Optimization of Ketobenzothiazole-Based Type II Transmembrane Serine Protease Inhibitors to Block H1N1 Influenza Virus Replication.

Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug-resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR-Kbt (IN-1, aka N-0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N-terminal groups, leading to significant improvements (from µM to nM, 60 times more potent than IN-1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu-3 cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance.

Modulating the selectivity of matriptase-2 inhibitors with unnatural amino acids.

Matriptase-2, a type II transmembrane serine protease (TTSP), is expressed in the liver and regulates iron homeostasis via the cleavage of hemojuvelin. Matriptase-2 emerges as an attractive target for the treatment of conditions associated with iron overload, such as hemochromatosis or beta-thalassemia. Starting from the crystal structure of its closest homolog matriptase, we constructed a homology model of matriptase-2 in order to further optimize the selectivity of serine trap peptidomimetic inhibitors for matriptase-2 vs matriptase. Careful modifications of the P4, P3 and P2 positions with the help of unnatural amino acids led to a thorough understanding of Structure-Activity Relationship and a >60-fold increase in selectivity for matriptase-2 vs matriptase. Additionally, the introduction of unnatural amino acids led to significant increases in plasma stability. Such compounds represent useful pharmacological tools to test matriptase-2 inhibition in a context of iron overload.

Matriptase regulates c-Met mediated proliferation and invasion in inflammatory breast cancer.

The poor prognosis for patients with inflammatory breast cancer (IBC) compared to patients with other types of breast cancers emphasizes the need to better understand the molecular underpinnings of this disease with the goal of developing effective targeted therapeutics. Dysregulation of matriptase expression, an epithelial-specific member of the type II transmembrane serine protease family, has been demonstrated in many different cancer types. To date, no studies have assessed the expression and potential pro-oncogenic role of matriptase in IBC. We examined the functional relationship between matriptase and the HGF/c-MET signaling pathway in the IBC cell lines SUM149 and SUM190, and in IBC patient samples. Matriptase and c-Met proteins are localized on the surface membrane of IBC cells and their expression is strongly correlated in infiltrating cancer cells and in the cancer cells of lymphatic emboli in patient samples. Abrogation of matriptase expression by silencing with RNAi or inhibition of matriptase proteolytic activity with a synthetic inhibitor impairs the conversion of inactive pro-HGF to active HGF and subsequent c-Met-mediated signaling, leading to efficient impairment of proliferation and invasion of IBC cells. These data show the potential of matriptase inhibitors as a novel targeted therapy for IBC, and lay the groundwork for the development and testing of such drugs.

Targeting matriptase in breast cancer abrogates tumour progression via impairment of stromal-epithelial growth factor signalling.

Matriptase is an epithelia-specific membrane-anchored serine protease that has received considerable attention in recent years because of its consistent dysregulation in human epithelial tumours, including breast cancer. Mice with reduced levels of matriptase display a significant delay in oncogene-induced mammary tumour formation and blunted tumour growth. The abated tumour growth is associated with a decrease in cancer cell proliferation. Here we demonstrate by genetic deletion and silencing that the proliferation impairment in matriptase-deficient breast cancer cells is caused by their inability to initiate activation of the c-Met signalling pathway in response to fibroblast-secreted pro-HGF. Similarly, inhibition of matriptase catalytic activity using a selective small-molecule inhibitor abrogates the activation of c-Met, Gab1 and AKT, in response to pro-HGF, which functionally leads to attenuated proliferation in breast carcinoma cells. We conclude that matriptase is critically involved in breast cancer progression and represents a potential therapeutic target in breast cancer.

Analysis of subpocket selectivity and identification of potent selective inhibitors for matriptase and matriptase-2.

We studied the factors affecting the selectivity of peptidomimetic inhibitors of the highly homologous proteases matriptase and matriptase-2 across subpockets using docking simulations. We observed that the farther away a subpocket is located from the catalytic site, the more pronounced its role in selectivity. As a result of our exhaustive virtual screening, we biochemically validated novel potent and selective inhibitors of both enzymes.

Matriptase proteolytically activates influenza virus and promotes multicycle replication in the human airway epithelium.

Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

Design and synthesis of potent, selective inhibitors of matriptase.

Matriptase is a member of the type II transmembrane serine protease family. Several studies have reported deregulated matriptase expression in several types of epithelial cancers, suggesting that matriptase constitutes a potential target for cancer therapy. We report herein a new series of slow, tight-binding inhibitors of matriptase, which mimic the P1-P4 substrate recognition sequence of the enzyme. Preliminary structure-activity relationships indicate that this benzothiazole-containing RQAR-peptidomimetic is a very potent inhibitor and possesses a good selectivity for matriptase versus other serine proteases. A molecular model was generated to elucidate the key contacts between inhibitor 1 and matriptase.