Apicomplexan Parasites Co-Opt Host Calpains to Facilitate Their Escape from Infected Cells

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii (the causative agents of malaria and toxoplasmosis, respectively), are responsible for considerable morbidity and mortality worldwide. These pathogenic protozoa replicate within an intracellular vacuole inside of infected host cells, from which they must escape to initiate a new lytic cycle. By integrating cell biological, pharmacological, and genetic approaches, we provide evidence that both Plasmodium and Toxoplasma hijack host cell calpain proteases to facilitate parasite egress. Immunodepletion or inhibition of calpain-1 in hypotonically lysed and resealed erythrocytes prevented the escape of P. falciparum parasites, which was restored by adding purified calpain-1. Similarly, efficient egress of T. gondii from mammalian fibroblasts was blocked by either small interfering RNA– mediated suppression or genetic deletion of calpain activity and could be restored by genetic complementation

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

Malaria causes worldwide morbidity and mortality, and while chemotherapy remains an excellent means of malaria control, drug-resistant parasites necessitate the discovery of new antimalarials. Peptidases are a promising class of drug targets and perform several important roles during the Plasmodium falciparum erythrocytic life cycle. Herein, we report a multidisciplinary effort combining activity-based protein profiling, biochemical, and peptidomic approaches to functionally analyze two genetically essential P. falciparum metallo-aminopeptidases (MAPs), PfA-M1 and Pf-LAP. Through the synthesis of a suite of activity-based probes (ABPs) based on the general MAP inhibitor scaffold, bestatin, we generated specific ABPs for these two enzymes. Specific inhibition of PfA-M1 caused swelling of the parasite digestive vacuole and prevented proteolysis of hemoglobin (Hb)-derived oligopeptides, likely starving the parasite resulting in death. In contrast, inhibition of Pf-LAP was lethal to parasites early in the life cycle, prior to the onset of Hb degradation suggesting that Pf-LAP has an essential role outside of Hb digestion

Targeting the ERAD Pathway via Inhibition of Signal Peptide Peptidase for Antiparasitic Therapeutic Design

Early secretory and endoplasmic reticulum (ER)-localized proteins that are terminally misfolded or misassembled are degraded by a ubiquitin- and proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan pathogens, including the causative agents of malaria, toxoplasmosis, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells. Herein, we exploit this fact to show that the malaria parasite Plasmodium falciparum is highly sensitized to the inhibition of components of this protein quality control system. Using a variety of approaches, including activity-based protein profiling, mammalian cell- and yeast-based assays, and resistance selection, we show small molecule inhibitors of a protease component of malarial ERAD, PfSPP, simultaneously disrupt the protein’s ability to facilitate degradation of unstable proteins and inhibit its proteolytic function, resulting in lethality for the parasite. Collectively, these data validate ER quality control as a novel vulnerability for the parasite and specifically, confirm the suitability of PfSPP as an antimalarial target

Synthesis of new (-)-Bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase.

The essential malarial PfA-M1 metalloaminopeptidase is a validated drug target that functions in the terminal stages of hemoglobin digestion. The natural product dipeptide mimetic, bestatin, is a potent inhibitor of PfA-M1 and provides an excellent scaffold for the development of novel research tools as well as more effective PfA-M1 inhibitors. Here we present a new, efficient and high yielding protocol for the synthesis of bestatin-derivatives from commercially available natural and unnatural N-Boc-D-amino acids. We developed a diverse library of bestatin derivatives with variants at the sidechain of either the α-hydroxy-β-amino acid or the adjacent natural α-amino acid. Surprisingly we found that large aromatic rings at the P1 position resulted in potent inhibition against PfA-M1, while small hydrophobic sidechains were favored at the P1’ position. These data contrast previous studies that suggested the primary substrate specificity (S1) pocket of the PfA-M1 enzyme is unable to accommodate side-chains much larger than a P1 phenylalanine. To understand these apparently contradictory data, we determined the X-ray crystal structure of the PfA-M1 / bestatin-Tyr(OBzl) complex. The structure revealed a substantial inhibitor-induced rearrangement of the primary loop that forms the S1 pocket that permits accommodation of the bestatin-Tyr(OBzl) inhibitor. These findings are in contrast to most proteases where the S1 pocket is considered to define primary enzyme specificity through substantial rigidity. Taken together, our data provide important insights for the rational design of more potent and selective inhibitors of this enzyme, which may eventually be of therapeutic value for the treatment of malaria

Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis

Although the federal estate tax is imposed on the transfer of property, the amount of the levy is a specified percentage of the taxable estate. Correct computation of the tax depends, therefore, upon accurate identification of the property included in the taxable estate. However, the determination and application of the rules governing identification are relatively uncharted areas