Figures & data
Table 1. Genomic validation techniques applied to antimalarial target validation.
Figure 1. Schematic representation of genomic validation techniques applied to Plasmodium. (a) Single crossover [Citation57]. (b) Double crossover [Citation59]. (c) Zinc Finger Nucleases (ZNFs). The parasites are transformed with a construct that encodes for pairs of engineered zinc-finger proteins linked to an endonuclease. After endogenous expression of the ZFNs (showed in the picture as ZFNL and ZFNR) the zinc finger proteins bind to their target sequence on each side of the chromosome and induce nuclease activity, causing a double-strand break (DSB). This allows for alteration of the target DNA sequence by taking advantage of the homology-directed DNA repair mechanisms at the site of nuclease action [Citation64]. (d) Clustered Regularly Interspaced Short Palindromic Repeats-associated proteins (CRISPR-Cas) system. The parasites are co-transfected with two plasmids; one contains the Cas9 nuclease gene, and the other contains the DNA fragment encoding the sgRNA and the donor template DNA. After endogenous expression, Cas9 nuclease forms an RNA-protein complex with the sgRNA. Binging of the sgRNA to the specific genomic locus induces Cas9 to perform a double-strand break (DSB) on the genomic DNA. Homologous recombination between the cleaved genomic locus and a donor DNA present in the sgRNA construct leads to the integration of the latest to the genome [Citation63,Citation91].
![Figure 1. Schematic representation of genomic validation techniques applied to Plasmodium. (a) Single crossover [Citation57]. (b) Double crossover [Citation59]. (c) Zinc Finger Nucleases (ZNFs). The parasites are transformed with a construct that encodes for pairs of engineered zinc-finger proteins linked to an endonuclease. After endogenous expression of the ZFNs (showed in the picture as ZFNL and ZFNR) the zinc finger proteins bind to their target sequence on each side of the chromosome and induce nuclease activity, causing a double-strand break (DSB). This allows for alteration of the target DNA sequence by taking advantage of the homology-directed DNA repair mechanisms at the site of nuclease action [Citation64]. (d) Clustered Regularly Interspaced Short Palindromic Repeats-associated proteins (CRISPR-Cas) system. The parasites are co-transfected with two plasmids; one contains the Cas9 nuclease gene, and the other contains the DNA fragment encoding the sgRNA and the donor template DNA. After endogenous expression, Cas9 nuclease forms an RNA-protein complex with the sgRNA. Binging of the sgRNA to the specific genomic locus induces Cas9 to perform a double-strand break (DSB) on the genomic DNA. Homologous recombination between the cleaved genomic locus and a donor DNA present in the sgRNA construct leads to the integration of the latest to the genome [Citation63,Citation91].](/cms/asset/d7e3cf0b-bb72-4f09-a26a-31950a4adede/iedc_a_1691996_f0001_oc.jpg)
Table 2. Conditional and inducible validation techniques applied to antimalarial target validation.
Figure 2. Schematic representation of conditional/inducible validation techniques applied to Plasmodium. (a) Post-translational knockdown principle in Plasmodium. Integration of FK506‑binding protein (FKBP)-based destabilization domain (DD) or an Escherichia coli DHFR destabilizing domain (DDD) in fusion with the target protein leads to protein degradation by Plasmodium degradation machinery when stabilizing compounds are removed from the culture [Citation65,Citation89]. (b) Tet-OFF system. Integration of episomal DNA upstream the target gene introduces a transcriptional transactivator domain (TRAD) gene sequence and an inducible promoter (TetO). In the absence of Anhydrotetracycline (ATc), the expressed TRAD binds to the inducible promoter, stimulating the mRNA expression of the target gene. Addition of ATc prevents binding of the TRAD to the TetO promoter reducing the expression of the target gene [Citation92]. (c) Riboswitch system. Integration of Glms coding sequence to 3ʹUTR of target gene coding sequence leads to degradation of its transcribed mRNA in the presence of glucosamine-6-phosphate (GlcN) due Glms ribozyme activity [Citation97,Citation98]. (d) Conditional knockout. Recombinase target sequences (loxP for Cre recombinase and frt for FLP recombinase) are integrated to the endogenous locus flanking the target gene coding region. The conditional expression of the recombinase encoded by the transfected construct or induction of diCre dimerization results in recombination of the two loxP or frt sites leading to target gene sequence excision [Citation101,Citation102].
![Figure 2. Schematic representation of conditional/inducible validation techniques applied to Plasmodium. (a) Post-translational knockdown principle in Plasmodium. Integration of FK506‑binding protein (FKBP)-based destabilization domain (DD) or an Escherichia coli DHFR destabilizing domain (DDD) in fusion with the target protein leads to protein degradation by Plasmodium degradation machinery when stabilizing compounds are removed from the culture [Citation65,Citation89]. (b) Tet-OFF system. Integration of episomal DNA upstream the target gene introduces a transcriptional transactivator domain (TRAD) gene sequence and an inducible promoter (TetO). In the absence of Anhydrotetracycline (ATc), the expressed TRAD binds to the inducible promoter, stimulating the mRNA expression of the target gene. Addition of ATc prevents binding of the TRAD to the TetO promoter reducing the expression of the target gene [Citation92]. (c) Riboswitch system. Integration of Glms coding sequence to 3ʹUTR of target gene coding sequence leads to degradation of its transcribed mRNA in the presence of glucosamine-6-phosphate (GlcN) due Glms ribozyme activity [Citation97,Citation98]. (d) Conditional knockout. Recombinase target sequences (loxP for Cre recombinase and frt for FLP recombinase) are integrated to the endogenous locus flanking the target gene coding region. The conditional expression of the recombinase encoded by the transfected construct or induction of diCre dimerization results in recombination of the two loxP or frt sites leading to target gene sequence excision [Citation101,Citation102].](/cms/asset/90e2a57f-92f0-44b7-8ed5-a72501ccf8ed/iedc_a_1691996_f0002_oc.jpg)
Table 3. Proteomic approaches applied to antimalarial target validation.
Figure 3. Schematic representation of protein-based validation techniques applied to Plasmodium. (a) SELEX methodology. After incubation of the aptamer library with culture or lysate of parasites, unbound nucleic acids are separated from bound ones. Nucleic acid-protein complexes are then dissociated and the nucleic acid pool is amplified and enriched. The new generated nucleic acid pool serves as a starting library for a new SELEX cycle composed of identical steps as the first round. The number of SELEX repetition depends on the library type used and on specific enrichment achieved per a selection cycle. After the last round of aptamer selection, the PCR products are cloned and sequenced [Citation107,Citation108]. (b) and (c) Overview of two independent strategies for the use of Protein Interference Assay (PIA) in Plasmodium. In the first, the transfection of parasites with a construct encoding for a copy of the target gene containing one or more inactivating mutations on the oligomeric interface leads to endogenous expression of this mutant. The formation of oligomeric complexes between the native active protein and the mutant protein results in inactive heterocomplexes, leading to a knockdown effect at protein level. In the second, clashing mutation(s) are introduced to one oligomeric interface while other(s) oligomeric interface(s) is preserved. The endogenous expression of the mutant allows for heterocomplex formation with the native protein, preventing the formation of the full oligomeric complex. The presence of the cashing mutation in at least one monomer leads to inactivation of proteins which function depend on the oligomeric state, thus, resulting in a knockdown effect [Citation10,Citation122].
![Figure 3. Schematic representation of protein-based validation techniques applied to Plasmodium. (a) SELEX methodology. After incubation of the aptamer library with culture or lysate of parasites, unbound nucleic acids are separated from bound ones. Nucleic acid-protein complexes are then dissociated and the nucleic acid pool is amplified and enriched. The new generated nucleic acid pool serves as a starting library for a new SELEX cycle composed of identical steps as the first round. The number of SELEX repetition depends on the library type used and on specific enrichment achieved per a selection cycle. After the last round of aptamer selection, the PCR products are cloned and sequenced [Citation107,Citation108]. (b) and (c) Overview of two independent strategies for the use of Protein Interference Assay (PIA) in Plasmodium. In the first, the transfection of parasites with a construct encoding for a copy of the target gene containing one or more inactivating mutations on the oligomeric interface leads to endogenous expression of this mutant. The formation of oligomeric complexes between the native active protein and the mutant protein results in inactive heterocomplexes, leading to a knockdown effect at protein level. In the second, clashing mutation(s) are introduced to one oligomeric interface while other(s) oligomeric interface(s) is preserved. The endogenous expression of the mutant allows for heterocomplex formation with the native protein, preventing the formation of the full oligomeric complex. The presence of the cashing mutation in at least one monomer leads to inactivation of proteins which function depend on the oligomeric state, thus, resulting in a knockdown effect [Citation10,Citation122].](/cms/asset/c63e87de-2d19-443f-8d14-5d6fc55fd396/iedc_a_1691996_f0003_oc.jpg)
Figure 4. (a) The dimeric structure of plasmodial aspartate aminotransferase (PfAspAT). (b) and (c) The oligomeric interface of the WT-PfAspAT dimer. Residues Tyr68 and Arg257 from both subunits are shown in sticks[Citation122].
![Figure 4. (a) The dimeric structure of plasmodial aspartate aminotransferase (PfAspAT). (b) and (c) The oligomeric interface of the WT-PfAspAT dimer. Residues Tyr68 and Arg257 from both subunits are shown in sticks[Citation122].](/cms/asset/98c25a24-a714-4133-992d-25fe8026c8a6/iedc_a_1691996_f0004_oc.jpg)
Figure 5. Mutations designed for the protein interference assay (PIA) approach targeting plasmodial malate dehydrogenase (PfMDH). (a) The tetrameric structure of PfMDH, the different subunits are labeled A-D. (b) The A-B interface of the wild-type (WT)-PfMDH tetramer. Residues Glu118 from both subunits are shown in sticks. (c) The steric clash generated by the introduction of a Tryptophan in position 118. The mutant monomer is shown in green and WT monomer is shown in yellow. (d) A-C interface of WT-PfMDH tetramer. Residues Val190 from both subunits are shown in sticks. (e) The steric clash generated by the introduction of a tryptophan in position 190. The mutant monomer is shown in green and WT monomer is shown in magenta [Citation122,Citation124].
![Figure 5. Mutations designed for the protein interference assay (PIA) approach targeting plasmodial malate dehydrogenase (PfMDH). (a) The tetrameric structure of PfMDH, the different subunits are labeled A-D. (b) The A-B interface of the wild-type (WT)-PfMDH tetramer. Residues Glu118 from both subunits are shown in sticks. (c) The steric clash generated by the introduction of a Tryptophan in position 118. The mutant monomer is shown in green and WT monomer is shown in yellow. (d) A-C interface of WT-PfMDH tetramer. Residues Val190 from both subunits are shown in sticks. (e) The steric clash generated by the introduction of a tryptophan in position 190. The mutant monomer is shown in green and WT monomer is shown in magenta [Citation122,Citation124].](/cms/asset/b76c43b3-c295-4096-b6b5-f1c42f69bd72/iedc_a_1691996_f0005_oc.jpg)