N-Benzyl-indolo carboxylic acids: Design and synthesis of potent and selective adipocyte fatty-acid binding protein (A-FABP) inhibitors

Small molecule inhibitors of adipocyte fatty-acid binding protein (A-FABP) have gained renewed interest following the recent publication of pharmacologically beneficial effects of such inhibitors. Despite the potential utility of selective A-FABP inhibitors within the fields of metabolic disease, inflammation and atherosclerosis, there are few examples of useful A-FABP inhibitors in the public domain. Herein, we describe the optimization of N-benzyl-tetrahydrocarbazole derivatives through the use of co-crystal structure guided medicinal chemistry efforts. This led to the identification of a potent and selective class of A-FABP inhibitors as illustrated by N-benzyl-hexahydrocyclohepta[b]indole 30.     

Cocaine and exercise: alpha-1 receptor blockade does not alter muscle glycogenolysis or blood lactacidosis.

In our previous work, we routinely observed that a combined cocaine-exercise challenge results in an abnormally rapid muscle glycogen depletion and excessive blood lactacidosis. These phenomena occur simultaneously with a rapid rise in norepinephrine and in the absence of any rise in epinephrine. We postulated that norepinephrine may cause vasoconstriction of the muscle vasculature through activation of alpha-1 receptors during cocaine-exercise, thus inducing hypoxia and a concomitant rise in glycogenolysis and lactate accumulation. To test this hypothesis, rats were pretreated with the selective alpha-1-receptor antagonist prazosin (P) (0.1 mg/kg iv) or saline (S). Ten minutes later, the animals were treated with cocaine (-C) (5 mg/kg iv) or saline (-S) and run for 4 or 15 min at 22 m/min at 10% grade. In the S-S group, glycogen content of the white vastus lateralis muscle was unaffected by exercise at both time intervals, whereas in S-C rats glycogen was reduced by 47%. This effect of cocaine-exercise challenge was not attenuated by P. Similarly, blood lactate concentration in S-C rats was threefold higher than that of S-S after exercise, a response also not altered by pretreatment with P. On the basis of these observations, we conclude that the excessive glycogenolysis and lactacidosis observed during cocaine-exercise challenge is not the result of vasoconstriction secondary to norepinephrine activation of alpha-1 receptors.     

The N-Terminal Sequence of Tyrosine Hydroxylase Is a Conformationally Versatile Motif That Binds 14-3-3 Proteins and Membranes

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the synthesis of catecholamine neurotransmitters, and a reduction in TH activity is associated with several neurological diseases. Human TH is regulated, among other mechanisms, by Ser19-phosphorylation-dependent interaction with 14-3-3 proteins. The N-terminal sequence (residues 1–43), which corresponds to an extension to the TH regulatory domain, also interacts with negatively charged membranes. By using X-ray crystallography together with molecular dynamics simulations and structural bioinformatics analysis, we have probed the conformations of the Ser19-phosphorylated N-terminal peptide [THp-(1-43)] bound to 14-3-3γ, free in solution and bound to a phospholipid bilayer, and of the unphosphorylated peptide TH-(1-43) both free and bilayer bound. As seen in the crystal structure of THp-(1-43) complexed with 14-3-3γ, the region surrounding pSer19 adopts an extended conformation in the bound state, whereas THp-(1-43) adopts a bent conformation when free in solution, with higher content of secondary structure and higher number of internal hydrogen bonds. TH-(1-43) in solution presents the highest mobility and least defined structure of all forms studied, and it shows an energetically more favorable interaction with membranes relative to THp-(1-43). Cationic residues, notably Arg15 and Arg16, which are the recognition sites of the kinases phosphorylating at Ser19, are also contributing to the interaction with the membrane. Our results reveal the structural flexibility of this region of TH, in accordance with the functional versatility and conformational adaptation to different partners. Furthermore, this structural information has potential relevance for the development of therapeutics for neurodegenerative disorders, through modulation of TH–partner interactions.     

Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome

Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.     

Early recurrence of thoracolumbar intervertebral disc extrusion after surgical decompression: a report of three cases

Thoracolumbar disc extrusions were diagnosed in three chondrodystrophic dogs with paraparesis of up to three days duration. All cases were managed by hemilaminectomy and removal of extruded disc material. In one dog, fenestration of the herniated disc space was also performed. Initially neurological function improved or was unchanged, but from two to ten days postoperatively clinical signs of deterioration became apparent. In all the dogs, recurrence of disc extrusion at the same location as the initial extrusion was diagnosed by computer tomography and at a second surgery abundant disc material was found at the hemilaminectomy site between the dura and an implanted graft of autogenous fat.     

Identification of a Novel Staphylococcus aureus Two-Component Leukotoxin Using Cell Surface Proteomics

Staphylococcus aureus is a prominent human pathogen and leading cause of bacterial infection in hospitals and the community. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 are highly virulent and, unlike hospital strains, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin–herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.