A Novel Mode of Intervention with Serine Protease Activity: TARGETING ZYMOGEN ACTIVATION*

Serine proteases are secreted from cells as single-chain zymogens, typically having activities orders of magnitude lower than those of the mature two-chain enzymes. Activation occurs by a conformational change initiated by cleavage of a specific peptide bond. We have derived a monoclonal antibody (mAb-112) which binds with subnanomolar affinity to pro-uPA, the zymogen form of urokinase-type plasminogen activator (uPA). We mapped the epitope of the antibody to the autolysis loop, one of the structural elements known to change conformation during zymogen activation. A mechanistic evaluation with biophysical methods elucidated a novel bifunctional inhibitory mechanism whereby mAb-112 not only delays the proteolytic conversion of single-chain pro-uPA into the two-chain form but also subsequently averts the conformational transition to a mature protease by sequestering the two-chain form in a zymogen-like, noncatalytic state. Functional studies employing two variants of human HT-1080 cells, exhibiting high and low levels of dissemination in a chorioallantoic membrane assay, demonstrate that mAb-112 is an effective inhibitor of tumor cell intravasation. These findings show that pharmacological interference with zymogen activation is a plausible and robust means to regulate uPA activity and the downstream effects of plasminogen activation in the spread of cancer and other processes of pathological tissue remodeling. A strategy that targets regions related to pro-enzyme activation likely provide a unique inhibitor-protease interaction surface and is, thus, expected to enhance the chances of engineering high inhibitor specificity. Our results provide new information about the structural flexibility underlying the equilibrium between active and inactive forms of serine proteases.In nature a key mechanism for regulation of serine proteases with a trypsin-like fold is the activation of secreted zymogens or proenzymes, which typically have activities orders of magnitude lower than the mature enzymes. Zymogen activation is the central step in natural protease cascade regulation, allowing for rapid amplification of the activation signal. The catalytic activity of a zymogen relative to the mature protease can generally be thought of as a problem of equilibrium between active and inactive conformational states of the protease domain. Zymogen activation generally occurs by cleavage of the bond between amino acid residues 15 and 16.² The liberated amino terminus inserts into a hydrophobic binding cleft of the catalytic domain forming, in addition to hydrophobic interactions, a salt bridge to the side chain of Asp¹⁹⁴ which stabilizes the substrate binding pocket and oxyanion hole in a catalytically productive conformation. Conformational changes after cleavage involves four disordered regions of the activation domain, including the activation loop (residues 16-21), the autolysis loop (residues 142-152), the oxyanion stabilizing loop (residues 184-194), and the S1 entrance frame (residues 216-223) (Fig. 1A) (for reviews, see Refs. 1-3).Open in a separate windowFIGURE 1.Three-dimensional structure of uPA. A, overview of the three-dimensional structure of the serine protease domain of active uPA, displayed as ribbons. Depicted as sticks are the residues Ile¹⁶, Asp¹⁹⁴, and Ser¹⁹⁵. The activation domain, i.e. the activation loop (residues 16-21), the autolysis loop (residues 142-152), the oxyanion stabilizing loop (residues 184-193), and the S1 entrance frame (residues 216-223) are colored green. B, the epitope of mAb-112, displayed on a surface presentation of the serine protease domain of active uPA. Alanine substitution of residues depicted in red resulted in a significant change in the affinity to mAb-112, whereas alanine substitution of residues depicted in blue did not. C, a close up view of the autolysis loop (residues Gly¹⁴¹ to Lys¹⁵⁶) and residues implicated in the binding of mAb-112. All figures were constructed with Pymol on the basis of the coordinates given in the PDB entry 1C5W.Several proteases contribute to a variety of pathophysiological states, thus stimulating considerable interest in the design of specific inhibitors for pharmacological intervention. Nonetheless, classical development of small molecule inhibitors of serine proteases has proved a daunting task, with only limited success in engineering inhibitors with high affinity and specificity for related proteases possessing conserved active site architecture and P1³ specificity (4, 5). Thus far targeting zymogen activation instead of the mature protease has been a greatly under-exploited strategy in therapeutic regulation of protease activity. This approach provides an opportunity to target more unique interaction surfaces, thereby increasing inhibitor specificity, and ultimately offering novel inhibitory mechanisms. In addition, a more efficient inhibition is expected by targeting enzymes functioning high up in a catalytic cascade.A serine protease of particular relevance for pursuing therapeutic intervention is urokinase-type plasminogen activator (uPA),⁴ which catalyzes the conversion of plasminogen to the active protease plasmin, which in turn acts directly on the degradation of extracellular matrix proteins (6). Abnormal expression of uPA is implicated in tissue remodeling in several pathological conditions, including rheumatoid arthritis, allergic vasculitis, and xeroderma pigmentosum. In particular, uPA is central to the invasive capacity of malignant tumors (6). As with all trypsin-like proteases, uPA has a catalytic serine protease domain with surface-exposed loops around residues 37, 60, 97, 110, 170, and 185. Besides the catalytic domain, uPA has an amino-terminal extension consisting of a kringle domain and an epidermal growth factor domain. The latter domain functions in binding to the cell surface-anchored uPA receptor (uPAR) (6). Several proteases including plasmin (6), glandular kallikrein (7), matriptase (8), and hepsin (9) can catalyze the activation of the zymogen, pro-uPA.A number of inhibitors targeting the proteolytic activity of uPA have been developed, such as small organochemical molecules, peptides, and monoclonal antibodies, with a perspective on their use for elucidating the pathophysiological functions of its various molecular interactions and generating leads during drug development. The most specific inhibitors to date appear to be those derived from antibodies and peptidyl inhibitors, which utilize binding sites involving surface loops of uPA and extended exosite interactions to drive selectivity and specificity (for reviews, see Refs. 4 and 5).Here we present evidence that targeting zymogen activation is an effective means to regulate protease activity. This conclusion was realized through the development and biochemical analysis of an inhibitory monoclonal antibody, referred to as monoclonal antibody (mAb)-112, which not only delays cleavage of pro-uPA but acts to stabilize the activated two-chain protease in a non-catalytic conformation by restricting the conformational mobility of the activation domain. Characterization of mAb-112 further provides new insights into the flexibility of protease domains and uPA zymogen activation mechanisms. Moreover, mAb-112 was shown to efficiently inhibit human tumor cell intravasation, a step in the metastatic cascade in which activation of pro-uPA was previously implicated as a key event (10).