A fluorescence polarization assay for the experimental validation of an in silico model of the chemokine CXCL8 binding to receptor-derived peptides.

Peptide based inhibitors of protein-protein interactions are of great interest in proteomics, structural biology and medicinal chemistry. Optimized inhibitors can be designed by experimental approaches or by computational prediction. Ideally, computational models are adjusted to the peptide-protein complex of interest according to experimental data obtained in specific binding experiments. The chemokine CXCL8 (interleukin-8) is an interesting target for drug discovery due to its role in inflammatory diseases. Given the available structural data and information on its receptor interactions it constitutes a basis for the rational design of inhibitor peptides. Starting from the reported structure of CXCL8 in complex with a peptide derived from its receptor CXCR1 we developed a computational docking procedure to estimate the changes in binding energy as a function of individual amino acid exchanges. This indicates whether the respective amino acid residue must be preserved or can be substituted to maintain or improve affinity, respectively. To validate and improve the assumptions made in this docking simulation we established a fluorescence polarization assay for receptor-derived peptides binding to CXCL8. A peptide library was tested comprising selected mutants characterized by docking simulations. A number of predictions regarding electrostatic interactions were confirmed by these experiments and it was revealed that the model needed to be corrected for backbone flexibility. Therefore, the assay presented here is a promising tool to systematically improve the computational model by iterative cycles of modeling, experimental validation and refinement of the algorithm, leading to a more reliable model and peptides with improved affinity.

WDR11, a WD protein that interacts with transcription factor EMX1, is mutated in idiopathic hypogonadotropic hypogonadism and Kallmann syndrome.

By defining the chromosomal breakpoint of a balanced t(10;12) translocation from a subject with Kallmann syndrome and scanning genes in its vicinity in unrelated hypogonadal subjects, we have identified WDR11 as a gene involved in human puberty. We found six patients with a total of five different heterozygous WDR11 missense mutations, including three alterations (A435T, R448Q, and H690Q) in WD domains important for ß propeller formation and protein-protein interaction. In addition, we discovered that WDR11 interacts with EMX1, a homeodomain transcription factor involved in the development of olfactory neurons, and that missense alterations reduce or abolish this interaction. Our findings suggest that impaired pubertal development in these patients results from a deficiency of productive WDR11 protein interaction.

Mutations in CHD7, encoding a chromatin-remodeling protein, cause idiopathic hypogonadotropic hypogonadism and Kallmann syndrome.

CHARGE syndrome and Kallmann syndrome (KS) are two distinct developmental disorders sharing overlapping features of impaired olfaction and hypogonadism. KS is a genetically heterogeneous disorder consisting of idiopathic hypogonadotropic hypogonadism (IHH) and anosmia, and is most commonly due to KAL1 or FGFR1 mutations. CHARGE syndrome, a multisystem autosomal-dominant disorder, is caused by CHD7 mutations. We hypothesized that CHD7 would be involved in the pathogenesis of IHH and KS (IHH/KS) without the CHARGE phenotype and that IHH/KS represents a milder allelic variant of CHARGE syndrome. Mutation screening of the 37 protein-coding exons of CHD7 was performed in 101 IHH/KS patients without a CHARGE phenotype. In an additional 96 IHH/KS patients, exons 6-10, encoding the conserved chromodomains, were sequenced. RT-PCR, SIFT, protein-structure analysis, and in situ hybridization were performed for additional supportive evidence. Seven heterozygous mutations, two splice and five missense, which were absent in > or = 180 controls, were identified in three sporadic KS and four sporadic normosmic IHH patients. Three mutations affect chromodomains critical for proper CHD7 function in chromatin remodeling and transcriptional regulation, whereas the other four affect conserved residues, suggesting that they are deleterious. CHD7's role is further corroborated by specific expression in IHH/KS-relevant tissues and appropriate developmental expression. Sporadic CHD7 mutations occur in 6% of IHH/KS patients. CHD7 represents the first identified chromatin-remodeling protein with a role in human puberty and the second gene to cause both normosmic IHH and KS in humans. Our findings indicate that both normosmic IHH and KS are mild allelic variants of CHARGE syndrome and are caused by CHD7 mutations.

Probing hot spots on protein-protein interfaces with all-atom free-energy simulation.

Modulation of protein-protein interactions by competitive small-molecule binding emerges as a promising avenue for drug discovery. Hot spots, i.e., amino acids with important contributions to the overall interaction energy, provide useful targets within these interfaces. To avoid time-consuming mutagenesis experiments, computational alanine screening has been developed for the prediction of hot spots based on existing structural information. Here we use the all-atom free-energy force field PFF02 to identify important amino acid residues in the complexes of the chemokine interleukin-8 (CXCL8) and an N-terminal peptide of its cognate receptor CXCR1, and of ERBIN, a molecular marker of the basolateral membrane in epithelial cells, in complex with the ERBIN-binding domain of tyrosin kinase ERBB2. The results of our analysis agree with available experimental functional assays, indicating that this approach is suitable for computational alanine screening and may help to identify competitive peptides as starting points for the development of inhibitors of protein-protein interactions for pharmaceutically relevant targets.

7-Alkyl-3-benzylcoumarins: a versatile scaffold for the development of potent and selective cannabinoid receptor agonists and antagonists.

A series of 7-alkyl-3-benzylcoumarins was designed, synthesized, and tested at cannabinoid CB(1) and CB(2) receptors in radioligand binding and cAMP accumulation studies. 7-Alkyl-3-benzylcoumarins were found to constitute a versatile scaffold for obtaining potent CB receptor ligands with high potency at either CB(1) or CB(2) and a broad spectrum of efficacies. Fine-tuning of compound properties was achieved by small modifications of the substitution pattern. The most potent compounds of the present series include 5-methoxy-3-(2-methylbenzyl)-7-pentyl-2H-chromen-2-one (19a, PSB-SB-1201), a selective CB(1)antagonist (K(i) CB(1) 0.022 µM), 5-methoxy-3-(2-methoxybenzyl)-7-pentyl-2H-chromen-2-one (21a, PSB-SB-1202), a dual CB(1)/CB(2)agonist (CB(1)K(i) 0.032 µM, EC(50) 0.056 µM; CB(2)K(i) 0.049 µM, EC(50) 0.014 µM), 5-hydroxy-3-(2-hydroxybenzyl)-7-(2-methyloct-2-yl)-2H-chromen-2-one (25b, PSB-SB-1203), a dual CB(1)/CB(2) ligand that blocks CB(1) but activates CB(2) receptors (CB(1)K(i) 0.244 µM; CB(2)K(i) 0.210 µM, EC(50) 0.054 µM), and 7-(1-butylcyclopentyl)-5-hydroxy-3-(2-hydroxybenzyl)-2H-chromen-2-one (27b, PSB-SB-1204), a selective CB(2) receptor agonist (CB(1)K(i) 1.59 µM; CB(2)K(i) 0.068 µM, EC(50) 0.048 µM).

Unique phenotype in a patient with CHARGE syndrome.

CHARGE is a phenotypically heterogeneous autosomal dominant disorder recognized as a cohesive syndrome since the identification of CHD7 as a genetic etiology. Classic features include: Coloboma, Heart defects, Atresia choanae, Retarded growth and development, Genitourinary abnormalities, and Ear anomalies and/or deafness. With greater accessibility to genetic analysis, a wider spectrum of features are emerging, and overlap with disorders such as DiGeorge syndrome, Kallmann syndrome, and Hypoparathyroidism Sensorineural Deafness and Renal Disease syndrome, is increasingly evident. We present a patient with a unique manifestation of CHARGE syndrome, including primary hypoparathyroidism and a limb anomaly; to our knowledge, he is also the first CHARGE subject reported with bilateral multicystic dysplastic kidneys. Furthermore, with structural modeling and murine expression studies, we characterize a putative CHD7 G744S missense mutation. Our report continues to expand the CHARGE phenotype and highlights that stringent fulfillment of conventional criteria should not strictly guide genetic analysis.

Nasal embryonic LHRH factor (NELF) mutations in patients with normosmic hypogonadotropic hypogonadism and Kallmann syndrome.

OBJECTIVE: To determine if mutations in NELF, a gene isolated from migratory GnRH neurons, cause normosmic idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS). DESIGN: Molecular analysis correlated with phenotype. SETTING: Academic medical center. PATIENT(S): A total of 168 IHH/KS patients as well as unrelated control subjects were studied for NELF mutations. INTERVENTION(S): NELF coding regions/splice junctions were subjected to polymerase chain reaction (PCR)-based DNA sequencing. Eleven additional IHH/KS genes were sequenced in three patients with NELF mutations. MAIN OUTCOME MEASURE(S): Mutations were confirmed by sorting intolerant from tolerant, reverse-transcription (RT)-PCR, and Western blot analysis. RESULT(S): Three novel NELF mutations absent in 372 ethnically matched control subjects were identified in 3/168 (1.8%) IHH/KS patients. One IHH patient had compound heterozygous NELF mutations (c.629-21G>C and c.629-23C>G), and he did not have mutations in 11 other known IHH/KS genes. Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: NELF/KAL1 (c.757G>A; p.Ala253Thr of NELF and c.488_490delGTT; p.Cys163del of KAL1) and NELF/TACR3 (c.1160-13C>T of NELF and c.824G>A; p.Trp275X of TACR3). In vitro evidence of these NELF mutations included reduced protein expression and splicing defects. CONCLUSION(S): Our findings suggest that NELF is associated with normosmic IHH and KS, either singly or in combination with a mutation in another gene.