Oleinikov, Andrew

The current work investigates the identification of novel drugs that have the potential to be suitable anti-malarials against Plasmodium falciparum-infected erythrocytes. The growing resistance to current therapeutic modalities necessitates the development of new emerging and effective compounds. The target of these compounds in this work will be PfGARP (P. falciparum glutamic-acid-rich protein), a surface antigen of infected erythrocytes (IEs) found only in P. falciparum that has been recently recognized as a valuable drug target and vaccine candidate. Using a two-step approach designed in our lab, we were able to efficiently screen large libraries of small molecules provided by ChemBridge and the Torrey Pines Institute for Molecular Studies (TPIMS). We base the current work on our preliminary results, obtained with a subset of 6,400 compounds of DIVERSet library, reasoning that there may be other individual compounds that can be identified as having equal or greater parasiticidal activity. In this work, initial screening of the ChemBridge DIVERSet library subset of other 3,600 compounds organized into compound mixtures using Bio-Plex technology resulted in the identification of the most active mixtures (HITS-1), which were further deconvoluted into simpler mixtures (HITS-2). Screenings of HITS-2 yielded two mixtures of interest that did not portray any noticeable binding inhibition, and the deconvolution process was thus forfeited.

A hallmark trait of P. falciparum malaria is sequestration, in which parasite infected erythrocytes (IEs) adhere to the vasculature, causing organ failure and death. Current antimalarials only kill the parasites, necessitating development of anti-adhesion drugs. Using our two-step approach, we can efficiently screen for anti-adhesion small molecules. Screenings of 75libraries using Bio-Plex 200 identified the most active TPI libraries, which were deconvoluted to single compounds. Screenings library TPI 1319 yielded 3 inhibiting non-optimized compounds, each of which inhibits binding between two receptors, CSA and ICAM1, and their binding PfEMP1 domains. Two compounds deconvoluted from TPI 2103 prevent binding between PfEMP1 and ICAM1. Cytoadhesion assays with live IEs support the results seen with Bio-Plex, with best hits showing inhibition below 200 nM. Cytotoxicity testing of active compounds showed minimaltoxicity. Identified hits appear to be amenable to Structure Activity Relationship studies to develop powerful anti-adhesion drugs to treat severe malaria.

Placental malaria infection, during which infected red blood cells sequester in the placenta, is a substantial cause of pregnancy-related complications in areas where malaria is endemic. Accumulation of infected red blood cells creates an inflammatory environment and induces an immune response that can be deleterious to the placenta. This response can cause complications that include low birth weight, which is a major risk factor for neonatal and infant death. A decrease in the megalin transport and signaling system has been demonstrated to be linked with placental malaria infection and to be connected with low birth weight pathology. In this study we analyze the abundance of a protein related to megalin, LRP1 (LDL receptor related protein 1) in pregnancy malaria. Protein expression was analyzed in placental tissue samples by immunofluorescence staining. A statistically significant decrease was observed in the expression of LRP1 in placental samples of patients stratified by presence of placental malaria infection and infants born with low birth weight. Findings were supported using an in vitro cell model of placental syncytial trophoblast during malarial infection. In this model BeWo cell line was incubated with erythrocytes infected with malaria parasite CS2 line that is known for binding to malaria placental receptor. LRP1 expression in BeWo cells was analyzed by immunostaining and Western Blot, and a reduction was found by both methods. Analysis of LRP1 mRNA levels by RT-qPCR revealed no difference compared to control samples, indicating that changes happen at the protein level.

Malaria is a severe global health problem that causes approximately 435,000 deaths per year. Any non-immune individual traveling to malaria endemic regions can be affected too, including humanitarian volunteers, travelers, and US troops.
Under physiological conditions, damaged or malaria-infected RBCs would be removed within the spleen, but Plasmodium falciparum infected RBCs (iRBCs) sequester to microvascular endothelial cells to avoid entering the spleen. Adhesion interactions and parasite sequestration to endothelial cells are mediated by Plasmodium falciparum erythrocyte membrane protein 1 family (PfEMP1) proteins expressed on the iRBC’s surface. The PfEMP1 proteins bind to existing endothelial cell surface receptors that already serve primary functions, including ICAM-1, integrin αVβ3, and CD36.
Traditionally, these receptors are explored in the context of endothelial cell sequestration, but this project examines the consequence of receptor::PfEMP1 interaction on immune cells, namely monocyte-like THP-1 cells.