Scientists report on an approach to treating inflammatory bowel diseases that uses small synthetic molecules to trigger the production of antibodies against two metalloproteinases (MMPs). The Weizmann Institute of Science team was looking to mimic the activity of the body’s own tissue inhibitors of metalloproteinases (TIMPs) by generating molecules that would specifically inhibit MMP-9 (gelatinase) and the closely related MMP-2 (gelatinase A).
To this end they designed synthetic molecules that mimic the conserved structure of the metalloenzyme catalytic zinc-histidine complex that lies within the active site of each target MMP enzyme. Their hypothesis was that such molecules would elicit an immune response and hence the production of mAbs targeting the synthetic molecules, which would effectively also target the natural zinc-protein motif of the MMPs.
Describing their work in Nature Medicine, Irit Sagi, Ph.D., and colleagues report that immunizing experimental mice with the organic metal-ligand tris-imidazole zinc complex (Zn-tripod) molecules triggered the production of antibodies against the closely related mouse MMPs, MMP-9 (gelatinase) and MMP-2 (gelatinase A). Importantly, the resulting antibodies significantly reduced colon inflammation, ulceration, and other colonic injury resulting from dextran sodium sulphate (DSS)-induced colitis.
The team’s findings are reported in a paper titled “Antibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential.”
Initial studies demonstrated that repeated administration of candidate Zn-tripod molecules in mice triggered the production of ‘metallobody’ antibodies (designated SDS3 and SDS4) that were specific to the MMP-9 surface epitopes and were similar to TIMPS. In vitro tests confirmed that SDS3 and SDS4 inhibited the enzyme activity of both mouse and human MMP-9 and MMP-2 but not other members of the MMP family. Surface plasmon resonance (SPR) and x-ray absorption spectral analyses along with immunioprecipitation studies confirmed that the inhibitor antibodies bound directly to the metal-protein complex on activated MMP-9. X-ray crystallographic data further indicated that the variable domain (Fv) of SDS3 (from the Fab SDS3 crystal structure) was docked onto the catalytic domain of MMP-9 protein and bound directly to the catalytic zinc ion.
The inflammatory bowel disease model was chosen for in vivo therapeutic studies because previous work had demonstrated that MMP-9 plays a key role in the pathogenesis of chronic inflammatory diseases including ulcerative colitis and Crohn disease and is upregulated in colonic tissues during active flares of inflammatory bowel disease in humans. Prior work in mice had similarly demonstrated that colitis is attenuated in MMP-2 and MMP-9 double knockout animals.
In agreement with data from human samples, the Weizmann team found elevated MMP-2 and MMP-9 protein expression and activation in inflamed colonic tissue in mouse models of inflammatory bowel disease. The amelioration of disease in animals treated using SDS3 was associated with preserved mucosal architecture, limited inflammation, reduced crypt loss, and more complete re-epithelialization. Fluorescence studies confirmed that SDS3 reached the colon of DSS-treated mice within 24 hours after intravenous administration.
The molecule accumulated in focal inaflammatory-like patches, suggesting that metallobodies specifically target the activated form of the two gelatinases. Encouragingly, treatment with both SDS3 and SDS4 also resulted in protective effects in a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model. “We report a general molecular mimicry-based immunization strategy for generating antibodies targeting metal-protein motifs residing within metalloprotein clefts,” the authors state.
Indeed, they point out, while catalytic metal-protein clefts have previously been thought to be nonimmunogenic, the reported studies suggest that immunization with a small synthetic metal-baring mimicry complex is highly effective in targeting antibodies to these epitopes.
“This molecular mechanism of recognition shows noticeable similarities to the mode by which the endogenous inhibitors of MMPs (TIMPs). As different MMPs may have a protective role in inflammatory bowel disease, targeting the active conformation of key individual MMPs by these inhibitory antibodies may prove therapeutically effective in treating inflammatory disease,” they conclude.