Systematic Down-Selection of Repurposed Drug Candidates for COVID-19.

SARS-CoV-2 is the cause of the COVID-19 pandemic which has claimed more than 6.5 million lives worldwide, devastating the economy and overwhelming healthcare systems globally. The development of new drug molecules and vaccines has played a critical role in managing the pandemic; however, new variants of concern still pose a significant threat as the current vaccines cannot prevent all infections. This situation calls for the collaboration of biomedical scientists and healthcare workers across the world. Repurposing approved drugs is an effective way of fast-tracking new treatments for recently emerged diseases. To this end, we have assembled and curated a database consisting of 7817 compounds from the Compounds Australia Open Drug collection. We developed a set of eight filters based on indicators of efficacy and safety that were applied sequentially to down-select drugs that showed promise for drug repurposing efforts against SARS-CoV-2. Considerable effort was made to evaluate approximately 14,000 assay data points for SARS-CoV-2 FDA/TGA-approved drugs and provide an average activity score for 3539 compounds. The filtering process identified 12 FDA-approved molecules with established safety profiles that have plausible mechanisms for treating COVID-19 disease. The methodology developed in our study provides a template for prioritising drug candidates that can be repurposed for the safe, efficacious, and cost-effective treatment of COVID-19, long COVID, or any other future disease. We present our database in an easy-to-use interactive interface (CoviRx that was also developed to enable the scientific community to access to the data of over 7000 potential drugs and to implement alternative prioritisation and down-selection strategies.

Probing the correlation between insulin activity and structural stability through introduction of the rigid A6-A11 bond.

The development of fast-acting and highly stable insulin analogues is challenging. Insulin undergoes structural transitions essential for binding and activation of the insulin receptor (IR), but these conformational changes can also affect insulin stability. Previously, we substituted the insulin A6-A11 cystine with a rigid, non-reducible C=C linkage ("dicarba" linkage). A cis-alkene permitted the conformational flexibility of the A-chain N-terminal helix necessary for high-affinity IR binding, resulting in surprisingly rapid activity in vivo Here, we show that, unlike the rapidly acting LysB28ProB29 insulin analogue (KP insulin), cis-dicarba insulin is not inherently monomeric. We also show that cis-dicarba KP insulin lowers blood glucose levels even more rapidly than KP insulin, suggesting that an inability to oligomerize is not responsible for the observed rapid activity onset of cis-dicarba analogues. Although rapid-acting, neither dicarba species is stable, as assessed by fibrillation and thermodynamics assays. MALDI analyses and molecular dynamics simulations of cis-dicarba insulin revealed a previously unidentified role of the A6-A11 linkage in insulin conformational dynamics. By controlling the conformational flexibility of the insulin B-chain helix, this linkage affects overall insulin structural stability. This effect is independent of its regulation of the A-chain N-terminal helix flexibility necessary for IR engagement. We conclude that high-affinity IR binding, rapid in vivo activity, and insulin stability can be regulated by the specific conformational arrangement of the A6-A11 linkage. This detailed understanding of insulin's structural dynamics may aid in the future design of rapid-acting insulin analogues with improved stability.

Modulation of the aggregation of an amyloidogenic sequence by flanking-disordered region in the intrinsically disordered antigen merozoite surface protein 2.

The abundant Plasmodium falciparum merozoite surface protein MSP2, a potential malaria vaccine candidate, is an intrinsically disordered protein with some nascent secondary structure present in its conserved N-terminal region. This relatively ordered region has been implicated in both membrane interactions and amyloid-like aggregation of the protein, while the significance of the flanking-disordered region is unclear. In this study, we show that aggregation of the N-terminal conserved region of MSP2 is influenced in a length- and sequence-dependent fashion by the disordered central variable sequences. Intriguingly, MSP2 peptides containing the conserved region and the first five residues of the variable disordered regions aggregated more rapidly than a peptide corresponding to the conserved region alone. In contrast, MSP2 peptides extending 8 or 12 residues into the disordered region aggregated more slowly, consistent with the expected inhibitory effect of flanking-disordered sequences on the aggregation of amyloidogenic ordered sequences. Computational analyses indicated that the helical propensity of the ordered region of MSP2 was modulated by the adjacent disordered five residues in a sequence-dependent manner. Nuclear magnetic resonance and circular dichroism spectroscopic studies with synthetic peptides confirmed the computational predictions, emphasizing the correlation between aggregation propensity and conformation of the ordered region and the effects thereon of the adjacent disordered region. These results show that the effects of flanking-disordered sequences on a more ordered sequence may include enhancement of aggregation through modulation of the conformational properties of the more ordered sequence.

The Single Disulfide-Directed ß-Hairpin Fold. Dynamics, Stability, and Engineering.

Grafting bioactive peptide sequences onto small cysteine-rich scaffolds is a promising strategy for enhancing their stability and value as novel peptide-based therapeutics. However, correctly folded disulfide-rich peptides can be challenging to produce by either recombinant or synthetic means. The single disulfide-directed ß-hairpin (SDH) fold, first observed in contryphan-Vc1, provides a potential alternative to complex disulfide-rich scaffolds. We have undertaken recombinant production of full-length contryphan-Vc1 (rCon-Vc1[Z1Q]) and a truncated analogue (rCon-Vc11-22[Z1Q]), analyzed the backbone dynamics of rCon-Vc1[Z1Q], and probed the conformational and proteolytic stability of these peptides to evaluate the potential of contryphan-Vc1 as a molecular scaffold. Backbone 15N relaxation measurements for rCon-Vc1[Z1Q] indicate that the N-terminal domain of the peptide is ordered up to Thr19, whereas the remainder of the C-terminal region is highly flexible. The solution structure of truncated rCon-Vc11-22[Z1Q] was similar to that of the full-length peptide, indicating that the flexible C-terminus does not have any effect on the structured domain of the peptide. Contryphan-Vc1 exhibited excellent proteolytic stability against trypsin and chymotrypsin but was susceptible to pepsin digestion. We have investigated whether contryphan-Vc1 can accept a bioactive epitope while maintaining the structure of the peptide by introducing peptide sequences based on the DINNN motif of inducible nitric oxide synthase. We show that sCon-Vc11-22[NNN12-14] binds to the iNOS-binding protein SPSB2 with an affinity of 1.3 µM while maintaining the SDH fold. This study serves as a starting point in utilizing the SDH fold as a peptide scaffold.

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.

Applications of (19)F-NMR in Fragment-Based Drug Discovery.

(19)F-NMR has proved to be a valuable tool in fragment-based drug discovery. Its applications include screening libraries of fluorinated fragments, assessing competition among elaborated fragments and identifying the binding poses of promising hits. By observing fluorine in both the ligand and the target protein, useful information can be obtained on not only the binding pose but also the dynamics of ligand-protein interactions. These applications of (19)F-NMR will be illustrated in this review with studies from our fragment-based drug discovery campaigns against protein targets in parasitic and infectious diseases.

Structure and dynamics of apical membrane antigen 1 from Plasmodium falciparum FVO.

Apical membrane antigen 1 (AMA1) interacts with RON2 to form a protein complex that plays a key role in the invasion of host cells by malaria parasites. Blocking this protein-protein interaction represents a potential route to controlling malaria and related parasitic diseases, but the polymorphic nature of AMA1 has proven to be a major challenge to vaccine-induced antibodies and peptide inhibitors exerting strain-transcending inhibitory effects. Here we present the X-ray crystal structure of AMA1 domains I and II from Plasmodium falciparum strain FVO. We compare our new structure to those of AMA1 from P. falciparum 3D7 and Plasmodium vivax. A combination of normalized B factor analysis and computational methods has been used to investigate the flexibility of the domain I loops and how this correlates with their roles in determining the strain specificity of human antibody responses and inhibitory peptides. We also investigated the domain II loop, a key region involved in inhibitor binding, by comparison of multiple AMA1 crystal structures. Collectively, these results provide valuable insights that should contribute to the design of strain-transcending agents targeting P. falciparum AMA1.

RASP: rapid and robust backbone chemical shift assignments from protein structure.

Chemical shift prediction has an unappreciated power to guide backbone resonance assignment in cases where protein structure is known. Here we describe Resonance Assignment by chemical Shift Prediction (RASP), a method that exploits this power to derive protein backbone resonance assignments from chemical shift predictions. Robust assignments can be obtained from a minimal set of only the most sensitive triple-resonance experiments, even for spectroscopically challenging proteins. Over a test set of 154 proteins RASP assigns 88 % of residues with an accuracy of 99.7 %, using only information available from HNCO and HNCA spectra. Applied to experimental data from a challenging 34 kDa protein, RASP assigns 90 % of manually assigned residues using only 40 % of the experimental data required for the manual assignment. RASP has the potential to significantly accelerate the backbone assignment process for a wide range of proteins for which structural information is available, including those for which conventional assignment strategies are not feasible.

Lipid interactions of the malaria antigen merozoite surface protein 2.

With more than half the world's population living at risk of malaria infection, there is a strong demand for the development of an effective malaria vaccine. One promising vaccine candidate is merozoite surface protein 2 (MSP2), which is among the most abundant antigens of the blood stage of the Plasmodium falciparum parasite. In solution, MSP2 is intrinsically unstructured, but little is known about the conformation of native MSP2, which is GPI-anchored to the merozoite surface, or of the implications of that conformation for the immune response induced by MSP2. Initial NMR studies have shown that MSP2 interacts with lipid micelles through a highly conserved N-terminal domain. We have further developed these findings by investigating how different lipid environments affect the protein structure. All of the tested lipid preparations perturbed only the N-terminal part of MSP2. In DPC micelles this region adopts an α-helical structure which we have characterized in detail. Our findings suggest a possible mechanism by which lipid interactions might modulate immune recognition of the conserved N-terminus of MSP2, potentially explaining the apparent immunodominance of the central variable region of this important malaria antigen.

Antigenic characterization of an intrinsically unstructured protein, Plasmodium falciparum merozoite surface protein 2.

Merozoite surface protein 2 (MSP2) is an abundant glycosylphosphatidylinositol (GPI)-anchored protein of Plasmodium falciparum, which is a potential component of a malaria vaccine. As all forms of MSP2 can be categorized into two allelic families, a vaccine containing two representative forms of MSP2 may overcome the problem of diversity in this highly polymorphic protein. Monomeric recombinant MSP2 is an intrinsically unstructured protein, but its conformational properties on the merozoite surface are unknown. This question is addressed here by analyzing the 3D7 and FC27 forms of recombinant and parasite MSP2 using a panel of monoclonal antibodies raised against recombinant MSP2. The epitopes of all antibodies, mapped using both a peptide array and by nuclear magnetic resonance (NMR) spectroscopy on full-length recombinant MSP2, were shown to be linear. The antibodies revealed antigenic differences, which indicate that the conserved N- and C-terminal regions, but not the central variable region, are less accessible in the parasite antigen. This appears to be an intrinsic property of parasite MSP2 and is not dependent on interactions with other merozoite surface proteins as the loss of some conserved-region epitopes seen using the immunofluorescence assay (IFA) on parasite smears was also seen on Western blot analyses of parasite lysates. Further studies of the structural basis of these antigenic differences are required in order to optimize recombinant MSP2 constructs being evaluated as potential vaccine components.

A new mass spectral library for high-coverage and reproducible analysis of the Plasmodium falciparum-infected red blood cell proteome.

BACKGROUND: Plasmodium falciparum causes the majority of malaria mortality worldwide, and the disease occurs during the asexual red blood cell (RBC) stage of infection. In the absence of an effective and available vaccine, and with increasing drug resistance, asexual RBC stage parasites are an important research focus. In recent years, mass spectrometry-based proteomics using data-dependent acquisition has been extensively used to understand the biochemical processes within the parasite. However, data-dependent acquisition is problematic for the detection of low-abundance proteins and proteome coverage and has poor run-to-run reproducibility. RESULTS: Here, we present a comprehensive P. falciparum-infected RBC (iRBC) spectral library to measure the abundance of 44,449 peptides from 3,113 P. falciparum and 1,617 RBC proteins using a data-independent acquisition mass spectrometric approach. The spectral library includes proteins expressed in the 3 morphologically distinct RBC stages (ring, trophozoite, schizont), the RBC compartment of trophozoite-iRBCs, and the cytosolic fraction from uninfected RBCs. This spectral library contains 87% of all P. falciparum proteins that have previously been reported with protein-level evidence in blood stages, as well as 692 previously unidentified proteins. The P. falciparum spectral library was successfully applied to generate semi-quantitative proteomics datasets that characterize the 3 distinct asexual parasite stages in RBCs, and compared artemisinin-resistant (Cam3.IIR539T) and artemisinin-sensitive (Cam3.IIrev) parasites. CONCLUSION: A reproducible, high-coverage proteomics spectral library and analysis method has been generated for investigating sets of proteins expressed in the iRBC stage of P. falciparum malaria. This will provide a foundation for an improved understanding of parasite biology, pathogenesis, drug mechanisms, and vaccine candidate discovery for malaria.

Peroxide Antimalarial Drugs Target Redox Homeostasis in Plasmodium falciparum Infected Red Blood Cells.

Plasmodium falciparum causes the most lethal form of malaria. Peroxide antimalarials based on artemisinin underpin the frontline treatments for malaria, but artemisinin resistance is rapidly spreading. Synthetic peroxide antimalarials, known as ozonides, are in clinical development and offer a potential alternative. Here, we used chemoproteomics to investigate the protein alkylation targets of artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, artefenomel. We greatly expanded the list of proteins alkylated by peroxide antimalarials and identified significant enrichment of redox-related proteins for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed by dynamic live imaging of the glutathione redox potential using a genetically encoded redox-sensitive fluorescence-based biosensor. Targeted liquid chromatography-mass spectrometry (LC-MS)-based thiol metabolomics also confirmed changes in cellular thiol levels. This work shows that peroxide antimalarials disproportionately alkylate proteins involved in redox homeostasis and that disrupted redox processes are involved in the mechanism of action of these important antimalarials.

Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway.

Plasmodium falciparum, the causative agent of malaria, remains a global health threat as parasites continue to develop resistance to antimalarial drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the host's main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here, we use both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that loss of PfA-M17 results in parasites exhibiting multiple digestive vacuoles at the trophozoite stage. In contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.

Malaria is a disease spread by mosquitoes. When infected insects bite the skin, they inject parasites called Plasmodium into the host. The symptoms of the disease then develop when Plasmodium infect host red blood cells. These parasites cannot make the raw materials to build their own proteins, so instead, they digest haemoglobin ­ the protein used by red blood cells to carry oxygen ­ and use its building blocks to produce proteins. Blocking the digestion of haemoglobin can stop malaria infections in their tracks, but it is unclear how exactly Plasmodium parasites break down the protein. Researchers think that a group of four enzymes called aminopeptidases are responsible for the final stage in this digestion, releasing the amino acids that make up haemoglobin. However, the individual roles of each of these aminopeptidases are not yet known. To start filling this gap, Edgar et al. set out to study one of these aminopeptidases, called PfA-M17. First, they genetically modified Plasmodium falciparum parasites so that the levels of this aminopeptidase were reduced during infection. Without the enzyme, the parasites were unable to grow. The next step was to confirm that this was because PfA-M17 breaks down haemoglobin, and not for another reason. To test this, Edgar et al. designed a new molecule that could stop PfA-M17 from releasing amino acids. This molecule, which they called 'compound 3', had the same effect as reducing the levels of PfA-M17. Further analysis showed that the amino acids that PfA- M17 releases match the amino acids found in haemoglobin. Malaria causes hundreds of thousands of deaths per year. Although there are treatments available, the Plasmodium parasites are starting to develop resistance. Confirming the role of PfA-M17 provides a starting point for new studies by parasitologists, biologists, and drug developers. This could lead to the development of chemicals that block this enzyme, forming the basis for new treatments.

Use of Human Lung Tissue Models for Screening of Drugs against SARS-CoV-2 Infection.

The repurposing of licenced drugs for use against COVID-19 is one of the most rapid ways to develop new and alternative therapeutic options to manage the ongoing pandemic. Given circa 7817 licenced compounds available from Compounds Australia that can be screened, this paper demonstrates the utility of commercially available ex vivo/3D airway and alveolar tissue models. These models are a closer representation of in vivo studies than in vitro models, but retain the benefits of rapid in vitro screening for drug efficacy. We demonstrate that several existing drugs appear to show anti-SARS-CoV-2 activity against both SARS-CoV-2 Delta and Omicron Variants of Concern in the airway model. In particular, fluvoxamine, as well as aprepitant, everolimus, and sirolimus, has virus reduction efficacy comparable to the current standard of care (remdesivir, molnupiravir, nirmatrelvir). Whilst these results are encouraging, further testing and efficacy studies are required before clinical use can be considered.

Interaction between Plasmodium falciparum apical membrane antigen 1 and the rhoptry neck protein complex defines a key step in the erythrocyte invasion process of malaria parasites.

Invasion of host cells by apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, is a multistep process. Central to invasion is the formation of a tight junction, an aperture in the host cell through which the parasite pulls itself before settling into a newly formed parasitophorous vacuole. Two protein groups, derived from different secretory organelles, the micronemal protein AMA1 and the rhoptry proteins RON2, RON4, and RON5, have been shown to form part of this structure, with antibodies targeting P. falciparum AMA1 known to inhibit invasion, probably via disruption of its association with the PfRON proteins. Inhibitory AMA1-binding peptides have also been described that block P. falciparum merozoite invasion of the erythrocyte. One of these, R1, blocks invasion some time after initial attachment to the erythrocyte and reorientation of the merozoite to its apical pole. Here we show that the R1 peptide binds the PfAMA1 hydrophobic trough and demonstrate that binding to this region prevents its interaction with the PfRON complex. We show that this defined association between PfAMA1 and the PfRON complex occurs after reorientation and engagement of the actomyosin motor and argue that it precedes rhoptry release. We propose that the formation of the AMA1-RON complex is essential for secretion of the rhoptry contents, which then allows the establishment of parasite infection within the parasitophorous vacuole.

EGCG disaggregates amyloid-like fibrils formed by Plasmodium falciparum merozoite surface protein 2.

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically unstructured and forms amyloid-like fibrils in solution. As this propensity of MSP2 to form fibrils in solution has the potential to impede its development as a vaccine candidate, finding an inhibitor that inhibits fibrillogenesis may enhance vaccine development. We have shown previously that EGCG inhibits the formation of MSP2 fibrils. Here we show that EGCG can alter the ß-sheet-like structure of the fibril and disaggregate pre-formed fibrils of MSP2 into soluble oligomers. The fibril remodelling effects of EGCG and other flavonoids were characterised using Thioflavin T fluorescence assays, electron microscopy and other biophysical methods.

Merozoite surface protein 2 adsorbed onto acetalated dextran microparticles for malaria vaccination.

Malaria remains a global health threat, with significant morbidity and mortality worldwide despite current interventions. The human disease is caused by five different parasitic species, with Plasmodium falciparum being the deadliest. As a result, vaccine research against P. falciparum is a global priority. Merozoite surface protein 2 (MSP2) is a promising vaccine antigen as MSP2-specific antibodies have been shown previously to be protective against malaria infection. In this study, the formulation of an MSP2 vaccine was explored to enhance antigen uptake and achieve both an antibody and Th1 immune response by adsorbing MSP2 antigen onto a biomaterial carrier system. Specifically, MSP2 antigen was adsorbed onto acetalated dextran (Ace-DEX) microparticles (MPs). IgG and IgG2a titers elicited by the Ace-DEX MP platform were compared to titer levels elicited by MSP2 adsorbed to an FDA-approved alum adjuvant, MSP2 alone, and PBS alone. Both adsorption of MSP2 to Ace-DEX MPs and to alum elicited antibody responses in vivo, but only the formulation containing Ace-DEX MPs was able to elicit a significant Th1-biased response needed to combat the intracellular pathogen. As such, MSP2 adsorbed to Ace-DEX MPs demonstrates promise as a malaria vaccine.

Guiding the Immune Response to a Conserved Epitope in MSP2, an Intrinsically Disordered Malaria Vaccine Candidate.

The malaria vaccine candidate merozoite surface protein 2 (MSP2) has shown promise in clinical trials and is in part responsible for a reduction in parasite densities. However, strain-specific reductions in parasitaemia suggested that polymorphic regions of MSP2 are immuno-dominant. One strategy to bypass the hurdle of strain-specificity is to bias the immune response towards the conserved regions. Two mouse monoclonal antibodies, 4D11 and 9H4, recognise the conserved C-terminal region of MSP2. Although they bind overlapping epitopes, 4D11 reacts more strongly with native MSP2, suggesting that its epitope is more accessible on the parasite surface. In this study, a structure-based vaccine design approach was applied to the intrinsically disordered antigen, MSP2, using a crystal structure of 4D11 Fv in complex with its minimal binding epitope. Molecular dynamics simulations and surface plasmon resonance informed the design of a series of constrained peptides that mimicked the 4D11-bound epitope structure. These peptides were conjugated to keyhole limpet hemocyanin and used to immunise mice, with high to moderate antibody titres being generated in all groups. The specificities of antibody responses revealed that a single point mutation can focus the antibody response towards a more favourable epitope. This structure-based approach to peptide vaccine design may be useful not only for MSP2-based malaria vaccines, but also for other intrinsically disordered antigens.

Liposome engraftment and antigen combination potentiate the immune response towards conserved epitopes of the malaria vaccine candidate MSP2.

Merozoite surface protein 2 (MSP2) is a highly abundant, GPI-anchored surface antigen on merozoites of the malaria parasite Plasmodium falciparum. It consists of highly conserved N- and C-terminal domains, and a central polymorphic region that allows all MSP2 alleles to be categorized into the 3D7 or FC27 family. Previously it has been shown that epitope accessibility differs between lipid-bound and lipid-free MSP2, suggesting that lipid interactions modulate the conformation and antigenicity in a way that may better mimic native MSP2 on the merozoite surface. Therefore, we have immunised mice with MSP2 engrafted onto liposomes using a C-terminal tether that mimics the native GPI anchor. To improve the immunogenicity of the formulated antigen, liposomes were supplemented with Pathogen Associated Molecular Pattern molecules, specifically agonists of the Toll-like receptor 4 (TLR4) or TLR2. Induced antibodies were directed mostly towards conserved epitopes, predominantly in the conserved C-terminal region of MSP2. We also found that immunisation with a combination of 3D7 and FC27 MSP2 enhanced antibody responses to conserved epitopes, and that the overall responses of mice immunised with MSP2-engrafted liposomes were comparable in magnitude to those of mice immunised with MSP2 formulated in Montanide ISA720. The antibodies elicited in mice by immunising with MSP2-engrafted liposomes recognised the native form of parasite MSP2 on western blots and were found to be cross-reactive with isolated 3D7 and FC27 merozoites when investigated by ELISA. The liposome-tethered MSP2 induced higher titres of complement-fixing antibodies to 3D7 and FC27 MSP2 than did MSP2 formulated in Montanide ISA720. Our results indicate that liposomal formulation represents a viable strategy for eliciting a strong immune response that favours conserved epitopes in MSP2 and thus a strain-transcendent immune response.

Discovery and development of 2-aminobenzimidazoles as potent antimalarials.

The emergence of Plasmodium falciparum resistance to frontline antimalarials, including artemisinin combination therapies, highlights the need for new molecules that act via novel mechanisms of action. Herein, we report the design, synthesis and antimalarial activity of a series of 2-aminobenzimidazoles, featuring a phenol moiety that is crucial to the pharmacophore. Two potent molecules exhibited IC50 values against P. falciparum 3D7 strain of 42 ± 4 (3c) and 43 ± 2 nM (3g), and high potency against strains resistant to chloroquine (Dd2), artemisinin (Cam3.IIC580Y) and PfATP4 inhibitors (SJ557733), while demonstrating no cytotoxicity against human cells (HEK293, IC50 > 50 µM). The most potent molecule, possessing a 4,5-dimethyl substituted phenol (3r) displayed an IC50 value of 6.4 ± 0.5 nM against P. falciparum 3D7, representing a 12-fold increase in activity from the parent molecule. The 2-aminobenzimidazoles containing a N1-substituted phenol represent a new class of molecules that have high potency in vitro against P. falciparum malaria and low cytotoxicity. They possessed attractive pharmaceutical properties, including low molecular weight, high ligand efficiency, high solubility, synthetic tractability and low in vitro clearance in human liver microsomes.