Host-Erythrocytic Sphingosine-1-Phosphate Regulates Plasmodium Histone Deacetylase Activity and Exhibits Epigenetic Control over Cell Death and Differentiation.

The evolution of resistance to practically all antimalarial drugs poses a challenge to the current malaria elimination and eradication efforts. Given that the epigenome of Plasmodium falciparum governs several crucial parasite functions, pharmaceutical interventions with transmission-blocking potential that target epigenetic molecular markers and regulatory mechanisms are likely to encounter drug resistance. In the malaria parasite, histone deacetylases (HDACs) are essential epigenetic modulators that regulate cellular transcriptional rearrangements, notably the molecular mechanisms underlying parasite proliferation and differentiation. We establish "lipid sequestration" as a mechanism by which sphingolipids, specifically Sphingosine-1-Phosphate (S1P) (a metabolic product of Sphingosine Kinase 1 [SphK-1]), regulate epigenetic reprogramming in the parasite by interacting with, and modulating, the histone-deacetylation activity of PfHDAC-1, thereby regulating Plasmodium pathogenesis. Furthermore, we demonstrate that altering host S1P levels with PF-543, a potent and selective Sphk-1 inhibitor, dysregulates PfHDAC-1 activity, resulting in a significant increase in the global histone acetylation signals and, consequently, transcriptional modulation of genes associated with gametocytogenesis, virulence, and proliferation. Our findings point to a hitherto unrecognized functional role for host S1P-mediated sphingolipid signaling in modulating PfHDAC-1's enzymatic activity and, as a result, the parasite's dynamic genome-wide transcriptional patterns. The epigenetic regulation of parasite proliferation and sexual differentiation offers a novel approach for developing host-targeted therapeutics to combat malaria resistance to conventional regimens. IMPORTANCE Sphingolipid is an 18-carbon amino-alcohol-containing lipid with a sphingosine backbone, which when phosphorylated by sphingosine kinase 1 (SphK-1), generates sphingosine-1-phosphate (S1P), an essential lipid signaling molecule. Dysregulation of S1P function has been observed in a variety of pathologies, including severe malaria. The malaria parasite Plasmodium acquires a host S1P pool for its growth and survival. Here, we describe the molecular attuning of histone deacetylase-1 (PfHDAC-1), a crucial epigenetic modulator that contributes to the establishment of epigenetic chromatin states and parasite survival, in response to S1P binding. Our findings highlight the host lipid-mediated epigenetic regulation of malaria parasite key genes.

Designing and development of phthalimides as potent anti-tubulin hybrid molecules against malaria.

Constant emergence of drug-resistant Plasmodium falciparum warrants urgent need for effective and inexpensive drugs. Herein, phthalimide (Pht) analogs possessing the bioactive scaffolds, benzimidazole and 1,2,3-triazole, were evaluated for in vitro and in vivo anti-plasmodial activity without any apparent hemolysis, or cytotoxicity. Analogs 4(a-e) inhibited the growth of 3D7 and RKL-9 strains at submicromolar concentrations. Defects were observed during parasite egress from or invasion of the red blood cells. Mitochondrial membrane depolarization was measured as one of the causes of cell death. Phts 4(a-e) in combination with artemisinin exhibited two-to three-fold increased efficacy. Biophysical and biochemical analysis suggest that Pht analogs mediate plasmodial growth inhibition by interacting with tubulin protein of the parasite. Lastly, Phts 4(a-e) significantly decreased parasitemia and extended host survival in murine model Plasmodium berghei ANKA infection. Combined, the data indicate that Pht analogs should be further explored, which could offer novel value to the antimalarial drug development pipeline.

Highly potent anti-malarial activity of benzopyrano(4,3-b)benzopyran derivatives and in silico interaction analysis with putative target Plasmodium falciparum lactate dehydrogenase.

Malaria infection caused by Plasmodium falciparum is majorly responsible for millions of deaths in humans every year. Moreover, a rapid increase in resistance to existing drugs has posed an urgent need for new anti-malarials. Herein, we report the highly potent anti-malarial activity of benzopyrano(4,3-b)benzopyran derivatives, inspired from naturally occurring dependensin against chloroquine (CQ) sensitive and resistant P. falciparum strains. Chemically synthesized, four dependensin analogs 85(A-D) exhibited growth inhibition at nanomolar concentrations ranging from 63.96 to 725.8 nM by blocking the parasite development at the ring and early trophozoite stages. The growth inhibitory activity of dependensin analogs was correlated with their anti-plasmodial lactate dehydrogenase activity by computational analysis. Molecular docking, 50 ns simulation and a 2D-Quantitative Structure-Activity Relationship (2D-QSAR) modelling revealed the interaction with their putative target P. falciparum lactate dehydrogenase (PfLDH). Here, developing the predictive 2D descriptors such as thermodynamic, spatial, electronic, and topological with multiple linear regression analysis (MLRA), the structural requirements for potent and selective PfLDH inhibitory activity has been identified. The strong binding of compound 85D to the catalytic Nicotinamide adenine dinucleotide (NADH) binding pocket of the PfLDH further supported the PfLDH targeting potential of dependensin analogs. Overall, this study revealed a highly potent anti-malarial activity of benzopyrano(4,3-b)benzopyran derivatives with their putative anti-PfLDH activity.Communicated by Ramaswamy H. Sarma.

Pathogenic Pore Forming Proteins of Plasmodium Triggers the Necrosis of Endothelial Cells Attributed to Malaria Severity.

Severe malaria caused by Plasmodium falciparum poses a major global health problem with high morbidity and mortality. P. falciparum harbors a family of pore-forming proteins (PFPs), known as perforin like proteins (PLPs), which are structurally equivalent to prokaryotic PFPs. These PLPs are secreted from the parasites and, they contribute to disease pathogenesis by interacting with host cells. The severe malaria pathogenesis is associated with the dysfunction of various barrier cells, including endothelial cells (EC). Several factors, including PLPs secreted by parasites, contribute to the host cell dysfunction. Herein, we have tested the hypothesis that PLPs mediate dysfunction of barrier cells and might have a role in disease pathogenesis. We analyzed various dysfunctions in barrier cells following rPLP2 exposure and demonstrate that it causes an increase in intracellular Ca2+ levels. Additionally, rPLP2 exposed barrier cells displayed features of cell death, including Annexin/PI positivity, depolarized the mitochondrial membrane potential, and ROS generation. We have further performed the time-lapse video microscopy of barrier cells and found that the treatment of rPLP2 triggers their membrane blebbing. The cytoplasmic localization of HMGB1, a marker of necrosis, further confirmed the necrotic type of cell death. This study highlights the role of parasite factor PLP in endothelial dysfunction and provides a rationale for the design of adjunct therapies against severe malaria.

Identification and structure-activity relationship (SAR) studies of carvacrol derivatives as potential anti-malarial against Plasmodium falciparum falcipain-2 protease.

In an effort to develop a potent anti-malarial agent against Plasmodium falciparum, a structure-guided virtual screening using an in-house library comprising 652 compounds was performed. By docking studies, we identified two compounds (JMI-105 and JMI-346) which formed significant non-covalent interactions and fit well in the binding pocket of PfFP-2. We affirmed this observation by MD simulation studies. As evident by the biochemical analysis, such as enzyme inhibition assay, Surface Plasmon Resonance (SPR), live-cell imaging and hemozoin inhibition, JMI-105 and JMI-346 at 25 µM concentration showed an inhibitory effect on purified PfFP-2. JMI-105 and JMI-346 inhibited the growth of CQS (3D7; IC50 = 8.8 and 13 µM) and CQR (RKL-9; IC50 = 14.3 and 33 µM) strains of P. falciparum. Treatment with compounds resulted in defect in parasite growth and development. No significant hemolysis or cytotoxicity towards human cells was observed suggesting that these molecules are non-toxic. We pursued, structural optimization on JMI-105 and in the process, SAR oriented derivatives (5a-5l) were synthesized and evaluated for growth inhibition potential. JMI-105 significantly decreased parasitemia and prolonged host survival in a murine model with P. berghei ANKA infection. The compounds (JMI-105 and JMI-346) against PfFP-2 have the potential to be used as an anti-malarial agent.

Plasmodium Perforin-Like Protein Pores on the Host Cell Membrane Contribute in Its Multistage Growth and Erythrocyte Senescence.

The pore forming Plasmodium Perforin Like Proteins (PPLP), expressed in all stages of the parasite life cycle are critical for completion of the parasite life cycle. The high sequence similarity in the central Membrane Attack Complex/ Perforin (MACPF) domain among PLPs and their distinct functional overlaps define them as lucrative target for developing multi-stage antimalarial therapeutics. Herein, we evaluated the mechanism of Pan-active MACPF Domain (PMD), a centrally located and highly conserved region of PPLPs, and deciphered the inhibitory potential of specifically designed PMD inhibitors. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to hemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence which can be hypothesized to account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing malaria anemia. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress processes and protected erythrocytes against rPMD induced senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage, transmission-blocking potential of these inhibitors. Concievably, our study has introduced a novel set of anti-PMD inhibitors with pan-inhibitory activity against all the PPLPs members which can be developed into potent cross-stage antimalarial therapeutics along with erythrocyte senescence protective potential to occlude PPLPs mediated anemia in severe malaria.

Inhibition of Hemoglobin Degrading Protease Falcipain-2 as a Mechanism for Anti-Malarial Activity of Triazole-Amino Acid Hybrids.

BACKGROUND: Novel drug development against malaria parasite over old conventional antimalarial drugs is essential due to rapid and indiscriminate use of drugs, which led to the emergence of resistant strains. METHODS: In this study, previously reported triazole-amino acid hybrids (13-18) are explored against Plasmodium falciparum as antimalarial agents. Among six compounds, 15 and 18 exhibited antimalarial activity against P. falciparum with insignificant hemolytic activity and cytotoxicity towards HepG2 mammalian cells. In molecular docking studies, both compounds bind into the active site of PfFP-2 and block its accessibility to the substrate that leads to the inhibition of target protein further supported by in vitro analysis. RESULTS: Antimalarial half-maximal inhibitory concentration (IC50) of 15 and 18 compounds were found to be 9.26 µM and 20.62 µM, respectively. Blood stage specific studies showed that compounds, 15 and 18 are effective at late trophozoite stage and block egress pathway of parasites. Decreased level of free monomeric heme was found in a dose dependent manner after the treatment with compounds 15 and 18, which was further evidenced by the reduction in percent of hemoglobin hydrolysis. Compounds 15 and 18 hindered hemoglobin degradation via intra- and extracellular cysteine protease falcipain-2 (PfFP-2) inhibitory activity both in in vitro and in vivo in P. falciparum. CONCLUSION: We report antimalarial potential of triazole-amino acid hybrids and their role in the inhibition of cysteine protease PfFP-2 as its mechanistic aspect.