Jamie S. Simpson

The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.

TERPENE METABOLITES FROM THE TROPICAL MARINE SPONGE AXINYSSA SP. NOV.

A new sesquiterpene isothiocyanate, ( – )-9-isothiocyanatopupukeanane (4), has been isolated along with the known sesquiterpene metabolites ( – )-9-isocyanopupukeanane (1), ( – )-2-thiocyanatoneopupukeanane (2) and ( – )-epipolasin-A (3) from the sponge Axinyssa sp. nov. Metabolites (1), (2) and (4) showed modest in vitro antimalarial activity.

Targeted delivery of a model immunomodulator to the lymphatic system: comparison of alkyl ester versus triglyceride mimetic lipid prodrug strategies.

A lipophilic prodrug approach has been used to promote the delivery of a model immunomodulator, mycophenolic acid (MPA), to the lymphatic system after oral administration. Lymphatic transport was employed to facilitate enhanced drug uptake into lymphocytes, as recent studies demonstrate that targeted drug delivery to lymph resident lymphocytes may enhance immunomodulatory effects. Two classes of lymph-directing prodrugs were synthesised. Alkyl chain derivatives (octyl mycophenolate, MPA-C8E; octadecyl mycophenolate, MPA-C18E; and octadecyl mycophenolamide, MPA-C18AM), to promote passive partitioning into lipids in lymphatic transport pathways, and a triglyceride mimetic prodrug (1,3-dipalmitoyl-2-mycophenoloyl glycerol, 2-MPA-TG) to facilitate metabolic integration into triglyceride deacylation-reacylation pathways. Lymphatic transport, lymphocyte uptake and plasma pharmacokinetics were assessed in mesenteric lymph and carotid artery cannulated rats following intraduodenal infusion of lipid-based formulations containing MPA or MPA prodrugs. Patterns of prodrug hydrolysis in rat digestive fluid, and cellular re-esterification in vivo, were evaluated to examine the mechanisms responsible for lymphatic transport. Poor enzyme stability and low absorption appeared to limit lymphatic transport of the alkyl derivatives, although two of the three alkyl chain prodrugs - MPA-C18AM (6-fold) and MPA-C18E (13-fold) still increased lymphatic drug transport when compared to MPA. In contrast, 2-MPA-TG markedly increased lymphatic drug transport (80-fold) and drug concentrations in lymphocytes (103-fold), and this was achieved via biochemical incorporation into triglyceride deacylation-reacylation pathways. The prodrug was hydrolysed rapidly to 2-mycophenoloyl glycerol (2-MPA-MG) in the presence of rat digestive fluid, and 2-MPA-MG was subsequently re-esterified in the enterocyte with oleic acid (most likely originating from the co-administered formulation) prior to accessing the lymphatics and lymphocytes. Importantly, after administration of 2-MPA-TG, the concentrations of free MPA in the mesenteric lymph nodes were significantly enhanced (up to 28 fold) when compared to animals administered equimolar quantities of MPA, suggesting the efficient conversion of the esterified prodrug back to the pharmacologically active parent drug. The data suggest that triglyceride mimetic prodrugs have potential as a means of enhancing immunotherapy via drug targeting to lymphocytes and lymph nodes.

Metallocyclodextrin catalysts for hydrolysis of phosphate triesters

Abstract The copper complexes of 6A-(2-aminoethylamino)- and 6A-(3-aminopropylamino)-6A-deoxy-β-cyclodextrin catalyse the hydrolysis of 4-tert-butyl-2-nitrophenyl dimethyl phosphate, with kinc=3.1×10−2 and 2.3×10−2 s−1, and Kd=4.3×10−4 and 1.2×10−3 M, respectively, in 0.05 M pH 7.0 HEPES buffer at 298 K. This corresponds to rate accelerations of more than 95 000 and 70 000 times for reaction of the cyclodextrin-bound species.

THIOCYANATE BIOSYNTHESIS IN THE TROPICAL MARINE SPONGE AXINYSSA N.SP.

The biosynthetic origin of the thiocyanate carbon in 2- thiocyanatoneopupukeanane (1) is defined by incorporation of sodium [C] cyanide and [C] thiocyanate into Axinyssa n.sp. The specificity of incorporation is demonstrated by reduction of (1) to the thiol (7).

Structure and Function of the Oxidoreductase DsbA1 from Neisseria meningitidis

Neisseria meningitidis encodes three DsbA oxidoreductases (NmDsbA1-NmDsbA3) that are vital for the oxidative folding of many membrane and secreted proteins, and these three enzymes are considered to exhibit different substrate specificities. This has led to the suggestion that each N. meningitidis DsbA (NmDsbA) may play a specialized role in different stages of pathogenesis; however, the molecular and structural bases of the different roles of NmDsbAs are unclear. With the aim of determining the molecular basis for substrate specificity and how this correlates to pathogenesis, we undertook a biochemical and structural characterization of the three NmDsbAs. We report the 2.0-A-resolution crystal structure of the oxidized form of NmDsbA1, which adopted a canonical DsbA fold similar to that observed in the structures of NmDsbA3 and Escherichia coli DsbA (EcDsbA). Structural comparisons revealed variations around the active site and candidate peptide-binding region. Additionally, we demonstrate that all three NmDsbAs are strong oxidases with similar redox potentials; however, they differ from EcDsbA in their ability to be reoxidized by E. coli DsbB. Collectively, our studies suggest that the small structural differences between the NmDsbA enzymes and EcDsbA are functionally significant and are the likely determinants of substrate specificity.

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief

Therapeutic targeting of the neurokinin 1 receptor in endosomes provides efficacious and prolonged pain relief. Targeting the enemy within endosomes With opioid addiction on the rise, there is a great need for effective nonopioid approaches to treat pain. Jensen et al. examined the function of substance P neurokinin 1 receptor, which plays a role in the transmission of pain signals in the central nervous system. The authors demonstrated that endocytosis of this receptor is required for the transmission of pain signals. Although systemic inhibition of endocytosis would not be feasible in a living organism, the authors discovered another way to take advantage of this information. They conjugated neurokinin 1 receptor antagonists to cholestanol, promoting their incorporation into endosomes, where they successfully inhibited their target to block pain transmission. Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)–coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK1R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK1R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane–targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and β-arrestin blocked SP-induced NK1R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal–regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK1R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK1R antagonists. These results reveal a critical role for endosomal signaling of the NK1R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.

New Chlorinated Metabolites from the Tropical Marine Sponge Dysidea herbacea

Three new chlorinated metabolites, namely 3-(3,3-dichloro-2-methylpropyl)-6-(3,3-dichloro-2-methylpropylidene)-1,4- dimethylpiperazine-2,5-dione (3), 1-methyl-3,3,3-trichloro-2-methylpropyl)-6-(3,3,3-trichloro-2-methylpropylidene) piperazine-2,5-dione (4) and 5,5-dichloro-4-methyl-2-[methyl(4,4-dichloro-3-methyl-l-oxobutyl)amino]-N- (thiazol-2-ylmethyl)pentanamide (8), have been isolated from collections of Dysidea herbacea from the southern region of the Great Barrier Reef. The relative and absolute stereochemistry of (4) was determined by X-ray crystallographic analysis.

Biosynthesis of dichloroimines in the tropical marine sponge Stylotella aurantium

Abstract The biosynthetic orgin of the dichloroimine carbon in the stylotellanes A and B, (1) and (2), is defined by specific incorporation of sodium [ 14 C] cyanide. Sodium [ 14 C] thiocyanate is also involved in their biosynthesis. A mechanistic scheme is presented for the formation of these bioactive metabolites.

Design and Evaluation of the Performance of an NMR Screening Fragment Library

The design of a suitable library is an essential prerequisite to establish a fragment-based screening capability. Several pharmaceutical companies have described their approaches to establishing fragment libraries; however there are few detailed reports of both design and analysis of performance for a fragment library maintained in an academic setting. Here we report our efforts towards the design of a fragment library for nuclear magnetic resonance spectroscopy-based screening, demonstrate the performance of the library through analysis of 14 screens, and present a comparison to previously reported fragment libraries.