The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ⌬H ϩ . In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

An oligodeoxynucleotide-dependent method to generate nascent polypeptide chains was adopted for use in a cell-free translation system prepared from Escherichia coli. In this way, NH 2 -terminal pOmpA fragments of distinct sizes were synthesized. Because most of these pOmpA fragments could be covalently linked to puromycin, precipitated with cetyltrimethylammonium bromide, and were enriched by sedimentation, they represent a population of elongation-arrested, ribosomeassociated nascent chains. Translocation of these nascent pOmpA chains into inside-out membrane vesicles of E. coli required SecA and (depending on size) SecB. Whereas their translocation was strictly dependent on the H ؉ -motive force of the vesicles, no indication for the involvement of the bacterial signal recognition particle was obtained. SecA and SecB, although required for translocation, did not mediate binding of the ribosome-associated pOmpA to membrane vesicles. However, SecA and SecB cotranslationally associated with nascent pOmpA, since they could be co-isolated with the ribosome-associated nascent chains and as such catalyzed translocation subsequent to the release of the ribosome. These results indicate that in E. coli, SecA also functionally interacts with preproteins before they are targeted to the translocase of the plasma membrane.Protein export across the plasma (cytoplasmic, inner) membrane of Escherichia coli is achieved by the concerted action of a distinct set of Sec proteins (summarized in Ref. 1). Most of them are integral membrane proteins of the plasma membrane. SecY and SecE most likely are core constituents of the translocation pore in the membrane; SecG appears to change its membrane topography during polypeptide translocation (2); the exact roles of SecD, SecF, and YajC are yet to be established. SecA on the other hand, is a peripheral membrane protein that binds to SecYE (3) probably serving as a membrane receptor for a preprotein-SecB complex. SecB functions as a chaperone (4) and as a targeting factor (5). Due to its ATPase activity, SecA inserts in, and deinserts from, the membrane in a cyclic manner (6, 7), which leads to the stepwise translocation of the precursor across the membrane bilayer (8, 9). In addition to ATP, the H ϩ -motive force (⌬H ϩ ) is utilized as an energy source for translocation.This model does not ascribe a function to the soluble form of SecA, which in E. coli partitions roughly equally between cytosol/ribosomes and the plasma membrane (10, 11). The occurrence of cytosolic complexes between precursor proteins and SecA (12, 13) suggests that SecA actually might interact with its protein substrate well before it has been targeted to the membrane. Besides the Sec proteins, E. coli possesses a signal recognition particle (SRP) 1 /SRP receptor system whose eukaryotic equivalents mediate the cotranslational targeting of nascent secretory and membrane proteins to the endoplasmic reticulum (14). Whereas more recent results (15-18) indicate that the bacterial SRP has a specialized role in the integrati...

PurposePanobinostat is partly metabolized by CYP3A4 in vitro. This study evaluated the effect of a potent CYP3A inhibitor, ketoconazole, on the pharmacokinetics and safety of panobinostat.MethodsPatients received a single panobinostat oral dose on day 1, followed by 4 days wash-out period. On days 5–9, ketoconazole was administered. On day 8, a single panobinostat dose was co-administered with ketoconazole. Panobinostat was administered as single agent three times a week on day 15 and onward.ResultsIn the presence of ketoconazole, there was 1.6- and 1.8-fold increase in Cmax and AUC of panobinostat, respectively. No substantial change in Tmax or half-life was observed. No difference in panobinostat-pharmacokinetics between patients carrying CYP3A5*1/*3 and CYP3A5*3/*3 alleles was observed. Most frequently reported adverse events were gastrointestinal related. Patients had asymptomatic hypophosphatemia (64%), and urine analysis suggested renal phosphate wasting.ConclusionsCo-administration of panobinostat with CYP3A inhibitors is feasible as the observed increase in panobinostat PK parameters was not considered clinically relevant. Considering the variability in exposure following enzyme inhibition and the fact that chronic dosing of panobinostat was not studied with CYP3A inhibitors, close monitoring of panobinostat-related adverse events is necessary.

The endothelin receptor antagonist avosentan may cause fluid overload at doses of 25 and 50 mg, but the actual mechanisms of this effect are unclear. We conducted a placebo-controlled study in 23 healthy subjects to assess the renal effects of avosentan and the dose dependency of these effects. Oral avosentan was administered once daily for 8 days at doses of 0.5, 1.5, 5, and 50 mg. The drug induced a dose-dependent median increase in body weight, most pronounced at 50 mg (0.8 kg on day 8). Avosentan did not affect renal hemodynamics or plasma electrolytes. A dose-dependent median reduction in the fractional renal excretion of sodium was found (up to 8.7% at avosentan 50 mg); this reduction was paralleled by a dose-related increase in proximal sodium reabsorption. It is suggested that avosentan dose-dependently induces sodium retention by the kidney, mainly through proximal tubular effects. The potential clinical benefits of avosentan should therefore be investigated at doses of

Purpose Panobinostat is a novel oral pan-deacetylase inhibitor with promising anti-cancer activity. The study aimed to determine the influence of food on the oral bioavailability of panobinostat. Methods This multicenter study consisted of a randomized, three-way crossover, food-effect study period (cycle 1) followed by single-agent panobinostat continual treatment phase in patients with advanced cancer. Patients received panobinostat 20 mg twice weekly, and panobinostat pharmacokinetics was investigated on days 1, 8, and 15 with a randomly assigned sequence of three prandial states (fasting, high-fat, and normal breakfast). Results Thirty-six patients were assessed for the food effect on pharmacokinetics and safety in cycle 1, after which 29 patients continued treatment, receiving singleagent panobinostat. Safety and antitumor activity were assessed during the extension period. Panobinostat systemic exposure was marginally reduced (14–16%) following food [geometric mean ratio (GMR) of the AUC0–∞/high-fat breakfast/fasting, 0.84 (90% confidence interval {CI}, 0.74–0.96); normal breakfast/fasting, 0.86 (90% CI, 0.75–1.00)], and interpatient variability (coefficient of variation, 59%) remained essentially unchanged with or without food. Panobinostat Cmax was reduced by 44% (high-fat) and 36% (normal) with median Tmax prolonged by 1–1.5 h following food. Panobinostat was well tolerated, with thrombocytopenia, fatigue, nausea, and vomiting as common adverse events, and demonstrated antitumor activity with one patient with a partial response and six patients with stable disease as best response. Conclusions Food produced minor changes in oral panobinostat exposure; thus, panobinostat can be given without regard to food intake in future clinical studies.