Stacie A. Dalrymple

Embryonic Lethality, Liver Degeneration, and Impaired NF-κB Activation in IKK-β-Deficient Mice

IkappaB kinase-alpha and -beta (IKK-alpha and IKK-beta), the catalytic subunits of the IKK complex, phosphorylate IkappaB proteins on specific serine residues, thus targeting IkappaB for degradation and activating the transcription factor NF-kappaB. To elucidate the in vivo function of IKK-beta, we generated IKK-beta-deficient mice. The homozygous mouse embryo dies at approximately 14.5 days of gestation due to liver degeneration and apoptosis. IKK-beta-deficient embryonic fibroblasts have both reduced basal NF-kappaB activity and impaired cytokine-induced NF-kappaB activation. Similarly, basal and cytokine-inducible kinase activities of the IKK complex are greatly reduced in IKK-beta-deficient cells. These results indicate that IKK-beta is crucial for liver development and regulation of NF-kappaB activity and that IKK-alpha can only partially compensate for the loss of IKK-beta.

A small molecule ubiquitination inhibitor blocks NF-κB-dependent cytokine expression in cells and rats

A small molecule inhibitor of NF-κB-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) α-induced IκBα degradation in MM6 cells but not the degradation of β-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNFα, interleukin-1β, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC50 values below 1 μm. Ro106-9920 also blocked TNFα production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require βTRCP but associates with IκBα and will ubiquitinate IκBα S32E,S36E (IκBαee) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IκBαee ubiquitination with an IC50 value of 2.3 ± 0.09 μm. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCFβTRCP, the putative E3 for IκBα ubiquitination. Ro106-9920 was observed to be selective for IκBαee ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPS- and TNFα-induced IκBα degradation and NF-κB activation.

The cytokine stew and innate resistance to L. monocytogenes.

Summary: The Listeria monocytogenes (L. monocytogenes) infection model has been a useful system to evaluate the cellular interactions leading to host immunity. The initiation of the innate immune response in naive animals and subsequent progression to acquired immunity represent an integrated system with numerous layers of complexity. Coincident with experimental infection is the induction of cytokines. Cytokines, which are soluble mediators of cell growth, maintenance and function, form a network of pleiotropic stimuli that serve as one of the main driving forces for the progressive development of cellular responses. A variety of in vivo approaches, such as injection of the recombinant cytokines themselves or antibodies lo neutralize their activity, have been used to define these stimuli. Perhaps one of the most useful tools is that of germline‐manipulated animals. One of the many cytokines implicated in resistance to L. monocytogenes Infection is interleukin (IL)‐6, a molecule associated with diverse infectious and pathophysiological disease states. This review concentrates on various cytokines (IL‐1, TNFα, IFN‐γ. IL‐12, IL‐10 and the colony‐stimulating factors (CSF)) thought to play a role during the innate host response to L. monocytogenes infection, with a special emphasis on studies using IL‐6‐deficient mice. Additionally, we show unpublished data obtained when the concepts learned from L. monocytogenes infection in IL‐6‐deficieni mice were applied :o other infection models.

Targeting B cells for the treatment of rheumatoid arthritis.

The role of B cells in rheumatoid arthritis (RA) has been debated for decades. However, recent clinical trial data indicating that depletion of B cells in RA patients is of therapeutic benefit has validated the importance of this cell type in the pathogenesis of the disease. Elucidation of the molecular basis of B cell development and activation has allowed the identification of a number of possible therapeutic targets that are appealing for drug development. This review discusses briefly a number of these molecules and the rationale for targeting them for the treatment of RA.

p38 mitogen activated protein kinase as a therapeutic target for Alzheimer’s disease

The characteristic pathological hallmarks of Alzheimer’s disease (AD) include neuritic plaques, neurofibrillary tangles, and inflammatory changes. Current therapies, such as molecules that target enhancing cholinergic activity, can improve cognitive function in the short term but, unfortunately, have no impact on progression of the disease. Although many molecular targets have been suggested to play a causative role in AD progression, clinical data demonstrating a link between the blockade of such targets and amelioration or halting of disease progression are lacking. Even so, there are many interesting candidate targets, and current research efforts in these areas promises to deliver a wealth of new possibilities for treating AD in the future. This brief review will focus on p38 mitogen-activated protein kinase as a possible target for therapeutic intervention in AD.

Discovery of 6-(2,4-Difluorophenoxy)-2-[3-hydroxy-1-(2-hydroxyethyl)propylamino]-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one (Pamapimod) and 6-(2,4-Difluorophenoxy)-8-methyl-2-(tetrahydro-2H-pyran-4-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (R1487) as Orally Bioavailable and Highly Selective Inhibitors of p38α Mitogen-Activated Protein Kinase

The development of a new series of p38α inhibitors resulted in the identification of two clinical candidates, one of which was advanced into a phase 2 clinical study for rheumatoid arthritis. The original lead, an lck inhibitor that also potently inhibited p38α, was a screening hit from our kinase inhibitor library. This manuscript describes the optimization of the lead to p38-selective examples with good pharmacokinetic properties.

Models of Inflammation: Adjuvant‐Induced Arthritis in the Rat

Injection of adjuvant (Mycobacterium butyricum suspended in mineral oil) into rats produces an immune reaction that characteristically involves inflammatory destruction of cartilage and bone of the distal joints with concomitant swelling of surrounding tissues. Adjuvant‐induced arthritis in rats is commonly used to evaluate compounds that might be of potential use as drugs for treatment of rheumatoid arthritis and other chronic inflammatory conditions. This unit describes a method for inducing arthritis by injecting adjuvant into the tail and evaluating a test compound for the ability to inhibit the inflammatory response.

A novel series of IKKβ inhibitors part I: Initial SAR studies of a HTS hit.

A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKβ. In this Letter we document our early efforts at optimization of the quinoline core, the imidazole and the semithiocarbazone moiety. Most potency gains came from substitution around the 6- and 7-positions of the quinoline ring. Replacement of the semithiocarbazone with a semicarbazone decreased potency but led to some measurable exposure.

A novel series of IKKβ inhibitors part II: description of a potent and pharmacologically active series of analogs.

A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKβ. In this Letter we document our efforts at further optimization of this series, culminating in 2 with submicromolar potency in a HWB assay and efficacy in a CIA mouse model.

Models of Inflammation: Measuring Gastrointestinal Ulceration in the Rat

In rats, nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin induce gastrointestinal ulcerations. This unit describes a procedure to elicit and measure indomethacin-induced gastrointestinal lesions. This approach can be used to test for gastrointestinal side effects of potential anti-inflammatory and other agents.