ISSN: 2469-6706 Vol. 5 2018 Bifunctional inhibitors of urokinase and metalloproteinase-9 for cancer treatment - in silico evaluation. Shawn P. Brewer a and Jerzy Jankun a , 1 aDepartment of Urology, The University of Toledo - Heath Science Campus, 3000 Arlington Ave., Toledo, OH 43614, USA. Matrix metalloproteinase-9 (MMP-9), and urokinase plasmino- gen activator (uPA) overexpression or/and increased activity are considered causative elements for cancer invasion and metas- tasis. These enzymes are degrading the extracellular matrix (ECM) providing space for cancer progression and cancer cell mobility. Process of angiogenesis, in which microvascular en- dothelial cells form blood vessels, requires local degradation of the underlying basal lamina to invade into the stroma proximal to cancer, and it strongly depends on the activity of MMP-9 and uPA as well. Malignant tumor invasion, cancer metastasis and angiogenesis have been documented as a fundamental factors in the morbidity and mortality among cancer patients, thus their inhibition can be exploited therapeutically. Numerous in vivo and in vitro studies have demonstrated that inhibition of prote- olytic activity can reduce cancer invasion, tumor size and limit angiogenesis. Consequently human clinical studies were de- signed inhibiting urokinase or MMPs, but these target-specific inhibitors produce mixed results. One of the possible explana- tions could be that cancers are overexpressing more than one enzyme simultaneously; for instance urokinase and MMPs. Thus upregulated net proteolytic activity should be normalized rather than trying to inhibit single proteolytic enzyme. Therefore, start- ing from specific inhibitors we have created - in silico - several hybrid molecules that could inhibit both uPA and MMP-9. The best hybrid (UI1xAGB) had theoretical affinities of Ki = 1.61−9 mol for MMP-9 and Ki = 1.36−9 mol for uPA. In the future each individual hybrid would need to be successfully synthesized and checked in the in vitro and in vivo analyses. metalloproteinase-9 | urokinase | inhibitor | molecular modeling MMatrix metalloproteinase-9 (MMP-9), and urokinase plas-minogen activator (uPA) overexpression or/and increased ac- tivity are considered causative elements for cancer invasion and metastasis. These enzymes are degrading the extracellular matrix (ECM) providing space for cancer progression and cancer cell mo- bility (1, 2). Process of angiogenesis, in which microvascular en- dothelial cells form blood vessels, depends on local degradation of the underlying basal lamina to invade into the stroma proximal to cancer, and it strongly depends on the activity of MMP-9 and uPA as well (3-5). Since malignant tumor invasion, metastasis and can- cer angiogenesis have been documented as fundamental factors in the morbidity and mortality among cancer patients and their inhibi- tion can be exploited therapeutically (5-7). Urokinase is an activator of plasminogen that upon cleavage is converted into plasmin, which can degrade a broad spectrum of proteins. Urokinase is expressed in tissues, contrary to tissue plas- minogen activator (tPA) which is present predominantly in the blood (8, 9). Therefore targeting uPA only will preserve plasmin activity necessary for dissolving fibrin blood clots and some other physio- logical processes (10-13). There are few possible approaches to inhibit urokinase. One is use of plasminogen activator inhibitor-1 (PAI-1). PAI-1, also known as endothelial plasminogen activator inhibitor or serpin E1, is a protein that functions as the principal inhibitor of urokinase and tissue plasminogen activator. Plasminogen activator inhibitor-1 exists as an active, nonactive-latent, and cleaved form. It converts spontaneously from active form into latent form in physiological conditions with half life time equal to t1/2=2 hours. Only active PAI-1 is therapeutically relevant. Thus, to use PAI-1 in therapy half-life must be extended (14-16). Several mutants of PAI-1 were produced extending its activity up to more than 700 hours (17-19). The other approach is to use antibodies against active site of uPA to restrict plasmin driven proteolytic activity (20-23). Although small molecule binding into specificity pocket or proximity of catalytic triad might be the easiest to produce. Among the large number of small molecular inhibitors amiloride was found to be uPA specific (24-27). Moreover, optimization of amiloride’s structure to poten- tiate inhibitory activity and loss of diuretic effects resulted in few novel anticancer compounds (25, 26, 28). Several clinical studies were conducted to evaluate inhibition of urokinase activity or ex- pression on cancer cells (29-34). Also, limited number of studies were monitoring prevention of cancer related angiogenesis. These reports show potential benefit of anti urokinase therapy in cancer patients and emphasize needs for additional trials (29-34). Pro-MMP-9 is activated by protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin cleaves MMP-3 zymogen to form active MMP-3 that cleaves the propeptide from the 92-kDa pro-MMP-9, generating an 82-kDa enzymatically active enzyme (35). The active MMP-9 domain contains two zinc and three cal- cium ions necessary for its function. The catalytic zinc is coordi- nated by only three histidines while the other metal co-factors (zinc and the three calcium) have their coordination spheres fulfilled by the components of surrounding protein structure (36). Inhibition of MMP-9 by small molecular chemicals lies on al- teration of its activity or/and reduction of protein expression by act- ing on DNA or RNA (37, 38). Like in the case of urokinase, MMP-9 can be inhibited by antibodies. For example GS-5745 antibody in- hibits MMP-9 by binding to pro-MMP-9 preventing activation of All authors contributed to this paper. 1To whom correspondence should be sent: jerzy.jankun@utoledo.edu The authors declare no conflict of interest. Submitted: 06/9/2018, published: 07/13//2018. Freely available online through the UTJMS open access option 6–11 UTJMS 2018 Vol. 5 utdc.utoledo.edu/Translation this metalloproteinase, or binding allosterically to active MMP-9 reducing its activity (39-42). Several clinical studies have been con- ducted in over 25 years (43-51). Overwhelming evidence from ani- mal studies warranted these studies, but unfortunately were plagued with side-effects of orally-dosed MMP-9 inhibitors. Fingleton (52) stated that for chronic dosing, agents with MMP inhibitory efficacy are needed that show minimal toxicity at low concentration. Given the well-known function for urokinase and MMP-9 in cancer cell invasion, metastasis and angiogenesis, a novel tactic to cancer therapy could be invented by testing inhibition of these proteases by one small molecular inhibitor. To inhibit both pro- teins at the same time, we have constructed in silico a novel hybrid compounds and evaluated their activity using Vina AutoDock pro- gram (53). This approach was used previously by constructing hy- brid protein consisting of the tissue inhibitor of metalloproteinases (TIMP-1) linked to the ATF domain of u-PA (54, 55). Materials and Methods Chemicals. The following chemical structures were used in molecular simulations: 1. Amiloride, AMR (Urokinase inhibitor); 3,5-diamino-6-chloro- N-(diaminomethylidene)pyrazine-2-carboxamide. 2. Ab145190 (MMP-9 inhibitor); N-[(1,1’-Biphenyl)-4-ylsulfonyl]- D-phenylalanine. 3. UI1 (Urokinase inhibitor); N-[4-(aminomethyl)phenyl]-6- carbamimidoyl-4-(pyrimidin-2yl amino)naphthalene-2- carboxam- ide. 4. 7IN (Urokinase inhibitor); rac-(1Z,2R)-2-(benzylsulfonylamino)- 3-hydroxy-N-[rac-(1S,2Z)-2-[(4-carbamimidoylphenyl) methylim- ino] -2-hydroxy-1-(hydroxymethyl)ethyl]propanimidic acid. 5. UI1xAGB (hybrid inhibitor); N-[4-[[2-[N-[4-[(1-adamantylcarb amoylamino)methyl] phenyl]carbamimidoyl] hydrazino] methyl] phenyl]-6-carbamimidoyl-4-(pyrimidin-2-ylamino)naphthalene -2- carboxamide. 6. UI1xAMR (hybrid inhibitor); N-[4-[[2-[6-amino-3-chloro- 5-[(diaminoamino)carbamoyl]pyrazin-2-yl] hydrazino] methyl ] phenyl ]-6-carbamimidoyl-4-(pyrimidin-2-ylamino)naphthalene-2- carboxamide. 7. 7INxAMR (hybrid inhibitor); 3-amino-5-[4-[[2-[[2-[(4- carbamimidoyl phenyl ) methylamino]-1-(hydroxymethyl)-2-oxo- ethyl] amino]-1-(hydroxymethyl)-2-oxo-ethyl]sulfamoylmethyl ] anilino ]-6-chloro-N-(diaminomethylene)pyrazine-2-carboxamide. 8. Hybrid3 (hybrid inhibitor); [4-[4-[2,4,6-trioxo-5-(4-pyrimidin-2- ylpiperazin-1-yl)hexahydropyrimidin-5-yl]phenoxy]phenyl]methyl N-(7-carbamimidoyl-1-naphthyl)carbamate. 9. AGB (Urokinase inhibitor); N-(1-adamantyl)-N’-(4-guanidino benzyl)urea 10. Pp3-3 [(2S)-3-[[(1S)-2-amino-1-(1H-indol-3-ylmethyl) - 2 - oxo - ethyl]amino ] - 3 -oxo - 2 - [(3-phenylisoxazol-5- yl)methyl]propyl]-phenyl-phosphinic acid. 11. Pp3 3xAMR (hybrid inhibitor) [(2S)-3-[[(1S)-2-amino-1-(1H- indol-3-ylmethyl)-2-oxo-ethyl]amino]-2-[[3-[4- [ [ 5- (carb amimi- doyl carbamoyl)-3-chloro - pyrazin - 2 -yl] amino]phenyl ] isoxazol- 5-yl]methyl]-3-oxo-propyl]-phenyl-phosphinic acid. 12. Pp3 3xp4 4 (hybrid inhibitor) [(2S)-3-[[(1S)-2-amino-1-(1H- indol-3-ylmethyl)-2-oxo-ethyl]amino]-2-[[3-[4-[(7-carbamimidoyl- 1-naphthyl)carbamoyloxymethyl]phenyl]isoxazol-5-yl] methyl] - 3 - oxo-propyl]-phenyl-phosphinic acid. Conversion of two-dimensional to three-dimensional chemical structure. When PDB 3D structure of chemicals existed it was used for molecular modeling and converted to PDBQT files through ADT. In some cases the ligand files were not in the proper format (SDF instead of PDB) or only a visual image of the structure was present. Files that were present in SDF format were converted to PDB using an online SMILES translator and structure file generator (https://cactus.nci.nih.gov/translate/). For visual models only, the inhibitors were built in 2D using Biovia draw (http://accelrys.com/). The 2D structure was then translated to a SMILES string and text was then translated by the online SMILES translator and structure file generator to the 3D PDB file. The PDB files generated through these alternative methods were then uploaded to ADT and converted to PDBQT files. Protein structure preparation and Autodock analysis. The structures of uPA (1F5L) (56) and MMP-9 (1GKC) (57) were downloaded as PDB files from RCSB Protein Data Bank. Each enzyme was open individually as a text file and the codes for wa- ter, bound ligands, and other compounds present in the file were deleted. Prior to deletion of the code, the coordinates of an individ- ual atom in the center of a ligand (present in the active site of each enzyme) was recorded for later use. For urokinase the coordinates used were: x=30.502, y=6.741, z=28.432. For MMP-9 the coordi- nates used were: x=-0.135, y=22.280, z=13.282. The isolated en- zymes were then uploaded to Autodock Tools (ADT). Using ADT, the coordinates and dimensions for the active sites of each enzyme were set. Urokinase active site size was set to 30 A on the x, y, and z axes, while for MMP-9 active site was set to 40 A on the all axes from the center defined by the above coordinates. Each enzyme was then saved as a PDBQT file as required for analysis by Autodock Vina. Each PDBQT inhibitor file was analyzed using the Autodock Vina program which calculates the inhibitors affinity (kcal/mol) for a specified enzyme binding site. For each analysis Autodock Vina generated an output file with 9 potential 3D configurations of a lig- and in an enzyme active site. Inhibitors were fitted in each enzyme and their respective output files were viewed in PyMol to ensure the best configuration was represented. The computed highest affinity as well as the observed best structure were considered as most prob- able final structure and corresponding affinity was recorded for each inhibitor. Ki = exp (∆G / (R ∗ T )) where: Ki is the inhibitory constant. T is temperature in Kelvin (calculations done at 298K). R is universal gas constant. Generation and evaluation of hybrid molecules. The inhibitors with the highest affinities for each enzyme were then used as templates for the production of a hybrid inhibitor (in this case a hybrid inhibitor refers to one that inhibits both Uroki- nase and MMP-9). The two inhibitors were bound through carbon- carbon, carbon-oxygen, or nitrogen-carbon bonds. The location of fusion of the two inhibitors aimed to leave the high affinity aspects of each on opposing ends of the new structure in order to maximize affinity for both urokinase and MMP-9 active sites. Structures were converted into PDBQT files and analyzed by Autodock Vina as de- scribed above. Brewer et al. UTJMS 2018 Vol. 5 7 Results and Discussion To validate the Vina Autodock docking protocol we redock the ligands of urokinase (amiloride and p-aminobenzamidine) to crys- tallographic protein structure after removing ligands. Ligands with lowest free energy or highest calculated affinity were used for com- parison. It is considered that a docking protocol should give RMSD < 2.0 A of crystallographic structure and that cutoff is frequently used as a criterion of the correct bound structure prediction (53). As it can be seen in Fig. 1 amiloride binds closely to its structure deter- mined by X-ray crystallography (56). P-aminobenzamidine showed similarities to its crystal structure and RMSD where below 2 A for these two controls as determined in this study and in our previous work (data not shown) (25, 56, 58, 59). Fig. 1. A: carton model of urokinase (1F5L), amino acids of cat- alytic triad (His57, Asp 102 and Ser 195) are shown as sticks model and colored: carbon in green, oxygen in red, nitrogen in blue. B: surface of uPA is shown in semitransparent gray, amilorides po- sition in specificity pocket are shown as stick model and colored: amiloride from crystallographic structure in red, best model calcu- lated by Vina Autodock colored as amino acids. Only hydrogens of amiloride calculated by Vina Autodock are shown for clarity. After testing of 21 potential inhibitors eight hybrid inhibitors were created from the best inhibitors and analyzed in silico. We have found that all the hybrids created had higher affinities for urokinase and MMP-9 than the control inhibitors (amiloride and STN) as can be seen in Table 1. The calculated affinity for amiloride bind to uPA was -7.8 kcal/mol while amiloride affinity bind to MMP-9 was -5.3 kcal/mol. The best hybrid (UI1xAGB) had affini- ties of -12.1 kcal/mol (or Ki = 1.61−9 mol) for MMP-9 and -12 kcal/mol ( (or Ki = 1.36−9 mol)) for urokinase (Fig. 2). Analyzing the binding of each individual hybrid in the target enzymes through PyMol demonstrates the potential efficacy of each hybrid. Each hybrid binds to, or in close proximity to, the catalytic triad of the urokinase active site, and the catalytic zinc and corresponding histi- dine residues of the MMP-9 active site. Binding this way makes the enzymes inaccessible to other potential ligands resulting in the ef- fective inhibition of the catalytic and/or metastatic activity of these enzymes. Table 1. Calculated affinity for proteins inhibitors complexes shown as kcal/mol or as Ki Inhibitor MMP-9a MMP-9b uPAa uPAb ab14519 -10.2 3.35−8 -6.5 2.42 −5 AGB -9.9 5.57−8 -8.2 9.81−7 Ul1 -9.6 9.24−8 -8.5 5.91−7 7lN -8.7 4.22−7 -7.5 3.19−6 AMRxab145190 -8.5 5.91−7 -9.5 1.08−8 2AMRxab145190 -8.9 4.22−7 -9.7 7.81−8 UI1xAGB -12.1 1.36−9 -12.0 1.61−9 UI1xAMR -10.1 3.97−8 -8.6 4.09−7 7INxAMR -10.3 2.83−8 -8.5 5.99−7 Pp3 3 -10.0 1.29−7 -9.4 8.28−7 Pp3 3xAMR -10.6 1.71−8 -10.8 1.22−8 Pp3 3xp4 4 -10.5 2.83−8 -9.7 7.81−8 affinity in a: kcal/mol, b: Ki mol. During the process of binding and generation of 3D structures in silico there is variance in the affinity scores as well as 3D structure orientation. A test done multiple times will almost never generate identical results. This variance can be attributed to the programs attempt at an authentic binding simulation. When running a binding analysis, the program attempts to imitate the random motion of a ligand about the binding site coordinates that have been assigned. By doing so, each test results in different affinities, but the differ- ences are so small that they are negligible. Moving forward, each individual hybrid would need to be suc- cessfully synthesized for in vitro analysis in the lab. The newly synthesized hybrids would be tested using ligand binding assays to determine the degree of affinity, equilibrium constant, reliability and validity of linked reactions, etc. Further tests would need to be run to test the hybrids ability to effectively inhibit the target enzymes function as well as other potential interactions with non-target en- zymes. Trials with animals induced with metastatic tumors would allow insight into the toxicity of the hybrid as well as its ability to control metastasis. From there, the goal would be clinical trials where it would hopefully be deemed safe and effective enough for commercial use against cancer metastasis. Numerous in vivo and in vitro studies have demonstrated that inhibition of proteolytic activity can reduce cancer invasion, tumor size and limit angiogenesis (59-63). Consequently human clini- cal studies were designed inhibiting urokinase or MMPs, but these 8 utdc.utoledo.edu/Translation Brewer et al. target specific inhibitors producing mixed results (64-67). One of the possible explanations is that cancers are overexpressing at least urokinase and MMPs simultaneously (5, 68-70). Thus upregulated net proteolytic activity should be normalized rather that inhibiting single proteolytic enzyme. Conclusion Therapy of the malignances preventing invasion, metastasis and pathological angiogenesis should include downregulation of the va- riety of proteolytic enzymes. Creating bifunctional inhibitors of urokinase and metalloproteinase could provide an alternative to ex- isting anticancer therapies. Conflict of interest Authors declare no conflict of interest. Authors’ contributions SPB, JJ conceived and designed the experiments; SPB per- formed the calculations and formal analysis; JJ reviewed and re- vised the manuscript. Both authors have wrote the manuscript, read and approved the final document. Asp 102 Ser 195 His 57 Inhibitor Inhibitor His 401 His 411 His 405 Glu 402 A B Fig 2. Best inhibitor of urokinase and MMP-9 (UI1xAGB). A: uroki- nase surface amino acids of catalytic triad (His57, Asp 102 and Ser 195) are shown as sticks model and colored: carbon i green, oxygen in red, nitrogen in blue, surface of uPA is shown in semitransparent gray. B: the catalytic center of MMP-9 is composed of the active- site zinc ion (shown as blue sphere), co-ordinated by three histidine residues (401, 405 and 411) and the essential glutamic acid residue (402) shown as a stick model colored: carbon - green, oxygen - red, nitrogen - blue), surface of uPA is shown in semitransparent gray. 1. No JH, et al. (2009) Expression of MMP-2, MMP-9, and urokinase-type plas- minogen activator in cervical intraepithelial neoplasia.Annals of the New York Academy of Sciences 1171:100-104. 2. Timoshenko OS, et al. (2015) [Matrix metalloproteinases 2 and 9, their endoge- nous regulators, and angiotensin-converting enzyme in cervical squamous cell carcinoma]. Arkhiv patologii 77(5):31-35. 3. Mignatti P & Rifkin DB (1996) Plasminogen activators and matrix metallopro- teinases in angiogenesis. Enzyme & protein 49(1-3):117-137. 4. Su SC, Lin CW, Yang WE, Fan WL, & Yang SF (2016) The urokinase-type plas- minogen activator (uPA) system as a biomarker and therapeutic target in human malignancies.Expert opinion on therapeutic targets 20(5):551-566. 5. Wong MS, Sidik SM, Mahmud R, & Stanslas J (2013) Molecular targets in the discovery and development of novel antimetastatic agents: current progress and future prospects. Clinical and experimental pharmacology & physiology 40(5):307-319. 6. Jankun J, Keck RW, & Selman SH (2014) Epigallocatechin-3-gallate prevents tumor cell implantation/growth in an experimental rat bladder tumor model. In- ternational journal of oncology 44(1):147-152. 7. Jankun J & Skrzypczak-Jankun E (2009) Yin and yang of the plasminogen acti- vator inhibitor.Polskie Archiwum Medycyny Wewnetrznej 119(6):410-417. 8. Jankun J, Selman SH, Swiercz R, & Skrzypczak-Jankun E (1997) Why drinking green tea could prevent cancer. Nature 387(6633):561. 9. Rabieian R, et al. (2018) Plasminogen Activator Inhibitor Type-1 as a Regulator of Fibrosis. Journal of cellular biochemistry 119(1):17-27. Brewer et al. UTJMS 2018 Vol. 5 9 10. Weisel JW & Litvinov RI (2017) Fibrin Formation, Structure and Properties. Sub- cellular biochemistry 82:405-456. 11. Afosah DK, Al-Horani RA, Sankaranarayanan NV, & Desai UR (2017) Potent, Se- lective, Allosteric Inhibition of Human Plasmin by Sulfated Non-Saccharide Gly- cosaminoglycan Mimetics. Journal of medicinal chemistry 60(2):641-657. 12. Godier A, Parmar K, Manandhar K, & Hunt BJ (2017) An in vitro study of the effects of t-PA and tranexamic acid on whole blood coagulation and fibrinolysis. Journal of clinical pathology 70(2):154-161. 13. Dietrich K, Ball GD, & Mitchell LG (2016) Increased plasminogen activator in- hibitor results in a hypofibrinolytic state in adolescents with obesity: in vivo and ex vivo evidence. British journal of haematology 175(2):300-307. 14. Kindell DG, Keck RW, & Jankun J (2015) Comparison between the clot-protecting activity of a mutant plasminogen activator inhibitor-1 with a very long half-life and 6-aminocaproic acid. Experimental and therapeutic medicine 9(6):2339-2343. 15. Qureshi T & Peterson CB (2016) Single fluorescence probes along the reactive center loop reveal site-specific changes during the latency transition of PAI-1. Protein science : a publication of the Protein Society 25(2):487-498. 16. Shahrour K, Keck R, & Jankun J (2015) Application of long-acting VLHL PAI-1 dur- ing sutureless partial nephrectomy in mice reduces bleeding. BioMed research international 2015:392862. 17. Berkenpas MB, Lawrence DA, & Ginsburg D (1995) Molecular evolution of plas- minogen activator inhibitor-1 functional stability. The EMBO journal 14(13):2969- 2977. 18. Chorostowska-Wynimko J, Skrzypczak-Jankun E, & Jankun J (2004) Plasmino- gen activator inhibitor type-1: its structure, biological activity and role in tumori- genesis (Review). International journal of molecular medicine 13(6):759-766. 19. Chorostowska-Wynimko J, et al. (2003) A novel form of the plasminogen acti- vator inhibitor created by cysteine mutations extends its half-life: relevance to cancer and angiogenesis. Molecular cancer therapeutics 2(1):19-28. 20. Zhou H, et al. (2017) Synergistic inhibitory effects of an engineered antibody-like molecule ATF-Fc and trastuzumab on tumor growth and invasion in a human breast cancer xenograft mouse model. Oncology letters 14(5):5189-5196. 21. Xu X, et al. (2014) Identification of a new epitope in uPAR as a target for the cancer therapeutic monoclonal antibody ATN-658, a structural homolog of the uPAR binding integrin CD11b (alphaM). PloS one 9(1):e85349. 22. Jakobsche CE, McEnaney PJ, Zhang AX, & Spiegel DA (2012) Reprogramming urokinase into an antibody-recruiting anticancer agent. ACS chemical biology 7(2):316-321. 23. Rabbani SA, et al. (2010) An anti-urokinase plasminogen activator receptor anti- body (ATN-658) blocks prostate cancer invasion, migration, growth, and experi- mental skeletal metastasis in vitro and in vivo. Neoplasia 12(10):778-788. 24. Ding Y, et al. (2012) u-PA inhibitor amiloride suppresses peritoneal metastasis in gastric cancer. World journal of surgical oncology 10:270. 25. Jankun J & Skrzypczak-Jankun E (2001) Binding site of amiloride to urokinase plasminogen activator depends on species. International journal of molecular medicine 8(4):365-371. 26. Klinghofer V, et al. (2001) Species specificity of amidine-based urokinase in- hibitors. Biochemistry 40(31):9125-9131. 27. Towle MJ, et al. (1993) Inhibition of urokinase by 4-substituted benzo[b]thiophene-2-carboxamidines: an important new class of selective synthetic urokinase inhibitor. Cancer research 53(11):2553-2559. 28. Matthews H, Ranson M, Tyndall JD, & Kelso MJ (2011) Synthesis and preliminary evaluation of amiloride analogs as inhibitors of the urokinase-type plasminogen activator (uPA). Bioorganic & medicinal chemistry letters 21(22):6760-6766. 29. Heinemann V, et al. (2013) Phase II randomised proof-of-concept study of the urokinase inhibitor upamostat (WX-671) in combination with gemcitabine com- pared with gemcitabine alone in patients with non-resectable, locally advanced pancreatic cancer. British journal of cancer 108(4):766-770. 30. Kantelhardt EJ, et al. (2011) Prospective evaluation of prognostic factors uPA/PAI-1 in node-negative breast cancer: phase III NNBC3-Europe trial (AGO, GBG, EORTC-PBG) comparing 6xFEC versus 3xFEC/3xDocetaxel. BMC cancer 11:140. 31. Ghamande SA, et al. (2008) A phase 2, randomized, double-blind, placebo- controlled trial of clinical activity and safety of subcutaneous A6 in women with asymptomatic CA125 progression after first-line chemotherapy of epithelial ovar- ian cancer. Gynecologic oncology 111(1):89-94. 32. Balasubramanian L & Evens AM (2006) Targeting angiogenesis for the treatment of sarcoma. Current opinion in oncology 18(4):354-359. 33. Kobayashi H, et al. (2004) Therapeutic efficacy of once-daily oral administration of a Kunitz-type protease inhibitor, bikunin, in a mouse model and in human cancer. Cancer 100(4):869-877. 34. Calvo FA, et al. (1992) Urokinase combination chemotherapy in small cell lung cancer. A phase II study. Cancer 70(11):2624-2630. 35. Ramos-DeSimone N, et al. (1999) Activation of matrix metalloproteinase-9 (MMP- 9) via a converging plasmin/stromelysin-1 cascade enhances tumor cell invasion. The Journal of biological chemistry 274(19):13066-13076. 36. Bode W, Gomis-Ruth FX, & Stockler W (1993) Astacins, serralysins, snake venom and matrix metalloproteinases exhibit identical zinc-binding environ- ments (HEXXHXXGXXH and Met-turn) and topologies and should be grouped into a common family, the ’metzincins’. FEBS letters 331(1-2):134-140. 37. Vandooren J, et al. (2017) Differential inhibition of activity, activation and gene expression of MMP-9 in THP-1 cells by azithromycin and minocycline versus bortezomib: A comparative study. PloS one 12(4):e0174853. 38. Kruger A, et al. (2005) Antimetastatic activity of a novel mechanism-based gelati- nase inhibitor. Cancer research 65(9):3523-3526. 39. Appleby TC, et al. (2017) Biochemical characterization and structure determina- tion of a potent, selective antibody inhibitor of human MMP9. The Journal of biological chemistry 292(16):6810-6820. 40. Gossage DL, et al. (2018) Phase 1b Study of the Safety, Pharmacokinetics, and Disease-related Outcomes of the Matrix Metalloproteinase-9 Inhibitor Andecalix- imab in Patients With Rheumatoid Arthritis. Clinical therapeutics 40(1):156-165 e155. 41. Marshall DC, et al. (2015) Selective Allosteric Inhibition of MMP9 Is Efficacious in Preclinical Models of Ulcerative Colitis and Colorectal Cancer. PloS one 10(5):e0127063. 42. Sandborn WJ, et al. (2016) Randomised clinical trial: a phase 1, dose-ranging study of the anti-matrix metalloproteinase-9 monoclonal antibody GS-5745 ver- sus placebo for ulcerative colitis. Alimentary pharmacology & therapeutics 44(2):157-169. 43. Sessa C, et al. (2013) Phase I safety, pharmacokinetic and pharmacodynamic evaluation of the vascular disrupting agent ombrabulin (AVE8062) in patients with advanced solid tumors. Clinical cancer research : an official journal of the American Association for Cancer Research 19(17):4832-4842. 44. Stephenson K, Neuenschwander PF, & Kurdowska AK (2013) The effects of com- pounded bioidentical transdermal hormone therapy on hemostatic, inflamma- tory, immune factors; cardiovascular biomarkers; quality-of-life measures; and health outcomes in perimenopausal and postmenopausal women. International journal of pharmaceutical compounding 17(1):74-85. 45. Reckamp KL, et al. (2008) Tumor response to combination celecoxib and er- lotinib therapy in non-small cell lung cancer is associated with a low baseline matrix metalloproteinase-9 and a decline in serum-soluble E-cadherin. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 3(2):117-124. 46. Chiappori AA, et al. (2007) A phase I pharmacokinetic and pharmacodynamic study of s-3304, a novel matrix metalloproteinase inhibitor, in patients with ad- vanced and refractory solid tumors. Clinical cancer research : an official journal of the American Association for Cancer Research 13(7):2091-2099. 47. Rizvi NA, et al. (2004) A phase I study of oral BMS-275291, a novel nonhydrox- amate sheddase-sparing matrix metalloproteinase inhibitor, in patients with ad- vanced or metastatic cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 10(6):1963-1970. 48. Falardeau P, Champagne P, Poyet P, Hariton C, & Dupont E (2001) Neovastat, a naturally occurring multifunctional antiangiogenic drug, in phase III clinical trials. Seminars in oncology 28(6):620-625. 49. Erlichman C, et al. (2001) Phase I study of the matrix metalloproteinase inhibitor, BAY 12-9566. Annals of oncology : official journal of the European Society for Medical Oncology 12(3):389-395. 50. Duivenvoorden WC, Hirte HW, & Singh G (2001) Quantification of matrix metallo- proteinase activity in plasma of patients enrolled in a BAY 12-9566 phase I study. International journal of cancer 91(6):857-862. 51. Wojtowicz-Praga S, et al. (1998) Phase I trial of Marimastat, a novel matrix metal- loproteinase inhibitor, administered orally to patients with advanced lung cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 16(6):2150-2156. 52. Fingleton B (2008) MMPs as therapeutic targets–still a viable option? Seminars in cell & developmental biology 19(1):61-68. 53. Trott O & Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of computational chemistry 31(2):455-461. 54. Eefting D, et al. (2010) A novel urokinase receptor-targeted inhibitor for plasmin and matrix metalloproteinases suppresses vein graft disease. Cardiovascular research 88(2):367-375. 55. Lamfers ML, et al. (2002) Gene transfer of the urokinase-type plasminogen activa- tor receptor-targeted matrix metalloproteinase inhibitor TIMP-1.ATF suppresses neointima formation more efficiently than tissue inhibitor of metalloproteinase-1. Circulation research 91(10):945-952. 56. Zeslawska E, et al. (2000) Crystals of the urokinase type plasminogen activa- tor variant beta(c)-uPAin complex with small molecule inhibitors open the way towards structure-based drug design. Journal of molecular biology 301(2):465- 475. 57. Rowsell S, et al. (2002) Crystal structure of human MMP9 in complex with a reverse hydroxamate inhibitor. Journal of molecular biology 319(1):173-181. 58. Jankun J & Skrzypczak-Jankun E (1999) Molecular basis of specific inhibition of urokinase plasminogen activator by amiloride. Cancer biochemistry biophysics 17(1-2):109-123. 59. Swiercz R, Keck RW, Skrzypczak-Jankun E, Selman SH, & Jankun J (2001) Re- combinant PAI-1 inhibits angiogenesis and reduces size of LNCaP prostate can- cer xenografts in SCID mice. Oncology reports 8(3):463-470. 60. Wiecikowski A, Cabral K, Almeida MDS, & Carvalho RS (2018) Ligand-free method to produce the anti-angiogenic recombinant Galectin-3 carbohydrate recognition domain. Protein expression and purification 144:19-24. 61. Tadbir AA, et al. (2012) Serum level of MMP-3 in patients with oral squamous cell carcinoma–lack of association with clinico-pathological features. Asian Pacific journal of cancer prevention : APJCP 13(9):4545-4548. 10 utdc.utoledo.edu/Translation Brewer et al. 62. Chakraborty S, Kaur S, Guha S, & Batra SK (2012) The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer. Biochimica et biophysica acta 1826(1):129-169. 63. Levchenko T, Bratt A, Arbiser JL, & Holmgren L (2004) Angiomotin expression promotes hemangioendothelioma invasion. Oncogene 23(7):1469-1473. 64. Lu JJ, et al. (2018) Prognostic value of urokinase plasminogen activator system in non-small cell lung cancer: A systematic review and meta-analysis. Molecular and clinical oncology 8(1):127-132. 65. Sasaki T, et al. (2014) A retrospective study of urokinase-type plasminogen ac- tivator receptor (uPAR) as a prognostic factor in cancer of the uterine cervix. International journal of clinical oncology 19(6):1059-1064. 66. Decock J, Paridaens R, & Cufer T (2005) Proteases and metastasis: clinical relevance nowadays? Current opinion in oncology 17(6):545-550. 67. Gandolfo GM, Conti L, & Vercillo M (1996) Fibrinolysis components as prog- nostic markers in breast cancer and colorectal carcinoma. Anticancer research 16(4B):2155-2159. 68. Santibanez JF, Obradovic H, Kukolj T, & Krstic J (2018) Transforming growth factor-beta, matrix metalloproteinases, and urokinase-type plasminogen activa- tor interaction in the cancer epithelial to mesenchymal transition. Developmen- tal dynamics : an official publication of the American Association of Anatomists 247(3):382-395. 69. Jiang WG, et al. (2015) Tissue invasion and metastasis: Molecular, biological and clinical perspectives. Seminars in cancer biology 35 Suppl:S244-S275. 70. Lah TT, Duran Alonso MB, and Van Noorden CJ (2006) Antiprotease therapy in cancer: hot or not? Expert opinion on biological therapy 6(3):257-279. Brewer et al. UTJMS 2018 Vol. 5 11 Cover Volume 5 1037 v5