Linear alpha-human atrial natriuretic peptide analogs display receptor binding activity and inhibit alpha-hANP-induced cGMP accumulation

We have synthesized a series of [Cys(R)7,23]alpha-hANP analogs, in which the two Cys residues were modified with various alkyl groups(R); i.e., R=Acm, Pe, Qe, Cam, Me, Ae, Bzl, Cm, Ocam and sulfo. The Acm-, Cam-, and Me-analogs exhibited binding activity as potent as alpha-hANP in rat vascular smooth muscle cells (VSMC). Binding activity of the analogs decreased progressively as the bulkiness of the R group increased. None of the analogs caused accumulation of cGMP in VSMC and vasorelaxant activity in rat aorta. Acm-, Cam- and Me-analogs substantially antagonized alpha-hANP-induced cGMP accumulation, but did not antagonize vasorelaxation induced by alpha-hANP in vitro.     

Divergence of ANF analogs in smooth muscle cell cGMP response and aorta vasorelaxation: evidence for receptor subtypes

ANF analog potencies in stimulating smooth muscle cell cGMP were compared with the ability to relax histamine-constricted rabbit aorta in vitro. ANF[1-28], [5-28], [5-27] and Lys-11[5-28] elevated cGMP and were potent vasorelaxants. ANF[7-23] and Lys-11[7-23] were potent cGMP stimulators but 1000-fold weaker relaxants. Tyr-8[5-27] did not stimulate cGMP synthesis or antagonize the response of the other peptides, yet was a potent vasorelaxant. Crosslinking with 125I-ANF identified bands at 150 and 65 KD by SDS-PAGE. ANF[1-28], Lys-11[7-23] and Tyr-8[5-27] blocked crosslinking at low concentration despite disparate activities. These data support the existence of ANF receptor subtypes and suggest that cGMP elevation alone is not sufficient to promote atrial peptide-induced vasorelaxation.     

Truncated atrial natriuretic factor analogs retain full agonist activity.

The synthesis, receptor binding, and agonist activity of a series of truncated atrial natriuretic analogs (ANF) are described. These analogs incorporate two portions of the native 28 amino peptide, the eight amino acids C-terminal to Cys7, and two amino acids from the C-terminus (phenylalanine and arginine), into disulfide-bonded cyclic peptides. The inclusion of the C-terminal amino acids converted the ANF analogs from receptor ligands to full agonists, as measured by several methods, including the stimulation of cGMP biosynthesis in endothelial cells, inhibition of aldosterone biosynthesis in rat adrenal cells, and natriuretic-hypotensive activity in vivo. The most potent analogs have cyclohexylalanine (Cha) at position 8. The lead compound (Arg6,Cha8 ANF 6-15 Phe-Arg-Cys-NH2) is a tridecapeptide that integrates the C-terminal amino acids inside the disulfide ring. This peptide, designated as A-68828, has a binding affinity of IC50 = 120 nM, approximately 1/400 of ANF 1-28. However, this analog, in vivo, is only slightly less natriuretic (1/20-1/50) than ANF 1-28. Unlike the native peptide, A-68828 is only mildly hypotensive and at the highest concentration tested reduced blood pressure less than 15 mmHg (1 mmHg = 133.322 Pa). A-68828 inhibited ACTH-induced aldosterone release to a greater extent than ANF 1-28: 100 vs. 50%. The selective natriuretic activity of A-66828, relative to ANF, suggests clinical utility for the treatment of acute renal failure.     

The role of the C-terminal region of rat brain natriuretic peptide in receptor selectivity.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have different C-terminal tail structures compared with the rather conservative ring structures which consist of 17 amino acid residues. To examine the different effects of the tail structures of ANP and BNP on their interaction with receptors, we synthesized several peptide analogs and measured their biological actions in three different assay systems. Deletion of the C-terminal tail from rat BNP did not effect the vasorelaxation activity against rat aorta, but it promoted cGMP production in cultured rat aortic smooth muscle cells (RASMC). Deletion of the C-terminal tail from rat ANP diminished both vasorelaxant and cGMP producing activities. In a binding competition assay with RASMC and [125I]rat ANP-(1-28), the competition activities of both ANP and BNP were greatly reduced by C-terminal deletion. In addition, we obtained agonists with novel receptor selectivity.     

Vascular receptor binding activities and cyclic GMP responses by synthetic human and rat atrial natriuretic peptides (ANP) and receptor down-regulation by ANP

Biological activities of a variety of synthetic human (h) and rat (r) atrial natriuretic peptide (ANP) and related peptides as assessed by receptor binding and cyclic GMP response, and regulation of vascular ANP receptors were studied in rat aortic vascular smooth muscle cells (VSMC) in culture. alpha-hANP1-28 and alpha-hANP7-28 equally inhibited the binding of 125I-labeled-alpha-hANP to its vascular receptors, whereas Met(O)12-alpha-hANP1-28 was less potent and reduced and carboxymethylated (RCM)-alpha-hANP1-28 was ineffective. rANP5-27 and rANP5-28 were equipotent in receptor binding, whereas rANP5-25 had somewhat less potent effect and rANP8-28 fragment was ineffective. alpha-hANP1-28, alpha-hANP7-28, rANP5-27 and rANP5-28 similarly stimulated intracellular cyclic GMP formation, whereas rANP5-25 showed less stimulatory effect, and RCM-alpha-hANP1-28, Met12-sulfoxide and rANP fragment were ineffective. Pretreatment with unlabeled alpha-hANP (3.2 X 10(-9) and 3.2 X 10(-8)M) for 24 hrs resulted in a substantial reduction (55 and 75%) of total receptor number without changing the affinity of ANP receptors. These results suggest that the common ring structure formed by the disulfide bond in the molecule is critical for receptor binding and subsequent biological actions, and that a hydrophobic amino acid located at the position of 12, and (24-26) residues at the C-terminal side, but not (1-6) at the N-terminal side, of the disulfide bridge may play a part in modulating receptor binding and/or biological functions. The present study also indicates "down-regulation" of vascular ANP receptors by homologous ligand.     

Inhibition of analgesia by C-terminal deletion analogs of human beta-endorphin

Human beta-endorphin (beta h-EP) analogs of variable chain lengths have been investigated for their potency in inhibiting analgesia induced by beta h-EP or by the potent opiate etorphine. It was found that beta h-EP-(1-28) inhibits the analgesic effect of beta h-EP and etorphine when co-injected intracerebroventricularly into mice. Antagonism by competition at same opioid receptor subtypes is suggested from parallel shifts of the dose-response curve of etorphine or beta h-EP in the presence of increasing doses of beta h-EP-(1-28). On a molar basis, beta h-EP-(1-28) is nearly 10 times more potent than naloxone. The reduction of the chain length from residues 1-28 to 1-27 lowered the antagonist potency while further reduction of the peptide chain led to a complete loss of inhibitory activity. From comparison of the opioid-receptor binding affinity, analgesic activity and antagonist potency, it is concluded that the C-terminus of beta-EP is critical to the biological efficacy of the molecule and that the antagonist activity of C-terminal deletion analogs is probably mediated through residues 27 and 28.     

Structural requirements of C-type natriuretic peptide for elevation of cyclic GMP in cultured vascular smooth muscle cells.

C-type natriuretic peptide (CNP), which was recently found to be a selective ligand for one of the two known natriuretic peptide receptor guanylyl cyclases (NPR-B), potently stimulates cGMP production in cultured rat vascular smooth muscle cells (VSMC) and exerts potent antiproliferative effects on the cells. To investigate the structural requirements of CNP for stimulation of cGMP accumulation via NPR-B, we prepared CNP analogs and tested them on cultured rat VSMC. Our results indicate that only the ring portion of CNP with a disulfide bond (CNP(6-22)) participates in stimulation of cGMP accumulation, especially the sequence Leu9-Lys10-Leu11 in the ring portion executes essential roles for both elevation of cGMP and selectivity of the ligand for NPR-B. We also found a good correlation between the activities of the CNP analogs for stimulation of cGMP accumulation and inhibition of DNA synthesis.     

Endothelin inhibits the atrial natriuretic factor stimulated cGMP production by activating the protein kinase C in rat aortic smooth muscle cells.

Preincubation of rat thoracic aortic smooth muscle cells with endothelin inhibits the atrial natriuretic factor (ANF)-induced cGMP accumulation in these cells in a concentration dependent manner. The maximal inhibition of 64% was afforded by 1 x 10(-6) M endothelin and the half maximal inhibition (IC50) was achieved with 1 x 10(-9) M endothelin. Endothelin (1 x 10(-6) M) also increased the plasma membrane bound protein kinase C (PKC) activity by 4 fold. Hormone-dependent increase in PKC activity was limited to plasma membranes only and some decrease in cytosolic PKC activity was observed. However, phorbol 12-myristate 13-acetate (PMA) (1 x 10(-6)M) provoked a total loss of cytosolic PKC activity and a net gain in membranous PKC activity indicative of the translocation of the enzyme. Pretreatment of these cells with H-7, a PKC inhibitor, released the endothelin and PMA-mediated attenuation of ANF-stimulated cGMP formation. These results suggest that PKC is involved in the regulation of ANF-induced cGMP accumulation and that the vasoconstrictor activity of endothelin might involve inhibition of the vasorelaxant activity of ANF through the inhibition of cGMP accumulation in smooth muscle cells (SMCs) of the rat aorta.     

Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities.

Interleukin-8 (IL-8) is an inflammatory mediator that stimulates neutrophil migration and functional activation. Analogs of human IL-8 were chemically synthesized, purified, and compared with the full-length 72-residue synthetic IL-8 for their ability to stimulate neutrophil chemotaxis and exocytosis as measured by assaying for release of elastase, as well as their binding to specific receptors in competition assays. Analogs corresponding to the less abundant natural forms, 3-72, 4-72, and 77-residue IL-8, were evaluated and the 3-72 and 4-72 had 2-5-fold higher potencies, whereas the 77-residue IL-8 was 2-fold less potent. A major finding was that NH2-terminal residues 4, 5, and 6 were absolutely essential for IL-8 activity and receptor binding. Quantitative dissociation of elastase release and chemotaxis activity was detected with 5-72, which compared with 1-72, was 80-fold less potent in the elastase assay, but was only slightly less potent in stimulating chemotaxis. IL-8 6-72 lacked all the biological activities tested but had detectable receptor binding activity. The NH2-terminal peptide, AVLPRSAKEL, lacked activity and receptor binding, suggesting that the NH2-terminal region alone is not sufficient for function. Comparison of analogs shortened at the COOH terminus showed that potency was progressively reduced as the COOH-terminal residues were excluded. However activity was retained in an analog (1-51) with the entire COOH-terminal alpha helix and beta turn missing. A peptide corresponding to the COOH-terminal 22 residues, although inactive alone, synergized with the 1-51 analog in stimulating elastase release. The results suggest that the NH2-terminal residues 4, 5, and 6, which are disordered in the IL-8 solution structure, are directly involved in receptor binding, but the COOH-terminal alpha helix is probably important for stabilizing the three-dimensional structure. Other regions within residues 7-51 are also functionally important.     

Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1.

The three-dimensional structure of stromal cell-derived factor-1 (SDF-1) was determined by NMR spectroscopy. SDF-1 is a monomer with a disordered N-terminal region (residues 1-8), and differs from other chemokines in the packing of the hydrophobic core and surface charge distribution. Results with analogs showed that the N-terminal eight residues formed an important receptor binding site; however, only Lys-1 and Pro-2 were directly involved in receptor activation. Modification to Lys-1 and/or Pro-2 resulted in loss of activity, but generated potent SDF-1 antagonists. Residues 12-17 of the loop region, which we term the RFFESH motif, unlike the N-terminal region, were well defined in the SDF-1 structure. The RFFESH formed a receptor binding site, which we propose to be an important initial docking site of SDF-1 with its receptor. The ability of the SDF-1 analogs to block HIV-1 entry via CXCR4, which is a HIV-1 coreceptor for the virus in addition to being the receptor for SDF-1, correlated with their affinity for CXCR4. Activation of the receptor is not required for HIV-1 inhibition.     

Importance of the central region of lamprey gonadotropin-releasing hormone III in the inhibition of breast cancer cell growth

Naturally occurring isoforms of the decapeptide gonadotropin-releasing hormone (GnRH) share residues 1-4 and 9-10. lGnRH-III, the third isoform isolated in the sea lamprey has no endocrine effect in mammals but shows a direct antiproliferative effect on human breast, prostate and endometrial cancer cell lines. To investigate these features, residues 5-8 of lGnRH-III were systematically replaced with Ala. The ability of the synthetic analogs to interact with receptors on MDA-MB 231 human breast cancer cells and their effect on the growth of the same cell line were investigated. [Ala6]lGnRH-III and [Ala7]lGnRH-III have neither receptor binding nor antiproliferative activity. Replacement of His5 with Ala resulted in an analog that binds to the receptor but does not have antiproliferative activity. The results are in agreement with previous reports that modifications of Lys at position 8 are well tolerated.     

Synthetic position 5 analogs of adrenocorticotropin fragments and their in vitro lipolytic activity.

Twenty-three analogs of the ACTH-(4-10)-heptapeptide sequence, which forms the "active core" of adrenocorticotropin (ACTH) and related hormones, have been synthesized by the solid-phase method. These analogs all contain structural modifications at or near the 5-glutamic acid residue of ACTH. The peptides were purified to electrophoretic and chromatographic homogeneity. The peptides were assayed for lipolytic activity in an isolated cell system derived from rabbit adipose tissue. In this system, it was determined that residue 5 plays a very important "spacer" role in the peptide, but that this spacer function is not very dependent on the nature of the side chain of the position 5 amino acid. It was found, however, that a number of analogs containing basic residues (arginine or lysine) in position 3 and/or position 5 of ACTH-(3-10) and ACTH-(4-10) fragments have 5 to 10 times the activity of the respective parent peptides. The presence of a latent anionic locus in the rabbit fat-cell receptor for ACTH is suggested by this study.     

Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. Inhibition of cAMP-dependent protein kinase

The minimal structure in the heat-stable inhibitor protein of cAMP-dependent protein kinase required for a low nanomolar potency of inhibition is the peptide Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-+ ++Ile22-NH2 (PKI-(6-22)-amide). While primary structural determinants for interaction with the protein kinase are distributed throughout the 17 residues of this peptide, we have previously shown that phenylalanine 10 in the NH2-terminal portion is a particularly important determinant for high affinity binding (Glass, D. B., Cheng, H.-C., Mende-Mueller, L., Reed, J., and Walsh, D. A. (1989) J. Biol. Chem. 264, 8802-8810). To investigate this requirement further, peptide analogs of PKI-(6-22)-amide in which various natural and nonstandard amino acids are substituted for phenylalanine 10 have been synthesized and tested for inhibitory potency against the catalytic subunit of the protein kinase. Consistent with the importance of the hydrophobicity of phenylalanine, an alanine 10 substitution analog exhibited a 270-fold decrease in inhibitory potency, whereas the leucine 10 analog lost only 33-fold in activity as compared to the parent peptide PKI-(6-22)-amide. Peptides containing the spatial conformation analogs D-phenylalanine, homophenylalanine, or phenylglycine were 60-120-fold less potent than the parent peptide. Peptides containing various para-substituted phenylalanines at position 10 were only 5-11-fold less potent. One exception to this was (4'-azidophenylalanine 10)PKI-(6-22)-amide, which was nearly equipotent with the parent inhibitor. The most potent analogs were those peptides containing highly aromatic residues at position 10. The 2'-thienylalanine 10, tryptophan (formyl) 10, tryptophan 10, and the 1'-naphthylalanine 10 analogs were 3-fold less potent, equipotent, slightly more potent, and 4-fold more potent than the parent peptide inhibitor, respectively. We conclude that phenylalanine 10 in PKI-(6-22)-amide, and presumably in the native protein inhibitor, interacts through specific hydrophobic and/or aromatic binding to a hydrophobic pocket or cleft near the active site of the protein kinase.     

Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb

Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two α-helices and a model α-helical sequence at the C-terminus (3× RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two α-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F(6)] and [V(8)]) in the proline hinge region with other hydrophobic residues ([W(6)] and [I(8)]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model α-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR](3) and KLLKQWPIG[KLLK](3), respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents.     

Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN. Design of a potent endozepine antagonist.

The octadecaneuropeptide (ODN; QATVGDVNTDRPGLLDLK) and its C-terminal octapeptide (OP; RPGLLDLK), which exert anxiogenic activity, have been previously shown to increase intracellular calcium concentration ([Ca2+]i) in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C. It has also been found that the [d-Leu5]OP analog possesses a weak antagonistic activity. The aim of the present study was to synthesize and characterize cyclic analogs of OP and [d-Leu5]OP. On-resin homodetic backbone cyclization of OP yielded an analog, cyclo1-8 OP, which was three times more potent and 1.4-times more efficacious than OP to increase [Ca2+]i in cultured rat astrocytes. Cyclo1-8 OP also mimicked the effect of both OP and ODN on polyphosphoinositide turnover. Conversely, the cyclo1-8 [d-Leu5]OP analog was totally devoid of agonistic activity but suppressed the effect of OP and ODN on [Ca2+]i and phosphoinositide metabolism in astrocytes. The structure of these cyclic analogs has been determined by two-dimensional 1H-NMR and molecular dynamics. Cyclo1-8 OP exhibited a single conformation characterized by a gamma turn comprising residues Pro2-Leu4 and a type III beta turn encompassing residues Leu5-Lys8. Cyclo1-8 [d-Leu5]OP was present as two equimolar conformers resulting from cis/trans isomerization of the Arg-Pro peptide bond. These pharmacological and structural data should prove useful for the rational design of non peptidic ODN analogs.     

Binding mode prediction of conformationally restricted anandamide analogs within the CB1 receptor

Background

CB₁ cannabinoid receptors are G-protein coupled receptors for endocannabinoids including anandamide and 2-arachidonoylglycerol. Because these arachidonic acid metabolites possess a 20-carbon polyene chain as the alkyl terminal moiety, they are highly flexible with the potential to adopt multiple biologically relevant conformations, particularly those in a bent form. To better understand the molecular interactions associated with binding and steric trigger mechanisms of receptor activation, a series of conformationally-restricted anandamide analogs having a wide range of affinity and efficacy were evaluated.

Results

A CB₁ receptor model was constructed to include the extracellular loops, particularly extracellular loop 2 which possesses an internal disulfide linkage. Using both Glide (Schrödinger) and Affinity (Accelrys) docking programs, binding conformations of six anandamide analogs were identified that conform to rules applicable to the potent, efficacious and stereoselective non-classical cannabinoid CP55244. Calculated binding energies of the optimum structures from both procedures correlated well with the reported binding affinity values. The most potent and efficacious of the ligands adopted conformations characterized by interactions with both the helix-3 lysine and hydrophobic residues that interact with CP55244. The other five compounds formed fewer or less energetically favorable interactions with these critical residues. The flexibility of the tested anandamide analogs, measured by torsion angles around the benzene as well as the stretch between side chain moieties, could contribute to the differences in ability to interact with the CB₁ receptor.

Conclusion

Analyses of multiple poses of conformationally-restricted anandamide analogs permitted identification of favored amino acid interactions within the CB₁ receptor binding pocket. A ligand possessing both high affinity and cannabinoid agonist efficacy was able to interact with both polar and hydrophobic interaction sites utilized by the potent and efficacious non-classical cannabinoid CP55940. In contrast, other analogs characterized by reduced affinity or efficacy exhibited less favorable interactions with those key residues.     

Structural requirements for the biological activity of thymopentin analogs

Thymopentin, the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr, corresponds to residues 32-36 of the thymic hormone thymopoietin. Thymopentin, like thymopoietin, induces intracellular cGMP elevations in the human T-cell line, CEM. Thymopentin also displaces radiolabeled thymopoietin from a receptor glycoprotein prepared from CEM cells, provided that a nonapeptide corresponding to thymopoietin is added to block thymopoietin binding to an additional binding site. Twenty nine analogs with single position substitutions were synthesized by solid-phase or classical solution synthesis, and are evaluated in these assays. All analogs that were active gave positive effects in both assays. A number of substitutions were tolerated at positions 2, 4, and 5, but there was an absolute requirement for L- or D-Arg at position 1 and L- or D-Asp at position 3 to maintain biological activity.     

心钠素的结构与功能的研究

心钠素的结构与功能的研究王进,施薄涛（中国科学院上海生物化学研究所,２０００３１）关键词心钠素ＡＰＩＩＩ;类似物;舒张;利尿;降血压心钻素（ＡＮＰ,ａｔｒｉａｌｎａｔｒｉｕｒｅｔｉｃＰｅＰｔｉｄｅ）是ＤｅＢｏｌｄ在１９８１年发现的一种由大鼠心肌细胞分...     

Development of potent glucagon antagonists: structure-activity relationship study of glycine at position 4.

We examined the functional role of glycine at position 4 in the potent glucagon antagonist [desHis(1), Glu(9)]glucagon amide, by substituting the L- and D-enantiomers of alanine and leucine for Gly(4) in this antagonist. The methyl and isobutyl side-chain substituents were introduced to evaluate the preference shown by the glucagon receptor, if any, for the orientation of the N-terminal residues. The L-amino acids demonstrated only slightly better receptor recognition than the D-enantiomers. These results suggest that the Gly(4) residue in glucagon antagonists may be exposed to the outside of the receptor. The enhanced binding affinities of analogs 1 and 3 compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide, may have resulted from the strengthened hydrophobic patch in the N-terminal region and/or the increased propensity for a helical conformation due to the replacement of alanine and leucine for glycine. Thus, as a result of the increased receptor binding affinities, antagonist activities of analogs 1-4 were increased 10-fold compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide. These potent glucagon antagonists have among the highest pA(2) values of any glucagon analogs reported to date.