A combinatorial disulfide cross-linking strategy was used to prepare a stalled complex of human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase with a DNA template:primer and a deoxynucleoside triphosphate (dNTP), and the crystal structure of the complex was determined at a resolution of 3.2 angstroms. The presence of a dideoxynucleotide at the 3'-primer terminus allows capture of a state in which the substrates are poised for attack on the dNTP. Conformational changes that accompany formation of the catalytic complex produce distinct clusters of the residues that are altered in viruses resistant to nucleoside analog drugs. The positioning of these residues in the neighborhood of the dNTP helps to resolve some long-standing puzzles about the molecular basis of resistance. The resistance mutations are likely to influence binding or reactivity of the inhibitors, relative to normal dNTPs, and the clustering of the mutations correlates with the chemical structure of the drug.

Genetic alterations that activate the mitogen-activated protein kinase (MAP kinase) pathway occur commonly in cancer. For example, the majority of melanomas harbor mutations in the BRAF oncogene, which are predicted to confer enhanced sensitivity to pharmacologic MAP kinase inhibition (e.g., RAF or MEK inhibitors). We investigated the clinical relevance of MEK dependency in melanoma by massively parallel sequencing of resistant clones generated from a MEK1 random mutagenesis screen in vitro, as well as tumors obtained from relapsed patients following treatment with AZD6244, an allosteric MEK inhibitor. Most mutations conferring resistance to MEK inhibition in vitro populated the allosteric drug binding pocket or ␣-helix C and showed robust (Ϸ100-fold) resistance to allosteric MEK inhibition. Other mutations affected MEK1 codons located within or abutting the Nterminal negative regulatory helix (helix A), which also undergo gain-of-function germline mutations in cardio-facio-cutaneous (CFC) syndrome. One such mutation, MEK1(P124L), was identified in a resistant metastatic focus that emerged in a melanoma patient treated with AZD6244. Both MEK1(P124L) and MEK1(Q56P), which disrupts helix A, conferred cross-resistance to PLX4720, a selective B-RAF inhibitor. However, exposing BRAF-mutant melanoma cells to AZD6244 and PLX4720 in combination prevented emergence of resistant clones. These results affirm the importance of MEK dependency in BRAF-mutant melanoma and suggest novel mechanisms of resistance to MEK and B-RAF inhibitors that may have important clinical implications.BRAF ͉ drug resistance ͉ MAP kinase ͉ melanoma A pproximately one-third of all cancers harbor genetic alterations that aberrantly upregulate mitogen-activated protein kinase (MAPK)-dependent signal transduction (1). In the MAPK pathway, RAS oncoproteins activate RAF, MEK, and ERK kinases to direct key cell proliferative and survival signals. When rendered constitutively active by genetic mutation, the MAP kinase pathway is believed to confer ''oncogene dependency'' (2), an excessive reliance on its dysregulated activity for tumor viability. Therefore, protein kinases within this signaling cascade offer promising targets for novel anticancer therapeutics.In melanoma, uncontrolled MAP kinase pathway activity is nearly ubiquitous and occurs most commonly through gain-offunction mutations involving codon 600 of the B-RAF kinase (3) (BRAF V600E ; 50-70% of cases). Considerable preclinical evidence has associated the BRAF V600E mutation with heightened sensitivity to pharmacologic inhibition of RAF or MEK kinases (4, 5). Although early clinical trials of RAF and MEK inhibitors failed to show a substantial benefit (6, 7), recent phase I studies of selective RAF inhibitors have shown promising results in patients with BRAF-mutant tumors (8, 9). Thus, optimizing therapeutic efficacy while avoiding or bypassing the emergence of resistance to MAP kinase pathway inhibition will likely gain increasing importance in melanoma and other MAP kinasedriven cancers.He...

Immunotherapy for Alzheimer's disease (AD) relies on antibodies directed against toxic amyloid-beta peptide (Aβ), which circulate in the bloodstream and remove Aβ from the brain [1], [2]. In mouse models of AD, the administration of anti-Aβ antibodies directly into the brain, in comparison to the bloodstream, was shown to be more efficient at reducing Aβ plaque pathology [3], [4]. Therefore, delivering anti-Aβ antibodies to the brain of AD patients may also improve treatment efficiency. Transcranial focused ultrasound (FUS) is known to transiently-enhance the permeability of the blood-brain barrier (BBB) [5], allowing intravenously administered therapeutics to enter the brain [6]–[8]. Our goal was to establish that anti-Aβ antibodies delivered to the brain using magnetic resonance imaging-guided FUS (MRIgFUS) [9] can reduce plaque pathology. To test this, TgCRND8 mice [10] received intravenous injections of MRI and FUS contrast agents, as well as anti-Aβ antibody, BAM-10. MRIgFUS was then applied transcranially. Within minutes, the MRI contrast agent entered the brain, and BAM-10 was later found bound to Aβ plaques in targeted cortical areas. Four days post-treatment, Aβ pathology was significantly reduced in TgCRND8 mice. In conclusion, this is the first report to demonstrate that MRIgFUS delivery of anti-Aβ antibodies provides the combined advantages of using a low dose of antibody and rapidly reducing plaque pathology.