With resistance to current frontline antimalarial treatments rapidly emerging in malaria endemic regions, there is an urgent need to identify new antimalarial compounds with novel mechanisms of action. Currently, no clinically used antimalarials target erythrocyte invasion, the process by which the short-lived, extracellular merozoite form of Plasmodium falciparum parasites enters a host erythrocyte. Following invasion, the parasite grows and then divides to form a schizont, containing merozoites that exit the host cell and invade new erythrocytes. To identify novel egress and invasion inhibitors, we screened the Medicines for Malaria Venture COVID Box (160 compounds) and Global Health Priority Box (240 compounds). Parasites were treated with the compounds at 1 μM during the egress and invasion period and across both libraries, 20 invasion inhibitors and two partial egress inhibitors were identified. A comparison of the growth IC50 and invasion IC50 of each hit compound revealed that invasion inhibition is likely central to the parasiticidal mechanism of six of the compounds, as their IC50s for growth and invasion were similar. Of these six inhibitors, two (MMV006931 and MMV024850) were found to directly block the invasion of mechanically purified merozoites. We sought to identify the protein targets of these compounds through resistance selection and subsequent whole genome sequencing. We were able to select parasites with robust resistance to MMV006931 but not MMV024850. Genome sequencing of clonal MMV006931-resistant parasites identified mutations in the P. falciparum sterol exporter PfNCR1, which helps eliminate excess cholesterol from the parasite's plasma membrane. By demonstrating that MMV006931 sensitises parasites to lysis by a sterol-dependent detergent, we confirmed that PfNCR1 is the likely target of MMV006931.

New antimalarials are needed due to the threat of emerging resistance against existing antimalarial therapies. A phenotypic screen uncovered the N-aryl acetamide class that inhibits the development of P. falciparum asexual ring-stage parasites. The structure-activity relationship of this class was investigated, and key modifications were introduced that produced WEHI-326 with potent antimalarial activity. Enhancing the metabolic stability of this class will be a future challenge to achieve efficacy in a malaria mouse model. WEHI-326 was found to have a moderate barrier to resistance and a moderate rate of asexual kill, potently inhibited gametocyte and gamete development, and in turn, blocked the transmission of parasites to the mosquito. Forward genetics and cross-resistance profiling determined that parasites resistant to N-aryl acetamides had mutations in rhomboid protease 8 (ROM8) and the putative cation channel, CSC1. WEHI-326 will be an important tool in unraveling the role of ROM8 and CSC1 in P. falciparum development.

Plasmepsin V (PMV), an essential aspartyl protease, plays a critical role during the asexual blood stage of infection of Plasmodium by enabling the export of parasite proteins into the host red blood cell. This export is vital for parasite survival and pathogenesis, making PMV an attractive target for antimalarial drug development. Peptidomimetic inhibitors designed to mimic the natural substrate of PMV have demonstrated potent parasite-killing activity by blocking protein export. While these compounds have been instrumental in validating PMV as a bona fide antimalarial target, inconsistencies between their biochemical potency and cellular activity have raised questions regarding their precise mechanism of action. In this study, we employed chemoproteomic approaches, including solvent-induced protein precipitation and intact-cell thermal profiling, to demonstrate PMV target engagement by the peptidomimetics. To further support these findings, we generated parasite lines exhibiting reduced sensitivity to peptidomimetics. Through whole-genome sequencing of these parasite lines, a single nucleotide variant within the pmv gene was revealed. This mutation was later validated using reverse genetics, confirming its role in mediating resistance. Together, these data provide strong evidence that the peptidomimetics exert their antimalarial activity by directly targeting PMV. These findings further support the potential of PMV as a validated and promising target for future antimalarial drug development.

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.

Apicomplexan parasites that reside within a parasitophorous vacuole harbor a conserved pore-forming protein that enables small-molecule transfer across the parasitophorous vacuole membrane (PVM). In Plasmodium parasites that cause malaria, this nutrient pore is formed by EXP2 which can complement the function of GRA17, an orthologous protein in Toxoplasma gondii. EXP2, however, has an additional function in Plasmodium parasites, serving also as the pore-forming component of the protein export machinery PTEX. To examine how EXP2 can play this additional role, transgenes that encoded truncations of EXP2, GRA17, hybrid GRA17-EXP2, or EXP2 under the transcriptional control of different promoters were expressed in EXP2 knockdown parasites to determine which could complement EXP2 function. This revealed that EXP2 is a unique pore-forming protein, and its protein export role in P. falciparum cannot be complemented by T. gondii GRA17. This was despite the addition of the EXP2 assembly strand and part of the linker helix to GRA17, which are regions necessary for the interaction of EXP2 with the other core PTEX components. This indicates that the body region of EXP2 plays a critical role in PTEX assembly and/or that the absence of other T. gondii GRA proteins in P. falciparum leads to its reduced efficiency of insertion into the PVM and complementation potential. Altering the timing and abundance of EXP2 expression did not affect protein export but affected parasite viability, indicating that the unique transcriptional profile of EXP2 when compared to other PTEX components enables it to serve an additional role in nutrient exchange.

With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite’s lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action.

Based on HER2 expression using immunohistochemistry (IHC) and HER2 gene amplification using in situ hybridization (ISH), breast cancers (BC) are usually classified, into HER2 positive (IHC 3+ or IHC 2+/ISH+) and HER2 negative (IHC 0, IHC 1+ or IHC 2+/ISH-). Recently, HER2-low breast cancers (HER2-low BC) emerged as a new subtype defined as IHC1+ or IHC2+/ISH- tumors. HER2-low BC represent more than half of all BC. Consistently with the clinical activity of HER2 antibody-directed chemotherapy (ADC) in HER2-low BC, this subtype should be identified to enable early set-up of therapy. Since HER2-low BC identification could be equivocal using conventional IHC and ISH, we evaluated the performance of NGS for integrated diagnostic including HER2 copy number analysis. BC tumor specimens were analyzed using IHC and ISH as performed routinely. NGS was performed using a custom capture based 51-gene panel, including BC molecular target genes such as ESR1, PIK3CA, AKT1, ERBB2, TP53, BRCA1 and BRCA2. Gene mutations as well as copy number variation (CNV) and Microsatellite Instability (MSI) were determined. Thirty-one FFPE BC tumor specimens were classified using IHC and ISH into 11 HER2-positive (IHC2+ ISH+ and IHC3+), 10 HER2-negative (IHC 0) and 10 HER2-low cancers (IHC1+ and IHC2+ ISH-). Using NGS, CNV values for ERBB2 gene were significantly (p<0.001) different between HER2 negative, HER2low and HER2- positive tumors with mean CNV values of 2.0 (SD=0.3), 1.9 (SD=0.3) and 7.8 (SD=6.8), respectively. Using 3.25 as cutoff value for CNV, 90% concordance of HER2 amplification status was achieved between ISH and NGS. Using NGS, additional drug-targetable gene mutations as well as amplifications were detected in 68% (21/31) and 19% (6/31) of the cases, respectively. One case of MSI was detected in a HER2-negative and ISH unamplified case. These results show that in HER2 IHC positive (scores 1+ to 3+) BC, CNV determination using NGS allows the identification of HER2-low status simultaneously with the detection of multiple molecular target genes while sparing tissue samples. This workflow could support molecular board decision between HER2-ADC or other targeted therapies.

The malaria parasite uses actin-based mechanisms throughout its lifecycle to control a range of biological processes including intracellular trafficking, gene regulation, parasite motility and invasion. In this work we assign functions to the Plasmodium falciparum formins 1 and 2 (FRM1 and FRM2) proteins in asexual and sexual blood stage development. We show that FRM1 is essential for merozoite invasion and FRM2 is required for efficient cell division. We also observed divergent functions for FRM1 and FRM2 in gametocyte development. Conditional deletion of FRM1 leads to a delay in gametocyte stage progression. We show that FRM2 controls the actin and microtubule cytoskeletons in developing gametocytes, with premature removal of the protein resulting in a loss of transmissible stage V gametocytes. Lastly, we show that targeting formin proteins with the small molecule inhibitor of formin homology domain 2 (SMIFH2) leads to a multistage block in asexual and sexual stage parasite development.

Based on immunohistochemistry (IHC) and in situ hybridization (ISH), HER2-low breast cancers (BC) subtype-defined as IHC1+ or IHC2+/ISH- tumors-emerged and represent more than half of all BC. We evaluated the performance of NGS for integrated molecular characterization of HER2-low BC, including identification of actionable molecular targets, copy number variation (CNV), and microsatellite instability (MSI) analysis. Thirty-one BC specimens (11 HER2+, 10 HER2-, and 10 HER2-low) were routinely analyzed using IHC and ISH, and were selected and analyzed using NGS for gene mutations including ESR1, PIK3CA, AKT1, ERBB2, TP53, BRCA1, and BRCA2, CNV, and MSI. CNV values for the ERBB2 gene were significantly (p < 0.001) different between HER2+, and either HER2-low or HER2- tumors with mean values of 7.8 (SD = 6.8), 1.9 (SD = 0.3), and 2.0 (SD = 0.3), respectively. Using 3.25 as the cutoff value, 96.8% overall concordance of HER2 status was achieved between IHC and NGS compared to IHC and ISH. Using NGS, gene mutations and amplifications were detected in 68% (21/31) and 19% (6/31) of the cases, respectively. One case of MSI was detected in a HER2-negative and ISH unamplified case. Beside IHC, NGS allows the identification of HER2-low subtype simultaneously, with the detection of multiple actionable gene mutations being helpful for molecular board treatment selection.

A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the parasite-encasing parasitophorous vacuole membrane, exported proteins utilise a channel-forming protein complex termed the Plasmodium translocon of exported proteins (PTEX). PTEX is obligatory for parasite survival, both in vitro and in vivo , suggesting that at least some exported proteins have essential metabolic functions. However, to date only one essential PTEX-dependent process, the new permeability pathways, has been described. To identify other essential PTEX-dependant proteins/processes, we conditionally knocked down the expression of one of its core components, PTEX150, and examined which pathways were affected. Surprisingly, the food vacuole mediated process of haemoglobin (Hb) digestion was substantially perturbed by PTEX150 knockdown. Using a range of transgenic parasite lines and approaches, we show that two major Hb proteases; falcipain 2a and plasmepsin II, interact with PTEX core components, implicating the translocon in the trafficking of Hb proteases. We propose a model where these proteases are translocated into the PV via PTEX in order to reach the cytostome, located at the parasite periphery, prior to food vacuole entry. This work offers a second mechanistic explanation for why PTEX function is essential for growth of the parasite within its host RBC.

Hormonal BCs are characterized by the expression of hormonal receptors (estrogen or progesterone). They can be treated with endocrine therapy (ET) including anti-aromatase inhibitors (AI) and/or anti-cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. However, such treatments can cause cancer cells to mutate and impact genes such as ESR1, PIK3CA and/or AKT1, which are associated with ET resistance. By establishing the baseline level of these mutations in Formalin-Fixed Paraffin-Embedded (FFPE) DNA from patients with advanced or metastatic BC treated with AI and CDK4/6 inhibitors, the CICLADES-CE study, ancillary to CICLADES trial aims to identify genomic signatures to be monitored during follow-up of clonal evolution. Twenty FFPE samples from female patients diagnosed with advanced BCs and treated with AI were qualified, selected and microdissected. The AllPrep® DNA/RNA FFPE kit (Qiagen) was used to extract DNA. A method of Hybridization Capture-based Target Enrichment with a 516-gene panel was used on the NextSeq 550® (Illumina) for sequencing. Single nucleotide variants, copy number variants and telomere length were detected. Data obtained were then analyzed to identify highly mutated genes, specific mutations in genes of interest and genomic signatures as described by Alexandrov et al, 2013. Among 20 samples, 19 reached the quality criteria to be sequenced. We showed that several genes involved in the PI3-Kinase pathway were mutated across several samples, but no relevant mutations of AKT and ESR1 were found at baseline. Among the samples, 3 genomic signatures were detected, corresponding to validated COSMIC (Catalogue of Somatic Mutations in Cancer) signatures 5, 6 and 30. Those signatures are associated with DNA damage repair and deficiency of the base excision repair system. The signatures found are a mirror of the samples origin and the type of fixation used. With this information we have created a focused gene panel to be used for ctDNA follow-up analysis. It also provides us with a clear baseline mutational landscape for the profiling of the rest of the CICLADES cohort.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the current leading blood-stage malaria vaccine candidate. PfRH5 functions as part of the pentameric PCRCR complex containing PTRAMP, CSS, PfCyRPA and PfRIPR, all of which are essential for infection of human red blood cells (RBCs). To trigger RBC invasion, PfRH5 engages with RBC protein basigin in a step termed the RH5-basigin binding stage. Although we know increasingly more about how antibodies specific for PfRH5 can block invasion, much less is known about how antibodies recognizing other members of the PCRCR complex can inhibit invasion. To address this, we performed live cell imaging using monoclonal antibodies (mAbs) which bind PfRH5 and PfCyRPA. We measured the degree and timing of the invasion inhibition, the stage at which it occurred, as well as subsequent events. We show that parasite invasion is blocked by individual mAbs, and the degree of inhibition is enhanced when combining a mAb specific for PfRH5 with one binding PfCyRPA. In addition to directly establishing the invasion-blocking capacity of the mAbs, we identified a secondary action of certain mAbs on extracellular parasites that had not yet invaded where the mAbs appeared to inactivate the parasites by triggering a developmental pathway normally only seen after successful invasion. These findings suggest that epitopes within the PfCyRPA-PfRH5 sub-complex that elicit these dual responses may be more effective immunogens than neighboring epitopes by both blocking parasites from invading and rapidly inactivating extracellular parasites. These two protective mechanisms, prevention of invasion and inactivation of uninvaded parasites, resulting from antibody to a single epitope indicate a possible route to the development of more effective vaccines.

AbstractWith resistance to most antimalarials increasing, it is imperative that new antimalarial drugs are developed to replace or complement front-line artemisinin therapies. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited ring development of newly invaded merozoites. Here, we selected parasites resistant to M-833 and identified independent mutations arising in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introduction of the identified PfSTART1 mutations into wildtype parasites reproduced resistance to both M-833 and highly potent analogues, confirming PfSTART1 mutations were sufficient to confer resistance. The analogues bound to recombinant PfSTART1 with nanomolar affinity. We also demonstrated selective PfSTART1 engagement by the analogues using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assay for the first time inPlasmodium.Imaging of newly invaded merozoites showed the inhibitors prevented the conversion into larger amoeboid ring-stage parasites potentially through the inhibition of phospholipid transfer from the parasite to the encasing parasitophorous vacuole membrane (PVM) and/or within the parasite. We show that these PfSTART1 inhibitors also block transmission. With multiple stages of the parasite’s lifecycle being targeted by PfSTART1 inhibitors, this protein therefore represents a novel drug target with a new mechanism of action.

Conditional gene expression is a powerful tool to investigate putative vaccine and drug targets, especially in a haploid organism such as Plasmodium falciparum. Inducible systems based on regulation of either transcription, translation, protein or mRNA stability, among others, allow switching on an off the expression of any desired gene causing specific gain or loss of function phenotypes. However, those systems can be cumbersome involving the construction of large plasmids and generation of multiple transgenic parasite lines. In addition, the dynamic range of regulation achieved is not predictable for each individual gene and can be insufficient to generate detectable phenotypes when the genes of interest are silenced. Here, we combined up to three distinct inducible systems to regulate the expression of a single gene. Expression of the reporter NanoLuc luciferase was regulated over 40-fold, which correlates to the regulation achieved by each individual system multiplied by each other. We applied the conditionally expressed NanoLuc to evaluate the effect of fast-acting antimalarials such as chloroquine and artesunate as well as of slower-acting ones such as atovaquone. The conditionally expressed reporter allowed faster and more reliable detection of toxicity to the parasite, which correlated to the expected action of each compound. Bioluminescence achieved by the expression of this inducible highly sensitive reporter is therefore a promising tool to investigate the temporal effect of potential new antimalarials. This single plasmid combination system might also prove useful to achieve sufficient regulation of genes of interest to produce loss-of-function phenotypes.

Plasmodium falciparum and P. vivax are the major causes of human malaria, and P. knowlesi is an important additional cause in SE Asia. Binding of apical membrane antigen 1 (AMA1) to rhoptry neck protein 2 (RON2) was thought to be essential for merozoite invasion of erythrocytes by Plasmodium spp. Our findings reveal that P. falciparum and P. vivax have diverged and show species-specific binding of AMA1 to RON2, determined by a β-hairpin loop in RON2 and specific residues in AMA1 Loop1E. In contrast, cross-species binding of AMA1 to RON2 is retained between P. vivax and P. knowlesi . Mutation of specific amino acids in AMA1 Loop1E in P. falciparum or P. vivax ablated RON2 binding without impacting erythrocyte invasion. This indicates that the AMA1–RON2-loop interaction is not essential for invasion and additional AMA1 interactions are involved. Mutations in AMA1 that disrupt RON2 binding also enable escape of invasion inhibitory antibodies. Therefore, vaccines and therapeutics will need to be broader than targeting only the AMA1–RON2 interaction. Antibodies targeting AMA1 domain 3 had greater invasion-inhibitory activity when RON2-loop binding was ablated, suggesting this domain is a promising additional target for vaccine development. Targeting multiple AMA1 interactions involved in invasion may enable vaccines that generate more potent inhibitory antibodies and address the capacity for immune evasion. Findings on specific residues for invasion function and species divergence and conservation can inform novel vaccines and therapeutics against malaria caused by three species, including the potential for cross-species vaccines.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum . We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P . falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P . falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

M5717 is a promising antimalarial drug under development that acts against multiple stages of the life cycle of Plasmodium parasites by inhibiting the translation elongation factor 2 (PfeEF2), thereby preventing protein synthesis. The parasite clearance profile after drug treatment in preclinical studies in mice, and clinical trials in humans showed a notable delayed clearance phenotype whereby parasite infected red blood cells (iRBCs) persisted in the bloodstream for a significant period before eventual clearance. In a normal P. falciparum infection iRBCs sequester in the deep circulation by cytoadherence, allowing them to avoid surveillance and clearance in the spleen. We found that M5717 blocks parasite modification of their host red blood cells (RBCs) by preventing synthesis of new exported proteins, rather than by directly blocking the export of these proteins into the RBC compartment. Using in vitro models, we demonstrated that M5717 treated ring/trophozoite stage iRBCs became less rigid, and cytoadhered less well compared to untreated iRBCs. This indicates that in vivo persistence of M5717 treated iRBCs in the bloodstream is likely due to reduced cytoadherence and splenic clearance.