Identification of Small Molecules That Suppress Ricin-Induced Stress-Activated Signaling Pathways

Wahome, Paul G.; Ahlawat, Sarita; Mantis, Nicholas J.

doi:10.1371/journal.pone.0049075

Cited by 17 publications

(20 citation statements)

References 38 publications

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“…We next examined the sensitivity of primary KCs and LSECs to ricin intoxication. J774E cells, a well‐characterized mouse macrophage cell line reported to express the MR (CD206), were included as a control for these experiments . Ricin cytotoxicity was initially evaluated across a range of KC and LSEC densities (1 × 10 4 to 1 × 10 5 cells total), as described in the section Materials and Methods .…”

Section: Resultsmentioning

confidence: 99%

Sensitivity of Kupffer cells and liver sinusoidal endothelial cells to ricin toxin and ricin toxin–Ab complexes

Mooney

Torres-Vélez

Doering

et al. 2019

Journal of Leukocyte Biology

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Ricin toxin is a plant‐derived, ribosome‐inactivating protein that is rapidly cleared from circulation by Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs)—with fatal consequences. Rather than being inactivated, ricin evades normal degradative pathways and kills both KCs and LSECs with remarkable efficiency. Uptake of ricin by these 2 specialized cell types in the liver occurs by 2 parallel routes: a “lactose‐sensitive” pathway mediated by ricin's galactose/N‐acetylgalactosamine‐specific lectin subunit (RTB), and a “mannose‐sensitive” pathway mediated by the mannose receptor (MR; CD206) or other C‐type lectins capable of recognizing the mannose‐side chains displayed on ricin's A (RTA) and B subunits. In this report, we investigated the capacity of a collection of ricin‐specific mouse MAb and camelid single‐domain (VHH) antibodies to protect KCs and LSECs from ricin‐induced killing. In the case of KCs, individual MAbs against RTA or RTB afforded near complete protection against ricin in ex vivo and in vivo challenge studies. In contrast, individual MAbs or VHHs afforded little (<40%) or even no protection to LSECs against ricin‐induced death. Complete protection of LSECs was only achieved with MAb or VHH cocktails, with the most effective mixtures targeting RTA and RTB simultaneously. Although the exact mechanisms of protection of LSECs remain unknown, evidence indicates that the Ab cocktails exert their effects on the mannose‐sensitive uptake pathway without the need for Fcγ receptor involvement. In addition to advancing our understanding of how toxins and small immune complexes are processed by KCs and LSECs, our study has important implications for the development of Ab‐based therapies designed to prevent or treat ricin exposure should the toxin be weaponized.

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Section: Resultsmentioning

confidence: 99%

Sensitivity of Kupffer cells and liver sinusoidal endothelial cells to ricin toxin and ricin toxin–Ab complexes

Mooney

Torres-Vélez

Doering

et al. 2019

Journal of Leukocyte Biology

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“…In an effort to circumvent the issue of solubility, high-throughput, cell-based screens were performed with commercially available small molecule libraries. While compounds were identified that were capable of protecting cells from ricin-induced killing, the molecules invariably targeted host cell processes, rather than the toxin itself ( 13, 14, 30 ).…”

Section: Discussionmentioning

confidence: 99%

“…However, issues related to solubility, limited potency and/or biodistribution have severely 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 ).…”

Section: Introductionmentioning

confidence: 99%

Intracellular Neutralization of Ricin Toxin by Single Domain Antibodies Targeting the Active Site Pocket

Rudolph

Czajka

Davis

et al. 2019

Preprint

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25The extreme potency of the plant toxin, ricin, is due to its enzymatic subunit, RTA, which 26 inactivates mammalian ribosomes with near perfect efficiency. Here we characterized, at the 27 functional and structural levels, seven alpaca single-domain antibodies (VHHs) previously 28 reported to recognize epitopes in proximity to RTA's active site. Three of the VHHs, V2A11, 29 V8E6 and V2G10, were potent inhibitors of RTA in vitro and protected Vero cells from ricin 30 when expressed as intracellular antibodies ("intrabodies"). Crystal structure analysis revealed 31 that the complementarity-determining region 3 (CDR3) elements of V2A11 and V8E6 penetrate 32 RTA's active site and interact with key catalytic residues. V2G10, in contrast, sits atop the 33 enzymatic pocket and occludes substrate accessibility. The other four VHHs also 34 penetrated/occluded RTA's active site, but lacked sufficient binding affinities to outcompete 35 RTA-ribosome interactions. Intracellular delivery of high-affinity, single-domain antibodies may 36 offer a new avenue in the development of countermeasures against ricin toxin. 37 38 3 39 65 Glu177 stabilizes the actual cleavage of the N-glycosidic bond. The role of Trp211 in catalysis 66 remains unknown. These catalytic residues, as well as the chemistry of the SRL depurination 67 reaction is conserved among other members of the ribosome-inactivating protein (RIP) 68 superfamily of toxins, including Shiga toxins 1 (Stx1) and 2 (Stx2) from foodborne Escherichia 69 coli (9). 70 With the capacity to inactivate >1500 ribosomes per minute (10), RTA's active site is an 71 obvious target to consider when designing therapeutics to arrest the effects of ricin toxin 72 exposure. In fact, early efforts successfully identified substrate analogues (e.g., pteroic acid, 73 131 performed Spearman correlation analysis to examine the relationship between in vitro RTA 132 inhibition and VHH binding affinity at both high and low antibody:RTA molar ratios (Figure 133 1E,F). There was indeed a correlation between RTA inhibitory activity and VHH dissociation 134 constants (KD), although a stronger relationship with RTA inhibitory activity and dissociation 135 rates (Koff). These results indicate that the stability of the antibody-antigen complex is critical for 136 potent inhibition of ricin in cell based killing assays and RTA in in vitro assays. 137 138

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“…Using a cell-based high-throughput screening (HTS), Wahome et al (2012) have identified three compounds, PW66, PW69, and PW72, which significantly delayed ricin-induced cell death without any demonstrable effect on ricin's ability to arrest protein synthesis in cells or on its A-chain enzymatic activity. Using a cell-based high-throughput screening (HTS), Wahome et al (2012) have identified three compounds, PW66, PW69, and PW72, which significantly delayed ricin-induced cell death without any demonstrable effect on ricin's ability to arrest protein synthesis in cells or on its A-chain enzymatic activity.…”

Section: Targeted Therapy and Intervention Points In Ricin-mediated Cmentioning

confidence: 99%

Biological Toxins and Bioterrorism

Gopalakrishnakone¹,

Balali-Mood²,

Llewellyn³

et al. 2015

Toxinology

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In recent years, the field of toxinology has expanded substantially. On the one hand it studies venomous animals, plants and micro organisms in detail to understand their mode of action on targets. While on the other, it explores the biochemical composition, genomics and proteomics of toxins and venoms to understand their three interaction with life forms (especially humans), development of antidotes and exploring their pharmacological potential. Therefore, toxinology has deep linkages with biochemistry, molecular biology, anatomy and pharmacology. In addition, there is a fast-developing applied subfield, clinical toxinology, which deals with understanding and managing medical effects of toxins on human body. Given the huge impact of toxin-based deaths globally, and the potential of venom in generation of drugs for so-far incurable diseases (for example, diabetes, chronic pain), the continued research and growth of the field is imminent. This has led to the growth of research in the area and the consequent scholarly output by way of publications in journals and books. Despite this ever-growing body of literature within biomedical sciences, there is still no all-inclusive reference work available that collects all of the important biochemical, biomedical and clinical insights relating to toxinology. Composed of 11 volumes, Toxinology provides comprehensive and authoritative coverage of the main areas in toxinology, from fundamental concepts to new developments and applications in the field. Each volume comprises a focused and carefully chosen collection of contributions from leading names in the subject.

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Identification of Small Molecules That Suppress Ricin-Induced Stress-Activated Signaling Pathways

Cited by 17 publications

References 38 publications

Sensitivity of Kupffer cells and liver sinusoidal endothelial cells to ricin toxin and ricin toxin–Ab complexes

Sensitivity of Kupffer cells and liver sinusoidal endothelial cells to ricin toxin and ricin toxin–Ab complexes

Intracellular Neutralization of Ricin Toxin by Single Domain Antibodies Targeting the Active Site Pocket

Biological Toxins and Bioterrorism

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