ABSTRACT
Upregulation of TGFß and Cox2 in the tumor microenvironment results in blockade of T-cell penetration into the tumor. Without access to tumor antigens, the T-cell response will not benefit from administration of the immune checkpoint antibodies. We created an intravenous polypeptide nanoparticle that can deliver two siRNAs (silencing TGFß and Cox2). Systemic administration in mice, bearing a syngeneic orthotopic hepatocellular carcinoma (HCC), delivers the siRNAs to various cells in the liver, and significantly reduces the tumor. At 2 mg/kg (BIW) the nanoparticle demonstrated a single agent action and induced tumor growth inhibition to undetectable levels after five doses. Reducing the siRNAs to 1mg/kg BIW demonstrated greater inhibition in the presence of PD-L1 mAbs. After only three doses BIW, we could still recover a smaller tumor and, in tumor sections, showed an increase in penetration of CD4+ and CD8+ T-cells deeper into the remaining tumor that was not evident in animals treated with non-silencing siRNA. The combination of TGFß and Cox2 siRNA co-administered in a polypeptide nanoparticle can act as a novel therapeutic alone against HCC and may augment the activity of the immune checkpoint antibodies. Silencing TGFß and Cox2 converts an immune excluded (cold) tumor into a T-cell inflamed (hot) tumor.
ABSTRACT
(2'R)-Ethynyl uridine 3, and its (2'S)-diastereomer 10, are synthesised in a divergent fashion from the inexpensive parent nucleoside. Both nucleoside analogues are obtained from a total of 5 simple synthetic steps and 3 trivial column chromatography purifications. To evaluate their effectiveness against HCV NS5B polymerase, the nucleosides were converted to their respective 5'-O-triphosphates. Subsequently, this lead to the discovery of the 2'-ß-ethynyl 18 and -propynyl 20 nucleotides having significantly improved potency over Sofosbuvir triphosphate 24.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Nucleosides/pharmacology , Uridine/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Molecular Conformation , Nucleosides/chemical synthesis , Nucleosides/chemistry , Structure-Activity Relationship , Uridine/analogs & derivatives , Uridine/chemistryABSTRACT
Microtubules are a well-validated target for anticancer therapy. Molecules that bind tubulin affect dynamic instability of microtubules causing mitotic arrest of proliferating cells, leading to cell death and tumor growth inhibition. Natural antitubulin agents such as taxanes and Vinca alkaloids have been successful in the treatment of cancer; however, several limitations have encouraged the development of synthetic small molecule inhibitors of tubulin function. We have previously reported the discovery of two novel chemical series of tubulin polymerization inhibitors, triazoles (Ouyang et al. Synthesis and structure-activity relationships of 1,2,4-triazoles as a novel class of potent tubulin polymerization inhibitors. Bioorg Med Chem Lett. 2005; 15:5154-5159) and oxadiazole derivatives (Ouyang et al. Oxadiazole derivatives as a novel class of antimitotic agents: synthesis, inhibition of tubulin polymerization, and activity in tumor cell lines. Bioorg Med Chem Lett. 2006; 16:1191-1196). Here, we report on the anticancer effects of a lead oxadiazole derivative in vitro and in vivo. In vitro, IMC-038525 caused mitotic arrest at nanomolar concentrations in epidermoid carcinoma and breast tumor cells, including multidrug-resistant cells. In vivo, IMC-038525 had a desirable pharmacokinetic profile with sustained plasma levels after oral dosing. IMC-038525 reduced subcutaneous xenograft tumor growth with significantly greater efficacy than the taxane paclitaxel. At efficacious doses, IMC-038525 did not cause substantial myelosuppression or peripheral neurotoxicity, as evaluated by neutrophil counts and changes in myelination of the sciatic nerve, respectively. These data indicate that IMC-038525 is a promising candidate for further development as a chemotherapeutic agent.
ABSTRACT
We report further expansion of the structure activity relationship (SAR) on the triaryl bis sulfone class of compounds (I), which are potent CB(2) receptor ligands with excellent selectivity over the CB(1) receptor. This study was extended to B ring changes, followed by simultaneous optimization of the A-, B-, and C-rings. Compound 42 has excellent CB(2) potency, selectivity and rat exposure.
Subject(s)
Receptor, Cannabinoid, CB2/drug effects , Sulfones/chemistry , Sulfones/pharmacology , Animals , Ligands , Rats , Receptor, Cannabinoid, CB2/metabolism , Structure-Activity Relationship , Sulfones/metabolismABSTRACT
The triaryl bis-sulfone 1 was modified by converting the aryl A-ring to a piperidine ring. The piperidine ring was further elaborated to a spirocyclopropyl piperidine moiety. The effect on CB2 binding potency, rat calcium channel affinity, and CYP 2C9 inhibition is described.
Subject(s)
Receptor, Cannabinoid, CB2/antagonists & inhibitors , Sulfonamides/chemical synthesis , Sulfones/chemistry , Sulfones/chemical synthesis , Animals , Calcium Channels/metabolism , Cytochrome P-450 Enzyme System , Drug Inverse Agonism , Humans , Piperidines/chemistry , Rats , Receptor, Cannabinoid, CB2/metabolism , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacokinetics , Sulfones/pharmacokineticsABSTRACT
A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 microg/mL). The results show that mass spectrometry-based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/analysis , Enzyme Inhibitors/analysis , Enzyme Inhibitors/pharmacology , High-Throughput Screening Assays/methods , Mass Spectrometry/methods , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Dimethyl Sulfoxide/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Stability/drug effects , Escherichia coli/drug effects , Fluorescence , Microbial Sensitivity Tests , Reproducibility of Results , Small Molecule Libraries/analysis , Small Molecule Libraries/pharmacology , Substrate Specificity/drug effects , TemperatureABSTRACT
The authors have characterized a set of cannabinoid CB(2) receptor ligands, including triaryl bis sulfone inverse agonists, in a cell-based receptor/beta-arrestin interaction assay (DiscoveRx PathHunter). The results were compared with results using a competitive ligand binding assay, and with effects on forskolin-stimulated cAMP levels (PerkinElmer LANCE). The authors show good correlation between the 3 assay systems tested, with the beta-arrestin protein complementation assay exhibiting a more robust signal than the cAMP assay for cannabinoid CB(2) agonists. Further assay validation shows that DiscoveRx PathHunter HEK293 CB(2) beta-arrestin assay can be carried out from cryopreserved cell suspensions, eliminating variations caused by the need for multiple cell pools during live cell screening campaigns. These results, and the authors' results evaluating a test set of random library compounds, validate the use of ligand-induced interaction between the human cannabinoid CB(2) receptor and beta-arrestin as an appropriate and valuable screening platform for compounds specific for the cannabinoid CB(2) receptor.
Subject(s)
Arrestins/analysis , Arrestins/metabolism , Receptor, Cannabinoid, CB2/metabolism , Animals , Cell Line , Cricetinae , Humans , Ligands , Receptor, Cannabinoid, CB2/genetics , beta-ArrestinsABSTRACT
Oxadiazole derivatives were synthesized and evaluated for their ability to inhibit tubulin polymerization and to cause mitotic arrest in tumor cells. The most potent compounds inhibited tubulin polymerization at concentrations below 1 microM. Lead analogs caused mitotic arrest of A431 human epidermoid cells and cells derived from multi-drug resistant tumors (10, EC(50)=7.8 nM). Competition for the colchicine binding site and pharmacokinetic properties of selected potent compounds were also investigated and are reported herein, along with structure-activity relationships for this novel series of antimitotic agents.
Subject(s)
Antimitotic Agents/chemical synthesis , Antimitotic Agents/pharmacology , Oxadiazoles/chemistry , Oxadiazoles/pharmacology , Tubulin/chemistry , Tubulin/metabolism , Animals , Antimitotic Agents/chemistry , Antimitotic Agents/classification , Biopolymers/chemistry , Biopolymers/metabolism , Cell Line, Tumor , Humans , Inhibitory Concentration 50 , Mice , Molecular Structure , Oxadiazoles/chemical synthesis , Oxadiazoles/classification , Protein Conformation/drug effects , Structure-Activity RelationshipABSTRACT
A novel triazole-containing chemical series was shown to inhibit tubulin polymerization and cause cell cycle arrest in A431 cancer cells with EC(50) values in the single digit nanomolar range. Binding experiments demonstrated that representative active compounds of this class compete with colchicine for its binding site on tubulin. The syntheses and structure-activity relationship studies for the triazole derivatives are described herein.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Triazoles/chemistry , Triazoles/pharmacology , Tubulin Modulators/chemistry , Tubulin Modulators/pharmacology , Antineoplastic Agents/chemical synthesis , Humans , Microtubules/drug effects , Molecular Structure , Structure-Activity Relationship , Triazoles/chemical synthesis , Tubulin Modulators/chemical synthesis , Tumor Cells, CulturedABSTRACT
Platelet-derived growth factor receptor alpha (PDGFRalpha) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRalpha, which did not cross-react with the beta form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRalpha. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRalpha. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P=0.0004) and SKLMS-1 (P <0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRalpha.
Subject(s)
Antibodies, Monoclonal/chemistry , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Animals , Becaplermin , Biological Assay , Cell Line, Tumor , Dose-Response Relationship, Immunologic , Flow Cytometry , Humans , Kinetics , Ligands , MAP Kinase Signaling System , Mice , Mice, Nude , Mice, Transgenic , Neoplasm Transplantation , Phosphorylation , Platelet-Derived Growth Factor/chemistry , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-sis , Receptor, Platelet-Derived Growth Factor alpha/immunology , Time Factors , TransfectionABSTRACT
The post-translational modifications of histones are key to the modulation of chromatin structure. Distinct patterns of modifications established by histone-modifying enzymes control diverse chromosomal processes. Here, we report the purification and molecular characterization of the fission yeast Clr6 histone deacetyl ase involved in higher order chromatin assembly. We show that a chromodomain protein Alp13, which belongs to the conserved MRG protein family linked to cellular senescence in humans, is associated with Clr6. In addition, Clr6 interacts with homologs of the mammalian transcriptional co-repressors Sin3, Pst1 and Pst2, and a WD40 repeat-containing protein, Prw1. Alp13, Pst2 and Prw1 form a stable complex with Clr6 in the nucleus. Deletion of any of these factors causes progressive loss of viability and sensitivity to DNA-damaging agents, and impairs condensation/resolution of chromosomes during mitosis. This is accompanied by hyperacetylation of histones and a reduction in histone H3 Ser10 phosphorylation, which correlates with chromosome condensation during mitosis. These results link the MRG family protein Alp13 to histone deacetylation, and suggest that Clr6 and its associated factors are essential for fundamental chromosomal events.