Mechanism-Based Macrocyclic Inhibitors of Serine Proteases.

Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery.

Small Molecule Antagonists of the DNA Repair ERCC1/XPA Protein-Protein Interaction.

The DNA excision repair protein ERCC1 and the DNA damage sensor protein, XPA are highly overexpressed in patient samples of cisplatin-resistant solid tumors including lung, bladder, ovarian, and testicular cancer. The repair of cisplatin-DNA crosslinks is dependent upon nucleotide excision repair (NER) that is modulated by protein-protein binding interactions of ERCC1, the endonuclease, XPF, and XPA. Thus, inhibition of their function is a potential therapeutic strategy for the selective sensitization of tumors to DNA-damaging platinum-based cancer therapy. Here, we report on new small-molecule antagonists of the ERCC1/XPA protein-protein interaction (PPI) discovered using a high-throughput competitive fluorescence polarization binding assay. We discovered a unique structural class of thiopyridine-3-carbonitrile PPI antagonists that block a truncated XPA polypeptide from binding to ERCC1. Preliminary hit-to-lead studies from compound 1 reveal structure-activity relationships (SAR) and identify lead compound 27 o with an EC50 of 4.7 µM. Furthermore, chemical shift perturbation mapping by NMR confirms that 1 binds within the same site as the truncated XPA67-80 peptide. These novel ERCC1 antagonists are useful chemical biology tools for investigating DNA damage repair pathways and provide a good starting point for medicinal chemistry optimization as therapeutics for sensitizing tumors to DNA damaging agents and overcoming resistance to platinum-based chemotherapy.

Inhibition of autocrine HGF maturation overcomes cetuximab resistance in colorectal cancer.

Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC.

Macrocyclic Inhibitors of HGF-Activating Serine Proteases Overcome Resistance to Receptor Tyrosine Kinase Inhibitors and Block Lung Cancer Progression.

Hepatocyte growth factor (HGF), the ligand for the MET receptor tyrosine kinase, is a tumor-promoting factor that is abundant in the tumor microenvironment. Proteolytic activation of inactive pro-HGF by one or more of the serine endopeptidases matriptase, hepsin, and HGF activator is the rate-limiting step in HGF/MET signaling. Herein, we have rationally designed a novel class of side chain cyclized macrocyclic peptide inhibitors. The new series of cyclic tripeptides has superior metabolic stability and significantly improved pharmacokinetics in mice relative to the corresponding linear peptides. We identified the lead compound VD2173 that potently inhibits matriptase and hepsin, which was tested in parallel alongside the acyclic inhibitor ZFH7116 using both in vitro and in vivo models of lung cancer. We demonstrated that both compounds block pro-HGF activation, abrogate HGF-mediated wound healing, and overcome resistance to EGFR- and MET-targeted therapy in lung cancer models. Furthermore, VD2173 inhibited HGF-dependent growth of lung cancer tumors in mice.

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254).

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.

Piperidine carbamate peptidomimetic inhibitors of the serine proteases HGFA, matriptase and hepsin.

Matriptase and hepsin are type II transmembrane serine proteases (TTSPs). Along with related S1 trypsin like serine protease HGFA (hepatocyte growth factor activator), their unregulated proteolytic activity has been associated with cancer including tumor progression and metastasis. These three proteases have two substrates in common, hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), the ligands for MET and recepteur d'origine nantais (RON) receptor tyrosine kinases. Mechanism-based tetrapeptide and benzamidine inhibitors of these proteases have been shown to block HGF/MET and MSP/RON cancer cell signaling. Herein, we have rationally designed a new class of peptidomimetic hybrid small molecule piperidine carbamate dipeptide inhibitors comparable in potency to much larger tetrapeptides. We have identified multiple compounds which have potent activity against matriptase and hepsin and with excellent selectivity over the off-target serine proteases factor Xa and thrombin.

Identification of small molecule enzyme inhibitors as broad-spectrum anthelmintics.

Targeting chokepoint enzymes in metabolic pathways has led to new drugs for cancers, autoimmune disorders and infectious diseases. This is also a cornerstone approach for discovery and development of anthelmintics against nematode and flatworm parasites. Here, we performed omics-driven knowledge-based identification of chokepoint enzymes as anthelmintic targets. We prioritized 10 of 186 phylogenetically conserved chokepoint enzymes and undertook a target class repurposing approach to test and identify new small molecules with broad spectrum anthelmintic activity. First, we identified and tested 94 commercially available compounds using an in vitro phenotypic assay, and discovered 11 hits that inhibited nematode motility. Based on these findings, we performed chemogenomic screening and tested 32 additional compounds, identifying 6 more active hits. Overall, 6 intestinal (single-species), 5 potential pan-intestinal (whipworm and hookworm) and 6 pan-Phylum Nematoda (intestinal and filarial species) small molecule inhibitors were identified, including multiple azoles, Tadalafil and Torin-1. The active hit compounds targeted three different target classes in humans, which are involved in various pathways, including carbohydrate, amino acid and nucleotide metabolism. Last, using representative inhibitors from each target class, we demonstrated in vivo efficacy characterized by negative effects on parasite fecundity in hamsters infected with hookworms.

Recent progress on inhibitors of the type II transmembrane serine proteases, hepsin, matriptase and matriptase-2.

Members of the type II transmembrane serine proteases (TTSP) family play a vital role in cell growth and development but many are also implicated in disease. Two of the well-studied TTSPs, matriptase and hepsin proteolytically process multiple protein substrates such as the inactive single-chain zymogens pro-HGF and pro-macrophage stimulating protein into the active heterodimeric forms, HGF and macrophage stimulating protein. These two proteases also have many other substrates which are associated with cancer and tumor progression. Another related TTSP, matriptase-2 is expressed in the liver and functions by regulating iron homoeostasis through the cleavage of hemojuvelin and thus is implicated in iron overload diseases. In the present review, we will discuss inhibitor design strategy and Structure activity relationships of TTSP inhibitors, which have been reported in the literature.

Hepatocyte growth factor activator inhibitor-2 stabilizes Epcam and maintains epithelial organization in the mouse intestine.

Mutations in SPINT2 encoding the epithelial serine protease inhibitor hepatocyte growth factor activator inhibitor-2 (HAI-2) are associated with congenital tufting enteropathy. However, the functions of HAI-2 in vivo are poorly understood. Here we used tamoxifen-induced Cre-LoxP recombination in mice to ablate Spint2. Mice lacking Spint2 died within 6 days after initiating tamoxifen treatment and showed severe epithelial damage in the whole intestinal tracts, and, to a lesser extent, the extrahepatic bile duct. The intestinal epithelium showed enhanced exfoliation, villous atrophy, enterocyte tufts and elongated crypts. Organoid crypt culture indicated that Spint2 ablation induced Epcam cleavage with decreased claudin-7 levels and resulted in organoid rupture. These organoid changes could be rescued by addition of serine protease inhibitors aprotinin, camostat mesilate and matriptase-selective α-ketobenzothiazole as well as by co-deletion of Prss8, encoding the serine protease prostasin. These results indicate that HAI-2 is an essential cellular inhibitor for maintaining intestinal epithelium architecture.

Discovery of Selective Matriptase and Hepsin Serine Protease Inhibitors: Useful Chemical Tools for Cancer Cell Biology.

Matriptase and hepsin belong to the family of type II transmembrane serine proteases (TTSPs). Increased activity of these and the plasma protease, hepatocyte growth factor activator (HGFA), is associated with unregulated cell signaling and tumor progression through increased MET and RON kinase signaling pathways. These proteases are highly expressed in multiple solid tumors and hematological malignancies. Herein, we detail the synthesis and structure-activity relationships (SAR) of a dipeptide library bearing Arg α-ketobenozothiazole (kbt) warheads as novel inhibitors of HGFA, matriptase, and hepsin. We elucidated the substrate specificity for HGFA using positional scanning of substrate combinatorial libraries (PS-SCL), which was used to discover selective inhibitors of matriptase and hepsin. Using these selective inhibitors, we have clarified the specific role of hepsin in maintaining epithelial cell membrane integrity, known to be lost in breast cancer progression. These selective compounds are useful as chemical biology tools and for future drug discovery efforts.

Adventures in Scaffold Morphing: Discovery of Fused Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors.

Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.

Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation.

Targeted therapeutic agents, such as inhibitors of epithelial growth factor receptor (EGFR), have transformed the management of non-small cell lung cancer (NSCLC) patients. MET-amplified NSCLC cells display resistance to EGFR-targeting agents, but are addicted to MET signaling for survival and proliferation and are sensitive to MET inhibition. However, responsive cancer cells invariably develop resistance to MET-targeted treatment. The tumor microenvironment plays a major role in resistance to anticancer therapy. We demonstrated that fibroblasts block the response of MET-amplified NSCLC cells to the MET kinase inhibitor, JNJ38877605 in an HGF-dependent manner. Thus, MET-amplified NSCLC cells become addicted to HGF upon pharmacological inhibition of MET. HGF restored phosphorylation of MET, EGFR and RON, and maintained pro-survival AKT and ERK signaling in MET-inhibited cells. We developed a small molecule inhibitor of pro-HGF activation, SRI31215, which acts as a triplex inhibitor of the pro-HGF activating proteases matriptase, hepsin and HGF activator (HGFA). SRI31215 blocked crosstalk between tumor cells and fibroblasts and overcame fibroblast-mediated resistance to MET inhibition by preventing fibroblast-mediated reactivation of AKT and ERK signaling. Structurally unrelated triplex inhibitors of matriptase, hepsin and HGFA that we developed in parallel showed similar biological activity. Our data suggest that simultaneous inhibition of HGF and MET is required to overcome resistance to MET inhibitors in MET-amplified NSCLC cells. This provides a rationale for the development of novel combination therapeutic strategies for the treatment of NSCLC patients with MET amplification.

Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.

Rational design strategies for FimH antagonists: new drugs on the horizon for urinary tract infection and Crohn's disease.

INTRODUCTION: The bacterial adhesin FimH is a virulence factor and an attractive therapeutic target for urinary tract infection (UTI) and Crohn's Disease (CD). Located on type 1 pili of uropathogenic E. coli (UPEC), the FimH adhesin plays an integral role in the pathogenesis of UPEC. Recent efforts have culminated in the development of small-molecule mannoside FimH antagonists that target the mannose-binding lectin domain of FimH, inhibiting its function and preventing UPEC from binding mannosylated host cells in the bladder, thereby circumventing infection. Areas covered: The authors describe the structure-guided design of mannoside ligands, and review the structural biology of the FimH lectin domain. Additionally, they discuss the lead optimization of mannosides for therapeutic application in UTI and CD, and describe various assays used to measure mannoside potency in vitro and mouse models used to determine efficacy in vivo. Expert opinion: To date, mannoside optimization has led to a diverse set of small-molecule FimH antagonists with oral bioavailability. With clinical trials already initiated in CD and on the horizon for UTI, it is the authors, opinion that mannosides will be a 'first-in-class' treatment strategy for UTI and CD, and will pave the way for treatment of other Gram-negative bacterial infections.

Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins.

Identifying sites of protein-ligand interaction is important for structure-based drug discovery and understanding protein structure-function relationships. Mass spectrometry (MS) has emerged as a useful tool for identifying residues covalently modified by ligands. Current methods use database searches that are dependent on acquiring interpretable fragmentation spectra (MS2) of peptide-ligand adducts. This is problematic for identifying sites of hydrophobic ligand incorporation in integral membrane proteins (IMPs), where poor aqueous solubility and ionization of peptide-ligand adducts and collision-induced adduct loss hinder the acquisition of quality MS2 spectra. To address these issues, we developed a fast ligand identification (FLI) tag that can be attached to any alkyne-containing ligand via Cu(I)-catalyzed cycloaddition. The FLI tag adds charge to increase solubility and ionization, and utilizes stable isotope labeling for MS1 level identification of hydrophobic peptide-ligand adducts. The FLI tag was coupled to an alkyne-containing neurosteroid photolabeling reagent and used to identify peptide-steroid adducts in MS1 spectra via the stable heavy isotope pair. Peptide-steroid adducts were not identified in MS2-based database searches because collision-induced adduct loss was the dominant feature of collision-induced dissociation (CID) fragmentation, but targeted analysis of MS1 pairs using electron transfer dissociation (ETD) markedly reduced adduct loss. Using the FLI tag and ETD, we identified Glu73 as the site of photoincorporation of our neurosteroid ligand in the IMP, mouse voltage-dependent anion channel-1 (mVDAC1), and top-down MS confirmed a single site of photolabeling.

Characterization of parasite-specific indels and their proposed relevance for selective anthelminthic drug targeting.

Insertions and deletions (indels) are important sequence variants that are considered as phylogenetic markers that reflect evolutionary adaptations in different species. In an effort to systematically study indels specific to the phylum Nematoda and their structural impact on the proteins bearing them, we examined over 340,000 polypeptides from 21 nematode species spanning the phylum, compared them to non-nematodes and identified indels unique to nematode proteins in more than 3000 protein families. Examination of the amino acid composition revealed uneven usage of amino acids for insertions and deletions. The amino acid composition and cost, along with the secondary structure constitution of the indels, were analyzed in the context of their biological pathway associations. Species-specific indels could enable indel-based targeting for drug design in pathogens/parasites. Therefore, we screened the spatial locations of the indels in the parasite's protein 3D structures, determined the location of the indel and identified potential unique drug targeting sites. These indels could be confirmed by RNA-Seq data. Examples are presented illustrating the close proximity of some indels to established small-molecule binding pockets that can potentially facilitate selective targeting to the parasites and bypassing their host, thus reducing or eliminating the toxicity of the potential drugs. This study presents an approach for understanding the adaptation of pathogens/parasites at a molecular level, and outlines a strategy to identify such nematode-selective targets that remain essential to the organism. With further experimental characterization and validation, it opens a possible channel for the development of novel treatments with high target specificity, addressing both host toxicity and resistance concerns.

α-Ketobenzothiazole Serine Protease Inhibitors of Aberrant HGF/c-MET and MSP/RON Kinase Pathway Signaling in Cancer.

Upregulation of the HGF and MSP growth-factor processing serine endopeptidases HGFA, matriptase and hepsin is correlated with increased metastasis in multiple tumor types driven by c-MET or RON kinase signaling. We rationally designed P1' α-ketobenzothiazole mechanism-based inhibitors of these proteases. Structure-activity studies are presented, which resulted in the identification of potent inhibitors with differential selectivity. The tetrapeptide inhibitors span the P1-P1' substrate cleavage site via a P1' amide linker off the benzothiazole, occupying the S3' pocket. Optimized inhibitors display sub-nanomolar enzyme inhibition against one, two, or all three of HGFA, matriptase, and hepsin. Several compounds also have good selectivity against the related trypsin-like proteases, thrombin and Factor Xa. Finally, we show that inhibitors block the fibroblast (HGF)-mediated migration of invasive DU145 prostate cancer cells. In addition to prostate cancer, breast, colon, lung, pancreas, gliomas, and multiple myeloma tumors all depend on HGF and MSP for tumor survival and progression. Therefore, these unique inhibitors have potential as new therapeutics for a diverse set of tumor types.

Mannose-derived FimH antagonists: a promising anti-virulence therapeutic strategy for urinary tract infections and Crohn's disease.

INTRODUCTION: Type 1 pili are utilized by Gram-negative bacteria to adhere to host tissue and thus are a key virulence factor in urinary tract infections (UTIs) and Crohn's disease (CD). This adhesion is mediated through specific binding of the terminal adhesin, FimH, to mannosylated host glycoproteins. FimH is essential for UTI pathogenesis and thus is a promising therapeutic target. AREAS COVERED: Herein, we review the structural frameworks of FimH antagonists disclosed in the patent literature. X-ray crystallographic binding studies of D-mannose and early FimH antagonists have uncovered key molecular interactions. Exploiting this knowledge, mannosides with extraordinarily high binding affinities have been designed. Structure-activity relationships (SAR) and structure-property relationship (SPR) studies have resulted in the rapid development of orally bioavailable FimH antagonists with promising therapeutic potential for UTI and CD. EXPERT OPINION: It is our opinion that biaryl or 'two-ring' mannosides, which represent the largest and most thoroughly tested class of FimH antagonists, also hold the most promise as a novel treatment for UTIs. These antagonists have also been shown to have efficacy in treating CD. Judging from the strong preclinical data, we predict that one or more FimH antagonists will be entering the clinic within the next 1-2 years.

Structure-based discovery of small molecule hepsin and HGFA protease inhibitors: Evaluation of potency and selectivity derived from distinct binding pockets.

Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA.