The Aryl Hydrocarbon Receptor Undergoes Chaperone-Mediated Autophagy in Triple-Negative Breast Cancer Cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule expressed in many cell types, including triple-negative and non-triple-negative breast cancer cells. It affects breast cancer growth and crosstalk with estrogen receptor signaling. Normally, this receptor is degraded shortly after ligand activation via the 26S proteasome. Here, we report that AHR undergoes chaperone-mediated autophagy in MDA-MB-468 triple-negative breast cancer cells. This lysosomal degradation of AHR exhibits the following characteristics: (1) it is triggered by 6 amino-nicotinamide, starvation, and piperazinylpyrimidine compound Q18; (2) it is not observed in non-triple-negative breast cancer cells (MCF-7, T47D, and MDA-MB-361); (3) it can be inhibited by progesterone receptor B but not estrogen receptor alpha; (4) it can be reversed by chloroquine but not MG132; (5) it requires LAMP2A; and (6) it involves AHR-HSC70 and AHR-LAMP2A interactions. The NEKFF sequence localized at amino acid 558 of human AHR appears to be a KFERQ-like motif of chaperone-mediated autophagy, responsible for the LAMP2A-mediated AHR protein degradation.

Pharmacological Inhibition of CDK8 in Triple-Negative Breast Cancer Cell Line MDA-MB-468 Increases E2F1 Protein, Induces Phosphorylation of STAT3 and Apoptosis.

Cyclin-dependent kinase 8 (CDK8) has been identified as a colon cancer oncogene. Since this initial observation, CDK8 has been implicated as a potential driver of other cancers including acute myelogenous leukemia (AML) and some breast cancers. Here, we observed different biological responses to CDK8 inhibition among colon cancer cell lines and the triple-negative breast cancer (TNBC) cell line MDA-MB-468. When treated with CDK8 inhibitor 4, all treated cell lines responded with decreased cell viability and increased apoptosis. In the MDA-MB-468 cell line, the decrease in cell viability was dependent on increased phosphorylation of signal transducer and activator of transcription 3 (STAT3), which is not observed in the colon cancer cell lines. Furthermore, increased STAT3 phosphorylation in 4 treated MDA-MB-468 cells was dependent on increased transcription factor E2F1 protein. These results are consistent with previous reports of exogenous expression of E2F1-induced apoptosis in MDA-MB-468 cells.

Preliminary in vitro and in vivo investigation of a potent platelet derived growth factor receptor (PDGFR) family kinase inhibitor.

Aberrant expression of wild-type and mutant forms of the platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases has been implicated in various oncologic indications such as leukemias, gliomas, and soft tissue sarcomas. Clinically used kinase inhibitors imatinib and sunitinib are potent inhibitors of wild-type PDGFR family members, but show reduced binding to mutant forms. Here we describe compound 5 which binds to both wild-type and oncogenic mutant forms of PDGFR family members, and demonstrates both cellular and in vivo activity.

Molecular docking and synthesis of novel quinazoline analogues as inhibitors of transcription factors NF-κB activation and their anti-cancer activities.

NF-kB is a transcription factor protein complex that can be found in almost all animal cell types and is a key player in some cancers and inflammatory responses. It can enhance the proliferation rate, reduce apoptosis, as well as creating more blood flow to ensure the survival of cancer, thus blocking the NF-kB pathway has potential therapeutic benefit. We designed a series of compounds based on a quinazoline scaffold pharmacophore model which may have high binding affinity with the p50 subunit of NF-kB. The compound series with phenyl substitution at the 2 position of the quinazoline proved to be more effective at inhibiting NF-kB function both theoretically and experimentally. These compounds also reduce the proliferation of numerous tumor cell lines and the mean GI(50) for compound 2a is 2.88 µM against the NCI-60 cell line. At the same time, compound 2a can induce significant apoptosis in EKVX cell line at the concentration of 1 µM.

Novel integrin-targeted binding-triggered drug delivery system for methotrexate.

PURPOSE: To design a binding-induced conformation change drug delivery system for integrin-targeted delivery of methotrexate and prove the feasibility of using hairpin peptide structure for binding triggered drug delivery. METHODS: Methotrexate prodrugs were synthesized using solid phase peptide synthesis techniques by conjugating methotrexate to Arg-Gly-Asp (RGD) or a hairpin peptide, RWQYV(D)PGKFTVQRGD (hairpin-RGD). Levels of integrin α(V)ß(3) in HUVEC were up-regulated using adenoviral system and knocked down using siRNA. Stability of prodrugs and methotrexate release from prodrugs were evaluated in plasma, in presence or absence of integrin α(V)ß(3)-expressing cells. Molecular modeling was performed to support experimental results using MOE. RESULTS: Prodrugs recognized and bound to integrin α(V)ß(3)-expressing cells in integrin α(V)ß(3) expression level-dependent manner. Prodrug with hairpin peptide could resist Streptomyces griseus-derived glutamic acid-specific endopeptidase (SGPE) and plasma enzyme hydrolysis. Drug release was triggered in presence of HUVEC cells and SGPE. Analysis of conformation energy supported that conformational change in MTX-hairpin-RGD led to exposure of labile link upon binding to integrin α(V)ß(3)-expressing cells. CONCLUSIONS: Binding-induced conformation change of hairpin peptide can be used to design integrin-targeted drug delivery system.

Anti-tumor pyrimidinylpiperazines bind to the prosurvival Bcl-2 protein family member Bcl-XL.

Overexpression of prosurvival or underexpression of pro-death Bcl-2 family proteins can lead to cancer cell resistance to chemotherapy and radiation treatment. Inhibition of the prosurvival Bcl-2 family proteins has become a strategy for cancer therapy and inhibitors are currently being evaluated in the clinic both as single agents and in combination with established drugs. Here we describe the design, synthesis, and evaluation of pyrimidylpiperazines that were discovered to be inhibitors of the prosurvival Bcl-2 protein family member Bcl-XL. This study identified compound 21 which demonstrated a GI(50) value of 8.4 µM against A549 lung adenocarcinoma cells and a binding affinity K(i) value for Bcl-XL of 127 nM.

Preliminary studies of 3,4-dichloroaniline amides as antiparasitic agents: structure-activity analysis of a compound library in vitro against Trichomonas vaginalis.

Trichomonas vaginalis, a human-infectious protozoan, can display resistance to treatment by metronidazole. A library of 3,4-dichloroaniline amides based on propanil, an herbicide, has been synthesized and screened to test susceptibility to these analogs. From this preliminary study, the most effective compound 15, inhibits growth of the organism by 66% and 69% on the two strains tested, T1 and G3, respectively.

Cell recognition enhanced enzyme hydrolysis of a model peptide-drug conjugate.

A model peptide-drug conjugate designed upon a beta-hairpin peptide with the alpha4beta1 integrin recognition sequence LDV appended to the N-terminus and a fluorescent model drug appended to the C-terminus. This model recognizes and binds to alpha4beta1 expressing cells and displays an enhanced rate of enzyme catalyzed hydrolytic model drug release in the presence of the cells compared to the rate in the absence of cells. The present work suggests that peptide-drug conjugate conformation change due to receptor binding may be a viable approach to targeted drug release.

Thiazolidinedione anti-cancer activity: Is inhibition of microtubule assembly implicated?

An hypothesis is presented which seeks to explain the anti-cancer activity of thiazolidinediones (TZDs), a class of drugs currently used to treat type 2 diabetes mellitus. Empirical data from the scientific literature is used to support the hypothesis that TZDs are inhibitors of microtubule assembly. The similarities between the affects of TZDs on cellular processes and known inhibitors of tubulin polymerization are identified. Similarities between TZDs and currently used inhibitors of microtubule assembly, such as cell cycle arrest in G1 phase, anti-angiogenesis activity, and inhibition of cell motility, are striking. In addition to the similarities in biological function, certain molecular structure similarities are also identified. The possibility that TZDs inhibit the polymerization of actin is presented as an alternative interpretation of the available data. Finally suggestions for testing the hypothesis, by using commercially available tubulin polymerization assays and fluorescence based binding assays, as well as isothermal titration calorimetry, are given. Considering TZD position as third-line therapy for treatment of type 2 diabetes mellitus and the potential loss of market share to newly introduced inhalable insulin, a better understanding of TZD anti-cancer activity may lead to revival for this drug class in cancer treatment.

Discovery, synthesis, and investigation of the antitumor activity of novel piperazinylpyrimidine derivatives.

Protein kinases play several pertinent roles in cell proliferation, and targeting these proteins has been shown to be a successful strategy toward controlling different malignancies. Despite the great discovery stories during the last two decades, there is still a demand for anticancer small molecules with the potential of being selective on both the protein kinase and/or the cellular level. A series of novel piperazinylpyrimidine compounds were synthesized and tested for their potential to selectively inhibit the growth of certain tumor cell lines included within the NCI-60 cell line panel. MDA-MB-468, a triple-negative/basal-like breast carcinoma, cell line was among the most sensitive cell lines towards compounds 4 and 15. The three most interesting compounds identified in cellular screens (4, 15, and 16) were subjected to kinase profiling and found to have an interesting selective tendency to target certain kinase subfamily members; PDGFR, CK1, RAF and others. Compound 4 showed a selective tendency to bind to and/or inhibit the function of certain KIT and PDGFRA mutants compared to their wild-type isoforms. Piperazinylpyrimidine based derivatives represent a new class of selective kinase inhibitors. Significantly 4 is more potent at inhibiting oncogenic mutant forms of PDGFR family kinases, which is relevant in terms of its potential use in treating tumors that have become resistant to treatment or driven by such mutations. The clinical demand for agents useful in the control of triple-negative/basal-like breast cancer justifies our interest in compound 15 which is a potent growth inhibitor of MDA-MB-468 cell line.

Novel design and synthesis of modified structure of carvedilol.

ß-adrenergic blocking agents have been in use for nearly 40 years. ß-blockers have been more thoroughly studied in the past twenty years as they have become commonly prescribed to heart failure patients. The class of ß-blockers has grown considerably and has many pharmaceutical applications in patients with heart failure. Carvedilol has been the most effective beta-blocker in the treatment of the systolic heart failure. Carvedilol is a non-selective ß- and α-blocker enantiomer with antioxidant effects that are attributed to its carbazole moiety. Carvedilol is taken twice daily because it is extensively metabolized and therefore loses its effectiveness due to a short half-life. Recently a long acting carvedilol has become available, as Coreg CR. Coreg CR is available for once-a-day administration as controlled-release oral capsules containing 10, 20, 40, or 80 mg carvedilol phosphate. The subject of the current report is to design a new structural analog of carvedilol that incorporates a protecting group such as a fluorine atom at position 8 of the carbazole ring for the purpose of blocking a critical metabolic pathway thus increasing its half life. This will follow discussion regarding current carvedilol patents. We believe that carvedilol activity will remain unchanged. The synthesis of 8-Fluoro-1, 2, 3, 9- tetrahydro-4H-carbazol-4-one, a key synthetic intermediate of the designed carvedilol analog, was carried out and successfully characterized.

Cyclic modular beta-sheets.

The development of peptide beta-hairpins is problematic, because folding depends on the amino acid sequence and changes to the sequence can significantly decrease folding. Robust beta-hairpins that can tolerate such changes are attractive tools for studying interactions involving protein beta-sheets and developing inhibitors of these interactions. This paper introduces a new class of peptide models of protein beta-sheets that addresses the problem of separating folding from the sequence. These model beta-sheets are macrocyclic peptides that fold in water to present a pentapeptide beta-strand along one edge; the other edge contains the tripeptide beta-strand mimic Hao [JACS 2000, 122, 7654] and two additional amino acids. The pentapeptide and Hao-containing peptide strands are connected by two delta-linked ornithine (deltaOrn) turns [JACS 2003, 125, 876]. Each deltaOrn turn contains a free alpha-amino group that permits the linking of individual modules to form divalent beta-sheets. These "cyclic modular beta-sheets" are synthesized by standard solid-phase peptide synthesis of a linear precursor followed by solution-phase cyclization. Eight cyclic modular beta-sheets 1a-1h containing sequences based on beta-amyloid and macrophage inflammatory protein 2 were synthesized and characterized by 1H NMR. Linked cyclic modular beta-sheet 2, which contains two modules of 1b, was also synthesized and characterized. 1H NMR studies show downfield alpha-proton chemical shifts, deltaOrn delta-proton magnetic anisotropy, and NOE cross-peaks that establish all compounds but 1c and 1g to be moderately or well folded into a conformation that resembles a beta-sheet. Pulsed-field gradient NMR diffusion experiments show little or no self-association at low (