Crystals of the urokinase type plasminogen activator variant beta(c)-uPAin complex with small molecule inhibitors open the way towards structure-based drug design.

Urokinase is a serine protease involved in cancer growth and metastasis. Here we present the first urokinase crystal structure in complex with reversible inhibitors at 2.1 and 2.6 A resolution. These inhibitor complex structures have been obtained from crystals of engineered urokinase type plasminogen activator designed to obtain a crystal form open for inhibitor soaking. The mutant C122S loses its flexible A-chain upon activation cleavage and crystallizes in the presence of benzamidine, which was later displaced by the desired inhibitor. This new soakable crystal form turned out to be of great value in the process of structure-based drug design. The evaluated binding mode of amiloride, and UKI-1D revealed a new subsite of the primary specificity pocket of urokinase that will be employed in the future ligand optimisation process.

Interference with the urokinase plasminogen activator system: a promising therapy concept for solid tumours.

There is abundant evidence that the plasminogen activator (PA) system with its key components uPA (urokinase-type plasminogen activator), its cell surface receptor uPA-R (CD87) and its inhibitor PAI-1 plays a key role in tumour invasion and metastasis. Elevated levels of these factors in tumour tissue are associated with tumour aggressiveness and poor patient outcome. Animal models suggest that the PA system is not essential for fertility or survival under physiological conditions. Thus, it seems well suited as a therapeutic target for patients with solid malignant tumours. Novel therapy concepts targeting the uPA system are currently being explored. A variety of different synthetic uPA inhibitor classes have been developed over the last decades. First generation inhibitors displayed a low uPA inhibitory potency combined with broad specificity. More recently, structure based design, x-ray crystallographic screening or NMR based screening have revealed a large number of new, potent and selective uPA-inhibitors. A few modern compounds have shown promising results in preclinical testing and are now ready for Phase I clinical studies. Other therapeutic strategies such as antagonists of uPA/uPA-R interaction or gene therapeutic approaches to suppress the uPA-system are still being evaluated in in vitro and in vivo models. For clinical application, a combination therapy targeting more than one of the interacting proteolytic pathways may be required for effective antiproteolytic therapy. In addition, antiproteolytic agents may provide additive or synergistic treatment benefits if used in combination together with conventional therapeutics, in particular in those solid tumours for which potent conventional regimens already exist.

The uPA/uPA receptor system as a target for tumor therapy.

Invasiveness of a variety of tumors depends on the regulated expression of proteolytic enzymes that degrade the surrounding extracellular matrix and dissociate cell-cell and/or cell-matrix attachments. The tumor cell surface-associated urokinase-type plasminogen activator (uPA) system plays an especially important role in tumor cell invasion and metastasis. It consists of the serine protease uPA, its membrane-bound receptor (uPAR, CD87) and one of the natural inhibitors PAI-1 or PAI-2. There are strong indications based on animal experiments that interference with this system by inhibiting the enzymatic activity of uPA and/or antagonizing its binding to the receptor is of therapeutic relevance. With the recent solution of various X-ray structures of uPA/inhibitor complexes, structural information is available for optimizing existing lead compounds in their affinity and selectivity for uPA. Furthermore, peptide compounds capable of mimicking the structural epitope of uPA responsible for binding to the receptor efficiently antagonize this recognition process. Thus, both approaches prove to be well suited for the design of highly promising drugs in human medicine.

Urethanyl-3-amidinophenylalanine derivatives as inhibitors of factor Xa. X-ray crystal structure of a trypsin/inhibitor complex and modeling studies.

Hydrophobic urethanyl derivatives of 3-amidinophenylalanine methyl ester were found to be relatively potent and selective factor Xa inhibitors. These compounds consist of the arginine-mimetic 3-benzamidino group as P1 residue and of hydrophobic residues as potential interaction partners for the S3/S4 aryl binding site of the enzyme. Attempts to possibly identify their binding mode to factor Xa via the X-ray crystal structure of a trypsin/inhibitor complex and analogy modeling on the crystal structure of factor Xa failed. However, synthesis of enantiomerically pure (R)- and (S)-derivatives, combined with modeling experiments, led to an hypothetical non-substrate like binding mode, which was fully confirmed by the remarkably enhanced inhibitory potency of derivatives in which the methyl ester was replaced by arylamides for interactions with the S3/S4 enzyme binding subsites. With adamantyloxycarbonyl-(R)-3-amidinophenylalanine-phenethylamide+ ++ a nanomolar inhibiton was obtained, thus indicating this new class of factor Xa inhibitors as a highly promising lead structure.

(4-aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase.

Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure-activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N'-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1' enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1'/S2' subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.