Discovery of a Novel Thienopyrimidine Compound as a Urate Transporter 1 and Glucose Transporter 9 Dual Inhibitor with Improved Efficacy and Favorable Druggability.

Gout and hyperuricemia are metabolic diseases characterized with high serum uric acid (SUA) levels that significantly impact human health. Lesinurad, a uricosuric agent, is limited to concurrent use with xanthine oxidase inhibitors (XOIs) in clinical practice due to its restricted efficacy and potential nephrotoxicity. Herein, extensive structural modifications of lesinurad were conducted through scaffold hopping and substituent modification strategies, affording 54 novel derivatives containing pyrimidine-fused cyclic structures. Notably, the thienopyrimidine compound 29 demonstrated a remarkable 2-fold increase in SUA-lowering in vivo activity compared to lesinurad, while exhibiting potent inhibitory activity against the urate transporter 1 (URAT1, IC50 = 2.01 µM) and glucose transporter 9 (GLUT9, IC50 = 18.21 µM). Furthermore, it possessed a lower effective dosage of 0.5 mg/kg, favorable safety profile without any apparent acute toxicity at doses of 1000 mg/kg, and improved pharmacokinetic properties. Overall, we have discovered an efficacious URAT1/GLUT9 dual inhibitor for inhibiting urate reabsorption with favorable pharmacokinetic profiles.

Synthesis, toxicological and in silico evaluation of novel spiro pyrimidines against Culex pipiens L. referring to chitinase enzyme.

The exponential development of resistance to conventional chemical insecticides adds another important motive for the creation of novel insecticidal active agents. One of the keys to meeting this challenge is the exploration of novel classes of insecticidal molecules with different modes of action. Herein, a novel series of spiro pyrimidine derivatives was prepared using some green synthetic methodologies such as microwave irradiation, and sonication under ultrasound waves. Spiro pyrimidine aminonitrile 1 is a key starting material for the synthesis of targets 2-9 by reaction with different carbon electrophiles and nitrogen nucleophiles. The structures of all the newly synthesized compounds were approved using spectral data. The toxicological efficiency and biological impacts of the synthesized spiro pyrimidine derivatives were assessed against Culex pipiens L. larvae. The toxicity of synthesized compounds showed remarkable variations against the C. pipiens larvae. Where, 3, 4 and 2 were the most efficient compounds with LC50 values of 12.43, 16.29 and 21.73 µg/mL, respectively. While 1 was the least potent compound with an LC50 value of 95.18 µg/mL. As well, other compounds were arranged according to LC50 values as follows 5 > 7 > 6 > 9 > 8. In addition, 3 and 4 exhibited significant prolongation of the developmental duration and greatly inhibited adult emergence. Moreover, many morphological deformities were observed in all developmental stages. Furthermore, cytotoxicity of the most effective compounds was assessed against the normal human cells (WI-38) as non-target organisms, where compounds 2, 4 and 3 showed weak to non-toxic effects. The study of binding affinity and correlation between chemical structure and reactivity was carried out using molecular docking study and DFT calculations to investigate their mode of action. This study shed light on promising compounds with larvicidal activity and biological impacts on the C. pipiens life cycle.

Nilotinib exhibits less toxicity than imatinib and influences the immune state by modulating iNOS, p-p38 and p-JNK in LPS/IFN gamma-activated macrophages.

In this study, we aimed to analyze the effects of first and second-generation Bcr-Abl tyrosine kinase inhibitors, imatinib and nilotinib on LPS/IFN gamma activated RAW 264.7 macrophages. Our data revealed that imatinib was less effective on nitrite levels and more toxic on macrophages compared to nilotinib. Therefore, we further analysed the effect of nilotinib on various inflammatory markers including iNOS, COX-2, NFkB, IL-6, p-ERK, p-p38 and p-JNK in LPS/IFN gamma activated RAW264.7 macrophages. Spectrophotometric viability test and Griess assay,western blot, RT-PCR and luciferase reporter assays were used to analyze the biological activity of nilotinib. Our findings revealed that nilotinib decreases nitrite levels, iNOS mRNA, iNOS and p-p38 protein expressions significantly whereas induces IL-6 mRNA and p-JNK protein expressions at particular doses. We did not find significant effect of nilotinib on COX-2, p-ERK and nuclear p65 proteins and NFkB transcriptional activity. In addition, the binding mode of nilotinib to iNOS protein was predicted by molecular docking. According to the docking analyses, nilotinib exhibited hydrophobic interactions between MET349, ALA191, VAL346, PHE363, TYR367, MET368, CYS194, TRP366 residues at the binding pocket and the molecule as well as van der Waals interactions at specific residues. In conclusion, our results reveal that, in addition to its anticancer activity, nilotinib can exhibit immune modulatory effects on macrophages through its effects on iNOS, IL-6, p-p38 and p-JNK.

Salmonella mutant strains resistant to herbicides - Acetohydroxyacid synthase inhibitors and their use at the Ames test.

Cytotoxicity of some pesticides is a disadvantage for the Salmonella/microsome assay with regard to the equivalence assessment of pesticide technical grade active ingredients to the original products and detection of low-level impurities. The technical grade active ingredients (TGAIs) of pesticides from certain chemical classes were found to be toxic for Salmonella typhimurium strains. Among the highly cytotoxic compounds were sulfonylureas, which include 20 active ingredients. In addition, this class includes active pharmaceutical ingredients used for the manufacture of antidiabetics drugs. A traditional selection methodology was applied using the cultivation of S. typhimurium TA100 in the presence of high concentrations of thifensulfuronmethyl (TFSM) to obtain a resistant test strain insusceptible to sulfonylurea toxic effect. Two strains resistant not only to sulfonylureas (SFU) but also triazolepyrimidines were received. The first mutant strain (deposited as S. typhimurium VKPM B-14099 in the Russian National Collection of Industrial Microorganisms) demonstrated the TA100 phenotypic characteristics: hisG46, rfa, ΔuvrB-bio, pKM101. The second strain (deposited as S. typhimurium VKPM B-14359) showed the TA1535 phenotypic characteristics and probably lost the R-factor due to the selection using the poor Gm-media with TFSM. Positive controls caused pronounced mutagenic effects (±S9) in both strains, consequently the mutants did not lose the ability to respond to induction of the reverse gene mutations. The maximum non-cytotoxic concentrations of SFUs and triazole-pyrimidines for the Ames test strains did not exceed 0.05-0.125 mg/plate, while no evidence of cytotoxicity was observed for the mutants up to 5.0 mg/plate. Electron microscopy of the ultrathin sections of Salmonella cells grown with and without TFSM showed an obvious difference in the structure of the cell wall and cytoplasm in mutant and parental cultures. The concurrent resistance both to SFU and triazolepyrimidines was assumed to be mediated by the same mechanism of action of the pesticides from these classes - inhibition of acetohydroxyacid synthase. To confirm this hypothesis, the tests in the presence of branched-chain amino acids were carried out. The enrichment of agar with isoleucine prevented the toxic effects of SFU and triazolepyrimidines for all Ames test strains used in the study, while strong cytotoxicity was observed in the presence of valine and leucine. Considering the tolerance of strains both to SFU and triazolpyrimidines and the results with branched-chain amino acids, the modification of target acetohydroxyacid synthase was supposed the key to the acquired resistance. The new strains resistant to sulfonylureas and triazole-pyrimidines expands the possibilities to reveal mutagenic impurities that may occur in TGAIs in small amounts.

Adverse Toxic Effects of Tyrosine Kinase Inhibitors on Non-Target Zebrafish Liver (ZFL) Cells.

Over the past 20 years, numerous tyrosine kinase inhibitors (TKIs) have been introduced for targeted therapy of various types of malignancies. Due to frequent and increasing use, leading to eventual excretion with body fluids, their residues have been found in hospital and household wastewaters as well as surface water. However, the effects of TKI residues in the environment on aquatic organisms are poorly described. In the present study, we investigated the cytotoxic and genotoxic effects of five selected TKIs, namely erlotinib (ERL), dasatinib (DAS), nilotinib (NIL), regorafenib (REG), and sorafenib (SOR), using the in vitro zebrafish liver cell (ZFL) model. Cytotoxicity was determined using the MTS assay and propidium iodide (PI) live/dead staining by flow cytometry. DAS, SOR, and REG decreased ZFL cell viability dose- and time-dependently, with DAS being the most cytotoxic TKI studied. ERL and NIL did not affect viability at concentrations up to their maximum solubility; however, NIL was the only TKI that significantly decreased the proportion of PI negative cells as determined by the flow cytometry. Cell cycle progression analyses showed that DAS, ERL, REG, and SOR caused the cell cycle arrest of ZFL cells in the G0/G1 phase, with a concomitant decrease of cells in the S-phase fraction. No data could be obtained for NIL due to severe DNA fragmentation. The genotoxic activity of the investigated TKIs was evaluated using comet and cytokinesis block micronucleus (CBMN) assays. The dose-dependent induction of DNA single strand breaks was induced by NIL (≥2 µM), DAS (≥0.006 µM), and REG (≥0.8 µM), with DAS being the most potent. None of the TKIs studied induced micronuclei formation. These results suggest that normal non-target fish liver cells are sensitive to the TKIs studied in a concentration range similar to those previously reported for human cancer cell lines. Although the TKI concentrations that induced adverse effects in exposed ZFL cells are several orders of magnitude higher than those currently expected in the aquatic environment, the observed DNA damage and cell cycle effects suggest that residues of TKIs in the environment may pose a hazard to non-intentionally exposed organisms living in environments contaminated with TKIs.

Lack of direct bone effect of tofacitinib, a JAK inhibitor, in juvenile rats and evaluation of the association between offspring growth and femur length.

Bone has recently emerged as a target organ for some Janus kinase (JAK) inhibitors in adult and/or juvenile animal toxicity studies. Oral administration of tofacitinib, a JAK inhibitor, was not associated with clinical or macroscopic effects on bone growth and development in a rat juvenile animal study (JAS) with tofacitinib dosing starting on postnatal day (PND) 21. However, given that previous JAS did not include a targeted evaluation of bone, inclusive of microscopic examination, an additional rat JAS was conducted to further assess this risk. In this subsequent JAS, administration of tofacitinib from PND 7-49 or from PND 21-49 did not result in any direct effects on bone, with no histologic effects on developing bone. The only bone effect in this JAS was nonadverse shorter femur length, which was not considered to be a direct effect of tofacitinib, but rather an indicator of growth delay, as this was associated with lower body weights. There were no effects on femur length or body weight after a 2-month recovery period. To further explore the relationship between body weight and femur length, historical control data were analyzed from control rats in other JAS. This analysis clearly demonstrated that shorter femur length can occur as an indirect effect that is highly associated with lower body weight, consistent with what was observed in the JAS with tofacitinib. These analyses provide a robust and valuable data set to support the interpretation of such data in JAS, and further support the lack of direct effects of tofacitinib on bone growth and development. As with the previously conducted juvenile studies with tofacitinib, the additional JAS did not identify any special JAS-based concerns for use in pediatric patients as young as 2 years of age.

Subacute sarin exposure disrupted the homeostasis of purine and pyrimidine metabolism in guinea pig striatum studied by integrated metabolomic, lipidomic and proteomic analysis.

Sarin was used as a chemical weapon due to its high neurotoxicity and mortality. Subacute sarin induced cognitive and behavioral disorder. However, the underlying mechanism is still unclear. Here we offered a multi-omic approach for the analysis of altered metabolites, lipids, and proteins to explore the neurotoxicity of subacute sarin. Guinea pigs were administered between the shoulder blades 16.8 µg/kg of sarin in a volume of 1.0 ml/kg body weight by subcutaneous injection once daily for 14 days. At the end of the final injection, guinea pigs were sacrificed, and striatum were dissected for analysis. A total of 138 different metabolites were identified in the metabolome analysis. Lipids and lipid-like molecules is the largest group (38.41%). For lipidomic analysis, a total of 216 lipids were identified. In proteomic study, over 4300 proteins were identified and quantified. By integrating these enriched components, we demonstrated that the joint pathways disturbed by subacute sarin mainly involving lipid, purine and pyrimidine metabolism in guinea pig striatum. Overall, this study highlights the powerfulness of omics platforms to deepen the understanding of nerve agents caused neurotoxicity.

Australindolones, New Aminopyrimidine Substituted Indolone Alkaloids from an Antarctic Tunicate Synoicum sp.

Five new alkaloids have been isolated from the lipophilic extract of the Antarctic tunicate Synoicum sp. Deep-sea specimens of Synoicum sp. were collected during a 2011 cruise of the R/V Nathanial B. Palmer to the southern Scotia Arc, Antarctica. Crude extracts from the invertebrates obtained during the cruise were screened in a zebrafish-based phenotypic assay. The Synoicum sp. extract induced embryonic dysmorphology characterized by axis truncation, leading to the isolation of aminopyrimidine substituted indolone (1-4) and indole (5-12) alkaloids. While the primary bioactivity tracked with previously reported meridianins A-G (5-11), further investigation resulted in the isolation and characterization of australindolones A-D (1-4) and the previously unreported meridianin H (12).

Identification of 2-Aminopyrimidine Derivatives as FLT3 Kinase Inhibitors with High Selectivity over c-KIT.

Herein, we report two promising compounds 30 and 36 possessing nanomolar FLT3 inhibitory activities (IC50 = 1.5-7.2 nM), high selectivity over c-KIT (>1000-fold), and excellent anti-AML activity (MV4-11 IC50 = 0.8-3.2 nM). Furthermore, these two compounds efficiently inhibited the growth of multiple mutant BaF3 cells expressing FLT3-ITD, FLT3-D835V/F, FLT3-F691L, FLT3-ITD-F691L, and FLT3-ITD-D835Y. Oral administration of 30 and 36 at 6 mg/kg/d could significantly suppress tumor growth in the MV4-11 cell-inoculated xenograft model, exhibiting tumor growth inhibitory rates of 83.5% and 95.1%, respectively. Importantly, 36 could prolong the mouse survival time in the FLT3-ITD-TKD dual mutation syngeneic mouse model (BaF3-FLT3-ITD-D835Y) at a dose of 6 mg/kg p.o. bid/4W. No clear myelosuppression was observed in the treated group of 36 in the MPO strain of zebrafish, even at 10 µM. In summary, our data demonstrated that 36 may represent a promising candidate for the treatment of FLT3 mutant AML.

Synthesis and Evaluation of Structurally Diverse C-2-Substituted Thienopyrimidine-Based Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase.

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.

Novel Bile Acid-Dependent Mechanisms of Hepatotoxicity Associated with Tyrosine Kinase Inhibitors.

Drug-induced liver injury (DILI) is the leading cause of acute liver failure and a major concern in drug development. Altered bile acid homeostasis via inhibition of the bile salt export pump (BSEP) is one mechanism of DILI. Dasatinib, pazopanib, and sorafenib are tyrosine kinase inhibitors (TKIs) that competitively inhibit BSEP and increase serum biomarkers for hepatotoxicity in ∼25-50% of patients. However, the mechanism(s) of hepatotoxicity beyond competitive inhibition of BSEP are poorly understood. This study examined mechanisms of TKI-mediated hepatotoxicity associated with altered bile acid homeostasis. Dasatinib, pazopanib, and sorafenib showed bile acid-dependent toxicity at clinically relevant concentrations, based on the C-DILI assay using sandwich-cultured human hepatocytes (SCHH). Among several bile acid-relevant genes, cytochrome P450 (CYP) 7A1 mRNA was specifically upregulated by 6.2- to 7.8-fold (dasatinib) and 5.7- to 9.3-fold (pazopanib), compared with control, within 8 hours. This was consistent with increased total bile acid concentrations in culture medium up to 2.3-fold, and in SCHH up to 1.4-fold, compared with control, within 24 hours. Additionally, protein abundance of sodium taurocholate co-transporting polypeptide (NTCP) was increased up to 2.0-fold by these three TKIs. The increase in NTCP protein abundance correlated with increased function; dasatinib and pazopanib increased hepatocyte uptake clearance (CLuptake) of taurocholic acid, a probe bile acid substrate, up to 1.4-fold. In conclusion, upregulation of CYP7A1 and NTCP in SCHH constitute novel mechanisms of TKI-associated hepatotoxicity. SIGNIFICANCE STATEMENT: Understanding the mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors (TKIs) is fundamental to development of effective and safe intervention therapies for various cancers. Data generated in sandwich-cultured human hepatocytes, an in vitro model of drug-induced hepatotoxicity, revealed that TKIs upregulate bile acid synthesis and alter bile acid uptake and excretion. These findings provide novel insights into additional mechanisms of bile acid-mediated drug-induced liver injury, an adverse effect that limits the use and effectiveness of TKI treatment in some cancer patients.

Evaluation of acetamiprid and azoxystrobin residues and their hormonal disrupting effects on male rats using liquid chromatography-tandem mass spectrometry.

Endocrine-disrupting compounds as pesticides affect the hormonal balance, and this can result in several diseases. Therefore, the analysis of representative hormones with acetamiprid (AC) and azoxystrobin (AZ) was a good strategy for the investigation of the endocrine-disrupting activity of pesticides. Hence, a sensitive and rapid analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method was validated for the analysis of AC, AZ, estriol, estrone, progesterone, and testosterone in the serum, testis, and liver of rats. The correlation between the residues of pesticides and the disturbance of the endocrine system was evaluated. The different mass parameters, mobile phase types, analytical columns, injection volumes, and extraction solvents were compared to get the lowest limit of detection of the studied compounds. The detection limits of AC, AZ, estriol, estrone, progesterone, and testosterone were 0.05, 0.05, 1.0, 10, and 1.0 ng/ml, respectively. The method developed was applied to evaluate the changes in these hormones induced by the duration of exposure to AC and AZ in rat testis and serum. The hormones level in rat serum and testis had a significant decrease as they were oral gavage treated with different high concentrations of studied pesticides. Both pesticides were distributed in the body of rats by the multi-compartment model (liver, testis, and serum).

Co-formulant in a commercial fungicide product causes lethal and sub-lethal effects in bumble bees.

Pollinators, particularly wild bees, are suffering declines across the globe, and pesticides are thought to be drivers of these declines. Research into, and regulation of pesticides has focused on the active ingredients, and their impact on bee health. In contrast, the additional components in pesticide formulations have been overlooked as potential threats. By testing an acute oral dose of the fungicide product Amistar, and equivalent doses of each individual co-formulant, we were able to measure the toxicity of the formulation and identify the ingredient responsible. We found that a co-formulant, alcohol ethoxylates, caused a range of damage to bumble bee health. Exposure to alcohol ethoxylates caused 30% mortality and a range of sublethal effects. Alcohol ethoxylates treated bees consumed half as much sucrose as negative control bees over the course of the experiment and lost weight. Alcohol ethoxylates treated bees had significant melanisation of their midguts, evidence of gut damage. We suggest that this gut damage explains the reduction in appetite, weight loss and mortality, with bees dying from energy depletion. Our results demonstrate that sublethal impacts of pesticide formulations need to be considered during regulatory consideration, and that co-formulants can be more toxic than active ingredients.

Azoxystrobin Impairs Neuronal Migration and Induces ROS Dependent Apoptosis in Cortical Neurons.

Fungicides often cause genotoxic stress and neurodevelopmental disorders such as autism (ASD). Fungicide-azoxystrobin (AZOX) showed acute and chronic toxicity to various organisms, and remained a concern for ill effects in developing neurons. We evaluated the neurotoxicity of AZOX in developing mouse brains, and observed prenatal exposure to AZOX reduced neuronal viability, neurite outgrowth, and cortical migration process in developing brains. The 50% inhibitory concentration (IC50) of AZOX for acute (24 h) and chronic (7 days) exposures were 30 and 10 µM, respectively. Loss in viability was due to the accumulation of reactive oxygen species (ROS), and inhibited neurite outgrowth was due to the deactivation of mTORC1 kinase activity. Pretreatment with ROS scavenger- N-acetylcysteine (NAC) reserved the viability loss and forced activation of mTORC1 kinase revived the neurite outgrowth in AZOX treated neurons. Intra-amniotic injection of AZOX coupled with in utero electroporation of GFP-labelled plasmid in E15.5 mouse was performed and 20 mg/kg AZOX inhibited radial neuronal migration. Moreover, the accumulation of mitochondria was significantly reduced in AZOX treated primary neurons, indicative of mitochondrial deactivation and induction of apoptosis, which was quantified by Bcl2/Bax ratio and caspase 3 cleavage assay. This study elucidated the neurotoxicity of AZOX and explained the possible cure from it.

Effects of pyrimethanil fungicide on Chironomidae community structure.

The use of agrochemicals in agriculture may impact aquatic ecosystems, particularly influencing the stream insect communities. Among aquatic insects, the family Chironomidae is the most abundant and species-diverse insect group found in freshwater ecosystems. However, in the southern hemisphere, studies with Chironomidae are still sparse, compared to Europe and North America. The present study evaluates the responses of Chironomidae species (Insecta: Diptera) to pyrimethanil fungicide in a mesocosm experiment. Water contamination and chironomid community were monitored over 10 months. After five months of monitoring, the pyrimethanil fungicide was completely degraded and there was a statistically significant increase in the Margalef Richness and Shannon-Wiener Index (H') in the control units when compared with the contaminated mesocosms (p = 0.003). Our results point out that the utilization of agrochemicals can be a harmful factor influencing negatively the Chironomidae populations. This finding has key implications for insect conservation strategies and ecological management environments.

Discovery of Novel Dihydrothiopyrano[4,3-d]pyrimidine Derivatives as Potent HIV-1 NNRTIs with Significantly Reduced hERG Inhibitory Activity and Improved Resistance Profiles.

Enlightened by the available structural biology information, a novel series of dihydrothiopyrano[4,3-d]pyrimidine derivatives were rationally designed via scaffold hopping and molecular hybridization strategies. Notably, compound 20a yielded exceptionally potent antiviral activities (EC50 = 4.44-54.5 nM) against various HIV-1 strains and improved resistance profiles (RF = 0.5-5.6) compared to etravirine and rilpivirine. Meanwhile, 20a exhibited reduced cytotoxicity (CC50 = 284 µM) and higher SI values (SI = 5210-63992). Molecular dynamics simulations were performed to rationalize the distinct resistance profiles. Besides, 20a displayed better solubility (sol. = 12.8 µg/mL) and no significant inhibition of the main CYP enzymes. Furthermore, 20a was characterized for prominent metabolic stability and in vivo safety properties. Most importantly, the hERG inhibition profile of 20a (IC50 = 19.84 µM) was a remarkable improvement. Overall, 20a possesses huge potential to serve as a promising drug candidate due to its excellent potency, low toxicity, and favorable drug-like properties.

A Chemoproteomics Approach to Determine the Mechanism of Testicular Toxicity for the Bruton's Tyrosine Kinase Inhibitor CC-292.

During drug development, potential safety issues can occur at any time. Understanding the cause of a toxicity can help with deciding on how to advance the drug development program. Chemoproteomics provides a way to help understand the cause of a toxicity wherein the affected tissue is accessible and can be probed with a covalently binding compound that is analogous to the offending drug. In this case, N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292), a covalently binding Bruton's tyrosine kinase inhibitor, had produced testicular toxicity in rodents. Experiments were conducted using a CC-292 analog that could be chemically modified with biotin to probe rodent testes homogenates for potential binding sites that were subsequently recovered with avidin beads. These biotin-tagged proteins undergo trypsin digest on the avidin beads to yield peptides that are identified using mass spectrometry. Two proteins were identified from the testicular homogenates of both rats and mice, namely retinal dehydrogenase 1 (ALDH1A1) and retinal dehydrogenase 2 (ALDH1A2). Literature confirmed a link between inhibition of these enzymes and testicular toxicity. Subsequently, molecular modeling was used to demonstrate that CC-292 can be docked into both the nicotinamide adenine dinucleotide and retinal binding pockets of the analogous human ALDH1A2 enzyme. These data suggest that the off-target binding site for CC-292 on retinal dehydrogenase enzymes may provide a mechanistic explanation to the testicular toxicity observed in rodents and that there may be a potential concern for human male fertility. SIGNIFICANCE STATEMENT: Biotinylated covalently binding drug analogues are used to enrich bound proteins from tissue homogenates wherein toxicity was observed in rodents. Bound proteins were subsequently identified by mass spectroscopy. Competition of the analog binding with the parent inhibitor itself and three-dimensional molecular modeling were used to establish a likely link between the off-targets of CC-292, ALDH1A1, and ALDH1A2 with potential testicular toxicity.

Discovery and In Vivo Anti-inflammatory Activity Evaluation of a Novel Non-peptidyl Non-covalent Cathepsin C Inhibitor.

Cathepsin C (Cat C) participates in inflammation and immune regulation by affecting the activation of neutrophil serine proteases (NSPs). Therefore, cathepsin C is an attractive target for treatment of NSP-related inflammatory diseases. Here, the complete discovery process of the first potent "non-peptidyl non-covalent cathepsin C inhibitor" was described with hit finding, structure optimization, and lead discovery. Starting with hit 14, structure-based optimization and structure-activity relationship study were comprehensively carried out, and lead compound 54 was discovered as a potent drug-like cathepsin C inhibitor both in vivo and in vitro. Also, compound 54 (with cathepsin C Enz IC50 = 57.4 nM) exhibited effective anti-inflammatory activity in an animal model of chronic obstructive pulmonary disease. These results confirmed that the non-peptidyl and non-covalent derivative could be used as an effective cathepsin C inhibitor and encouraged us to continue further drug discovery on the basis of this finding.

Structure activity relationships and the binding mode of quinolinone-pyrimidine hybrids as reversal agents of multidrug resistance mediated by P-gp.

P-gp-associated multidrug resistance is a major impediment to the success of chemotherapy. With the aim of finding non-toxic and effective P-gp inhibitors, we investigated a panel of quinolin-2-one-pyrimidine hybrids. Among the active compounds, two of them significantly increased intracellular doxorubicin and rhodamine 123 accumulation by inhibiting the efflux mediated by P-gp and restored doxorubicin toxicity at nanomolar range. Structure-activity relationships showed that the number of methoxy groups, an optimal length of the molecule in its extended conformation, and at least one flexible methylene group bridging the quinolinone to the moiety bearing the pyrimidine favored the inhibitory potency of P-gp. The best compounds showed a similar binding pattern and interactions to those of doxorubicin and tariquidar, as revealed by MD and hybrid QM/MM simulations performed with the recent experimental structure of P-gp co-crystallized with paclitaxel. Analysis of the molecular interactions stabilizing the different molecular complexes determined by MD and QTAIM showed that binding to key residues from TMH 4-7 and 12 is required for inhibition.

PPARα agonist WY-14,643 induces adipose atrophy and fails to blunt chronic ethanol-induced hepatic fat accumulation in mice lacking adipose FGFR1.

Fibroblast growth factor 21 (FGF21) is mainly regulated by peroxisome proliferator-activated receptor α (PPARα) in liver. The PPARα-FGF21 axis protects against alcohol-related liver disease (ALD). FGF21 exerts its effect via FGF receptor 1 (FGFR1). However, liver specific FGFR1 abrogation had no effect on ALD. Adipose tissues highly express FGFR1. When adipocyte specific FGFR1 knockout (fgfr1adipoQ-cre) mice and corresponding normal control (fgfr1fl/fl) mice were fed with Lieber-DeCarli ethanol liquid diet for 3 weeks, liver triglyceride (TG) accumulation was increased in the fgfr1fl/fl mice to a greater extent than in the fgfr1adipoQ-cre mice. When PPARα agonist WY-14,643 was added in the liquid ethanol diet at 10 mg/L, the ethanol-induced liver TG accumulation was blunted in the fgfr1fl/fl mice but not in the fgfr1adipoQ-cre mice. There was no significant difference in WY-14,643-induced fatty acid oxidation, ethanol metabolism, and oxidative stress between the fgfr1fl/fl and fgfr1adipoQ-cre mice. Interestingly, adipose atrophy was induced by WY-14,643 in the fgfr1adipoQ-cre mice but not in the fgfr1fl/fl mice. Serum free fatty acid was also decreased by WY-14,643 in the fgfr1adipoQ-cre mice but not in the fgfr1fl/fl mice. These results suggest that WY-14,643 inhibits alcoholic fatty liver and regulates adipose tissue mass and fat mobilization from adipose tissues to liver in an adipocyte FGFR1-dependent manner.