Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening

Aminopeptidases have emerged as new promising drug targets for the development of novel anti-parasitic drugs. An aspartyl aminopeptidase-like gene has been identified in the Toxoplasma gondii genome (TgAAP), although its function remains unknown. In this study, we characterized TgAAP and performed functional analysis of the gene product. Firstly, we expressed a functional recombinant TgAAP (rTgAAP) protein in Escherichia coli, and found that it required metal ions for activity and showed a substrate preference for N-terminal acidic amino acids Glu and Asp. Then, we evaluated the function and drug target potential of TgAAP using the CRISPR/Cas9 knockout system. Western blotting demonstrated the deletion of TgAAP in the knockout strain. Indirect immunofluorescence analysis showed that TgAAP was localized in the cytoplasm of the wild-type parasite, but was not expressed in the knockout strain. Phenotype analysis revealed that TgAAP knockout inhibited the attachment/invasion, replication, and substrate-specific activity in T. gondii. Finally, the activity of drug CID 23724194, previously described as targeting Plasmodium and malarial parasite AAP, was tested against rTgAAP and the parasite. Overall, TgAAP knockout affected the growth of T. gondii but did not completely abolish parasite replication and growth. Therefore, TgAAP may comprise a useful adjunct drug target of T. gondii.

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“…In vitro inhibition assay of r Tg AAP using CID 23724194, identified as a Pf M18AAP inhibitor 21 , is shown in Fig. 6a .…”

Section: Resultsmentioning

confidence: 99%

Characterization of aspartyl aminopeptidase from Toxoplasma gondii

Zheng

Cheng

Jia

et al. 2016

Sci Rep

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“…5. Using well established and previously utilized insilico tools such as PAINs and promiscuity filtering, we identified 666 compounds active in < 5 other screens that were also free from PAINs 17,[21][22][23] . Of these, 662 of these compounds were available for cherry-picking (Table 1).…”

Section: Resultsmentioning

confidence: 99%

Rescue of mutant gonadotropin-releasing hormone receptor function independent of cognate receptor activity

Smith

Bannister

Shumate

et al. 2020

Sci Rep

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Molecules that correct the folding of protein mutants, restoring their functional trafficking, are called pharmacoperones. Most are clinically irrelevant and possess intrinsic antagonist or agonist activity. Here, we identify compounds capable of rescuing the activity of mutant gonadotropinreleasing hormone receptor or GnRHR which, is sequestered within the cell and if dysfunctional leads to Hypogonadotropic Hypogonadism. To do this we screened the E90K GnRHR mutant vs. a library of 645,000 compounds using a cell-based calcium detection system. Ultimately, we identified 399 compounds with EC 50 ≤ 5 µM with no effect in counterscreen assays. Medicinal chemistry efforts confirmed activity of 70 pure samples and mode of action studies, including radioligand binding, inositol phosphate, and toxicity assays, proved that we have a series of tractable compounds that can be categorized into structural clusters. These early lead molecules rescue mutant GnRHR function and are neither agonist nor antagonists of the GnRHR cognate receptor, a feature required for potential clinical utility. The gonadotropin-releasing hormone receptor (GnRHR) belongs to a super family of G-protein coupled receptors (GPCRs). There are many mutations across the GnRHR that cause this protein to misfold, not traffic to the plasma membrane, and remain in the endoplasmic reticulum (ER). The quality control system (QCS) of the cell is responsible for the proper production, folding and transport of proteins from the ER to the cell membrane. Endogenous "chaperones" are present to protect the proteins from misfolding but, they are not protein-specific. Many misfolded protein mutants are able to retain (or regain) substantial biological activity but are viewed as inactive in the cell only due to their incorrect cellular location, not because of loss of function 1. Often times, these misfolded proteins are unable to traffic to the cell membrane. When this occurs, ligands cannot bind to nor activate these proteins, and a physiologic defect occurs 1,2. G-protein-coupled receptors are maintained under the QCS machinery. Normally they are produced in the ER and shuttled to the plasma membrane where they become functional with the appropriate ligands. After ligand binding, the WT GnRHR activates the Gα q /11 G protein, which activates the inositol phosphate pathway, leading to the release of intracellular calcium which affords us the ability to easily interrogate this target for drug screening 3. While GnRHR signals primarily through Gα q /11 to activate phospholipase C β (PLCβ) it has also been described as coupling through Gα s which drives adenylate cyclase and ultimately cAMP formation thus stimulating PKA activation of CREB. Although gonadotropin promoter subunits contain cAMP response elements, it appears that the MAPK cascade is favored over cAMP pathway for driving gonadotropin promoter activation. This is an important distinction because the MAPK cascade is directly associated to the Gα q /11 signaling pathway which is the foundation basis of our assa...

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“…However, the involvement of this metallopeptidase in parasite survival remains controversial since Dalal and Klemba (2007) [ 14 ] were able to delete the gene without finding any deleterious effects, concluding that the protein function was not essential. This metallopeptidase is, however, considered a potential therapeutic target [ 58 ]. Pf M18AAP has also been shown to bind in vitro to human erythrocyte spectrin (spectrin binding region of 33 amino acids only present in P. falciparum ), showing multiple enzymatic functions in the parasite and the erythrocytic host [ 36 ].…”

Section: Resultsmentioning

confidence: 99%

Metallopeptidases ofToxoplasma gondii:in silicoidentification and gene expression

et al. 2018

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Metallopeptidases are a family of proteins with domains that remain highly conserved throughout evolution. These hydrolases require divalent metal cation(s) to activate the water molecule in order to carry out their catalytic action on peptide bonds by nucleophilic attack. Metallopeptidases from parasitic protozoa, including Toxoplasma, are investigated because of their crucial role in parasite biology. In the present study, we screened the T. gondii database using PFAM motifs specific for metallopeptidases in association with the MEROPS peptidase Database (release 10.0). In all, 49 genes encoding proteins with metallopeptidase signatures were identified in the Toxoplasma genome. An Interpro Search enabled us to uncover their domain/motif organization, and orthologs with the highest similarity by BLAST were used for annotation. These 49 Toxoplasma metallopeptidases clustered into 15 families described in the MEROPS database. Experimental expression analysis of their genes in the tachyzoite stage revealed transcription for all genes studied. Further research on the role of these peptidases should increase our knowledge of basic Toxoplasma biology and provide opportunities to identify novel therapeutic targets. This type of study would also open a path towards the comparative biology of apicomplexans.

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Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening

Cited by 16 publications

References 25 publications

Characterization of aspartyl aminopeptidase from Toxoplasma gondii

Characterization of aspartyl aminopeptidase from Toxoplasma gondii

Rescue of mutant gonadotropin-releasing hormone receptor function independent of cognate receptor activity

Metallopeptidases ofToxoplasma gondii:in silicoidentification and gene expression

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