Research ArticleMalaria

Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay

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Science Translational Medicine  02 Jan 2019:
Vol. 11, Issue 473, eaau3174
DOI: 10.1126/scitranslmed.aau3174
  • Fig. 1 Schematic illustration of P. falciparum CETSA protocol.

    Lysate and intact-cell CETSA experiments are conducted using soluble parasite protein lysate or magnetic cell sorting–enriched infected RBCs (iRBCs), respectively. In both scenarios, the samples are separated into 10 identical fractions, subjected to drug treatment, and exposed to the thermal challenge to denature and irrevocably precipitate unstable proteins. Two alternative CETSA variants, ITDR or melt curve, can be performed. ITDR involves treating samples with a drug concentration gradient and exposing them to a single temperature, whereas melt curve CETSA relies on the presence or absence of the drug in corresponding samples, which are then heated to 10 distinct temperatures along 37° to 73°C thermal gradient. The soluble protein is isolated by centrifugation and, in case of whole-cell approach, preceded by cell lysis, and samples are reduced, alkylated, digested by lysyl-endopeptidase (Lys-C) and trypsin and then labeled with distinct TMT10 isobaric peptide tags. Peptide abundance across 10 fractions is then quantified through multiplexed MS. After mapping to the plasmodium proteome database, values are translated to protein quantities along the thermal/drug gradient.

  • Fig. 2 P. falciparum proteome melting behavior.

    (A) Global protein melting behavior in lysate (green) and intact-cell (purple) melt curve CETSA in the absence of drugs. The box plot is drawn on the basis of independent protein melting profiles within each dataset representing remaining soluble protein abundance across the temperature gradient (x axis) relative to the nondenaturing 37°C condition (black band) on the y axis. Median (second quartile) protein levels are indicated with a black band, first and third quartiles with colored boxes, whereas the lowest/highest datum within 1.5 × IQR (interquartile range) of the lower/higher quartile is represented with whiskers. Data outliers not included within whiskers are plotted as dots. (B) Proteome-wide Tm distribution in melt curve CETSA lysate (green) and intact cell (purple). Only proteins achieving >50% denaturation within the thermal gradient, detected at >3 peptide-spectrum matches (PSMs), and with <5°C Tm SD between replicates were included in the analysis. Number of proteins (left, y axis) exhibiting Tm at a given temperature (x axis) is plotted as bar chart, whereas their cumulative number including proteins with lower Tm is indicated on the right (y axis). (C) Tm comparison of 964 proteins identified in lysate and intact-cell melt curve. The correlation coefficient (R2) of linear regression analysis is indicated for each dataset pair. Dashed black trend-line indicates perfect overlap of values in two conditions.

  • Fig. 3 Protein target engagement by pyrimethamine and E64d.

    (A) Whole proteome analysis in lysate ITDR experiments under 0 to 100 μM pyrimethamine (PM) treatment with thermal challenges at 59°C (blue dots) or 65°C (red triangles). Distribution of protein stabilization is plotted as a function of R2 value (which quantifies the adherence of protein stabilization profile to the dose-response trend) against ΔAUC (area under the curve of heat-challenged sample normalized against nondenaturing 37°C control) for all proteins detected in the assay and the reference condition (“n”). Three times of median absolute deviation (MAD) of ΔAUC in each dataset (MAD × 3) and R2 = 0.8 cutoffs are indicated on the graph. Significant hit − PfDHFR is highlighted in red. (B) Protein stabilization curve of PfDHFR identified in (A). The extent of stabilization under thermal denaturation conditions [59°C (blue) or 65°C (red)] is plotted relative to no-drug control with nondenaturing control condition plotted in black. (C) Whole proteome analysis in intact-cell ITDR experiments under 0 to 125 μM E64d treatment with thermal challenges at 51°C (blue dots) or 57°C (red triangles). Distribution of protein stabilization is plotted as a function of R2 value against ΔAUC, similarly as in (A). (D) Protein stabilization curves of hits identified in (C). The extent of stabilization under thermal denaturation conditions [51°C (blue) or 57°C (red)] is plotted relative to no-drug control with nondenaturing control condition plotted in black.

  • Fig. 4 Deorphanization of quinine and mefloquine protein targets.

    (A) Whole proteome ITDR analysis of quinine-treated (10 to 0 μM) lysate samples, plotted as a function of R2 against ΔAUC for all proteins (“n”) detected in the 51°C thermal challenge condition dataset (blue dots) and the reference. The MAD × 3 of ΔAUC and R2 = 0.8 cutoffs are indicated, and stabilized proteins are highlighted. (B) Protein stabilization profile of the top drug-binding candidate identified in the preceding panel. The extent of stabilization (y axis), depicted as the remaining soluble protein abundance after thermal challenge [51°C (blue)] relative to the no-drug control is plotted along the drug gradient (x axis). The nondenaturing 37°C control condition is plotted in black. Subsequent panel pairs represent the results of corresponding ITDR analyses, depicted and described as in (A) and (B), but conducted for: mefloquine (0 to 100 μM) in lysate in (C) and (D); quinine (0 to 10 μM) in intact-cell in (E) and (F); mefloquine (0 to 10 μM) in intact-cell in (G) and (H). All intact-cell analyses have an additional 57°C thermal challenge condition (red triangles). The stabilization curves for the two additional hits identified in mefloquine intact-cell ITDR 57°C but not represented in (H) are listed in the fig. S4.

  • Fig. 5 Validation of PfPNP as a target of quinine and mefloquine.

    (A) DSF analysis of PfPNP stabilization by ImmH, mefloquine (MFQ), quinine (QN), quinidine (QD), lumefantrine (LUM), chloroquine (CQ), and primaquine (PMQ) in a concentration gradient (0 to 100μM). The change in PfPNP Tm under drug exposure relative to untreated sample is represented on the y axis in relation to drug concentration. (B and C) Sensorgrams, double-referenced binding data (black traces) and fitted (blue traces) from SPR analysis of PfPNP binding affinity to three drugs. ImmH and quinine (B) were analyzed using single-cycle experiments and kinetic 1-to-1 model, whereas mefloquine (C) was analyzed in a multicycle experiment and fitted using a steady-state model. Mefloquine binding isotherm is represented below the sensorgram. (D) PfPNP enzymatic activity inhibition by quinine and mefloquine across 0 to 50 μM and 0 to 250 μM drug concentration gradients, respectively. (E) Fractional inhibitory concentration 50 (FIC) analysis for the combinations of ImmH with mefloquine, quinine, and E64d. Isoboles representing FIC index of each drug in combination are plotted across a range of drug pair concentrations. Reference isobole indicating Loewe additivity model is presented as a black dashed line.

  • Fig. 6 Cocrystal structures of PfPNP with quinine and mefloquine.

    (A) Overlay of PFPNP-quinine⋅PO4 and PfPNP-mefloquine⋅PO4 cocrystal structures. Quinine is represented as pink sticks, mefloquine as green sticks, and the two corresponding protein structures in yellow and blue, respectively. Oxygen, blue; phosphorus, orange; oxygen, red; fluorine, light cyan. (B) Surface representation of both structures, showing hydrophobic (red) regions and both ligands bound to PfPNP. (C and D) Magnified binding pockets of structures presented in (A); major interactions between ligands and binding pocket amino acids are represented by black dashed lines. (E) Fo-Fc electron density maps of quinine and mefloquine residing within binding pockets of the two cocrystal structures contoured at 1σ level.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/473/eaau3174/DC1

    Materials and Methods

    Fig. S1. Coaggregation of protein complexes in P. falciparum melt curve CETSA.

    Fig. S2. The comparison of protein melting behavior between RBC, P. falciparum, and K562 cells.

    Fig. S3. The effect of pyrimethamine on thermal stability of PfDHFR-TS across four CETSA experimental variants.

    Fig. S4. Mefloquine-induced stabilization profiles of Mge1 and Hsp70-3 in intact-cell ITDR assay.

    Fig. S5. Western blot detection of PfPNP stabilization in mefloquine- and quinine-treated ITDR datasets.

    Fig. S6. Human protein engagement by antimalarial drugs in intact-cell ITDR assays.

    Fig. S7. ITC analysis of quinine and mefloquine binding to PfPNP.

    Fig. S8. Cocrystal structure overlay with 2bsx and 1nw4 reference PfPNP structures.

    Table S1. High- and low-confidence protein stabilizations observed in quinine/mefloquine ITDR assays.

    Table S2. SPR measurements of PfPNP-mefloquine/quinine/ImmH KD.

    Table S3. ITC binding affinities for PfPNP-mefloquine/quinine interaction.

    Table S4. PfPNP in vitro enzymatic activity inhibition by mefloquine and quinine.

    Table S5. Combinatory effect of fixed-ratio drug combinations against P. falciparum 3D7 strain.

    Table S6. Data collection and refinement statistics of PfPNP-mefloquine and PfPNP-quinine cocrystals.

    Data S1. Intact-cell and lysate CETSA melt curve analysis of P. falciparum proteome.

    Data S2. Intact-cell CETSA melt curve analysis of RBC proteome.

    Data S3. PM lysate ITDR–plasmodium proteome.

    Data S4. E64d intact-cell ITDR–plasmodium proteome.

    Data S5. Quinine lysate ITDR–plasmodium proteome.

    Data S6. Mefloquine lysate ITDR–plasmodium proteome.

    Data S7. Quinine intact-cell ITDR–plasmodium proteome.

    Data S8. Quinine intact-cell ITDR–human proteome.

    Data S9. Mefloquine intact-cell ITDR–plasmodium proteome.

    Data S10. Mefloquine intact-cell ITDR–human proteome.

    References (7381)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Coaggregation of protein complexes in P. falciparum melt curve CETSA.
    • Fig. S2. The comparison of protein melting behavior between RBC, P. falciparum, and K562 cells.
    • Fig. S3. The effect of pyrimethamine on thermal stability of PfDHFR-TS across four CETSA experimental variants.
    • Fig. S4. Mefloquine-induced stabilization profiles of Mge1 and Hsp70-3 in intact-cell ITDR assay.
    • Fig. S5. Western blot detection of PfPNP stabilization in mefloquine- and quinine-treated ITDR datasets.
    • Fig. S6. Human protein engagement by antimalarial drugs in intact-cell ITDR assays.
    • Fig. S7. ITC analysis of quinine and mefloquine binding to PfPNP.
    • Fig. S8. Cocrystal structure overlay with 2bsx and 1nw4 reference PfPNP structures.
    • Table S1. High- and low-confidence protein stabilizations observed in quinine/mefloquine ITDR assays.
    • Table S2. SPR measurements of PfPNP-mefloquine/quinine/ImmH KD.
    • Table S3. ITC binding affinities for PfPNP-mefloquine/quinine interaction.
    • Table S4. PfPNP in vitro enzymatic activity inhibition by mefloquine and quinine.
    • Table S5. Combinatory effect of fixed-ratio drug combinations against P. falciparum 3D7 strain.
    • Table S6. Data collection and refinement statistics of PfPNP-mefloquine and PfPNP-quinine cocrystals.
    • Legends for data S1 to S10
    • References (7381)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data S1 (.pdf format). Intact-cell and lysate CETSA melt curve analysis of P. falciparum proteome.
    • Data S2 (.pdf format). Intact-cell CETSA melt curve analysis of RBC proteome.
    • Data S3 (.pdf format). PM lysate ITDR–plasmodium proteome.
    • Data S4 (.pdf format). E64d intact-cell ITDR–plasmodium proteome.
    • Data S5 (.pdf format). Quinine lysate ITDR–plasmodium proteome.
    • Data S6 (.pdf format). Mefloquine lysate ITDR–plasmodium proteome.
    • Data S7 (.pdf format). Quinine intact-cell ITDR–plasmodium proteome.
    • Data S8 (.pdf format). Quinine intact-cell ITDR–human proteome.
    • Data S9 (.pdf format). Mefloquine intact-cell ITDR–plasmodium proteome.
    • Data S10 (.pdf format). Mefloquine intact-cell ITDR–human proteome.

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