Regulation of osteoclasts by calcitonin and amphiphilic calcitonin conjugates: role of cytosolic calcium.

The peptide hormone calcitonin is a potent inhibitor of osteoclastic resorption, but it is unstable and poorly absorbed following oral administration. Conjugates of salmon calcitonin covalently linked to low-molecular-weight amphiphilic polymers show improved stability and absorption. The purpose of this study was to investigate the biological activity of these conjugates in vitro using rat osteoclasts and HEK-293 cells transfected with the C1a isoform of the calcitonin receptor. Salmon calcitonin or its conjugates (10 pM-10 nM) caused rapid arrest of osteoclast membrane ruffling and subsequent retraction. The same amphiphilic polymer attached to an unrelated protein had no effect on osteoclast morphology or motility. Since calcitonin-induced retraction of osteoclasts is thought to be mediated by Ca2+ signaling, we investigated the effects of calcitonin and its conjugates on cytosolic free Ca2+ concentration ([Ca24]i). In HEK-293 cells transfected with the calcitonin receptor, these agents induced transient elevations of [Ca2+]i. However, the rise of [Ca2+]i in HEK-293 cells occurred at concentrations 100-1000-fold higher than those required to elicit osteoclast retraction. To investigate the role of Ca2+ in osteoclast retraction, we preloaded cells with BAPTA to buffer changes in [Ca2+]i. BAPTA decreased the initial rate of calcitonin-induced osteoclast retraction, but it did not affect the degree of retraction 2-3 hours following calcitonin, indicating that retraction is mediated primarily by Ca(2+)-independent processes. We conclude that calcitonin conjugates cause osteoclast retraction and [Ca2+]i signaling in a manner similar to that elicited by calcitonin. Thus, orally bioavailable calcitonin conjugates show potential for use as antiresorptive agents.

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries.

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.

Comparative analyses of mechanistic differences among antiestrogens.

Antiestrogens such as tamoxifen are one of the most effective methods of treating estrogen receptor (ERalpha) positive breast cancers; however, the effectiveness of this therapy is limited by the almost universal development of resistance to the drug. If antiestrogens are recognized differently by the cell as it has been suggested, then in disease conditions where tamoxifen fails to function effectively, a mechanistically different antiestrogen might yield successful results. Although many antiestrogens have been developed, a direct comparison of their mechanisms of action is lacking, thus limiting their utility. Therefore, to determine if there are mechanistic differences among available antiestrogens, we have carried out a comprehensive analysis of the molecular mechanisms of action of 4-hydroxy-tamoxifen (40HT), idoxifene, raloxifene, GW7604, and ICI 182,780. Using a novel set of peptides that recognize different surfaces on ERalpha, we have found that following binding to ERalpha, each ligand induces a distinct ERalpha-ligand conformation. Furthermore, transcriptional assays indicate that each ERalpha-ligand complex is recognized distinctly by the transcription machinery, and consequently, antiestrogens vary in their ability to inhibit estradiol- and 40HT-mediated activities. Relative binding assays have shown that the affinity of these ligands for ERalpha is not always representative of their inhibitory activity. Using this assay, we have also shown that the pharmacology of each antiestrogen is influenced differently by hormone binding proteins. Furthermore, GW7604, like ICI 182,780, but unlike the other antiestrogens evaluated, decreases the stability of the receptor. Overall, our results indicate that there are clear mechanistic distinctions among each of the antiestrogens studied. However, GW7604 and ICI 182,780 differ more significantly from tamoxifen than idoxifene and raloxifene. These data, which reveal differences among antiestrogens, should assist in the selection of compounds for the clinical regulation of ERalpha function.

Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta.

Recruitment of transcriptional coactivators following ligand activation is a critical step in nuclear receptor-mediated target gene expression. Upon binding an agonist, the receptor undergoes a conformational change which facilitates the formation of a specific coactivator binding pocket within the carboxyl terminus of the receptor. This permits the alpha-helical LXXLL motif within some coactivators to interact with the nuclear receptors. Until recently, the LXXLL motif was thought to function solely as a docking module; however, it now appears that sequences flanking the core motif may play a role in determining receptor selectivity. To address this issue, we used a combinatorial phage display approach to evaluate the role of flanking sequences in influencing these interactions. We sampled more than 10(8) variations of the core LXXLL motif with estradiol-activated estrogen receptor alpha (ERalpha) as a target and found three different classes of peptides. All of these peptides interacted with ERalpha in an agonist-dependent manner and disrupted ERalpha-mediated transcriptional activity when introduced into target cells. Using a series of ERalpha-mutants, we found that these three classes of peptides showed different interaction patterns from each other, suggesting that not all LXXLL motifs are the same and that receptor binding selectivity can be achieved by altering sequences flanking the LXXLL core motif. Most notable in this regard was the discovery of a peptide which, when overexpressed in cells, selectively disrupted ERbeta- but not ERalpha-mediated reporter gene expression. This novel ERbeta-specific antagonist may be useful in identifying and characterizing the ERbeta-regulated process in estradiol-responsive cells. In conclusion, using a combinatorial approach to define cofactor-receptor interactions, we have clearly been able to demonstrate that not all LXXLL motifs are functionally equivalent, a finding which suggests that it may be possible to target receptor-LXXLL interactions to develop receptor-specific antagonists.

Peptide antagonists of the human estrogen receptor.

Estrogen receptor alpha transcriptional activity is regulated by distinct conformational states that are the result of ligand binding. Phage display was used to identify peptides that interact specifically with either estradiol- or tamoxifen-activated estrogen receptor alpha. When these peptides were coexpressed with estrogen receptor alpha in cells, they functioned as ligand-specific antagonists, indicating that estradiol-agonist and tamoxifen-partial agonist activities do not occur by the same mechanism. The ability to regulate estrogen receptor alpha transcriptional activity by targeting sites outside of the ligand-binding pocket has implications for the development of estrogen receptor alpha antagonists for the treatment of tamoxifen-refractory breast cancers.

Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta.

Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs alpha and beta and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER alpha and ER beta ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.

Synthesis and biological activity of DNA damaging agents that form decoy binding sites for the estrogen receptor.

It is a goal of cancer chemotherapy to achieve the selective killing of tumor cells while minimizing toxicity to normal tissues. We describe the design of selective toxins forming DNA adducts that attract the estrogen receptor (ER), a transcription factor that is overexpressed in many human breast and ovarian tumors. The compounds consist of 4-(3-aminopropyl)-N,N-(2-chloroethyl)-aniline linked to 2-(4'-hydroxyphenyl)-3-methyl-5-hydroxy-indole. The former moiety is a DNA damaging nitrogen mustard and the latter is a ligand for the ER. The connection between these groups was refined to permit DNA adducts formed by the mustard portion of the molecule to present the ligand domain so that it was able to interact efficiently with the ER. By using 16-mers containing specific DNA adducts, it was determined that monoadducts and putative intrastrand crosslinks were preferred targets for the ER over interstrand crosslinks. A series of structurally related 2-phenylindole mustards was prepared, some of which were selectively toxic to the ER-positive breast cancer cell line MCF-7, as compared with the ER(-) negative line MDA-MB231. The ability both to bind to DNA and to interact significantly with the ER were essential to achieve selective lethality toward ER(+) cells. Compounds forming DNA adducts without the ability to bind receptor showed similar toxicities in the two cell lines. Several models could explain the selective toxicity of the mustard-phenylindole compounds toward ER(+) cells. The favored model suggests that a mustard-DNA adduct is shielded by the ER from DNA repair enzymes and hence cells possessing an abundance of the ER selectively retain the adduct and are killed.

Synthesis of myristoyl CoA analogues and myristoyl peptides as inhibitors of myristoyl CoA:protein N-myristoyltransferase.

To develop inhibitors of myristoyl CoA:protein N-myristoyltransferase (NMT), a series of myristoyl coenzyme A analogues and myristoyl peptides were synthesized, including S-(2-oxopentadecyl)-CoA (1), S-(2-hydroxypentadecyl)-CoA (2), S-(2-oxopentadecyl)-pantetheine (3), Myr-N-Gly-(L)-Phe (4), Myr-N-Gly-(L)-Tyr (5), and Myr-N-Gly-(L)-Asn-Ala- Ala-Ser-Ala-Arg-(NH2) (6). Biological evaluation of these compounds in an in vitro NMT enzyme assay revealed that the nonhydrolyzable acyl CoA analogue 1 was the most potent inhibitor [inhibitor dissociation constant (Ki) = 24 nM]. A preliminary structure-activity relationship study showed that the adenosine moiety and the 2-keto group in this nonhydrolyzable analogue were necessary for inhibitory activity. A possible mechanism for the inhibition of NMT by 1 was proposed, in which 1 might block the reaction at the stage of an acyl-CoA-NMT-peptide complex. Product analogues such as the myristoylated peptides 4-6 were poor inhibitors of NMT.

Protein tyrosine phosphatase 1B undergoes mitosis-specific phosphorylation on serine.

We have investigated the regulation of protein tyrosine phosphatase 1B (PTP1B) through the cell cycle of HeLa cells. PTP1B from HeLa cells arrested in mitosis migrated more slowly during sodium dodecyl sulfate-polyacrylamide gel electrophoresis than did PTP1B from unsynchronized HeLa cells. To explore whether this mobility shift was caused by phosphorylation, PTP1B was immunoprecipitated from 32Pi-labeled unsynchronized and mitotic HeLa cells. PTP1B from mitotic cells incorporated significantly more 32Pi than did PTP1B from unsynchronized cells. Alkaline phosphatase treatment of mitotic HeLa cell lysates resulted in the conversion of PTP1B to its more rapidly migrating form, confirming that the mobility shift was a result of the mitotic phosphorylation. Phosphoamino acid analysis of PTP1B from mitotic cells revealed that PTP1B became phosphorylated on serine. Dephosphorylation of PTP1B occurred following the release of cells from nocodazole synchronization and was independent of new protein synthesis. This dephosphorylation was inhibited by okadaic acid, a potent inhibitor of types 1 and 2A serine/threonine phosphatases. The mitotic phosphorylation had no apparent effect on the activity of PTP1B as measured in in vitro phosphatase assays using 32P-labeled Raytide as substrate. p34cdc2 appears not to be the mitotic PTP1B kinase, as mapping experiments showed that this enzyme phosphorylated PTP1B on a site different from that on which it was phosphorylated in vivo. These observations suggest that PTP1B may be differentially regulated through the cell cycle.

Reversible palmitoylation of the protein-tyrosine kinase p56lck.

The myristoylated protein-tyrosine kinase, p56lck, is expressed predominantly in T cells where it is believed to play a role in T cell activation. We observed a 56-kDa protein that became metabolically labeled in intact T lymphoid cells that were incubated with either [3H]myristate or [3H]palmitate. This protein was identified as p56lck based on its specific immunoprecipitation with polyclonal antisera to p56lck, by induction of a shift in its electrophoretic mobility following treatment of cells with 12-O-tetradecanoylphorbol-13-acetate and by co-chromatography with p56lck on protamine-agarose. Characterization of the two acylation events revealed that, in contrast to the p56lck-associated radioactivity from [3H]myristate-labeled cells, the p56lck-associated radioactivity from [3H]palmitate-labeled cells was susceptible to cleavage by neutral hydroxylamine and was not blocked by inhibitors of protein synthesis. Pulse-chase analyses revealed that the labeling of p56lck with [3H]palmitate, but not [3H]myristate, was reversible. The presence of covalently attached palmitate on p56lck from [3H]palmitate-labeled cells was verified by thin-layer chromatography following acid hydrolysis of the acylated protein. 2-Hydroxymyristate, which is metabolically activated to form a potent inhibitor of protein myristoylation, specifically inhibited the acylation of p56lck with [3H]myristate without affecting its labeling with [3H]palmitate. These studies indicate that p56lck is both a cotranslationally myristoylated and post-translationally palmitoylated protein.

A general method for preparation of peptides biotinylated at the carboxy terminus.

A method for the preparation of a biotinylated resin that can be elongated by standard methods of solid-phase peptide synthesis to give peptides biotinylated at the carboxy terminus is described. This methodology is particularly important for the preparation of biotinylated peptides in which a free amino terminus is required. Coupling of N epsilon-9-fluorenylmethoxycarbonyl-(Fmoc)-N alpha-tert-butyloxycarbonyl(Boc)-L- lysine to p-methylbenzhydrylamine resin, followed by removal of the Fmoc protecting group and reaction with (+)-biotin-4-nitrophenyl ester yielded N alpha-Boc-biocytin-p-methyl-benzhydrylamine resin. The utility of this resin was tested by the synthesis of a biotinylated peptide, Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-biocytin-NH2, for use as an in vitro substrate for myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation. Analysis of the peptide derivative by HPLC and mass spectrometry revealed a single major product of the expected mass, indicating that the biotin group survived cleavage and deprotection with HF. The biotinylated peptide served as a substrate for NMT, and the resulting myristoylated peptide could be quantitatively recovered by adsorption to immobilized avidin.

Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA:protein N-myristoyltransferase.

The importance of myristoylation for the proper biological functioning of many acylated proteins has generated interest in the enzymes of the myristoylation pathway and their interactions with substrates and inhibitors. Previous observations that S-(2-oxopentadecyl)-CoA, a nonhydrolyzable methylene-bridged analogue of myristoyl-CoA, was a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT) [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1989) J. Med. Chem. 32, 1665] prompted a closer examination of the effect of substituents at the 2-position on the interactions of myristic acid and myristoyl-CoA analogues with NMT. As an initial approach, three myristic acid derivatives bearing different substituents at the 2-position, 2-fluoromyristic acid, 2-bromomyristic acid, and 2-hydroxymyristic acid, were selected for study. Both 2-bromomyristic acid and 2-hydroxymyristic acid were available commercially; 2-fluoromyristic acid was prepared synthetically. All three compounds were found to be only weak inhibitors of NMT in vitro. Of the three, 2-bromomyristic acid was the most potent (Ki = 100 microM). In cultured cells, however, 2-hydroxymyristic acid was by far the more effective inhibitor of protein myristoylation. Neither 2-hydroxymyristic acid nor 2-bromomyristic acid significantly inhibited protein palmitoylation in cultured cells, indicating that inhibition was not occurring at the level of acyl-CoA synthetase. Activation of the 2-substituted myristic acid derivatives to their corresponding acyl-CoA thioesters by acyl-CoA synthetase resulted in inhibitors of greatly increased potency. The 2-substituted acyl-CoA analogues, 2-hydroxymyristoyl-CoA, 2-bromomyristoyl-CoA, and 2-fluoromyristoyl-CoA, were synthesized and shown to be competitive inhibitors of NMT in vitro (Ki's = 45, 450, and 200 nM, respectively). These data suggested that the enhanced inhibitory potency of 2-hydroxymyristic acid seen in cells was most probably a result of its metabolic activation to the CoA thioester. The presence of substituents at the 2-position also affected the ability of the acyl group to be transferred by NMT to a peptide substrate. Of the three acyl-CoA analogues, only 2-fluoromyristoyl-CoA served as a substrate for NMT.

Detection of myristoyl CoA:protein N-myristoyltransferase activity by ion-exchange chromatography.

Several proteins of viral and cellular origins are myristoylated on an amino-terminal glycine residue during biosynthesis. The enzyme responsible for this modification, myristoyl CoA:protein N-myristoyltransferase (NMT), can be measured in cell-free systems by following the transfer of [3H]myristate from [3H]myristoyl CoA to a synthetic peptide substrate. We report here a procedure for the analysis of NMT activity using ion-exchange chromatography on CM-Sepharose to separate [3H]myristoyl peptide from radiolabeled reactants. This technique provides a convenient method for assaying multiple samples that is much more rapid and sensitive than procedures that rely on reversed-phase HPLC for the separation of reaction components. Characterization of this assay indicates that it is suitable for the kinetic analysis of NMT activity and for the rapid analysis of column fractions generated during the purification of NMT.