The Effect of a Novel Serine Protease Inhibitor on Inflammation and Intestinal Permeability in a Murine Colitis Transfer Model.

Background: A protease/antiprotease disbalance is observed in inflammatory bowel diseases (IBD). We therefore studied the effect of the novel serine protease inhibitor UAMC-00050 on intestinal inflammation and permeability in a chronic colitis T cell transfer mouse model to get further insight into the regulation of T cell-mediated immunopathology. Methods: Colitis was induced in severe combined immunodeficient (SCID) mice, by the adoptive transfer of CD4+CD25-CD62L+ T cells. Animals were treated intraperitoneally (i.p.) 2x/day with vehicle or UAMC-00050 (5 mg/kg) from week 2 onwards. Colonic inflammation was assessed by clinical parameters, colonoscopy, macroscopy, microscopy, myeloperoxidase activity and cytokine expression levels. At week 4, 4 kDa FITC-dextran intestinal permeability was evaluated and T helper transcription factors, protease-activated receptors and junctional proteins were quantified by RT-qPCR. Results: Adoptive transfer of CD4+CD25-CD62L+ T cells resulted in colonic inflammation and an altered intestinal permeability. The serine protease inhibitor UAMC-00050 ameliorated both the inflammatory parameters and the intestinal barrier function. Furthermore, a decrease in colonic mRNA expression of Tbet and PAR4 was observed in colitis mice after UAMC-00050 treatment. Conclusion: The beneficial effect of UAMC-00050 on inflammation was apparent via a reduction of Tbet, IFN-γ, TNF-α, IL-1ß and IL-6. Based on these results, we hypothesize a pivotal effect of serine protease inhibition on the Th1 inflammatory profile potentially mediated via PAR4.

Local Colonic Administration of a Serine Protease Inhibitor Improves Post-Inflammatory Visceral Hypersensitivity in Rats.

Dysregulation of the protease-antiprotease balance in the gastrointestinal tract has been suggested as a mechanism underlying visceral hypersensitivity in conditions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). We aimed to study the potential therapeutic role of an intracolonically administered serine protease inhibitor for the treatment of abdominal pain in a post-inflammatory rat model for IBS. An enema containing 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to induce colitis in male Sprague-Dawley rats, whereas controls received a saline solution. Colonoscopies were performed to confirm colitis and follow-up mucosal healing. In the post-inflammatory phase, the serine protease inhibitor UAMC-00050 (0.1-5 mg/kg) or its vehicle alone (5% DMSO in H2O) was administered in the colon. Thirty minutes later, visceral mechanosensitivity to colorectal distensions was quantified by visceromotor responses (VMRs) and local effects on colonic compliance and inflammatory parameters were assessed. Specific proteolytic activities in fecal and colonic samples were measured using fluorogenic substrates. Pharmacokinetic parameters were evaluated using bioanalytical measurements with liquid chromatography-tandem mass spectrometry. Post-inflammatory rats had increased trypsin-like activity in colonic tissue and elevated elastase-like activity in fecal samples compared to controls. Treatment with UAMC-00050 decreased trypsin-like activity in colonic tissue of post-colitis animals. Pharmacokinetic experiments revealed that UAMC-00050 acted locally, being taken up in the bloodstream only minimally after administration. Local administration of UAMC-00050 normalized visceral hypersensitivity. These results support the role of serine proteases in the pathophysiology of visceral pain and the potential of locally administered serine protease inhibitors as clinically relevant therapeutics for the treatment of IBS patients with abdominal pain.

A novel serine protease inhibitor as potential treatment for dry eye syndrome and ocular inflammation.

Dry eye syndrome (DES), a multifactorial disorder which leads to ocular discomfort, visual disturbance and tear film instability, has a rising prevalence and limited treatment options. In this study, a newly developed trypsin-like serine protease inhibitor (UAMC-00050) in a tear drop formulation was evaluated to treat ocular inflammation. A surgical animal model of dry eye was employed to investigate the potential of UAMC-00050 on dry eye pathology. Animals treated with UAMC-00050 displayed a significant reduction in ocular surface damage after evaluation with sodium fluorescein, compared to untreated, vehicle treated and cyclosporine-treated animals. The concentrations of IL-1α and TNF-α were also significantly reduced in tear fluid from UAMC-00050-treated rats. Additionally, inflammatory cell infiltration in the palpebral conjunctiva (CD3 and CD45), was substantially reduced. An accumulation of pro-MMP-9 and a decrease in active MMP-9 were found in tear fluid from animals treated with UAMC-00050, suggesting that trypsin-like serine proteases play a role in activating MMP-9 in ocular inflammation in this animal model. Comparative qRT-PCR analyses on ocular tissue indicated the upregulation of tryptase, urokinase plasminogen activator receptor (uPAR) and protease-activated receptor 2 (PAR2). The developed UAMC-00050 formulation was stable up to 6 months at room temperature in the absence of light, non-irritating and sterile with compatible pH and osmolarity. These results provide a proof-of-concept for the in vivo modifying potential of UAMC-00050 on dry eye pathology and suggest a central role of trypsin-like serine proteases and PAR2 in dry eye derived ocular inflammation.

Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post-inflammatory rat model for irritable bowel syndrome.

BACKGROUND AND PURPOSE: Serine proteases have been re suggested as important mediators of visceral pain. We investigated their effect by using newly developed serine protease inhibitors with a well-characterized inhibitory profile in a rat model of post-inflammatory irritable bowel syndrome (IBS). EXPERIMENTAL APPROACH: Colitis was induced in rats receiving intrarectal trinitrobenzenesulphonic acid; controls received 0.9% NaCl. Colonoscopies were performed on day 3, to confirm colitis, and later until mucosal healing. Visceral hypersensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30 min after i.p. injection of the serine protease inhibitors nafamostat, UAMC-00050 or UAMC-01162. Serine proteases, protease-activated receptors (PARs) and TRP channels were quantified by qPCR and immunohistochemistry. Proteolytic activity was characterized using fluorogenic substrates. KEY RESULTS: VMR was significantly elevated in post-colitis rats. Nafamostat normalized VMRs at the lowest dose tested. UAMC-00050 and UAMC-01162 significantly decreased VMR dose-dependently. Expression of mRNA for tryptase-αß-1and PAR4, and tryptase immunoreactivity was significantly increased in the colon of post-colitis animals. Trypsin-like activity was also significantly increased in the colon but not in the faeces. PAR2 and TRPA1 immunoreactivity co-localized with CGRP-positive nerve fibres in control and post-colitis animals. CONCLUSIONS AND IMPLICATIONS: Increased expression of serine proteases and activity together with increased expression of downstream molecules at the colonic and DRG level and in CGRP-positive sensory nerve fibres imply a role for serine proteases in post-inflammatory visceral hypersensitivity. Our results support further investigation of serine protease inhibitors as an interesting treatment strategy for IBS-related visceral pain.

Selective Glucocorticoid Receptor Properties of GSK866 Analogs with Cysteine Reactive Warheads.

Synthetic glucocorticoids (GC) are the mainstay therapy for treatment of acute and chronic inflammatory disorders. Due to the high adverse effects associated with long-term use, GC pharmacology has focused since the nineties on more selective GC ligand-binding strategies, classified as selective glucocorticoid receptor (GR) agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs). In the current study, GSK866 analogs with electrophilic covalent-binding warheads were developed with potential SEGRA properties to improve their clinical safety profile for long-lasting topical skin disease applications. Since the off-rate of a covalently binding drug is negligible compared to that of a non-covalent drug, its therapeutic effects can be prolonged and typically, smaller doses of the drug are necessary to reach the same level of therapeutic efficacy, thereby potentially reducing systemic side effects. Different analogs of SEGRA GSK866 coupled to cysteine reactive warheads were characterized for GR potency and selectivity in various biochemical and cellular assays. GR- and NFκB-dependent reporter gene studies show favorable anti-inflammatory properties with reduced GR transactivation of two non-steroidal GSK866 analogs UAMC-1217 and UAMC-1218, whereas UAMC-1158 and UAMC-1159 compounds failed to modulate cellular GR activity. These results were further supported by GR immuno-localization and S211 phospho-GR western analysis, illustrating significant GR phosphoactivation and nuclear translocation upon treatment of GSK866, UAMC-1217, or UAMC-1218, but not in case of UAMC-1158 or UAMC-1159. Furthermore, mass spectrometry analysis of tryptic peptides of recombinant GR ligand-binding domain (LBD) bound to UAMC-1217 or UAMC-1218 confirmed covalent cysteine-dependent GR binding. Finally, molecular dynamics simulations, as well as glucocorticoid receptor ligand-binding domain (GR-LBD) coregulator interaction profiling of the GR-LBD bound to GSK866 or its covalently binding analogs UAMC-1217 or UAMC-1218 revealed subtle conformational differences that might underlie their SEGRA properties. Altogether, GSK866 analogs UAMC-1217 and UAMC-1218 hold promise as a novel class of covalent-binding SEGRA ligands for the treatment of topical inflammatory skin disorders.

Sorafenib tosylate inhibits directly necrosome complex formation and protects in mouse models of inflammation and tissue injury.

Necroptosis contributes to the pathophysiology of several inflammatory, infectious and degenerative disorders. TNF-induced necroptosis involves activation of the receptor-interacting protein kinases 1 and 3 (RIPK1/3) in a necrosome complex, eventually leading to the phosphorylation and relocation of mixed lineage kinase domain like protein (MLKL). Using a high-content screening of small compounds and FDA-approved drug libraries, we identified the anti-cancer drug Sorafenib tosylate as a potent inhibitor of TNF-dependent necroptosis. Interestingly, Sorafenib has a dual activity spectrum depending on its concentration. In murine and human cell lines it induces cell death, while at lower concentrations it inhibits necroptosis, without affecting NF-κB activation. Pull down experiments with biotinylated Sorafenib show that it binds independently RIPK1, RIPK3 and MLKL. Moreover, it inhibits RIPK1 and RIPK3 kinase activity. In vivo Sorafenib protects against TNF-induced systemic inflammatory response syndrome (SIRS) and renal ischemia-reperfusion injury (IRI). Altogether, we show that Sorafenib can, next to the reported Braf/Mek/Erk and VEGFR pathways, also target the necroptotic pathway and that it can protect in an acute inflammatory RIPK1/3-mediated pathology.

Regulation of intestinal permeability: The role of proteases.

The gastrointestinal barrier is - with approximately 400 m2 - the human body's largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extra-intestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases.

Inhibitor screening and enzymatic activity determination for autophagy target Atg4B using a gel electrophoresis-based assay.

Atg4B is a cysteine hydrolase that plays a key role in autophagy. Although it has been proposed as an attractive drug target, inhibitor discovery has proven highly challenging. The absence of a standardized, easily implementable enzyme activity/inhibition assay for Atg4B most likely contributes to this situation. Therefore, three different assay types for Atg4B activity/inhibition quantification were first compared: (1) an approach using fluorogenic Atg4B-substrates, (2) an in-gel densitometric quantification assay and (3) a thermal shift protocol. The gel-based approach showed the most promising results and was validated for screening of potential Atg4B inhibitors. A set of 8 literature inhibitors was included. Remarkably, in our hands only 2 literature references were found to have measurable Atg4B affinity. Furthermore, a fragment library (n = 182) was tested for Atg4B inhibition. One library member showed inhibition at high micromolar concentration and was found fit for further, fragment-based inhibitor design.

Preclinical evaluation of [111 In]MICA-401, an activity-based probe for SPECT imaging of in vivo uPA activity.

Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 are key players in cancer invasion and metastasis. Both uPA and PAI-1 have been described as prognostic biomarkers; however, non-invasive methods measuring uPA activity are lacking. We developed an indium-111 (111 In)-labelled activity-based probe to image uPA activity in vivo by single photon emission computed tomography (SPECT). A DOTA-conjugated uPA inhibitor was synthesized and radiolabelled with 111 In ([111 In]MICA-401), together with its inactive, hydrolysed form ([111 In]MICA-402). A biodistribution study was performed in mice (healthy and tumour-bearing), and tumour-targeting properties were evaluated in two different cancer xenografts (MDA-MB-231 and HT29) with respectively high and low levels of uPA expression in vitro, with either the active or hydrolysed radiotracer. MicroSPECT was performed 95 h post injection followed by ex vivo biodistribution. Tumour uptake was correlated with human and murine uPA expression determined by ELISA and immunohistochemistry (IHC). Biodistribution data with the hydrolysed probe [111 In]MICA-402 showed almost complete clearance 95 h post injection. The ex vivo biodistribution and SPECT data with [111 In]MICA-401 demonstrated similar tumour uptakes in the two models: ex vivo 5.68 ± 1.41%ID/g versus 5.43 ± 1.29%ID/g and in vivo 4.33 ± 0.80 versus 4.86 ± 1.18 for MDA-MB-231 and HT-29 respectively. Human uPA ELISA and IHC showed significantly higher uPA expression in the MDA-MB-231 tumours, while mouse uPA staining revealed similar staining intensities of the two tumours. Our data demonstrate non-invasive imaging of uPA activity in vivo, although the moderate tumour uptake and hence potential clinical translation of the radiotracer warrants further investigation. Copyright © 2016 John Wiley & Sons, Ltd.

Optimization and validation of an existing, surgical and robust dry eye rat model for the evaluation of therapeutic compounds.

The aim of this research was to optimize and validate an animal model for dry eye, adopting clinically relevant evaluation parameters. Dry eye was induced in female Wistar rats by surgical removal of the exorbital lacrimal gland. The clinical manifestations of dry eye were evaluated by tear volume measurements, corneal fluorescein staining, cytokine measurements in tear fluid, MMP-9 mRNA expression and CD3(+) cell infiltration in the conjunctiva. The animal model was validated by treatment with Restasis(®) (4 weeks) and commercial dexamethasone eye drops (2 weeks). Removal of the exorbital lacrimal gland resulted in 50% decrease in tear volume and a gradual increase in corneal fluorescein staining. Elevated levels of TNF-α and IL-1α have been registered in tear fluid together with an increase in CD3(+) cells in the palpebral conjunctiva when compared to control animals. Additionally, an increase in MMP-9 mRNA expression was recorded in conjunctival tissue. Reference treatment with Restasis(®) and dexamethasone eye drops had a positive effect on all evaluation parameters, except on tear volume. This rat dry eye model was validated extensively and judged appropriate for the evaluation of novel compounds and therapeutic preparations for dry eye disease.

Resistance and cross-resistance profile of the diaryltriazine NNRTI and candidate microbicide UAMC01398.

OBJECTIVES: The resistance development, cross-resistance to other NNRTIs and the impact of resistance on viral replicative fitness were studied for the new and potent NNRTI UAMC01398. METHODS: Resistance was selected by dose escalation and by single high-dose selection against a comprehensive panel of NNRTIs used as therapeutics and NNRTIs under investigation for pre-exposure prophylaxis of sexual HIV transmission. A panel of 27 site-directed mutants with single mutations or combinations of mutations involved in reverse transcriptase (RT) inhibitor-mediated resistance was developed and used to confirm resistance to UAMC01398. Cross-resistance to other NNRTIs was assessed, as well as susceptibility of UAMC01398-resistant HIV to diarylpyrimidine-resistant viruses. Finally, the impact of UAMC01398 resistance on HIV replicative fitness was studied. RESULTS: We showed that UAMC01398 has potent activity against dapivirine-resistant HIV, that at least four mutations in the RT are required in concert for resistance and that the resistance profile is similar to rilpivirine, both genotypically and phenotypically. Resistance development to UAMC01398 is associated with a severe fitness cost. CONCLUSIONS: These data, together with the enhanced safety profile and good solubility in aqueous gels, make UAMC01398 an excellent candidate for HIV topical prevention.

Visceral hypersensitivity in inflammatory bowel diseases and irritable bowel syndrome: The role of proteases.

Proteases, enzymes catalyzing the hydrolysis of peptide bonds, are present at high concentrations in the gastrointestinal tract. Besides their well-known role in the digestive process, they also function as signaling molecules through the activation of protease-activated receptors (PARs). Based on their chemical mechanism for catalysis, proteases can be classified into several classes: serine, cysteine, aspartic, metallo- and threonine proteases represent the mammalian protease families. In particular, the class of serine proteases will play a significant role in this review. In the last decades, proteases have been suggested to play a key role in the pathogenesis of visceral hypersensitivity, which is a major factor contributing to abdominal pain in patients with inflammatory bowel diseases and/or irritable bowel syndrome. So far, only a few preclinical animal studies have investigated the effect of protease inhibitors specifically on visceral sensitivity while their effect on inflammation is described in more detail. In our accompanying review we describe their effect on gastrointestinal permeability. On account of their promising results in the field of visceral hypersensitivity, further research is warranted. The aim of this review is to give an overview on the concept of visceral hypersensitivity as well as on the physiological and pathophysiological functions of proteases herein.

Novel Ferroptosis Inhibitors with Improved Potency and ADME Properties.

Ferroptosis is a nonapoptotic, iron-catalyzed form of regulated necrosis that is critically dependent on glutathione peroxidase 4 (GPX4). It has been shown to contribute to liver and kidney ischemia reperfusion injury in mice. A chemical inhibitor discovered by high-throughput screening displayed inhibition of ferroptosis with nanomolar activity and was dubbed ferrostatin-1 (fer-1). Ferrostatins inhibit oxidative lipid damage, but suffer from inherent stability problems due to the presence of an ester moiety. This limits the application of these molecules in vivo, due to rapid hydrolysis of the ester into the inactive carboxylic acid. Previous studies highlighted the importance of the ethyl ester and suggested steric modifications of the ester for generating improved molecules. In this study, we report the synthesis of novel ferroptosis inhibitors containing amide and sulfonamide moieties with improved stability, single digit nanomolar antiferroptotic activity, and good ADME properties suitable for application in in vivo disease models.

Discovery and SAR of Novel and Selective Inhibitors of Urokinase Plasminogen Activator (uPA) with an Imidazo[1,2-a]pyridine Scaffold.

Urokinase plasminogen activator (uPA) is a biomarker and therapeutic target for several cancer types. Its inhibition is regarded as a promising, noncytotoxic approach in cancer therapy by blocking growth and/or metastasis of solid tumors. Earlier, we reported the modified substrate activity screening (MSAS) approach and applied it for the identification of fragments with affinity for uPA's S1 pocket. Here, these fragments are transformed into a novel class of uPA inhibitors with an imidazo[1,2-a]pyridine scaffold. The SAR for uPA inhibition around this scaffold is explored, and the best compounds in the series have nanomolar uPA affinity and selectivity with respect to the related trypsin-like serine proteases (thrombin, tPA, FXa, plasmin, plasma kallikrein, trypsin, FVIIa). Finally, the approach followed for translating fragments into small molecules with a decorated scaffold architecture is conceptually straightforward and can be expected to be broadly applicable in fragment-based drug design.

Synthesis and preclinical evaluation of an 18F labeled PDE7 inhibitor for PET neuroimaging.

INTRODUCTION: Phosphodiesterase 7 (PDE7) hydrolyzes selectively cyclic adenosine monophosphate (cAMP) which is an intracellular second messenger. PDE7 is expressed by 2 genes which are both present in the brain. To date there is no radiotracer for PDE7 imaging described and detection of PDE7 has only been performed by ex vivo techniques. In this report we describe the radiosynthesis of a novel fluorine-18 labeled radiotracer for PDE7 as well as the in vivo evaluation in mice to verify whether it has potential for imaging of PDE7 in the brain. METHODS: We have synthesized a potent fluorinated PDE7 inhibitor, [(18)F]MICA-003 (PDE7 IC(50)=17 nM) and the corresponding tosylate precursor for radiolabeling. [(18)F]MICA-003 was injected in C57BL/6J mice (n=5) and in vivo images were acquired by µPET imaging. Radiometabolite analysis in plasma and brain was performed to determine the stability of the radioligand. RESULTS: [(18)F]MICA-003 was synthesized by direct fluorination of the tosylate and produced in high decay corrected radiochemical yield (40%), high radiochemical purity (>98%) and high specific activity (86-497 GBq/µmol). µPET imaging revealed that [(18)F]MICA-003 crosses the blood brain barrier and has a homogenous distribution over the brain which washes out after the initial peak uptake. [(18)F]MICA-003 was quickly metabolized in plasma with 8.9%±0.59% of intact [(18)F]MICA-003 remaining at 5m in post injection. We observed the formation of three distinct radiometabolites of which the main radiometabolite was also detected in the brain in a proportion of 25.7±2.57% at this same time point. CONCLUSION: We have described the synthesis and in vivo evaluation of a novel radioligand for PDE7 imaging. Despite high uptake in the brain and favorable kinetics in vivo, the presence of a brain penetrant radiometabolite makes [(18)F]MICA-003 unfavorable for the accurate quantification of PDE7 and more stable spiroquinazolinones analogs are in development.

Repositioning the substrate activity screening (SAS) approach as a fragment-based method for identification of weak binders.

Fragment-based drug discovery (FBDD) has evolved into an established approach for "hit" identification. Typically, most applications of FBDD depend on specialised cost- and time-intensive biophysical techniques. The substrate activity screening (SAS) approach has been proposed as a relatively cheap and straightforward alternative for identification of fragments for enzyme inhibitors. We have investigated SAS for the discovery of inhibitors of oncology target urokinase (uPA). Although our results support the key hypotheses of SAS, we also encountered a number of unreported limitations. In response, we propose an efficient modified methodology: "MSAS" (modified substrate activity screening). MSAS circumvents the limitations of SAS and broadens its scope by providing additional fragments and more coherent SAR data. As well as presenting and validating MSAS, this study expands existing SAR knowledge for the S1 pocket of uPA and reports new reversible and irreversible uPA inhibitor scaffolds.

Synthesis and in vivo preclinical evaluation of an (18)F labeled uPA inhibitor as a potential PET imaging agent.

INTRODUCTION: The urokinase plasminogen activator (uPA) system is a proteolytic cascade involved in tumor invasion and metastasis. uPA and its inhibitor PAI-1 are described as biomarkers for breast cancer with the highest level of evidence. The present study describes the synthesis and first in vivo application of an activity based uPA PET probe. METHODS: Based on the design of a small irreversible and selective uPA inhibitor we developed an (18)F-labeled activity based probe for uPA imaging. Human uPA expressing MDA-MB-231-luc2-GFP cells were inoculated in the mammary fat pads of nude mice and treated with the probe once tumors reached a volume of 150mm(3). Scans were performed at 0.25, 0.75, 1.5, 4 and 6h post injection. To evaluate tumor uptake in vivo and ex vivo data were gathered. Biodistribution data of the organs and tissues of interest were collected at all time points. Due to a relatively low tumor uptake, probe stability was further evaluated. RESULTS: The uPA targeting PET tracer was produced in high purity and with good specific radioactivity. In vivo PET data showed a maximum tumor uptake of 2,51±0,32 %ID/g at 4h p.i. A significant correlation between in vivo and ex vivo tumor uptake calculation was found (R=0.75; p<0.01). Due to a high blood signal at all time points, probe stability was further examined revealing high plasma protein binding and low plasma stability. CONCLUSIONS: In vivo and ex vivo results clearly demonstrate that uPA expressing tumors can be detected with non-invasive PET imaging. Stability tests suggest that further optimization is needed to provide a better tumor-to-background contrast.

Extended structure-activity relationship and pharmacokinetic investigation of (4-quinolinoyl)glycyl-2-cyanopyrrolidine inhibitors of fibroblast activation protein (FAP).

Fibroblast activation protein (FAP) is a serine protease related to dipeptidyl peptidase IV (DPPIV). It has been convincingly linked to multiple disease states involving remodeling of the extracellular matrix. FAP inhibition is investigated as a therapeutic option for several of these diseases, with most attention so far devoted to oncology applications. We previously discovered the N-4-quinolinoyl-Gly-(2S)-cyanoPro scaffold as a possible entry to highly potent and selective FAP inhibitors. In the present study, we explore in detail the structure-activity relationship around this core scaffold. We report extensively optimized compounds that display low nanomolar inhibitory potency and high selectivity against the related dipeptidyl peptidases (DPPs) DPPIV, DPP9, DPPII, and prolyl oligopeptidase (PREP). The log D values, plasma stabilities, and microsomal stabilities of selected compounds were found to be highly satisfactory. Pharmacokinetic evaluation in mice of selected inhibitors demonstrated high oral bioavailability, plasma half-life, and the potential to selectively and completely inhibit FAP in vivo.

Diaryltriazine non-nucleoside reverse transcriptase inhibitors are potent candidates for pre-exposure prophylaxis in the prevention of sexual HIV transmission.

OBJECTIVES: Pre-exposure prophylaxis and topical microbicides are important strategies in the prevention of sexual HIV transmission, especially since partial protection has been shown in proof-of-concept studies. In search of new candidate drugs with an improved toxicity profile and with activity against common non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV, we have synthesized and investigated a library of 60 new diaryltriazine analogues. METHODS: From this library, 15 compounds were evaluated in depth using a broad armamentarium of in vitro assays that are part of a preclinical testing algorithm for microbicide development. Antiviral activity was assessed in a cell line, and in primary human cells, against both subtype B and subtype C HIV-1 and against viruses resistant to therapeutic NNRTIs and the candidate NNRTI microbicide dapivirine. Toxicity towards primary blood-derived cells, cell lines originating from the female reproductive tract and female genital microflora was also studied. RESULTS AND CONCLUSIONS: We identified several compounds with highly potent antiviral activity and toxicity profiles that are superior to that of dapivirine. In particular, compound UAMC01398 is an interesting new candidate that warrants further investigation because of its superior toxicity profile and potent activity against dapivirine-resistant viruses.

Selective Inhibitors of Fibroblast Activation Protein (FAP) with a (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine Scaffold.

Fibroblast activation protein (FAP) is a serine protease that is generally accepted to play an important role in tumor growth and other diseases involving tissue remodeling. Currently there are no FAP inhibitors with reported selectivity toward both the closely related dipeptidyl peptidases (DPPs) and prolyl oligopeptidase (PREP). We present the discovery of a new class of FAP inhibitors with a N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold. We have explored the effects of substituting the quinoline ring and varying the position of its sp(2) hybridized nitrogen atom. The most promising inhibitors combined low nanomolar FAP inhibition and high selectivity indices (>10(3)) with respect to both the DPPs and PREP. Preliminary experiments on a representative inhibitor demonstrate that plasma stability, kinetic solubility, and log D of this class of compounds can be expected to be satisfactory.