Advances in protease inhibition-based chemotherapy: A decade of insights from Malaria research.

Over the last decade, research on the most studied parasite, Plasmodium falciparum, has disclosed significant findings in protease research. Detailed descriptions of the individual roles of protease isoenzymes from various protease classes encoded by the parasite genome have been elucidated, along with their functional and biochemical characterizations. These insights have enabled the development of innovative chemotherapy using low molecular weight inhibitors targeting specific molecular sites. Progress has been made in understanding the proteolytic cascade associated with the apical complex, particularly the roles of aspartyl proteases plasmepsins IX and X as master regulators. Additionally, advancements in direct and alternative methods of proteasome inhibition and expression regulation have been achieved. Research on digestive/food vacuole-associated proteases, with a focus on essential metalloproteases, has also seen significant developments. The rise of extensive genomic datasets and functional genomic tools for other parasitic organisms now allows these approaches to be applied to the study and treatment of other, less known parasitic diseases, aiming to uncover specific biological mechanisms and develop innovative, less toxic chemotherapies.

Evolution of Pfdhps and Pfdhfr mutations before and after adopting seasonal malaria chemoprevention in Nanoro, Burkina Faso.

Seasonal Malaria Chemoprevention consisting of monthly administration of amodiaquine/sulfadoxine-pyrimethamine to children aged 3-59 months during the transmission season could promote SP-resistance. Mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes were assessed before and after SMC adoption in Burkina Faso. A total of 769 dried blood spots were selected from studies conducted in Nanoro, Burkina Faso, between 2010 and 2020. Of those, 299 were pre-SMC (2010-2012) and 470 were post-SMC-samples. Pfdhps and Pfdhfr genes were PCR-amplified and sequenced. A systematic review/meta-analysis of published studies conducted in Burkina Faso (2009-2023) was additionally performed. In Nanoro, the prevalence of Pfdhfr triple mutations (CIRNI) rose from 43.6% pre-SMC to 89.4% post-SMC (p < 0.0001). There was no mutation in Pfdhfr 164 and Pfdhps 540; Pfdhps A437G mutation increased from 63.9% (2010-2012) to 84.7% (2020) (p < 0.0001). The VAGKGS haplotype was 2.8% (2020). Pfdhfr/Pfdhps quintuple mutant IRN-436A437G rose from 18.6% (2010-2012) to 58.3% (2020) (p < 0.0001). Meta-analysis results from Burkina Faso showed an increase in mutations at Pfdhfr N51I, C59R, S108N, and Pfdhps A437G after SMC adoption. Post-SMC, the pyrimethamine-resistance marker prevalence increased, while the sulfadoxine-resistance marker prevalence remained stable. Detection of emerging PfdhpsVAGKGS haplotypes in 2020 underscores the importance of continuous SP-resistance monitoring.

The three Plasmodium falciparum Aurora-related kinases display distinct temporal and spatial associations with mitotic structures in asexual blood stage parasites and gametocytes.

Aurora kinases are crucial regulators of mitotic cell cycle progression in eukaryotes. The protozoan malaria parasite Plasmodium falciparum replicates via schizogony, a specialized mode of cell division characterized by consecutive asynchronous rounds of nuclear division by closed mitosis followed by a single cytokinesis event producing dozens of daughter cells. P. falciparum encodes three Aurora-related kinases (PfARKs) that have been reported essential for parasite proliferation, but their roles in regulating schizogony have not yet been explored in great detail. Here, we engineered transgenic parasite lines expressing GFP-tagged PfARK1-3 to provide a systematic analysis of their expression timing and subcellular localization throughout schizogony as well as in the non-dividing gametocyte stages, which are essential for malaria transmission. We demonstrate that all three PfARKs display distinct and highly specific and exclusive spatiotemporal associations with the mitotic machinery. In gametocytes, PfARK3 is undetectable, and PfARK1 and PfARK2 show male-specific expression in late-stage gametocytes, consistent with their requirement for endomitosis during male gametogenesis in the mosquito vector. Our combined data suggest that PfARK1 and PfARK2 have non-overlapping roles in centriolar plaque maturation, assembly of the mitotic spindle, kinetochore-spindle attachment and chromosome segregation, while PfARK3 seems to be exquisitely involved in daughter cell cytoskeleton assembly and cytokinesis. These important new insights provide a reliable foundation for future research aiming at the functional investigation of these divergent and possibly drug-targetable Aurora-related kinases in mitotic cell division of P. falciparum and related apicomplexan parasites.IMPORTANCEMalaria parasites replicate via non-conventional modes of mitotic cell division, such as schizogony, employed by the disease-causing stages in the human blood or endomitosis during male gametogenesis in the mosquito vector. Understanding the molecular mechanisms regulating cell division in these divergent unicellular eukaryotes is not only of scientific interest but also relevant to identify potential new antimalarial drug targets. Here, we carefully examined the subcellular localization of all three Plasmodium falciparum Aurora-related kinases (ARKs), distantly related homologs of Aurora kinases that coordinate mitosis in model eukaryotes. Detailed fluorescence microscopy-based analyses revealed distinct, specific, and exclusive spatial associations for each parasite ARK with different components of the mitotic machinery and at different phases of the cell cycle during schizogony and gametocytogenesis. This comprehensive set of results closes important gaps in our fragmentary knowledge on this important group of kinases and offers a valuable source of information for future functional studies.

Design and synthesis of pyrano[2,3-c]pyrazole-4-aminoquinoline hybrids as effective antimalarial compounds.

In this work, a series of nineteen novel pyrano[2,3-c]pyrazole-4-aminoquinoline hybrids were synthesized as potent antimalarial agents by covalently linking the scaffolds of 4-aminoquinoline and pyrano[2,3-c]pyrazoles via an ethyl linker and characterized using Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). Molecular docking was used to test each hybrid's and standard chloroquine's ability to bind to Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH), an important enzyme in the parasite's glycolytic pathway. The hybrid compounds had a stronger binding affinity than the standard chloroquine (CQ). The schizontical antimalarial test of pyrano[2,3-c]pyrazole-4-aminoquinoline hybrid compound shows that all nineteen hybrid compounds were potent with the IC50 values ranging from 0.0151 to 0.301 µM against the CQ-sensitive 3D7 P. falciparum strain, and were active against the CQ-resistant K1 P. falciparum strain with the IC50 values ranging from 0.01895 to 2.746 µM. All the tested hybrid compounds were less potent than the standard drug chloroquine dipaspate (CQDP) against the CQ-sensitive 3D7 strain. In contrast, nine of the nineteen hybrids (16d, 16g, 16h, 16i, 16l, 16n, 16o, 16r, and 16s) displayed superior antimalarial activity than the CQDP against the CQ-resistant K1 P. falciparum strain. Among all the tested hybrids, 16c against the 3D7 strain and 16h against the K1 strain were the most promising antimalarial agents with 0.0151 and 0.01895 µM of IC50 values, respectively. In addition, the compounds were selective, showing moderate to low cytotoxic activity against a human normal liver WRL68 cell line. The synthesis of pyrano[2,3-c]pyrazole-4-aminoquinoline hybrids introduces new chemical entities that have the potential to exhibit potent antimalarial activity. It could address the ongoing challenge of drug resistance in malaria treatment.

Mixed alkyl/aryl phosphonates identify metabolic serine hydrolases as antimalarial targets.

Malaria, caused by Plasmodium falciparum, remains a significant health burden. One major barrier for developing antimalarial drugs is the ability of the parasite to rapidly generate resistance. We previously demonstrated that salinipostin A (SalA), a natural product, potently kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism that results in a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a small library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent antiparasitic potencies that enabled the identification of therapeutically relevant targets. The active compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor orlistat and shows synergistic killing with orlistat. Our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are promising, synthetically tractable antimalarials.

Novel molecular inhibitor design for Plasmodium falciparum Lactate dehydrogenase enzyme using machine learning generated library of diverse compounds.

Generative machine learning models offer a novel strategy for chemogenomics and de novo drug design, allowing researchers to streamline their exploration of the chemical space and concentrate on specific regions of interest. In cases with limited inhibitor data available for the target of interest, de novo drug design plays a crucial role. In this study, we utilized a package called 'mollib,' trained on ChEMBL data containing approximately 365,000 bioactive molecules. By leveraging transfer learning techniques with this package, we generated a series of compounds, starting from five initial compounds, which are potential Plasmodium falciparum (Pf) Lactate dehydrogenase inhibitors. The resulting compounds exhibit structural diversity and hold promise as potential novel Pf Lactate dehydrogenase inhibitors.

ResMAP-a saturation mutagenesis platform enabling parallel profiling of target-specific resistance-conferring mutations in Plasmodium.

New and improved drugs are required for the treatment and ultimate eradication of malaria. The efficacy of front-line therapies is now threatened by emerging drug resistance; thus, new tools to support the development of drugs with a lower propensity for resistance are needed. Here, we describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium drug targets. Proof-of-concept studies focused on interrogating the antimalarial drug target, Plasmodium falciparum lysyl tRNA synthetase (PfKRS). Saturation mutagenesis was used to construct a plasmid library encoding all conceivable mutations within a 20-residue span at the base of the PfKRS ATP-binding pocket. The superior transfection efficiency of Plasmodium knowlesi was exploited to generate a high coverage parasite library expressing PfKRS bearing all possible amino acid changes within this region of the enzyme. The selection of the library with PfKRS inhibitors, cladosporin and DDD01510706, successfully identified multiple resistance-conferring substitutions. Genetic validation of a subset of these mutations confirmed their direct role in resistance, with computational modeling used to dissect the structural basis of resistance. The application of ResMAP to inform the development of resistance-resilient antimalarials of the future is discussed. IMPORTANCE: An increase in treatment failures for malaria highlights an urgent need for new tools to understand and minimize the spread of drug resistance. We describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium spp, the causative agent of malaria. Saturation mutagenesis was used to generate a mutation library containing all conceivable mutations for a region of the antimalarial-binding site of a promising drug target, Plasmodium falciparum lysyl tRNA synthetase (PfKRS). Screening of this high-coverage library with characterized PfKRS inhibitors revealed multiple resistance-conferring substitutions including several clinically relevant mutations. Genetic validation of these mutations confirmed resistance of up to 100-fold and computational modeling dissected their role in drug resistance. We discuss potential applications of this data including the potential to design compounds that can bypass the most serious resistance mutations and future resistance surveillance.

A multistage Plasmodium CRL4WIG1 ubiquitin ligase is critical for the formation of functional microtubule organization centers in microgametocytes.

Malaria is a mosquito-borne infectious disease caused by unicellular eukaryotic parasites of the Plasmodium genus. Protein ubiquitination by E3 ligases is a critical post-translational modification required for various cellular processes during the lifecycle of Plasmodium parasites. However, little is known about the repertoire and function of these enzymes in Plasmodium. Here, we show that Plasmodium expresses a conserved cullin RING E3 ligase (CRL) complex that is functionally related to CRL4 in other eukaryotes. In P. falciparum asexual blood stages, a cullin-4 scaffold interacts with the RING protein RBX1, the adaptor protein DDB1, and a set of putative receptor proteins that may determine substrate specificity for ubiquitination. These receptor proteins contain WD40-repeat domains and include WD-repeat protein important for gametogenesis 1 (WIG1). This CRL4-related complex is also expressed in P. berghei gametocytes, with WIG1 being the only putative receptor detected in both the schizont and gametocyte stages. WIG1 disruption leads to a complete block in microgamete formation. Proteomic analyses indicate that WIG1 disruption alters proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. Further analysis by ultrastructure expansion microscopy (U-ExM) indicates that WIG1-dependent depletion of ciliary proteins is associated with impaired the formation of the microtubule organization centers that coordinate mitosis with axoneme formation and altered DNA replication during microgametogenesis. This work identifies a CRL4-related ubiquitin ligase in Plasmodium that is critical for the formation of microgametes by regulating proteostasis of ciliary and DNA replication proteins.IMPORTANCEPlasmodium parasites undergo fascinating lifecycles with multiple developmental steps, converting into morphologically distinct forms in both their mammalian and mosquito hosts. Protein ubiquitination by ubiquitin ligases emerges as an important post-translational modification required to control multiple developmental stages in Plasmodium. Here, we identify a cullin RING E3 ubiquitin ligase (CRL) complex expressed in the replicating asexual blood stages and in the gametocyte stages that mediate transmission to the mosquito. WIG1, a putative substrate recognition protein of this ligase complex, is essential for the maturation of microgametocytes into microgametes upon ingestion by a mosquito. More specifically, WIG1 is required for proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. This requirement is linked to DNA replication and microtubule organization center formation, both critical to the development of flagellated microgametes.

Cryo-EM structure of 1-deoxy-D-xylulose 5-phosphate synthase DXPS from Plasmodium falciparum reveals a distinct N-terminal domain.

Plasmodium falciparum is the main causative agent of malaria, a deadly disease that mainly affects children under five years old. Artemisinin-based combination therapies have been pivotal in controlling the disease, but resistance has arisen in various regions, increasing the risk of treatment failure. The non-mevalonate pathway is essential for the isoprenoid synthesis in Plasmodium and provides several under-explored targets to be used in the discovery of new antimalarials. 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) is the first and rate-limiting enzyme of the pathway. Despite its importance, there are no structures available for any Plasmodium spp., due to the complex sequence which contains large regions of high disorder, making crystallisation a difficult task. In this manuscript, we use cryo-electron microscopy to solve the P. falciparum DXPS structure at a final resolution of 2.42 Å. Overall, the structure resembles other DXPS enzymes but includes a distinct N-terminal domain exclusive to the Plasmodium genus. Mutational studies show that destabilization of the cap domain interface negatively impacts protein stability and activity. Additionally, a density for the co-factor thiamine diphosphate is found in the active site. Our work highlights the potential of cryo-EM to obtain structures of P. falciparum proteins that are unfeasible by means of crystallography.

Development of peptoid-based heteroaryl-decorated histone deacetylase (HDAC) inhibitors with dual-stage antiplasmodial activity.

Dynamics of epigenetic modifications such as acetylation and deacetylation of histone proteins have been shown to be crucial for the life cycle development and survival of Plasmodium falciparum, the deadliest malaria parasite. In this study, we present a novel series of peptoid-based histone deacetylase (HDAC) inhibitors incorporating nitrogen-containing bicyclic heteroaryl residues as a new generation of antiplasmodial peptoid-based HDAC inhibitors. We synthesized the HDAC inhibitors by an efficient multicomponent protocol based on the Ugi four-component reaction. The subsequent screening of 16 compounds from our mini-library identified 6i as the most promising candidate, demonstrating potent activity against asexual blood-stage parasites (IC50Pf3D7 = 30 nM; IC50PfDd2 = 98 nM), low submicromolar activity against liver-stage parasites (IC50PbEEF = 0.25 µM), excellent microsomal stability (t1/2 > 60 min), and low cytotoxicity to HEK293 cells (IC50 = 136 µM).

Inhibition of falcilysin from Plasmodium falciparum by interference with its closed-to-open dynamic transition.

In the absence of an efficacious vaccine, chemotherapy remains crucial to prevent and treat malaria. Given its key role in haemoglobin degradation, falcilysin constitutes an attractive target. Here, we reveal the mechanism of enzymatic inhibition of falcilysin by MK-4815, an investigational new drug with potent antimalarial activity. Using X-ray crystallography, we determine two binary complexes of falcilysin in a closed state, bound with peptide substrates from the haemoglobin α and ß chains respectively. An antiparallel ß-sheet is formed between the substrate and enzyme, accounting for sequence-independent recognition at positions P2 and P1. In contrast, numerous contacts favor tyrosine and phenylalanine at the P1' position of the substrate. Cryo-EM studies reveal a majority of unbound falcilysin molecules adopting an open conformation. Addition of MK-4815 shifts about two-thirds of falcilysin molecules to a closed state. These structures give atomic level pictures of the proteolytic cycle, in which falcilysin interconverts between a closed state conducive to proteolysis, and an open conformation amenable to substrate diffusion and products release. MK-4815 and quinolines bind to an allosteric pocket next to a hinge region of falcilysin and hinders this dynamic transition. These data should inform the design of potent inhibitors of falcilysin to combat malaria.

Peptidic Boronic Acid Plasmodium falciparum SUB1 Inhibitors with Improved Selectivity over Human Proteasome.

Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P3 amino acid side chains as well as N-capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.

Functionality of the V-type ATPase during asexual growth and development of Plasmodium falciparum.

Vacuolar type ATPases (V-type ATPases) are highly conserved hetero-multisubunit proton pumping machineries found in all eukaryotes. They utilize ATP hydrolysis to pump protons, acidifying intracellular or extracellular compartments, and are thus crucial for various biological processes. Despite their evolutionary conservation in malaria parasites, this proton pump remains understudied. To understand the localization and biological functions of Plasmodium falciparum V-type ATPase, we employed CRISPR/Cas9 to endogenously tag the subunit A of the V1 domain. V1A (PF3D7_1311900) was tagged with a triple hemagglutinin epitope and the TetR-DOZI-aptamer system for conditional expression under the regulation of anhydrotetracycline. Via immunofluorescence assays, we identified that V-type ATPase is expressed throughout the intraerythrocytic developmental cycle and is mainly localized to the digestive vacuole and parasite plasma membrane. Immuno-electron microscopy further revealed that V-type ATPase is also localized on secretory organelles in merozoites. Knockdown of V1A led to cytosolic pH imbalance and blockage of hemoglobin digestion in the digestive vacuole, resulting in an arrest of parasite development in the trophozoite-stage and, ultimately, parasite demise. Using bafilomycin A1, a specific inhibitor of V-type ATPases, we found that the P. falciparum V-type ATPase is likely involved in parasite invasion but is not critical for ring-stage development. Further, we detected a large molecular weight complex in blue native-PAGE (∼1.0 MDa), corresponding to the total molecular weights of V1 and Vo domains. Together, we show that V-type ATPase is localized to multiple subcellular compartments in P. falciparum, and its functionality throughout the asexual cycle varies depending on the parasite developmental stages.

Identification of potent and reversible piperidine carboxamides that are species-selective orally active proteasome inhibitors to treat malaria.

Malaria remains a global health concern as drug resistance threatens treatment programs. We identified a piperidine carboxamide (SW042) with anti-malarial activity by phenotypic screening. Selection of SW042-resistant Plasmodium falciparum (Pf) parasites revealed point mutations in the Pf_proteasome ß5 active-site (Pfß5). A potent analog (SW584) showed efficacy in a mouse model of human malaria after oral dosing. SW584 had a low propensity to generate resistance (minimum inoculum for resistance [MIR] >109) and was synergistic with dihydroartemisinin. Pf_proteasome purification was facilitated by His8-tag introduction onto ß7. Inhibition of Pfß5 correlated with parasite killing, without inhibiting human proteasome isoforms or showing cytotoxicity. The Pf_proteasome_SW584 cryoelectron microscopy (cryo-EM) structure showed that SW584 bound non-covalently distal from the catalytic threonine, in an unexplored pocket at the ß5/ß6/ß3 subunit interface that has species differences between Pf and human proteasomes. Identification of a reversible, species selective, orally active series with low resistance propensity provides a path for drugging this essential target.

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy.

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome.

PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.

The malaria parasite egress protease SUB1 is activated through precise, plasmepsin X-mediated cleavage of the SUB1 prodomain.

BACKGROUND: The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31. METHODS: Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX. RESULTS: We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity. CONCLUSIONS: Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity. GENERAL SIGNIFICANCE: Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.

Structure-function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum.

Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure-function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography-multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the ß-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum.

Malate dehydrogenase in parasitic protozoans: roles in metabolism and potential therapeutic applications.

The role of malate dehydrogenase (MDH) in the metabolism of various medically significant protozoan parasites is reviewed. MDH is an NADH-dependent oxidoreductase that catalyzes interconversion between oxaloacetate and malate, provides metabolic intermediates for both catabolic and anabolic pathways, and can contribute to NAD+/NADH balance in multiple cellular compartments. MDH is present in nearly all organisms; isoforms of MDH from apicomplexans (Plasmodium falciparum, Toxoplasma gondii, Cryptosporidium spp.), trypanosomatids (Trypanosoma brucei, T. cruzi) and anaerobic protozoans (Trichomonas vaginalis, Giardia duodenalis) are presented here. Many parasitic species have complex life cycles and depend on the environment of their hosts for carbon sources and other nutrients. Metabolic plasticity is crucial to parasite transition between host environments; thus, the regulation of metabolic processes is an important area to explore for therapeutic intervention. Common themes in protozoan parasite metabolism include emphasis on glycolytic catabolism, substrate-level phosphorylation, non-traditional uses of common pathways like tricarboxylic acid cycle and adapted or reduced mitochondria-like organelles. We describe the roles of MDH isoforms in these pathways, discuss unusual structural or functional features of these isoforms relevant to activity or drug targeting, and review current studies exploring the therapeutic potential of MDH and related genes. These studies show that MDH activity has important roles in many metabolic pathways, and thus in the metabolic transitions of protozoan parasites needed for success as pathogens.

Mutation of the Plasmodium falciparum Flavokinase Confers Resistance to Roseoflavin and 8-Aminoriboflavin.

The riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilization. To determine their mechanism of action, we generated roseoflavin-resistant parasites by in vitro evolution. Relative to wild-type, these parasites were 4-fold resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Whole genome sequencing of the resistant parasites revealed a missense mutation leading to an amino acid change (L672H) in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene. The IC50 values for roseoflavin and 8-aminoriboflavin against the roseoflavin-resistant parasites created through in vitro evolution were indistinguishable from those against parasites in which the missense mutation was introduced into the native PfFK. We also generated two parasite lines episomally expressing GFP-tagged versions of either the wild-type or mutant forms of PfFK. We found that PfFK-GFP localizes to the parasite cytosol and that immunopurified PfFK-GFP phosphorylated riboflavin, roseoflavin, and 8-aminoriboflavin. The L672H mutation increased the KM for roseoflavin, explaining the resistance phenotype. Mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonized by increasing the extracellular concentration of riboflavin, consistent with it also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore, are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite proliferation by inhibiting riboflavin phosphorylation and via the generation of toxic flavin cofactor analogues.