Validation of inhibitors of an ABC transporter required to transport lipopolysaccharide to the cell surface in Escherichia coli

The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria creates a permeability barrier that prevents the entry of most currently available antibiotics. The seven lipopolysaccharide transport (Lpt) proteins involved in transporting and assembling this glycolipid are essential for growth and division in Escherichia coli; therefore, inhibiting their functions leads to cell death. LptB, the ATPase that provides energy for LPS transport and assembly, forms a complex with three other inner membrane (IM) components, LptC, F, and G. We demonstrate that inhibitors of pure LptB can also inhibit the full IM complex, LptBFGC, purified in detergent. We also compare inhibition of LptB and the LptBFGC complex with the antibiotic activity of these compounds. Our long-term goal is to develop tools to study inhibitors of LPS biogenesis that could serve as potentiators by disrupting the OM permeability barrier, facilitating entry of clinically used antibiotics not normally used to treat Gram-negative infections, or that can serve as antibiotics themselves.     

Palmitoylation of GAP-43 by the ER-Golgi intermediate compartment and Golgi apparatus.

Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [(35)S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation.     

3,5-Dialkoxypyridine analogues of bedaquiline are potent antituberculosis agents with minimal inhibition of the hERG channel

Bedaquiline is a new drug of the diarylquinoline class that has proven to be clinically effective against drug-resistant tuberculosis, but has a cardiac liability (prolongation of the QT interval) due to its potent inhibition of the cardiac potassium channel protein hERG. Bedaquiline is highly lipophilic and has an extremely long terminal half-life, so has the potential for more-than-desired accumulation in tissues during the relatively long treatment durations required to cure TB. The present work is part of a program that seeks to identify a diarylquinoline that is as potent as bedaquiline against Mycobacterium tuberculosis, with lower lipophilicity, higher clearance, and lower risk for QT prolongation. Previous work led to the identification of compounds with greatly-reduced lipophilicity compounds that retain good anti-tubercular activity in vitro and in mouse models of TB, but has not addressed the hERG blockade. We now present compounds where the C-unit naphthalene is replaced by a 3,5-dialkoxy-4-pyridyl, demonstrate more potent in vitro and in vivo anti-tubercular activity, with greatly attenuated hERG blockade. Two examples of this series are in preclinical development.     

1H NMR study of the binding of bis(acridines) to d(AT)5.d(AT)5. 2. Dynamic aspects

Measurements of the 1H NMR spectra and relaxation rates were used to study the dynamic properties of 9-aminoacridine (9AA) and four bis(acridine) complexes with d(AT)5.d(AT)5. The behavior of the 9AA (monointercalator) and that of C8 (bisintercalator containing an eight-carbon atom linker chain) are entirely similar. For both compounds, the lifetime of the drug in a particular binding site is 2-3 ms at approximately 20 degrees C, and neither affects the A.T base pair opening rates. The complex with C10 (bisintercalator containing a 10-carbon atom linker chain) is slightly more stable than the C8 complex since its estimated binding site lifetime is 5-10 ms at 29 degrees C. Base pairs adjacent to the bound C10 are destabilized, relative to free d(AT)5.d(AT)5, but other base pairs in the C10 complex are little affected. Bis(acridine) pyrazole (BAPY) and bis(acridine) spermine (BAS) considerably stabilize those base pairs that are sandwiched between the two acridine chromophores, but in the BAS complex proton exchange from the two flanking base pairs appears to be accelerated, relative to free d(AT)5.d(AT)5. The lifetime of these drugs in specific binding sites is too long (>10 ms) to be manifested in increased line widths, at least up to 41 degrees C. An important conclusion from this study is that certain bisintercalators rapidly migrate along DNA, despite having large binding constants (K>10(6) M-1). For C8 and C10 complexes, migration rates are little different from those deduced for 9AA. The rigid linker chain in BAPY and the charge interactions in BAS retard migration of these two bisintercalators. These results provide new parameters that are useful in understanding the biochemical and biological properties of these and other bisintercalating drugs.     

Mouse as the measure of man?

The humble house mouse's cohabitation with humans has been noted since the birth of agriculture, about 10 000 years ago, in the fertile flood plains of the Middle East. In recent times, however, the mouse has been elevated from pest to model for the study of human health and disease. Recent genomics and genetics initiatives will ensure the continued growth of the house mouse as a disease model.     

Complement receptor 1 gene variants are associated with erythrocyte sedimentation rate

The erythrocyte sedimentation rate (ESR), a commonly performed test of the acute phase response, is the rate at which erythrocytes sediment in vitro in 1 hr. The molecular basis of erythrocyte sedimentation is unknown. To identify genetic variants associated with ESR, we carried out a genome-wide association study of 7607 patients in the Electronic Medical Records and Genomics (eMERGE) network. The discovery cohort consisted of 1979 individuals from the Mayo Clinic, and the replication cohort consisted of 5628 individuals from the remaining four eMERGE sites. A nonsynonymous SNP, rs6691117 (Val→IIe), in the complement receptor 1 gene (CR1) was associated with ESR (discovery cohort p = 7 × 10(-12), replication cohort p = 3 × 10(-14), combined cohort p = 9 × 10(-24)). We imputed 61 SNPs in CR1, and a "possibly damaging" SNP (rs2274567, His→Arg) in linkage disequilibrium (r(2) = 0.74) with rs6691117 was also associated with ESR (discovery p = 5 × 10(-11), replication p = 7 × 10(-17), and combined cohort p = 2 × 10(-25)). The two nonsynonymous SNPs in CR1 are near the C3b/C4b binding site, suggesting a possible mechanism by which the variants may influence ESR. In conclusion, genetic variation in CR1, which encodes a protein that clears complement-tagged inflammatory particles from the circulation, influences interindividual variation in ESR, highlighting an association between the innate immunity pathway and erythrocyte interactions.     

Crystal structure of 9-amino-N-[2-(4-morpholinyl)ethyl]-4-acridinecarboxamide bound to d(CGTACG)2: implications for structure–activity relationships of acridinecarboxamide topoisomerase poisons

The structure of the complex formed between d(CGTACG)₂ and 9-amino-N-[2-(4-morpholinyl)ethyl]-4-acridinecarboxamide, an inactive derivative of the antitumour agents N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and 9-amino-DACA, has been solved to a resolution of 1.8 Å using X-ray crystallography. The complex crystallises in the space group P6₄ and the final structure has an overall R factor of 21.9%. A drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the major groove, and its protonated morpholino nitrogen partially occupying positions close to the N7 and O6 atoms of guanine G2. The morpholino group is disordered, the major conformer adopting a twisted boat conformation that makes van der Waals contact with the O4 oxygen of thymine T3. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of guanine G2. Sugar rings are found in alternating C3′-exo/C2′-endo conformations except for cytosine C1 which is C3′-endo. Intercalation perturbs helix winding throughout the hexanucleotide compared with B-DNA, steps 1 and 2 being unwound by 10 and 8°, respectively, while the central TpA step is overwound by 11°. An additional drug molecule lies at the end of each DNA helix linking it to the next duplex to form a continuously stacked structure. The protonated morpholino nitrogen of this ‘end-stacked’ drug hydrogen bonds to the N7 atom of guanine G6, and its conformationally disordered morpholino ring forms a C–H···O hydrogen bond with the guanine O6 oxygen. In both drug molecules the 4-carboxamide group is internally hydrogen bonded to the protonated N10 atom of the acridine ring. We discuss our findings with respect to the potential role played by the interaction of the drug side chain and the topoisomerase II protein in the poisoning of topoisomerase activity by the acridinecarboxamides.     

Interspecies determinants of Friend leukemia virus antigens involved in cytolysis of virus-pfoducing cells.

The cytolytic reactivity of a complex goat anti-feline leukemia virus (FeLV) antiserum for mouse cells (Eveline) releasing large quantities of Friend leukemia virus (FLV) was analyzed by the sensitive [14C]nicotinamide release microcytotoxicity assay. Whereas this interspecies killing reactivity could be blocked by absorption of the goat anti-FeLV serum with a preparation of disrupted FLV, absorption with purified FLV gp71, the major envelope glycoprotein, had no effect. Subsequent serum absorptions with the individual FLV structural polypeptides revealed that the lysis of the Eveline cells by the goat anti-FeLV serum is mediated by antibodies recognizing the interspecies determinant of p30, the major internal capsid protein. The expression of this internal viral component at the surface of virus-producing cells is discussed further. The results also demonstrated that removal of approximately 70% of the carbohydrate portion of gp71 with a preparation of glycosidases did not affect the integrity of its interspecies determinant; these results are in agreement with an earlier study (Bolgnesi et al., 1975) that examined primarily the group- and type-specific sites.     

Effects of Prolonged Exposure to Hypobaric Hypoxia on Oxidative Stress,Inflammation and Gluco-Insular Regulation: The Not-So-Sweet Price for Good Regulation

Objectives

The mechanisms by which low oxygen availability are associated with the development of insulin resistance remain obscure. We thus investigated the relationship between such gluco-insular derangements in response to sustained (hypobaric) hypoxemia, and changes in biomarkers of oxidative stress, inflammation and counter-regulatory hormone responses.

Methods

After baseline testing in London (75 m), 24 subjects ascended from Kathmandu (1,300 m) to Everest Base Camp (EBC;5,300 m) over 13 days. Of these, 14 ascended higher, with 8 reaching the summit (8,848 m). Assessments were conducted at baseline, during ascent to EBC, and 1, 6 and 8 week(s) thereafter. Changes in body weight and indices of gluco-insular control were measured (glucose, insulin, C-Peptide, homeostasis model assessment of insulin resistance [HOMA-IR]) along with biomarkers of oxidative stress (4-hydroxy-2-nonenal-HNE), inflammation (Interleukin-6 [IL-6]) and counter-regulatory hormones (glucagon, adrenalin, noradrenalin). In addition, peripheral oxygen saturation (SpO₂) and venous blood lactate concentrations were determined.

Results

SpO₂ fell significantly from 98.0% at sea level to 82.0% on arrival at 5,300 m. Whilst glucose levels remained stable, insulin and C-Peptide concentrations increased by >200% during the last 2 weeks. Increases in fasting insulin, HOMA-IR and glucagon correlated with increases in markers of oxidative stress (4-HNE) and inflammation (IL-6). Lactate levels progressively increased during ascent and remained significantly elevated until week 8. Subjects lost on average 7.3 kg in body weight.

Conclusions

Sustained hypoxemia is associated with insulin resistance, whose magnitude correlates with the degree of oxidative stress and inflammation. The role of 4-HNE and IL-6 as key players in modifying the association between sustained hypoxia and insulin resistance merits further investigation.