Gingipains from Porphyromonas gingivalis drive Alzheimer’s pathology and can be blocked with small-molecule inhibitors.

Purpose: Bortezomib (Velcade), a dipeptide boronate 20S proteasome inhibitor and an approved treatment option for multiple myeloma, is associated with a treatment-emergent, painful peripheral neuropathy (PN) in more than 30% of patients. Carfilzomib, a tetrapeptide epoxyketone proteasome inhibitor, currently in clinical investigation in myeloma, is associated with low rates of PN. We sought to determine whether PN represents a target-mediated adverse drug reaction (ADR).Experimental Design: Neurodegenerative effects of proteasome inhibitors were assessed in an in vitro model utilizing a differentiated neuronal cell line. Secondary targets of both inhibitors were identified by a multifaceted approach involving candidate screening, profiling with an activity-based probe, and database mining. Secondary target activity was measured in rats and patients receiving both inhibitors.Results: Despite equivalent levels of proteasome inhibition, only bortezomib reduced neurite length, suggesting a nonproteasomal mechanism. In cell lysates, bortezomib, but not carfilzomib, significantly inhibited the serine proteases cathepsin G (CatG), cathepsin A, chymase, dipeptidyl peptidase II, and HtrA2/Omi at potencies near or equivalent to that for the proteasome. Inhibition of CatG was detected in splenocytes of rats receiving bortezomib and in peripheral blood mononuclear cells derived from bortezomib-treated patients. Levels of HtrA2/Omi, which is known to be involved in neuronal survival, were upregulated in neuronal cells exposed to both proteasome inhibitors but was inhibited only by bortezomib exposure.Conclusion: These data show that bortezomib-induced neurodegeneration in vitro occurs via a proteasome-independent mechanism and that bortezomib inhibits several nonproteasomal targets in vitro and in vivo, which may play a role in its clinical ADR profile. Clin Cancer Res; 17(9); 2734-43. Ó2011 AACR.

Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) coimmunoprecipitates with the Plasmodium homologue of TgRON4, a secreted rhoptry neck protein of Toxoplasma gondii that migrates at the moving junction in association with TgAMA1 during invasion. PfRON4 also originates in the rhoptry necks, suggesting that this unusual collaboration of micronemes and rhoptries is a conserved feature of Apicomplexa.Apicomplexa is a protozoan class of obligate intracellular parasites that includes many important human and animal pathogens. Apical membrane antigen 1 (AMA1) was first identified in Plasmodium knowlesi, and since then homologues have been seen in all Plasmodium species so far examined as well as at least two other apicomplexan genera, Toxoplasma and Babesia (homologues have not been detected in organisms outside Apicomplexa) (9,12,14,25). This essential membrane protein is stored in the micronemes of the asexual stages and transported to the parasite surface prior to and during host cell invasion (3). Antibodies to AMA1 directly interfere with invasion by Toxoplasma sp. tachyzoites (14) and Plasmodium falciparum merozoites (11,20), suggesting a key role in the invasion process. A similar function for the P. falciparum AMA1 protein (PfAMA1) has been described during sporozoite invasion of hepatocytes (27), indicating PfAMA1 might be an effective vaccine target for both the preerythrocytic and the asexual blood stages (17,28).One of the most distinctive features of apicomplexan invasion is the moving junction (MJ) that occurs at the site where the parasite invades into the developing parasitophorous vacuole (PV) (1, 2, 21). The appearance of electron-dense structures at the MJ is consistent with the organization of a secreted parasite complex at the interface with the host membrane. In Toxoplasma gondii, a complex minimally composed of TgAMA1 and the rhoptry neck protein, TgRON4, specifically localizes to the ring-like MJ (2,19). This ring marks the boundary where specific surface antigen complexes are removed from the parasite surface as it enters into the nascent PV (10). Host membrane proteins are also sorted at the MJ, and many that are found in complexes or associated with the extracellular matrix are excluded from the developing PV membrane (8). Thus, the MJ marks a site of intimate attachment by the parasite to the host and a sieve at which parasite and host surface proteins are selectively sorted, allowing some but not others to pass into the nascent vacuole.AMA1 has been presumed to function similarly in all Apicomplexa organisms. Given our findings in Toxoplasma, we asked whether similar immunoprecipitation experiments might reveal previously undetected binding partners for PfAMA1. P. falciparum strains 3D7 and D10 were cultured in human erythrocytes according to standard protocols (5,18). Synchronous cultures containing a majority of the parasites in the mature schizont stage were harvested ϳ40 h postinfection by lysis in 0.15% saponin (to disrupt the erythrocyte and PV membrane [4]) and stored at Ϫ80°C...

Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum cysteine proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum cysteine proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.