Meet the Authors: Christopher R. Mansfield and Emily R. Derbyshire.

In an interview with Samantha Nelson, a scientific editor of Cell Chemical Biology, the first and corresponding authors of the research article entitled Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome share more about the project and their perspectives on the field.

Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.

Toxoplasma and Plasmodium associate with host Arfs during infection.

The apicomplexans Toxoplasma gondii and Plasmodium are intracellular parasites that reside within a host-derived compartment termed the parasitophorous vacuole (PV). During infection, the parasites must acquire critical host resources and transport them across their PV for development. However, the mechanism by which host resources are trafficked to and across the PV remains uncertain. Here, we investigated host ADP ribosylation factors (Arfs), a class of proteins involved in vesicular trafficking that may be exploited by T. gondii and Plasmodium berghei for nutrient acquisition. Using overexpressed Arf proteins coupled with immunofluorescence microscopy, we found that all Arfs were internalized into the T. gondii PV, with most vacuoles containing at least one punctum of Arf protein by the end of the lytic cycle. We further characterized Arf1, the most abundant Arf inside the T. gondii PV, and observed that active recycling between its GDP/GTP-bound state influenced Arf1 internalization independent of host guanine nucleotide exchange factors (GEFs). In addition, Arf1 colocalized with vesicle coat complexes and exogenous sphingolipids, suggesting a role in nutrient acquisition. While Arf1 and Arf4 were not observed inside the PV during P. berghei infection, our gene depletion studies showed that liver stage development and survival depended on the expression of Arf4 and the host GEF, GBF1. Collectively, these observations indicate that apicomplexans use distinct mechanisms to subvert the host vesicular trafficking network and efficiently replicate. The findings also pave the way for future studies to identify parasite proteins critical to host vesicle recruitment and the components of vesicle cargo. IMPORTANCE: The parasites Toxoplasma gondii and Plasmodium live complex intracellular lifestyles where they must acquire essential host nutrients while avoiding recognition. Although previous work has sought to identify the specific nutrients scavenged by apicomplexans, the mechanisms by which host materials are transported to and across the parasite vacuole membrane are largely unknown. Here, we examined members of the host vesicular trafficking network to identify specific pathways subverted by T. gondii and Plasmodium berghei. Our results indicate that T. gondii selectively internalizes host Arfs, a class of proteins involved in intracellular trafficking. For P. berghei, host Arfs were restricted by the parasite's vacuole membrane, but proteins involved in vesicular trafficking were identified as essential for liver stage development. A greater exploration into how and why apicomplexans subvert host vesicular trafficking could help identify targets for host-directed therapeutics.

Characterization of 2,4-Dianilinopyrimidines Against Five P. falciparum Kinases PfARK1, PfARK3, PfNEK3, PfPK9, and PfPKB.

Plasmodium kinases are increasingly recognized as potential novel antiplasmodial targets for the treatment of malaria, but only a small subset of these kinases have had structure-activity relationship (SAR) campaigns reported. Herein we report the discovery of CZC-54252 (1) as an inhibitor of five P. falciparum kinases PfARK1, PfARK3, PfNEK3, PfPK9, and PfPKB. 39 analogues were evaluated against all five kinases to establish SAR at three regions of the kinase active site. Nanomolar inhibitors of each kinase were discovered. We identified common and divergent SAR trends across all five kinases, highlighting substituents in each region that improve potency and selectivity for each kinase. Potent analogues were evaluated against the P. falciparum blood stage. Eight submicromolar inhibitors were discovered, of which 37 demonstrated potent antiplasmodial activity (EC50 = 0.16 µM). Our results provide an understanding of features needed to inhibit each individual kinase and lay groundwork for future optimization efforts toward novel antimalarials.

Exploring the Chain Release Mechanism from an Atypical Apicomplexan Polyketide Synthase.

Polyketide synthases (PKSs) are megaenzymes that form chemically diverse polyketides and are found within the genomes of nearly all classes of life. We recently discovered the type I PKS from the apicomplexan parasite Toxoplasma gondii, TgPKS2, which contains a unique putative chain release mechanism that includes ketosynthase (KS) and thioester reductase (TR) domains. Our bioinformatic analysis of the thioester reductase of TgPKS2, TgTR, suggests differences compared to other systems and hints at a possibly conserved release mechanism within the apicomplexan subclass Coccidia. To evaluate this release module, we first isolated TgTR and observed that it is capable of 4 electron (4e-) reduction of octanoyl-CoA to the primary alcohol, octanol, utilizing NADH. TgTR was also capable of generating octanol in the presence of octanal and NADH, but no reactions were observed when NADPH was supplied as a cofactor. To biochemically characterize the protein, we measured the catalytic efficiency of TgTR using a fluorescence assay and determined the TgTR binding affinity for cofactor and substrates using isothermal titration calorimetry (ITC). We additionally show that TgTR is capable of reducing an acyl carrier protein (ACP)-tethered substrate by liquid chromatography mass spectrometry and determine that TgTR binds to holo-TgACP4, its predicted cognate ACP, with a KD of 5.75 ± 0.77 µM. Finally, our transcriptional analysis shows that TgPKS2 is upregulated ∼4-fold in the parasite's cyst-forming bradyzoite stage compared to tachyzoites. Our study identifies features that distinguish TgPKS2 from well-characterized systems in bacteria and fungi and suggests it aids the T. gondii cyst stage.

Synthesis and activity of ß-carboline antimalarials targeting the Plasmodium falciparum heat shock 90 protein.

A collection of ß-carbolines based on the natural product harmine, a compound known to target the heat shock 90 protein of Plasmodium falciparum, was synthesized and tested for antimalarial activity and potential toxicity. Several of these novel compounds display promising bioactivity, providing a new potential therapeutic with a mode of action that differs versus any currently available clinical treatment.

Labeling strategies to track protozoan parasite proteome dynamics.

Intracellular protozoan parasites are responsible for wide-spread infectious diseases. These unicellular pathogens have complex, multi-host life cycles, which present challenges for investigating their basic biology and for discovering vulnerabilities that could be exploited for disease control. Throughout development, parasite proteomes are dynamic and support stage-specific functions, but detection of these proteins is often technically challenging and complicated by the abundance of host proteins. Thus, to elucidate key parasite processes and host-pathogen interactions, labeling strategies are required to track pathogen proteins during infection. Herein, we discuss the application of bioorthogonal non-canonical amino acid tagging and proximity-dependent labeling to broadly study protozoan parasites and include outlooks for future applications to study Plasmodium, the causative agent of malaria. We highlight the potential of these technologies to provide spatiotemporal labeling with selective parasite protein enrichment, which could enable previously unattainable insight into the biology of elusive developmental stages.

Examination of Secondary Metabolite Biosynthesis in Apicomplexa.

Natural product discovery has traditionally relied on the isolation of small molecules from producing species, but genome-sequencing technology and advances in molecular biology techniques have expanded efforts to a wider array of organisms. Protists represent an underexplored kingdom for specialized metabolite searches despite bioinformatic analysis that suggests they harbor distinct biologically active small molecules. Specifically, pathogenic apicomplexan parasites, responsible for billions of global infections, have been found to possess multiple biosynthetic gene clusters, which hints at their capacity to produce polyketide metabolites. Biochemical studies have revealed unique features of apicomplexan polyketide synthases, but to date, the identity and function of the polyketides synthesized by these megaenzymes remains unknown. Herein, we discuss the potential for specialized metabolite production in protists and the possible evolution of polyketide biosynthetic gene clusters in apicomplexan parasites. We then focus on a polyketide synthase from the apicomplexan Toxoplasma gondii to discuss the unique domain architecture and properties of these proteins when compared to previously characterized systems, and further speculate on the possible functions for polyketides in these pathogenic parasites.

Characterization of Unexpected Self-Acylation Activity of Acyl Carrier Proteins in a Modular Type I Apicomplexan Polyketide Synthase.

Natural products play critical roles as antibiotics, anticancer therapeutics, and biofuels. Polyketides are a distinct natural product class of structurally diverse secondary metabolites that are synthesized by polyketide synthases (PKSs). The biosynthetic gene clusters that encode PKSs have been found across nearly all realms of life, but those from eukaryotic organisms are relatively understudied. A type I PKS from the eukaryotic apicomplexan parasite Toxoplasma gondii,TgPKS2, was recently discovered through genome mining, and the functional acyltransferase (AT) domains were found to be selective for malonyl-CoA substrates. To further characterize TgPKS2, we resolved assembly gaps within the gene cluster, which confirmed that the encoded protein consists of three distinct modules. We subsequently isolated and biochemically characterized the four acyl carrier protein (ACP) domains within this megaenzyme. We observed self-acylation─or substrate acylation without an AT domain─for three of the four TgPKS2 ACP domains with CoA substrates. Furthermore, CoA substrate specificity and kinetic parameters were determined for all four unique ACPs. TgACP2-4 were active with a wide scope of CoA substrates, while TgACP1 from the loading module was found to be inactive for self-acylation. Previously, self-acylation has only been observed in type II systems, which are enzymes that act in-trans with one another, and this represents the first report of this activity in a modular type I PKS whose domains function in-cis. Site-directed mutagenesis of specific TgPKS2 ACP3 acidic residues near the phosphopantetheinyl arm demonstrated that they influence self-acylation activity and substrate specificity, possibly by influencing substrate coordination or phosphopantetheinyl arm activation. Further, the lack of TgPKS2 ACP self-acylation with acetoacetyl-CoA, which is utilized by previously characterized type II PKS systems, suggests that the substrate carboxyl group may be critical for TgPKS2 ACP self-acylation. The unexpected properties observed from T. gondii PKS ACP domains highlight their distinction from well-characterized microbial and fungal systems. This work expands our understanding of ACP self-acylation beyond type II systems and helps pave the way for future studies on biosynthetic enzymes from eukaryotes.

Discovery of potent Plasmodium falciparum protein kinase 6 (PfPK6) inhibitors with a type II inhibitor pharmacophore.

Malaria is a devastating disease that causes significant global morbidity and mortality. The rise of drug resistance against artemisinin-based combination therapy demonstrates the necessity to develop alternative antimalarials with novel mechanisms of action. We report the discovery of Ki8751 as an inhibitor of essential kinase PfPK6. 79 derivatives were designed, synthesized and evaluated for PfPK6 inhibition and antiplasmodial activity. Using group efficiency analyses, we established the importance of key groups on the scaffold consistent with a type II inhibitor pharmacophore. We highlight modifications on the tail group that contribute to antiplasmodial activity, cumulating in the discovery of compound 67, a PfPK6 inhibitor (IC50 = 13 nM) active against the P. falciparum blood stage (EC50 = 160 nM), and compound 79, a PfPK6 inhibitor (IC50 < 5 nM) with dual-stage antiplasmodial activity against P. falciparum blood stage (EC50 = 39 nM) and against P. berghei liver stage (EC50 = 220 nM).

Cut it out! A CRISPR-Cas9 screen identifies host regulators of the Plasmodium liver stage.

In this issue of Cell Chemical Biology, Vijayan and colleagues identify host factors integral for Plasmodium liver-stage infection using a whole-genome CRISPR-Cas9 knockout screen. Their efforts reveal that liver-stage parasites redistribute host microtubules to the parasite membrane in a process dependent on the host Golgi.

Identification of Novel 2,4,5-Trisubstituted Pyrimidines as Potent Dual Inhibitors of Plasmodial PfGSK3/PfPK6 with Activity against Blood Stage Parasites In Vitro.

Essential plasmodial kinases PfGSK3 and PfPK6 are considered novel drug targets to combat rising resistance to traditional antimalarial therapy. Herein, we report the discovery of IKK16 as a dual PfGSK3/PfPK6 inhibitor active against blood stage Pf3D7 parasites. To establish structure-activity relationships for PfPK6 and PfGSK3, 52 analogues were synthesized and assessed for the inhibition of PfGSK3 and PfPK6, with potent inhibitors further assessed for activity against blood and liver stage parasites. This culminated in the discovery of dual PfGSK3/PfPK6 inhibitors 23d (PfGSK3/PfPK6 IC50 = 172/11 nM) and 23e (PfGSK3/PfPK6 IC50 = 97/8 nM) with antiplasmodial activity (23d Pf3D7 EC50 = 552 ± 37 nM and 23e Pf3D7 EC50 = 1400 ± 13 nM). However, both compounds exhibited significant promiscuity when tested in a panel of human kinase targets. Our results demonstrate that dual PfPK6/PfGSK3 inhibitors with antiplasmodial activity can be identified and can set the stage for further optimization efforts.

Elucidating the path to Plasmodium prolyl-tRNA synthetase inhibitors that overcome halofuginone resistance.

The development of next-generation antimalarials that are efficacious against the human liver and asexual blood stages is recognized as one of the world's most pressing public health challenges. In recent years, aminoacyl-tRNA synthetases, including prolyl-tRNA synthetase, have emerged as attractive targets for malaria chemotherapy. We describe the development of a single-step biochemical assay for Plasmodium and human prolyl-tRNA synthetases that overcomes critical limitations of existing technologies and enables quantitative inhibitor profiling with high sensitivity and flexibility. Supported by this assay platform and co-crystal structures of representative inhibitor-target complexes, we develop a set of high-affinity prolyl-tRNA synthetase inhibitors, including previously elusive aminoacyl-tRNA synthetase triple-site ligands that simultaneously engage all three substrate-binding pockets. Several compounds exhibit potent dual-stage activity against Plasmodium parasites and display good cellular host selectivity. Our data inform the inhibitor requirements to overcome existing resistance mechanisms and establish a path for rational development of prolyl-tRNA synthetase-targeted anti-malarial therapies.

A single amino acid residue controls acyltransferase activity in a polyketide synthase from Toxoplasma gondii.

Type I polyketide synthases (PKSs) are multidomain, multimodule enzymes capable of producing complex polyketide metabolites. These modules contain an acyltransferase (AT) domain, which selects acyl-CoA substrates to be incorporated into the metabolite scaffold. Herein, we reveal the sequences of three AT domains from a polyketide synthase (TgPKS2) from the apicomplexan parasite Toxoplasma gondii. Phylogenic analysis indicates these ATs (AT1, AT2, and AT3) are distinct from domains in well-characterized microbial biosynthetic gene clusters. Biochemical investigations revealed that AT1 and AT2 hydrolyze malonyl-CoA but the terminal AT3 domain is non-functional. We further identify an "on-off switch" residue that controls activity such that a single amino acid change in AT3 confers hydrolysis activity while the analogous mutation in AT2 eliminates activity. This biochemical analysis of AT domains from an apicomplexan PKS lays the foundation for further molecular and structural studies on PKSs from T. gondii and other protists.

Random Forest Model Predictions Afford Dual-Stage Antimalarial Agents.

The need for novel antimalarials is apparent given the continuing disease burden worldwide, despite significant drug discovery advances from the bench to the bedside. In particular, small-molecule agents with potent efficacy against both the liver and blood stages of Plasmodium parasite infection are critical for clinical settings as they would simultaneously prevent and treat malaria with a reduced selection pressure for resistance. While experimental screens for such dual-stage inhibitors have been conducted, the time and cost of these efforts limit their scope. Here, we have focused on leveraging machine learning approaches to discover novel antimalarials with such properties. A random forest modeling approach was taken to predict small molecules with in vitro efficacy versus liver-stage Plasmodium berghei parasites and a lack of human liver cell cytotoxicity. Empirical validation of the model was achieved with the realization of hits with liver-stage efficacy after prospective scoring of a commercial diversity library and consideration of structural diversity. A subset of these hits also demonstrated promising blood-stage Plasmodium falciparum efficacy. These 18 validated dual-stage antimalarials represent novel starting points for drug discovery and mechanism of action studies with significant potential for seeding a new generation of therapies.

Erratum: A single amino acid residue controls acyltransferase activity in a polyketide synthase from Toxoplasma gondii.

[This corrects the article DOI: 10.1016/j.isci.2022.104443.].

Plasmodium's fight for survival: escaping elimination while acquiring nutrients.

Plasmodium parasites extensively alter their host hepatocyte to evade host detection and support an unprecedented replication rate. Host cell manipulation includes association with the host early and late endomembrane systems, where Plasmodium accesses nutrients while suppressing cellular immune processes. Early endomembrane organelles provide an opportunity to sequester an abundance of lipids and proteins, but the association with late endomembrane organelles also risks autophagy-mediated elimination. While not all parasites survive, those that do benefit from a plethora of nutrients provided through this pathway. In this review, we discuss recent advances in our understanding of how Plasmodium parasites balance the need for host nutrients while avoiding elimination during the liver stage.

Characterization of the Tubovesicular Network in Plasmodium vivax Liver Stage Hypnozoites and Schizonts.

Plasmodium is a genus of apicomplexan parasites which replicate in the liver before causing malaria. Plasmodium vivax can also persist in the liver as dormant hypnozoites and cause clinical relapse upon activation, but the molecular mechanisms leading to activation have yet to be discovered. In this study, we use high-resolution microscopy to characterize temporal changes of the P. vivax liver stage tubovesicular network (TVN), a parasitophorous vacuole membrane (PVM)-derived network within the host cytosol. We observe extended membrane clusters, tubules, and TVN-derived vesicles present throughout P. vivax liver stage development. Additionally, we demonstrate an unexpected presence of the TVN in hypnozoites and observe some association of this network to host nuclei. We also reveal that the host water and solute channel aquaporin-3 (AQP3) associates with TVN-derived vesicles and extended membrane clusters. AQP3 has been previously shown to localize to the PVM of P. vivax hypnozoites and liver schizonts but has not yet been shown in association to the TVN. Our results highlight host-parasite interactions occur in both dormant and replicating liver stage P. vivax forms and implicate AQP3 function during this time. Together, these findings enhance our understanding of P. vivax liver stage biology through characterization of the TVN with an emphasis on the presence of this network in dormant hypnozoites.

Chemoproteomics for Plasmodium Parasite Drug Target Discovery.

Emerging Plasmodium parasite drug resistance is threatening progress towards malaria control and elimination. While recent efforts in cell-based, high-throughput drug screening have produced first-in-class drugs with promising activities against different Plasmodium life cycle stages, most of these antimalarial agents have elusive mechanisms of action. Though challenging to address, target identification can provide valuable information to facilitate lead optimization and preclinical drug prioritization. Recently, proteome-wide methods for direct assessment of drug-protein interactions have emerged as powerful tools in a number of systems, including Plasmodium. In this review, we will discuss current chemoproteomic strategies that have been adapted to antimalarial drug target discovery, including affinity- and activity-based protein profiling and the energetics-based techniques thermal proteome profiling and stability of proteins from rates of oxidation. The successful application of chemoproteomics to the Plasmodium blood stage highlights the potential of these methods to link inhibitors to their molecular targets in more elusive Plasmodium life stages and intracellular pathogens in the future.

Activation of GPR37 in macrophages confers protection against infection-induced sepsis and pain-like behaviour in mice.

GPR37 was discovered more than two decades ago, but its biological functions remain poorly understood. Here we report a protective role of GPR37 in multiple models of infection and sepsis. Mice lacking Gpr37 exhibited increased death and/or hypothermia following challenge by lipopolysaccharide (LPS), Listeria bacteria, and the mouse malaria parasite Plasmodium berghei. Sepsis induced by LPS and Listeria in wild-type mice is protected by artesunate (ARU) and neuroprotectin D1 (NPD1), but the protective actions of these agents are lost in Gpr37-/- mice. Notably, we found that ARU binds to GPR37 in macrophages and promotes phagocytosis and clearance of pathogens. Moreover, ablation of macrophages potentiated infection, sepsis, and their sequelae, whereas adoptive transfer of NPD1- or ARU-primed macrophages reduced infection, sepsis, and pain-like behaviors. Our findings reveal physiological actions of ARU in host cells by activating macrophages and suggest that GPR37 agonists may help to treat sepsis, bacterial infections, and malaria.