Abstract
Understanding the mechanisms of modulators’ action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regulated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM17′s broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New ‘exosite’ (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modulators as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibition mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity.
Highlights
A disintegrin and metalloproteinases (ADAMs) are multidomain proteins consisting of an N-terminal metalloproteinase domain, a disintegrin domain, an epidermal growth factor (EGF)-like domain, a cysteine-rich domain, a transmembrane domain, and an intracellular C-terminal domain
A minimum of approximately 0.1 mg/mL total protein was required to detect proteolysis and measure above the detection limit. These facts indicate that the measurements were performed at substrate saturation and that the amount of total protein, including ADAM17 is the rate-limiting process
We previously described a structurally related ADAM17 inhibitor that can potentially bind across domains due to the spatial proximity of the non-catalytic and catalytic domains, which corroborates with the evidence presented [30]
Summary
A disintegrin and metalloproteinases (ADAMs) are multidomain proteins consisting of an N-terminal metalloproteinase domain, a disintegrin domain, an epidermal growth factor (EGF)-like domain, a cysteine-rich domain, a transmembrane domain, and an intracellular C-terminal domain. As ADAM10 and ADAM17 lack the EGF-like domain, the membrane-proximal domain is referred to as stalk region [1] Both proteases have been shown to contribute to multiple pathophysiological processes, including acute and chronic inflammatory diseases, autoimmune diseases, and cancer formation (for review see [2,3,4]). There exist several approaches to address ADAMs as therapeutic targets, including targeting expression, maturation, and activation, the use of conformation-specific antibodies, inhibition of the active site by small molecule inhibitors, or changes of the conformation by so-called exosite inhibitors [8,9,10].
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