Jérôme Cabana

Impact of guideline-consistent therapy on outcome of patients with healthcare-associated and community-acquired pneumonia

BACKGROUND A new category of healthcare-associated pneumonia (HCAP) has been added in the most recent American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) guidelines, since multidrug-resistant (MDR) pathogens are more common in patients with HCAP than in those with community-acquired pneumonia (CAP). The optimal empirical management of patients with HCAP remains controversial and adherence to guidelines is inconsistent. METHODS A retrospective cohort study of 3295 adults admitted for pneumonia in an academic centre of Canada, between 1997 and 2008. RESULTS MDR pathogens were more common among patients with HCAP than in those with CAP, but less so than in other studies. Compared with patients with CAP, those with HCAP had a higher all-cause 30 day mortality [68/563 (12%) versus 201/2732 (7%); P < 0.001] and more frequent need for mechanical ventilation [78/563 (14%) versus 276/2732 (10%); P = 0.01]. In patients with CAP, mortality was lower when treatment was concordant with guidelines [86/1557 (6%) versus 109/1097 (10%) if discordant; adjusted odds ratio 0.6 (0.4-0.8); P < 0.001]. In HCAP, mortality was similar whether or not empirical treatment was concordant with guidelines [6/35 (17%) versus 18/148 (12%) if discordant; P = 0.4]. However, 30 day mortality tended to be higher when the empirical treatment was microbiologically ineffective [4/22 (18%) versus 17/187 (9%) when effective; P = 0.3]. CONCLUSIONS HCAP is associated with worse outcomes than CAP. MDR pathogens were implicated in only a small fraction of HCAP cases. In our study, unlike CAP, non-respect of current HCAP guidelines had no adverse effect on the ultimate outcome. Strategies for the empirical management of HCAP should be tailored to the local epidemiological context.

Structure of the Human Angiotensin II Type 1 (AT1) Receptor Bound to Angiotensin II from Multiple Chemoselective Photoprobe Contacts Reveals a Unique Peptide Binding Mode

Background: G protein-coupled receptors (GPCRs) modulate a plethora of physiological processes. Results: The determination of 38 ligand/receptor contacts enabled the evidence-based modeling of a GPCR structure. Conclusion: The proposed GPCR structure assumes four interaction clusters with its ligand, which adopts a vertical binding mode. Significance: Elucidating the structure of GPCRs is central to their understanding and has several implications, e.g. rational drug design. Breakthroughs in G protein-coupled receptor structure determination based on crystallography have been mainly obtained from receptors occupied in their transmembrane domain core by low molecular weight ligands, and we have only recently begun to elucidate how the extracellular surface of G protein-coupled receptors (GPCRs) allows for the binding of larger peptide molecules. In the present study, we used a unique chemoselective photoaffinity labeling strategy, the methionine proximity assay, to directly identify at physiological conditions a total of 38 discrete ligand/receptor contact residues that form the extracellular peptide-binding site of an activated GPCR, the angiotensin II type 1 receptor. This experimental data set was used in homology modeling to guide the positioning of the angiotensin II (AngII) peptide within several GPCR crystal structure templates. We found that the CXC chemokine receptor type 4 accommodated the results better than the other templates evaluated; ligand/receptor contact residues were spatially grouped into defined interaction clusters with AngII. In the resulting receptor structure, a β-hairpin fold in extracellular loop 2 in conjunction with two extracellular disulfide bridges appeared to open and shape the entrance of the ligand-binding site. The bound AngII adopted a somewhat vertical binding mode, allowing concomitant contacts across the extracellular surface and deep within the transmembrane domain core of the receptor. We propose that such a dualistic nature of GPCR interaction could be well suited for diffusible linear peptide ligands and a common feature of other peptidergic class A GPCRs.

Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5

STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA>LCA>CDCA>GDCA>TDCA>CA>UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.

Photolabeling identifies transmembrane domain 4 of CXCR4 as a T140 binding site.

CXCR4, a G-protein-coupled receptor, which binds the chemokine stromal cell-derived factor 1 alpha (SDF-1alpha, CXCL12), is one of two co-receptors most frequently used by HIV-1 to infect CD4+ lymphocytes. The SDF-1alpha/CXCR4 axis is also involved in angiogenesis, in stem cell homing to bone marrow, in rheumatoid arthritis and in cancer. Here, we directly determined the binding site of the inverse agonist T140 on CXCR4 using photoaffinity labeling. Two T140 photoanalogs were synthesized containing the photoreactive amino acid p-benzoyl-l-phenylalanine (Bpa) in positions 5 or 10, yielding [Bpa(5)]T140 and [Bpa(10)]T140. Binding experiments on HEK293 cells stably expressing the wild-type CXCR4 receptor using 125I-SDF-1alpha demonstrated that T140 and both photoanalogs had affinities in the nanomolar range, similar to SDF-1alpha. Photolabeling led to the formation of specific, covalent 42 kDa T140-CXCR4 complexes. V8 protease digestion of both CXCR4/125I-[Bpa(5)]T140 and CXCR4/125I-[Bpa(10)]T140 adducts generated a fragment of 6kDa suggesting that the T140 photoanalogs labeled a fragment corresponding to Lys(154)-Glu(179) of the receptors 4th transmembrane domain. Further digestion of this 6kDa fragment with endo Asp-N led to the generation of a shorter fragment validating the photolabeled region. Our results demonstrate that T140 interacts with residues of the fourth transmembrane domain of the CXCR4 receptor and provide new structural constraints enabling us to model the complex between T140 and CXCR4.

A single-nucleotide polymorphism of alanine to threonine at position 163 of the human angiotensin II type 1 receptor impairs Losartan affinity.

Background and objective AT1 is the principal receptor for angiotensin II (AngII), which regulates blood pressure and osmotic homeostasis. Earlier studies have shown that position 163 interacts with the antihypertensive nonpeptide antagonist, Losartan. A recently discovered polymorphism found in humans (rs12721226) coding for residue 163 led us to determine whether this polymorphism would affect Losartan antihypertensive therapies. The pharmacological properties of the A163T hAT1 variant are described. Method and results The A163T hAT1 mutation was evaluated by testing its affinity by dose displacement of AngII analogs in COS-7 cells expressing either wild-type hAT1 or the A163T hAT1. The expressions of the receptors were evaluated by saturation binding and the efficacies were assessed by measuring the 3H-inositol phosphate production. The results showed that the A163T hAT1 receptor is comparable with the affinity, expression, and efficacy of native hAT1 towards peptide ligands. The affinities were also tested with nonpeptide antagonists Losartan, L-158 809, valsartan, telmisartan, irbesartan, candesartan, and EXP3174. Losartan and EXP3174 displayed a 7-fold loss in affinity towards A163T hAT1. The ability of Losartan to inhibit AngII-induced inositol triphosphate production also confirmed a loss in efficacy. Molecular modeling showed a higher steric and hydrophilic hindrance of the A163T hAT1-Losartan complex. Conclusion The polymorphism that codes for the A163T hAT1 variant results in a receptor with normal physiological properties toward the endogenous hormone. However, the significant reduction in affinity to Losartan and its active metabolite, EXP3174, could significantly impair the clinical effectiveness of an antihypertensive therapy using Losartan with patients bearing the A163T polymorphism.

Mode of Binding of the Cyclic Agonist Peptide TC14012 to CXCR7: Identification of Receptor and Compound Determinants

The chemokine receptor CXCR7 is an atypical CXCL12 receptor that, as opposed to the classical CXCL12 receptor CXCR4, signals preferentially via the β-arrestin pathway and does not mediate chemotaxis. We previously reported that the cyclic peptide TC14012, a potent CXCR4 antagonist, also engaged CXCR7, albeit with lower potency. Surprisingly, the compound activated the CXCR7-arrestin pathway. The reason underlying the opposite effects of TC14012 on CXCR4 and CXCR7, and the mode of binding of TC14012 to CXCR7, remained unclear. The mode of binding of TC14012 to CXCR4 is known from cocrystallization of its analogue CVX15 with CXCR4. We here report the the mode of binding of TC14012 to CXCR7 by combining the use of compound analogues, receptor mutants, and molecular modeling. We find that the mode of binding of TC14012 to CXCR7 is indeed similar to that of CVX15 to CXCR4, with compound positions Arg2 and Arg14 engaging CXCR7 key residues D179(4.60) (on the tip of transmembrane domain 4) and D275(6.58) (on the tip of transmembrane domain 6), respectively. Interestingly, the TC14012 parent compound T140 is not a CXCR7 agonist, because of conformational constraints in its pharmacophore, which in TC14012 are relieved through C-terminal amidation. However, an engineered salt bridge between the CXCR7 ECL2 substitution R197D and compound residue Arg1 permitted T140 agonism by repositioning the compound in the binding pocket. In conclusion, our results show that the opposite effect of TC14012 on CXCR4 and CXCR7 is not explained by different binding modes. Rather, engagement of the interface between transmembrane domains and extracellular loops readily triggers CXCR7, but not CXCR4, activation.

Critical Hydrogen Bond Formation for Activation of the Angiotensin II Type 1 Receptor

Background: The N111G and N111W mutations make the AT1 receptor constitutively active and inactivable, respectively. Results: The orientation and interactions of D742.50 are influenced by the residue at position 1113.35. Conclusion: H-bond formation between D742.50 and N461.50 is critical for AT1 receptor activation. Significance: This novel molecular switch could be involved in the GPCR activation mechanism as it involves highly conserved residues D2.50 and N1.50. G protein-coupled receptors contain selectively important residues that play central roles in the conformational changes that occur during receptor activation. Asparagine 111 (N1113.35) is such a residue within the angiotensin II type 1 (AT1) receptor. Substitution of N1113.35 for glycine leads to a constitutively active receptor, whereas substitution for tryptophan leads to an inactivable receptor. Here, we analyzed the AT1 receptor and two mutants (N111G and N111W) by molecular dynamics simulations, which revealed a novel molecular switch involving the strictly conserved residue D742.50. Indeed, D742.50 forms a stable hydrogen bond (H-bond) with the residue in position 1113.35 in the wild-type and the inactivable receptor. However, in the constitutively active mutant N111G-AT1 receptor, residue D74 is reoriented to form a new H-bond with another strictly conserved residue, N461.50. When expressed in HEK293 cells, the mutant N46G-AT1 receptor was poorly activable, although it retained a high binding affinity. Interestingly, the mutant N46G/N111G-AT1 receptor was also inactivable. Molecular dynamics simulations also revealed the presence of a cluster of hydrophobic residues from transmembrane domains 2, 3, and 7 that appears to stabilize the inactive form of the receptor. Whereas this hydrophobic cluster and the H-bond between D742.50 and W1113.35 are more stable in the inactivable N111W-AT1 receptor, the mutant N111W/F77A-AT1 receptor, designed to weaken the hydrophobic core, showed significant agonist-induced signaling. These results support the potential for the formation of an H-bond between residues D742.50 and N461.50 in the activation of the AT1 receptor.

Temperature dependent photolabeling of the human angiotensin II type 1 receptor reveals insights into its conformational landscape and its activation mechanism

We present a photoaffinity labeling study of the human Angiotensin II (AngII) type 1 receptor (hAT(1)) and a constitutively active mutant (CAM) N111G hAT(1) at multiple temperatures using a p-benzoyl-l-phenylalanine (Bpa) containing AngII analogue (125)I-[Sar(1), Bpa(8)] AngII and the Methionine Proximity Approach (MPA). By introducing Met residues, which react selectively with Bpa, by mutagenesis in hAT(1) and its CAM, we were able to identify the position of residues that surround the Bpa moiety in the receptor-ligand complexes. Here we refined this characterization by controlling and varying (from -20 to 50 degrees C) the temperature at which the photolabeling was carried out. The hAT(1) Met mutant, as well as CAM double mutant, photolabeled receptors were digested with CNBr and the fragmentation patterns were quantified by radioactive and densitometric analysis. Many important and significant changes in the fragmentation patterns were observed as function of both the temperature of photolysis and the context of constitutive activation. The ligand-receptor complex was increasingly flexible as temperature was increased, i.e. that the Bpa moiety could more easily label increasingly distant residues. These fragmentation patterns were converted into distance constraints that were included into a simulated annealing protocol in order to explore the extent of these conformational changes. In the context of constitutive activation, the 6th transmembrane domain (TM6) was found to exhibit a relative outward movement while TM2 and 5 were found to move closer to the ligand binding site. TM3 showed a slight displacement.

Activation induces structural changes in the liganded angiotensin II type 1 receptor

The octapeptide hormone angiotensin II (AngII) binds to and activates the human angiotensin II type 1 receptor (hAT1) of the G protein-coupled receptor class A family. Several activation mechanisms have been proposed for this family, but they have not yet been experimentally validated. We previously used the methionine proximity assay to show that 11 residues in transmembrane domain (TMD) III, VI, and VII of the hAT1 receptor reside in close proximity to the C-terminal residue of AngII. With the exception of a single change in TMD VI, the same contacts are present on N111G-hAT1, a constitutively active mutant; this N111G-hAT1 is a model for the active form of the receptor. In this study, two series of 53 individual methionine mutations were constructed in TMD I, II, IV, and V on both receptor forms. The mutants were photolabeled with a neutral antagonist, 125I-[Sar1,p-benzoyl-l-Phe8]AngII, and the resulting complexes were digested with cyanogen bromide. Although no new contacts were found for the hAT1 mutants, two were found in the constitutively active mutants, Phe-77 in TMD II and Asn-200 in TMD V. To our knowledge, this is the first time that a direct ligand contact with TMD II and TMD V has been reported. These contact point differences were used to identify the structural changes between the WT-hAT1 and N111G-hAT1 complexes through homology-based modeling and restrained molecular dynamics. The model generated revealed an important structural rearrangement of several TMDs from the basal to the activated form in the WT-hAT1 receptor.

STARD6 on steroids: solution structure, multiple timescale backbone dynamics and ligand binding mechanism.

START domain proteins are conserved α/β helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through 15N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6.