Transient Blockade of Endothelin-1 Mitigates Amiodarone-Induced Pulmonary Fibrosis.

INTRODUCTION: A number of studies indicate that endothelin-1 (ET-1) may act as an inflammatory cell "gatekeeper," by regulating the influx of neutrophils following pulmonary injury. To further examine the role of ET-1 in modulating lung inflammation, hamsters were treated with an endothelin receptor antagonist (ERA), HJP272, either 1 h prior to intratracheal instillation of amiodarone (AM) or 24 h afterwards. METHODS: In both cases, the extent of lung injury and repair was determined by (1) histopathological changes; (2) neutrophil content in bronchoalveolar lavage fluid (BALF); (3) lung collagen content; (4) tumor necrosis factor receptor 1 expression by BALF macrophages; (5) BALF levels of (a) transforming growth factor beta-1, (b) stromal cell-derived factor 1 (commonly referred to as CXCL12), and (c) platelet-derived growth factor BB; (6) alveolar septal cell apoptosis. RESULTS: For each parameter, pretreatment with HJP272 resulted in a significant reduction compared to AM alone, whereas post-treatment was either ineffective or produced only a marginally significant change, suggesting that the course of lung inflammation and repair is programmed at a very early stage. CONCLUSIONS: This finding may explain why ERAs are not an effective treatment for human pulmonary fibrosis. Nevertheless, they may be useful as an adjunct to therapeutic regimens involving drugs that have fibrogenic potential.

Time-dependent effects of HJP272, an endothelin receptor antagonist, in bleomycin-induced pulmonary fibrosis.

Using a lipopolysaccharide (LPS) model of acute lung injury, we have previously shown that endothelin-1 (ET-1), a potent mediator of vasoconstriction, may act as a "gatekeeper" for the influx of inflammatory cells into the lung. To further investigate the potential of ET-1 to limit the progression of lung injury, hamsters were treated with an endothelin receptor antagonist (ERA), HJP272, either 1 h prior to intratracheal instillation of bleomycin (BLM) or 24 h afterwards. Lung injury and repair were examined by measuring the following parameters: 1) histopathological changes; 2) neutrophil content in bronchoalveolar lavage fluid (BALF); 3) lung collagen content; 4) tumor necrosis factor receptor 1 (TNFR1) expression by BALF macrophages; 5) BALF levels of: a) transforming growth factor beta-1 (TGF-ß1), b) stromal cell-derived factor 1 (commonly referred to as CXCL12), and c) platelet-derived growth factor BB (PDGF-BB); 6) alveolar septal cell apoptosis (as measured by the TUNEL assay). For each of these parameters, animals pretreated with HJP272 showed significant reductions compared to those receiving BLM alone. In contrast, post-treatment with HJP272 was either ineffective or produced only marginally significant changes. The efficacy of a single pretreatment with HJP272 prior to induction of lung injury suggests that subsequent features of the disease are determined at a very early stage. This may explain why ERAs are not an effective treatment for human pulmonary fibrosis. Nevertheless, our findings suggest that they may be useful as prophylactic agents when given in combination with drugs that have fibrogenic potential.

Design, synthesis and evaluation of 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acid derivatives as ETA receptor selective antagonists using FRET assay.

The endothelin axis and in particular the two receptor subtypes, ETA and ETB, are under investigation for the treatment of various diseases such as pulmonary arterial hypertension, fibrosis, renal failure and cancer. Previous work in our lab has shown that 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acid derivatives exhibit noteworthy endothelin receptor antagonist activity. A series of analogues with modifications centered around position 6 of the heterocyclic quinolone core and replacement of the aryl carboxylic acid group with an isosteric tetrazole ring was designed and synthesized to further optimize the structure activity relationship. The endothelin receptor antagonist activity was determined by in vitro Förster resonance energy transfer (FRET) using GeneBLAzer® assay technology. The most potent member of this series exhibited ETA receptor antagonist activity in the subnanomolar range with an IC50 value of 0.8nM, and was 1000-fold selective for the ETA receptor compared to the ETB receptor. Its activity and selectivity profile resembles that of the most recently approved drug, macitentan.

Synthesis of N-1', N-3'-disubstituted spirohydantoins and their anticonvulsant activities in pilocarpine model of temporal lobe epilepsy.

Herein we report the synthesis and anticonvulsant activity of a library of eighteen new compounds that are structural mimics of phenytoin. These class of compounds contain a N-1', N-3'-disubstituted spirohydantoin scaffold, where the N-1' and N-3' positions are modified with an alkyl group or aryl group. Of the eighteen compounds synthesized and tested, compound 5c showed the best anticonvulsant activity. It completely prevented the precursor events of motor seizure in the pilocarpine model of temporal lobe epilepsy. Additionally, ten of the analogs were more effective than phenytoin when compared using the Racine's score in the pilocarpine model. Based on the structure activity relationship (SAR), we concluded that alkyl groups (ethyl, propyl or cyclopropyl) at N-3' position and 4-nitro phenyl group at N-1' position are desirable.

Structure-activity relationship in a purine-scaffold compound series with selectivity for the endoplasmic reticulum Hsp90 paralog Grp94.

Grp94 is involved in the regulation of a restricted number of proteins and represents a potential target in a host of diseases, including cancer, septic shock, autoimmune diseases, chronic inflammatory conditions, diabetes, coronary thrombosis, and stroke. We have recently identified a novel allosteric pocket located in the Grp94 N-terminal binding site that can be used to design ligands with a 2-log selectivity over the other Hsp90 paralogs. Here we perform extensive SAR investigations in this ligand series and rationalize the affinity and paralog selectivity of choice derivatives by molecular modeling. We then use this to design 18c, a derivative with good potency for Grp94 (IC50 = 0.22 µM) and selectivity over other paralogs (>100- and 33-fold for Hsp90α/ß and Trap-1, respectively). The paralog selectivity and target-mediated activity of 18c was confirmed in cells through several functional readouts. Compound 18c was also inert when tested against a large panel of kinases. We show that 18c has biological activity in several cellular models of inflammation and cancer and also present here for the first time the in vivo profile of a Grp94 inhibitor.

HJP272, a novel endothelin receptor antagonist, attenuates lipopolysaccharide-induced acute lung injury in hamsters.

INTRODUCTION: Previous studies from this laboratory indicate that endothelin-1 (ET-1), a potent vasoconstrictor, may play an important role in lipopolysaccharide (LPS)-induced release of neutrophils from the pulmonary microvasculature. To further test this concept, Syrian hamsters were treated with a novel endothelin receptor A (ETA) antagonist (HJP272) prior to intratracheal instillation of LPS. METHODS: The effect of HJP272 on the LPS-induced inflammatory reaction was determined by measuring: (1) lung histopathological changes, (2) total neutrophils in bronchoalveolar lavage fluid (BALF), (3) expression of tumor necrosis factor receptor 1 (TNFR1) by BALF macrophages, and (4) alveolar septal cell apoptosis. RESULTS: Treatment with HJP272 significantly reduced each of these parameters during a 24-hr period following LPS instillation, supporting the concept that limiting the activity of ET-1 may reduce the extent of lung injury. This hypothesis was further tested by giving ET-1 prior to LPS instillation, which resulted in a marked enhancement of LPS-induced lung inflammation, as measured by BALF neutrophils and TNFR1-positive macrophages. Furthermore, the increase in neutrophils resulting from treatment with ET-1 was significantly reduced by HJP272, again demonstrating the ability of ETA receptor antagonists to limit the influx of these cells into the lung. CONCLUSIONS: These findings suggest a potential therapeutic role for these agents in diseases where neutrophils are a significant cause of lung injury, such as bronchopneumonia, respiratory distress syndrome, and chronic obstructive pulmonary disease.

Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2.

Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90ß, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralog-specific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers.

N,N-dimethylacetamide regulates the proinflammatory response associated with endotoxin and prevents preterm birth.

The proinflammatory response leads to various types of pathologic pathways, including the development of preterm birth. Preterm birth occurs in 12% of deliveries in the United States and causes more than 70% of perinatal morbidity and mortality. The most common cause of spontaneous preterm birth is intrauterine infection in the mother. There is accumulating evidence indicating that the release of proinflammatory cytokines plays a critical role in the pathogenesis of inflammation-associated premature delivery. We found that the common organic solvent, N,N-dimethylacetamide (DMA), prevents endotoxin-induced preterm birth in timed pregnant C57BL/6 embryonic day (E)15.5 mice and rescues their pups from spontaneous abortion at doses many-fold lower than those currently used clinically and in a dose-dependent fashion. We also provide histologic evidence that DMA suppresses the endotoxin-triggered proinflammatory response by significantly attenuating inflammatory cell infiltration of placental tissue. Furthermore, immunoblotting analysis of placental tissue harvested from our murine models revealed DMA-mediated regulation of expression of the proinflammatory cytokines IL-1ß, tumor necrosis factor α, and IL-6, and increased expression of the regulatory inflammatory cytokine IL-10. By using in vitro studies, we provide evidence that DMA suppresses macrophage function and that this small molecule prevents nuclear translocation of nuclear factor-kB. These results suggest that DMA represents a newly discovered, nontoxic therapy for a broad range of inflammatory disorders.

PD173074, a selective FGFR inhibitor, reverses ABCB1-mediated drug resistance in cancer cells.

PURPOSE: Specific tyrosine kinase inhibitors were recently reported to modulate the activity of ABC transporters, leading to an increase in the intracellular concentration of their substrate drugs. In this study, we determine whether PD173074, a specific fibroblast growth factor receptor (FGFR) inhibitor, could reverse ABC transporter-mediated multidrug resistance. METHODS: 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyllapatinibrazolium bromide assay was used to determine the effect of PD173074 on reversal of ABC transporter-mediated multidrug resistance (MDR). In addition, [³H]-paclitaxel accumulation/efflux assay, western blotting analysis, ATPase, and photoaffinity labeling assays were done to study the interaction of PD173074 on ABC transporters. RESULTS: PD173074 significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to substrate anticancer drugs colchicine, paclitaxel, and vincristine. This effect of PD173074 is specific to ABCB1, as no significant interaction was detected with other ABC transporters such as ABCC1 and ABCG2. The observed reversal effect seems to be primarily due to the decreased active efflux of [³H]-paclitaxel in ABCB1 overexpressing cells observed in efflux assay. In addition, no significant change in the ABCB1 expression was observed when ABCB1 overexpressing cells were exposed to 5 µM PD173074 for up to 3 days, thereby further suggesting its role in modulating the function of the transporter. In addition, PD173074 stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Interestingly, PD173074 did not inhibit photolabeling of ABCB1 with [¹²5I]-iodoarylazidoprazosin (IAAP), showing that it binds at a site different from that of IAAP in the drug-binding pocket. CONCLUSIONS: Here, we report for the first time, PD173074, an inhibitor of the FGFR, to selectively reverse ABCB1 transporter-mediated MDR by directly blocking the efflux function of the transporter.

The novel ETA receptor antagonist HJP-272 prevents cerebral microvascular hemorrhage in cerebral malaria and synergistically improves survival in combination with an artemisinin derivative.

AIM: To investigate the association between vasculopathy and survival during experimental cerebral malaria (ECM), and to determine whether targeting the endothelin-1 (ET-1) pathway alone or in combination with the anti-malaria drug artemether (a semi-synthetic derivative of artemisinin) will improve microvascular hemorrhage and survival. MAIN METHODS: C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) were randomly assigned to four groups: no treatment, artemether treated, ET(A) receptor antagonist (HJP-272) treated, or HJP-272 and artemether treated. The uninfected control mice were treated with HJP-272 and artemether. We analyzed survival, cerebral hemorrhage, weight change, blood glucose levels and parasitemia. KEY FINDINGS: Our studies demonstrated decreased brain hemorrhage in PbA-infected (ECM) mice treated when HJP-272, a 1,3,6-trisubstituted-2-carboxy-quinol-4-one novel ET(A) receptor antagonist synthesized by our group, is used in conjunction with artemether, an anti-malarial agent. In addition, despite adversely affecting parasitemia and weight in non-artemether treated infected mice, HJP-272, seemed to confer some survival benefit when used as adjunctive therapy, though this did not reach significance. SIGNIFICANCE: Previous studies demonstrate that the endothelin pathway is associated with vasculopathy, neuronal injury and inflammation in ECM. As demonstrated here, components of the ET-1 pathway may be important targets for adjunctive therapy in ECM, and may help in preventing hemorrhage and in improving survival when used as adjunctive therapy during malaria infection. The data presented suggest that our novel agent, HJP-272, may ameliorate alterations in the vasculature which can potentially lead to inflammation, neurological dysfunction, and subsequent death in mice with ECM.

Synthesis, resolution and anticonvulsant activity of chiral N-1'-ethyl,N-3'-(1-phenylethyl)-(R,S)-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-trione diastereomers.

Four new N-1',N-3'-disubstituted-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-triones bearing a chiral N-3' substituent were synthesized, resolved and their anticonvulsant activity was obtained and determined that the activity was not stereoselective.

Synthesis and pharmacological activity of 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acids as selective ET(A) antagonists.

A series of 1,3,6-trisubsituted-4-oxo-1,4-dihyroquinoline-2-carboxylic acid analogs (2a-m) were designed and synthesized and their pharmacological activity determined, with the objective to better understand their SAR as potential ET(A) selective inhibitors. Most of the compounds displayed significant ET(A) antagonist activity having IC(50) for inhibition of binding of the [(125)I]ET-1 to ET(A) receptor <10 nM, with good selectivity for ET(A) antagonism over ET(B) receptor. Based on the in vitro results, SAR of this series of compounds requires an alkoxy substituent at the 6-position to be a straight and saturated chain up to three carbons long, since substitution of unsaturated and branched alkyloxy groups results in decrease in ET(A) antagonist activity. In this series, compound 2c (6-O-n-propyl analog) was found to be most potent (IC(50)=0.11 nM) with ET(B)/ET(A) selectivity of 8303.

Blockade of endothelin-1 with a novel series of 1,3,6-trisubstituted-2-carboxy-quinol-4-ones controls infection-associated preterm birth.

Preterm birth (PTB) currently accounts for 13% of all births in the United States, with the leading cause of PTB being maternal infection. Endothelin-1, an extremely potent vasoconstrictor capable of increasing myometrial smooth muscle tone, has been shown to be up-regulated in the setting of infection in pregnancy, ultimately leading to PTB. In previous work, we have shown that infection-associated PTB is controlled in our murine model by using phospharamidon, an endothelin-converting enzyme-1 inhibitor; knocking down endothelin-converting enzyme-1 mRNA; or blocking the binding of endothelin-1 to the endothelin-A (ET(A)) receptor with either BQ-123 or with HJP-272, the 6-OH compound of our series of novel synthetic (ET(A)) receptor antagonists. In the current study, we show that HJP-272, a highly selective ET(A) receptor antagonist with an IC(50) of 70.1 nmol/L, binds in a noncompetitive manner to the ET(A) receptor. Additionally, we introduce n-propyl (HJP-286) and n-butyl (HJP-278) analogs of HJP-272. We find that the LD(50) of HJP-272, the analog in the series most effective in controlling preterm birth, is more than 20-fold higher than its therapeutic dose. Acute exposure to high doses of these compounds produces no histological changes in any organ, while chronic exposure produces only a rare hepatotoxic effect. These findings may be of clinical significance, as there is currently no FDA-approved therapy for women presenting with threatened preterm delivery.

Preferential recruitment of neutrophils by endothelin-1 in acute lung inflammation induced by lipopolysaccharide or cigarette smoke.

This study examined the role of endothelin-1 (ET-1) in recruiting inflammatory cells to the lung after induction of injury with either lipopolysaccharide (LPS) or cigarette smoke. Hamsters injected with either ET-1 or its precursor peptide (Big ET-1) prior to treatment with LPS or cigarette smoke had markedly increased concentrations of neutrophils in bronchoalveolar lavage fluid (BALF) despite a reduction in total numbers of BALF leukocytes. Furthermore, the effect of ET-1 on smoke-exposed animals was reversed by addition of an endothelin-A receptor antagonist. These results are consistent with preferential recruitment of neutrophils by ET-1, and suggest that inhibition of this proinflammatory mediator may decrease acute pulmonary inflammation associated with cigarette smoke and other pulmonary toxins.

Effect of the putative novel selective ETA-receptor antagonist HJP272, a 1,3,6-trisubstituted-2-carboxy-quinol-4-one, on infection-mediated premature delivery.

Preterm birth (PTB), defined as any birth occurring before 37 weeks of gestation, occurs in only 12% of all births, yet accounts for nearly half of long-term neurological morbidity, and 60%-80% of perinatal mortality. The single most common cause of PTB is intrauterine infection. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that is both upregulated by inflammatory cytokines and capable of increasing myometrial smooth muscle tone. We hypothesized, therefore, that ET-1 is a critical component of the parturition cascade in the setting of infection-associated PTB. In our previous work, we have shown that blockade of ET-1 synthesis through the use of the metalloproteinase inhibitor phosphoramidon results in control of preterm labor. In the current work, we showed that blockade of ET-1 action with 5-50 mg/kg i.p. 3-(3-carboxybenzyl)-1-((6-ethylbenzo[d][1,3]dioxol-5-yl)methyl)-6-hydroxy-4-oxo-1,4-dihydroquinoline-2-carboxylic acid (HJP272), a putative novel selective ETA-receptor antagonist (IC50, 70 nmol/L), prevents PTB induced with up to 50 mg/kg of i.p. lipopolysaccharide in a mouse model. This is the first report, to our knowledge, of control of infection-associated PTB with a specific ETA-receptor antagonist. The identification of a novel effective therapy for PTB could have important clinical implications.

Synthesis and anticonvulsant activity of new N-1',N-3'-disubstituted-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-triones.

Thirteen new N-1',N-3'-disubstituted-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-triones were synthesized and their pharmacological activity determined with the objective to better understand their SAR for anticonvulsant activity. The anticonvulsant effects of these compounds were evaluated by standard pentylenetetrazol (scPTZ test) and maximum electroshock seizure (MES test) models in mice. Most of the compounds showed ability to protect against the pentylenetetrazol-induced convulsions. Compound 3o (the N-1'-p-nitrophenyl, N-3'-ethyl derivative) in the N-1'-aryl, N-3'-alkyl disubstituted series exhibited maximum activity with ED(50) of 41.8 mg/kg in scPTZ convulsion model.