKGROUND: During erythrocytic schizogony, Plasmodium falciparum interacts with the human erythrocyte membrane when it enters into, grows within and escapes from the erythrocyte. An interaction between the P. falciparum M18 aspartyl aminopeptidase (PfM18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) PfM18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed. METHODS: rPfM18AAP was produced as a hexahistidine-fusion protein in Escherichia coli and purified using magnetic bead technology. The pI of the enzyme was determined by two-dimensional gel electrophoresis and the number of subunits in the native enzyme was estimated from Ferguson plots. The enzymatic activity over a pH and temperature range was tested by a coupled enzyme assay. Blot overlays were performed to validate the spectrin-PfM18AAP interaction, as well as identify additional interactions between the enzyme and other erythrocyte membrane proteins. Sequence analysis identified conserved amino acids that are expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization. RESULTS: rPfM18AAP has a molecular weight of ~67 kDa and the enzyme separated as three entities with pI 6.6, 6.7 and 6.9. Non-denaturing gel electrophoresis indicated that rPfM18AAP aggregated into oligomers. An in vitro coupled enzyme assay showed that rPfM18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37 degrees C. The spectrin-binding region of PfM18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and otherPlasmodium species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with rPfM18AAP against spectrin and erythrocyte membrane proteins indicated that rPfM18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase. CONCLUSION: Studies characterizing rPfM18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, PfM18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions.

BACKGROUND: The development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised. METHODS: P. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database. RESULTS: Following four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library. CONCLUSION: The construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.