Submitted Abstracts

Molecular Modeling Study of 6-[(Z) (4-Chlorophenyl) Diazenyl]-N-(3, 4-Dichlorophenyl)-2-Oxo-2H-Chromene-3-Carboxamide on HIV-1 Protease: Virtual Screening, Molecular Docking, ADME & Tox Study

S. Devda1, V. Saini1, P. Rao1, S. Mali1, P. Bafna1, T. Sahu1, S. Parveen1, Dr. K. Sharma1 S.A.H. Naqvi2

1Department of Biotechnology, MLSU, Udaipur, India; 2BioDiscovery Group LifeSciences, India

Presenting Author: S. Devda
Phone: 9457860087
Email: ceo.biodiscovery@gmail.com
Pushpanjali Puram, Phase 4
Agra, UP 282001
India

Retroviral protease (PR) from the human immunodeficiency virus type 1 (HIV-1) was identified over a decade ago as a potential target for structure-based drug design and is an important target for treatment of AIDS. Antiviral inhibitors of HIV-1 protease are a notable success of structure-based drug design and have dramatically improved AIDS therapy. However, the resistance and negative side effects of the current drugs has necessitated the development of new compounds with different binding patterns. The study conducted for the development of new HIV-1 PR inhibitors based on the biding mode of around 3500 different classes of 6-[(Z) (4-chlorophenyl) diazenyl]-N-(3,4-dichlorophenyl)-2-oxo-2H-chromene-3-carboxamide, an anti-HIV. Molecular docking approach using Lamarckian Genetic Algorithm was carried out to find out the compelling inhibitors for HIV-1 PR on the basis of calculated ligand-protein pairwise interaction energies. The grid maps representing the protein were calculated using auto grid and grid size was set to 60*60*60 points with grid spacing of 0.375 Ǻ. Docking was carried out with standard docking protocol on the basis of a population size of 150 randomly placed individuals; a maximum number of 2.5 *107 energy evaluations, a mutation rate of 0.02, a crossover rate of 0.80 and an elitism value of 1. Ten independent docking runs were carried out for each ligand and results were clustered according to the 1.0 Ǻ rmsd criteria. The docking result of the study of 1800 molecules demonstrated that the binding energies were in the range of -3.80 kcal/mol to -9.83 kcal/mol with the minimum binding energy of -9.83 kcal/mol. 12 molecules showed promising ADMET properties. Further in-vitro and in-vivo study is required on these molecules as the binding mode provided hints for the future design of new inhibitors for HIV-1 PR.


Alpha-2-Macroglobulin (A2M) Variants for Treatments of Osteoarthritis

Shawn Browning, Amiee M. Weiser, Naruewaan Woolf, Gaetano J Scuderi, and Lewis S. Hanna

Cytonics Corp

Presenting Author: Lewis S. Hanna
Phone: 239-272-0742
Email: shawn.browning@cytonics.com
555 Heritage Dr. Suite 115
Jupiter, FL 33458
United States

Alpha-2-Macroglobulin (A2M) is a protease inhibitor effective against metalloproteinases (MMPs), Disintegrins (ADAMTSs), and inflammatory proteases, all of which contribute to osteoarthritis (OA). Intraarticular injection of wild type A2M (wt-A2M) inhibits cartilage breakdown due to protease activity in an OA rabbit and rat models. We have developed several recombinant A2M Variants (rAV) that are more effective than wt-A2M at inhibiting the proteases implicated in OA. Recombinant wt-A2M and 85 rAV were tested for their in vitro inhibition of OA proteases, including MMP-1, -2, -8, 9 and -13, ADAMTS-4 and -5, Cathepsin-G, Elastase, and Trypsin. Fresh bovine cartilage explants (BCE) were treated with TNFa and IL-1β to activate chondrocytes and release proteases responsible for cartilage degradation. Wt-A2M and the 6 most efficacious rAV were tested for their ability to inhibit cartilage catabolism. Some of the variants were >400% more effective than wt-A2M in inhibiting ADAMTS-4 and 5, some of these variants also inhibited the other proteases by 200%. In the BCE model, wt-A2M and all A2M variants were potently chondroprotective against cytokine-induced damage. Cyt-108 was the most promising and showed 208% increased inhibition relative to wt-A2M. Four A2M variants were equally inhibitory at approximately 175% of wt-A2M (Cyt-86, 98, 102 and 107), while Cyt-105 was equivalent to wt-A2M. We have been successful in producing recombinant A2M variants that are 175 - 208% more potent than wt-A2M at inhibiting cartilage degradation in ex vivo BCE models. An approved device that concentrates A2M from the patient’s own blood can produce 4 – 6mg of A2M to be injected intra-articularly. Great results in relieving pain due to OA are observed. A2M variants designed to be more chondroprotective than wt-A2M have the potential to be the first biological therapy that will address the underlying cartilage degradation in OA.


In vitro Selection of RNA Aptamers as Potential Inhibitors of ADAM10 and ADAM17

Jaroslaw Jucha, Anna Grybos, Joanna Bereta

Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland

Presenting Author: Jaroslaw Jucha
Phone: 0048 12 664 6405
Email: jaroslaw.jucha@uj.edu.pl
Gronostajowa 7
Krakow, Malopolska 30-387
Poland

Two transmembrane proteins of “a disintegrin and metalloprotease” family, ADAM10 and ADAM17, are involved in embryonic development and immunity. The lists of ADAM10 and ADAM17 substrates partially overlap and include growth factors, cytokines, receptors of both as well as adhesion proteins, thus it is not surprising that the two enzymes play an important role in the development of chronic inflammation and cancer through influencing cell-cell communication and signaling. Both ADAM10 and ADAM17 are therefore promising targets of new therapeutics that would slow down progression of the serious and often incurable diseases. Apart from small molecular drugs and monoclonal antibodies also aptamers could play a role of the protease inhibitors. Aptamers are synthetic, single-stranded oligonucleotides selected from a pool of random sequences that can bind to a wide range of targets with high affinity and specificity. In this study, the systematic evolution of ligands by exponential enrichment (SELEX) was used to select RNA oligonucleotides specific to ADAM10 and ADAM17. Whole cells expressing ADAM10 or ADAM17 protein were used for selection of aptamers (cell-SELEX) from a library of 2’-F-pyrimidine modified RNA sequences with a complexity of around 1014. Following 18 rounds of cell-SELEX, the selected aptamers were cloned and sequenced. To determine inhibitory properties of distinct aptamers, an active recombinant catalytic ectodomains of murine ADAM10 and ADAM17 were produced in an insect cell expression system. Preliminary results confirmed that obtained aptamers are able to reduce the level of soluble TNF, shed from cells surface in vitro by ADAM10 or ADAM17.


Protease Inhibitor Support in Oral Insulin Delivery

Miriam Kidron, Ehud Arbit, Joel Neutel

Oramed Pharmaceuticals

Presenting Author: Miriam Kidron
Phone: 97225660001
Email: ariella@oramed.com
Hi-Tech Park 2/4
Jerusalem 91390
Israel

Orally delivered protein and peptide (P/P) drugs have been described to better mimic physiological gradients and natural sites of action. In addition, such delivery options promote patient compliance and adherence, typically supporting early intervention and improved clinical results. However, P/P drugs are intrinsically poorly absorbable owing to their high molecular weight and hydrophilicity. Furthermore, they are susceptible to mechanical and enzymatic degradation along the gastrointestinal tract (GIT). Numerous works have demonstrated the protective role of protease inhibitors (PIs) against degradative threats along the GIT, when incorporated in drug formulations. In pursuit of a universally applicable oral protein delivery platform tailored to address GIT challenges, Oramed Pharmaceuticals has developed the innovative PODTM technology, which provides both a protective environ for active ingredients and promotes their absorption across the intestinal epithelium. The technology, integrates essential, species-specific protease inhibitors, is currently being tested for its capacity to orally deliver insulin (ORMD-0801) and maintain its bioefficacy. In a randomized, double-blind, placebo-controlled study, 10 type 2 diabetes mellitus (T2DM) patients, received 16 mg insulin (two 8 mg capsules) at bedtime for 7 days; 10 patients received placebo capsules. On the last day of treatment, ORMD-0801-treated patients showed consistently higher mean plasma insulin levels throughout the 180 min postdosing period, when compared to baseline. Moreover, in the first 60 min postdosing, plasma insulin exposure was 20.53 μIU*h/mL greater among ORMD-0801-treated patients when compared to the placebo arm and followed a concentration-time course similar to that of plasma c-peptide. Fasting CGM data demonstrated a mean -30.24 mg/dL difference between the last two days of active versus placebo treatment. When tested in 18 healthy, fasting volunteers, a mean decline of -105.7 pmol/L c-peptide from the baseline phase (minutes 0-40 of treatment) values was observed among treated volunteers. No serious adverse events or adverse events were reported in either study. Overall, the PI-supported ORMD-0801 formulation led to a stable, consistent and short-acting rise in plasma insulin levels, which was reflected in a decline in c-peptide levels in both T2DM patients and healthy volunteers and positively impacted FBG concentrations in diabetic patients.


New Principles for Pharmacological Intervention with Trypsin-Like Proteases: Stabilization of an Inactive Conformation of Urokinase-Type Plasminogen Activator by a Camelid Derived Antibody Fragment

Tobias Kromann-Hansen1, Eva Louise Lange1, Hans Peter Sørensen1, Jan K. Jensen1, Gholamreza H. Ghassabeh2,3, Serge Muyldermans2,3, Paul J. Declerck4, Peter A. Andreasen1

1Department of Molecular Biology and Genetics, Aarhus University, Denmark; 2Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium; 3Department of Structural Biology, Flanders Institute for Biotechnology (VIB), Brussels, Belgium; 4Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Science, KU Leuven, Belgium.

Presenting Author: Tobias Kromann-Hansen
Phone: 004524452009
Email: tobiaskh@mbg.au.dk
Gustav Wieds Vej 10C
Aarhus C 8000
Denmark

Trypsin-like proteases exist in allosteric equilibria between maximally active and less active conformations. By differential binding to different conformations, ligands to allosteric sites can shift the pre-existing equilibrium and thus regulate activity. The allosteric equilibrium thereby offers opportunities for the development of therapeutic strategies aiming at stabilizing active or inactive conformations. One trypsin-like protease of particular interest is urokinase-type plasminogen activator (uPA). uPA is responsible for tissue remodeling in a variety of pathological conditions, including rheumatoid arthritis and cancer. There is therefore extensive interest in generating specific inhibitors of uPA’s enzymatic activity. We have worked with a new type of agents for regulating the activity of trypsin-like proteases, i.e., Camelid derived antibody fragments, also termed nanobodies. We have selected a nanobody (Nb7) that inhibits the activity of murine uPA against the natural protein substrate plasminogen as well as against a small chromogenic substrate. Using biochemical analysis, site-directed mutagenesis, and X-ray crystal structure analysis, we show that Nb7 binds murine uPA at a site situated more than 20 Å from the active site, between the 37 and the 70 loops. On the basis of our data, we suggest that Nb7 inhibits uPA activity through stabilization of a pre-existing inactive conformation with a highly distorted conformation, incompatible with substrate binding due to increased flexibility of functionally important regions. Together, our analyses cast new light on molecular principles for allosteric regulation of the catalytic activity of trypsin-like proteases and new principles for pharmacological intervention with their pathophysiological functions.


Determining Binding Modes of Allosteric Human Herpesvirus Protease Inhibitors

Gregory M. Lee, Jonathan E. Gable, Timothy M. Acker, Charles S. Craik

Department of Pharmaceutical Chemistry University of California - San Francisco

Presenting Author: Gregory M. Lee
Phone: 415-476-8147
Email: gregory.lee@ucsf.edu
600 - 16th St. Box 2280
San Francisco, CA 94158-2280
United States

Human herpesviruses (HHV) are known to be the cause of numerous diseases ranging from herpes simplex infections to chicken pox/shingles to Kaposi's sarcoma and consist of nine viruses separated into three subfamilies. Viral capsid formation of all HHVs is dependent upon a homologous family of dimeric serine proteases (HHV Pr). Similar to HIV proteases, whose activity has been successfully inhibited by antivirals, HHV proteases have been a target for potential therapeutics. However, there are currently no effective treatments which specifically target HHV proteases, as traditional high-throughput screening has thus far been unable to identify active-site inhibitors that can be developed into clinically useful drugs. Using a combination of high-throughput fluorescence and lower-throughput nuclear magnetic resonance (NMR) assays, we identified a small molecule helical mimetic (DD2) which allosterically inhibits HHV proteases by disrupting enzyme dimerization. We initially determined the binding mode of this lead compound against Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr, HHV-8 Pr, gamma subfamily). X-ray crystallography studies of the KSHV Pr-DD2 complex demonstrate that DD2 binds to a hydrophobic hot-spot located at the dimer interface. Successive NMR studies indicate that DD2 and its optimized scaffolds employ similar modes of binding to human simplex virus 2 protease (HSV-2 Pr, HHV-2 Pr, alpha subfamily) and cytomegalovirus protease (CMV Pr, HHV-5 Pr, beta subfamily). Hits from other high-throughput small-molecule and fragment screening campaigns have also provided new scaffolds. These results suggest that targeting protein-protein interactions is a viable route in developing pan-specific allosteric inhibitors for HHV proteases.


Effects of the Cajanus Cajan Trypsin Inhibitor (CCTI) and Lima Bean Trypsin Inhibitor (LBTI) on Normal and Cancer Cell Line

Tooba Naz Shamsi and Sadaf Fatima

Department of Biotechnology, Jamia Millia Islamia, New Delhi-110025, India

Presenting Author: Tooba Naz Shamsi
Email: amu.jmi@rediffmail.co
Jamia Millia Islamia
New Delhi-110025
India

Protease Inhibitors (PIs) are widespread in nature, produced by animals, plants and microorganisms. They play vital role in various biological activities by keeping a check on activity of proteases. Present study is an advancement of our preliminary studies which included direct in-vitro antioxidant and anti-inflammatory assays and antimicrobial activity. This study aims to investigate antioxidant and anti-proliferative properties of CCTI and LBTI on cell lines. PPI was purified from C. cajan (PUSA-992) by ammonium sulfate precipitation followed by ion exchange chromatography. The anti-proliferative activity was checked on cell lines procured from NCCS, Pune i.e. Human Embryonic Kidney (HEK) and adeno-carcinomic human alveolar basal epithelial cells (A549). The MTT endpoints showed low cytotoxic effects of PPIs extracts in HEK; LBTI showing maximum cytotoxicity of ̴ 31% followed by CCTI with cytotoxicty 27%. There was enhanced cyotoxicity in A549 cells with 58% cytotoxicity by LBTI and 50% by CCTI. The anti-oxidant potential was analyzed by various assays including Nitric Oxide (NO Assay), Superoxide dismutase (SOD) assay and Glutathione-S-Transferase (GST) Assay. The CCTI and LBTI showed free radical scavenging activity in dose dependent manner where LBTI had higher scavenging potential as compared to CCTI. The results showed more production of NO enzyme in presence of PPIs than in control. The results explain the increased SOD concentration in A549 cell line in presence of LBTI and CCTI were comparatively higher than in HEK cell line. The GST activity was highest in presence of LBTI followed by CCTI followed by control. Therefore, results conclude that CCTI and LBTI could be a useful agent for the free-radical scavenging and cancer treatment.


CM-352, A New Matrix Metalloproteinase Inhibitor, is a Potent and Safe Antifibrinolytic Agend for the Prevention and Treatment of Hemorrhage

Josune Orbe, José A. Rodríguez, Juan A. Sánchez, Agustina Salicio, Miriam Belzunce, Ana Ugarte, Haisul C.Y. Chang, Obdulia Rabal, José A. Páramo, and Julen Oyarzabal

Center for Applied Medical Research (CIMA) University of Navarra

Presenting Author: Julen Oyarzabal
Phone: +34 948 194700 ext. 2044
Email: julenoyarzabal@unav.es
Avda Pio XII, 55
Pamplona, Navarra 31008
Spain

New therapeutic strategies are required for the prevention of hemorrhaging and blood transfusions, associated with increased morbidity and mortality in surgical and trauma patients. It has been challenging to discover potent and safe agents that improve upon currently available antifibrinolytics (e.g., tranexamic acid/TXA and aprotinin). Matrix metalloproteinases (MMP) participate in thrombus dissolution through direct fibrin targeting or by enhancing tissue plasminogen activator-induced fibrinolysis. Hypothesizing that MMP inhibition may reduce bleeding by delaying fibrinolysis, we designed and synthesized a novel series of optimized MMP inhibitors that underwent a phenotypic screening consisting of thromboelastometry and mouse tail-bleeding. Our lead compound, CM-352, inhibited fibrinolysis in human whole blood functional assays and was more effective than TXA in the tail-bleeding model, using a 30,000 times lower dose. Moreover, CM-352 reduced blood loss during liver hepatectomy, while TXA and aprotinin had no effect, as well as intracranial haemorrhage induced by collagenase. CM-352 displayed optimal pharmacokinetic and safety profiles with no evidence of thrombosis or coagulation impairment; thus, CM-352 represents a promising new pre-clinical candidate for the acute treatment of bleeding. In summary, we provide evidence that fibrinolysis can be inhibited by targeting MMP, acute treatment overcoming the musculoskeletal toxicity due to chronic treatment, and this novel mechanism of action defines a new class of antihemorrhagic agents that may also lead to drug repositioning: known MMP inhibitors that reached clinical phases. In fact, Tanomastat is the most promising candidate for this novel indication; however, this molecule has poorer efficacy than CM-352.


Selective Inhibition of Bone Resorption by Cathepsin K Exosite Inhibitor

Preety Panwar1,2,3, Kent Soe3, Rafael V. C. Guido4, Renata V. Bueno3, Jean-Marie Delaisse3 and Dieter Brömme1,2,5

1Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia; 2Center for Blood Research, Vancouver, British Columbia V6T1Z3, Canada; 3Clinical Cell Biology, Vejle Hospital/Lillebaelt Hospital, Institute of Regional Health Research, University of Southern Denmark, Kabbeltoft 25, 7100 Vejle, Denmark; 4Laboratório de Química Medicinal e Computacional, Centro de Inovação em Biodiversidade e Fármacos, Instituto de Física de São Carlos, Universidade de São Paulo, 13563-120, São Carlos-SP, Brazil;  5Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, V6T 1Z3, Canada

Presenting Author: Preety Panwar
Phone: 7788720275
Email: preity.pan@gmail.com
4540-2350 Health Sciences Centre
Vancouver, British Columbia V6T1Z3
Canada

Cathepsin K (CatK) is a major drug target for the treatment of osteoporosis. Potent active site-targeting CatK inhibitors have been developed and several of them were in clinical trials with variable success. Active site inhibitors block the entire activity of the target protease and thus may interfere with other pathways counting for some of the observed adverse effects. Here, we demonstrate for the first time the antiresorptive potency of an exosite inhibitor, dihydrotanshinone I (DHTI) that specifically inhibits the collagenase activity of CatK without affecting other activities. In osteoclast bone resorption assays, the action of DHTI is comparable to that of the selective active site CatK inhibitor, odanacatib (ODN). 1 µM DHTI reduced the resorption surface by 41% when compared to 33 % in the presence of 500 nM ODN. The resorption depth of the pits was reduced by 46 and 48% and the CTx release by 83 and 61% for DHTI and ODN, respectively. Both inhibitors abolished the formation of resorption trenches, without affecting the metabolic activity and the number of OCs. Ultrastructural and microchemical analysis of resorption cavities revealed the accumulation of demineralized collagen fibrils indicating the inhibition of osteoclast-mediated collagenolysis in the presence of the both DHTI and ODN. In contrast, 10 µM DHTI did not block TGF-ß1 degradation by fibroblasts, whereas 1 µM ODN was effective. Collectively, our study shows that an exosite inhibitor of CatK can specifically block bone resorption without interfering with other pathways and thus introduce an alternative approach for the treatment of bone disorders.


Differential Role of Serine Protease Inhibitor and PAR-2 Antagonist in Attenuation of Mucosal Allergic Sensitization

Sanjay Saw, Naveen Arora

CSIR-Institute of Genomics and Integrative Biology, Delhi

Presenting Author: Sanjay Saw
Phone: +919873436443
Email: sanjay.saw84@gmail.com
Lab -509, Mall road
Delhi 110007
India

Proteases augment allergic sensitization by disrupting epithelial barrier and activating innate cells to release inflammatory mediators. The present study, aimed at elucidating the effect of serine protease inhibitor 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) and PAR-2 antagonist in allergen sensitization. Methodology: The effect of protease inhibitor in allergen uptake and processing was evaluated in BMDCs; pulsed with cockroach extract and labeled ovalbumin with AEBSF or PAR-2 antagonist. In an animal experiment AEBSF and PAR-2 antagonist was given to the mice prior to sensitization. After last challenge, airway resistance was evaluated and mice were euthanized to collect BALF, blood and lung for analysis. Results: Allergen uptake by DCs is an initial step in allergen sensitization which was reduced by protease inhibitor whereas PAR-2 antagonist showed moderate effect. Further, AEBSF given to mice lowered the airway resistance as compared to PAR-2 antagonist. AEBSF also reduced the cellular infiltration into lungs but couldn’t reduce eosinophil significantly whereas; PAR-2 antagonist specifically lowered eosinophil infiltration. The effect of AEBSF in reducing IgE, IL-4 and TNFα was PAR-2 mediated though its effect on IL-13 was partially dependent. AEBSF also reduced IgG1 and GM-CSF and induced IL-12 independent of PAR-2. Conclusion: Reduced allergic sensitization by serine protease inhibitor was partially dependent on PAR-2 mediated processes.


Isolation, Optimization and Characterization of Protease Producing Bacteria from Soil and Water

Gampa Vijaya Kumar, B.Agaiah, S.Murali, and N.Rajendraprasad

CVM College of Pharmacy, SRR College of Pharmaceutical Sciences

Presenting Author: Gampa Vijaya Kumar
Phone: 9849171547
Email: vijaytanu71@rediffmail.com
CVM College of Pharmacy
Velichala-Post, via; Kathapalli, Ramadugu-Mandal
Karimnagar, Telangana 505451
India

Protease can produce from Microorganisms that share nearly 48% of the total worldwide enzyme market. Proteases have many applications in different industries and environmental bioremediations. The objective of this study was to isolate, optimize and characterize protease producing bacteria. One gram soil or 1ml water from each collected sample was added in test tube containing 9 ml saline water and mixed well using vortex mixer. Each test tube containing the suspensions was used as stock culture. Serial dilution was made up to 10-6 for each sample. Hundred micro-liter suspensions were taken from each test tube containing 10-6, spread over milk agar plates and incubated at 370C for 24 hrs. Morphologically distinct colonies were subcultured in to nutrient agar slant for incubation at 370C for 24 hrs and maintained at 40C for further analysis. Isolated colonies were screened on tryptone yeast extract dexterous agar plate for proteolytic activity. The promising isolates were selected for optimization and characterization. From a total of 32 isolates, 9.5 % (ATCSs, DSs and DWs) isolates were shown proteolytic activity. The optimum pH and temperature for growth and protease productions by the isolates were found to be 8-10 and 370C-450C, respectively. Based on morphological and biochemical characterization isolate ATCSs was identified as genus Staphylococcus. Proteolytic activity is influenced by the type of microorganism used chemical and physical parameters. Therefore, a significant task should be done to get good proteolytic activity on identification and selection of potential microorganisms and optimization of physical and chemical conditions for protease producing isolates. Keywords: Proteolytic activity, protease producing microorganisms,Isolation, optimization, characterization


Development of Potent and Selective Legumain Inhibitors

Hughes, K.A., Parkinson, S.E., Higgins, C.A., D’Costa, Z., Gaddadle, K., Buozzaoui, S., Jordan, L., Janssen, D., Harrsion, T., Burkamp, F., Young, A., Scott, C., Mullan, P.B., Williams, R.

Centre for Cancer Research and Cell Biology, Queen’s University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK

Presenting Author: Rich Williams
Phone: (0)2890972791
Email: rich.williams@qub.ac.uk
CCRCB, 97 Lisburn Road
Belfast, County Down, Northern Ireland BT9 7BL
United Kingdom

Legumain, or Asparaginyl endopeptidase 1 (AEP1), was first identified as a Cysteine protease in leguminous seeds. Analysis of the cleavage substrates of Legumain revealed that this protease had a specific requirement for Asparagine in the P1 position adjacent to the cleaved C-terminus. Mammalian Legumain has shown to be expressed within the lysosome and plays an important role in MHC class II-mediated antigen presentation. It has also been shown to play a role in regulating the activity of the papain family cathepsins, specifically B, L and H. In addition, Legumain has been reported to play a major role in the conversion and activation of pro-MMP2 into the active zymogen within the tumour microenvironment. Recently there have been a number of publications that have described the use of Legumain as a marker of prognosis in many human cancers including breast, ovarian, glioma, pancreatic and prostate. In each of these cancer settings the expression level can be correlated a poor rate of patient survival. Those patients that have higher tumour Legumain expression have reported to have a worse overall outcome than those with low expression. Ohno recently published clinical data showing that Legumain expression increased with disease progression and with the highest expression being observed in patients with a Gleason score 8. We have observed similar findings in the analysis of breast cancer tumour sections within the CCRCB. In 2006, Stamenkovic reported increased expression of Legumain with disease progression in a transgenic murine model of prostate cancer. The expression of Legumain was observed to significantly increased during the transition from PIN-foci to adjacent invasive carcinomas. This published data strongly implicates that Legumain may be playing an important role in cancer progression and that further research is required. Within our group we became interested in Legumain as a drug target after studies revealed that cancer cells, and not normal cells, have an addiction to expression and activity. During studies with the natural inhibitor, CST6, and Legumain siRNA knock downs we observed that loss of this protease has significant impact of many of the hallmarks of cancer, such as proliferation, invasion and metastasis. With these results in hand we initiated a drug discovery programme with the aim of identifying a potent, selective and in-vivo compatible drug-like Legumain inhibitor.