Applications of combinatorial technologies to drug discovery. 1. Background and peptide combinatorial libraries.
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Amino Acid Sequence↗
Biomedical subjects
R W Barrett
Publications and source records attributed to R W Barrett.
Generation and screening of an oligonucleotide-encoded synthetic peptide library.
We have prepared a library of approximately 10(6) different peptide sequences on small, spherical (10-microns diameter) beads by the combinatorial chemical coupling of both L- and D-amino acid building blocks. To each bead is covalently attached many copies of a single peptide sequence and, additionally, copies of a unique single-stranded oligonucleotide that codes for that peptide sequence. The oligonucleotide tags are synthesized through a parallel combinatorial procedure that effectively records the process by which the encoded peptide sequence is assembled. The collection of beads was screened for binding to a fluorescently labeled anti-peptide antibody using a fluorescence-activated cell sorting instrument. Those beads to which the antibody bound tightly were isolated by fluorescence-activated sorting, and the oligonucleotide identifiers attached to individual sorted beads were amplified by the PCR. Sequences of the amplified DNAs were determined to reveal the identity of peptide sequences that bound to the antibody with high affinity. By combining the capacity for information storage in an oligonucleotide code with the tremendous level of amplification possible through the PCR, we have devised a means for specifying the identity of each member of a vast library of molecules synthesized from both natural and unnatural chemical building blocks. In addition, we have shown that the use of flow cytometry instrumentation permits facile isolation of individual beads that bear high-affinity ligands for biological receptors.
Amino Acid Sequence↗
Selective enrichment and characterization of high affinity ligands from collections of random peptides on filamentous phage.
Large collections of random peptides can be expressed on the N-terminus of the pIII protein of filamentous phage and screened for binding to antibodies and other receptors. In our previous work with a monoclonal antibody (3E7) (Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-6382, 1990), we showed that a high proportion of the selected peptides had relatively low affinity (Kd's greater than 1 microM). Here we describe conditions for selective enrichment of phage expressing high affinity peptides. This is done by allowing the phage to interact with a low concentration of 3E7 Fab followed by extensive washing to allow dissociation of phage-bearing peptides with low affinity. These affinity selection conditions were applied to the pool of phage previously selected using a high concentration of IgG. A phage clone with the known high affinity ligand YGGFL (Kd 7.1 nM) and several other closely related peptides were isolated. The dissociation rate of 125I-3E7 Fab from several phage clones approximated that of phage expressing YGGFL. A good correlation was found between the dissociation rate of the peptides found on phage and the equilibrium binding constants of chemically synthesized peptides. The strategy of using a low concentration of receptor and extensive washing to select phage-bearing high affinity peptides, combined with assays to determine the specificity and relative affinity of peptides on isolated phage clones, should be generally applicable in using the peptides-on-phage system for discovery of high affinity receptor ligands.
Amino Acid Sequence↗
Dopamine D1 receptor stimulation of cyclic AMP accumulation in COS-1 cells.
Dopamine is shown to stimulate cAMP accumulation in COS-1 cells via endogenously expressed dopamine D1 receptors. A dissociation of dopamine and beta-adrenoceptor responses is demonstrated by the use of selective antagonists and different desensitization patterns following exposure of the cells to dopamine or the beta-adrenoceptor agonist, isoproterenol. The dopamine response in COS-1 cells exhibits a pharmacological profile similar to that found in dopamine D1 tissues such as rat striatum and fish retina. The presence of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr 32,000) immunoreactivity in COS-1 cells is shown by Western blotting and is consistent with the endogenous expression of a dopamine D1 receptor in these cells. It is concluded that a dopamine D1 receptor is expressed in COS-1 cells and the implications of this are discussed.
2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
A chimeric D2 dopamine/m1 muscarinic receptor with D2 binding specificity mobilizes intracellular calcium in response to dopamine.
Using PCR methodology, a chimeric receptor cDNA was constructed in which the entire third cytoplasmic loop of the human D2 dopamine receptor was replaced by the analogous portion of the human m1 muscarinic receptor. When expressed in CHO cells, the chimeric D2/m1 receptor bound dopaminergic ligands with affinities similar to the native D2(414) receptor. Intracellular calcium levels (measured with Fura-2) were not altered when CHO cells expressing the D2(414) receptor were exposed to dopamine. In contrast, dopamine elevated intracellular calcium levels in cells expressing the D2/m1 chimeric receptor in a dose-dependent manner which was blocked by the D2 antagonist, fluphenazine. The ability to construct G-protein-linked receptor chimeras which mobilize calcium with nearly unaltered pharmacologic specificity raises the possibility of a generic strategy for creating non-radioisotopic reporter systems for use in drug discovery.
Animals↗
Peptides on phage: a vast library of peptides for identifying ligands.
We have constructed a vast library of peptides for finding compounds that bind to antibodies and other receptors. Millions of different hexapeptides were expressed at the N terminus of the adsorption protein (pIII) of fd phage. The vector fAFF1, derived from the tetracycline resistance-transducing vector fd-tet, allows cloning of oligonucleotides in a variety of locations in the 5' region of gene III. A library of 3 x 10(8) recombinants was generated by cloning randomly synthesized oligonucleotides. The library was screened for high-avidity binding to a monoclonal antibody (3-E7) that is specific for the N terminus of beta-endorphin (Tyr-Gly-Gly-Phe). Fifty-one clones selected by three rounds of the affinity purification technique called panning were sequenced and found to differ from previously known ligands for this antibody. The striking finding is that all 51 contained tyrosine as the N-terminal residue and that 48 contained glycine as the second residue. The binding affinities of six chemically synthesized hexapeptides from this set range from 0.35 microM (Tyr-Gly-Phe-Trp-Gly-Met) to 8.3 microM (Tyr-Ala-Gly-Phe-Ala-Gln), compared with 7.1 nM for a known high-affinity ligand (Tyr-Gly-Gly-Phe-Leu). These results show that ligands can be identified with no prior information concerning antibody specificity. Peptide libraries are also likely to be useful in finding ligands that bind to other classes of receptors and in discovering pharmacologic agents.
Amino Acid Sequence↗
Biochemical characterization of the cholecystokinin receptor on CHP212 human neuroblastoma cells.
Cholecystokinin (CCK) receptors reside on a large number of cell types along the digestive tract and in the nervous system. A human neuroblastoma cell line (CHP212) has recently been described to express a type A receptor, with structural specificity similar to that on pancreatic acinar cells and gall bladder smooth muscle cells but different from the predominant type of binding site found in brain (type B). In this work, we have performed photoaffinity labeling and protease peptide mapping of the CHP212 receptor and have compared it to other type A CCK receptors. 125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)-CCK-26-33], a probe that possesses a photolabile residue at position 33 within the theoretical receptor-binding domain of this hormone, specifically labeled a Mr = 80,000-90,000 glycoprotein on this cell line, while labeling larger proteins (Mr = 85,000-95,000) on rat pancreas and human gall bladder. Deglycosylation with endo-beta-N-acetylglucosaminidase F yielded bands of Mr = 43,000 from CHP212 and gall bladder and Mr = 42,000 from pancreas. Peptide mapping of the deglycosylated bands using Staphylococcus aureus V8 protease demonstrated identical patterns in CHP212 and gall bladder and a similar but different pattern in pancreas. Thus, although possessing heterogeneity in their carbohydrate domains, CCK receptors on human neuroblastoma cells (CHP212) and human gall bladder smooth muscle cells have highly similar or identical protein cores. The core protein on another type A CCK receptor, from rat pancreas, appears to differ from these, likely representing molecular heterogeneity between species.
Affinity Labels↗
Biochemical evidence for somatostatin receptors in murine neuroblastoma clone N1E-115.
Radioligand binding and functional assays were employed to demonstrate the existence of somatostatin receptors in the murine neuroblastoma clone N1E-115. Saturation experiments with [125I][Tyr11]somatostatin-14 indicated the presence of a single class of binding sites in membranes prepared from N1E-115 cells (Kd = 83 pM; Bmax = 21,000 receptors/cell). Somatostatin-14, somatostatin-28 and L363586 (cyclo(N-Me-ALA-TYR-D-TRP-LYS-VAL-PHE] all displaced the 125I-ligand monophasically in N1E-115 cells (Ki values were 28, 82 and 34 pM, respectively), which contrasted with the binding heterogeneity apparent with L363586 in rat brain membranes. The binding of [125I][Tyr11]somatostatin-14 was reduced by GppNHp, indicating that N1E-115 somatostatin receptors interacted with guanine nucleotide binding protein(s). Somatostatin agonists decreased by 30-50% the levels of [3H]cyclic AMP induced in intact cells by forskolin, prostaglandin E1, or vasoactive intestinal polypeptide. The EC50 values for inhibition of the [3H]cyclic AMP response to PGE1 by L363586, somatostatin-14, and somatostatin-28 were 0.24, 0.63 and 1.0 nM, respectively. Pertussis toxin treatment of N1E-115 cells reduced both binding to the receptor and the functional response to somatostatin-14. These data suggest that a single class of somatostatin receptors in N1E-115 cells are linked to the inhibition of adenylate cyclase through a Gi protein.
Adenylate Cyclase Toxin↗
Type-A cholecystokinin receptors in CHP212 neuroblastoma cells: evidence for association with G protein and activation of phosphoinositide hydrolysis.
125I-Bolton Hunter-cholecystokinin octapeptide (BH-CCK8) and (-)-[3H]L-364718 membrane binding assays were used to identify and characterize cholecystokinin (CCK) receptors in CHP212 human neuroblastoma cells. The ligand binding properties of CCK receptors in these cells are similar to those found in pancreas (CCK-A sites) and differ from the predominant type of CCK binding site found in brain (CCK-B sites). The specific binding of 125I-BH-CCK8 but not (-)-[3H]L-364718 was reduced by the metabolically stable GTP analog guanosine 5'-(beta-delta-imido)trisphosphate. A substantial difference in the Bmax for the radiolabeled agonist (125I-BH-CCK8) and antagonist [(-)-[3H]L-364718] was noted. These observations are consistent with CCK receptors existing in guanine nucleotide-binding protein-coupled and -uncoupled states. Similar to its action in pancreatic acinar cells, CCK8(S) stimulated the accumulation of [3H]inositol phosphates in cells prelabeled with [3H]myo-inositol (EC50 = 3.2 +/- 0.4 nM; maximum response = 4.5 +/- 0.4 x basal). The intrinsic activity of CCK analogues in stimulating phosphoinositide hydrolysis was substantially less than their reported intrinsic activity in stimulating phosphoinositide hydrolysis in pancreatic acinar cells. The CHP212 neuroblastoma cell may serve as a useful model for the recently reported CCK-A binding site found in the central nervous system.
Cholecystokinin↗
Distinct requirements for activation at CCK-A and CCK-B/gastrin receptors: studies with a C-terminal hydrazide analogue of cholecystokinin tetrapeptide (30-33).
We describe here the properties of tert-butyloxycarbonyl-Trp-Leu-Asp-Phe-NHNH2 (A-57696), a C-terminal hydrazide analogue of tert-butyloxycarbonyl-CCK4 (Boc-Trp-Met-Asp-Phe-NH2), at four cholecystokinin (CCK) receptor-bearing tissues, the guinea pig pancreas and gall bladder (Type A), guinea pig cortex (Type B), and NCI-H345 cells, a human small cell lung cancer cell line that expresses CCK-B/gastrin receptors. Using 125I-Bolton-Hunter-cholecystokinin octapeptide (26-33) (125I-Bolton-Hunter-CCK8) as the radioligand, A-57696 was found to be selective for cortical CCK-B receptors (IC50 = 25 nM), compared with pancreatic CCK-A receptors (IC50 = 15 microM). A-57696 behaved as a competitive antagonist in reversing CCK8-stimulated pancreatic amylase secretion and phosphoinositide breakdown. By Schild analysis, its Kd was determined to be 4.7 and 6.8 microM in amylase and phosphoinositide assays, respectively. A-57696 (100 microM) did not elicit gall bladder contraction, and it inhibited contractions induced by CCK8. The Kd of A-57696 at gall bladder CCK-A receptors was 19 microM. In contrast, A-57696 behaved as a partial agonist (80% of maximal CCK8 response) in stimulating calcium mobilization at CCK-B/gastrin receptors on NCI-H345 cells. A-57696 and CCK8 inhibited each other in calcium mobilization experiments utilizing the fluorescent dye Indo-1. Stimulatory actions of CCK8 and A-57696 were reversed by the CCK-B-selective (R)-L-365,260 (100 nM), whereas at the same concentration, the CCK-A-selective (S)-L-365,260 was ineffective. Binding studies using 125I-Bolton-Hunter-CCK8 and 125I-gastrin indicated that binding sites labeled by these two ligands displayed similar affinities for CCK8, desulfated CCK8, gastrin, A-57696, and both enantiomers of L-365,260. A-57696 represents a new class of CCK-A peptide antagonist at guinea pig pancreas a new class of CCK-A peptide antagonist at guinea pig pancreas and gall bladder. Its contrasting functional activities at guinea pig CCK-A and CCK-B/gastrin receptors in a human tumor cell demonstrate that, in addition to the previously described differences in binding specificity for selective agonists and antagonists, CCK-A receptors and CCK-B/gastrin receptors have different requirements for activation.
Amino Acid Sequence↗
Type-A cholecystokinin binding sites in cow brain: characterization using (-)-[3H]L364718 membrane binding assays.
(-)-[3H]L364718 membrane binding assays were employed to localize and characterize cholecystokinin (CCK)-A binding sites in rat and cow brain. Specific binding was detected in all brain areas tested, but in all areas of rat brain and most areas of cow brain the level was too low to allow characterization of the ligand binding specificity of these sites. Membranes prepared from cow nucleus accumbens and striatum contained higher levels of (-)-[3H]L364718 specific binding which represented 55-70% of total binding. Characterization of the ligand binding properties of (-)-[3H]L364718 binding sites in cow nucleus accumbens revealed that these sites are similar to CCK-A sites found in pancreatic membranes. Binding of (-)-[3H]L364718 was saturable and had high affinity (Kd = 45 pm). Sites labeled by (-)-[3H]L364718 displayed stereospecificity for the stereoisomers of CR1409. The competition curve for CCK8 was shallow and was steepened and shifted to the right by the presence of the stable GTP analog guanosine 5'-(beta,delta-imido)triphosphate. The potency of CCK8, but not (-)-L36478, was also affected by the buffer in which the assay was conducted. Future use of (-)-[3H]L364718 membrane binding assays using cow nucleus accumbens and/or striatum will help explore the possibility of differences in ligand recognition among CCK-A sites found in brain and peripheral tissues.
Animals↗
Modulation of morphine analgesia by opioid peptides. Implications for antinociceptive processes.
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Analgesia↗
Ligand dissociation constants from competition binding assays: errors associated with ligand depletion.
The dissociation constant of a ligand at a binding site (e.g., receptor, antibody) is often determined indirectly by competitive displacement of a radioligand. It is well known that such a determination may be seriously in error unless the free concentrations of both the radioligand and the unlabeled ligand can be measured. By means of computer simulations we have explored the conditions under which this error may occur and its magnitude. We offer guidelines for recognizing a probably inaccurate dissociation constant, and we show how, in some cases, a correction can be made. The problem addressed here is not only a theoretical one; it can arise in the ordinary performance of competition binding assays.
Binding, Competitive↗
Comparative immuno-removal: a novel method for estimating the concentration of an unknown immunoreactive compound.
We describe a method for estimating the molar concentration of an unknown immunoreactive compound. The amount of antibody required to bind half of a standard is compared to the amount required to bind half of an equal number of immunoreactive equivalents of the unknown. We demonstrate the utility of the method using a morphine antibody affinity resin and compounds structurally related to morphine.
Animals↗
Artifactual high-affinity and saturable binding of [3H]5-hydroxytryptamine induced by radioligand oxidation.
The binding of [3H]5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM [3H]5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (butylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of [3H]5-HT in the absence of ascorbate. The increase in apparent specific [3H]5-HT binding is time-dependent and plateaus after 4-6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of [3H]5-HT binding reveals a KD value of 3.0 +/- 0.3 nM and a Bmax value of 1.9 +/- 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially unchanged at 3.6 +/- 0.1 nM but the Bmax is significantly increased to 36.5 +/- 7 pmol/g tissue. Drug competition studies reveal that the apparent specific "[3H]5-HT binding" in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22 degrees C in the absence of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
A monoclonal antibody specific for a dynorphin precursor.
Murine monoclonal antibodies were produced against dynorphin-32 (D32), a naturally occurring opioid peptide containing the sequence of dynorphin A (Dyn A) at its amino terminus, followed by Lys-Arg, then by the sequence of dynorphin B (Dyn B). Initial characterization using an enzyme-linked immunosorbent assay revealed that one clone (17.M) recognized D32 but not Dyn A or Dyn B. A solid-phase radioimmunoassay using 125I-labeled antibody 17.M was developed and showed a maximum sensitivity of 45 fmol for synthetic D32. Dyn B crossreacted only 0.05%; Dyn A and other related opioid peptides crossreacted less than 0.001%. Immunoreactive D32 was detected in extracts of rat brain and anterior pituitary but not neurointermediate lobe of pituitary. The unique specificity of monoclonal antibody 17.M should make it useful in investigations of the differential localization and release of Dyn A and Dyn B and the precursor D32.
Animals↗
Morphine and other opiates from beef brain and adrenal.
We describe nonpeptide opioids found in extracts of beef hypothalamus and adrenal, which are recognized by antisera raised against morphine. Four have been purified to homogeneity. One is morphine. The structures of the other three have not been determined yet. None of them are derived from morphine or normorphine after extraction from the tissues. It is not known whether the opiates described here are of endogenous or exogenous origin.
Adrenal Glands↗
Evaluation of the interactions of mu and delta selective ligands with [3H]D-Ala2-D-Leu5-enkephalin binding to mouse brain membranes.
The interactions of putative mu and delta selective ligands with [3H]D-ala2-D-leu5 enkephalin (DADLE) binding to mouse brain membranes were investigated. Computerized curve fitting of displacement curves performed at three different concentrations of 3H-DADLE indicated that a one site competitive model was sufficient to explain the interactions of leu-enkephalin (LE) and D-ser2-thr6-leucine enkephalin with 3H-DADLE binding. Similar experiments with morphine and morphiceptin were unique in that the multiple displacement curves crossed over one another. A two-site competitive model was required to adequately describe the interactions of these mu selective ligands with 3H-DADLE. This two-site model was one in which the inhibitor had higher affinity for the site labeled with lower affinity by 3H-DADLE. However, this two site model did not correctly predict the interaction of LE with 3H-DADLE in the presence of morphiceptin. These data indicate that: 1) putative mu and delta selective ligands do not bind to a common high affinity site; 2) mu selective ligands are not simple mixed inhibitors of a single site labeled by 3H-DADLE; and 3) competitive binding models may not explain the interaction of mu ligands with 3H-DADLE binding.
Animals↗