Synthesis and Potent Antimalarial Activity of Kalihinol B

Of the 50+ kalihinane diterpenoids reported to date, only five had been tested for antimalarial activity, in spite of the fact that kalihinol A is the most potent among the members of the larger family of antimalarial isocyanoterpenes. We have validated a strategy designed to access many of the kalihinanes with a 12-step enantioselective synthesis of kalihinol B, the tetrahydrofuran isomer of kalihinol A (a tetrahydropyran). Kalihinol B shows similarly high potency against chloroquine-resistant Plasmodium falciparum

Additional file 3: of The mRNA-bound proteome of the human malaria parasite Plasmodium falciparum

Document containing supplementary Figures S1–S10 and their legends. (PDF 1225 kb

Isodon trichocarpus Kudo

原著和名: クロバナヒキオコシ科名: シソ科 = Labiatae採集地: 長野県 白馬岳 (信濃 白馬岳)採集日: 1963/8/22採集者: 萩庭丈壽整理番号: JH042870国立科学博物館整理番号: TNS-VS-99287

Domain architecture of the ubiquitylating components of the <i>Plasmodium</i> ERAD system and cf-C3HC4 domain alignment of PF14_0215.

<p>(<b>A</b>) Proteins are represented as black boxes of length proportional to their sizes in amino acids (aa). SP =  signal peptide; TM = transmembrane domain; ThiF/UBACT = repeats found in E1 ubiquitin-activating enzymes; UBA_e1_C = E1 ubiquitin-activating enzyme catalytic domain; UQ_con = E2 ubiquitin-conjugating enzyme domain; zf-C3HC4 = E3 ubiquitin ligase RING finger domain. Catalytic cysteines are marked in yellow. (<b>B</b>) Using MUSCLE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043477#pone.0043477-Edgar1" target="_blank">[43]</a>, we performed protein alignments on PF14_0215 and found that its zf-C3HC4 domain had high homology to other previously characterized HRD1 of various model systems.</p

Graphical depiction of the <i>Plasmodium</i> ERAD pathway in protein quality control and its potential for antimalarial strategies.

<p>Similar to their human and yeast counterparts, the components of the <i>Plasmodium</i> ERAD system likely serve to recognize and translocate misfolded ER luminal proteins to the cytosol for degradation by the 26S proteasome. A simplified flowchart is presented: (1) misfolded proteins are recognized and recruited to the ERAD complex, (2) where they are inserted across the ER membrane through a pore formed by a complex of ERAD proteins. (3) During translocation, the aberrant proteins are poly-ubiquitylated by the concerted action of the E1 ubiquitin-activating enzyme <i>Pf</i>UBA1 (PFL1245w), the E2 ubiquitin-conjugating enzyme PfUBC (PFL0190w), and the E3 ligase <i>Pf</i>HRD1 (PF14_0215). (4) The ubiquitylated aberrant proteins are extracted by the CDC48-UFD1-NPL4 AAA-ATPase complex and shuttled to the 26S proteasome (5) for degradation. <i>Plasmodium</i> ERAD drug target candidates are highlighted with an orange star.</p

<i>In vitro</i> ubiquitylation assay of <i>Pf</i>HRD1 (PF14_0215).

<p>(<b>A</b>) Epitope-tagged recombinant <i>Pf</i>HRD1 was expressed in <i>E. coli</i> and purified. Anti-GST immunoblot on purified protein extracts reveals the presence of detached free GST tags (MW = 29 kDa) and of <i>Pf</i>HRD1-GST (MW = 42.6 kDa). (<b>B</b>) <i>In vitro</i> ubiquitylation assays were performed with commercial ubiquitin (Ub), human E1 UBA1, varying human E2 UBC enzymes (<i>i.e.,</i> UBCH2, UBCH3, UBCH5a, UBCH6, UBCH7, UBCH8, UBCH10, and UBCH13), and our purified extract of epitope-tagged recombinant <i>Pf</i>HRD1. Ubiquitylation patterns were revealed on anti-ubiquitin immunoblots. Different patterns of auto-ubiquitylation are observed with UBCH5a, UBCH13, and UBCH6. (<b>C</b>) <i>In vitro</i> ubiquitylation assays were performed in the presence (+) or absence (-) of commercial ubiquitin (UB), human E1 UBA1, human E2 UBCH5a, and our purified extract of epitope-tagged recombinant <i>Pf</i>HRD. Ubiquitylation patterns were revealed on anti-ubiquitin immunoblots. Ubiquitylation is specifically observed only when all components are mixed together, including <i>Pf</i>HRD1. (<b>D</b>) <i>In vitro</i> ubiquitylation assays were performed with commercial ubiquitin (Ub), human E1 UBA1, the human E2 enzymes UBCH5a, UBCH6, and UBCH13, and our purified extract of epitope-tagged recombinant <i>Pf</i>HRD1. The attachment of ubiquitin on the recombinant <i>Pf</i>HRD1 itself was detected by anti-GST immuno-detection. <i>Pf</i>HRD1 is poly-ubiquitylated in the presence of UBCH5a and mono-ubiquitylated in the presence of UBCH6 whereas UBCH13 does not permit <i>Pf</i>HRD1 auto-ubiquitylation.</p

<i>In vitro</i> ubiquitylation assay of <i>Pf</i>UBA1 and <i>Pf</i>UBC.

<p>(<b>A</b>) 6xHIS-tagged recombinant <i>Pf</i>UBA1 (E1) and <i>Pf</i>UBC (E2) are depicted and anti-HIS blots reveal purification. (<b>B</b>) <i>In vitro</i> ubiquitylation assays were performed with recombinant <i>Pf</i>UBC (E2), which revealed an attachment of a single ubiquitin when incubated with human UBA1 (E1) (second lane from the right) and an increased number of ubiquitylation products when both UBA1 and recombinant <i>Pf</i>HRD1(E3) was added (far right lane). (<b>C</b>) When incubated with human UBA1 (E1), recombinant <i>Pf</i>UBC (E2) (along with the other necessary reagents) attaches polymers of ubiquitin to recombinant <i>Pf</i>HRD1 (E3) (far right lane). (<b>D</b>) Recombinant <i>Pf</i>UBA1 (E1) is capable of attaching a single ubiquitin to recombinant <i>Pf</i>UBC (E2), which is depicted by the appearance of a 27 kDa band (second lane from the right). Extra banding was not detected with the addition of recombinant <i>Pf</i>HRD1 (E3) (far right lane).</p

Cellular localization of PfHRD1.

<p>(<b>A</b>) <i>Pf</i>HRD1 and plasmepsin V (PMV), an ER membrane marker, co-localize in <i>P. falciparum</i>. (<b>B</b>) At various stages of the parasite life cycle, <i>Pf</i>HRD1 was co-stained with the nuclei (DAPI) and the apicoplast (ACP-GFP) in the <i>P. falciparum</i> strain D10.</p

ERAD inhibitor Eeyarestatin I inhibits <i>P. falciparum in vitro</i> and increases the amount of ubiquitylated <i>Plasmodium</i> proteins.

<p>(<b>A</b>) Eeyarestatin I (ES₁) inhibits <i>P. falciparum</i> 3D7 strain with an IC₅₀ value of 3.5415±1.0399 µM (mean value ± standard error). Dose-response curve is plotted with increasing concentrations of ES₁ (<b>B</b>) ES₁ treatment leads to an accumulation of ubiquitylated proteins in <i>P. falciparum</i>. Anti-ubiquitin immunoblots show higher levels of ubiquitylated protein levels in <i>P. falciparum</i> parasites when treated with ES₁ (lane 2) compared to the control (DMSO, lane 1). As a comparison, parasite were also treated with proteasome inhibitor MG132 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043477#pone.0043477-Melikova1" target="_blank">[48]</a> (lane 3) and exhibited slightly higher amounts of ubiquitylated proteins. When parasites were treated with both ES₁ and MG132 (lane 4), significant increases in ubiquitylated products were detected.</p

Localization studies of PfUba and Pfubc7.

<p>(<b>A,B</b>) IFA experiments, using different parasite stages, show that <i>Pf</i>UBA1 and <i>Pf</i>UBC localize mainly to the cytosol. (<b>C</b>) When co-immunostained with plasmepsin V (PMV), a <i>Plasmodium</i> ER membrane protein marker, there was noticeable overlap between the two proteins, suggesting <i>Pf</i>UBA1 recruitment to the ER as well.</p