As recently discovered, matriptase-2, a type II transmembrane serine protease, plays a crucial role in body iron homeostasis by down-regulating hepcidin expression, which results in increased iron levels. Thus, matriptase-2 represents a novel target for the development of enzyme inhibitors potentially useful for the treatment of systemic iron overload (hemochromatosis). A comparative three-dimensional model of the catalytic domain of matriptase-2 was generated and utilized for structure-based virtual screening in combination with similarity searching and knowledge-based compound design. Two N-protected dipeptide amides containing a 4-amidinobenzylamide as P1 residue (compounds 1 and 3) were identified as the first small molecule inhibitors of matriptase-2 with K i values of 170 and 460 nM, respectively. An inhibitor of the closely related protease matriptase (compound 2, K i = 220 nM), with more than 50-fold selectivity over matriptase-2, was also identified.

Matriptase-2 is a member of the TTSPs (type II transmembrane serine proteases), an emerging class of cell surface proteases involved in tissue homoeostasis and several human disorders. Matriptase-2 exhibits a domain organization similar to other TTSPs, with a cytoplasmic N-terminus, a transmembrane domain and an extracellular C-terminus containing the non-catalytic stem region and the protease domain. To gain further insight into the biochemical functions of matriptase-2, we characterized the subcellular localization of the monomeric and multimeric form and identified cell surface shedding as a defining point in its proteolytic processing. Using HEK (human embryonic kidney)-293 cells, stably transfected with cDNA encoding human matriptase-2, we demonstrate a cell membrane localization for the inactive single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric, as well as multimeric, forms covalently linked by disulfide bonds. Furthermore, matriptase-2 undergoes shedding into the conditioned medium as an activated two-chain form containing the catalytic domain, which is cleaved at the canonical activation motif, but is linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by MS, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active-site serine residue is dependent on the catalytic activity of co-expressed or co-incubated wild-type matriptase-2, indicating a transactivation and trans-shedding mechanism.

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Matriptase-2, a recently identified cell surface protease, is the key enzyme of iron homoeostasis modulating the expression of the liver peptide hormone hepcidin. HAI (hepatocyte growth factor activator inhibitor) types 1 and 2 (HAI-1 and HAI-2 respectively) have been shown to inhibit the close homologue, i.e. matriptase. By co-expressing matriptase-2 and the inhibitor HAI-2 we have identified HAI-2 displaying high inhibitory potential against matriptase-2 at the cell surface as well as in conditioned medium. Accordingly, complex formation between matriptase-2 and HAI-2 was demonstrated by isolation of the complex via immobilizing either HAI-2 or matriptase-2 from lysates and conditioned medium of co-expressing cells. Furthermore, HAI-2 indirectly influences the expression of the hepcidin-encoding gene HAMP. The inhibitor abrogates the matriptase-2-mediated suppression of HAMP expression, presumably by inhibiting the supposed potential of matriptase-2 to cleave membrane-bound HJV (haemojuvelin). Taken together, the results of the present study have characterized HAI-2 as an inhibitor of matriptase-2 that modulates the synthesis of hepcidin and provides new insights into the regulatory mechanism of iron homoeostasis, with clinical importance for a treatment of iron overload diseases.