Angiotensin II cyclic analogs as tools to investigate AT1R biased signaling mechanisms.

G protein coupled receptors (GPCRs) produce pleiotropic effects by their capacity to engage numerous signaling pathways once activated. Functional selectivity (also called biased signaling), where specific compounds can bring GPCRs to adopt conformations that enable selective receptor coupling to distinct signaling pathways, continues to be significantly investigated. However, an important but often overlooked aspect of functional selectivity is the capability of ligands such as angiotensin II (AngII) to adopt specific conformations that may preferentially bind to selective GPCRs structures. Understanding both receptor and ligand conformation is of the utmost importance for the design of new drugs targeting GPCRs. In this study, we examined the properties of AngII cyclic analogs to impart biased agonism on the angiotensin type 1 receptor (AT1R). Positions 3 and 5 of AngII were substituted for cysteine and homocysteine residues ([Sar1Hcy3,5]AngII, [Sar1Cys3Hcy5]AngII and [Sar1Cys3,5]AngII) and the resulting analogs were evaluated for their capacity to activate the Gq/11, G12, Gi2, Gi3, Gz, ERK and ß-arrestin (ßarr) signaling pathways via AT1R. Interestingly, [Sar1Hcy3,5]AngII exhibited potency and full efficacy on all pathways tested with the exception of the Gq pathway. Molecular dynamic simulations showed that the energy barrier associated with the insertion of residue Phe8 of AngII within the hydrophobic core of AT1R, associated with Gq/11 activation, is increased with [Sar1Hcy3,5]AngII. These results suggest that constraining the movements of molecular determinants within a given ligand by introducing cyclic structures may lead to the generation of novel ligands providing more efficient biased agonism.

Design, synthesis, and biological evaluation of CXCR4 ligands.

A combination of the CXCR4 inverse agonist T140 with N-terminal CXCL12 oligopeptides has produced the first nanomolar synthetic CXCR4 agonists. In these agonists, the inverse agonistic portion provides affinity whereas the N-terminal CXCL12 sequence induces receptor activation. Several CXCR4 crystal structures exist with either CVX15, an inverse agonist closely related to T140 and IT1t, a small molecule; we therefore attempted to produce another CXCL12 oligopeptide combination with IT1t. For this purpose, a primary amino group was introduced by total synthesis into one of the methyl groups of IT1t, serving as an anchoring point for the oligopeptide graft. The introduction of the oligopeptides on this analog however yielded antagonists, one compound displaying high affinity. On the other hand, the amino-substituted analogue itself proved to be an inverse agonist with a binding affinity of 2.6 nM compared to 11.5 nM for IT1t. This IT1t-like analog is hitherto one of the most potent non-peptidic CXCR4 inverse agonists reported.

Structure-Activity Relationship and Signaling of New Chimeric CXCR4 Agonists.

The CXCR4 receptor binds with meaningful affinities only CXCL12 and synthetic antagonists/inverse agonists. We recently described high affinity synthetic agonists for this chemokine receptor, obtained by grafting the CXCL12 N-terminus onto the inverse agonist T140. While those chimeric molecules behave as agonists for CXCR4, their binding and activation mode are unknown. The present SAR of those CXCL12-oligopeptide grafts reveals the key determinants involved in CXCR4 activation. Position 3 (Val) controls affinity, whereas position 7 (Tyr) acts as an efficacy switch. Chimeric molecules bearing aromatic residues in position 3 possess high binding affinities for CXCR4 and are Gαi full agonists with robust chemotactic properties. Fine-tuning of electron-poor aromatic rings in position 7 enhances receptor activation. To rationalize these results, a homology model of a receptor-ligand complex was built using the published crystal structures of CXCR4. Molecular dynamics simulations reveal further details accounting for the observed SAR for this series.

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

START domain proteins are conserved α/ß 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 (15)N 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.

Identification of Distinct Conformations of the Angiotensin-II Type 1 Receptor Associated with the Gq/11 Protein Pathway and the ß-Arrestin Pathway Using Molecular Dynamics Simulations.

Biased signaling represents the ability of G protein-coupled receptors to engage distinct pathways with various efficacies depending on the ligand used or on mutations in the receptor. The angiotensin-II type 1 (AT1) receptor, a prototypical class A G protein-coupled receptor, can activate various effectors upon stimulation with the endogenous ligand angiotensin-II (AngII), including the Gq/11 protein and ß-arrestins. It is believed that the activation of those two pathways can be associated with distinct conformations of the AT1 receptor. To verify this hypothesis, microseconds of molecular dynamics simulations were computed to explore the conformational landscape sampled by the WT-AT1 receptor, the N111G-AT1 receptor (constitutively active and biased for the Gq/11 pathway), and the D74N-AT1 receptor (biased for the ß-arrestin1 and -2 pathways) in their apo-forms and in complex with AngII. The molecular dynamics simulations of the AngII-WT-AT1, N111G-AT1, and AngII-N111G-AT1 receptors revealed specific structural rearrangements compared with the initial and ground state of the receptor. Simulations of the D74N-AT1 receptor revealed that the mutation stabilizes the receptor in the initial ground state. The presence of AngII further stabilized the ground state of the D74N-AT1 receptor. The biased agonist [Sar(1),Ile(8)]AngII also showed a preference for the ground state of the WT-AT1 receptor compared with AngII. These results suggest that activation of the Gq/11 pathway is associated with a specific conformational transition stabilized by the agonist, whereas the activation of the ß-arrestin pathway is linked to the stabilization of the ground state of the receptor.

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 ß-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.

Identification of transmembrane domain 1 & 2 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor.

The vasoactive urotensin-II (UII), a cyclic undecapeptide widely distributed in cardiovascular, renal and endocrine systems, specifically binds the UII receptor (UT receptor), a G protein-coupled receptor (GPCR). The involvement of this receptor in numerous pathophysiological conditions including atherosclerosis, heart failure, hypertension, renal impairment and diabetes potentially makes it an interesting therapeutic target. To elucidate how UII binds the UT receptor through the identification of specific residues in transmembrane domains (TM) one (TM1) and two (TM2) that are involved in the formation of the receptor's binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue of TM1 (V49((1.30)) to M76((1.57))) and TM2 (V88((2.41)) to H117((2.70))) was mutated, one by one, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant binding inhibition of (125)I-UII to mutant I54C((1.35)) in TM1 and mutants Y100C((2.53)), S103C((2.56)), F106C((2.59)), I107C((2.60)), T110C((2.63)) and Y111C((2.64)) in TM2. These results identify key structural residues in TM1 and TM2 that participate in the formation of the UT receptor binding pocket. Together with previous SCAM analysis of TM3, TM4, TM5, TM6 and TM7, these results have led us to identify residues within all 7 TMs that participate in UT's binding pocket and have enabled us to propose a model of this receptor's orthosteric binding site.

Identification of transmembrane domain 3, 4 & 5 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor.

Urotensin-II (UII), a cyclic undecapeptide, selectively binds the urotensin-II receptor (UT receptor), a G protein-coupled receptor (GPCR) involved in cardiovascular effects and associated with numerous pathophysiological conditions including hypertension, atherosclerosis, heart failure, pulmonary hypertension and others. In order to identify specific residues in transmembrane domains (TM) three (TM3), four (TM4) and five (TM5) that are involved in the formation of the UT receptor binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue in the F118((3.20)) to S146((3.48)) fragment of TM3, the L168((4.44)) to G194((4.70)) fragment of TM4 and the W203((5.30)) to V232((5.59)) fragment of TM5, was mutated, individually, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the positively charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant reduction in the binding of (125)I-UII to TM3 mutants L126C((3.28)), F127C((3.29)), F131C((3.33)) and M134C((3.36)) and TM4 mutants M184C((4.60)) and I188C((4.64)). No loss of binding was detected following treatment by MTSEA for all TM5 mutants tested. In absence of a crystal structure of UT receptor, these results identify key determinants in TM3, TM4 and TM5 that participate in the formation of the UT receptor binding pocket and has led us to propose a homology model of the UT receptor.

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.

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.

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 ß-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.

Critical hydrogen bond formation for activation of the angiotensin II type 1 receptor.

G protein-coupled receptors contain selectively important residues that play central roles in the conformational changes that occur during receptor activation. Asparagine 111 (N111(3.35)) is such a residue within the angiotensin II type 1 (AT(1)) receptor. Substitution of N111(3.35) for glycine leads to a constitutively active receptor, whereas substitution for tryptophan leads to an inactivable receptor. Here, we analyzed the AT(1) receptor and two mutants (N111G and N111W) by molecular dynamics simulations, which revealed a novel molecular switch involving the strictly conserved residue D74(2.50). Indeed, D74(2.50) forms a stable hydrogen bond (H-bond) with the residue in position 111(3.35) in the wild-type and the inactivable receptor. However, in the constitutively active mutant N111G-AT(1) receptor, residue D74 is reoriented to form a new H-bond with another strictly conserved residue, N46(1.50). When expressed in HEK293 cells, the mutant N46G-AT(1) receptor was poorly activable, although it retained a high binding affinity. Interestingly, the mutant N46G/N111G-AT(1) 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 D74(2.50) and W111(3.35) are more stable in the inactivable N111W-AT(1) receptor, the mutant N111W/F77A-AT(1) 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 D74(2.50) and N46(1.50) in the activation of the AT(1) receptor.

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.

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.

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.

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 receptor's 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.

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 (hAT(1)) 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 hAT(1) 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-hAT(1), a constitutively active mutant; this N111G-hAT(1) 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, (125)I-[Sar(1),p-benzoyl-L-Phe(8)]AngII, and the resulting complexes were digested with cyanogen bromide. Although no new contacts were found for the hAT(1) 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-hAT(1) and N111G-hAT(1) 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-hAT(1) receptor.