• hydroxamate
• molrep
• mutant form
• proteolytic events
• reverse screening
• selectivity
• sitting drop

The selectivity of zinc-metalloproteases inhibitors is crucial as broad spectrum inhibitors can cause severe adverse effects. In both laboratories where I have carried out my thesis work, the development of new selective inhibitors is a major endeavor. The work has involved the synthesis of inhibitors for ADAM-10 and the crystal structure determination of these ligands complexed to the catalytic domains of different MMPs with the aim to design inhibitors with enhanced specificity.
The ADAMs family of transmembrane proteins belongs to the zinc protease superfamily. Members of this family have a modular design, characterized by metalloprotease and integrin receptor-binding activities. The cytoplasmic domain in many family members specifies binding sites for various signal transducing proteins. The ADAMs family has been implicated in the control of membrane fusion, cytokine and growth factor shedding, cell migration, as well as processes such as muscle development, fertilization, and cell fate determination. Pathologies such as inflammation and cancer may involve ADAMs family members. Zinc proteases are classified according to the primary structure of their catalytic sites and include gluzincin, metzincin, inuzincin and certain carboxypeptidases. The ADAMs belong to the metzincin subgroup that is subdivided into serralysins, astacins, matrixins, and adamalysins. The matrixins comprise the matrix metalloproteases, or MMPs. These enzymes are the main agents responsible for extracellular matrix degradation and remodeling, and play relevant roles in development, wound healing and in the pathology of diseases such as arthritis and cancer. Adamalysins differ from the matrixins by the presence of a unique integrin receptor-binding disintegrin domain but have similar metalloprotease domains. ADAMs derive their name from the presence of these two domains (A Disintegrin And Metalloprotease). Their involvement in such a broad spectrum of diseases is due to the large variety of substrates that ADAMs are able to cleave. They can activate growth factors or inactivate receptors by shedding their extracellular domain from the cell membrane. Similarly, they can break off cells by cleaving cell adhesion molecules. Some of these proteolytic events are part of cleavage cascades known as Regulated Intramembrane Proteolysis that leads to intracellular signaling. Thus, ADAMs can fulfill a key role in diverse processes and pathologies, making them prime targets for developing therapies. This is the main reason for pharmaceutical research investment in zinc protease inhibitors (MMP and ADAM inhibitors).
My thesis started with the aim to synthesize and test in crystallographic studies on some MMPs two selective ADAM10 inhibitors LT2 (a carboxylate) and LT4, its hydroxamate analogue.
These new inhibitors, were tested in vitro on human recombinant ADAM-10 and ADAM-17 (TACE1) and on MMPs (MMP-8, MMP-9, MMP-12, MMP-13) by fluorometric assay. The inhibitors' activity was in the micro-molar range, except for LT4 that resulted nanomolar (45 nM on ADAM-10). Interestingly this inhibitor was potent and selective for ADAM-10 (as expected from the design) and able to discriminate against TACE. The reason why this hydroxamate-based inhibitor has such a good selectivity needs to be understood.
During the course of my thesis, I carried out crystallographic studies only using the catalytic domains of MMP-8, MMP-9, MMP-12 and MMP-13 since MMP-14, ADAM-10 and TACE were not yet available for crystallographic studies. This has given an initial idea regarding how the ligands are stabilized in the metzincin catalytic sites and of the interactions made by the substituents within the pockets. This allows a comparison with theoretical predictions and other crystallographic studies.