Synthesis and SAR of novel imidazoquinoxaline-based Lck inhibitors: improvement of cell potency

A series of anilino(imidazoquinoxaline) analogues bearing solubilizing side chains at the 6- and 7-positions of the fused phenyl ring has been prepared and evaluated for inhibition against Lck enzyme and of T-cell proliferation. Significant improvement of the cellular activity was achieved over the initial lead, compound 2.     

Sequence comparison of mitochondrial tRNA genes and origin of light strand replication in Bos taurus and Nellore (Bos indicus) breeds

Although the complete bovine mitochondrial DNA molecule has been previously sequencedand sequence comparisons of the mitochondrial displacement loop have been performed, detailed sequence information is limited on coding regions of mitochondrial DNA within and among breeds of Bos taurus and Bos indicus. This study analysed polymorphism of the mitochondrial DNA transfer RNA genes for trypto-phan, alanine, asparagine, cysteine, tyrosine and the origin of light strand replication among Ayrshire, Canadian, Belgium Blue, Brown Swiss, Hereford, Jersey, Limousine, Piedmon-taise, Red Angus, Simmental (Bos taurus) and a Nellore (Bos indicus). Nucleotide sequence analysis of a 420-bp fragment of mitochondrial DNA comprising the five transfer RNA genes showed 100% homology among single individuals of the Bos taurus breeds. The Nellore breed showed guanine to adenine substitutions in the DHU arm of asparagine tRNA and in the origin of light-strand replication. This equates to a 0.5% sequence difference between the Nellore andBos taurus breeds and may reflect an independent evolutionary origin of the species.     

Novel tricyclic inhibitors of IKK2: discovery and SAR leading to the identification of 2-methoxy-N-((6-(1-methyl-4-(methylamino)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-7-yl)pyridin-2-yl)methyl)acetamide (BMS-066)

The synthesis, structure-activity relationships (SAR), and biological results of pyridyl-substituted azaindole based tricyclic inhibitors of IKK2 are described. Compound 4m demonstrated potent in vitro potency, acceptable pharmacokinetic and physicochemical properties, and efficacy when dosed orally in a mouse model of inflammatory bowel disease.     

High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment

Stem cells in vivo are housed within a functional microenvironment termed the “stem cell niche.” As the niche components can modulate stem cell behaviors like proliferation, migration and differentiation, evaluating these components would be important to determine the most optimal platform for their maintenance or differentiation. In this review, we have discussed methods and technologies that have aided in the development of high throughput screening assays for stem cell research, including enabling technologies such as the well-established multiwell/microwell plates and robotic spotting, and emerging technologies like microfluidics, micro-contact printing and lithography. We also discuss the studies that utilized high throughput screening platform to investigate stem cell response to extracellular matrix, topography, biomaterials and stiffness gradients in the stem cell niche. The combination of the aforementioned techniques could lay the foundation for new perspectives in further development of high throughput technology and stem cell research.     

CYLD Deubiquitinates RIP1 in the TNFα-Induced Necrosome to Facilitate Kinase Activation and Programmed Necrosis

Background

Necroptosis/programmed necrosis is initiated by a macro-molecular protein complex termed the necrosome. Receptor interacting protein kinase 1 (RIPK1/RIP1) and RIP3 are key components of the necrosome. TNFα is a prototypic inducer of necrosome activation, and it is widely believed that deubiquitination of RIP1 at the TNFR-1 signaling complex precedes transition of RIP1 into the cytosol where it forms the RIP1-RIP3 necrosome. Cylindromatosis (CYLD) is believed to promote programmed necrosis by facilitating RIP1 deubiquitination at this membrane receptor complex.

Methodology/Principal Findings

We demonstrate that RIP1 is indeed the primary target of CYLD in TNFα-induced programmed necrosis. We observed that CYLD does not regulate RIP1 ubiquitination at the TNF receptor. TNF and zVAD-induced programmed necrosis was highly attenuated in CYLD^-/- cells. However, in the presence of cycloheximide or SMAC mimetics, programmed necrosis was only moderately reduced in CYLD^-/- cells. Under the latter conditions, RIP1-RIP3 necrosome formation is only delayed, but not abolished in CYLD^-/- cells. We further demonstrate that RIP1 within the NP-40 insoluble necrosome is ubiquitinated and that CYLD regulates RIP1 ubiquitination in this compartment. Hence, RIP1 ubiquitination in this late-forming complex is greatly increased in CYLD^-/- cells. Increased RIP1 ubiquitination impairs RIP1 and RIP3 phosphorylation, a signature of kinase activation.

Conclusions/Significance

Our results show that CYLD regulates RIP1 ubiquitination in the TNFα-induced necrosome, but not in the TNFR-1 signaling complex. In cells sensitized to programmed necrosis with SMAC mimetics, CYLD is not essential for necrosome assembly. Since SMAC mimetics induces the loss of the E3 ligases cIAP1 and cIAP2, reduced RIP1 ubiquitination could lead to reduced requirement for CYLD to remove ubiquitin chains from RIP1 in the TNFR-1 complex. As increased RIP1 ubiquitination in the necrosome correlates with impaired RIP1 and RIP3 phosphorylation and function, these results suggest that CYLD controls RIP1 kinase activity during necrosome assembly.     

Discovery and SAR of 2-amino-5-[(thiomethyl)aryl]thiazoles as potent and selective Itk inhibitors

A series of structurally novel aminothiazole based small molecule inhibitors of Itk were prepared to elucidate their structure-activity relationships (SARs), selectivity and cell activity in inhibiting IL-2 secretion in a Jurkat T-cell assay. Compound 2 is identified as a potent and selective Itk inhibitor which inhibits anti-TCR antibody induced IL-2 production in mice in vivo.     

5-amino-pyrazoles as potent and selective p38α inhibitors

The synthesis and structure-activity relationships (SAR) of p38α MAP kinase inhibitors based on a 5-amino-pyrazole scaffold are described. These studies led to the identification of compound 2j as a potent and selective inhibitor of p38α MAP kinase with excellent cellular potency toward the inhibition of TNFα production. Compound 2j was highly efficacious in vivo in inhibiting TNFα production in an acute murine model of TNFα production. X-ray co-crystallography of a 5-amino-pyrazole analog 2f bound to unphosphorylated p38α is also disclosed.     

Viral Cell Death Inhibitor MC159 Enhances Innate Immunity against Vaccinia Virus Infection

Viral inhibitors of host programmed cell death (PCD) are widely believed to promote viral replication by preventing or delaying host cell death. Viral FLIPs (Fas-linked ICE-like protease [FLICE; caspase-8]-like inhibitor proteins) are potent inhibitors of death receptor-induced apoptosis and programmed necrosis. Surprisingly, transgenic expression of the viral FLIP MC159 from molluscum contagiosum virus (MCV) in mice enhanced rather than inhibited the innate immune control of vaccinia virus (VV) replication. This effect of MC159 was specifically manifested in peripheral tissues such as the visceral fat pad, but not in the spleen. VV-infected MC159 transgenic mice mounted an enhanced innate inflammatory reaction characterized by increased expression of the chemokine CCL-2/MCP-1 and infiltration of γδ T cells into peripheral tissues. Radiation chimeras revealed that MC159 expression in the parenchyma, but not in the hematopoietic compartment, is responsible for the enhanced innate inflammatory responses. The increased inflammation in peripheral tissues was not due to resistance of lymphocytes to cell death. Rather, we found that MC159 facilitated Toll-like receptor 4 (TLR4)- and tumor necrosis factor (TNF)-induced NF-κB activation. The increased NF-κB responses were mediated in part through increased binding of RIP1 to TNFRSF1A-associated via death domain (TRADD), two crucial signal adaptors for NF-κB activation. These results show that MC159 is a dual-function immune modulator that regulates host cell death as well as NF-κB responses by innate immune signaling receptors.Successful immunity against pathogenic challenges is central to the survival of all organisms. Metazoans employ a wide array of innate and adaptive immune responses to control various pathogens. In response, pathogens have developed various strategies to evade detection and elimination by the immune system. Programmed cell death (PCD) plays an important role in host defense against pathogens by directly eliminating infected cells to limit the viral factory. A role for host cell death in antiviral responses is highlighted by the identification of viral inhibitors of apoptosis (3). In addition to apoptosis, nonapoptotic PCD pathways, such as necrosis and autophagy have recently been shown to participate in host defense against pathogens (31, 44). For instance, we recently showed that genetic ablation of an essential necrosis mediator, RIP3, resulted in severely impaired innate immune responses against vaccinia virus (VV) infection characterized by the lack of virus-induced tissue necrosis and inflammation (11). In addition, certain vFLIPs (viral Fas-linked ICE-like protease [FLICE; caspase-8]-like inhibitor proteins) are potent inhibitors of programmed necrosis (6, 8). These results indicate that host PCD machineries play important roles in controlling the viral factory and dissemination of the virus within the infected host.Despite the widely accepted view that inhibition of host cell death is an important viral immune evasion strategy, relatively few in vivo studies have been performed to directly test this hypothesis. This is due partly to the lack of suitable animal models in which specific components of host apoptotic machinery are inhibited. For instance, germ line inactivation of many of the components of the PCD machinery, such as Fas-associated via death domain (FADD) and caspase-8, resulted in embryonic lethality (50, 55, 57), thus preventing in vivo virus infection studies from using these animal models. Another approach that was widely used was transgenic expression of viral apoptosis inhibitors, such as poxvirus CrmA, baculovirus p35, and vFLIPs. However, since expression of these inhibitors was restricted mostly to the lymphoid compartment (26, 28, 34, 46, 51, 54, 58), they do not permit evaluation of the role of host cell death in the parenchyma in antiviral responses. Cell death in the stromal compartment could impact the innate inflammatory reaction, cross-priming of antigens, and viral dissemination to other tissues. Because cells in the parenchyma are the primary targets for many virus infections, it is important to determine the contribution of cell death in the parenchyma in antiviral responses.The vFLIPs were first identified as inhibitors of caspase-dependent apoptosis. They share homology with caspase-8 and caspase-10 in the tandem death effector domains (DEDs) at the amino termini. However, vFLIPs lack the caspase enzyme domain at the carboxyl termini. Thus, binding of vFLIPs to FADD and caspase-8/-10 via DED-mediated homotypic interaction led to inhibition of FasL-, tumor necrosis factor (TNF)-, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis (18, 19). Importantly, certain vFLIPs, including MC159 and E8, are also potent inhibitors of programmed necrosis induced by TNF-like death cytokines (8). These results suggest that viral inhibitors could inhibit multiple host PCD pathways to avoid elimination by the host immune system.In order to determine the effect of vFLIPs on host responses against viral infections, we generated transgenic mice expressing vFLIP MC159 under the control of the ubiquitous H2-K^b promoter (53). We previously showed that transgenic expression of MC159 did not alter lymphocyte functions and development, but rather caused a mild form of lymphoproliferation that resembled that seen with the lpr mice with Fas/CD95/Apo-1 mutations (53). Here, we show that MC159 transgenic mice exhibited enhanced innate immune responses to VV infections, which led to enhanced viral clearance in peripheral tissues. Surprisingly, the enhanced control of VV production was not due to enhanced lymphocyte survival. Rather, VV-induced expression of the chemokine CCL-2/MCP-1 was highly elevated in MC159 transgenic mice and was accompanied by enhanced recruitment of γδ T cells to peripheral tissues. MC159 promotes the binding between TRADD and RIP1, two crucial signal adaptors for NF-κB activation. Consequently, MC159 transgenic fibroblasts exhibited enhanced NF-κB activation to TNF and Toll-like receptor 4 (TLR4) stimulation. These results reveal a previously unappreciated effect of MC159 on NF-κB activation and indicate that viral cell death inhibitors could impact innate immune responses through mechanisms beyond cell death regulation.     

B cells promote resistance to heterosubtypic strains of influenza via multiple mechanisms

Immunity to heterosubtypic strains of influenza is thought to be mediated primarily by memory T cells, which recognize epitopes in conserved proteins. However, the involvement of B cells in this process is controversial. We show in this study that influenza-specific memory T cells are insufficient to protect mice against a lethal challenge with a virulent strain of influenza in the absence of B cells. B cells contribute to protection in multiple ways. First, although non-neutralizing Abs by themselves do not provide any protection to challenge infection, they do reduce weight loss, lower viral titers, and promote recovery of mice challenged with a virulent heterosubtypic virus in the presence of memory T cells. Non-neutralizing Abs also facilitate the expansion of responding memory CD8 T cells. Furthermore, in cooperation with memory T cells, naive B cells also promote recovery from infection with a virulent heterosubtypic virus by generating new neutralizing Abs. These data demonstrate that B cells use multiple mechanisms to promote resistance to heterosubtypic strains of influenza and suggest that vaccines that elicit both memory T cells and Abs to conserved epitopes of influenza may be an effective defense against a wide range of influenza serotypes.     

An antigen capture assay for the measurement of serum clusterin concentrations

We have developed and validated a robust antigen capture assay for the measurement of serum clusterin. Increased clusterin expression, and alterations in serum clusterin levels have been associated with a number of disease states. In particular, clusterin has been shown to be associated with tissue regression and apoptosis in the rat ventral prostate in response to androgen ablation or administration of anti-androgens. The object of this study was to determine if changes in human serum clusterin can be used as a diagnostic or prognostic marker to monitor the response to hormonal therapy in patients with prostate cancer, and to determine if clusterin concentrations increase with the progression towards androgen independence. The antigen capture assay was used for an extensive analysis of human serum clusterin concentration in fasting males, and to determine if there is any relationship between clusterin and age or cholesterol levels. The average clusterin level in serum is 101+/-42 microg/ml (n=96). There is no correlation to age or serum cholesterol levels. Analysis of serum clusterin levels in patients with newly diagnosed prostate cancer (n=5), hormone responsive tumors (n=5), and hormone refractory disease (n=5), demonstrates that no significant changes in serum clusterin levels accompany the progression of prostatic disease, or response to hormone therapy.