Discovery and Development of Inhibitors of the Plasmodial FNT-Type Lactate Transporter as Novel Antimalarials

Plasmodium spp. malaria parasites in the blood stage draw energy from anaerobic glycolysis when multiplying in erythrocytes. They tap the ample glucose supply of the infected host using the erythrocyte glucose transporter 1, GLUT1, and a hexose transporter, HT, of the parasite’s plasma membrane. Per glucose molecule, two lactate anions and two protons are generated as waste that need to be released rapidly from the parasite to prevent blockage of the energy metabolism and acidification of the cytoplasm. Recently, the missing Plasmodium lactate/H+ cotransporter was identified as a member of the exclusively microbial formate–nitrite transporter family, FNT. Screening of an antimalarial compound selection with unknown targets led to the discovery of specific and potent FNT-inhibitors, i.e., pentafluoro-3-hydroxy-pent-2-en-1-ones. Here, we summarize the discovery and further development of this novel class of antimalarials, their modes of binding and action, circumvention of a putative resistance mutation of the FNT target protein, and suitability for in vivo studies using animal malaria models.

Lactate activates hypothalamic POMC neurons by intercellular signaling

Previous studies indicate that the activity of hypothalamic POMC neurons can be regulated by glucose via intracellular mechanisms, but its regulation by lactate is poorly understood. In addition to its energetic role, lactate acts as a signaling molecule. In this study, we evaluated the function and location of the lactate receptor, hydroxycarboxylic acid receptor 1 (HCAR1). We used a conditional genetic approach to label POMC neurons and evaluated their sensitivity to lactate using patch-clamp recordings. l-Lactate and 3-chloro-5-hydroxybenzoic acid (3Cl-HBA), HCAR1 specific agonist depolarized POMC neurons and the increase in excitability was abolished by pertussis toxin (PTX), indicating the involvement of Gαi/o-protein-coupled receptors. In addition, the depolarization of a subset of POMC neurons was sensitive to α-cyano-4-hydroxycinnamate (4-CIN), a lactate transporter blocker, suggesting that the depolarization induced by l-lactate can also occur by direct intracellular action. Surprisingly, HCAR1 was not detected in POMC neurons, but instead localized in astrocytes. These results suggest a new lactate-mediated mechanism for astrocyte-neuron intercellular communication.

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

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.

Catastrophic ATP loss underlies a metabolic combination therapy tailored for MYCN-amplified neuroblastoma

MYCN-amplified neuroblastoma is a lethal subset of pediatric cancer. MYCN drives numerous effects in the cell, including metabolic changes that are critical for oncogenesis. The understanding that both compensatory pathways and intrinsic redundancy in cell systems exists implies that the use of combination therapies for effective and durable responses is necessary. Additionally, the most effective targeted therapies exploit an “Achilles’ heel” and are tailored to the genetics of the cancer under study. We performed an unbiased screen on select metabolic targeted therapy combinations and correlated sensitivity with over 20 subsets of cancer. We found that MYCN-amplified neuroblastoma is hypersensitive to the combination of an inhibitor of the lactate transporter MCT1, AZD3965, and complex I of the mitochondrion, phenformin. Our data demonstrate that MCT4 is highly correlated with resistance to the combination in the screen and lowly expressed in MYCN-amplified neuroblastoma. Low MCT4 combines with high expression of the MCT2 and MCT1 chaperone CD147 in MYCN-amplified neuroblastoma, altogether conferring sensitivity to the AZD3965 and phenformin combination. The result is simultaneous disruption of glycolysis and oxidative phosphorylation, resulting in dramatic disruption of adenosine triphosphate (ATP) production, endoplasmic reticulum stress, and cell death. In mouse models of MYCN-amplified neuroblastoma, the combination was tolerable at concentrations where it shrank tumors and did not increase white-blood-cell toxicity compared to single drugs. Therefore, we demonstrate that a metabolic combination screen can identify vulnerabilities in subsets of cancer and put forth a metabolic combination therapy tailored for MYCN-amplified neuroblastoma that demonstrates efficacy and tolerability in vivo.

Identifying the major lactate transporter of Toxoplasma gondii tachyzoites

Toxoplasma gondii and Plasmodium falciparum parasites both extrude l-lactate, a byproduct of glycolysis. The P. falciparum Formate Nitrite Transporter, PfFNT, mediates l-lactate transport across the plasma membrane of P. falciparum parasites and has been validated as a drug target. The T. gondii genome encodes three FNTs that have been shown to transport l-lactate, and which are proposed to be the targets of several inhibitors of T. gondii proliferation. Here, we show that each of the TgFNTs localize to the T. gondii plasma membrane and are capable of transporting l-lactate across it, with TgFNT1 making the primary contribution to l-lactate transport during the disease-causing lytic cycle of the parasite. We use the Xenopus oocyte expression system to provide direct measurements of l-lactate transport via TgFNT1. We undertake a genetic analysis of the importance of the tgfnt genes for parasite proliferation, and demonstrate that all three tgfnt genes can be disrupted individually and together without affecting the lytic cycle under in vitro culture conditions. Together, our experiments identify the major lactate transporter in the disease causing stage of T. gondii, and reveal that this transporter is not required for parasite proliferation, indicating that TgFNTs are unlikely to be targets for anti-Toxoplasma drugs.

Lactate Secreted by PKM2 Upregulation Promotes Galectin-9-Mediated Immunosuppression via Inhibiting NF-κB Pathway in HNSCC

Background: Pyruvate kinase M2 (PKM2) is a key rate-limiting enzyme in glycolysis, and which plays a critical role in tumor progression in various malignancies. However, whether PKM2 can promote head and neck squamous cell carcinoma (HNSCC) progression and immunosuppression remains unknown. Methods: PKM2 expression was evaluated using immunohistochemical staining. The biological functions of PKM2 were investigated in vitro and in vivo. Lactate production and the expression of galectin-9, a critical immunosuppression molecule, were detected after PKM2 knockdown and overexpression in HNSCC cells. Results: Overexpression of PKM2 correlates with poor prognosis in HNSCC patients. Silencing PKM2 markedly inhibits proliferation and metastasis capacity in vivo and in vitro, and vice versa. Furthermore, lactate production induced by PKM2 significantly promotes migration and invasion. A positive correlation between PKM2 and galectin-9 expression is observed in HNSCC tissues. Finally, the induction of galectin-9 expression by PKM2 can be affected by a lactate transporter inhibitor.Conclusion: Our findings demonstrate that PKM2 promotes tumor progression and galectin-9-mediated immunosuppression via NF-κB signaling inhibition in HNSCC, which bridges metabolism and immunosuppression. The novel PKM2-lactate-galectin-9 axis might be a potential therapeutic target in HNSCC.

The Arg/N-degron pathway targets transcription factors and regulates specific genes

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal or internal degrons. Our previous work produced double-knockout (2-KO) HEK293T human cell lines that lacked the functionally overlapping UBR1 and UBR2 E3 ubiquitin ligases of the Arg/N-degron pathway. Here, we studied these cells in conjunction with RNA-sequencing, mass spectrometry (MS), and split-ubiquitin binding assays. 1) Some mRNAs, such as those encoding lactate transporter MCT2 and β-adrenergic receptor ADRB2, are strongly (∼20-fold) up-regulated in 2-KO cells, whereas other mRNAs, including those encoding MAGEA6 (a regulator of ubiquitin ligases) and LCP1 (an actin-binding protein), are completely repressed in 2-KO cells, in contrast to wild-type cells. 2) Glucocorticoid receptor (GR), an immunity-modulating transcription factor (TF), is up-regulated in 2-KO cells and also physically binds to UBR1, strongly suggesting that GR is a physiological substrate of the Arg/N-degron pathway. 3) PREP1, another TF, was also found to bind to UBR1. 4) MS-based analyses identified ∼160 proteins whose levels were increased or decreased by more than 2-fold in 2-KO cells. For example, the homeodomain TF DACH1 and the neurofilament subunits NF-L (NFEL) and NF-M (NFEM) were expressed in wild-type cells but were virtually absent in 2-KO cells. 5) The disappearance of some proteins in 2-KO cells took place despite up-regulation of their mRNAs, strongly suggesting that the Arg/N-degron pathway can also modulate translation of specific mRNAs. In sum, this multifunctional proteolytic system has emerged as a regulator of mammalian gene expression, in part through conditional targeting of TFs that include ATF3, GR, and PREP1.