Crystal structure of HLA-DP2 and implications for chronic beryllium disease.

Chronic beryllium disease (CBD) is a fibrotic lung disorder caused by beryllium (Be) exposure and is characterized by granulomatous inflammation and the accumulation of Be-responsive CD4(+) T cells in the lung. Genetic susceptibility to CBD has been associated with certain alleles of the MHCII molecule HLA-DP, especially HLA-DPB1*0201 and other alleles that contain a glutamic acid residue at position 69 of the beta-chain (betaGlu69). The HLA-DP alleles that can present Be to T cells match those implicated in the genetic susceptibility, suggesting that the HLA contribution to disease is based on the ability of those molecules to bind and present Be to T cells. The structure of HLA-DP2 and its interaction with Be are unknown. Here, we present the HLA-DP2 structure with its antigen-binding groove occupied by a self-peptide derived from the HLA-DR alpha-chain. The most striking feature of the structure is an unusual solvent exposed acidic pocket formed between the peptide backbone and the HLA-DP2 beta-chain alpha-helix and containing three glutamic acids from the beta-chain, including betaGlu69. In the crystal packing, this pocket has been filled with the guanidinium group of an arginine from a neighboring molecule. This positively charged moiety forms an extensive H-bond/salt bridge network with the three glutamic acids, offering a plausible model for how Be-containing complexes might occupy this site. This idea is strengthened by the demonstration that mutation of any of the three glutamic acids in this pocket results in loss of the ability of DP2 to present Be to T cells.

Targeting heart failure therapeutics: a historical perspective.

This article reviews information on the role of genetic variability in the development, progression, and treatment of heart failure. It focuses primarily on genetic variation in neurohormonal systems, where adrenergic receptors and the renin-angiotensin-aldosterone system are the two most explored areas. The article also reviews the endothelin system and natriuretic peptides. Clinical trial design issues are examined, and suggestions for pharmacogenomic clinical trials are presented. Finally, lessons from the oncology field and the changing regulatory landscape are discussed.

The Sir4 C-terminal coiled coil is required for telomeric and mating type silencing in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Sir4p plays important roles in silent chromatin at telomeric and silent mating type loci. The C terminus of Sir4p (Sir4CT) is critical for its functions in vivo because over-expression or deletion of Sir4CT fragments disrupts normal telomeric structure and abolishes the telomere position effect. The 2.5A resolution X-ray crystal structure of an Sir4CT fragment (Sir4p 1217-1358) reveals a 72 residue homodimeric, parallel coiled coil, burying an extensive 3600A(2) of surface area. The crystal structure is consistent with results of protein cross-linking and analytical ultracentrifugation results demonstrating that Sir4CT exists as a dimer in solution. Disruption of the coiled coil in vivo by point mutagenesis results in total derepression of telomeric and HML silent mating marker genes, suggesting that coiled coil dimerization is essential for Sir4p-mediated silencing. In addition to the coiled coil dimerization interface (Sir4CC interface), a crystallographic interface between pairs of coiled coils is significantly hydrophobic and buries 1228A(2) of surface area (interface II). Remarkably, interface II mutants are deficient in telomeric silencing but not in mating type silencing in vivo. However, point mutants of interface II do not affect the oligomerization state of Sir4CT in solution. These results are consistent with the hypothesis that interface II mimics a protein interface between Sir4p and one of its protein partners that is essential for telomeric silencing but not mating type silencing.

Prevention of atrial fibrillation by bucindolol is dependent on the beta1389 Arg/Gly adrenergic receptor polymorphism.

OBJECTIVES: This study assessed the impact of bucindolol, a beta-blocker/sympatholytic agent, on the development of atrial fibrillation (AF) in advanced chronic heart failure with reduced left ventricular ejection fraction (HFREF) patients enrolled in the BEST (Beta-Blocker Evaluation of Survival Trial). BACKGROUND: ß-blockers have modest efficacy for AF prevention in HFREF patients. Bucindolol's effects on HF and ventricular arrhythmic endpoints are genetically modulated by ß1- and α(2c)-adrenergic receptor (AR) polymorphisms that can be used to subdivide HFREF populations into those with bucindolol effectiveness levels that are enhanced, unchanged, or lost. METHODS: BEST enrolled 2,708 New York Heart Association (NYHA) class III to IV HFREF patients. A substudy in which 1,040 patients' DNA was genotyped for the ß1-AR position 389 Arg/Gly and the α(2c)322-325 wild type (Wt)/deletion (Del) polymorphisms, and new-onset AF was assessed from adverse event case report forms or electrocardiograms at baseline and at 3 and 12 months. RESULTS: In the entire cohort, bucindolol reduced the rate of new-onset AF compared to placebo by 41% (hazard ratio [HR]: 0.59 [95% confidence interval (CI): 0.44 to 0.79], p = 0.0004). In the 493 ß1389 arginine homozygotes (Arg/Arg) in the DNA substudy, bucindolol reduced new-onset AF by 74% (HR: 0.26 [95% CI: 0.12 to 0.57]), with no effect in ß1389 Gly carriers (HR: 1.01 [95% CI: 0.56 to 1.84], interaction test = 0.008). When ß1389 Gly carriers were subdivided by α(2c) Wt homozygotes (n = 413, HR: 0.94 [95% CI: 0.48 to 1.82], p = 0.84) or Del variant carriers (n = 134, HR: 1.33 [95% CI: 0.32 to 5.64], p = 0.70), there was a positive interaction test (p = 0.016) when analyzed with ß1389 Arg homozygotes. CONCLUSIONS: Bucindolol prevented new-onset AF; ß1 and α(2c) polymorphisms predicted therapeutic response; and the 47% of patients who were ß1389 Arg homozygotes had an enhanced effect size of 74%. (Beta-Blocker Evaluation in Survival Trial [BEST]; NCT00000560)

Effect of bucindolol on heart failure outcomes and heart rate response in patients with reduced ejection fraction heart failure and atrial fibrillation.

AIMS: There is little evidence of beta-blocker treatment benefit in patients with heart failure and reduced left ventricular ejection fraction (HFREF) and atrial fibrillation (AF). We investigated the effects of bucindolol in HFREF patients with AF enrolled in the Beta-blocker Evaluation of Survival Trial (BEST). METHODS AND RESULTS: A post-hoc analysis of patients in BEST with and without AF was performed to estimate the effect of bucindolol on mortality and hospitalization. Patients were also evaluated for treatment effects on heart rate and the influence of beta1-adrenergic receptor position 389 (ß(1)389) arginine (Arg) vs. glycine (Gly) genotypes. In the 303/2708 patients in AF, patients receiving bucindolol were more likely to achieve a resting heart rate ≤ 80 b.p.m. at 3 months (P < 0.005) in the absence of treatment-limiting bradycardia. In AF patients and sinus rhythm (SR) patients who achieved a resting heart rate ≤ 80 b.p.m., there were beneficial treatment effects on cardiovascular mortality/cardiovascular hospitalization [hazard ratio (HR) 0.61, P = 0.025, and 0.79, P = 0.002]. Without achieving a resting heart rate ≤ 80 b.p.m., there were no treatment effects on events in either group. ß(1)389-Arg/Arg AF patients had nominally significant reductions in all-cause mortality/HF hospitalization and cardiovascular mortality/hospitalization with bucindolol (HR 0.23, P = 0.037 and 0.28, P = 0.039), whereas Gly carriers did not. There was no evidence of diminished heart rate response in ß(1)389-Arg homozygotes. CONCLUSION: In HFREF patients with AF, bucindolol was associated with reductions in composite HF endpoints in those who achieved a resting heart rate ≤ 80 b.p.m. and nominally in those with the ß(1)389-Arg homozygous genotype.

Combinatorial pharmacogenetic interactions of bucindolol and ß1, α2C adrenergic receptor polymorphisms.

BACKGROUND: Pharmacogenetics involves complex interactions of gene products affecting pharmacodynamics and pharmacokinetics, but there is little information on the interaction of multiple genetic modifiers of drug response. Bucindolol is a ß-blocker/sympatholytic agent whose efficacy is modulated by polymorphisms in the primary target (ß(1) adrenergic receptor [AR] Arg389 Gly on cardiac myocytes) and a secondary target modifier (α(2C) AR Ins [wild-type (Wt)] 322-325 deletion [Del] on cardiac adrenergic neurons). The major allele homozygotes and minor allele carriers of each polymorphism are respectively associated with efficacy enhancement and loss, creating the possibility for genotype combination interactions that can be measured by clinical trial methodology. METHODOLOGY: In a 1,040 patient substudy of a bucindolol vs. placebo heart failure clinical trial, we tested the hypothesis that combinations of ß(1)389 and α(2C)322-325 polymorphisms are additive for both efficacy enhancement and loss. Additionally, norepinephrine (NE) affinity for ß(1)389 AR variants was measured in human explanted left ventricles. PRINCIPAL FINDINGS: The combination of ß(1)389 Arg+α(2C)322-325 Wt major allele homozygotes (47% of the trial population) was non-additive for efficacy enhancement across six clinical endpoints, with an average efficacy increase of 1.70-fold vs. 2.32-fold in ß(1)389 Arg homozygotes+α(2C)322-325 Del minor allele carriers. In contrast, the minor allele carrier combination (13% subset) exhibited additive efficacy loss. These disparate effects are likely due to the higher proportion (42% vs. 8.7%, P = 0.009) of high-affinity NE binding sites in ß(1)389 Arg vs. Gly ARs, which converts α(2C)Del minor allele-associated NE lowering from a therapeutic liability to a benefit. CONCLUSIONS: On combination, the two sets of AR polymorphisms 1) influenced bucindolol efficacy seemingly unpredictably but consistent with their pharmacologic interactions, and 2) identified subpopulations with enhanced (ß(1)389 Arg homozygotes), intermediate (ß(1)389 Gly carriers+α(2C)322-325 Wt homozygotes), and no (ß(1)389 Gly carriers+α(2C)322-325 Del carriers) efficacy.

An alpha2C-adrenergic receptor polymorphism alters the norepinephrine-lowering effects and therapeutic response of the beta-blocker bucindolol in chronic heart failure.

BACKGROUND: Adrenergic activation is an important determinant of outcomes in chronic heart failure. Adrenergic activity is regulated in part by prejunctional alpha(2C)-adrenergic receptors (ARs), which exhibit genetic variation in humans. Bucindolol is a novel beta-AR blocking agent that also lowers systemic norepinephrine and thus is also a sympatholytic agent. This study investigated whether alpha(2C)-AR polymorphisms affect sympatholytic effects of bucindolol in patients with heart failure. METHODS AND RESULTS: In the beta-Blocker Evaluation of Survival Trial, adrenergic activation was estimated by systemic venous norepinephrine measured at baseline, 3 months, and 12 months posttreatment in patients treated with placebo or bucindolol. In the beta-Blocker Evaluation of Survival Trial AR polymorphism substudy, DNA was collected from 1040 of the 2708 randomized patients, and alpha(2C)-AR gene polymorphisms (alpha(2C) Del322-325 or the wild-type counterpart) were measured by polymerase chain reaction and gel electrophoresis. Patients who were alpha(2C) Del carriers (heterozygotes or homozygotes) exhibited a much greater sympatholytic response to bucindolol (decrease in norepinephrine at 3 months of 153+/-57 pg/mL, P=0.012 compared with placebo versus decrease of 50+/-13 pg/mL in alpha(2C) wild type, P=0.0005 versus placebo; P=0.010 by interaction test). alpha(2C) Del carriers had no evidence of a favorable survival benefit from bucindolol (mortality compared with placebo hazard ratio, 1.09; 95% CI, 0.57 to 2.08; P=0.80), whereas bucindolol-treated subjects who were wild type for the alpha(2C)-AR had a 30% reduction in mortality (hazard ratio, 0.70; 95% CI, 0.51 to 0.96; P=0.025). CONCLUSIONS: In the beta-Blocker Evaluation of Survival Trial AR polymorphism substudy, the norepinephrine lowering and clinical therapeutic responses to bucindolol were strongly influenced by alpha(2C) receptor genotype.