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Structural characterization of the Plasmodium falciparum lactate transporter PfFNT alone and in complex with antimalarial compound MMV007839 reveals its inhibition mechanism

['Xi Peng', 'Department Of Obstetrics', 'Key Laboratory Of Birth Defects', 'Related Disease Of Women', 'Children Of Moe', 'State Key Laboratory Of Biotherapy', 'West China Second Hospital', 'Sichuan University', 'Chengdu', 'Nan Wang']

Date: 2021-09

Plasmodium falciparum, the deadliest causal agent of malaria, caused more than half of the 229 million malaria cases worldwide in 2019. The emergence and spreading of frontline drug-resistant Plasmodium strains are challenging to overcome in the battle against malaria and raise urgent demands for novel antimalarial agents. The P. falciparum formate–nitrite transporter (PfFNT) is a potential drug target due to its housekeeping role in lactate efflux during the intraerythrocytic stage. Targeting PfFNT, MMV007839 was identified as a lead compound that kills parasites at submicromolar concentrations. Here, we present 2 cryogenic-electron microscopy (cryo-EM) structures of PfFNT, one with the protein in its apo form and one with it in complex with MMV007839, both at 2.3 Å resolution. Benefiting from the high-resolution structures, our study provides the molecular basis for both the lactate transport of PfFNT and the inhibition mechanism of MMV007839, which facilitates further antimalarial drug design.

Funding: This work was supported by National Key R&D Program of China 2016YFA0502700 from Ministry of Science and Technology of the People’s Republic of China ( https://service.most.gov.cn ) to DD and Beijing Nova Program Z201100006820039 from Beijing Municipal Science & Technology Commission ( https://mis.kw.beijing.gov.cn/out/typt/staticHTML/homePage.html ) to CY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Atomic coordinates of PfFNT in apo form or bound with MMV007839 have been deposited in the Protein Data Bank ( http://www.rcsb.org ) under the accession codes 7E26 and 7E27, respectively. The corresponding electron microscopy maps have been deposited in the Electron Microscopy Data Bank ( https://www.ebi.ac.uk/pdbe/ emdb/), under the accession codes EMD-30952 and EMD-30953, respectively.

Despite progress made in inhibitor screening and optimization, structural characterization of FNT family members was limited to prokaryotic homologs. Previously, structures of 5 prokaryotic FNT family members have been reported, including formate channels (FocAs) from Escherichia coli [ 19 ], Vibrio cholera [ 20 ], and Salmonella typhimurium [ 21 ]; nitrite channel (NirC) from S. typhimurium [ 22 ]; and hydrosulphide ion channel (HSC) from Clostridium difficile [ 23 ]. During our manuscript preparation, structures of nanodisc reconstituted PfFNT in apo form and bound with MMV007839 were reported at 2.6 Å and 2.8 Å, respectively [ 24 ]. Due to the relatively low resolution of these 2 structures, the unique N-terminal loop (NTL), which reveals a different transport mechanism than a previously reported prokaryotic model, was not fully characterized. Moreover, the model of MMV007839 was built as a cyclic hemiketal, the prodrug form, rather than the active, vinylogous acid, leading to an incorrect interpretation of the mode of action between PfFNT and the inhibitor. Consequently, we reported 2 cryo-electron microscopy (cryo-EM) structures of PfFNT in apo form or in complex with MMV007839, both at a resolution of 2.3 Å. Our unambiguous ligand density revealed the vinylogous acid model of MMV007839, leading to an accurate and comprehensive understanding of current inhibitor optimization. Our high-resolution structures also revealed the tight coordination between the protomers of the PfFNT pentamer. Combined with the local switching of a bulky residue in the intracellular constriction site, our results indicated an alternating access mechanism different from previously reported N-terminal domain movement in prokaryotic FNT family members.

The P. falciparum lactate transporter PfFNT, encoded by pf3D7_0316600, belongs to the formate–nitrite transporter (FNT) family. As an essential component of the glucose–lactate transport cycle, PfFNT, together with PfHT1, is vital for the energy supply and metabolic homeostasis of parasites [ 9 , 10 ]. The energy supply of parasites can be cut off by inhibiting glucose uptake, making PfHT1 a valuable drug target for next-generation antimalarial development [ 12 – 14 ]. PfFNT, hence, represents another Achilles’ heel of this transport cycle that can be exploited for chemotherapeutic development of malaria. As expected, screening of malaria box, collected by the Medicine for Malaria Venture (MMV), yielded 2 structurally similar PfFNT inhibitors, MMV007839 and MMV000972, which kill parasites at submicromolar and single-digit micromolar levels, respectively [ 15 – 17 ]. Structure–activity relation (SAR) studies and potent characterization of BH296, a derivative of MMV007839, revealed that vinylogous acid rather than cyclic hemiketal is the active form of MMV007839 [ 15 , 18 ].

(A) Hexose–monocarboxylate transport system of the P. falciparum–infected erythrocyte. Glucose and lactate are represented by orange hexagons and yellow triangles, respectively. Protons are presented by the blue circles. The magenta pentagon represents the PfFNT protein. Other reported structures, including human glucose transporter GLUT1 (PDB code: 4PYP), human monocarboxylate transporter MCT1 (PDB code: 6LZ0), and P. falciparum hexose transporter PfHT1 (PDB code: 6M2L), are presented as surface representations. Inhibitors of PfFNT (MMV007839) and PfHT1 (C3361) are displayed as sphere models. (B) Overall structure of pentameric PfFNT. The central tunnel of the pentamer and substrate translocation path in the protomer are indicated by pink circle and ellipse in the top view, respectively. (C) Topology diagram of a PfFNT protomer. The N-terminal and carboxyl-terminal TM segments are colored blue and green, respectively. Soluble helices, including the EH, NTH, and CTH, are colored gray. The NTL and Ω loops are colored sandy brown and salmon, respectively. Thr106 and His230 are presented as side chain models. (D) Cartoon representation of a PfFNT protomer. Components of the protomer are labeled in C. CTH, carboxyl-terminal helix; EH, extracellular helix; GLUT1, glucose transporter 1; MCT1, monocarboxylate transporter 1; NTH, N-terminal helix; NTL, N-terminal loop; PDB, Protein Data Bank; PfFNT, P. falciparum formate–nitrite transporter; PfHT1, P. falciparum hexose transporter 1; PVM, parasitophorous vacuole membrane; TM, transmembrane.

The asexual stage of P. falciparum takes up glucose from host erythrocytes as a primary energy source [ 5 ]. In addition, to adapt to the rapid growth and proliferation of asexual stage parasites, P. falciparum mainly relies on the glycolysis to maintain its energy supply, which results in the fast consumption of glucose and accumulation of lactate [ 6 ]. Rapid absorption of glucose and excretion of lactate are executed by the cooperation of 4 essential transporters: human glucose transporter 1 (hGLUT1), P. falciparum hexose transporter 1 (PfHT1), P. falciparum formate–nitrite transporter (PfFNT), and human monocarboxylate transporter 1 (hMCT1) ( Fig 1A ). Briefly, a high concentration of blood glucose enters erythrocytes through facilitated diffusion via hGLUT1 [ 7 ], followed by rapid diffusion into P. falciparum via PfHT1 [ 8 ]. After glucose is broken down into lactic acid by glycolysis inside the parasite, the end products, including lactate and protons, are extruded to the extraparasite milieu by PfFNT [ 9 , 10 ]. Finally, hMCT1 mediates the efflux of lactate and protons toward the extracellular space [ 11 ].

Malaria remains a worldwide life-threatening disease, leading to an estimated 229 million infections and 409,000 deaths worldwide in 2019 [ 1 ]. Plasmodium falciparum, one of the 5 agents of human malaria, is responsible for more than half of the total infections globally and nearly all the deaths in WHO African region, representing the deadliest form of Plasmodium spp. Despite advances in chemotherapies in past decades, efforts to eradicate malaria have been hampered by the emergence of multidrug-resistant parasites [ 2 , 3 ], which calls for novel antimalarial agents [ 4 ].

Results

Overall structure of PfFNT To solve the structure of PfFNT, full-length PfFNT was purified to homogeneity. The cryo-EM images exhibited monodispersed particles, revealing homogeneous protein behavior (S1A Fig). Two-dimensional classification results clearly display the pentameric assembly of PfFNTs that has been observed in structures of bacterial FNT family members (S1B Fig). Using C5 symmetry, we solved the structure of PfFNT to a final resolution of 2.3 Å (Fig 1B, S1 and S2 Figs, S1 Table). Owing to the high resolution, the polypeptide chain, including residues 7 to 293, of each protomer was unambiguously assigned to the density map (S2C Fig). Although 250 mM sodium lactate was incubated with PfFNT before grid preparation, no extra density for lactate was found, which might be attributed to the low affinity between lactate and PfFNT. The 5 protomers of PfFNT resemble as a pentagon-like homopentamer, with a width of approximately 80 Å and height of approximately 70 Å, with a 5-fold axis that is perpendicular to the membrane plane (Fig 1B). Although a hydrophobic central tunnel is encompassed by the 5 protomers, functional characterization of both PfFNT and other FNT family members implies that one tunnel of each protomer, rather than the central tunnel, acts as the pathway for substrate translocation [25,26]. Currently, the structures of 5 prokaryotic FNT family members have been elucidated [19–23,26], and they share approximately 20% identity and 40% similarity with the eukaryotic PfFNTs (S3 Fig). Similar to the reported prokaryotic homologs, each protomer of PfFNT, comprising 6 transmembrane (TM) segments, presents a typical 3+3 invert repeat that is conserved throughout FNT family members (Fig 1C and 1D). The intervening sequence between TM3 and TM4 forms an extracellular helix (EH) that lays on the outer leaflet of the membrane. Two discontinuous helices, TM2 and TM5, enwrap the central pore of each protomer and form constriction sites on both the intracellular and extracellular sides of the membrane. Together with tunnels from the cytoplasmic and extracellular sides, a pathway for substrate translocation is located in the center of each protomer (Fig 1B–1D). To facilitate the comparison with a previously proposed transport model, the loop region between TM2a and TM2b is designated to the Ω loop [20]. Notably, except for the common features of previous prokaryotic homologs, PfFNT processes 2 unprecedented structures, a long and unstructured NTL and an additional carboxyl-terminal helix (CTH) (Fig 1C and 1D). The sequences of the cytosolic NTL, N-terminal helix (NTH), and CTH of PfFNT are not similar to those of prokaryotic homologs but are highly conserved in all Plasmodium species, representing the unique features of lactate transporters of parasites (S3 Fig).

Mechanistic insight into the drug-resistant mutant G107S Two independent groups have reported that constantly exposing P. falciparum to MMV007839 results in mutagenesis of Gly107 to Ser in PfFNT, which leads to an inhibitor-insensitive parasite and a drug-resistant phenotype [15,16]. In the structure of inhibitor-bound PfFNT, an amide nitrogen directly coordinates with the carbonyl group of the vinylogous acid moiety and the hydroxyl group of the phenol moiety (Fig 2D). Additional hydroxymethyl side chain in the G107S replacement mutation substantially increase the steric hindrance of MMV007839, decreasing inhibitor affinity. To circumvent the drug-resistant mutation, a series of MMV007839 derivatives have been synthesized and characterized through SAR studies [15,18]. Notably, a pyridine substitution of the original phenol moiety yielded the most potent dual inhibitor, BH267.meta, to both wild type and PfFNT G107S . In addition to its high efficacy in killing cultured P. falciparum parasites, BH267.meta prevented the formation of drug resistance even when the parasite was treated with it for a long period [18]. Moreover, a recent study revealed that BH267.meta inhibits a broad spectrum of FNTs, including all causal agents of human malaria, indicating its potential application in further chemotherapeutic development [27]. To elucidate the mechanism by which BH267.meta circumvents the G107S resistance mutation, we docked MMV007839 and BH267.meta to both the wild-type and PfFNT G107S models (Fig 3). Consistent with the complex structure, MMV007839 docked into the inhibitor binding pocket of wild-type PfFNT with a slight shift in its location (Fig 3A). However, introduction of a hydroxymethyl side chain by mutating Gly107 to Ser inhibited MMV007839 by sterically blocking the entrance of the inhibitor to the central pocket (Fig 3B). Consistent with this model, the binding affinity between MMV007839 and PfFNT G107S was undetectable (Fig 2H). BH267.meta, by contrast, could be bound to both the wild-type PfFNT and the G107S mutant (Fig 3C and 3D). The major discrepancy in docking results between MMV007839 and BH267.meta is caused by 2 reasons: (1) the removal of the hydroxyl group of the aromatic moiety avoids the inhibitor clashing with the side chain of Ser107; and [2] a novel hydrogen bond is introduced between the pyridine group of BH267.meta and the hydroxymethyl group of Ser107. Nonetheless, the Gly107➔Ser mutation still increases steric hindrance of BH267.meta, leading to a slight increase in binding energy (Fig 3E). This result is consistent with the report that the affinity of BH267.meta to the G107S mutant is slightly lower than that of wild-type PfFNT [18]. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Molecular docking of MMV007839 and BH267.meta in the wild-type PfFNT or G107S resistant mutant. (A) The docking model of MMV007839 in the wild-type PfFNT. (B) The docking model of MMV007839 in PfFNT G107S . (C) The docking model of BH267.meta in the wild-type PfFNT. (D) The docking model of MMV007839 in PfFNT G107S . (E) Summary of binding energy of docking results. The transparent stick model of MMV007839 represents the real location in the inhibitor-bound structure. The structure of PfFNT is presented as cartoon representation and Gly107 or S107 are presented as sticks. The docking compounds are represented as sticks and colored gray. PfFNT, P. falciparum formate–nitrite transporter; WT, wild type. https://doi.org/10.1371/journal.pbio.3001386.g003

The unique intracellular region of PfFNT Consistent with other FNT family members, both the N-terminus and carboxyl terminus of PfFNT are located on in the cytosolic side. Nonetheless, compared with structures of prokaryotic homologs, a unique long NTL and an extra soluble CTH were characterized in our structures (Fig 5A). Although the NTL is unstructured, 5 NTLs of each protomer orderly tangle to each other (Fig 5B). To depict the sophisticated interaction between 5 NTLs, we illustrate the interaction between NTLs from protomers 1, 4, and 5 (Fig 5C). These unstructured NTLs are stabilized by tightly interactions, suggesting a relatively static state of the N-terminal structure. Therefore, the gating mechanism of PfFNT is unlikely to adopt to the previously reported N-terminal reorientation in S. typhimurium FocA (stFocA) [21], which further supports the aforementioned bulky residue swing model (Fig 5D). The extra CTH protrudes into the cytoplasm of the parasite and is stabilized by 3 hydrogen bonds (Tyr8-His284, Glu30-Tyr285, and Lys120-Glu289; Fig 5C and 5D). Sequence alignment between Plasmodium and prokaryotic FNTs reveals that the NTL, NTH, and CTH regions, especially for residues involved in NTL/CTH interactions, are highly conserved in Plasmodium spp. but vary in prokaryotic homologous (S3 Fig). In addition, the Lys120 from the loop region between TM2 and TM3 is also highly conserved throughout Plasmodium species. Therefore, the intracellular region of PfFNT, including the unstructured NTL and protruding CTH, represents the unique feature of FNTs in Plasmodium parasites. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Intracellular region of PfFNT. (A) A unique intracellular region is presented in a side view of PfFNT. The overall structure of PfFNT is shown as a cylinder cartoon. The region containing the intracellular region overlaps with a transparent light blue rectangle. (B) Overlap of NTLs in a bottom view of PfFNT. The overall structure of PfFNT is shown as a surface, and the 5 protomers are distinguished by different colors. (C) The interactions between NTLs. Each protomer is colored the same as in patterns A and B. Hydrogen bonds are shown as the gray dashed lines. (D) Coordination between the cytosolic CTH and the rest of the PfFNT protomer. The structure of the protomer of PfFNT is shown as cartoon and fitted to the cryo-EM density. The residues involved in the interactions are shown as pink sticks. Hydrogen bonds are shown as gray dashed lines. cryo-EM, cryo-electron microscopy; CTH, carboxyl-terminal helix; NTL, N-terminal loop; PfFNT, P. falciparum formate–nitrite transporter. https://doi.org/10.1371/journal.pbio.3001386.g005

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