Background VX-809, a cystic fibrosis transmembrane conductance regulator (CFTR) modulator, has been shown to increase the cell surface density of functional F508del-CFTR in vitro. Methods A randomised, double-blind, placebo-controlled study evaluated the safety, tolerability and pharmacodynamics of VX-809 in adult patients with cystic fibrosis (n=89) who were homozygous for the F508del-CFTR mutation. Subjects were randomised to one of four VX-809 28 day dose groups (25, 50, 100 and 200 mg) or matching placebo. Results The type and incidence of adverse events were similar among VX-809- and placebo-treated subjects. Respiratory events were the most commonly reported and led to discontinuation by one subject in each active treatment arm. Pharmacokinetic data supported a once-daily oral dosing regimen. Pharmacodynamic data suggested that VX-809 improved CFTR function in at least one organ (sweat gland). VX-809 reduced elevated sweat chloride values in a dose-dependent manner (p=0.0013) that was statistically significant in the 100 and 200 mg dose groups. There was no statistically significant improvement in CFTR function in the nasal epithelium as measured by nasal potential difference, nor were there statistically significant changes in lung function or patient-reported outcomes. No maturation of immature F508del-CFTR was detected in the subgroup that provided rectal biopsy specimens. Conclusions In this study, VX-809 had a similar adverse event profile to placebo for 28 days in F508del-CFTR homozygous patients, and demonstrated biological activity with positive impact on CFTR function in the sweat gland. Additional data are needed to determine how improvements detected in CFTR function secondary to VX-809 in the sweat gland relate to those measurable in the respiratory tract and to long-term measures of clinical benefit. Clinical trial number NCT00865904
Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed three previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry. We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dissociation constant (Kd) of 600 nM; a phosphopeptide based on one binding site from the platelet-derived growth factor receptor binds to the Nterminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subunit with a Kd of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kd of 4 ,uM. In addition, we demonstrate that avidity effects that result from the dimerization of glutathione S-transferase fusion proteins with SH2 domains could be responsible for overestimates of affinities for these interactions previously studied by surface plasmon resonance.The specificity of signaling pathways involving tyrosine kinases is thought to be defined, at least in part, by Src homology 2 (SH2) domain-mediated interactions. SH2 domains interact with tyrosine residues that have been phosphorylated by kinases in the pathway (1, 2). Mutagenesis studies have shown that particular phosphotyrosine [Tyr(P)] residues in activated receptors serve to recruit particular downstream signaling molecules via interactions with their SH2 domains (3-5). This indicates that the specificity of these SH2-Tyr(P) interactions in intracellular signaling pathways is defined by the sequence and structural context of the Tyr(P) residue. These interactions can be reconstituted by using short peptides that contain a Tyr(P) residue (6). Studies involving selection from randomized Tyr(P)-containing peptide libraries have shown that specific motifs, based on the amino acids proximal to the Tyr(P) residue, are recognized by different SH2 domains (7). Through use of such peptides in several techniques, relative affinities have been reported for various SH2-peptide interactions. Kd values from 10 ,uM to 0.1 nM have been reported, representing a wide range over which specificity can apply. There are, however, a number of discrepancies in the reported data.The Kd for the interaction between the SH2 domain of Src [in the form of a fusion protein with glutathione S-transferase (GST)] and a peptide based on the tyrosine phosphorylation siteThe publication costs of this article were defrayed in part by page charge paym...
Many proteins involved in intracellular signal transduction contain a small, 50‐60 amino acid domain, termed the Src homology 3 (SH3) domain. This domain appears to mediate critical protein‐protein interactions that are involved in responses to extracellular signals. Previous studies have shown that the SH3 domains from several proteins recognize short, contiguous amino acid sequences that are rich in proline residues. While all SH3 recognition sequences identified to date share a conserved P‐X‐X‐P motif, the sequence recognition specificity of individual SH3 domains is poorly understood. We have employed a novel modification of phage display involving biased libraries to identify peptide ligands of the Src, Fyn, Lyn, PI3K and Abl SH3 domains. With biased libraries, we probed SH3 recognition over a 12 amino acid window. The Src SH3 domain prefers the sequence XXXRPLPPLPXP, Fyn prefers XXXRPLPP(I/L)PXX, Lyn prefers RXXRPLPPLPXP, PI3K prefers RXXRPLPPLPP while the Abl SH3 domain selects phage containing the sequence PPPYPPPP(I/V)PXX. We have also analysed the binding properties of Abl and Src SH3 ligands. We find that although the phage‐displayed Abl and Src SH3 ligands are proline rich, they are distinct. In surface plasmon resonance binding assays, these SH3 domains displayed highly selective binding to their cognate ligands when the sequences were displayed on the surface of the phage or as synthetic peptides. The selection of these high affinity SH3 peptide ligands provides valuable information on the recognition motifs of SH3 domains, serve as new tools to interfere with the cellular functions of SH3 domain‐mediated processes and form the basis for the design of SH3‐specific inhibitors of disease pathways.
SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase-linked receptors.Recent three-dimensional structures suggest mechanisms by which tandem SH2 domains might confer higher specificity than individual SH2 domains. To test this, binding studies were conducted with tandem domains from the five signaling enzymes: phosphatidylinositol 3-kinase p85, ZAP-70, Syk, SHP-2, and phospholipase C-␥1. Bisphosphorylated TAMs (tyrosine-based activation motifs) were derived from biologically relevant sites in platelet-derived growth factor, T cell, B cell, and high affinity IgE receptors and the receptor substrates IRS-1 (insulin receptor substrate-1) and SHPS-1/ SIRP. Each tandem SH2 domain binds a distinct TAM corresponding to its appropriate biological partner with highest affinity (0.5-3.0 nM). Alternative TAMs bind the tandem SH2 domains with 1,000-to >10,000-fold lower affinity than biologically relevant TAMs. This level of specificity is significantly greater than the ϳ20 -50-fold typically seen for individual SH2 domains. We conclude that high biological specificity is conferred by the simultaneous interaction of two SH2 domains in a signaling enzyme with bisphosphorylated TAMs in activated receptors and substrates.SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase receptors (1). The SH2 domains bind phosphorylated receptor tyrosines, and, since many SH2 domain proteins also contain or associate with catalytic subunits, these interactions recruit the effector enzymes to activated receptors. Tyrosine kinase signaling pathways thus gain specificity from the intrinsic binding preferences of SH2 domains for short sequences that flank phosphotyrosine.A great deal has been learned by studying isolated, individual SH2 domains. Common mechanisms are used for phosphotyrosine recognition (2-12). Most notably, SH2 domain residues Arg␣A2 and ArgB5 chelate the phosphotyrosine phosphate. The latter is within the conserved FLVRES sequence. Binding site selectivity is conferred by interactions between two variable loops within SH2 domains (EF and BG) and peptide residues COOH-terminal to phosphotyrosine. The degree of selectivity varies, but phosphopeptides derived from biologically relevant sites typically bind with 20 -50-fold higher affinity than irrelevant or randomized sequences (e.g. Refs. 13-18). Although it is true that a small subset of SH2 domains shows greater selectivity (e.g. 1,000-fold for Grb2), 1 these are exceptions and not the rule.Nevertheless, biological specificity in intact cells is substantially greater than 50-fold, suggesting that more is involved than individual SH2 domain interactions. In fact, all SH2 domain proteins contain additional binding modules (e.g. SH2, SH3, PTB, and PH domains) or motifs. A fundamental concept may have been overlooked by the common tendency to evaluate specificity using isolated, individual domains. Simultaneous binding to multiple domains could enhance specificity through combinatorial effects, as 1) each ...
The Src homology 3 (SH3) domain is a 50-aa modular unit present in many cellular proteins involved in intracellular signal transduction. It functions to direct protein-protein interactions through the recognition of prolinerich motifs on associated proteins. SH3 domains are important regulatory elements that have been demonstrated to specify distinct regulatory pathways important for cell growth, migration, differentiation, and responses to the external milieu. By the use of synthetic peptides, ligands have been shown to consist of a minimum core sequence and to bind to SH3 domains in one of two pseudosymmetrical orientations, class I and class II. The class I sites have the consensus sequence ZP(L/P)PPIP whereas the class II consensus is PPIPPZ (where I is a hydrophobic residue and Z is a SH3 domain-specific residue). We previously showed by M13 phage display that the Src, Fyn, Lyn, and phosphatidylinositol 3-kinase (PI3K) SH3 domains preferred the same class I-type core binding sequence, RPLPPIP. These results failed to explain the specificity for cellular proteins displayed by SH3 domains in cells. In the current study, class I and class II core ligand sequences were displayed on the surface of bacteriophage M13 with five random residues placed either N-or C-terminal of core ligand residues. These libraries were screened for binding to the Src, Fyn, Lyn, Yes, and P13K SH3 domains. By this approach, additional ligand residue preferences were identified that can increase the affinity of SH3 peptide ligands at least 20-fold compared with core peptides. The amino acids selected in the flanking sequences were similar for Src, Fyn, and Yes SH3 domains; however, Lyn and P13K SH3 domains showed distinct binding specificities. These results indicate that residues that flank the core binding sequences shared by many SH3 domains are important determinants of SH3 binding affinity and selectivity.One feature common to many proteins involved in signal transduction is the capacity to associate specifically with other molecules, forming a complex that can alter cell activity. Such multiprotein associations are facilitated by domain(s) of conserved sequence which serve as noncatalytic modules that direct molecular interactions during signal transduction (1, 2). One such domain, termed the Src homology 2 (SH2) domain, binds to phosphotyrosine-containing proteins in a sequencespecific manner (3). Another module, termed the SH3 domain, has a preference for proline-rich binding sites (4-9). In a number of cell types, both SH2 and SH3 domains function to regulate cellular events such as protein localization, enzyme activity, and substrate recruitment (1, 2).SH2 and SH3 domains help determine the routes of intracellular communication (1, 2). The sequences that these domains recognize are important determinants of specific signal transduction pathways. Targeting these modules with pharmaceutical agents that mimic binding sites may provide a means to block specific events in the cell. To investigate SH3 binding specificity, S...
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