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SCIENCE CHINA Life Sciences
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SCIENCE CHINA Life Sciences, Volume 63 , Issue 7 : 1016-1025(2020) https://doi.org/10.1007/s11427-019-1581-2

Discovery of a natural fluorescent probe targeting the Plasmodium falciparum cysteine protease falcipain-2

Lili Zhu 1,†, Lei Shan 2,†, Junsheng Zhu 1, Li Li 2, Shiliang Li 1, Liyan Wang 1, Jiawei Wang 1, Shoude Zhang 1,2, Hongchang Zhou 3, Weidong Zhang 2,*, Honglin Li 1,*
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  • ReceivedSep 5, 2019
  • AcceptedNov 26, 2019
  • PublishedFeb 9, 2020
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Abstract


Funded by

the National Key Research and Development Program(2016YFA0502304,to,H.L.)

the National Natural Science Foundation of China(81825020)

the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program”

China(2018ZX09711002)

the Fundamental Research Funds for the Central Universities

Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund(the,second,phase)

Professor of Chang Jiang Scholars Program(to,W.Z.)

and the Natural Science Foundation of Zhejiang Province(LY15H190007)


Acknowledgment

We thank Prof. Hua Xie (Shanghai Insitute of Materia Medica, Chinese Academy of Sciences) for expert technical assistance with the target location experiments using confocal microscopy; Prof. Jin Huang (East China University of Science and Technology) for the enzyme inhibition assays and the effect evaluation of NP1024 on degradation of hemoglobin by FP-2; Profs. Huaimin Zhu, Weiqing Pan, Heng Peng, and Weibin Guan (The Secondary Military Medical University) for sharing Plasmodium parasite strains and providing technical assistance with the in vitro culture of Plasmodium parasites; Xianwen Yang (Third Institute of Oceanography, State Oceanic Administration) for providing natural compounds used in this study; Drs. Troy J. Smillie and Ikhlas A. khan (National Center for Natural Products Research, the University of Mississippi) for in vitro anti-malarial assay; We also thank Prof. Hualiang Jiang and Eric Xu (Shanghai Insitute of Materia Medica) for their technological assistance and helpful suggestions. This work was supported by the National Key Research and Development Program (2016YFA0502304 to H.L.), the National Natural Science Foundation of China (81825020), the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program”, China (2018ZX09711002), the Fundamental Research Funds for the Central Universities, Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase) (U1501501), Professor of Chang Jiang Scholars Program (to W.Z.), and the Natural Science Foundation of Zhejiang Province (LY15H190007). Honglin Li is also sponsored by the National Program for Special Supports of Eminent Professionals and National Program for Support of Top-notch Young Professionals.


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1 Structures and inhibitory activities of other FP-2 inhibitors identified in this study.

Figure S2 Two repeated Surface Plasmon Resonance measurements for analysis of the binding affinity between FP-2 and NP1024 (A and B). The adjusted (Fc2-Fc1) sensorgrams are shown (left); the binding affinity was obtained using the steady-state model-fitting (right).

Figure S3 Colocalization of NP1024 and FP-2 in one malaria parasite-infected erythrocyte.

Figure S4 Analysis of the accurate molecular weight of NP1024 by LC-TOF MS.

Figure S5 Analysis of the erythrocyte lysate of Plasmodium berghei-infected mice by LC-TOF MS.

Video S1 Target localization of NP1024 in erythrocytes of Plasmodium berghei-infected mice detected by XYZ scanning images. Points 1, 2, 3, 4, 5: parasite-infected erythrocytes at the trophozoite stage; point 6: parasite-infected erythrocytes at the ring stage. Detecting instrument: Olympus FV1000 Confocal; objective amplification: 100×; zoom: ×2; excitation wavelength: 488 nm; Z dimension: 26.8–24.0 mm; scanning step size: 0.40 mm/slice.

Video S2 Target localization of NP1024 in erythrocytes of Plasmodium berghei-infected mice detected by XYT scanning images. Points 1, 2, 3: parasite-infected erythrocytes at the trophozoite stage; point 4: uninfected erythrocytes. Detecting instrument: Olympus FV1000 Confocal; objective amplification: 100×; zoom: ×2; excitation wavelength: 488 nm; scanning temporal dimension: 0.1–213.8 s.

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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    Figure 1

    (Color online) Inhibitory activities of NP1024. A, Chemical structure of compound NP1024 and its inhibitory activities against FP-2 and parasite growth in vitro. PfD6, a chloroquine-sensitive P. falciparum strain; PfW2, a chloroquine-resistant P. falciparum strain. B, Effect of NP1024 on degradation of hemoglobin by FP-2. Lane 1, Hb, FP-2 and NP1024 (10 μmol L–1); lane 2, Hb, FP-2 and NP1024 (50 μmol L–1); lane 3, Hb, FP-2 and NP1024 (100 μmol L–1); lane 4, Hb, FP-2 and E-64 (10 μmol L–1); lane 5, Hb and FP-2; lane 6, Hb; lane 7, protein marker.

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    Figure 2

    (Color online) Analysis of the interaction mechanism between FP-2 and its inhibitors. A and B, Studies on the changes of the activities of FP-2 with inhibitors before (A) and after (B) dialysis. NP513 (Figure S1 in Supporting Information) and NP1024 are inhibitors of FP-2 identified in this study. E-64 is a covalent inhibitor of FP-2. NP1031 is a non-covalent inhibitor of FP-2 identified in our lab (data not shown). C, Lineweaver-Burk plots of FP-2 inhibited by NP1024. The panel shows the representative double-reciprocal plots of 1/V versus 1/[S] at different concentrations of NP1024. The concentrations of the substrate (Z-Leu-Arg-AMC) were 50, 40, 30, 20 and 10 µmol L–1.

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    Figure 3

    Analysis of the interaction between NP1024 and FP-2. A, Analysis of the binding affinity between FP-2 and NP1024 by surface plasmon resonance. The adjusted (Fc2-Fc1) sensorgrams are shown (left); the binding affinity was obtained using the steady-state model-fitting (right). B, Proposed binding mode of NP1024 in the active site of FP-2. The surface of subsites S1, S1’, S2 and S3, and the catalytic Cys42 are colored in green, purple, yellow, pink and orange, respectively (left, PDB code: 3BPF), and the key residues are shown as sticks in the same color as the corresponding subsite (right). NP1024 is presented as ball-and-sticks with carbon atoms in gray and oxygen atoms in red. Hydrogen bonds are displayed as black dashed lines.

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    Figure 4

    Colocalization of NP1024 and FP-2 in malaria parasite-infected erythrocytes in vitro. FP-2 (first column): images were collected with excitation at 405 nm and emission at 422 nm; NP1024 (second column): images were collected with excitation at 488 nm and emission at 520 nm; Merge (third column): co-localization was observed between NP1024 and the fluorescently labeled secondary antibody (points 1, 2, 4, 5, 6, 7 and 8).

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    Figure 5

    Target localization of NP1024 in erythrocytes of Plasmodiumberghei-infected mice. A–D and a–d, Treated group with compound NP1024 at a dose of 50 mg kg–1. E and e, Negative control injected with corn oil containing 10% alcohol. B, b, C, c, Parasite-infected erythrocytes at the trophozoite stage. a–e, Fluorescent images. A–E, Merge of the fluorescent images overlaid on the bright field images. D2: parasite-infected erythrocytes at the sporozoite stage; D1, E1: uninfected erythrocytes; E2: parasite-infected erythrocytes at the trophozoite stage without treatment with NP1024.