Binding site of amiloride to urokinase plasminogen activator depends on species
J Jankun1, E Skrzypczak-Jankun
1Urology Research Center, Medical College of Ohio, Toledo, OH 43614-5807, USA. jerzy@golemxiv.dh.mco.edu
Abstract:
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 animal phase disqualify it. Targeting specific enzymes by small chemicals raises the question about the appropriateness of this approach. As an example, the urokinase (uPA) is recognized as an important enzyme responsible for cancer metastasis and angiogenesis. It is therefore important to ask the question if a small chemical will inhibit uPA of different species with the same or different potency. Using DNA sequence and known structure of uPA we have modeled 3D structures of uPAs for several different species. By theoretical calculations we have determined most probable structure of amiloride/uPAs complexes. Catalytic triad (B57, B102, B195) and specificity pocket (B187-B197, B212-B229) are highly conserved in all cases, and are the regions responsible for proteolytic activity and recognition of the substrate. Significant differences were observed in a different region (loop B93-B101), that we identified as binding site of amiloride to the tissue plasminogen activator (tPA). Although tPA shares the same function of activating plasminogen 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 of 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-B101. This location could weaken affinity of binding or prevent inhibition of uPA. Therefore, drug screening and elimination process based solely on animal study, without careful structural analysis, could lead to the elimination of potential drugs for humans.
Insights
Drug candidates targeting urokinase (uPA) may show varied potency across species. Structural analysis is crucial, as animal studies alone might wrongly eliminate promising human drugs.
Area of Science:
- Biochemistry and Molecular Biology
- Pharmacology
- Computational Chemistry
Background:
- Urokinase (uPA) is a key enzyme in cancer metastasis and angiogenesis.
- Small molecule inhibitors targeting uPA are investigated as potential cancer therapeutics.
- Cross-species efficacy of drug candidates is a critical consideration in preclinical development.
Purpose of the Study:
- To investigate the species-specific binding and inhibitory potential of amiloride to urokinase (uPA).
- To evaluate the impact of structural variations in uPA across species on drug binding.
- To assess the reliability of animal models in predicting drug efficacy for human application.
Main Methods:
- 3D modeling of urokinase (uPA) structures from various species using DNA sequences.
- In silico docking simulations to predict amiloride binding sites and complex structures.
- Comparative analysis of conserved and variable regions in uPA structures.
Main Results:
- Highly conserved catalytic triad and specificity pocket across different species' uPA.
- Significant structural differences identified in the loop region (B93-B101) of uPA.
- Amiloride binding site prediction varied by species, with some locations potentially reducing inhibitory efficacy.
- Amiloride demonstrated species-dependent binding affinity to uPA, unlike its known specificity for human uPA.
Conclusions:
- Species-specific structural variations in uPA can significantly influence the binding affinity and inhibitory potency of small molecule drugs like amiloride.
- Relying solely on animal models for drug screening may lead to the premature elimination of potentially effective human therapeutics.
- Detailed structural analysis is essential for accurate prediction of drug efficacy across species and for optimizing drug development pipelines.
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