Efficacy of host cell serine protease inhibitor MM3122 against SARS-CoV-2 for treatment and prevention of COVID-19.

We developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases, including transmembrane protease serine 2 (TMPRSS2), matriptase, and hepsin. TMPRSS2 is a membrane-associated protease that is highly expressed in the upper and lower respiratory tracts and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell entry, replication, and dissemination of new virus particles. We have previously shown that compound MM3122 exhibited subnanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Furthermore, we evaluated MM3122 in a mouse model of COVID-19 and demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in proinflammatory cytokine and chemokine production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2-infected mice. Therefore, MM3122 is a promising lead candidate small-molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses. IMPORTANCE: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and a therapeutic drug for the treatment of COVID-19 given intraperitoneally in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research, but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.

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.

Discovery of Orally Bioavailable FmlH Lectin Antagonists as Treatment for Urinary Tract Infections.

FmlH, a bacterial adhesin of uropathogenic Escherichia coli (UPEC), has been shown to provide a fitness advantage in colonizing the bladder during chronic urinary tract infections (UTIs). Previously reported ortho-biphenyl glycosides based on ßGal and ßGalNAc have excellent binding affinity to FmlH and potently block binding to its natural carbohydrate receptor, but they lack oral bioavailability. In this paper, we outline studies where we have optimized compounds for improved pharmacokinetics, leading to the discovery of novel analogues with good oral bioavailability. We synthesized galactosides with the anomeric O-linker replaced with more stable S- and C-linked linkers. We also investigated modifications to the GalNAc sugar and modifications to the biphenyl aglycone. We identified GalNAc 69 with an IC50 of 0.19 µM against FmlH and 53% oral bioavailability in mice. We also obtained a FimlH-bound X-ray structure of lead compound 69 (AM4085) which has potential as a new antivirulence therapeutic for UTIs.

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.

Efficacy of Host Cell Serine Protease Inhibitor MM3122 against SARS-CoV-2 for Treatment and Prevention of COVID-19.

We have developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases including transmembrane protease serine 2 (TMPRSS2), matriptase and hepsin. TMPRSS2 is a membrane associated protease which is highly expressed in the upper and lower respiratory tract and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell receptor binding, entry, replication, and dissemination of new virion particles. We have previously shown that compound MM3122 exhibited sub nanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell-viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Further, we have evaluated MM3122 in a mouse model of COVID-19 and have demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion, and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in pro-inflammatory cytokines and chemokines production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2 infected mice. Therefore, MM3122 is a promising lead candidate small molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses. IMPORTANCE: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host-cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and therapeutic drug for the treatment of COVID-19 in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.

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.

Aspartyl Protease Inhibitors as Anti-Filarial Drugs.

The current treatments for lymphatic filariasis and onchocerciasis do not effectively kill the adult parasitic nematodes, allowing these chronic and debilitating diseases to persist in millions of people. Thus, the discovery of new drugs with macrofilaricidal potential to treat these filarial diseases is critical. To facilitate this need, we first investigated the effects of three aspartyl protease inhibitors (APIs) that are FDA-approved as HIV antiretroviral drugs on the adult filarial nematode, Brugia malayi and the endosymbiotic bacteria, Wolbachia. From the three hits, nelfinavir had the best potency with an IC50 value of 7.78 µM, followed by ritonavir and lopinavir with IC50 values of 14.3 µM and 16.9 µM, respectively. The three APIs have a direct effect on killing adult B. malayi after 6 days of exposure in vitro and did not affect the Wolbachia titers. Sequence conservation and stage-specific gene expression analysis identified Bm8660 as the most likely primary aspartic protease target for these drug(s). Immunolocalization using antibodies raised against the Bm8660 ortholog of Onchocerca volvulus showed it is strongly expressed in female B. malayi, especially in metabolically active tissues such as lateral and dorsal/ventral chords, hypodermis, and uterus tissue. Global transcriptional response analysis using adult female B. pahangi treated with APIs identified four additional aspartic proteases differentially regulated by the three effective drugs, as well as significant enrichment of various pathways including ubiquitin mediated proteolysis, protein kinases, and MAPK/AMPK/FoxO signaling. In vitro testing against the adult gastro-intestinal nematode Trichuris muris suggested broad-spectrum potential for these APIs. This study suggests that APIs may serve as new leads to be further explored for drug discovery to treat parasitic nematode infections.

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.

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 a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 ( 4 ) has an IC 50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC 50 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 EC 50 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 EC 50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours 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.

A host receptor enables type 1 pilus-mediated pathogenesis of Escherichia coli pyelonephritis.

Type 1 pili have long been considered the major virulence factor enabling colonization of the urinary bladder by uropathogenic Escherichia coli (UPEC). The molecular pathogenesis of pyelonephritis is less well characterized, due to previous limitations in preclinical modeling of kidney infection. Here, we demonstrate in a recently developed mouse model that beyond bladder infection, type 1 pili also are critical for establishment of ascending pyelonephritis. Bacterial mutants lacking the type 1 pilus adhesin (FimH) were unable to establish kidney infection in male C3H/HeN mice. We developed an in vitro model of FimH-dependent UPEC binding to renal collecting duct cells, and performed a CRISPR screen in these cells, identifying desmoglein-2 as a primary renal epithelial receptor for FimH. The mannosylated extracellular domain of human DSG2 bound directly to the lectin domain of FimH in vitro, and introduction of a mutation in the FimH mannose-binding pocket abolished binding to DSG2. In infected C3H/HeN mice, type 1-piliated UPEC and Dsg2 were co-localized within collecting ducts, and administration of mannoside FIM1033, a potent small-molecule inhibitor of FimH, significantly attenuated bacterial loads in pyelonephritis. Our results broaden the biological importance of FimH, specify the first renal FimH receptor, and indicate that FimH-targeted therapeutics will also have application in pyelonephritis.

An Integrated Approach to Identify New Anti-Filarial Leads to Treat River Blindness, a Neglected Tropical Disease.

Filarial worms cause multiple debilitating diseases in millions of people worldwide, including river blindness. Currently available drugs reduce transmission by killing larvae (microfilariae), but there are no effective cures targeting the adult parasites (macrofilaricides) which survive and reproduce in the host for very long periods. To identify effective macrofilaricides, we carried out phenotypic screening of a library of 2121 approved drugs for clinical use against adult Brugia pahangi and prioritized the hits for further studies by integrating those results with a computational prioritization of drugs and associated targets. This resulted in the identification of 18 hits with anti-macrofilaricidal activity, of which two classes, azoles and aspartic protease inhibitors, were further expanded upon. Follow up screening against Onchocerca spp. (adult Onchocerca ochengi and pre-adult O. volvulus) confirmed activity for 13 drugs (the majority having IC50 < 10 µM), and a counter screen of a subset against L. loa microfilariae showed the potential to identify selective drugs that prevent adverse events when co-infected individuals are treated. Stage specific activity was also observed. Many of these drugs are amenable to structural optimization, and also have known canonical targets, making them promising candidates for further optimization that can lead to identifying and characterizing novel anti-macrofilarial drugs.

Novel approaches to glycomimetic design: development of small molecular weight lectin antagonists.

Introduction: The direct binding of carbohydrates or those presented on glycoproteins or glycolipids to proteins is the primary effector of many biological responses. One class of carbohydrate-binding proteins, lectins are important in all forms of life. Their functions in animals include regulating cell adhesion, glycoprotein synthesis, metabolism, and mediating immune system response while in bacteria and viruses a lectin-mediated carbohydrate-protein interaction between host cells and the pathogen initiates pathogenesis of the infection.Areas covered: In this review, the authors outline the structural and functional pathogenesis of lectins from bacteria, amoeba, and humans. Mimics of a carbohydrate are referred to as glycomimetics, which are much smaller in molecular weight and are devised to mimic the key binding interactions of the carbohydrate while also allowing additional contacts with the lectin. This article emphasizes the various approaches used over the past 10-15 years in the rational design of glycomimetic ligands.Expert opinion: Medicinal chemistry efforts enabled by X-ray structural biology have identified small-molecule glycomimetic lectin antagonists that have entered or are nearing clinical trials. A common theme in these strategies is the use of biaryl ring systems to emulate the carbohydrate interactions with the lectin.

Optimizing Pyrazolopyrimidine Inhibitors of Calcium Dependent Protein Kinase 1 for Treatment of Acute and Chronic Toxoplasmosis.

Calcium dependent protein kinase 1 (CDPK1) is an essential Ser/Thr kinase that controls invasion and egress by the protozoan parasite Toxoplasma gondii. The Gly gatekeeper of CDPK1 makes it exquisitely sensitive to inhibition by small molecule 1H-pyrazolo[3,4-d]pyrimidine-4-amine (PP) compounds that are bulky ATP mimetics. Here we rationally designed, synthesized, and tested a series of novel PP analogs that were evaluated for inhibition of CDPK1 enzyme activity in vitro and parasite growth in cell culture. Optimal substitution on the PP scaffold included 2-pyridyl ethers directed into the hydrophobic pocket and small carbocyclic rings accessing the ribose-binding pocket. Further optimization of the series led to identification of the lead compound 3a that displayed excellent potency, selectivity, safety profile, and efficacy in vivo. The results of these studies provide a foundation for further work to optimize CDPK1 inhibitors for the treatment of acute and chronic toxoplasmosis.

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.

Biphenyl Gal and GalNAc FmlH Lectin Antagonists of Uropathogenic E. coli (UPEC): Optimization through Iterative Rational Drug Design.

The F9/Yde/Fml pilus, tipped with the FmlH adhesin, has been shown to provide uropathogenic Escherichia coli (UPEC) a fitness advantage in urinary tract infections (UTIs). Here, we used X-ray structure guided design to optimize our previously described ortho-biphenyl Gal and GalNAc FmlH antagonists such as compound 1 by replacing the carboxylate with a sulfonamide as in 50. Other groups which can accept H-bonds were also tolerated. We pursued further modifications to the biphenyl aglycone resulting in significantly improved activity. Two of the most potent compounds, 86 (IC50 = 0.051 µM) and 90 (IC50 = 0.034 µM), exhibited excellent metabolic stability in mouse plasma and liver microsomes but showed only limited oral bioavailability (<1%) in rats. Compound 84 also showed a good pharmacokinetic (PK) profile in mice after IP dosing with compound exposure above the IC50 for 6 h. These new FmlH antagonists represent new antivirulence drugs for UTIs.