Activity and conformation of a cyclic heptapeptide possessing the message sequence His-Phe-Arg-Trp of alpha-melanotropin.

Alpha-melanotropin (alpha-MSH, i.e. alpha-melanocyte stimulating hormone), tridecapeptide (Ac-Ser(1)-Tyr-Ser-Met-G1u(5)-His-Phe-Arg-Trp-Gly(10)-Lys-Pro-Val(13)-NH(2)), has been extensively studied to understand structure-activity relationships. The core sequence (His-Phe-Arg-Trp) is conserved in several species and is considered as the primary active site or "message sequence". Attempts have been made to design conformationally constrained cyclic analogs containing the message sequence to improve the activity. We had earlier reported that the cyclic analog--cyclo[Gly-His-D-Phe-Arg-Trp-Gly], a 18 membered ring system with two fused beta-turn structure, was less active than the corresponding linear peptide. It was suggested that ring size could be an important parameter in the activity of cyclic melanotropic analogs. To investigate the effect of ring size on biological activity, a cyclic heptapeptide, cyclo[Nle(1)-Gly-His-D-Phe-Arg(5)-Trp-Gly(7)], with 21 member ring system was synthesized. This peptide has three orders of magnitude higher biological activity than the cyclic hexapeptide. The conformational study of this cyclic heptapeptide in DMSO-d(6) by NMR and molecular dynamics simulations reveals a structure with two fused beta-turns running across the residues D-Phe(4)-Gly(7) (Type I) and Gly(7)-His(3) (Type II). These findings confirm that stabilization of beta-turns and a relatively larger ring size are essential determinants of activity for cyclic alpha-MSH analogs.

Antagonist and agonist activities of the mouse agouti protein fragment (91-131) at the melanocortin-1 receptor.

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91-131) (AP91-131) at the melanocortin type-1 receptor (MC1-R) were assessed using B 16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP91-131 was about 3-fold less potent than the natural agonist alpha-melanocyte-stimulating hormone (alpha-MSH) in displacing the radioligand [125I]-[Nle4, D-Phe7]-alpha-MSH (Ki 6.5 +/- 0.8 nmol/l). Alpha-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP9 l -131; the IC50 values for AP91-131 in thetwo assay systems were 91 +/- 22 nM and 95 +/- 15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP91-131 with IC50 values of 9.6+/-1.8 nM and 5.0+/-2.4 nM, respectively. This indicates inverse agonist activity of AP91-131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP91-131 in the adenylate cyclase and melanin assays. On the other hand, AP91-131 inhibited cell growth similar to alpha-MSH (IC50 11.0 +/- 2.1 nM; maximal inhibition 1.8-fold higher than that of alpha-MSH). Furthermore, MC1-R was down-regulated by AP91-131 with about the same potency and time-course as with alpha-MSH. These results demonstrate that AP91-131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different alpha-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.

An alternative solid phase peptide fragment condensation protocol with improved efficiency.

The success of solid phase peptide synthesis is often limited by the aggregation of the growing peptide chains on the resin. Working from the results of a study of model coupling reactions in solution between Z-Gly-Phe-OH and H-Phe-OBzl, we have achieved higher efficiency in the repetitive solid phase fragment condensation of VGVAPG, in a 3:1 chloroform-phenol solvent system, using diisopropylcarbodiimide (DIC) as coupling agent, and a combination of 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt) and its tetrabutyl ammonium salt as additive, than in DMF with DIC and HODhbt alone.

The effect of histidyl residues on the complexation of bis(imidazolyl) containing tripeptides with copper(II) ion.

Copper(II) complexes of tripeptide derivatives of bis(imidazol-2-yl) group have been studied by potentiometric, UV-visible and EPR spectroscopic methods. The peptide molecules correspond to the amino acid sequence of collagen containing histidyl residues in different locations and were connected to the bis(imidazol-2-yl) group either on the C-termini (BOC-Pro-Leu-His-BIMA, BOC-His-Leu-Gly-BIMA) or on the N-termini (BIP-His-Ala-Gly-OEt, BIP-Ile-Ala-His-OMe). It was concluded that the imidazole nitrogen donor atoms of the bis(imidazol-2-yl) moiety are the primary metal binding sites, but the histidyl imidazole nitrogens in the side chains have also some effect on the stability and the coordination mode of the complexes. All ligands can coordinate tridentately to copper(II) ion forming a six-membered chelate and a macrochelate in the [CuL]2+ complexes, which results in a slight distortion in the coordination geometry of [CuL2]2+ complexes. The deprotonation and coordination of amide nitrogens, however, were not observed in any cases.

Synthesis and cytotoxic activity of N-(2-chloroethyl)-N-nitroureas and N-(2-chloroethyl)-N-nitrocarbamates.

As analogs of the widely used anti-tumor agents, N-(2-chloroethyl)-N-nitrosoureas, N-(2-chloroethyl)-N-nitroureas and N-(2-chloroethyl)-N-nitrocarbamates were synthesized by nitration following the reaction of the appropriate amines or alcohols with 2-chloroethyl isocyanate. All tested compounds exert cytotoxic effect with IC50 values of 10(-4) to 10(-6) M and most of them show somewhat higher cytotoxicity in nitrogen than in air.

Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His-Phe-Arg-Trp of alpha-melanotropin by NMR.

Solution conformation of cyclo(Gly1-His2-Phe3-Arg4-Trp5-Gly6) and its D-Phe analog corresponding to the message sequence [Gly-alpha-MSH5-10] of alpha-MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)-d6 solution and in a DMSO-d6/H2O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only -NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross-peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the D-analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low-energy conformers with two gamma-turns, a gamma-turn and a beta-turn, two beta-turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a gamma-bend at Gly6, two gamma-bends at Phe3 and Gly6 and a conformer with a single beta-turn and a gamma-bend for the L-Phe analog. On the other hand, a conformation with two fused beta-turns around the two tetrads His2-D-Phe3-Arg4-Trp5 and Trp5-Gly6-Gly1-His2 dominates the equilibrium mixture for the D-Phe analog. For the D-Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.

The antiproliferative action of a melphalan hexapeptide with collagenase-cleavable site.

PURPOSE: The objective of the present study was to examine the relevance of collagenase in the antitumor action of a melphalan peptide (MHP) with a collagenase-cleavable sequence. The question was addressed as to whether collagenase may act as an activator or a target in the antiproliferative mechanism of MHP. METHODS: Melphalan was inserted into peptides representing the sequence Pro-Gln-Gly-Ile-Ala.Gly of the collagenase-cleavable site in collagens. Changes in growth and collagenase IV activities of HT-1080, HT-29, HT-168, and MCF-7 cell cultures were investigated. RESULTS: The present investigations provide data indicating that Pro-Gln-Gly-Ile-Mel-Gly (melphalan hexapeptide, MHP) is a substrate for both bacterial and 72-kDa type IV collagenases and that in this way it can generate Ile-Mel-Gly (melphalan tripeptide, MTP) of higher cytotoxic potency. Indeed, the formation of MTP was detected in the conditioned medium of HT-1080, a collagenase IV-producing human fibrosarcoma. In a comparison of equimolar concentrations of melphalan and its two peptide derivatives (MHP and MTP), superior antiproliferative action of MTP was seen in HT-29, HT-1080, and HT-168 tumor cell cultures. However, the relatively modest cytostatic actions of MHP were increased when bacterial collagenase was added to the cell cultures. After melphalan treatment, reduced levels of both 92 and 72-kDa type IV collagenases were seen in the HT-1080 cell cultures. However, the reduction of collagenase activity and the cell counts did not run parallel in the MTP- or MHP-treated cultures; indeed, collagenase activity related to cell numbers showed an elevated level. CONCLUSIONS: As the conversion of MHP to the more toxic MTP was detected in the presence of collagenases, it is possible that collagenase-directed activation of prodrugs may be a promising approach for the development of more selective cytostatic drugs against malignant tumors with high collagenase activities.

BCNU is a caspase-mediated inhibitor of drug-induced apoptosis.

BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea], a bifunctional (alkylating/carbamoylating) anticancer agent, in noncytotoxic doses (12-50 microM) inhibited drug-induced apoptosis in HT58 human lymphoma cells exposed to etoposide (ETO; 50 microM) as well as in mouse thymocytes exposed to dexamethasone (5 microg/ml) in vitro in 4-h cultures. The cytoplasmic extracts of ETO-treated HT58 cells cleaved both purified poly(ADP-ribose)polymerase and Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin fluorogenic caspase substrate, indicating the presence of active caspases, and these effects were inhibited by BCNU concentration dependently. The carbamoylating decomposite, 2-chloroethyl-isocyanate (6-25 microM), also decreased ETO-induced apoptosis in HT58 cells in vitro and their caspase 3-like activity ex vivo, whereas N-(2-chloroethyl)-N-nitrosocarbamoyl-valinamide, an alkylating and mainly intramolecularly carbamoylating nitrosourea derivative (400 microM), did not influence these phenomena. Furthermore, the activity of recombinant caspase 3 was also strongly inhibited by BCNU and 2-chloroethyl-isocyanate. These results indicate that BCNU, via its carbamoylating capacity, can inactivate cysteine protease(s) essential for ETO-induced apoptosis. This apoptosis-modulating property of BCNU, in turn, may influence the efficacy of chemotherapeutic protocols in the treatment of cancer.

In vitro cytotoxic effect of difluoromethylomithine increased nonspecifically by peptide coupling.

Difluoromethylomithine (DFMO)-peptide conjugates were synthesized as prodrugs to improve the cytotoxic efficacy of DFMO. All conjugates inhibited cell growth in different cell lines more effectively than DFMO itself. The best cytotoxic effect was achieved in all cell lines by DFMO-Glu-His-Phe-Arg-Trp-Gly-OMe, where the carrier peptide is a melanotropin hormone fragment. Although this conjugate is capable of displacing labeled melanotropin from its receptor, its cytotoxic effect on the receptor-positive human melanoma cell line has not been proven to be receptor-mediated. The differences in the cytotoxicities of the congeners seem to be influenced, at least in part, by the nature of the carrier molecule.

Effect of Ca2+ on the secondary structure of linear and cyclic collagen sequence analogs.

To investigate the role of secondary structure in the substrate specificity of human 72 kDa type IV collagenase, we synthesised linear and cyclic collagen sequence analogs. As Ca2+ plays a crucial role in the enzyme activity, the CD and FTIR spectra of the peptides were also measured in the presence of Ca2+. Most of the linear, but none of the cyclic peptides form stable 1:1 Ca2+ complexes. The cyclic hexapeptides adopt significantly different backbone conformations comprising not only beta-turns but also the less frequent gamma-turns. Consequently, in the cyclopeptides the scissile Gly-Ile(Leu) bond is embedded into a different conformational environment, but in spite of that none of them is a substrate or an inhibitor of the enzyme. The best substrate Ac-Pro-Leu-Gly-Leu-Ala-Gly-D-Lys-OH binds Ca2+, but does not form a stable 1:1 Ca2+ complex, which suggests that instead of a folded structure an extended flexible conformation is preferred by the enzyme.

Receptor-mediated cytotoxicity of alpha-MSH fragments containing melphalan in a human melanoma cell line.

Four alpha-MSH drug conjugates have been synthesized, 2 C-terminal (Pep 3 and 4) and 2 central fragments (Pep 1 and 2), the latter being the 4-10 sequence known to be the main alpha-MSH-receptor-recognition site. Melphalan was introduced into each sequence at different locations. Their ability to recognize alpha-MSH receptors as well as their cytotoxic effects were compared in 3 cell lines: melanoma, carcinoma and fibroblast cells. Pep 1 and 2 were able to specifically bind to MSH receptors on melanoma cells by displacing labelled alpha-MSH from its binding sites at concentrations similar to the 4-10 heptapeptide sequence known to contain the main receptor-recognition site. They subsequently penetrate the cell, most probably by a receptor internalization mechanism, since about half of their effect could be inhibited by competition at the receptor level. Significant and selective cytotoxic effects to melanoma cells could be observed after only 2 hr exposure to the drug conjugates. Interestingly, these 2 conjugates, differing only in melphalan position, showed completely different cytotoxicity in terms of IC50 values, Pep 1 being 24 times more toxic to all cells; but the 2 were equally specific to melanoma cells. However, they both were less toxic to all cells than melphalan itself. Furthermore, Pep 1 and 2 were able to block the receptor and, unlike Pep 3 and 4, their cytotoxic effect could be significantly inhibited by an alpha-MSH agonist. Pep 3 and 4 were 5 to 10 times less toxic than melphalan to melanoma and carcinoma cells and 50 times less to fibroblast cells, and did not show any cell-type selectivity. They were less toxic than Pep 1 to melanoma and carcinoma cells by a factor of 2, but equally toxic to fibroblasts. In contrast, they were more toxic than Pep 2 to fibroblasts, melanoma and carcinoma by a factor of 3, 10 and 24 respectively. Our data strongly suggest a receptor-mediated cytotoxicity mechanism occurring with alpha-MSH central fragments in human melanoma cells due to the presence of alpha-MSH-specific receptors. This mechanism appeared to be both peptide- and cell-type-specific.

The effect of N-terminal substitutions on the biological activity of MSH fragments.

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of alpha-melanotropin, [Glp5]alpha-MSH(5-10), [Glp5,D-Phe7]alpha-MSH(5-10), [Sar5,D-Phe7]alpha-MSH(5-10), [Nle4,D-Phe7]alpha-MSH(4-10), [N-carbamoyl]alpha-MSH(5-10), and formyl and acetyl derivatives of alpha-MSH(5-10), [Gly5]alpha-MSH(5-10) and [Gly5,D-Phe7]alpha-MSH(5-10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.

Synthesis and characterization of some collagen sequence analogs.

Some analogs of natural collagen sequences (773-779) were synthesized. The peptides were hydrolyzed at the Gly-Ile bond not only by crude collagenase isolated from normal rat liver, but also by the bacterial Clostridium histolyticum collagenase. The reason for this unusual cleavage site in the latter case may lie in the unordered secondary structure of the substrates measured by CD spectroscopy.

Investigations on the antitumor effect and mutagenicity of alpha-MSH fragments containing melphalan.

alpha-MSH fragments containing melphalan were tested in vivo on L1210 leukemia and on human amelanotic melanoma xenograft in mice and in vitro on human amelanotic melanoma cell lines. The compounds exhibit significant antitumor activity, but no selectivity in targeting of melanoma can be achieved. There is a difference between melphalan and the melphalyl-peptide in their action on protein synthesis. The peptide derivatives also are less mutagenic than melphalan, according to the SCE assay, furnishing further evidence for the positive effect of natural carrier molecules.

Structural determinants of the binding of gliadin fragments to human peripheral blood lymphocytes.

A series of alpha-gliadin fragments, structurally related to alpha-gliadin-(43-49), were synthesized. The effect of these fragments and beta-casomorphin and naloxone on the steady-state binding of [125I]-alpha-gliadin-(43-49) to human peripheral blood lymphocytes was investigated. In an attempt to correlate the binding data with the conformation of the peptides, their circular dichroism spectra were measured in both trifluorethanol and aqueous solution. It was found that there is a striking correlation between the results of the binding studies and the chiroptical properties of the gliadin fragments. The presence of N-terminal tyrosine and the tendency of the peptides to adopt periodic, 310 helix-like secondary structure appear to be crucial for the binding to human peripheral blood lymphocytes.

Synthesis of alpha-MSH fragments containing phenylalanine mustard for receptor studies.

For chemical affinity labeling of the melanotropin receptor several alpha-MSH fragments containing phenylalanine mustard were synthesized in solution. Tested in the frog skin bioassay the derivatives roughly preserved the biological activity of the corresponding natural sequences. Alkylating peptides with prolonged biological activity containing phenylalanine mustard in place of arginine, phenylalanine or methionine are inhibitors of alpha melanotropin, suggesting an irreversible binding to reactive nucleophiles on the part of the MSH receptor, where the Met-Glu-His-Phe-Arg sequence is attached.

Decomposition of N-(2-chloroethyl)-N-nitrosocarbamoyl amino acid amides.

The chemical decomposition of N-(2-chloroethyl)-N-nitrosocarbamoyl (Q(NO] prolinamide and valinamide were studied under physiological conditions. The volatile products were identified with GC. Q(NO)-Pro-NH2 gave twice the amount of ethylene glycol and only one-fifth of the 2-chloroethanol produced by Q(NO)-Val-NH2 or BCNU, pointing to different pathways of their decomposition. The carbamoylating activity was also investigated in the presence of cyclohexylamine, and it was found to lead mainly to intramolecular carbamoylation with the formation of hydantoin derivatives.

Synthesis and antitumor activity of N-terminal proline-containing peptide-(chloroethyl)nitrosoureas.

The N alpha-(2-chloroethyl)-N-nitrosocarbamoyl derivatives of H-Pro-Lys(X)-Pro-Val-NH2 (X: tert-butyloxycarbonyl, formyl, (2-chloroethyl)nitrosocarbamoyl) were synthesized. It was found that the bis-substitution of the urea N3 in these derivatives does not decrease the antitumor activity influenced mainly by the nature of the carrier molecule as a whole.