Norway pharmacy online: Kjøp av viagra uten resept i Norge på nett.

Jeg kan anbefale en god måte for å øke potens - Cialis. Fungerer mye bedre kjøp levitra Alltid interessant, disse pillene og andre ting i Generelle virkelig har helse til å handle.

Pq059901953p

Affinity modulation of small-molecule ligands by borrowing
endogenous protein surfaces

ROGER BRIESEWITZ*, GREGORY T. RAY†, THOMAS J. WANDLESS†‡, AND GERALD R. CRABTREE*‡*Howard Hughes Medical Institute and †Department of Chemistry, Stanford University, Stanford, CA 94305 Contributed by Gerald R. Crabtree, December 16, 1998 ABSTRACT
A general strategy is described for improving
unlike the FK506–FKBP complex, the rapamycin–FKBP com- the binding properties of small-molecule ligands to protein
plex establishes only few contacts with its target (10).
targets. A bifunctional molecule is created by chemically linking
Inspired by these natural examples, we envisioned that the a ligand of interest to another small molecule that binds tightly
specificity and affinity of a ligand–protein interaction or a to a second protein. When the ligand of interest is presented to
drug–protein interaction could be modulated deliberately by the target protein by the second protein, additional protein–
borrowing additional surface contacts from an endogenous protein interactions outside of the ligand-binding sites serve
protein. Thus, we set out to chemically link a ligand for an either to increase or decrease the affinity of the binding event. We
abundant presenting protein to a weak binder for a target have applied this approach to an intractable target, the SH2
protein. By using chemical linkers of different lengths, tilt, and domain, and demonstrate a 3-fold enhancement over the natural
rotation, one may expect the resulting protein–protein inter- peptide. This approach provides a way to modulate the potency
actions to be favorable or unfavorable so that binding occurs and specificity of biologically active compounds.
with enhanced or decreased affinity, respectively. As an ex- tension of traditional medicinal chemistry, such an approach In nature, certain small-molecule ligands use a remarkable may be useful to modulate the potency and specificity of mechanism to enhance the affinity for their targets. These ligands bind to an endogenous protein, forming a new com- As a first attempt to explore the feasibility of this approach, posite surface, which can bind then to a target protein.
we selected two members of the FK506-binding protein family, Presentation of the ligand by the larger endogenous protein FKBP12 and FKBP52, as presenter proteins. We chose the vastly enlarges the surface area available for interactions with SH2 domain of the Fyn tyrosine kinase as the target protein the target, facilitating additional protein–protein interactions because of its well established structure and the availability of ligands for SH2 domains (11). SH2 domains bind to peptides For example, the peptide ligands of the T cell receptor use and proteins that contain phosphotyrosine residues, and they this strategy of surface enlargement to promote a high-affinity- are commonly found in signaling proteins that regulate cell binding event. By themselves, peptides usually have only a very growth and differentiation. Ligands that bind to SH2 domains low affinity for the polymorphic T cell receptor (TCR). For a have been explored as possible therapeutics for cancer, osteo- high-affinity-binding event to occur, a peptide must be pre- porosis, and inflammation and as immunosuppressive agents sented by the major histocompatibility complex (MHC). As the (12, 13). However, the development of ligands that bind to SH2 crystal structures of the trimeric TCR–peptide–MHC complex domains with high affinity and selectivity has met with little have shown, the TCR makes contacts not only with the peptide success, and SH2 domains generally are considered to be good but also with the MHC (1, 2). The TCR-MHC contacts are examples of an intractable drug target.
important because mutations of these sites abolish the immune response and much of the remarkable specificity of the im- mune response is a result of this simple trimeric complex (3–5).
MATERIALS AND METHODS
Certain microorganisms also make use of endogenous pro- Synthetic Chemistry. Peptides were synthesized by using
teins to present and enhance the activity of their toxins. For conventional solid-phase peptide synthesis methods. The example, the immunosuppressive drugs cyclosporin and FK506 FK506-derived mixed carbonate was synthesized as described are presented by cyclophilin and human FK506-binding pro- by Spencer et al. (14). It was dissolved in dimethylformamide tein 12 (FKBP), respectively, to inhibit the activity of a with triethylamine and a 2-fold excess of phosphotyrosyl- common protein target, calcineurin (6). By themselves, FK506 glutamyl-glutamyl-isoleucine (pYEEI). The coupled product and cyclosporin have no measurable affinity for calcineurin.
was treated with hydrogen fluoride in acetonitrile to remove However, the FK506–FKBP and cyclosporin–cyclophilin com- the two silyl ether protecting groups, and the desired product plexes bind to calcineurin with high affinity (7). The cocrystal was purified by using reverse-phase HPLC. Synthetic ligand for structure of calcineurin–FK506–FKBP reveals that the protein FKBP (SLF) was synthesized according to the procedures of surfaces of calcineurin and FKBP make extensive contacts that Holt et al. (15). SLF was coupled to the N terminus of the promote the high affinity of the binding event (8, 9).
resin-bound protected pYEEI peptide by using PyBOP. The However, extensive protein–protein interactions between a bifunctional SLFpYEEI was simultaneously deprotected and presenting protein and the drug target are not always required cleaved from the Novasyn TGT resin (Calbiochem) by using to enhance the affinity of a ligand for its target protein. Like 25% trifluoroacetic acid and 2.5% triisopropylsilane in meth- FK506, the macrolide drug rapamycin has no measurable affinity for its target, the cell cycle control protein FRAP.
Instead, rapamycin forms a complex with FKBP to create a Abbreviations: FKBP, human FK506-binding protein 12; pYEEI, tetrapeptide phosphotyrosyl-glutamyl-glutamyl-isoleucine; FKpY- composite surface that binds to FRAP with high affinity. But, EEI, FK506 covalently linked to the peptide pYEEI; SLF, synthetic ligand for FKBP; SLFpYEEI, SLF covalently linked to pYEEI; GST, The publication costs of this article were defrayed in part by page charge glutathione S-transferase; ITC, isothermal titration calorimetry; TCR, payment. This article must therefore be hereby marked ‘‘advertisement’’ in A Commentary on this article begins on page 1826.
accordance with 18 U.S.C. §1734 solely to indicate this fact.
‡To whom reprint requests should be addressed. e-mail: hf.grc@ PNAS is available online at www.pnas.org.
forsythe.stanford.edu or wandless@chem.stanford.edu.
Proc. Natl. Acad. Sci. USA 96 (1999) ylene chloride, and the desired product was isolated by using Peptide Coupling to Beads. The peptide pYEEI (1 mg) was
dissolved in 1 ml of dimethyl sulfoxide and incubated with 1 ml of Affi-Gel 10 beads (Bio-Rad) for 6 hr at room temperature.
The reaction was stopped by incubating the beads in 5 ml of ethanolamine (1 M, pH 8.0) for 1 hr. The beads were washed and resuspended 1:2 in 20 mM Tris, pH 7.2͞150 mM NaCl.
Protein Expression. The human Fyn SH2 domain (residues
102–205, SIQA-LVVP) and human FKBP12 were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The cDNAs were cloned into the pGEX2TK expression vector (Pharmacia). When required, the recombinant proteins were labeled with [␥-32P]ATP on glutathione beads (Pharma- cia) using protein kinase A (PKA) and the pGEX2TK-derived PKA site at the N terminus of FKBP12 and the Fyn SH2 domain. FKBP12 and the Fyn SH2 domain were cleaved from GST by using thrombin (Sigma) and the thrombin cleavage site between the PKA site and GST. Human FKBP52 was ex- pressed in pET28c (Novagen) with a His tag at the N terminus.
The recombinant protein was purified with Ni2ϩ nitrilotriace- Isothermal Titration Calorimetry. The binding constants of
FKBP12, FKBP52, and the Fyn SH2 domain for SLF, SLF- pYEEI, and FKpYEEI were determined by using an Omega Isothermal Titration Calorimeter (Microcal, Northampton, MA). Protein in aqueous buffer (20–60 ␮M: 150 mM NaCl͞20 FIG. 1. Structures of FKpYEEI and SLFpYEEI. The C21 allyl mM Tris, pH 7.2) was equilibrated in the microcalorimeter cell group of FK506, which is required for calcineurin binding, was used to link the pYEEI peptide, effectively eliminating any immunosuppres- at 25°C for 1–2 hr after being degassed for 10 min. Ligand sive activity of the FKpYEEI molecule.
(170–600 ␮M) in identical buffer as protein was taken into a 250-␮l syringe after being degassed for 10 min. The syringe was A competition binding assay was used to determine whether loaded into the cell and spun at 400 rpm, and the system was the pYEEI peptide bound more tightly to the Fyn SH2 domain allowed to equilibrate for 1–2 hr. Ligand was then injected into when presented by FKBP (Fig. 3A). The pYEEI tetrapeptide the cell (25 ϫ 10 ␮l, 6-min intervals), and the heat evolved was was covalently attached to beads. The beads were incubated quantitated. Binding constants were calculated from a numer- with radioactively labeled Fyn SH2 domain, and the bound ical fit to the experimental data as described in ref. 16.
protein was quantified by centrifuging the beads and counting the associated radioactivity. When FKpYEEI or SLFpYEEI was added to the binding reaction, the pYEEI peptide of the bifunctional molecules competed with the peptide beads for Molecular Design. Beginning with a tetrapeptide that binds
binding to the 32P-labeled Fyn SH2 domain. As a consequence to the Fyn SH2 domain, pYEEI (17), we synthesized two of this competition, less 32P-labeled Fyn SH2 domain bound to bifunctional molecules. Both molecules are capable of simul- the beads. Hence, low levels of radioactivity bound to the taneously binding to FKBP and the Fyn SH2 domain. The beads reflect high occupancy of the Fyn SH2 domain by a pYEEI peptide was linked covalently to two FKBP ligands, competing ligand in the solution (Fig. 3B).
FK506 and SLF (15), to provide the desired bifunctional Using a Borrowed Protein Surface to Enhance Affinity. By
molecules, FKpYEEI and SLFpYEEI (Fig. 1). SLF is smaller using this assay, FKBP12 or FKBP52 was added at various than FK506 and does not project as far from the FKBP protein concentrations to a binding reaction to form a complex with surface. By using the three-dimensional structures of FKBP12 FKpYEEI or SLFpYEEI. When presented by FKBP12, FK- (15, 18), FKBP52 (19), and the Fyn SH2 domain (20), the pYEEI bound to the Fyn SH2 domain as well as free FKpYEEI linkers between the two halves of the bifunctional molecules (data not shown). However, the FKpYEEI–FKBP52 complex were designed to bring the FKBP surface into close proximity competed more effectively for binding to the Fyn SH2 domain to the SH2 domain surface. The affinities of FKpYEEI and than did FKpYEEI alone (Fig. 3B). To confirm that the SLFpYEEI for recombinant FKBP12, FKBP52, and the Fyn observed effect depends on binding of FKpYEEI to the SH2 domain were measured by using isothermal titration FK506-binding pocket of FKBP52, FK506 was added to the binding reaction. FK506 binds more tightly to FKBP12 and Binding Assay. To determine whether the bifunctional
FKBP52 than either bifunctional molecule (Table 1). In the molecules allow the formation of a trimeric complex between binding assay, as FK506 reaches an equimolar concentration Fyn and FKBP, GST-Fyn SH2 domain fusion proteins bound to glutathione beads were incubated with radioactively labeled Table 1. Dissociation constants (Kd) for FKBP12 and increasing concentrations of FKpYEEI or SLF- pYEEI (Fig. 2). After equilibrium was established, the beads were sedimented and the radioactively labeled FKBP12 asso- ciated with the beads was quantified. Both FKpYEEI and SLFpYEEI support the formation of a trimeric complex between the Fyn SH2 domain and FKBP12. However, FKpY- EEI forms the trimeric complex more efficiently. FKpYEEI and SLFpYEEI can also form a trimeric complex between immobilized FKBP52 and the radioactively labeled Fyn SH2 Proc. Natl. Acad. Sci. USA 96 (1999) effective competitor than SLFpYEEI alone. The effect is reversed by the addition of FK506, which indicates that SLFpYEEI binds to the FK506-binding pocket of FKBP12 (Fig. 4B). The IC50 of SLFpYEEI for the Fyn SH2 domain when presented by FKBP12 was increased 6-fold from 0.25 to 1.5 ␮M (Fig. 4C). This increase was confirmed by ITC (SLF- pYEEI plus Fyn SH2 domain: Kd ϭ 180 nM versus the SLFpYEEI–FKBP12 complex plus Fyn SH2 domain: Kd ϭ 1.0 ␮M; 5.5-fold increase). We interpret this decrease in affinity to indicate that the FKBP12 surface establishes unfavorable interactions with the Fyn SH2 domain surface in the trimeric complex (Fig. 4D). The SLFpYEEI–FKBP52 complex does not affect the binding of the Fyn SH2 domain to pYEEI (data not shown). Considering the four possible complexes for presentation of the pYEEI peptide (two FKBPs and two bifunctional molecules), one complex improves binding of the pYEEI peptide to the Fyn SH2 domain, one complex dimin- ishes binding, and two complexes have no measurable effect.
DISCUSSION
Borrowing Endogenous Proteins to Enhance the Charac-
teristics of Small-Molecule Ligands. Our findings demonstrate
that a phosphopeptide ligand for the Fyn SH2 domain can be FIG. 2. FKpYEEI and SLFpYEEI form trimeric complexes with engineered to bind more tightly to its protein target by the Fyn SH2 domain and FKBP12. Recombinant GST-Fyn SH2 domain was expressed, bound to glutathione beads (7.5 ␮l beads, 0.55 inducing the formation of a trimeric complex. In the case of nmol protein), and was incubated with 100 nM 32P-labeled FKBP12 FKBP52, FKpYEEI, and the Fyn SH2 domain, the trimeric (10,000 cpm) in 100 ␮l of binding buffer (20 mM Tris, pH 7.2͞150 mM complex is more stable than would be expected based on the NaCl). Increasing concentrations of FKpYEEI or SLFpYEEI were stabilities of individual bimolecular complexes (Table 1).
added and the reactions were rotated for 2 hr. The binding reactions Although our data do not definitively demonstrate new pro- were centrifuged in a pierced PCR tube to separate the beads from the tein–protein interactions resulting from the covalent linkage of supernatant. The radioactive protein associated with the beads was FK506 to the pYEEI peptide, the observation that the change resuspended in 100 ␮l of PBS, which was added to EcoLite scintillation in affinity is related to both the structure of the ligand (SLF vs.
fluid (ICN) and counted in an LS5000CE liquid scintillation counter FK506) and the borrowed endogenous protein (FKBP12 vs.
FKBP52) points to the surface between the SH2 domain and with respect to FKBP52, most of the FKBP52 is bound to the FKBP as the origin of the altered affinity.
FK506. Under these conditions, the affinity-enhancing effect One explanation for the increase in affinity is that additional of FKBP52 was abolished (Fig. 3C). These results demonstrate protein–protein interactions between FKBP52 and the SH2 that the enhanced affinity of FKpYEEI for the Fyn SH2 domain surface make a significant, direct energetic contribu- domain depends on FKpYEEI binding to the FK506-binding tion to the stability of the complex. Alternatively, the addi- tional distal interactions may indirectly enhance the free To quantify the increase in affinity of the Fyn SH2 domain energy of binding of the peptide ligand to the SH2 domain.
for pYEEI presented by FKBP52, we measured the binding of This possibility is based on the analysis of the energetic the 32P-labeled Fyn SH2 domain to peptide beads as a function contributions of single amino acid side chains to protein– of the concentration of FKpYEEI (Fig. 3D). FKBP52 en- protein interactions. The area of contact between two proteins hances the affinity by a factor of 3, which is reflected in a shift is often large and flat (21, 22), but, interestingly, a major part of the free energy of binding of two interacting protein 50 from 750 nM in the absence of FKBP52 to an IC50 of 250 nM in the presence of FKBP52. ITC measurements surfaces can be contributed by a limited number of clustered confirmed the approximately 3-fold affinity enhancement amino acids. These clusters have been designated as ‘‘hot spots’’ (23), and the surrounding contacts may serve to insulate FKpYEEI–FKBP52 complex plus Fyn SH2 domain: K the critical amino acids from bulk solvent. Thus, the SH2 domain-FKBP52 contacts may limit the accessibility of water to pYEEI and the binding pocket so that their energetic We considered the possibility that FKBP12 and FKBP52 interactions are increased. Ultimately, structural studies will present the pYEEI peptide in different orientations. However, be helpful for confirming the role of FKBP-SH2 domain the structures of the FK506-binding domains from both pro- contacts in the affinity modification demonstrated here.
teins are very similar (19), and it is unlikely that preorgani- Plasticity of Binding Surfaces. The establishment of favor-
zation of the relatively flexible pYEEI peptide is responsible able or, at least, nondetrimental contacts will depend on the for the observed binding enhancement. Thus, we conclude that juxtaposition of the presenter protein surface to the surface of when the Fyn SH2 domain binds to the FKpYEEI-FKBP52 the target protein. Recent studies of human growth hormone complex, the SH2 domain makes additional favorable protein– and the TCR suggest that the plasticity of protein surfaces can protein interactions with FKBP52 that enhance the overall act favorably in the attempt to establish beneficial protein– stability of the trimeric complex (Fig. 3E).
protein contacts between the presenter protein and the target.
Using a Borrowed Protein Surface to Reduce Affinity. In
Mutations in growth hormone that compensate for a mutation contrast to the FKpYEEI–FKBP52 complex, the SLFpYEEI– in the core region of its receptor have been shown to lead to FKBP12 complex displays decreased affinity for the Fyn SH2 major rearrangements at the protein–protein interface (24).
domain. In the competition binding assay, greater amounts of The crystal structure of the mutant proteins reveals that amino the Fyn SH2 domain bind to the peptide beads in response to acid side chains reorganize to establish new interactions or to increasing concentrations of FKBP12 (Fig. 4A). This result avoid unfavorable interactions. The plasticity of protein sur- indicates that the FKBP12–SLFpYEEI complex is a less faces is also apparent in the binding of the TCR to peptide– Proc. Natl. Acad. Sci. USA 96 (1999) FIG. 3. Fyn SH2 domain binding to FKpYEEI alone or in a complex with FKBP52. (A) Model of the competition binding assay. (B–D) In 100 ␮l of binding buffer, peptide beads (7.5 ␮l) were incubated with the 32P-labeled Fyn SH2 domain (200 nM, 14,000 cpm) and the molecules stated below. After a 2-hr incubation time, the beads were centrifuged and the radioactive Fyn SH2 domain bound was quantitated. (B) Addition of FKpYEEI (1.0 ␮M) and increasing concentrations of FKBP52 (0.25–4.0 ␮M). (C) Addition of FKpYEEI (1.0 ␮M), FKBP52 (2.0 ␮M), and increasing concentrations of FK506 (0.25–15 ␮M). The maximal radioactivity associated with the beads (ϭ100%) was 9,139 cpm for b and 10,467 cpm for C. (D) Competition binding curves: 32P-labeled Fyn SH2 domain (200 nM, 16,000 cpm) was incubated without or with FKBP52 (4.5 ␮M) in 100 ␮l of binding buffer. Increasing concentrations of FKpYEEI (0.1–20.0 ␮M) were added to the binding reactions. The maximal radioactivity associated with the beads was 12,569 cpm (ϭ100%) for the 32P-labeled Fyn SH2 domain alone and 10,250 cpm (ϭ100%) for the 32P-labeled Fyn SH2 domain in the presence of FKBP52. All data points were taken in triplicate, and the average is plotted. (Bars ϭ SE.) (E) Model for favorable, affinity-enhancing protein–protein interactions.
MHC, which can lead to large conformational changes in the a phosphopeptide with its SH2 domain. Steric hindrance or complementarity determining regions of the TCR to gain new electrostatic repulsion are probably the basis for the decreased stability of the SH2 domain–SLFpYEEI–FKBP12 complex. In We also have demonstrated that borrowing the surface of an general, the creation of unfavorable contacts should be easier endogenous protein can reduce the affinity of interactions of to achieve than favorable contacts, and it can be exploited to Proc. Natl. Acad. Sci. USA 96 (1999) FIG. 4. Fyn SH2 domain binding to SLFpYEEI in a complex with FKBP12. (A–C) In 100 ␮l of binding buffer, peptide beads (7.5 ␮l) were incubated with the 32P-labeled Fyn SH2 domain (200 nM, 18,000 cpm) and the molecules stated below. After a 2-hr incubation time, the beads were centrifuged and the radioactive Fyn SH2 domain bound was quantitated. (A) Addition of SLFpYEEI (1.0 ␮M) and increasing concentrations of FKBP12 (0.25–4.0 ␮M). (B) Addition of SLFpYEEI (1.0 ␮M), FKBP12 (2.0 ␮M), and increasing concentrations of FK506 (1.0–3.0 ␮M). The maximal radioactivity associated with the beads (ϭ100%) was 12,583 cpm for A and 12,187 cpm for B. (C) Competition binding curves: 32P-labeled Fyn SH2 domain (200 nM, 13,000 cpm) was incubated without or with FKBP12 (20 ␮M) in 100 ␮l of binding buffer. Increasing concentrations of FKpYEEI (0.1–20.0 ␮M) were added to the binding reactions. The maximal radioactivity associated with the beads was 9,284 cpm (ϭ100%) for the 32P-labeled Fyn SH2 domain alone and 10,492 cpm (ϭ100%) for the 32P-labeled Fyn SH2 domain in the presence of FKBP12. All data points were taken in triplicate, and the average was plotted. (Bars ϭ SE.) (D) Model for unfavorable, destabilizing protein–protein interactions.
enhance the specificity of a molecule of interest. If, for molecule drugs for their targets. It can also provide a useful example, a ligand binds to one desired and several undesired tool for the, so far, intractable problem of developing agonists targets, a bifunctional molecule that causes unfavorable pro- or antagonists of protein–protein interactions.
tein–protein interactions with the undesired targets may be selected. If the obtained bifunctional molecule shows favorable We thank Lewis Cantley for providing the Fyn SH2 domain or at least neutral interactions with the desired molecule, expression construct; David Smith for providing the FKBP52 expres- specificity for the desired molecule will be created.
sion construct; Jay Boniface, Dan Lyons, and Steve Biggar for helpful General Strategy to Modulate Ligand-Binding Affinities.
discussion; Bob Flowers for help designing and Lincoln Bickford for The introduction of secondary binding interactions through help executing the ITC measurements; and Kurt Vogel for assistance synthetic modifications to small-molecule ligands has been in the HPLC purification of compounds.
examined in some detail (26, 27). Our approach differs by borrowing surface area from presenting proteins. Features of 1. Garcia, K. C., Degano, M., Stanfield, R. L., Brunmark, A., our strategy include the ability to (i) recruit different endog- Jackson, M. R., Peterson, P. A., Teyton, L. & Wilson, I. A. (1996) enous presenting proteins by changing one-half of the bifunc- Science 274, 209–219.
tional molecule, (ii) vary the length and rigidity of the linker 2. Garboczi, D. N., Ghosh, P., Utz, U., Fan, Q. R., Biddison, W. E.
& Wiley, D. C. (1996) Nature (London) 384, 134–141.
that joins the two halves of the bifunctional molecule, and (iii) 3. Ajitkumar, P., Geier, S. S., Kesari, K. V., Borriello, F., Nakagawa, vary the affinity of the small-molecule ligand that binds to the M., Bluestone, J. A., Saper, M. A, Wiley, D. C. & Nathenson, presenting protein. Changes to any of these three variables S. G. (1988) Cell 54, 47–56.
have the potential to directly affect the overall stability of the 4. Sun, R., Shepherd, S. E., Geier, S. S., Thomson, C. T., Sheil, J. M.
trimeric complex. Ultimately, this general strategy may prove & Nathenson, S. G. (1995) Immunity 3, 573–582.
useful to improve the affinity and͞or specificity of small- 5. Smith, K. D. & Lutz, C. T. (1997) J. Immunol. 158, 2805–2812.
Proc. Natl. Acad. Sci. USA 96 (1999) 6. Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman, 18. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, I. & Schreiber, S. L. (1991) Cell 66, 807–815.
S. L. & Clardy, J. (1991) Science 252, 839–842.
7. Liu, J., Albers, M. W., Wandless, T. J., Luan, S., Alberg, D. G., 19. Craescu, C. T., Rouviere, N., Popescu, A., Cerpolini, E., Lebeau, Belshaw, P. J., Cohen, P., MacKintosh, C., Klee, C. B. & M. C., Baulieu, E. E. & Mispelter, J. (1996) Biochemistry 35,
Schreiber, S. L. (1992) Biochemistry 31, 3896–3901.
8. Griffith, J. P., Kim, J. L., Kim, E. E., Sintchak, M. D., Thomson, 20. Mulhern, T. D., Shaw, G. L., Morton, C. J., Day, A. J. & J. A., Fitzgibbon, M. J., Fleming, M. A., Caron, P. R., Hsiao, K.
Campbell, I. D. (1997) Structure 5, 1313–1323.
& Navia, M. A. (1995) Cell 82, 507–522.
21. Janin, J. & Chothia, C. (1990) J. Biol. Chem. 265, 16027–16030.
9. Kissinger, C. R., Parge, H. E., Knighton, D. R., Lewis, C. T., 22. Jones, S. & Thornton, J. M. (1996) Proc. Natl. Acad. Sci. USA 93,
Pelletier, L. A., Tempczyk, A., Kalish, V. J., Tucker, K. D., Showalter, R. E., Moomaw, E. W., et al. (1995) Nature (London) 23. Clackson, T. & Wells, J. A. (1995) Science 267, 383–386.
378, 641–644.
24. Atwell, S., Ultsch, M., De Vos, A. M. & Wells, J. A. (1997) 10. Choi, J., Chen, J., Schreiber, S. L. & Clardy, J. (1996) Science 273,
278, 1125–1128.
11. Koch, C. A., Anderson, D., Moran, M. F., Ellis, C. & Pawson, T.
25. Garcia, K. C., Degano, M., Pease, L. R., Huang, M., Peterson, (1991) Science 252, 668–674.
P. A., Teyton, L. & Wilson, I. A. (1998) Science 279, 1166–1172.
12. Pawson, T. (1995) Nature (London) 373, 573–580.
26. Jain, A., Huang, S. G. & Whitesides, G. M. (1994) J. Am. Chem. 13. Bolen, J. B. & Brugge, J. S. (1997) Annu. Rev. Immunol. 15,
Soc. 116, 5057–5062.
27. Jain, A., Whitesides, G. M., Alexander, R. S. & Christianson, 14. Spencer, D. M., Wandless, T. J., Schreiber, S. L. & Crabtree, D. W. (1994) J. Med. Chem. 37, 2100–2105.
G. R. (1993) Science 262, 1019–1024.
28. Bierer, B. E., Mattila, P. S., Standaert, R. F., Herzenberg, L. A., 15. Holt, D. A., Luengo, J. I., Yamashita, D. S., Oh, H.-J., Konialian, Burakoff, S. J., Crabtree, G. R. & Schreiber, S. L. (1990) Proc. A. L., Yen, H.-K., Rozamus, L. W., Brandt, M., Bossard, M. J., Natl. Acad. Sci. USA 87, 9231–9235.
Levy, M. A., et al. (1993) J. Am. Chem. Soc. 115, 9925–9938.
29. Siekierka, J. J., Hung, S. H., Poe, M., Lin, C. S. & Sigal, N. H.
16. Wiseman, T., Williston, S., Brandts, J. F. & Lin, L. N. (1989) Anal. (1989) Nature (London) 341, 755–757.
Biochem. 179, 131–137.
30. Tai, P. K., Albers, M. W., Chang, H., Faber, L. E. & Schreiber, 17. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, S. L. (1992) Science 256, 1315–1318.
T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, 31. Yem, A. W., Reardon, I. M., Leone, J. W., Heinrikson, R. L. & R. J., et al. (1993) Cell 72, 767–778.
Deibel, M. R., Jr. (1993) Biochemistry 32, 12571–12576.

Source: http://wandless.stanford.edu/documents/2-tjwPDFs/TJW16.pdf

cymbalta.de

Aktueller Stand Cymbalta-Rabattverträge der Lilly Deutschland GmbH Für Cymbalta (alle Formulierungen und Stärken) bestehen Rabattverträge zwischen der Lilly Deutschland GmbH und nachfolgend genannten Krankenkassen. Die Verträge beinhalten nur Originalware (deutsche Ware, keine Parallel- oder Reimporte). Zur besseren Übersichtlichkeit listen wir Ihnen die Top 10 Rabattkassen zusät

Drtp_azrob_proj

2005-2006 Field Guide to Antibiotic Therapy I N T R O D U C T I O N Proper antibiotic selection requires a knowledge of hostrosyphilis because of limited penetration into the centralfactors (e.g., immune function, comorbid diseases, andnervous system. Similarly, cefotaxime has activity againstage);

Copyright © 2010-2014 Drug Shortages pdf