Activity In Cell-based Assays Research Articles

Modular synthesis of cell-permeating 2-ketoglutarate esters.

Cell-permeating esters of 2-ketoglutarate (2-KG) have been synthesized through a convergent sequence from two modules in two and three steps, respectively. This route provides access to a full series of mono- and disubstituted 2-KG esters, enabling us to define the effect of regioisomeric masking on metabolite release and antihypoxic activity in cell-based assays. In addition to providing insight into the biological activity of cell permeable 2-KG esters, the straightforward and modular nature of this synthetic route may prove useful for the development of next-generation 2-KG analogues for diagnostic and therapeutic applications.

Fighting malaria: structure-guided discovery of nonpeptidomimetic plasmepsin inhibitors.

Plasmepsins (Plms) are aspartic proteases involved in the degradation of human hemoglobin by Plasmodium falciparum. Given that the parasite needs the resulting amino acid building blocks for its growth and development, plasmepsins are an important antimalarial drug target. Over the past decade, tremendous progress has been achieved in the development of inhibitors of plasmepsin using two strategies: structure-based drug design (SBDD) and structure-based virtual screening (SBVS). Herein, we review the inhibitors of Plms I-IV developed by SBDD or SBVS with a particular focus on obtaining selectivity versus the human Asp proteases cathepsins and renin and activity in cell-based assays. By use of SBDD, the flap pocket of Plm II has been discovered and constitutes a convenient handle to obtain selectivity. In SBVS, activity against Plms I-IV and selectivity versus cathepsins are not always taken into account. A combination of SBVS, SBDD, and molecular dynamics simulations opens up opportunities for future design cycles.

USP9X inhibition promotes radiation-induced apoptosis in non-small cell lung cancer cells expressing mid-to-high MCL1

Background and Purpose: Radiotherapy (RT) is vital for the treatment of locally advanced non-small cell lung cancer (NSCLC), yet its delivery is limited by tolerances of adjacent organs. We sought therefore to identify and characterize gene targets whose inhibition may improve RT. Materials and Methods: Whole genome pooled shRNA cytotoxicity screens were performed in A549 and NCI-H460 using a retroviral library of 74,705 sequences. Cells were propagated with or without daily radiation Monday–Friday. Radiosensitization by top differential dropout hits was assessed by clonogenic assays. Apoptosis was assessed using a caspase 3/7 cell-based activity assay and by annexin V-FITC and PI staining. MCL1 expression was assessed by qPCR and Western blotting. Results: USP9X, a deubiquitinase, was a top hit among druggable gene products. WP1130, a small molecule USP9X inhibitor, showed synergistic cytotoxicity with IR. MCL1, an anti-apoptotic protein deubiquitinated by USP9X, decreased with USP9X inhibition and IR. This was accompanied by increases in caspase 3/7 activity and apoptosis. In a panel of NSCLC lines, MCL1 and USP9X protein and gene expression levels were highly correlated. Lines showing high levels of MCL1 expression were the most sensitive to USP9X inhibition. Conclusions: These data support the use of MCL1 expression as a predictive biomarker for USP9X inhibitors in NSCLC therapy.

Structural basis for PPARγ transactivation by endocrine-disrupting organotin compounds.

Organotin compounds such as triphenyltin (TPT) and tributyltin (TBT) act as endocrine disruptors through the peroxisome proliferator–activated receptor γ (PPARγ) signaling pathway. We recently found that TPT is a particularly strong agonist of PPARγ. To elucidate the mechanism underlying organotin-dependent PPARγ activation, we here analyzed the interactions of PPARγ ligand-binding domain (LBD) with TPT and TBT by using X-ray crystallography and mass spectroscopy in conjunction with cell-based activity assays. Crystal structures of PPARγ-LBD/TBT and PPARγ-LBD/TPT complexes were determined at 1.95 Å and 1.89 Å, respectively. Specific binding of organotins is achieved through non-covalent ionic interactions between the sulfur atom of Cys285 and the tin atom. Comparisons of the determined structures suggest that the strong activity of TPT arises through interactions with helix 12 of LBD primarily via π-π interactions. Our findings elucidate the structural basis of PPARγ activation by TPT.

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.

Synthesis and biological evaluation of conformationally restricted adenine bicycloribonucleosides.

We prepared a novel series of conformationally restricted bicyclonucleosides and nucleotides. The synthetic approach employed a ring closing metathesis to provide access to both 6 and 7 membered saturated and unsaturated rings linking the 3' to 5' methylene groups of the sugar. The bicyclonucleosides were also transformed to the corresponding phosphoramidate prodrugs by an innovative one-pot protocol of boronate ester protection, coupling of the phosphoryl chloridate and deprotection of the boronate. A similar strategy was also employed for the synthesis of the corresponding monophosphates as crucial intermediates for the synthesis of selected triphosphates. The biological properties of the nucleosides and monophosphate prodrugs were assessed for antiviral and cytostatic activities in cell based assays whilst the triphosphates were evaluated in enzymatic assays. The lack of significant effects suggests that the linkage of the 3' to 5'via a ring system and the subsequent conformational restriction of the ribose ring to the South conformation are incompatible with the kinases and polymerases that recognize nucleosides and their metabolites.

The effect of quercetin and kaempferol aglycones and glucuronides on peroxisome proliferator-activated receptor-gamma (PPAR-γ).

The consumption of dietary flavonoids has been associated with a variety of health benefits, including effects mediated by the activation of peroxisome proliferator-activated receptor-gamma (PPAR-γ). Flavonoids are extensively metabolized during and after uptake and there is little known on the biological effects of these conjugated metabolites of flavonoids that are found in plasma. To investigate the effect of glucuronidation on the ability of flavonoids to activate PPAR-γ we studied and compared the activity of quercetin, kaempferol and their relevant plasma conjugates quercetin-3-O-glucuronide (Q3G) and kaempferol-3-O-glucuronide (K3G) on different PPAR-γ related endpoints. The flavonoid aglycones increased PPAR-γ mediated gene expression in a stably transfected reporter gene cell line and glucuronidation diminished their effect. To study the intrinsic activity of the test compounds to activate PPAR-γ we used a novel microarray technique to study ligand induced ligand binding domain (LBD) - nuclear receptor coregulator interactions. In this cell-free system we demonstrate that, unlike the known PPAR-γ agonist rosiglitazone, neither the flavonoid aglycones nor the conjugates are agonistic ligands of the receptor. The increases in reporter gene expression in the reporter cells were accompanied by increased PPAR-γ receptor-mRNA expression and quercetin synergistically increased the effect of rosiglitazone in the reporter gene assay. It is concluded that flavonoids affect PPAR-γ mediated gene transcription by a mode of action different from agonist binding. Increases in PPAR-γ receptor mRNA expression and synergistic effects with endogenous PPAR-γ agonists may play a role in this alternative mode of action. Glucuronidation reduced the activity of the flavonoid aglycones.

Evaluation of Cellular Antioxidant and Antiproliferative Activities of Five Main Phyllanthus Emblica L. Cultivars in China.

The cell-based antioxidant activity assay as more biological relevant assay was considered to be more accurate to predict antioxidant activity in vivo than chemical activity assays. In the present study, the five main Phyllanthus emblica L. cultivars in China were subjected for cellular antioxidant activity based on HepG2 cells as well as antiproliferative activity. Total phenolics, total flavonoids and oxygen radical absorbance capacity were also measured. The results showed that Qingyougan, Binggan and Boligan (832±100, 774±52 and 704±28 μmol of quercetin equivalents/100 g) had higher cellular antioxidant activity than Tianyougan and Yougan (553±50 and 457±24 μmol of quercetin equivalents/100 g) in phosphate buffered saline wash protocol whereas, Boligan (3735±217 μmol of quercetin equivalents/100 g) had the highest cellular antioxidant activity and Tianyougan (2025±171 μmol of quercetin equivalents/100 g) had the lowest cellular antioxidant activity in no phosphate buffered saline wash protocol. The highest and lowest antiproliferative activities were observed in Binggan and Tianyougan (median effective dose: 6.95±0.11 and 14.03±0.10 mg/ml), respectively. The significant correlation was only observed between total flavonoids and cellular antioxidant activity from no phosphate buffered saline wash protocol (R(2) =0.908, P<0.05), and total flavonoids and antiproliferative activity (R(2) =0.887, P<0.05), suggesting the major contribution of flavonoids to the bioactivities of emblica. Overall, the data obtained revealed that different Phyllanthus emblica L. cultivars had strong cellular antioxidant and antiproliferative activities, thus should be recommended to increase consumption for health.

Evaluation of the cellular antioxidant activity of 25 commonly consumed fruit species in China based on Caco-2 cells

Introduction. Determination of antioxidant activity using a biological method is important in predicting antioxidant capacity in vivo . The Caco-2 cell-based antioxidant activity (CAA) assay quantifies antioxidant activity in Caco-2 cells and has proved to possess good biological relevance. The aim of this study was to determine the CAA of 25 fruits commonly consumed in China, along with total phenolic content, total flavonoid content and oxygen radical absorbance capacity (ORAC). Materials and methods. The CAA was determined in Caco-2 cells by utilizing 2’,7’-dichlorofluorescin diacetate (DCFH-DA) as a probe and 2,2’-azobis(2-methylpropionamidine) dihydrochloride (AAPH) as the generator of peroxyl radicals. The ORAC values were measured using fluorescein and AAPH. The total phenolic content was determined by the Folin-Ciocalteu method and total flavonoid content by aluminum chloride-sodium nitrite method. Results and discussion. There was a large variation in CAA values among different fruit species with the highest value in pomegranate (96.3 ± 5.6 ¯mol quercetin equivalents (QE) 100 g-1 ) and the lowest value in papaya (0.453 ± 0.031 ¯mol QE 100 g-1 ). For melons (white muskmelon, hami melon and watermelon), pineapple, pitaya, nectarine and loquat, the CAA was too low to be measured. Data analysis showed that CAA values were significantly correlated to total phenolic content (R 2 = 0.159, P = 0.003, P > 0.05) or ORAC values (R 2 = 0.123, P > 0.05). Conclusion. Increasing the consumption of fruits, especially pomegranate, is an effective strategy to increase antioxidant intake, thus helpful for consumers to protect against the oxidative stressors.

A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants

A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2′,7′-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5×104/well, cell culture time of 24h, co-incubation of 60μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20min and fluorescence recorded for 90min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R2=0.815, p<0.01), demonstrating the good biological relevance of CAA assay.

Aromatase deficiency in a Chinese adult man caused by novel compound heterozygous CYP19A1 mutations: Effects of estrogen replacement therapy on the bone, lipid, liver and glucose metabolism

ObjectivesAromatase deficiency is a rare disorder resulting in estrogen insufficiency in humans. It has been reported in remarkably few men with loss-of-function mutations in the CYP19A1 gene encoding the aromatase, a cytochrome P450 enzyme that plays a crucial role in the biosynthesis of estrogens from androgens. We investigated a non-consanguineous family including an adult man with clinical features of aromatase deficiency, and studied the effects of estrogen replacement in the man. MethodsWe investigated the clinical and biochemical phenotype, performed CYP19A1 mutational analysis in the family and 50 unrelated persons, studied the effects of CYP19A1 mutations on aromatase protein structure, functionally characterized the mutations by cell-based aromatase activity assays, and studied the effects of estrogen replacement on the bone, lipid, liver and glucose metabolism. ResultsThe man with clinical features of aromatase deficiency had novel compound heterozygous CYP19A1 mutations (Y81C and L451P) that were not found in 50 unrelated persons. Three-dimensional modeling predicted that Y81C and L451P mutants disrupted protein structure. Functional studies on the basis of in vitro expression showed that Y81C and L45P mutants significantly decreased the aromatase activity and catalytic efficiency. Estrogen replacement in the man increased bone mineral density, accelerated bone maturation, improved lipid profile and liver steatosis, and improved glucose levels but not insulin resistance. ConclusionsWe have identified two novel CYP19A1 missense mutations in an aromatase-deficient man. Estrogen replacement in the man shows great impact on recovering the impairments in the bone, lipid, liver and glucose metabolism, but fails to improve insulin resistance.

Abstract 182: SPARC as a regulator of collagen signaling in pancreatic cancer

Abstract The extracellular matrix (ECM) is a principal component of pancreatic ductal adenocarcinoma (PDA), which is rich in fibrillar collagens, and provides structural support that facilitates tumor cell survival and chemoresistance. Collagen expression and deposition is a complex process that is orchestrated in part by the matricellular protein SPARC. SPARC expression in human patients with PDA correlates with improved chemoresponse; however, this mechanism is unclear. We propose that SPARC controls collagen binding to the cell surface and reduces collagen-mediated signaling via Discoidin domain receptors (DDR1, DDR2). DDR1/DDR2 are homologous receptor tyrosine kinases that bind specifically to fibrillar collagens and have been implicated regulating cell proliferation, migration, adhesion, ECM remodeling, and response to growth factors. Additionally, collagen has been shown to promote chemoresistance in pancreatic tumor cells. We hypothesize that SPARC promotes chemoresponse by reducing collagen-mediated DDR signaling. Structural studies identified that SPARC and DDRs share the same collagen binding site. We have demonstrated that SPARC inhibits collagen binding to DDR1/DDR2 via in vitro binding assays and cell based activity assays. To determine the functional relevance of SPARC expression and collagen-mediated DDR activation in PDA, Sparc-null (Sparc-/-) mice were crossed with KIC (LSL-KrasG12D; Cdkn2alox/lox; p48Cre/+) mice. Survival was reduced and tumors were more aggressive in Sparc-/- KIC along with an elevation in DDR-mediated signaling in the tumor microenvironment. Primary PDA cell lines isolated from Sparc+/+ and Sparc-/- KIC animals were used to probe collagen signaling cascades revealing that collagen activated Ddr1 and downstream intermediates including protein tyrosine kinase 2 (Pyk2) and pseudopodium-enriched atypical kinase 1 (Peak1) in Sparc-/- cell lines. Furthermore, recombinant SPARC or neutralizing antibodies to Ddr1 abrogated collagen induced Ddr1 signaling in vitro implicating Ddr1 as a putative therapeutic target in PDA. Pharmacologic inhibition of Ddr1 was evaluated with nilotinib, a BCR-Abl inhibitor that shows high activity against Ddr1, and 7rh, a novel inhibitor with high specificity for Ddr1. In vitro nilotinib and 7rh suppress collagen-induced Ddr1 signaling, cell migration, and colony formation. In vivo nilotinib reduced the growth of implanted pancreatic tumors in Sparc-/- animals with little effect on tumor growth in Sparc+/+ mice. Studies with 7rh in vivo demonstrate an inhibition of Ddr1 signaling in syngeneic pancreatic tumors and is well tolerated at doses up to 30 mg/kg given daily via oral gavage. Therapy studies combining standard chemotherapy (gemcitabine + Nab-paclitaxel) with 7rh are ongoing. Our results suggest that Ddr1 enhances tumor cell survival and tumor progression and that Sparc functions to reduce PDA progression by blunting collagen-induced DDR signaling. Citation Format: Kristina Y. Aguilera, Courtney D. Goldstein, Lee B. Rivera, Amy D. Bradshaw, Ke Ding, Rolf A. Brekken. SPARC as a regulator of collagen signaling in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 182. doi:10.1158/1538-7445.AM2014-182

Abstract 841: Therapeutic targeting of cancer cells in the hypoxic microenvironment using an orally bioavailable small molecule inhibitor of carbonic anhydrase IX

Abstract Carbonic anhydrase IX (CAIX) is a membrane-bound, hypoxia-inducible enzyme that is highly expressed in many types of solid tumors, is an independent marker of poor prognosis and functions as a critical component of the pH regulatory machinery required by hypoxic cancer cells for survival, invasion and metastasis. These attributes make CAIX an attractive therapeutic target for cancer therapy. The development of pharmacologic inhibitors that selectively target tumor-associated, extracellular carbonic anhydrases (CAs) without “off-target” inhibition of cytosolic isoforms is critical for their use as cancer therapeutics. Here, we characterize an orally bioavailable, highly selective small molecule inhibitor of CAIX and CAXII, SLC-0111, and investigate its efficacy when used as a monotherapy and in combination with conventional chemotherapy in vivo. SLC-0111 exhibited a favorable in vitro ADME profile, including IC50 values of &amp;gt;100 mM against most CYP isoforms, an IC50 for hERG inhibition of &amp;gt;30 mM and absence of mutagenic properties. Incubation of the compound with 67NR cells constitutively expressing human CAIX significantly suppressed, in a dose-dependent fashion, a drop in extracellular pH in a cell-based CAIX activity assay. The active compound was formulated as a self-emulsifying liquid for oral administration in vivo and evaluation of plasma levels showed good oral bioavailability of 40%. Toxicity studies involving repeat dosing by oral gavage for 7 days established maximum-tolerated doses in excess of 1,000 and 750 mg/kg for rats and dogs, respectively. Daily oral administration of SLC-0111 to mice harboring established MDA-MB-231 LM2-4 orthotopic human breast tumors, a model of hypoxic, CAIX-positive triple negative metastatic breast cancer, resulted in a significant, dose-dependent reduction in tumor growth, with 30% inhibition at a dose of 50 mg/kg, no toxicity and a 10-fold therapeutic window. Treatment also reduced viable tumor volume as determined by 18F-deoxyglucose uptake detected by positron emission tomography. Furthermore, treatment of mice with SLC-0111 in combination with paclitaxel resulted in significantly reduced tumor growth compared to either treatment administered alone, with no additional toxicity and plasma levels of SLC-0111 similar to those observed with its use as a monotherapy. Plasma levels of SLC-0111 showed dose proportionality and analysis of tumor levels demonstrated a greater than proportional relationship, with a 4-fold increase in dose (25-100 mg/kg) resulting in more than a 13-fold increase in tumor levels of the compound. Collectively, these data demonstrate the potential utility of SLC-0111 as a safe, target-specific inhibitor of CAIX for the treatment of cancer, particularly in combination with conventional chemotherapy. Citation Format: Paul C. McDonald, Jasbinder Sanghera, Madhu Singh, Yuanmei Lou, Marylou Vallejo, Claudiu T. Supuran, Shoukat Dedhar. Therapeutic targeting of cancer cells in the hypoxic microenvironment using an orally bioavailable small molecule inhibitor of carbonic anhydrase IX. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 841. doi:10.1158/1538-7445.AM2014-841

M08 Laquinimod Reduces Neuronal Caspase-6 Activation And Axonal Degeneration In Vitro

<h3>Background</h3> Laquinimod is an immunomodulatory compound that reduces relapse rate, brain atrophy and disability progression in multiple sclerosis. It has well-documented effects on inflammation, is widely distributed in the CNS and has been shown to ameliorate axonal damage <i>in vitro</i>and <i>in vivo</i>through an unknown mechanism. We have shown recently that caspase-6 is an important mediator in axonal degeneration, since sympathetic neurons derived from caspase-6 -/- mice do not degenerate when cultured in the absence of neuronal growth factor (NGF). We therefore investigated whether the beneficial effect of Laquinimod on axonal damage is mediated by caspase-6. <h3>Aim</h3> To investigate neuron-specific beneficial effects of Laquinimod. <h3>Methods</h3> Cell-free and cell-based enzymatic activity assays were used to determine whether Laquinimod directly inhibits caspase-6. To investigate an effect on intraneuronal caspase-6 activity, primary cortical neurons were treated with camptothecin in the presence or absence of Laquinimod, and cleavage of the caspase-6 specific substrate lamin A was quantified by ELISA. Axonal degeneration of primary sympathetic neurons from the superior cerebral ganglion was induced by NGF withdrawal in the presence or absence of Laquinimod. <h3>Results</h3> Laquinimod did not directly inhibit caspase-6 activity in cell-free or transfection-based cellular systems. However, the presence of Laquinimod in camptothecin-stressed primary cortical neuron cultures led to a significant decrease in caspase-6 activation. In cultures of primary superior cerebral ganglion neurons, Laquinimod partially protected axons from degeneration after NGF withdrawal, a process that is specifically dependent on caspase-6 activity, in agreement with the reduction of caspase-6 activity observed in stressed cortical neurons. <h3>Conclusions</h3> The beneficial effects of Laquinimod described so far involve neuroprotection through the downregulation of glial activation, whereas our findings represent a novel, purely neuronal mechanism of action for this drug. We propose that by preventing neuronal caspase-6 activation and axonal degeneration, Laquinimod might also provide benefits in other neurodegenerative disorders such as Huntington’s disease that are associated with excessive activation of caspase-6.

Native Hexameric Full-Length HIV-1 Capsid: Crystal Structure and Drug Targeting

The HIV-1 full length capsid protein (CA-FL) is increasingly viewed as an attractive therapeutic target since proper capsid formation is required for viral infection. CA-FL is synthesized as a central domain of a structural Gag polyprotein that is involved in both early and late stages of the viral life cycle. During the HIV-1 maturation process, Gag is cleaved by a viral protease to produce several discrete new proteins that include matrix, capsid (CA-FL), and nucleocapsid. After proteolytic cleavage, CA-FL forms hexamers and pentamers that rearrange into a fullerene cone-shaped structure, which surrounds the viral genome at the center of the mature virus. Crystal structures of the native unassembled hexameric CA-FL (without cross-linked residues that might prevent changes in the inter- or intra-subunit interactions) are of great interest, as they may provide insights relevant to the development of drugs that prevent or impede the transition from the preassembled to the assembled capsid states. Recently, we crystallized and solved the crystal structure of the first hexameric HIV-1 CA-FL in its native form (without engineered cross-linking cysteines). There is one molecule per asymmetric unit, and the P6 space group generates the native hexameric assembly. We have also identified a small molecule, 18E8, which exhibits broad anti-HIV activity in cell-based assays, and targets CA-FL. This was demonstrated by experiments that selected for viruses with drug resistance and revealed that an A105T mutation in CA-FL confers resistance to the compound. Time-of-inhibitor addition experiments showed that 18E8 targets an early step in the HIV replication cycle, after reverse transcription and before integration. Electron microscopy experiments suggest that 18E8 does not impart significant morphological changes in CA-FL tubular assemblies. Our structure of CA-FL and our ongoing work with the CA-FL/18E8 complex will provide a system for the investigation of molecular interactions between CA-FL and small molecule antivirals that work with a novel mechanism of action.

Design and discovery of 5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide inhibitors of HIV-1 integrase

Raltegravir (RAL) is a first clinically approved integrase (IN) inhibitor for the treatment of HIV but rapid mutation of the virus has led to chemo-resistant strains. Therefore, there is a medical need to develop new IN inhibitors to overcome drug resistance. At present, several IN inhibitors are in different phases of clinical trials and few have been discontinued due to toxicity and lack of efficacy. The development of potent second-generation IN inhibitors with improved safety profiles is key for selecting new clinical candidates. In this article, we report the design and synthesis of potent 5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide analogues as second-generation IN inhibitors. These compounds satisfy two structural requirements known for potent inhibition of HIV-1 IN catalysis: a metal chelating moiety and a hydrophobic functionality necessary for selectivity against the strand transfer reaction. Most of the new compounds described herein are potent and selective for the strand transfer reaction and show antiviral activity in cell-based assays. Furthermore, this class of compounds are drug-like and suitable for further optimization and preclinical studies.

Delivery of Suramin as an Antiviral Agent through Liposomal Systems

Norovirus RNA-dependent RNA polymerase (RdRp) is a promising target enzyme for the development of new antiviral drugs. Starting from the crystal structure of norovirus RdRp, we had previously performed an in silico docking search using a library of low-molecular-weight compounds that enabled us to select molecules with predicted enzyme inhibitory activity. Among these, the polysulfonated naphthylurea suramin proved to inhibit in vitro both murine and human norovirus polymerases, with IC50 values in the low micromolar range. The negatively charged inhibitor, however, displayed poor cell permeability in cell-based experiments. Therefore, we produced different suramin-loaded liposome formulations and evaluated their activities in cell-based assays using murine norovirus cultivated in RAW 264.7 macrophages, as a model for norovirus genus. The results obtained show that suramin, when delivered through liposomes, can effectively inhibit murine norovirus replication.

Individual letters of the RNA polymerase II CTD code govern distinct gene expression programs in fission yeast

The primary structure and phosphorylation pattern of the tandem Y(1)S(2)P(3)T(4)S(5)P(6)S(7) repeats of the RNA polymerase II carboxyl-terminal domain (CTD) comprise an informational code that coordinates transcription, chromatin modification, and RNA processing. To gauge the contributions of individual CTD coding "letters" to gene expression, we analyzed the poly(A)(+) transcriptomes of fission yeast mutants that lack each of the four inessential CTD phosphoacceptors: Tyr1, Ser2, Thr4, and Ser7. There was a hierarchy of CTD mutational effects with respect to the number of dysregulated protein-coding RNAs, with S2A (n = 227) >> Y1F (n = 71) > S7A (n = 58) >> T4A (n = 7). The majority of the protein-coding RNAs affected in Y1F cells were coordinately affected by S2A, suggesting that Tyr1-Ser2 constitutes a two-letter code "word." Y1F and S2A elicited increased expression of genes encoding proteins involved in iron uptake (Frp1, Fip1, Fio1, Str3, Str1, Sib1), without affecting the expression of the genes that repress the iron regulon, implying that Tyr1-Ser2 transduces a repressive signal. Y1F and S2A cells had increased levels of ferric reductase activity and were hypersensitive to phleomycin, indicative of elevated intracellular iron. The T4A and S7A mutations had opposing effects on the phosphate response pathway. T4A reduced the expression of two genes encoding proteins involved in phosphate acquisition (the Pho1 acid phosphatase and the phosphate transporter SPBC8E4.01c), without affecting the expression of known genes that regulate the phosphate response pathway, whereas S7A increased pho1(+) expression. These results highlight specific cellular gene expression programs that are responsive to distinct CTD cues.

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

Novel 2,4-dianilino-5-fluoropyrimidine derivatives possessing ALK inhibitory activities

A new series of 2,4-dianilino-5-fluoropyrimidine derivatives were designed and synthesized and their anaplastic lymphoma kinase (ALK) inhibitory activities were evaluated by biochemical and cell-based assays in order to discover a new ALK inhibitor. Most compounds synthesized showed good inhibitory activities against ALK and good cytotoxic activities in H3122 cell line. The best compound 6f showed good activity against wild-type ALK along with crizotinib-resistant mutant ALK, and it showed 6 times better activity in cell-based assay than crizotinib. Some SAR studies were performed by the comparisons of the activities between 6 and the designed-synthesized compounds.