Characterization of a new selective glucocorticoid receptor modulator with anorexigenic activity.

Obesity, a worldwide epidemic, leads to various metabolic disorders threatening human health. In response to stress or fasting, glucocorticoid (GC) levels are elevated to promote food intake. This involves GC-induced expression of the orexigenic neuropeptides in agouti-related protein (AgRP) neurons of the hypothalamic arcuate nucleus (ARC) via the GC receptor (GR). Here, we report a selective GR modulator (SGRM) that suppresses GR-induced transcription of genes with non-classical glucocorticoid response elements (GREs) such as Agrp-GRE, but not with classical GREs, and via this way may serve as a novel anti-obesity agent. We have identified a novel SGRM, 2-O-trans-p-coumaroylalphitolic acid (Zj7), a triterpenoid extracted from the Ziziphus jujube plant, that selectively suppresses GR transcriptional activity in Agrp-GRE without affecting classical GREs. Zj7 reduces the expression of orexigenic genes in the ARC and exerts a significant anorexigenic effect with weight loss in both high fat diet-induced obese and genetically obese db/db mouse models. Transcriptome analysis showed that Zj7 represses the expression of a group of orexigenic genes including Agrp and Npy induced by the synthetic GR ligand dexamethasone (Dex) in the hypothalamus. Taken together, Zj7, as a selective GR modulator, showed beneficial metabolic activities, in part by suppressing GR activity in non-classical GREs in orexigenic genes. This study demonstrates that a potential anorexigenic molecule may allow GRE-specific inhibition of GR transcriptional activity, which is a promising approach for the treatment of metabolic disorders.

Targeting HMG-CoA synthase 2 suppresses tamoxifen-resistant breast cancer growth by augmenting mitochondrial oxidative stress-mediated cell death.

AIMS: In this study, we aimed to investigate previously unrecognized lipid metabolic perturbations in tamoxifen-resistant breast cancer (BC) by conducting comprehensive metabolomics and transcriptomics analysis. We identified the role of 3-hydroxy-3-methylglutary-coenzyme-A-synthase 2 (HMGCS2), a key enzyme responsible for ketogenesis, in tamoxifen-resistant BC growth. MAIN METHODS: Comprehensive metabolomics (CE-TOFMS, LC-TOFMS) and transcriptiomics analysis were performed to characterize metabolic pathways in tamoxifen-resistant BC cells. The upregulation of HMGCS2 were verified thorugh immunohistochemistry (IHC) in clinical samples obtained from patients with recurrent BC. HMGCS2 inhibitor was discovered through surface plasmon resonance analysis, enzyme assay, and additional molecular docking studies. The effect of HMGCS2 suppression on tumor growth was studied thorugh BC xenograft model, and intratumoral lipid metabolites were analyzed via MALDI-TOFMS imaging. KEY FINDINGS: We revealed that the level of HMGCS2 was highly elevated in both tamoxifen-resistant T47D sublines (T47D/TR) and clinical refractory tumor specimens from patients with ER+ breast cancer, who had been treated with adjuvant tamoxifen. Suppression of HMGCS2 in T47D/TR resulted in the accumulation of mitochondrial reactive oxygen species (mtROS) and apoptotic cell death. Further, we identified alphitolic acid, a triterpenoid natural product, as a novel HMGCS2-specific inhibitor that elevated mtROS levels and drastically retarded the growth of T47D/TR in in vitro and in vivo experiments. SIGNIFICANCE: Enhanced ketogenesis with upregulation of HMGCS2 is a potential metabolic vulnerability of tamoxifen-resistant BC that offers a new therapeutic opportunity for treating patients with ER+ BC that are refractory to tamoxifen treatment.

Pharmacological perturbation of thiamine metabolism sensitizes Pseudomonas aeruginosa to multiple antibacterial agents.

New therapeutic concepts are critically needed for carbapenem-resistant Pseudomonas aeruginosa, an opportunistic pathogen particularly recalcitrant to antibiotics. The screening of around 230,000 small molecules yielded a very low hit rate of 0.002% after triaging for known antibiotics. The only novel hit that stood out was the antimetabolite oxythiamine. Oxythiamine is a known transketolase inhibitor in eukaryotic cells, but its antibacterial potency has not been reported. Metabolic and transcriptomic analyses indicated that oxythiamine is intracellularly converted to oxythiamine pyrophosphate and subsequently inhibits several vitamin-B1-dependent enzymes, sensitizing the bacteria to several antibiotic and non-antibiotic drugs such as tetracyclines, 5-fluorouracil, and auranofin. The positive interaction between 5-fluorouracil and oxythiamine was confirmed in a murine ocular infection model, indicating relevance during infection. Together, this study revealed a system-level significance of thiamine metabolism perturbation that sensitizes P. aeruginosa to multiple small molecules, a property that could inform on the development of a rational drug combination.

The ThiL enzyme is a valid antibacterial target essential for both thiamine biosynthesis and salvage pathways in Pseudomonas aeruginosa.

Thiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. Thiamine biosynthesis occurs in bacteria but not in humans; therefore, the enzymes in this pathway are attractive targets for antibiotic development. Among these enzymes, thiamine monophosphate kinase (ThiL) catalyzes the final step of this pathway, phosphorylating thiamine monophosphate to produce TPP. Here, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen responsible for hospital-acquired infections. We demonstrate that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability and results in growth defects of the ΔthiL strain even in the presence of thiamine derivatives, except for TPP. Most importantly, the pathogenesis of the ΔthiL strain was markedly attenuated, compared with that of WT cells, with lower inflammatory cytokine induction and 103-104-fold decreased bacterial loads in an in vivo infection model in which the intracellular TPP level was in the submicromolar range. To validate P. aeruginosa ThiL (PaThiL) as a drug target, we further characterized its biochemical properties, determining a Vmax of 4.0 ± 0.2 nmol·min-1 and Km values of 111 ± 8 and 8.0 ± 3.5 µm for ATP and thiamine monophosphate, respectively. An in vitro small-molecule screening assay identified PaThiL inhibitors including WAY213613, a noncompetitive inhibitor with a Ki value of 13.4 ± 2.3 µm and potential antibacterial activity against P. aeruginosa These comprehensive biological and biochemical results indicate that PaThiL represents a potential drug target for the development of an augmented repertoire of antibiotics against P. aeruginosa.

Kinome-Wide RNA Interference Screening Identifies Mitogen-Activated Protein Kinases and Phosphatidylinositol Metabolism as Key Factors for Rabies Virus Infection.

Throughout the rabies virus (RABV) infectious cycle, host-virus interactions define its capacity to replicate, escape the immune response, and spread. As phosphorylation is a key regulatory mechanism involved in most cellular processes, kinases represent a target of choice to identify host factors required for viral replication. A kinase and phosphatase small interfering RNA (siRNA) high-content screening was performed on a fluorescent protein-recombinant field isolate (Tha RABV). We identified 57 high-confidence key host factors important for RABV replication with a readout set at 18 h postinfection and 73 with a readout set at 36 h postinfection, including 24 common factors at all stages of the infection. Amongst them, gene clusters of the most prominent pathways were determined. Up to 15 mitogen-activated protein kinases (MAPKs) and effectors, including MKK7 (associated with Jun N-terminal protein kinase [JNK] signalization) and DUSP5, as well as 17 phosphatidylinositol (PI)-related proteins, including PIP5K1C and MTM1, were found to be involved in the later stage of RABV infection. The importance of these pathways was further validated, as small molecules Ro 31-8820 and PD 198306 inhibited RABV replication in human neurons.IMPORTANCE Rabies virus relies on cellular machinery for its replication while simultaneously evading the host immune response. Despite their importance, little is known about the key host factors required for rabies virus infection. Here, we focused on the human kinome, at the core of many cellular pathways, to unveil a new understanding of the rabies virus infectious cycle and to discover new potential therapeutic targets in a small interfering RNA screening. The mitogen-activated protein kinase pathway and phosphatidylinositol metabolism were identified as prominent factors involved in rabies virus infection, and those findings were further confirmed in human neurons. While bringing a new insight into rabies virus biology, we also provide a new list of host factors involved in rabies virus infection.

High-throughput drug screening using the Ebola virus transcription- and replication-competent virus-like particle system.

The massive epidemic of Ebola virus disease (EVD) in West Africa, followed in recent months by two outbreaks in the Democratic Republic of the Congo, underline the importance of this severe disease. Because Ebola virus (EBOV) must be manipulated under biosafety level 4 (BSL4) containment, the discovery and development of virus-specific therapies have been hampered. Recently, a transient transfection-based transcription- and replication competent virus-like particle (trVLP) system was described, enabling modeling of the entire EBOV life cycle under BSL2 conditions. Using this system, we optimized the condition for bulk co-transfection of multiple plasmids, developed a luciferase reporter-based assay in 384-well microtiter plates, and performed a high-throughput screening (HTS) campaign of an 8,354-compound collection consisting of U.S. Food & Drug Administration (FDA) -approved drugs, bioactives, kinase inhibitors, and natural products in duplicates. The HTS achieved a good signal-to-background ratio with a low percent coefficient of variation resulting in Z' = 0.7, and data points were reproducible with R2 = 0.89, indicative of a robust assay. After applying stringent hit selection criteria of ≥70% EBOV trVLP inhibition and ≥70% cell viability, 381 hits were selected targeting early, entry, and replication steps and 49 hits targeting late, maturation, and secretion steps in the viral life cycle. Of the total 430 hits, 220 were confirmed by dose-response analysis in the primary HTS assay. They were subsequently triaged by time-of-addition assays, then clustered and ranked according to their chemical structures, biological functions, therapeutic index, and maximum inhibition. Several novel drugs have been identified to very efficiently inhibit EBOV. Interestingly, most showed pharmacological activity in treatments for central nervous system-related diseases. We developed and screened an HTS assay using the novel EBOV trVLP system. Newly identified inhibitors are useful tools to study the poorly understood EBOV life cycle. In addition, they also provide opportunities to either repurpose FDA-approved drugs or develop novel viral interventions to combat EVD.

Identification of pyrrolo[3,2-c]pyridin-4-amine compounds as a new class of entry inhibitors against influenza viruses in vitro.

Various influenza virus entry inhibitors are being developed as therapeutic antiviral agents in ongoing preparation for emerging influenza viruses, particularly those that may possess drug resistance to the current FDA-approved neuraminidase inhibitors. In this study, small molecules having the pyrrolopyridinamine (PPA), aminothiadiazole (ATD), dihydrofuropyridine carboxamide (HPC), or imidazopyridinamine (IPA) moiety were selected from a target-focused chemical library for their inhibitory activity against influenza A virus by high-throughput screening using the PR8GFP assay. Activity was evaluated by measuring changes the proportion of GFP-expressing cells as a reflection of influenza virus infection. Among them, PPA showed broad-spectrum activity against multiple influenza A viruses and influenza B virus. PPA was found to block the early stages of influenza virus infection using a time-of-addition assay. Using additional phenotypic assays that dissect the virus entry process, it appears that the antiviral activity of PPA against influenza virus can be attributed to interference of the post-fusion process: namely, virus uncoating and nuclear import of viral nucleoprotein complexes. Based on these results, PPA is an attractive chemical moiety that can be used to develop new antiviral drug candidates against influenza viruses.

Benzothiazepinecarboxamides: Novel hepatitis C virus inhibitors that interfere with viral entry and the generation of infectious virions.

Upon screening synthetic small molecule libraries with the infectious hepatitis C virus (HCV) cell culture system, we identified a benzothiazepinecarboxamide (BTC) scaffold that inhibits HCV. A structure-activity relationship (SAR) study with BTCs was performed, and modifications that led to nanomolar antiviral activity and improved the selective index (CC50/EC50) by more than 1000-fold were identified. In addition, a pharmacophore modeling study determined that the tricyclic core and positive charge on the piperidine moiety were essential for antiviral activity. Furthermore, we demonstrated that BTC interferes with HCV glycoprotein E1/E2-mediated viral entry and the generation of infectious virions by using HCV pseudoparticle and cell culture supernatant transfer assays, respectively. BTC showed potent antiviral activity against HCV genotype 2 (EC50 = 0.01 ± 0.01 µM), but was less potent against a genotype 1/2 chimeric virus (EC50 = 2.71 ± 0.05 µM), which expressed the structural proteins of HCV genotype 1. In summary, we identified, optimized, and characterized novel BTC inhibitors that interfere with early and late steps of the HCV viral life cycle.

Serendipitous discovery of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine derivatives as novel HIV-1 replication inhibitors.

We identified a novel class of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds as potent HIV-1 replication inhibitors serendipitously during the process of evaluation of triazolothienopyrimidine (TTPM) compounds. Herein, we report synthesis and biological evaluation of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds using a cell-based full replication assay to identify thienopyrimidines 6 and 30, which could be further utilized as viable lead compounds.

Lead optimization of a novel series of imidazo[1,2-a]pyridine amides leading to a clinical candidate (Q203) as a multi- and extensively-drug-resistant anti-tuberculosis agent.

A critical unmet clinical need to combat the global tuberculosis epidemic is the development of potent agents capable of reducing the time of multi-drug-resistant (MDR) and extensively-drug-resistant (XDR) tuberculosis therapy. In this paper, we report on the optimization of imidazo[1,2-a]pyridine amide (IPA) lead compound 1, which led to the design and synthesis of Q203 (50). We found that the amide linker with IPA core is very important for activity against Mycobacterium tuberculosis H37Rv. Linearity and lipophilicity of the amine part in the IPA series play a critical role in improving in vitro and in vivo efficacy and pharmacokinetic profile. The optimized IPAs 49 and 50 showed not only excellent oral bioavailability (80.2% and 90.7%, respectively) with high exposure of the area under curve (AUC) but also displayed significant colony-forming unit (CFU) reduction (1.52 and 3.13 log10 reduction at 10 mg/kg dosing level, respectively) in mouse lung.

Anti-obesity phenotypic screening looking to increase OBR cell surface expression.

The leptin receptor, OBR, is involved in the regulation of whole-body energy homeostasis. Most obese people are resistant to leptin and do not respond to the hormone. The prevention and reversal of leptin resistance is one of the major current goals of obesity research. We showed previously that increased OBR cell surface expression concomitantly increases cellular leptin signaling and prevents obesity development in mice. Improvement of OBR cell surface expression can thus be considered as an interesting anti-obesity therapeutic strategy. To identify compounds that increase the surface expression of OBR, we developed a cell-based, phenotypic assay to perform a high-content screen (HCS) against a library of 50,000 chemical compounds. We identified 67 compounds that increased OBR cell surface expression with AC50 values in the low micromolar range and no effect on total OBR expression and cellular toxicity. Compounds were classified into 16 chemical clusters, of which 4 potentiated leptin-promoted signaling through the JAK2/STAT3 pathway. In conclusion, development of a robust phenotypic screening approach resulted in the discovery of four new scaffolds that demonstrate the desired biological activity and could constitute an original therapeutic solution against obesity and associated disorders.

Synthesis and evaluation of hexahydropyrimidines and diamines as novel hepatitis C virus inhibitors.

In order to identify novel anti-hepatitis C virus (HCV) agents we devised cell-based strategies and screened phenotypically small molecule chemical libraries with infectious HCV particles, and identified a hit compound (1) containing a hexahydropyrimidine (HHP) core. During our cell-based SAR study, we observed a conversion of HHP 1 into a linear diamine (6), which is the active component in inhibiting HCV and exhibited comparable antiviral activity to the cyclic HHP 1. In addition, we engaged into the biological characterization of HHP and demonstrated that HHP does not interfere with HCV RNA replication, but with entry and release of viral particles. Here we report the results of the preliminary SAR and mechanism of action studies with HHP.

Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis.

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.

Identification of a novel sulfonamide non-nucleoside reverse transcriptase inhibitor by a phenotypic HIV-1 full replication assay.

Classical target-based, high-throughput screening has been useful for the identification of inhibitors for known molecular mechanisms involved in the HIV life cycle. In this study, the development of a cell-based assay that uses a phenotypic drug discovery approach based on automated high-content screening is described. Using this screening approach, the antiviral activity of 26,500 small molecules from a relevant chemical scaffold library was evaluated. Among the selected hits, one sulfonamide compound showed strong anti-HIV activity against wild-type and clinically relevant multidrug resistant HIV strains. The biochemical inhibition, point resistance mutations and the activity of structural analogs allowed us to understand the mode of action and propose a binding model for this compound with HIV-1 reverse transcriptase.

Synthesis and biological evaluation of triazolothienopyrimidine derivatives as novel HIV-1 replication inhibitors.

We identified a novel class of triazolothienopyrimidine (TTPM) compounds as potent HIV-1 replication inhibitors during a high-throughput screening campaign that evaluated more than 200,000 compounds using a cell-based full replication assay. Herein, we report the optimization of the antiviral activity in a cell-based assay system leading to the discovery of aryl-substituted TTPM derivatives (38, 44, and 45), which exhibited significant inhibition of HIV-1 replication with acceptable safety margins. These novel and potent TTPMs could serve as leads for further development.

A novel 3,4-dihydropyrimidin-2(1H)-one: HIV-1 replication inhibitors with improved metabolic stability.

Following the previous SAR of a novel dihydropyrimidinone scaffold as HIV-1 replication inhibitors a detailed study directed towards optimizing the metabolic stability of the ester functional group in the dihydropyrimidinone (DHPM) scaffold is described. Replacement of the ester moiety by thiazole ring significantly improved the metabolic stability while retaining antiviral activity against HIV-1 replication. These novel and potent DHPMs with bioisosteres could serve as advanced leads for further optimization.

Discovery of Phenylaminopyridine Derivatives as Novel HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors.

We identified a novel class of aryl-substituted triazine compounds as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) during a high-throughput screening campaign that evaluated more than 200000 compounds for antihuman immunodeficiency virus (HIV) activity using a cell-based full replication assay. Herein, we disclose the optimization of the antiviral activity in a cell-based assay system leading to the discovery of compound 27, which possessed excellent potency against wild-type HIV-1 (EC50 = 0.2 nM) as well as viruses bearing Y181C and K103N resistance mutations in the reverse transcriptase gene. The X-ray crystal structure of compound 27 complexed with wild-type reverse transcriptase confirmed the mode of action of this novel class of NNRTIs. Introduction of a chloro functional group in the pyrazole moiety dramatically improved hERG and CYP inhibition profiles, yielding highly promising leads for further development.

Identification of (beta-carboxyethyl)-rhodanine derivatives exhibiting peroxisome proliferator-activated receptor gamma activity.

We applied an improved virtual screening scheme combining ligand-centric and receptor-centric methods for the identification of a new series of PPARgamma agonists known as (beta-carboxyethyl)-rhodanine derivatives which include a thiazolidin-based core structure, 2-thioxo-thiazolidine-4-one. An in vitro assay confirmed the nanomolar binding affinity in one of the (beta-carboxyethyl)-rhodanine derivatives, SP1818. It showed a PPARgamma agonistic activity similar to that of a known PPARgamma drug, pioglitazone, in a cell-based transactivation assay. Furthermore, the structure-activity relationships of the rhodanine derivatives were investigated through comparative molecular field analysis. We also characterized the inconsistency between the in vitro binding affinity and cell-based transactivation ability by using a set of property-based molecular descriptors. The binding mode analysis provided new insight concerning their agonistic effect on PPARgamma.

Cyanobacterial glucokinase complements the glucose sensing role of Arabidopsis thaliana hexokinase 1.

Unlike Arabidopsis hexokinase (AtHXK) 1, cyanobacterial glucokinase (cGlk, Sll0593) from Synechocystis sp. PCC6803 does not function endogenously as a glucose sensor for glucose repression of photosynthesis-related genes such as psbA2, psbD2, rbcS, and rbcL. However, when cGlk was constitutively expressed in the cytosol of the glucose insensitive AtHXK 1 null mutant gin2-1, transgenic plants showed glucose sensitive phenotypes similar to those of wild type plants, namely glucose-induced decreases in Chl content and transcript levels of genes encoding Chl binding proteins (CAB1) and Rubisco small subunit (RBCS). Therefore, we suggest that cGlk's ability to complement glucose sensing activity in higher plants is attributable to the presence of cGlk-interacting proteins present in Arabidopsis, but absent in Synechocystis.