The Role of Spermidine and Its Key Metabolites in Important, Pathogenic Human Viruses and in Parasitic Infections Caused by Plasmodium falciparum and Trypanosoma brucei.

The triamine spermidine is a key metabolite of the polyamine pathway. It plays a crucial role in many infectious diseases caused by viral or parasitic infections. Spermidine and its metabolizing enzymes, i.e., spermidine/spermine-N1-acetyltransferase, spermine oxidase, acetyl polyamine oxidase, and deoxyhypusine synthase, fulfill common functions during infection in parasitic protozoa and viruses which are obligate, intracellular parasites. The competition for this important polyamine between the infected host cell and the pathogen determines the severity of infection in disabling human parasites and pathogenic viruses. Here, we review the impact of spermidine and its metabolites in disease development of the most important, pathogenic human viruses such as SARS-CoV-2, HIV, Ebola, and in the human parasites Plasmodium and Trypanosomes. Moreover, state-of-the-art translational approaches to manipulate spermidine metabolism in the host and the pathogen are discussed to accelerate drug development against these threatful, infectious human diseases.

High-Throughput Screening to Identify Inhibitors of Plasmodium falciparum Importin α.

The global burden of malaria and toxoplasmosis has been limited by the use of efficacious anti-parasitic agents, however, emerging resistance in Plasmodium species and Toxoplasma gondii threatens disease control worldwide, implying that new agents/therapeutic targets are urgently needed. Nuclear localization signal (NLS)-dependent transport into the nucleus, mediated by members of the importin (IMP) superfamily of nuclear transporters, has shown potential as a target for intervention to limit viral infection. Here, we show for the first time that IMPα from P. falciparum and T. gondii have promise as targets for small molecule inhibitors. We use high-throughput screening to identify agents able to inhibit P. falciparum IMPα binding to a P. falciparum NLS, identifying a number of compounds that inhibit binding in the µM-nM range, through direct binding to P. falciparum IMPα, as shown in thermostability assays. Of these, BAY 11-7085 is shown to be a specific inhibitor of P. falciparum IMPα-NLS recognition. Importantly, a number of the inhibitors limited growth by both P. falciparum and T. gondii. The results strengthen the hypothesis that apicomplexan IMPα proteins have potential as therapeutic targets to aid in identifying novel agents for two important, yet neglected, parasitic diseases.

Synthesis, Molecular Docking and Antiplasmodial Activities of New Tetrahydro-ß-Carbolines.

Malaria is still one of the most dangerous infectious diseases and the emergence of drug resistant parasites only worsens the situation. A series of new tetrahydro-ß-carbolines were designed, synthesized by the Pictet-Spengler reaction, and characterized. Further, the compounds were screened for their in vitro antiplasmodial activity against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Moreover, molecular modeling studies were performed to assess the potential action of the designed molecules and toxicity assays were conducted on the human microvascular endothelial (HMEC-1) cell line and human red blood cells. Our studies identified N-(3,3-dimethylbutyl)-1-octyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b] indole-3-carboxamide (7) (a mixture of diastereomers) as the most promising compound endowed with the highest antiplasmodial activity, highest selectivity, and lack of cytotoxicity. In silico simulations carried out for (1S,3R)-7 provided useful insights into its possible interactions with enzymes essential for parasite metabolism. Further studies are underway to develop the optimal nanosized lipid-based delivery system for this compound and to determine its precise mechanism of action.

Screening of viral-vectored P. falciparum pre-erythrocytic candidate vaccine antigens using chimeric rodent parasites.

To screen for additional vaccine candidate antigens of Plasmodium pre-erythrocytic stages, fourteen P. falciparum proteins were selected based on expression in sporozoites or their role in establishment of hepatocyte infection. For preclinical evaluation of immunogenicity of these proteins in mice, chimeric P. berghei sporozoites were created that express the P. falciparum proteins in sporozoites as an additional copy gene under control of the uis4 gene promoter. All fourteen chimeric parasites produced sporozoites but sporozoites of eight lines failed to establish a liver infection, indicating a negative impact of these P. falciparum proteins on sporozoite infectivity. Immunogenicity of the other six proteins (SPELD, ETRAMP10.3, SIAP2, SPATR, HT, RPL3) was analyzed by immunization of inbred BALB/c and outbred CD-1 mice with viral-vectored (ChAd63 or ChAdOx1, MVA) vaccines, followed by challenge with chimeric sporozoites. Protective immunogenicity was determined by analyzing parasite liver load and prepatent period of blood stage infection after challenge. Of the six proteins only SPELD immunized mice showed partial protection. We discuss both the low protective immunogenicity of these proteins in the chimeric rodent malaria challenge model and the negative effect on P. berghei sporozoite infectivity of several P. falciparum proteins expressed in the chimeric sporozoites.

Sialic Acids as Receptors for Pathogens.

Carbohydrates have long been known to mediate intracellular interactions, whether within one organism or between different organisms. Sialic acids (Sias) are carbohydrates that usually occupy the terminal positions in longer carbohydrate chains, which makes them common recognition targets mediating these interactions. In this review, we summarize the knowledge about animal disease-causing agents such as viruses, bacteria and protozoa (including the malaria parasite Plasmodium falciparum) in which Sias play a role in infection biology. While Sias may promote binding of, e.g., influenza viruses and SV40, they act as decoys for betacoronaviruses. The presence of two common forms of Sias, Neu5Ac and Neu5Gc, is species-specific, and in humans, the enzyme converting Neu5Ac to Neu5Gc (CMAH, CMP-Neu5Ac hydroxylase) is lost, most likely due to adaptation to pathogen regimes; we discuss the research about the influence of malaria on this trait. In addition, we present data suggesting the CMAH gene was probably present in the ancestor of animals, shedding light on its glycobiology. We predict that a better understanding of the role of Sias in disease vectors would lead to more effective clinical interventions.

Conus venom fractions inhibit the adhesion of Plasmodium falciparum erythrocyte membrane protein 1 domains to the host vascular receptors.

Using high-throughput BioPlex assays, we determined that six fractions from the venom of Conus nux inhibit the adhesion of various recombinant PfEMP-1 protein domains (PF08_0106 CIDR1α3.1, PF11_0521 DBL2ß3, and PFL0030c DBL3X and DBL5e) to their corresponding receptors (CD36, ICAM-1, and CSA, respectively). The protein domain-receptor interactions permit P. falciparum-infected erythrocytes (IE) to evade elimination in the spleen by adhering to the microvasculature in various organs including the placenta. The sequences for the main components of the fractions, determined by tandem mass spectrometry, yielded four T-superfamily conotoxins, one (CC-Loop-CC) with I-IV, II-III connectivity and three (CC-Loop-CXaaC) with a I-III, II-IV connectivity. The 3D structure for one of the latter, NuxVA = GCCPAPLTCHCVIY, revealed a novel scaffold defined by double turns forming a hairpin-like structure stabilized by the two disulfide bonds. Two other main fraction components were a miniM conotoxin, and a O2-superfamily conotoxin with cysteine framework VI/VII. This study is the first one of its kind suggesting the use of conotoxins for developing pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as inhibitors of protein-protein interactions as treatment. BIOLOGICAL SIGNIFICANCE: Among the 850+ species of cone snail species there are hundreds of thousands of diverse venom exopeptides that have been selected throughout several million years of evolution to capture prey and deter predators. They do so by targeting several surface proteins present in target excitable cells. This immense biomolecular library of conopeptides can be explored for potential use as therapeutic leads against persistent and emerging diseases affecting non-excitable systems. We aim to expand the pharmacological reach of conotoxins/conopeptides by revealing their in vitro capacity to disrupt protein-protein and protein-polysaccharide interactions that directly contribute to pathology of Plasmodium falciparum malaria. This is significant for severe forms of malaria, which might be deadly even after treated with current parasite-killing drugs because of persistent cytoadhesion of P. falciparum infected erythrocytes even when parasites within red blood cells are dead. Anti-adhesion adjunct drugs would de-sequester or prevent additional sequestration of infected erythrocytes and may significantly improve survival of malaria patients. These results provide a lead for further investigations into conotoxins and other venom peptides as potential candidates for anti-adhesion or blockade-therapies. This study is the first of its kind and it suggests that conotoxins can be developed as pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as potential inhibitors of protein-protein interactions as treatment.