C3 Rho-Inhibitor for Targeted Pharmacological Manipulation of Osteoclast-Like Cells

Abstract

The C3 toxins from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) as well as C3-derived fusion proteins are selectively taken up into the cytosol of monocytes/macrophages where the C3-catalyzed ADP-ribosylation of Rho results in inhibition of Rho-signalling and characteristic morphological changes. Since the fusion toxin C2IN-C3lim was efficiently taken up into and inhibited proliferation of murine macrophage-like RAW 264.7 cells, its effects on RAW 264.7-derived osteoclasts were investigated. C2IN-C3lim was taken up into differentiated osteoclasts and decreased their resorption activity. In undifferentiated RAW 264.7 cells, C2IN-C3lim-treatment significantly decreased their differentiation into osteoclasts as determined by counting the multi-nucleated, TRAP-positive cells. This inhibitory effect was concentration- and time-dependent and most efficient when C2IN-C3lim was applied in the early stage of osteoclast-formation. A single-dose application of C2IN-C3lim at day 0 and its subsequent removal at day 1 reduced the number of osteoclasts in a comparable manner while C2IN-C3lim-application at later time points did not reduce the number of osteoclasts to a comparable degree. Control experiments with an enzymatically inactive C3 protein revealed that the ADP-ribosylation of Rho was essential for the observed effects. In conclusion, the results indicate that Rho-activity is crucial during the early phase of osteoclast-differentiation. Other bone cell types such as pre-osteoblastic cells were not affected by C2IN-C3lim. Due to their cell-type selective and specific mode of action, C3 proteins and C3-fusions might be valuable tools for targeted pharmacological manipulation of osteoclast formation and activity, which could lead to development of novel therapeutic strategies against osteoclast-associated diseases.

C3larvin Toxin, an ADP-ribosyltransferase from Paenibacillus larvae

“…Inhibition of C3larvin Transferase Activity-Previous work with C3 toxins has not produced any inhibitors of enzyme transferase activity (although at least one cell entry inhibitor has been identified for both C3bot1 and C3lim) (28). Because a number of inhibitors have been developed against mART toxin enzymatic function (29 -31), the lack of inhibitors reported for C3 toxins suggests that there are properties of the C3 subgroup which make them unique.…”

Spatiotemporally Controlled Release of Rho‐Inhibiting C3 Toxin from a Protein–DNA Hybrid Hydrogel for Targeted Inhibition of Osteoclast Formation and Activity

“…To address the excessive bone resorption by osteoclasts in osteoporosis, the targeted pharmacological modulation of osteogenesis or osteoclast activity represents a favorable strategy. The clostridial Rho‐inhibiting C3 toxins could be attractive candidates for this purpose, because the treatment of cultured osteoclast‐like cells with these enzymes effectively decreased both osteoclast formation from monocytic progenitor cells and their resorption activity in vitro . The C3 toxins (≈25 kDa) mono‐ADP‐ribosylate Rho in the cytosol inhibits Rho‐mediated signal‐transduction and results in reorganization of the actin cytoskeleton accompanied by a dramatic change in cell morphology and inhibition of central actin‐associated cellular functions .…”

A Supramolecular Approach toward Bioinspired PAMAM‐Dendronized Fusion Toxins

“…Bacterial exotoxins such as diphtheria toxins and botulinum neurotoxins have been engineered in nature as sophisticated weaponry to modify their target molecules in the cytosol of eukaryotic cells in a highly specific and extremely potent fashion that interfere with various host functions . Evidently, nature has provided a highly optimized toolbox, which offers structurally distinct protein domains exhibiting remarkable cell‐type selectivity and domains that affect intracellular signaling processes, leading to the innovation of toxin‐inspired macromolecular therapeutics through genetic engineering. The high affinity and specificity make toxins exemplary candidates as molecular Trojan Horses for mediating transport of therapeutic cargoes to specific cell types .…”

“…Evidently, nature has provided a highly optimized toolbox, which offers structurally distinct protein domains exhibiting remarkable cell‐type selectivity and domains that affect intracellular signaling processes, leading to the innovation of toxin‐inspired macromolecular therapeutics through genetic engineering. The high affinity and specificity make toxins exemplary candidates as molecular Trojan Horses for mediating transport of therapeutic cargoes to specific cell types . On the other hand, the enzymatic component of toxins could be redirected into other cells such as cancer cells to arrest their cell cycles, migration, and division.…”

“…In this context, the clostridial C3‐like exoenzymes (≈25 kDa) are therapeutically attractive cargo proteins. The C3bot1 protein (henceforth abbreviated as C3) from Clostridium botulinum , being a potent and specific Rho inhibitor, is an eminent candidate that can be utilized to address a broad spectrum of diseases, where Rho is the molecular target including cardiovascular disorder, cancer, nervous system disease, and asthma . However, the extremely low efficiency of C3 proteins to enter the cytosol of most mammalian cell types has presented significant limitation to their therapeutic value.…”