Serine protease inhibitors decrease metastasis in prostate, breast, and ovarian cancers.

Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti-metastatic activity. Consequently, there is an urgent need for new agents targeting the metastatic process. Emerging evidence correlates in vitro and in vivo cancer invasion and metastasis with increased activity of the proteases mesotrypsin (prostate and breast cancer) and kallikrein 6 (KLK6; ovarian cancer). Thus, mesotrypsin and KLK6 are attractive putative targets for therapeutic intervention. As potential therapeutics for advanced metastatic prostate, breast, and ovarian cancers, we report novel mesotrypsin- and KLK6-based therapies, based on our previously developed mutants of the human amyloid ß-protein precursor Kunitz protease inhibitor domain (APPI). These mutants, designated APPI-3M (prostate and breast cancer) and APPI-4M (ovarian cancer), demonstrated significant accumulation in tumors and therapeutic efficacy in orthotopic preclinical models, with the advantages of long retention times in vivo, high affinity and favorable pharmacokinetic properties. The applicability of the APPIs, as a novel therapy and for imaging purposes, is supported by their good safety profile and their controlled and scalable manufacturability in bioreactors.

Bioorthogonal PEGylation Prolongs the Elimination Half-Life of N-TIMP2 While Retaining MMP Inhibition.

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of the matrix metalloproteinase (MMP) family of proteins, whose members are key regulators of the proteolysis of extracellular matrix components and hence of multiple biological processes. In particular, imbalanced activity of matrix metalloproteinase-14 (MMP-14) may lead to the development of cancer and cardiovascular and other diseases. This study aimed to engineer TIMP2, one of the four homologous TIMPs, as a potential therapeutic by virtue of its ability to bind to the active-site Zn2+ of MMP-14. However, the susceptibility to degradation of TIMP2 and its small size, which results in a short circulation half-life, limit its use as a therapeutic. PEGylation was thus used to improve the pharmacokinetic profile of TIMP2. PEGylation of the MMP-targeting N-terminal domain of TIMP2 (N-TIMP2), via either cysteine or lysine residues, resulted in a significant decrease in N-TIMP2 affinity toward MMP-14 or multisite conjugation and conjugate heterogeneity, respectively. Our strategy designed to address this problem was based on incorporating a noncanonical amino acid (NCAA) into N-TIMP2 to enable site-specific mono-PEGylation. The first step was to incorporate the NCAA propargyl lysine (PrK) at position S31 in N-TIMP2, which does not interfere with the N-TIMP2-MMP-14 binding interface. Thereafter, site-specific PEGylation was achieved via a click chemistry reaction between N-TIMP2-S31PrK and PEG-azide-20K. Inhibition studies showed that PEGylated N-TIMP2-S31PrK did indeed retain its inhibitory activity toward MMP-14. The modified protein also showed improved serum stability vs non-PEGylated N-TIMP2. In vivo pharmacokinetic studies in mice revealed a significant 8-fold increase in the elimination half-life of PEGylated N-TIMP2 vs the non-PEGylated protein. This study shows that site-specific bioorthogonal mono-PEGylation extends the half-life of N-TIMP2 without impairing its biological activity, thereby highlighting the advantage of this strategy for generating potent PEGylated proteins.

A KLK4 proteinase substrate capture approach to antagonize PAR1.

Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind 'decoy' PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This 'substrate-capturing' KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 'proteinase substrate capture' approach to regulate proteinase-mediated PAR1 signaling.

Kallikrein-Related Peptidase 6 Is Associated with the Tumour Microenvironment of Pancreatic Ductal Adenocarcinoma.

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine.

Nanobodies Targeting Prostate-Specific Membrane Antigen for the Imaging and Therapy of Prostate Cancer.

The repertoire of methods for the detection and chemotherapeutic treatment of prostate cancer (PCa) is currently limited. Prostate-specific membrane antigen (PSMA) is overexpressed in PCa tumors and can be exploited for both imaging and drug delivery. We developed and characterized four nanobodies that present tight and specific binding and internalization into PSMA+ cells and that accumulate specifically in PSMA+ tumors. We then conjugated one of these nanobodies to the cytotoxic drug doxorubicin, and we show that the conjugate internalizes specifically into PSMA+ cells, where the drug is released and induces cytotoxic activity. In vivo studies show that the extent of tumor growth inhibition is similar when mice are treated with commercial doxorubicin and with a 42-fold lower amount of the nanobody-conjugated doxorubicin, attesting to the efficacy of the conjugated drug. These data highlight nanobodies as promising agents for the imaging of PCa tumors and for the targeted delivery of chemotherapeutic drugs.

Depsipeptides Featuring a Neutral P1 Are Potent Inhibitors of Kallikrein-Related Peptidase 6 with On-Target Cellular Activity.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins (KLKs). Many KLKs are investigated as potential biomarkers for cancer as well as therapeutic drug targets for a number of pathologies. KLK6, in particular, has been implicated in neurodegenerative diseases and cancer, but target validation has been hampered by a lack of selective inhibitors. This work introduces a class of depsipeptidic KLK6 inhibitors, discovered via high-throughput screening, which were found to function as substrate mimics that transiently acylate the catalytic serine of KLK6. Detailed structure-activity relationship studies, aided by in silico modeling, uncovered strict structural requirements for potency, stability, and acyl-enzyme complex half-life. An optimized scaffold, DKFZ-251, demonstrated good selectivity for KLK6 compared to other KLKs, and on-target activity in a cellular assay. Moreover, DKFZ-633, an inhibitor-derived activity-based probe, could be used to pull down active endogenous KLK6.

A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering.

Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPIM17L,I18F,S19F,F34V (APPI-4M), an APPI variant with a KLK6 inhibition constant (Ki ) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPIWT). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPIWT/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.

Strigolactone analogs act as new anti-cancer agents in inhibition of breast cancer in xenograft model.

Strigolactones (SLs) are a novel class of plant hormones. Previously, we found that analogs of SLs induce growth arrest and apoptosis in breast cancer cell lines. These compounds also inhibited the growth of breast cancer stem cell enriched-mammospheres with increased potency. Furthermore, strigolactone analogs inhibited growth and survival of colon, lung, prostate, melanoma, osteosarcoma and leukemia cancer cell lines. To further examine the anti-cancer activity of SLs in vivo, we have examined their effects on growth and viability of MDA-MB-231 tumor xenografts model either alone or in combination with paclitaxel. We show that strigolactone act as new anti-cancer agents in inhibition of breast cancer in xenograft model. In addition we show that SLs affect the integrity of the microtubule network and therefore may inhibit the migratory phenotype of the highly invasive breast cancer cell lines that were examined.