Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.

The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of compounds that selectively inhibit mTORC1 and not mTORC2. These 'bi-steric inhibitors' comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors.

Unlocking the Potential of High-Throughput Drug Combination Assays Using Acoustic Dispensing.

Assessment of synergistic effects of drug combinations in vitro is a critical part of anticancer drug research. However, the complexities of dosing and analyzing two drugs over the appropriate range of doses have generally led to compromises in experimental design that restrict the quality and robustness of the data. In particular, the use of a single dose response of combined drugs, rather than a full two-way matrix of varying doses, has predominated in higher-throughput studies. Acoustic dispensing unlocks the potential of high-throughput dose matrix analysis. We have developed acoustic dispensing protocols that enable compound synergy assays in a 384-well format. This experimental design is considerably more efficient and flexible with respect to time, reagent usage, and labware than is achievable using traditional serial-dilution approaches. Data analysis tools integrated in Genedata Screener were used to efficiently deconvolute the combination compound mapping scheme and calculate compound potency and synergy metrics. We have applied this workflow to evaluate interactions among drugs targeting different nodes of the mitogen-activated protein kinase pathway in a panel of cancer cell lines.

Combined inhibition of tumor necrosis factor α and interleukin-17 as a therapeutic opportunity in rheumatoid arthritis: development and characterization of a novel bispecific antibody.

OBJECTIVE: Rheumatoid arthritis therapies that are based on inhibition of a single cytokine, e.g., tumor necrosis factor α (TNFα) or interleukin-6 (IL-6), produce clinically meaningful responses in only about half of the treated patients. This study was undertaken to investigate whether combined inhibition of TNFα and IL-17 has additive or synergistic effects in the suppression of mesenchymal cell activation in vitro and inflammation and tissue destruction in arthritis in vivo. METHODS: Cultures of human fibroblast-like synoviocytes (FLS) were stimulated with TNFα, IL-17, or a combination of both. Single/combined neutralizing antibodies against TNFα and IL-17 were used to examine in vitro cytokine responses and in vivo development of arthritis and bone and cartilage destruction in TNFα-transgenic mice. Bispecific anti-TNFα/IL-17 antibodies were designed, and their potential to block cytokine responses in human FLS was tested. RESULTS: TNFα and IL-17 had additive/synergistic effects in promoting production of IL-6, IL-8, and granulocyte colony-stimulating factor, as well as matrix metalloproteinases, in FLS. Bispecific anti-TNFα/IL-17 antibodies showed superior efficacy in blocking cytokine and chemokine responses in vitro. Furthermore, dual versus single inhibition of both cytokines using neutralizing antibodies was more effective in inhibiting the development of inflammation and bone and cartilage destruction in arthritic mice. CONCLUSION: Combined blockade of TNFα and IL-17 was more effective than single blockade in inhibiting cytokine, chemokine, and matrix enzyme responses from human mesenchymal cells and in blocking tissue destruction associated with arthritis, and additionally showed a positive impact on rebalance of bone homeostasis. Bispecific anti-TNFα/IL-17 antibodies may have superior efficacy in the treatment of arthritis and may overcome the limited therapeutic responses obtained with single cytokine neutralization.

Monitoring membrane potential with second-harmonic generation.

This protocol describes the nonlinear optical phenomenon known as second-harmonic generation (SHG) and discusses its special attributes for imaging membrane-potential changes in single cells and multicellular preparations. Undifferentiated N1E-115 mouse neuroblastoma cells are used as a model cellular system for membrane electrophysiology. Styryl and naphthylstyryl dyes, also known as hemicyanines, are a class of electrochromic membrane-staining probes that have been used to monitor membrane potential by fluorescence; they also produce SHG images of cell membranes with SHG intensities that are sensitive to voltage. These experiments allow for the precise characterization of the voltage sensitivity of SHG and identification of the optimal wavelength for the incident laser fundamental light. This protocol presents the steps for the culture, staining, patching, and imaging of cells. The details of the imaging system and the measurements obtained are discussed, as are the prospects of this technology for imaging membrane potential changes in neuronal preparations.

Kinetic characterization of human JNK2alpha2 reaction mechanism using substrate competitive inhibitors.

Jun N-terminal kinase (JNK) is a stress activated serine/threonine protein kinase that phosphorylates numerous cellular protein substrates including the transcription factors c-Jun and ATF2. In this study, we defined the kinetic mechanism for the active form of JNK2alpha2. Double reciprocal plots of initial rates versus concentrations of substrate revealed the sequential nature of the JNK2alpha2 catalyzed ATF2 phosphorylation. Dead-end JNK inhibitors were then used to differentiate ordered and random kinetic mechanisms for the reaction. A peptide inhibitor containing the homology JNK docking sequence for substrate recognition, derived from amino acid residues 153-163 of JNK-interacting protein 1 (JIP-1), inhibited JNK activity via competition with ATF2. This peptide functioned as a noncompetitive inhibitor against ATP. In contrast, the anthrapyrazolone compound, SP600125, exhibited competitive inhibition for ATP and noncompetitive inhibition against ATF2. Furthermore, binding of one substrate had no significant effect on the affinity for the other substrate. The data in this study are consistent with a kinetic mechanism for activated JNK2alpha2 in which (1) substrate binding is primarily due to the distal contacts in the JNK2alpha2 docking groove that allow the delivery of the substrate phosphorylation sequence into the catalytic center, (2) there is minimal allosteric communication between the protein-substrate docking site and the ATP binding site in the catalytic center for activated JNK2alpha2, and (3) the reaction proceeds via a random sequential mechanism.

Selective and non-selective metalloproteinase inhibitors reduce IL-1-induced cartilage degradation and loss of mechanical properties.

Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulated with proinflammatory cytokines such as interleukin-1 (IL-1). Aggrecanases and matrix metalloproteinases (MMPs) are thought to be principal downstream effectors of cytokine-induced matrix catabolism, and aggrecanase- or MMP-selective inhibitors reduce or block matrix destruction in several model systems. The objective of this study was to use metalloproteinase inhibitors to perturb IL-1-induced matrix catabolism in bovine cartilage explants and examine their effects on changes in tissue compression and shear properties. Explanted tissue was stimulated with IL-1 for up to 24 days in the absence or presence of inhibitors that were aggrecanase-selective, MMP-selective, or non-selective. Analysis of conditioned media and explant digests revealed that aggrecanase-mediated aggrecanolysis was delayed to varying extents with all inhibitor treatments, but that aggrecan release persisted. Collagen degradation was abrogated by MMP- and non-selective inhibitors and reduced by the aggrecanase inhibitor. The inhibitors delayed but did not reduce loss of the equilibrium compression modulus, whereas the losses of dynamic compression and shear moduli were delayed and reduced. The data suggest that non-metalloproteinase mechanisms participate in IL-1-induced matrix degradation and loss of tissue material properties.

Results of aortic valve-sparing and restoration with autologous pericardial leaflet extensions in congenital heart disease.

BACKGROUND: The purpose of this study is to evaluate the efficacy of aortic valve-sparing repair with glutaraldehyde-treated autologous pericardium in congenital valvular pathology. METHODS: Sixty-two patients underwent reparative aortic valve surgery from January 1997 through December 2003. The mean age was 25 +/- 20 years (+/- standard deviation) (range, 10 days to 81 years). Fifty percent (31 of 62) were less than 19 years old at operation. The diagnoses included bicuspid aortic valve (39 patients), ventricular septal defect (14 patients), severe aortic stenosis (6 patients), subaortic stenosis (7 patients), bacterial endocarditis (7 patients), neonatal truncus arteriosus (2 patients), Shone's complex (2 patients), transposition complex (1 patient), double-chambered right ventricle (1 patient), and Marfan's syndrome (1 patient). Twelve patients (19 %) had prior sternotomy and cardiac operations. Valve-sparing techniques included pericardial leaflet extensions in 62 patients, creation of one or more pericardial neoaortic sinuses in 8, subcommissuroplasty in 8, pericardial patch of perforated leaflets in 9, Dacron mesh wrap (Boston Scientific, Wayne, NJ) of dilated ascending aorta in 12, and concomitant tricuspid and mitral valve repairs in 3 and 4 patients, respectively. RESULTS: There was one early death (1.6%). There were no late deaths at a mean follow-up of 25 +/- 16 (range, 0.1 to 72.5 months). Six patients required reoperation and prosthetic or homograft replacement for aortic valve incompetence. One out of 6 reoperations required re-repair. The remaining patients are well with a mean aortic regurgitation grade by echocardiography of 1.3 +/- 0.9 (scale, 0 to 4). CONCLUSIONS: Aortic valve repair with pericardial leaflet extension is a promising technique for the growing child.