Mineralocorticoid receptor degradation is promoted by Hsp90 inhibition and the ubiquitin-protein ligase CHIP.

The mineralocorticoid receptor (MR) plays a crucial role in the regulation of Na(+) balance and blood pressure, as evidenced by gain of function mutations in the MR of hypertensive families. In the kidney, aldosterone binds to the MR, induces its nuclear translocation, and promotes a transcriptional program leading to increased transepithelial Na(+) transport via the epithelial Na(+) channel. In the unliganded state, MR is localized in the cytosol and part of a multiprotein complex, including heat shock protein 90 (Hsp90), which keeps it ligand-binding competent. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin antibiotic that binds to Hsp90 and alters its function. We investigated whether 17-AAG affects the stability and transcriptional activity of MR and consequently Na(+) reabsorption by renal cells. 17-AAG treatment lead to reduction of MR protein level in epithelial cells in vitro and in vivo, thereby interfering with aldosterone-dependent transcription. Moreover, 17-AAG inhibited aldosterone-induced Na(+) transport, possibly by interfering with MR availability for the ligand. Finally, we identified the ubiquitin-protein ligase, COOH terminus of Hsp70-interacting protein, as a novel partner of the cytosolic MR, which is responsible for its polyubiquitylation and proteasomal degradation in presence of 17-AAG. In conclusion, 17-AAG may represent a novel pharmacological tool to interfere with Na(+) reabsorption and hypertension.

Tasosartan, enoltasosartan, and angiotensin II receptor blockade: the confounding role of protein binding.

Tasosartan is a long-acting angiotensin II (AngII) receptor blocker. Its long duration of action has been attributed to its active metabolite enoltasosartan. In this study we evaluated the relative contribution of tasosartan and enoltasosartan to the overall pharmacological effect of tasosartan. AngII receptor blockade effect of single doses of tasosartan (100 mg p.o. and 50 mg i.v) and enoltasosartan (2.5 mg i.v.) were compared in 12 healthy subjects in a randomized, double blind, three-period crossover study using two approaches: the in vivo blood pressure response to exogenous AngII and an ex vivo AngII radioreceptor assay. Tasosartan induced a rapid and sustained blockade of AngII subtype-1 (AT1) receptors. In vivo, tasosartan (p.o. or i.v.) blocked by 80% AT1 receptors 1 to 2 h after drug administration and still had a 40% effect at 32 h. In vitro, the blockade was estimated to be 90% at 2 h and 20% at 32 h. In contrast, the blockade induced by enoltasosartan was markedly delayed and hardly reached 60 to 70% despite the i.v. administration and high plasma levels. In vitro, the AT1 antagonistic effect of enoltasosartan was markedly influenced by the presence of plasma proteins, leading to a decrease in its affinity for the receptor and a slower receptor association rate. The early effect of tasosartan is due mainly to tasosartan itself with little if any contribution of enoltasosartan. The antagonistic effect of enoltasosartan appears later. The delayed in vivo blockade effect observed for enoltasosartan appears to be due to a high and tight protein binding and a slow dissociation process from the carrier.

T-cell activation by treatment of cancer patients with EMD 521873 (Selectikine), an IL-2/anti-DNA fusion protein.

ABSTRACT: BACKGROUND: EMD 521873 (Selectikine or NHS-IL2LT) is a fusion protein consisting of modified human IL-2 which binds specifically to the high-affinity IL-2 receptor, and an antibody specific for both single- and double-stranded DNA, designed to facilitate the enrichment of IL-2 in tumor tissue. METHODS: An extensive analysis of pharmacodynamic (PD) markers associated with target modulation was assessed during a first-in-human phase I dose-escalation trial of Selectikine. RESULTS: Thirty-nine patients with metastatic or locally advanced tumors refractory to standard treatments were treated with increasing doses of Selectikine, and nine further patients received additional cyclophosphamide. PD analysis, assessed during the first two treatment cycles, revealed strong activation of both CD4+ and CD8+ T-cells and only weak NK cell activation. No dose response was observed. As expected, Treg cells responded actively to Selectikine but remained at lower frequency than effector CD4+ T-cells. Interestingly, patient survival correlated positively with both high lymphocyte counts and low levels of activated CD8+ T-cells at baseline, the latter of which was associated with enhanced T-cell responses to the treatment. CONCLUSIONS: The results confirm the selectivity of Selectikine with predominant T-cell and low NK cell activation, supporting follow-up studies assessing the clinical efficacy of Selectikine for cancer patients.

A positive interaction between inhibitors of protein synthesis and cefepime in the fight against methicillin-resistant Staphylococcus aureus.

Quinupristin-dalfopristin (Q-D) synergizes with cefepime for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Here, we studied whether the synergism was restricted to MRSA and if it extended to non-beta-lactam cell wall inhibitors or to other inhibitors of protein synthesis. Three MRSA and two methicillin-susceptible S. aureus (MSSA) strains were tested, including an isogenic pair of mecA (-)/mecA (+) S. aureus Newman. The drug interactions were determined by fractional inhibitory concentration (FIC) indices and population analysis profiles. The antibacterial drugs that we used included beta-lactam (cefepime) and non-beta-lactam cell wall inhibitors (D-cycloserine, fosfomycin, vancomycin, teicoplanin), inhibitors of protein synthesis (Q-D, erythromycin, chloramphenicol, tetracycline, linezolid, fusidic acid), and polynucleotide inhibitors (cotrimoxazole, ciprofloxacin). The addition of each protein inhibitor to cefepime was synergistic (FIC ≤ 0.5) or additive (FIC > 0.5 but < 1) against MRSA, but mostly indifferent against MSSA (FIC ≥ 1 but ≤ 4). This segregation was not observed after adding cotrimoxazole or ciprofloxacin to cefepime. Population analysis profiles were performed on plates in the presence of increasing concentrations of the cell wall inhibitors plus 0.25 × minimum inhibitory concentration (MIC) of Q-D. Cefepime combined with Q-D was synergistic against MRSA, but D-cycloserine and glycopeptides were not. Thus, the synergism was specific to beta-lactam antibiotics. Moreover, the synergism was not lost against fem mutants, indicating that it acted at another level. The restriction of the beneficial effect to MRSA suggests that the functionality of penicillin-binding protein 2A (PBP2A) was affected, either directly or indirectly. Further studies are necessary in order to provide a mechanism for this positive interaction.

HAMAP in 2015: updates to the protein family classification and annotation system.

HAMAP (High-quality Automated and Manual Annotation of Proteins-available at http://hamap.expasy.org/) is a system for the automatic classification and annotation of protein sequences. HAMAP provides annotation of the same quality and detail as UniProtKB/Swiss-Prot, using manually curated profiles for protein sequence family classification and expert curated rules for functional annotation of family members. HAMAP data and tools are made available through our website and as part of the UniRule pipeline of UniProt, providing annotation for millions of unreviewed sequences of UniProtKB/TrEMBL. Here we report on the growth of HAMAP and updates to the HAMAP system since our last report in the NAR Database Issue of 2013. We continue to augment HAMAP with new family profiles and annotation rules as new protein families are characterized and annotated in UniProtKB/Swiss-Prot; the latest version of HAMAP (as of 3 September 2014) contains 1983 family classification profiles and 1998 annotation rules (up from 1780 and 1720). We demonstrate how the complex logic of HAMAP rules allows for precise annotation of individual functional variants within large homologous protein families. We also describe improvements to our web-based tool HAMAP-Scan which simplify the classification and annotation of sequences, and the incorporation of an improved sequence-profile search algorithm.