Crystallographic evidence suggests that there is a large hinged domain motion associated with substrate binding in adenylate kinase. To test this hypothesis, resonance energy transfer measurements of substrate binding were initiated. Adenylate kinase from Escherichia coli consists of three domains: the main body of the enzyme with alpha-helical and beta-sheet secondary structure, and domains that close over the AMP and ATP binding sites. Four single tryptophan mutants were constructed to map distances. Two tryptophan mutants were positioned at residues 133 (Y133W) and 137 (F137W), which are in the domain that closes over the ATP binding site. Mutant F86W that is located at the AMP binding site, and mutant S41W that is in the loop that close over AMP, complete the mapping library. Energy transfer was measured between each of these tryptophans and 5-[[2-(acetylamino)ethyl]amino]naphthalene-1-sulfonic acid (AEDANS) covalently bound to the single cysteine residue at position 77, which is located in the main body of adenylate kinase. The distance between the tryptophan of the F137W mutant adenylate kinase and the AEDANS-labeled Cys-77 decreased by 12.1 A upon the binding of the bisubstrate inhibitor P1, P5-bis(5'-adenosyl) pentaphosphate (AP5A). There were only small alterations in the tryptophan to Cys-77-AEDANS distances in the Y133W, F86W, and S41W mutants upon the binding of AP5A, ATP, or AMP, implying that movement of residues 133, 86, and 41 in relation to the Cys-77 residue was minimal. These results suggest that there is significant closure of the ATP binding domain upon the binding of ATP or AP5A. Unexpectedly, exposure of the enzyme to AMP also introduced a partial closure of the ATP hinged domain.

Researchers at Alnylam Pharmaceuticals Inc. and the Massachusetts Institute of Technology have developed a class of lipid-like carriers that are nearly 100 times more efficient at delivering small interfering RNA than previously studied lipid-based carriers.1 The parties are now scaling up production of one of the carriers-dubbed lipidoids-and are optimizing it in rodent and nonhuman primate disease models.If successful, lipidoids could address one of the most significant challenges facing RNAi therapeutics: safely and efficiently delivering the molecules to diseased tissue.2 Naked siRNA is the simplest option but has primarily been limited to diseases of the eye, in which direct local delivery is possible. For most other indications, systemic delivery is the only way to go, requiring a carrier to package and protect the molecules from serum nucleases and to selectively target the compounds to the desired tissue.Lipidoids and lipids share many of the physicochemical properties that drive the formation of liposomes for RNAi delivery. However, lipidoids require many fewer steps to synthesize and purify. Those advantages make high throughput combinatorial synthesis of lipidoids and rapid in vitro screening of thousands of potential drug delivery candidates possible.The Alnylam-MIT team previously evaluated a library of more than 1,200 lipidoids, the best of which delivered siRNA in three different animal models: mice, rats and nonhuman primates.3,4 However, the first-generation lipidoids had efficacies that were only comparable to those for existing stable nucleic acid lipid particle formulations, thus requiring siRNA loads in the mg/kg body weight range.The team hoped to improve the therapeutic index and reduce the off-target toxicity of the siRNA loads by lowering dosing to the µg/kg range, and they produced a second-generation library of 126 epoxidederived lipidoids.A cell-based screen of the library identified 12 lipidoids that delivered sufficient siRNA to reduce luciferase gene expression by at least 80% in a transfected cell line. In mouse livers, one of the lipidoids-C12-200-delivered 3, 10 and 30 µg/kg of an siRNA against Factor VII that reduced gene expression by about 25%, 50% and 80%, respectively.Most lipid-based delivery carriers require siRNA doses of at least 1 mg/kg to get more than 50% gene silencing."The lipidoid-siRNA formulation described in the PNAS paper showed an incredibly high efficacy, perhaps two or even three orders of magnitude higher than most previously reported lipid-based RNAi delivery formulations, " said Niren Murthy, associate professor of biomedical engineering at the Georgia Institute of Technology.Given the efficient gene silencing by siRNA delivered with C12-200, the researchers hypothesized that the lipidoid might be able to simultaneously deliver multiple siRNA molecules to the liver without generating toxicity.In mice, a single dose of C12-200 complexed with 0.1 mg/kg of five different siRNAs reduced expression of all five genes by at least 50% compared with C12-200 compl...

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American and French researchers have identified CXC chemokine receptor 1 as a new target for blocking the formation of breast cancer stem cells that drive tumor growth and metastasis. 1 The findings could represent a repurposing opportunity for Italian biotech Dompe Farmaceutici S.p.A., which is exploring new indications for its small molecule inhibitor of the receptor after the compound missed the primary endpoint in Phase II transplant dysfunction trials.The therapeutic rationale for targeting cancer stem cells is clear: deplete the subset of cancer cells with the ability to self-renew and generate the full range of cells that make up a bulk tumor 2 so the disease cannot progress or relapse. The challenge has been identifying druggable targets unique to cancer stem cells.Researchers at the University of Michigan and the Institut National de la Santé et de la Recherche Médicale (INSERM) have used gene expression profiling to identify a breast cancer stem cell signature. 3 In 2009, the team found that CXC chemokine receptor 1 (CXCR1) was among the genes almost exclusively expressed in the cancer stem cell population compared with its expression in bulk tumor cells.CXCR1 is a receptor for IL-8 (CXCL8), a proinflammatory chemokine that has been implicated in the metastasis and progression of multiple malignancies, including glioma and prostate, breast and ovarian cancers. 4-7 IL-8 has also been shown to stimulate self-renewal of breast cancer stem cells in vitro. 2 The UM-INSERM team now has taken the next step and studied the effects of small molecule and antibody antagonists of CXCR1.In a human breast cancer cell line, both the small molecule CXCR1 inhibitor reparixin and an anti-CXCR1 antibody reduced the number of breast cancer stem cells compared with no treatment.After four days, both molecules abolished the entire cancer cell population even though CXCR1-expressing stem cells comprised about 2% of the total. That result suggested that CXCR1 blockade directly decreased the survival of cancer stem cells and indirectly induced cell death in the bulk tumor population.Mechanistic studies showed that inhibiting CXCR1 induced the production of soluble Fas ligand (TNF superfamily, member 6; FASL) by cancer stem cells. The proapoptotic FASL then bound its receptor on bulk tumor cells, triggering apoptosis and cell death (see Figure 1, "Chemokine blockade in breast cancer").In mice with primary human breast cancer xenografts, reparixin alone or in combination with docetaxel reduced tumor growth compared with saline control. The combination had greater efficacy than either reparixin or docetaxel alone.Additional experiments showed that reparixin could reduce cancer metastasis. Three groups of mice were injected with breast cancer cell lines. The animals then received reparixin twice daily for 28 days. At the end of that period, two of the three groups showed significant reductions in metastasis compared with animals that received saline control (p<0.01).The team was led by Max Wicha, professor of oncology and intern...

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A Roche team has concluded that overreliance on simplistic, targetcentered screens early in drug development may inadvertently select for compounds destined to fail in late-stage trials. 1 The resulting high attrition rates and low R&D productivity could be reversed, the researchers believe, by refocusing discovery strategies on phenotypic screens that account for complex mechanisms of action and cell signaling pathways.

Researchers at Dynavax Technologies Corp. and the Baylor Institute for Immunology Research have found that toll-like receptor antagonists could offer a way to lower steroid dosage and thus reduce side effects in lupus patients. 1 Dynavax has a toll-like receptor 7 and toll-like receptor 9 antagonist poised to enter lupus trials within the year. Lupus is a chronic autoimmune disease characterized by excess production of autoantibodies and proinflammatory cytokines that cause severe damage to multiple tissues, including the skin, joints and kidneys. There are no targeted therapies marketed for lupus. Instead, depending on disease severity, patients receive varying doses of oral or i.v. glucocorticoids in combination with hydroxychloroquine and/or immunosuppressants such as cyclophosphamide and mycophenolate. Hydroxychloroquine is approved for malaria and for lupus, although its mechanism of action is not entirely clear in the latter. Compared with other autoimmune indications, lupus often requires high doses of glucocorticoids to reduce the frequency of flares and treat disease, putting patients at risk of a broad range of side effects including hypertension, osteoporosis, hyperglycemia, obesity and retinopathy. The Dynavax-Baylor team hypothesized that in lupus, unlike in other autoimmune diseases, the anti-inflammatory effects of glucocorticoids are offset by a proinflammatory pathway in immune cells, leading to the need for high-dose steroids. The group reasoned that blocking that unknown proinflammatory pathway could lead to steroid-sparing treatment regimens. The team first isolated plasmacytoid dendritic cells (pDCs) from lupus patients and tested the response of the cells to glucocorticoids in vitro. pDCs are a key contributor to lupus pathogenesis-they bind autoantigen nucleic acids at toll-like receptor 7 (TLR7) and TLR9 and then trigger downstream production of interferon-α (IFNA; IFN-α) and other proinflammatory cytokines as well as autoantibody-producing B cells (see Figure 1, "Targeting toll-like receptor 7 and 9 in lupus"). 2,3 In the pDCs, glucocorticoids failed to lower production of IFN-α in the presence of two different TLR7-and TLR9-activating ligands. In contrast, glucocorticoids lowered IFN-α production in the presence of a dual TLR7 and TLR9 antagonist. Those results suggested that signaling through TLR7 and TLR9 made pDCs resistant to glucocorticoids and thus suggested a way to counteract the resistance with dual TLR7 and TLR9 antagonists. In two mouse models of lupus, glucocorticoids plus a dual TLR7 and TLR9 antagonist significantly lowered pDC counts compared with glucocorticoids alone (p=0.001). Reductions in pDC levels also correlated with lower expression of IFN-α-regulated genes. The study, reported in Nature, was led by Franck Barrat, director of drug discovery at Dynavax, Robert Coffman, VP and CSO at the company, and Virginia Pascual, principal investigator at the Baylor Institute.