An Elite Scaffold and a Wonderful Pharmacophore in Drug Discovery: Styrylquinoline.

Styrylquinoline is a quinoline molecule linked to phenyl rings with an unsaturated ethylene linker, resulting in a flat and rigid conformation. The synthesis of the molecule was reported almost a century ago but was not much explored due to its adverse toxicity and poor selectivity. In the last two decades, a plethora of work was reported related to the synthesis and antiretroviral activity of several styrylquinoline derivatives. Later, other activities such as antimicrobial and anticancer abilities of these derivatives were also reported. In this review, we summarize the diverse steps of the development and analyze the spectrum of the activity of styrylquinolines and their utilization in drug design. Styrylquinolines are extensively explored for new pharmacological activities in recent years and this makes the moiety gain more visibility as a potential drug candidate and lead molecule in medicinal chemistry. The data obtained in vitro and ex vivo shed light on their different mechanism of action. Styrylquinoline has proved to be a potential lead molecule in medicinal chemist's toolkit due to the exploration of a variety of avenues of its activity as a drug candidate.

Classification models and SAR analysis on CysLT1 receptor antagonists using machine learning algorithms.

Cysteinyl leukotrienes 1 (CysLT1) receptor is a promising drug target for rhinitis or other allergic diseases. In our study, we built classification models to predict bioactivities of CysLT1 receptor antagonists. We built a dataset with 503 CysLT1 receptor antagonists which were divided into two groups: highly active molecules (IC50 < 1000 nM) and weakly active molecules (IC50 ≥ 1000 nM). The molecules were characterized by several descriptors including CORINA descriptors, MACCS fingerprints, Morgan fingerprint and molecular SMILES. For CORINA descriptors and two types of fingerprints, we used the random forests (RF) and deep neural networks (DNN) to build models. For molecular SMILES, we used recurrent neural networks (RNN) with the self-attention to build models. The accuracies of test sets for all models reached 85%, and the accuracy of the best model (Model 2C) was 93%. In addition, we made structure-activity relationship (SAR) analyses on CysLT1 receptor antagonists, which were based on the output from the random forest models and RNN model. It was found that highly active antagonists usually contained the common substructures such as tetrazoles, indoles and quinolines. These substructures may improve the bioactivity of the CysLT1 receptor antagonists.

Structure-Based Virtual Screening of Ultra-Large Library Yields Potent Antagonists for a Lipid GPCR.

Cysteinyl leukotriene G protein-coupled receptors, CysLT1R and CysLT2R, regulate bronchoconstrictive and pro-inflammatory effects and play a key role in allergic disorders, cardiovascular diseases, and cancer. CysLT1R antagonists have been widely used to treat asthma disorders, while CysLT2R is a potential target against uveal melanoma. However, very few selective antagonist chemotypes for CysLT receptors are available, and the design of such ligands has proved to be challenging. To overcome this obstacle, we took advantage of recently solved crystal structures of CysLT receptors and an ultra-large Enamine REAL library, representing a chemical space of 680 M readily available compounds. Virtual ligand screening employed 4D docking models comprising crystal structures of CysLT1R and CysLT2R and their corresponding ligand-optimized models. Functional assessment of the candidate hits yielded discovery of five novel antagonist chemotypes with sub-micromolar potencies and the best Ki = 220 nM at CysLT1R. One of the hits showed inverse agonism at the L129Q constitutively active mutant of CysLT2R, with potential utility against uveal melanoma.

Identification of cysteinyl-leukotriene-receptor 1 antagonists as ligands for the bile acid receptor GPBAR1.

The cysteinyl leukotrienes (CysLTs), i.e. LTC4, LTD4 and LTE4, are a family of proinflammatory agents synthesized from the arachidonic acid. In target cells, these lipid mediators bind to the cysteinyl leukotriene receptors (CysLTR), a family of seven transmembrane G-protein coupled receptors. The CysLT1R is a validated target for treatment of pulmonary diseases and several selective antagonists for this receptor, including montelukast, zafirlukast and pranlukast, have shown effective in the management of asthma. Nevertheless, others CysLT1R antagonists, such as the alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV5901), have been extensively characterized without reaching sufficient priority for clinical development. Since drug reposition is an efficient approach for maximizing investment in drug discovery, we have investigated whether CysLT1R antagonists might exert off-target effects. In the report we demonstrate that REV5901 interacts with GPBAR1, a well characterized cell membrane receptor for secondary bile acids. REV5901 transactivates GPBAR1 in GPBAR1-transfected cells with an EC50 of 2.5 µM and accommodates the GPBAR1 binding site as shown by in silico analysis. Exposure of macrophages to REV5901 abrogates the inflammatory response elicited by bacterial endotoxin in a GPBAR1-dependent manner. In vivo, in contrast to montelukast, REV5901 attenuates inflammation and immune dysfunction in rodent models of colitis. The beneficial effects exerted by REV5901 in these models were abrogated by GPBAR1 gene ablation, confirming that REV5901, a shelved CysLT1R antagonist, is a GPBAR1 ligand. These data ground the basis for the development of novel hybrid ligands designed for simultaneous modulation of CysTL1R and GPBAR1.

Structural basis of ligand selectivity and disease mutations in cysteinyl leukotriene receptors.

Cysteinyl leukotriene G protein-coupled receptors CysLT1 and CysLT2 regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT1R has been used for treating asthma and associated diseases for over two decades, CysLT2R has recently started to emerge as a potential drug target against atopic asthma, brain injury and central nervous system disorders, as well as several types of cancer. Here, we describe four crystal structures of CysLT2R in complex with three dual CysLT1R/CysLT2R antagonists. The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular determinants of CysLTR ligand selectivity and specific effects of disease-related single nucleotide variants.

Structure-based mechanism of cysteinyl leukotriene receptor inhibition by antiasthmatic drugs.

The G protein-coupled cysteinyl leukotriene receptor CysLT1R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT1R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT1R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue-coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs.

Computer-Assisted Selective Optimization of Side-Activities-from Cinalukast to a PPARα Modulator.

Automated computational analogue design and scoring can speed up hit-to-lead optimization and appears particularly promising in selective optimization of side-activities (SOSA) where possible analogue diversity is confined. Probing this concept, we employed the cysteinyl leukotriene receptor 1 (CysLT1 R) antagonist cinalukast as lead for which we discovered peroxisome proliferator-activated receptor α (PPARα) modulatory activity. We automatically generated a virtual library of close analogues and classified these roughly 8000 compounds for PPARα agonism and CysLT1 R antagonism using automated affinity scoring and machine learning. A computationally preferred analogue for SOSA was synthesized, and in vitro characterization indeed revealed a marked activity shift toward enhanced PPARα activation and diminished CysLT1 R antagonism. Thereby, this prospective application study highlights the potential of automating SOSA.

Leukotriene receptors as potential therapeutic targets.

Leukotrienes, a class of arachidonic acid-derived bioactive molecules, are known as mediators of allergic and inflammatory reactions and considered to be important drug targets. Although an inhibitor of leukotriene biosynthesis and antagonists of the cysteinyl leukotriene receptor are clinically used for bronchial asthma and allergic rhinitis, these medications were developed before the molecular identification of leukotriene receptors. Numerous studies using cloned leukotriene receptors and genetically engineered mice have unveiled new pathophysiological roles for leukotrienes. This Review covers the recent findings on leukotriene receptors to revisit them as new drug targets.

Leukotriene Receptor Antagonists Inhibit Mitogenic Activity in Triple Negative Breast Cancer Cells

Despite a discovery of hormonal pathways regulating breast cancer, a definitive cure for the disease requires further identification of alternative targets that provide a hormone-independent support. Apart from their role in inflammatory diseases, cysteinyl leukotriene (CysLT) receptor antagonists (LTRAs) decrease the risk of lung cancer in asthma patients and inhibit tumor progression in several malignancies. In the present study, we evaluate the effects of two chemically different, clinically relevant LTRAs (montelukast and zafirlukast) in a triple negative breast cancer cell line, MDAMB- 231. We found that these two LTRAs reduced breast cancer cell viability in a dose-dependent manner with the 50% inhibitory concentration (IC50) between 5-10 µM. Although both LTRAs have several pharmacological properties in common, we noticed that montelukast mainly induced apoptosis, while zafirlukast mainly exerted its action on cell cycle. However, the precise mechanisms responsible for such different effects remain unclear. In summary, our results suggest that CysLT plays a role in proliferation and survivability of breast cancer cells in the absence of hormonal stimuli.

Structure and Ligand-Binding Mechanism of a Cysteinyl Leukotriene-Binding Protein from a Blood-Feeding Disease Vector.

Blood-feeding disease vectors mitigate the negative effects of hemostasis and inflammation through the binding of small-molecule agonists of these processes by salivary proteins. In this study, a lipocalin protein family member (LTBP1) from the saliva of Rhodnius prolixus, a vector of the pathogen Trypanosoma cruzi, is shown to sequester cysteinyl leukotrienes during feeding to inhibit immediate inflammatory responses. Calorimetric binding experiments showed that LTBP1 binds leukotrienes C4 (LTC4), D4 (LTD4), and E4 (LTE4) but not biogenic amines, adenosine diphosphate, or other eicosanoid compounds. Crystal structures of ligand-free LTBP1 and its complexes with LTC4 and LTD4 reveal a conformational change during binding that brings Tyr114 into close contact with the ligand. LTC4 is cleaved in the complex, leaving free glutathione and a C20 fatty acid. Chromatographic analysis of bound ligands showed only intact LTC4, suggesting that cleavage could be radiation-mediated.

Protective effect of montelukast which is cysteinyl-leukotriene receptor antagonist on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney.

Nephrotoxicity is a major complication of gentamicin (GEN). We aimed to evaluate the potential protective effect of montelukast (MK) against GEN-induced nephrotoxicity in rats. Thirty-two rats were randomly divided into four groups, each consisting of eight animals as follows: (1) the rats were control; (2) intraperitoneally injected with GEN 14 consecutive days (100 mg/kg/day); (3) treated with GEN plus distilled water via nasogastric gavage for 14 days; and (4) treated with GEN plus MK (10 mg/kg/day) for 14 days. After 15 days, rats were killed and their kidneys were taken and blood analysis was performed. Twenty-four hours urine collections were obtained in standard metabolic cages a day before the rats were killed. Tubular necrosis and interstitial fibrosis scoring were determined histopathologically in a part of kidneys; nitric oxide (NO), malondialdehyde (MDA), and reduced glutathione (GSH) levels were determined in the other part of kidneys. Statistical analyses were made by the chi-square test and analysis of variance. Serum urea and creatinine levels were significantly higher in rats treated with GEN alone, than the rats in control and GEN + MK groups.The GSH levels in renal tissue of only GEN-treated rats were significantly lower than those in control group, and administration of MK to GEN-treated rats significantly increased the level of GSH. The group that was given GEN and MK had significantly lower MDA and NO levels in kidney cortex tissue than those that was given GEN alone. In rats treated with GEN + MK, despite the presence of mild tubular degeneration and tubular necrosis are less severe, and glomeruli maintained a better morphology when compared with GEN group. We can say that MK prevents kidney damage with antioxidant effect, independently of NO.

Montelukast abrogates rhabdomyolysis-induced acute renal failure via rectifying detrimental changes in antioxidant profile and systemic cytokines and apoptotic factors production.

In addition to antiasthmatic effect, the cysteinyl leukotriene receptor 1 (CysLT1) antagonist montelukast shows renoprotective effect during ischemia/reperfusion and cyclosporine-induced renal damage. Here, we proposed that montelukast protects against rhabdomyolysis-induced acute renal failure. Compared with saline-treated rats, at 48 h following the induction of rhabdomyolysis using intramuscular glycerol (10 ml 50% glycerol/kg), significant elevations in serum levels of urea, creatinine, phosphate and acute renal tubular necrosis were observed. This was associated with elevations in serum Fas, interleukin-10, tumor necrotic factor-alpha, and transforming growth factor-beta1 and renal malondialdehyde and nitrite and detrimental reductions in renal catalase and superoxide dismutase activities. The effects of rhabdomyolysis on renal functional, biochemical and structural integrity and the associated changes in cytokines and Fas levels were abolished upon concurrent administration of montelukast (10 mg/kg i.p.) for 3 days (1 day before and 2 days after induction of rhabdomyolysis). Alternatively, administration of the anti-oxidant, α-tocopherol (400 mg/kg i.m.) for 3 days, succeeded in alleviating renal oxidative stress, but had no significant effect on the circulating levels of most cytokines and partially restored kidney functional and structural damage. Serum level of interleukin-6 was not altered by rhabdomyolysis but showed significant elevations in rats treated with montelukast or α-tocopherol. Collectively, motelukast abrogated functional and structural renal damage induced by rhabdomyolysis via ameliorating renal oxidative stress and modulation of systemic cytokines and apoptotic factors production. The results of this work are expected to open new avenues for early prevention of rhabdomyolysis-induced acute renal failure using selective CysLT1 antagonists such as montelukast.

Inhibition of leukotriene receptors boosts neural progenitor proliferation.

Neural stem and progenitor cells serve as a reservoir for new neurons in the adult brain throughout lifetime. One of the critical steps determining the net production of new neurons is neural progenitor proliferation, which needs to be tightly controlled. Since inflammation has detrimental effects on neurogenesis and the 5-lipoxygenase/leukotriene pathway is involved in inflammatory processes, we investigated the effects of leukotrienes and montelukast, a small molecule inhibitor of the leukotriene receptors CysLT(1)R and GPR17, on neural stem and progenitor cell proliferation. We demonstrate expression of the leukotriene receptor GPR17 by neural progenitors and by neural stem cells. Stimulation with excess amounts of leukotrienes did not affect progenitor proliferation, whereas blockade of GPR17 with montelukast strongly elevated neural stem and progenitor proliferation, while maintaining their differentiation fate and potential. This effect was associated with increased ERK1/2 phosphorylation suggesting an involvement of the EGF signaling cascade. Based on our results, montelukast and the inhibition of the 5-LOX pathway might be potent candidates for future therapies employing neurogenesis to promote structural and functional improvement in neurodegeneration, neuropsychiatric disease and ageing.

International Union of Basic and Clinical Pharmacology. LXXXIV: leukotriene receptor nomenclature, distribution, and pathophysiological functions.

The seven-transmembrane G protein-coupled receptors activated by leukotrienes are divided into two subclasses based on their ligand specificity for either leukotriene B(4) or the cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)). These receptors have been designated BLT and CysLT receptors, respectively, and a subdivision into BLT(1) and BLT(2) receptors and CysLT(1) and CysLT(2) receptors has been established. However, recent findings have also indicated the existence of putative additional leukotriene receptor subtypes. Furthermore, other ligands interact with the leukotriene receptors. Finally, leukotrienes may also activate other receptor classes, such as purinergic receptors. The aim of this review is to provide an update on the pharmacology, expression patterns, and pathophysiological roles of the leukotriene receptors as well as the therapeutic developments in this area of research.

The approach of conformational chimeras to model the role of proline-containing helices on GPCR mobility: the fertile case of Cys-LTR1.

The homology modeling of GPCRs has benefitted vastly from the availability of some resolved structures, which allow the generation of many reliable GPCR models. However, the dynamic behavior of such receptors has been only minimally examined in silico, although several pieces of evidence have highlighted some conformational switches that can orchestrate the activation mechanism. Among such switches, Pro-containing helices play a key role in determining bending in TM helices and thereby the width of the TM bundle. The approach proposed herein involves the generation of a set of possible models (conformational chimeras) by exhaustively combining the two main conformations (straight and bent) that a Pro-containing helix can assume. This approach was validated by generating conformational chimeras for the Cys-LTR1 receptor, which is involved in contractile and inflammatory processes. The generated chimeras were then used for docking a small set of representative ligands. The results revealed the flexibility mechanisms of Cys-LTR1, showing how the docked agonists vary their stabilizing interactions, shifting from the open to closed state, and how the examined antagonists are able to block the receptor in an open and inactive conformation, thus behaving as inverse agonists. This study emphasizes the promising potential of chimera modeling, confirms the key role of proline residues in receptor activation, and suggests that docking results can be improved by considering the often-overlooked flexibility of receptors.

Pharmacophore identification, synthesis, and biological evaluation of carboxylated chalcone derivatives as CysLT1 antagonists.

The pharmacophore model (Hypo1) with a well prediction capacity for CysLT(1) antagonists was developed using Catalyst/HypoGen program. Virtual screening against an in-house database consisted of carboxylated chalcones using Hypo1 was performed. Retrieved hits 26a, 26b, 27a, and 27b were synthesized and biological evaluated, the results of which demonstrated that these compounds showed moderate to good CysLT(1) antagonistic activities. This study indicated that the generated model (Hypo1) is a reliable and useful tool in lead optimization for novel CysLT(1) antagonists.

Cysteinyl leukotrienes and their receptors: molecular and functional characteristics.

The cysteinyl leukotrienes (CysLTs) are a family of potent inflammatory lipid mediators synthesized from arachidonic acid by a variety of cells including mast cells, eosinophils, basophils and macrophages. The family includes leukotriene C(4) (LTC(4)), leukotriene D(4) (LTD(4)) and leukotriene E(4) (LTE(4)), which are potent biological mediators in the pathophysiology of inflammatory diseases and trigger contractile and inflammatory processes through the specific interaction with cell surface receptors, belonging to the superfamily of G-protein-coupled receptor. Pharmacological characterizations have suggested the existence of at least 2 types of CysLT receptors based on potency of agonist and antagonist, designated as CysLT(1) and CysLT(2). The CysLT(1) receptors are mostly expressed in lung smooth muscle cells, interstitial lung macrophages and the spleen, and it has been studied a lot elucidating its role in the etiology of airway inflammation and asthma. On the other hand, CysLT(2) receptors are present in the heart, brain and adrenal glands. This review discusses the role of CysLTs and their receptor in the pathophysiology of various inflammatory disorders. The understanding of CysLTs and their receptors in allergic airway disease is currently limited to CysLT(1)-receptor-mediated effects, and the role of the CysLT(2) receptors is pharmacologically less well defined, as there is no specific antagonist available yet. Specific CysLT(2)-receptor-selective antagonists would be very helpful to identify the precise role of CysLT and their receptors. Some recent evidence indicates the existence of additional receptor subtypes and requires further investigation for a better understanding of the role of the CysLT receptors. This review is an effort to summarize the localization, regulation and expression pattern along with the molecular and functional pharmacology of the CysLT receptors and to discuss their role in the pathophysiology of different diseases along with the recent update.

[Method for screening cysteinyl leukotriene receptor 2 antagonists and preliminary screening of compounds].

OBJECTIVE: To establish a method for screening cysteinyl leukotriene receptor 2 (CysLT(2)) antagonists and to preliminarily screen a series of synthetic compounds. METHODS: Rat glioma cell line (C6 cells) highly expressing CysLT(2) receptor was used. Intracellular calcium concentration was measured after stimulation with the agonist LTD(4),which was used to screen compounds with antagonist activity for CysLT(2) receptor. Bay u9773, a CysLT1/CysLT(2) receptor non-selective antagonist, and AP-100984, a CysLT(2) receptor antagonist, were used as control. RESULT: PT-PCR showed a higher expression of CysLT(2) receptor in C6 cells. LTD(4) at 1 mumol/L significantly increased intracellular calcium in C6 cells; the maximal effect was about 37.5% of ATP, a positive stimulus.LTD(4)-induced increase of intracellular calcium was blocked by CysLT(2) receptor antagonists, but not by CysLT(1) receptor antagonists. Among the synthetic compounds, D(XW-)1,2,13,23,29 and 30 inhibited LTD(4)-induced increase of intracellular calcium. CONCLUSION: LTD(4)-induced change in intracellular calcium in C6 cells can be used as a screening method for CysLT(2) receptor antagonists. The compounds, D(XW-)1,2,13,23,29 and 30, possess antagonist activity for CysLT(2) receptor.

Synthesis and structure-activity relationships of gamma-carboline derivatives as potent and selective cysLT(1) antagonists.

A gamma-carboline series of cysLT(1) receptor antagonists has been prepared. Some of the compounds show good potencies both, in vitro and in vivo, compared to the standard compounds.

Determination of structure and transcriptional regulation of CYSLTR1 and an association study with asthma and rhinitis.

Pharmacologic studies have revealed that cysteinyl leukotrienes (CYSLTs) act through two receptors, cysteinyl leukotriene receptor 1 (CYSLTR1) and CYSLTR2. CYSLTR1 antagonists are widely used to treat asthma and rhinitis. In this study, we characterized the genomic structure and transcriptional regulation of CYSLTR1 and examined associations between CYSLTR1 polymorphisms and asthma/rhinitis. The experiment of rapid amplification of cDNA end revealed that CYSLTR1 contains three exons and that the entire open reading frame is located in exon 3. Reverse transcriptase-polymerase chain reaction showed that there were multiple splice variants of CYSLTR1 and that the transcript expression patterns differed from tissues and cell types. The promoter region of CYSLTR1 is from -665 to -30 bp relative to the transcription start site. We identified four polymorphisms (c.-618-434T/C, c.-618-275C/A, c.-618-136G/A, and 927C/T), and transmission disequilibrium tests revealed that none of these polymorphisms was associated with the development of asthma/rhinitis. However, the TCG and CAA haplotypes in the promoter region caused different transcriptional activity. Our findings indicate that CYSLTR1 polymorphisms are not likely to be involved in the development of asthma/rhinitis, but it is possible that these polymorphisms could influence drug responses in individuals with atopic diseases.