Accumulation of very long chain fatty acids (VLCFAs) due to defects in ATP binding cassette protein D1 (ABCD1) is thought to underlie the pathologies observed in adrenoleukodystrophy (ALD). Pursuing a substrate reduction approach based on the inhibition of elongation of very long chain fatty acid 1 enzyme (ELOVL1), we explored a series of thiazole amides that evolved into compound 27─a highly potent, central nervous system (CNS)-penetrant compound with favorable in vivo pharmacokinetics. Compound 27 selectively inhibits ELOVL1, reducing C26:0 VLCFA synthesis in ALD patient fibroblasts, lymphocytes, and microglia. In mouse models of ALD, compound 27 treatment reduced C26:0 VLCFA concentrations to near-wild-type levels in blood and up to 65% in the brain, a disease-relevant tissue. Preclinical safety findings in the skin, eye, and CNS precluded progression; the origin and relevance of these findings require further study. ELOVL1 inhibition is an effective approach for normalizing VLCFAs in models of ALD.

There are currently no treatments for life-threatening infections caused by human polyomaviruses JCV and BKV. We therefore report herein the first crystal structure of the hexameric helicase of JCV large T antigen (apo) and its use to drive the structure-based design of dual JCV and BKV ATP-competitive inhibitors. The crystal structures obtained by soaking our early inhibitors into the JCV helicase allowed us to rapidly improve the biochemical activity of our inhibitors from 18 μM for the early 6-(2-methoxyphenyl)- and the 6-(2-ethoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole hits 1a and 1b to 0.6 μM for triazolopyridine 12i. In addition, we were able to demonstrate measurable antiviral activity in Vero cells for our thiazolopyridine series in the absence of marked cytotoxicity, thus confirming the usefulness of this approach.

UNLABELLED:The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5BΔ21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5BΔ21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5BΔ21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5BΔ21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication. IMPORTANCE:The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood, but viral and host proteins collaborate in this process. Using a rolling-circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A(S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5BΔ21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors.

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of two membrane proteins: calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a class B G-protein-coupled receptor (GPCR), possessing a characteristic large amino-terminal extracellular domain (ECD) important for ligand recognition and binding. Dimerization of CLR with RAMP1 provides specificity for CGRP versus related agonists. Here we report the expression, purification, and refolding of a soluble form of the CGRP receptor comprising a heterodimer of the CLR and RAMP1 ECDs. The extracellular protein domains corresponding to residues 23-133 of CLR and residues 26-117 of RAMP1 were shown to be sufficient for formation of a stable, monodisperse complex. The binding affinity of the purified ECD complex for the CGRP peptide was significantly lower than that of the native receptor (IC(50) of 12 microM for the purified ECD complex vs 233 pM for membrane-bound CGRP receptor), indicating that other regions of CLR and/or RAMP1 are important for peptide agonist binding. However, high-affinity binding to known potent and specific nonpeptide antagonists of the CGRP receptor, including olcegepant and telcagepant (K(D) < 0.02 muM), as well as N-terminally truncated peptides and peptide analogues (140 nM to 1.62 microM) was observed.

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

Structure-based drug design (SBDD) has played an integral role in the development of highly specific, potent protease inhibitors resulting in a number of drugs in clinical trials and on the market. Possessing biochemical assays and structural information on both the target protease and homologous family members helps ensure compound selectivity. We have redesigned the path from clone to protein eliminating many of the traditional bottlenecks associated with protein production to ensure a constant supply to feed many diverse protease drug discovery programs. The process was initiated with the design of a multi-system vector, capable of expression in both eukaryotic and prokaryotic hosts; this vector also facilitated high-throughput cloning, expression and purification. When combined into an expression screen, supplemented with salvage screens for detergent extraction and refolding, a route for protein production was established rapidly. Using this process-orientated approach we have successfully expressed and purified all mechanistic classes of active human and viral proteases for enzymatic assays and crystallization studies. While exploiting recent developments in high-throughput biochemistry, we still employ classical biophysical techniques such as light-scattering and analytical ultracentrifugation, to ensure the highest quality protein enters crystallization trials. We have drawn on examples from our own research programs to illustrate how these strategies have been successfully used in the production of proteases for SBDD.

Aurora-2 is a key member of a closely related subgroup of serine/threonine kinases that plays important roles in the completion of essential mitotic events. Aurora-2 is oncogenic and amplified in various human cancers and could be an important therapeutic target for inhibitory molecules that would disrupt the cell cycle and block proliferation. We report the first crystal structure of Aurora-2 kinase in complex with adenosine. Analysis of residues in the active site suggests differences with structurally and biologically related protein kinases. The activation loop, which contains residues specific to the Aurora family of kinases, has a unique conformation. These results provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of the Aurora kinases.

GSK3 beta was identified as the kinase that phosphorylates glycogen synthase but is now known to be involved in multiple signaling pathways. GSK3 beta prefers prior phosphorylation of its substrates. We present the structure of unphosphorylated GSK3 beta at 2.7 A. The orientation of the two domains and positioning of the activation loop of GSK3 beta are similar to those observed in activated kinases. A phosphate ion held by Arg 96, Arg 180 and Lys 205 occupies the same position as the phosphate group of the phosphothreonine in activated p38 gamma, CDK2 or ERK2. A loop from a neighboring molecule in the crystal occupies a portion of the substrate binding groove. The structure explains the unique primed phosphorylation mechanism of GSK3 beta and how GSK3 beta relies on a phosphoserine in the substrate for the alignment of the beta- and alpha -helical domains.

Background: Peptide inhibitors of caspases have helped define the role of these cysteine proteases in biology. Structural and biochemical characterization of the caspase enzymes may contribute to the development of new drugs for the treatment of caspase-mediated inflammation and apoptosis.Results: The crystal structure of the previously unpublished caspase-7 (Csp7; 2.35 Angstrom) bound to the reversible tetrapeptide aldehyde inhibitor acetyl-Asp-Glu-Val-Asp-CHO is compared with crystal structures of caspases-1 (2.3 Angstrom), -3 (2.2 Angstrom),and -8 (2.65 Angstrom) bound to the same inhibitor. Csp7 is a close homolog of caspase-3 (Csp3), and these two caspases possess some quarternary structural characteristics that support their unique role among the caspase family. However, although Csp3 and Csp7 are quite similar overall, they were found to have a significantly different substitution pattern of amino acids in and around the S4-binding site.Conclusions: These structures span all three caspase subgroups, and provide a basis for inferring substrate and inhibitor binding, as well as selectivity for the entire caspase family. This information will influence the design of selective caspase inhibitors to further elucidate the role of caspases in biology and hopefully lead to the design of therapeutic agents to treat caspase-mediated diseases, such as rheumatoid arthritis, certain neurogenerative diseases and stroke.

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G, T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation.

BACKGROUND:The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein (MAP) kinase family, and regulate signal transduction in response to environmental stress. Activation and nuclear localization of JNK3, a neuronal-specific isoform of JNK, has been associated with hypoxic and ischemic damage of CA1 neurons in the hippocampus. Knockout mice lacking JNK3 showed reduced apoptosis of hippocampal neurons and reduced seizure induced by kainic acid, a glutamate-receptor agonist. Thus, JNK3 may be important in the pathology of neurological disorders and is of significant medical interest.RESULTS:We report here the structure of unphosphorylated JNK3 in complex with adenylyl imidodiphosphate, an ATP analog. JNK3 has a typical kinase fold, with the ATP-binding site situated within a cleft between the N- and C-terminal domains. In contrast to other known MAP kinase structures, the ATP-binding site of JNK3 is well ordered; the glycine-rich nucleotide-binding sequence forms a beta-strand-turn-beta-strand structure over the nucleotide. Unphosphorylated JNK3 assumes an open conformation, in which the N- and C-terminal domains are twisted apart relative to their positions in cAMP-dependent protein kinase. The rotation leads to the misalignment of some of the catalytic residues. The phosphorylation lip of JNK3 partially blocks the substrate-binding site.CONCLUSIONS:This is the first JNK structure to be determined, providing a unique opportunity to compare structures from the three MAP kinase subfamilies. The structure reveals atomic-level details of the shape of JNK3 and the interactions between the kinase and the nucleotide. The misalignment of catalytic residues and occlusion of the active site by the phosphorylation lip may account for the low activity of unphosphorylated JNK3. The structure provides a framework for understanding the substrate specificity of different JNK isoforms, and should aid the design of selective JNK3 inhibitors.