Novel method to specifically determine the structures of non-N297 glycans in IgGs.

Monoclonal antibodies comprise a major class of biologic therapeutics and are also extensively studied in immunology. Given the importance of glycans on antibodies, fluorescent labeling of enzymatically released glycans and their LC/MS analysis is routinely used for in-depth characterization of antibody glycosylation. In this technical note, we propose a method for facile characterization of glycans in the variable region of antibodies using sequential enzymatic digests with Endoglycosidase-S2 and RapidTM Peptide-N-Glycosidase-F followed by labeling with a fluorescent dye carrying an NHS-carbamate moiety. The results and proposed mechanism also suggest that the choice of glycosidases along with the labeling chemistry is critical for accurate glycan analysis for a desired application.

PULSE SPR: A High Throughput Method to Evaluate the Domain Stability of Antibodies.

Downstream purification of therapeutic antibodies requires candidates to be stable under various stress conditions, such as low pH. Current approaches to assess the conformational or colloidal stability of biologics may require significant amounts of material, and the testing may occur over an extended period of time. Furthermore, typical methodologies for early stability testing often do not directly address low pH stability, but focus more on conditions suitable for long-term drug product storage. Here we report a high-throughput method that measures protonation-induced unfolding of ligand binding sites for stability evaluation by surface plasmon resonance or PULSE SPR. This method, which requires only several micrograms of sample, is highly sensitive to the structural integrity of ligand binding sites and correlates well with thermal and chemical conformational stability. Combined with ligands targeting different regions of antibodies, this method allows a comprehensive assessment of antibody domain stability. By applying PULSE SPR, we found that antibodies with different complementarity-determining regions (CDRs), frameworks, formats, and interchain disulfide bonds showed different stabilities under acidic conditions. Additionally, biologics that aggregated in solution also had poor low pH stability. Taken together, PULSE SPR is a high-throughput and low sample consumption method that can be adopted to evaluate antibody domain stability and aggregation at low pH, which are two important aspects of therapeutic biologics.

miR-155 Modifies Inflammation, Endothelial Activation and Blood-Brain Barrier Dysfunction in Cerebral Malaria.

miR-155 has been shown to participate in host response to infection and neuro-inflammation via negative regulation of blood-brain-barrier (BBB) integrity and T cell function. We hypothesized that miR-155 may contribute to the pathogenesis of cerebral malaria (CM). To test this hypothesis, we used a genetic approach to modulate miR-155 expression in an experimental model of cerebral malaria (ECM). In addition, an engineered endothelialized microvessel system and serum samples from Ugandan children with CM were used to examine an anti-miR-155 as a potential adjunctive therapeutic for severe malaria. Despite higher parasitemia, survival was significantly improved in miR-155-/- mice vs. wild-type littermate mice in ECM. Improved survival was associated with preservation of BBB integrity and reduced endothelial activation, despite increased levels of pro-inflammatory cytokines. Pre-treatment with antagomir-155 reduced vascular leak induced by human CM sera in an ex vivo endothelial microvessel model. These data provide evidence supporting a mechanistic role for miR-155 in host response to malaria via regulation of endothelial activation, microvascular leak and BBB dysfunction in CM.

Metalloproteinase-mediated Shedding of Integrin ß2 promotes macrophage efflux from inflammatory sites.

Macrophage exiting from inflammatory sites is critical to limit the local innate immune response. With tissue insult, resident tissue macrophages rapidly efflux to lymph nodes where they modulate the adaptive immune response, and inflammatory macrophages attracted to the site of injury then exit during the resolution phase. However, the mechanisms that regulate macrophage efflux are poorly understood. This study has investigated soluble forms of integrin ß2 whose levels are elevated in experimental peritonitis at times when macrophages are exiting the peritoneum, suggesting that its proteolytic shedding may be involved in macrophage efflux. Both constitutive and inducible metalloproteinase-dependent shedding of integrin ß2 from mouse macrophages are demonstrated. Soluble integrin ß2 is primarily released as a heterodimeric complex with αM that retains its ability to bind its ligands intracellular adhesion molecule-1, fibrin, and collagen and thus may serve as a soluble antagonist. In a model of accelerated exiting, administration of a metalloproteinase inhibitor prevents macrophage efflux by 50% and impedes loss of macrophage integrin ß2 from the cell surface. Exiting of peritoneal macrophages in mice lacking integrin ß2 is accelerated, and antibody disruption of integrin ß2-substrate interactions can reverse 50% of the metalloprotease inhibitor blockade of macrophage exiting. Thus, our study demonstrates the ability of metalloproteinase-mediated shedding of integrin ß2 to promote macrophage efflux from inflammatory sites, and the release of soluble integrin heterodimers may also limit local inflammation.

Hematopoietic Fas deficiency does not affect experimental atherosclerotic lesion formation despite inducing a proatherogenic state.

The Fas death receptor (CD95) is expressed on macrophages, smooth muscle cells, and T cells within atherosclerotic lesions. Given the dual roles of Fas in both apoptotic and nonapoptotic signaling, the aim of the present study was to test the effect of hematopoietic Fas deficiency on experimental atherosclerosis in low-density lipoprotein receptor-null mice (Ldlr(-/-)). Bone marrow from Fas(-/-) mice was used to reconstitute irradiated Ldlr(-/-) mice as a model for atherosclerosis. After 16 weeks on an 0.5% cholesterol diet, no differences were noted in brachiocephalic artery lesion size, cellularity, or vessel wall apoptosis. However, Ldlr(-/-) mice reconstituted with Fas(-/-) hematopoietic cells had elevated hyperlipidemia [80% increase, relative to wild-type (WT) controls; P < 0.001] and showed marked elevation of plasma levels of CXCL1/KC, CCL2/MCP-1, IL-6, IL-10, IL-12 subunit p70, and soluble Fas ligand (P < 0.01), as well as systemic microvascular inflammation. It was not possible to assess later stages of atherosclerosis because of increased mortality in Fas(-/-) bone marrow recipients. Our data indicate that hematopoietic Fas deficiency does not affect early atherosclerotic lesion development in Ldlr(-/-) mice.

On-column ligand exchange for structure-based drug design: a case study with human 11ß-hydroxysteroid dehydrogenase type 1.

Successfully forming ligand-protein complexes with specific compounds can be a significant challenge in supporting structure-based drug design for a given protein target. In this respect, an on-column ligand- and detergent-exchange method was developed to obtain ligand-protein complexes of an adamantane series of compounds with 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) after a variety of other complexation methods had failed. This report describes the on-column exchange method and an unexpected byproduct of the method in which artificial trimers were observed in the structures.

Very late antigen-4 function of myeloblasts correlates with improved overall survival for patients with acute myeloid leukemia.

Adhesion of acute myeloid leukemia (AML) blasts in the bone marrow microenvironment confers protection from chemotherapy-induced apoptosis. One mechanism for retention of blasts within the bone marrow is adhesion via very late antigen-4 (VLA-4), the alpha(4)beta(1) integrin heterodimer that binds to its main ligands, fibronectin, and vascular cell adhesion molecule-1 (VCAM-1). To examine the relationship of functional expression of VLA-4 to prognosis in AML, we studied marrow samples from 175 adult AML patients who underwent induction chemotherapy with anthracycline and cytarabine on Southwest Oncology Group trials. The studies included flow cytometry and functional in vitro assays for ligand binding and maximal beta(1) activation. VLA-4 expression varied widely, with mean expression 60.6% for alpha(4), and was not significantly associated with response to chemotherapy, relapse-free, or overall survival (OS). However, increased binding of soluble VCAM-1 via VLA-4 was significantly associated with longer OS, corrected for age (P = .033). Estimated 5-year OS was 31% (95% confidence interval, 14%-48%) in 30 patients with soluble VCAM-1 binding greater than or equal to 40%, compared with 10% (confidence interval, 3%-17%) in 72 patients with lower binding. Adhesion and migratory properties of AML blasts thus appear to influence chemosensitivity and therefore may be therapeutic targets.

FLIP (Flice-like inhibitory protein) suppresses cytoplasmic double-stranded-RNA-induced apoptosis and NF-κB and IRF3-mediated signaling.

BACKGROUND: Cytoplasmic viral double-stranded RNA (dsRNA) is detected by a class of ubiquitous cytoplasmic RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation antigen-5 (MDA5), which initiate a signaling cascade via their common adaptor called interferon-ß (IFN-ß) promoter stimulator-1 (IPS-1). This leads to the production of proinflammatory and antiviral cytokines, the type I Interferons, via mainly nuclear factor kappa B (NF-κB) and interferon response factor-3 (IRF3) transcription factors. Fas-associated death domain (FADD) protein, receptor-interacting protein (RIP1), caspase-8 and tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) protein, all traditionally associated with death receptor signaling, are also involved in RIG-I/MDA5 signaling pathway. We previously showed that FLIP (Flice-like inhibitory protein), also designated as cflar (CASP8 and FADD-like apoptosis regulator), negatively regulates lipopolysaccharide (LPS)-induced toll-like receptor 4 (TLR4) signaling in endothelial cells and mouse embryonic fibroblasts (MEFs) and protected against TLR4-mediated apoptosis. RESULTS: In this study, we investigated the role of FLIP in cellular response to cytoplasmic polyinosinic:polycytidylic acid, poly(I:C), a synthetic analog of dsRNA. Consistent with the previously described role of FADD in RIG-I/MDA5-mediated apoptosis, we found that FLIP-/- MEFs were more susceptible to killing by cytoplasmic poly(I:C). However, FLIP-/- MEFs also exhibited markedly increased expression of NF-κB-and IRF3- dependent genes in response to cytoplasmic poly(I:C). Importantly, reconstitution of FLIP in FLIP-/-MEFs reversed the hyper-activation of IRF3- and NF-κB-mediated gene expression. Further, we found that caspase-8 catalytic activity was not required for cytoplasmic poly(I:C)-mediated NF-κB and IRF3 signaling. CONCLUSIONS: These results provide evidence for a crucial dual role for FLIP in antiviral responses to cytoplasmic dsRNA: it protects from cytoplasmic dsRNA-mediated cell death while down-regulating IRF3-and NF-κB-mediated gene expression. Since the pathogenesis of several viral infections involves a heightened and dysregulated cytokine response, a possible therapy could involve modulating FLIP levels.

Haematological malignancies in sub-Saharan Africa: east Africa as an example for improving care.

Haematological malignancies account for almost 10% of all cancers diagnosed in sub-Saharan Africa, although the exact incidences and treatment outcomes are difficult to discern because population-based cancer registries in the region are still underdeveloped. More research on haematological malignancies in sub-Saharan Africa is required to establish whether these cancers have a natural history similar to those diagnosed in high-income countries, about which more is known. Several factors negatively affect the outcome of haematological malignancies in sub-Saharan Africa, showcasing a need for improved understanding of the clinicobiological profile of these cancers to facilitate prevention, early detection, diagnosis, and appropriate treatment through increased capacity building, infrastructure, community awareness, coordinated resource mobilisation, and collaboration across the world. The east African governments have pooled resources for common investments to tackle non-communicable diseases, developing the East Africa's Centres of Excellence for Skills and Tertiary Education project funded by the African Development Bank, an initiative that could be replicated for the care of haematological malignancies in other countries in sub-Saharan Africa. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Structural characterization of a soluble amyloid beta-peptide oligomer.

Alzheimer's disease (AD) is a neurodegenerative disorder that is linked to the presence of amyloid beta-peptides that can form insoluble fibrils or soluble oligomeric assemblies. Soluble forms are present in the brains and tissues of Alzheimer's patients, and their presence correlates with disease progression. Long-lived soluble forms can be generated in vitro by using small amounts of aliphatic hydrocarbon chains of detergents or fatty acids in preparations of amyloid beta-peptides. Using NMR, we have characterized soluble oligomers of Abeta preglobulomer and globulomer that are stable and alter synaptic activity. The NMR data indicate that these soluble forms have a mixed parallel and antiparallel beta-sheet structure that is different from fibrils which contain only parallel beta-sheets. Using the structural data, we engineered a disulfide bond into the soluble Abeta globulomer to give a "new" soluble antigen that is stable, homogeneous, and binds with the same affinity to selective antibodies as the parent wt globulomer.

Synthesis and evaluation of inhibitors of cytochrome P450 3A (CYP3A) for pharmacokinetic enhancement of drugs.

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2-P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.

Empowering therapeutic antibodies with IFN-α for cancer immunotherapy.

Type 1 IFNs stimulate secretion of IP-10 (CXCL10) which is a critical chemokine to recruit effector T cells to the tumor microenvironment and IP-10 knockout mice exhibit a phenotype with compromised effector T cell generation and trafficking. Type 1 IFNs also induce MHC class 1 upregulation on tumor cells which can enhance anti-tumor CD8 T cell effector response in the tumor microenvironment. Although type 1 IFNs show great promise in potentiating anti-tumor immune response, systemic delivery of type 1 IFNs is associated with toxicity thereby limiting clinical application. In this study, we fused tumor targeting antibodies with IFN-α and showed that the fusion proteins can be produced with high yields and purity. IFN fusions selectively induced IP-10 secretion from antigen positive tumor cells, which was critical in recruiting the effector T cells to the tumor microenvironment. Further, we found that treatment with the anti-PDL1-IFN- α fusion at concentrations as low as 1 pM exhibited potent activity in mediating OT1 CD8+ T cell killing against OVA expressing tumor cells, while control IFN fusion did not exhibit any activity at the same concentration. Furthermore, the IFN-α fusion antibody was well tolerated in vivo and demonstrated anti-tumor efficacy in an anti-PD-L1 resistant syngeneic mouse tumor model. One of the potential mechanisms for the enhanced CD8 T cell killing by anti-PD-L1 IFN fusion was up-regulation of MHC class I/tumor antigen complex. Our data supports the hypothesis of targeting type 1 IFN to the tumor microenvironment may enhance effector T cell functions for anti-tumor immune response.

CD40/anti-CD40 antibody complexes which illustrate agonist and antagonist structural switches.

BACKGROUND: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications. RESULTS: To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies. CONCLUSIONS: The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.

A Bispecific Molecule Targeting CD40 and Tumor Antigen Mesothelin Enhances Tumor-Specific Immunity.

Agonistic CD40 monoclonal antibodies (mAb) have demonstrated some clinical activity, but with dose-limiting toxicity. To reduce systemic toxicity, we developed a bispecific molecule that was maximally active in the presence of a tumor antigen and had limited activity in the absence of the tumor antigen. LB-1 is a bispecific molecule containing single-chain Fv domains targeting mouse CD40 and the tumor antigen mesothelin. LB-1 exhibited enhanced activity upon binding to cell-surface mesothelin but was less potent in the absence of mesothelin binding. In a mouse model implanted with syngeneic 4T1 tumors expressing cell-surface mesothelin, LB-1 demonstrated comparable antitumor activity as an agonistic CD40 mAb but did not cause elevation of serum cytokines and liver enzymes, as was observed in anti-CD40-treated mice. The results from our study of LB-1 were used to develop a human cross-reactive bispecific molecule (ABBV-428) that targeted human CD40 and mesothelin. ABBV-428 demonstrated enhanced activation of antigen-presenting cells and T cells upon binding to cell-surface mesothelin, and inhibition of cultured or implanted PC3 tumor cell growth after immune activation. Although expression of cell-surface mesothelin is necessary, the bispecific molecules induced immune-mediated antitumor activity against both mesothelin+ and mesothelin- tumor cells. ABBV-428 represents a class of bispecific molecules with conditional activity dependent on the binding of a tumor-specific antigen, and such activity could potentially maximize antitumor potency while limiting systemic toxicity in clinical studies.

Fas (CD95) induces macrophage proinflammatory chemokine production via a MyD88-dependent, caspase-independent pathway.

Activation of the prototypical death receptor, Fas (CD95), can induce both caspase-dependent cell death and production of proinflammatory chemokines, leading to neutrophil recruitment and end-organ injury. The precise mechanism(s) by which Fas up-regulates chemokine production and release, is currently unclear. We hypothesized that Fas-induced chemokine release by macrophages is dependent on the MyD88 adaptor molecule and independent of caspase activity. To test this hypothesis, we measured chemokine response to Fas activation both in RAW 264.7 cells with RNAi-attenuated MyD88 expression and in MyD88-deficient primary macrophages. We found that Fas-induced chemokine release was abrogated in the absence of MyD88. In vivo, MyD88(-/-) mice had impaired CXCL1/KC release and polymorphonuclear cell recruitment in response to intratracheal treatment with the Fas-activating monoclonal antibody, Jo-2. Furthermore, Fas-induced chemokine release was not dependent on either IL-1 receptor signaling or on caspase activity. We conclude that MyD88 plays an integral role in Fas-induced macrophage-mediated inflammation.

Whom to treat? Factors associated with chemotherapy recommendations and outcomes among patients with NHL at the Uganda Cancer Institute.

INTRODUCTION: Cancer treatment options in sub-Saharan Africa are scarce despite an increasing burden of disease. Identification of those cancer patients who would benefit most from the limited resources available would allow broader and more effective therapy. METHODS: We conducted a retrospective analysis of patients over the age of 18 at the time of a pathologic diagnosis of NHL between 2003 and 2010 who were residents of Kyandondo County (Uganda) and presented to the Uganda Cancer Institute for care. RESULTS: A total of 128 patients were included in this analysis. Chemotherapy was recommended to 117 (91.4%) of the patients; the odds of recommending chemotherapy decreased for each additional month of reported symptoms prior to diagnosis. Of the 117 patients to whom chemotherapy was recommended, 111 (86.7%) patients received at least 1 cycle of chemotherapy; HIV infected patients, as well as those with a lower hemoglobin and advanced disease at the time of diagnosis were significantly less likely to complete therapy. Among the patients who initiated chemotherapy, twenty patients died prior to treatment completion (including nine who died within 30 days). Hemoglobin level at the time of presentation was the only variable associated with early mortality in the adjusted model. CONCLUSION: In resource-poor areas, it is essential to align health care expenditures with interventions likely to provide benefit to affected populations. Targeting cancer therapy to those with a favorable chance of responding will not only save limited resources, but will also prevent harm in those patients unlikely to realize an effect of cancer-directed therapy.

Association Between HIV Infection and Cancer Stage at Presentation at the Uganda Cancer Institute.

PURPOSE: The HIV epidemic has contributed to the increasing incidence of cancer in sub-Saharan Africa, where most patients with cancer present at an advanced stage. However, improved access to HIV care and treatment centers in sub-Saharan Africa may facilitate earlier diagnosis of cancer among patients who are HIV positive. To test this hypothesis, we characterized the stage of cancer and evaluated the factors associated with advanced stage at presentation among patients in Uganda. METHODS: We conducted a retrospective analysis of adult patients with any of four specific cancers who presented for care in Kampala, Uganda, between 2003 and 2010. Demographic, clinical, and laboratory data were abstracted from the medical record, together with the outcome measure of advanced stage of disease (clinical stage III or IV). We identified measures for inclusion in a multivariate logistic regression model. RESULTS: We analyzed 731 patients with both AIDS-defining cancers (cervical [43.1%], and non-Hodgkin lymphoma [18.3%]), and non-AIDS-defining cancers (breast [30.0%] and Hodgkin lymphoma [8.6%]). Nearly 80% of all patients presented at an advanced stage and 37% had HIV infection. More than 90% of patients were symptomatic and the median duration of symptoms before presentation was 5 months. In the multivariate model, HIV-positive patients were less likely to present at an advanced stage as were patients with higher hemoglobin and fewer symptoms. CONCLUSION: Patients with limited access to primary care may present with advanced cancer because of a delay in diagnosis. However, patients with HIV now have better access to clinical care. Use of this growing infrastructure to increase cancer screening and referral is promising and deserves continued support, because the prognosis of HIV-positive patients with advanced cancer is characterized by poor survival globally.

The Fas-associated death domain protein suppresses activation of NF-kappa B by LPS and IL-1 beta.

Activation of NF-kappa B by bacterial LPS promotes the upregulation of proinflammatory cytokines that contribute to the pathogenesis of Gram-negative septic shock. LPS activation of NF-kappa B is dependent upon the interaction of two death domain-containing (DD-containing) proteins, MyD88 and IL-1 receptor-associated kinase IRAK. Another DD-containing protein, Fas-associated death domain (FADD), also binds MyD88 through respective DD-DD interactions. Although FADD has been classically described as a proapoptotic signaling molecule, several reports have implicated a role for FADD in mediating NF-kappa B activation. In the present report, we investigated whether FADD could mediate LPS activation of NF-kappa B. Overexpression of FADD blocked LPS-induced NF-kappa B activation, whereas absence of FADD enhanced activation of NF-kappa B by LPS. Further, LPS-induced expression of two NF-kappa B-dependent gene products, IL-6 and KC, was enhanced in FADD(-/-) mouse embryo fibroblasts (MEFs) compared with wild-type. This increase in NF-kappa B activity correlated with enhanced I kappa B degradation. FADD(-/-) MEFs were also resistant to NF-kappa B activation induced by IL-1 beta. Finally, reconstitution of full-length FADD in the FADD(-/-) MEFs completely reversed the enhanced activation of NF-kappa B elicited by either LPS or IL-1 beta. Together, these data indicate that FADD negatively regulates LPS- and IL-1 beta-induced NF-kappa B activation and that this regulation occurs upstream of I kappa B degradation.

GEA 3162, a peroxynitrite donor, induces Bcl-2-sensitive, p53-independent apoptosis in murine bone marrow cells.

Apoptosis may be regulated by oxidants such as peroxynitrite (ONOO(-)). The tumour suppressor, p53, has been reported to play a crucial role in apoptosis induced by oxidants, therefore we assessed the ability of a ONOO(-) donor, GEA 3162, to activate caspases and induce mitochondrial permeability in a p53-deficient murine bone marrow cell line, Jaws II. Furthermore, these cells were stably transfected with Bcl-2, in order to investigate the impact of this survival protein on ONOO(-)-induced apoptosis. GEA 3162 activated caspases and induced loss of mitochondrial membrane potential in Jaws II cells. In particular, caspases 3 and 2 were activated, alongside minor activation of caspases 8 and 9, and apoptosis was partially dependent upon p38 MAP kinase activation, with little or no role for JNK. Overexpression of Bcl-2 abolished activation of all caspases and reduced the change in mitochondrial membrane potential. Thus, we have demonstrated that the ONOO(-) donor, GEA 3162, induces apoptosis in Jaws II murine myeloid cells despite lacking functional p53, via a pathway that principally involves caspases 2 and 3 and mitochondrial changes. This is blocked by overexpression of Bcl-2 via a mechanism that does not appear to merely reflect stabilisation of the mitochondrial membrane.

A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.