Binding site of amiloride to urokinase plasminogen activator depends on species

A novel drug candidate is checked on its potency on animal models before it can advance to human phase of the research. Usually negative results on an imal phase disqualify it. Targeting specific enzymes by small chemicals rai ses the question about the appropriateness of this approach. As an example, the urokinase (uPA) is recognized as an important enzyme responsible for c ancer metastasis and angiogenesis. It is therefore important to ask the que stion if a small chemical will inhibit uPA of different species with the sa me or different potency. Using DNA sequence and known structure of uPA we h ave modeled 3D structures of uPAs for several different species. By theoret ical calculations we have determined most probable structure of amiloride/u PAs complexes. Catalytic triad (B57, B102, B195) and specificity pocket (B1 87-B197, B212-B229) are highly conserved in all cases, and are the regions responsible for proteolytic activity and recognition of the substrate. Sign ificant differences were observed in a different region (loop B93-B101), th at we identified as binding site of amiloride to the tissue plasminogen act ivator (tPA). Although tPA shares the same function of activating plasminog en and it is structurally similar to uPA. Amiloride is a specific inhibitor of uPA but does not inhibit tPA. Our study shows that predicted position o f amiloride depends on species and in some cases was located, as expected, in the specificity pocket, but in the other cases close to the loop B93-B10 1. This location could weaken affinity of binding or prevent inhibition of uPA. Therefore, drug screening and elimination process based solely on anim al study, without careful structural analysis, could lead to the eliminatio n of potential drugs for humans.