Vero cells were seeded in 96-well plates on day 1 and transiently transfected with VHH-encoding pcDNA3

Vero cells were seeded in 96-well plates on day 1 and transiently transfected with VHH-encoding pcDNA3.1 plasmids on day 2. Yet, ricin is equally renowned as a biothreat agent, especially if dispersed by aerosol (2). Ricins galactose/N-acetylgalactosamine-binding lectin subunit, RTB, mediates toxin endocytosis and retrograde transport to the endoplasmic reticulum (ER) of mammalian cells. In the ER lumen, ricins enzymatic subunit, RTA, is liberated from RTB and retro-translocated into the cytoplasm where it inactivates ribosomes with remarkable efficiency (3,4). Activation of the ribotoxic stress response (RSR) and multiple stress-activated protein kinase (SAPK) pathways ensue, resulting in the triggering of programed cell death pathways. In the context of the lung, ricin triggers acute injury characterized by a massive inflammatory response driven by IL-1, IL-6 and members of the tumor necrosis factor-(TNF-) superfamily, triggering destruction of the lung epithelium, vascular leak, and edema (5). The catalytic mechanism by which RTA disables mammalian ribosomes was elucidated three decades ago when the X-ray crystal structure of ricin and its enzymatic activities were resolved more or less simultaneously GNF-PF-3777 (6,7). Endo and colleagues demonstrated that RTA is an RNA N-glycosidase (EC 3.2.2.22) that depurinates a single adenosine residue within the sarcin-ricin loop (SRL) of the 28S rRNA, an activity measurable inin vitrotranslation assays (6). The SRL, one of the longest conserved stretches of rRNA sequence, makes direct interactions with the GTP-binding domains of elongation factors like EF-Tu and is therefore indispensable for peptide elongation. The depurination reaction is confined to RTAs active site, a large solvent-exposed cleft on one face of the molecule that accommodates the protruding adenine (A) within the conserved GNF-PF-3777 GAGA motif of the mammalian SRL. The five critical residues associated with RTAs enzymatic activity have been defined by site-directed mutagenesis and include Tyr80, Tyr123, Glu177, Arg180, and Trp211 (8). Tyr80 and Tyr123 serve to stabilize the adenine base substrate via a -stacking network. Arg180 is involved in protonation of the adenine leaving group while Glu177 stabilizes the actual cleavage of the N-glycosidic bond. The role of Trp211 in catalysis remains unknown. These catalytic residues, as well as the chemistry of the SRL depurination reaction is conserved among GNF-PF-3777 other members of the ribosome-inactivating protein (RIP) superfamily of toxins, including Shiga GNF-PF-3777 toxins 1 (Stx1) and 2 (Stx2) from foodborneEscherichia coli(9). With GNF-PF-3777 the capacity to inactivate >1500 ribosomes per minute (10), RTAs active site is an obvious target to consider when designing therapeutics to arrest the effects of ricin toxin exposure. In fact, early efforts successfully identified substrate analogues (e.g., pteroic acid, guanine-like compounds) with modest RTA inhibitory activityin vitro(9), while other groups identified molecules capable of trapping RTAs active site in a closed conformation (11). However, issues related to solubility, limited potency and/or biodistribution have severely Rabbit Polyclonal to LRG1 curtailed the use of those small molecule inhibitors in cell-based assays and animal models of ricin intoxication (12). High-throughput, cell-based screens run in parallel as a complementary means of identifying novel ricin inhibitors yielded compounds that targeted host proteins associated with toxin trafficking and SAPK pathways, but not ricin itself (13,14). In the past decade, camelid-derived, single-domain antibodies, commonly referred to as VHHs or nanobodies, have received enormous attention for their potential as therapeutics against emerging infectious disease and biothreat agents, including botulinum neurotoxin (BoNT), anthrax toxin, and Shiga toxin (1518). VHHs are small (1316 kDa) immunoglobulin elements amenable to expression inE. coliand surface display on bacteriophage M13. VHHs are also highly soluble and thermostable. Of particular relevance to RTA is the reported propensity of VHHs to target active site clefts and enzymatic pockets, as shown for lysozyme, a-amylase and others (19,20). We recently described a collection of 21 VHHs that bind in immediate.