Curcumin and curcumin-like molecules: from spice to drugs.

Curcumin is the major yellow pigment extracted from turmeric, a commonly used spice in Asian cuisine and extensively employed in ayurvedic herbal remedies. A number of studies have shown that curcumin can be a prevention and a chemotherapeutic agent for colon, skin, oral and intestinal cancers. Curcumin is also well known for its antiinflammatory and antioxidant properties, showing high reactivity towards peroxyl radicals, and thus acting as a free radical scavenger. Recently, experimental studies have demonstrated that curcumin might be used in the prevention and the cure of Alzheimer's disease. Indeed, curcumin injected peripherally in vivo into aged Tg mice crossed the blood-brain barrier and bound to amyloid plaques, reducing amyloid levels and plaque formation decisively. The present review will resume the most recent developments in the medicinal chemistry of curcumin and curcumin-like molecules.

(99m)Tc-radiolabelled peptides for tumour imaging: present and future.

Receptor-binding peptides have attracted an enormous interest in targeting molecules for the development of tumour specific radiopharmaceutical compounds. The overexpression of many receptors on human tumour makes such peptide-ligands attractive agents for diagnostic imaging and therapy of cancers. The use of solid-phase peptide synthesis and the availability of a wide range of bifunctional chelating agents for the radiolabelling of bioactive peptides with radionuclides have produced a wide variety of useful radiopharmaceutical molecules. For diagnostic purposes, techenetium - 99m is the ideal radionuclide thanks to its nuclear properties and the availability of a low cost portable generator system.

Antennapedia/HS1 chimeric phosphotyrosyl peptide: conformational properties, binding capability to c-Fgr SH2 domain and cell permeability.

With the aim of interfering with the signaling pathways mediated by the SH2 domains of Src-like tyrosine kinases, we synthesized a tyrosyl-phospho decapeptide, corresponding to the sequence 392-401 of HS1 protein, which inhibits the secondary phosphorylation of HS1 protein catalyzed by the Src-like kinases c-Fgr or Lyn. This phospho-peptide was modified to enter cells by coupling to the third helix of Antennapedia homeodomain, which is able to translocate across cell membranes. Here we present CD and fluorescence studies on the conformational behavior in membrane-mimicking environments and on lipid interactions of Antennapedia fragment and its chimeric phosphorylated and unphosphorylated derivatives. These studies evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption on lipids. Experiments performed with recombinant protein containing the SH2 domain of c-Fgr fused with GST and with isolated erythrocyte membranes demonstrated that the presence of the N-terminal Antennapedia fragment only slightly affects the binding of the phospho-HS1 peptide to the SH2 domain. In fact, it has been shown that in isolated erythrocyte membranes, both phospho-HS1 peptide and its chimeric derivative greatly affect either the SH2-mediated recruitment of the c-Fgr to the transmembrane protein band 3 and the following phosphorylation of the protein catalyzed by the Src-like kinase c-Fgr. The ability of the chimeric phospho-peptide to enter cells has been demonstrated by confocal microscopy analysis.

Ion-binding and pharmacological properties of Tyr6 and Tyr9 antamanide analogs.

In order to investigate the antiproliferative properties of antamanide, we have synthesized and studied two antamanide analogs where the phenylalanine residue in positions 6 or 9 is substituted by tyrosine, their corresponding linear forms and the cyclic and linear des Phe5,Phe6-Tyr9-analogs. Antamanide and its biologically active synthetic analogs are able to form highly stable complexes with metal ions, particularly Na+, K+ and Ca2+. We studied the ion-binding properties of the Tyr-antamanide analogs by CD and Tb3+ -mediated fluorescence in acetonitrile. In this medium the far-and near-UV CD spectra of the neat Tyr6-antamanide analog are very similar to that of the parent cyclic decapeptide. Substantial differences occur on the contrary in the CD spectra of the neat Tyr9-antamanide, particularly in the regions at 220 nm and 270-290 nm. In acetonitrile, as already found for antamanide, the interaction with the above-mentioned metal ions always produces evident changes in the far- and near-UV CD spectra of both analogs. On the contrary, the CD spectra of the linear deca- and octa- and of the cyclic octa-analogs are affected by the presence of metal ions only in the near-UV region. In the same solvent the Tb3+ -mediated fluorescence spectra of all the synthetic peptides are remarkably affected by the addition of ions. On the basis of the spectral total changes, by using either or both the spectroscopic techniques, it has been possible to determine the ion binding constants for all the linear and cyclic Tyr-antamanide analogs and to compare them with that of the parent peptide. The antitoxic and antiproliferative activities of these antamanide analogs have been tentatively correlated to their ion-binding properties. A preliminary account of this work was given in (1).

Linear and cyclic peptides as substrates for Lyn tyrosine kinase.

Two Tyr residues are supposed to play a crucial role in the regulation of protein tyrosine kinases of the Src family. Autophosphorylation of Src Tyr416 correlates with enzyme activation, while phosphorylation of C-terminal Tyr527 by Csk gives rise to inactive forms of Src kinases. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a particularly high affinity (Km 20 microm) toward the dimeric linear and cyclic derivatives of the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH which reproduces the main autophosphorylation site of most of the Src enzymes. Under the experimental conditions used only one Tyr residue of the dimeric sequence can be phosphorylated [P. Ruzza, A. Calderan, B.Filippi, B. Biondi, A. Donella Deana, L. Cesaro, L. A. Pinna & G. Borin (1995) Int. J. Peptide Protein Res. 45, 529-539]. The present study addresses the problem of the efficiency displayed by Lyn towards the two Tyr residues located at positions 5 and 12 of the dimeric peptide. To this purpose, two tetradecapeptides were synthesized by the classical solution method, each containing one of the two Tyr residues alternatively replaced by Phe, and the corresponding univocal cyclic form. A possible correlation between the different structural properties induced by the modifications of the native sequence and the ability of the peptides to act as Lyn substrates was noted. The kinetic data obtained indicate that Lyn phosphorylates the residues located at different positions in the two linear analogues differently. In particular, while the Tyr5, Phe12 derivative presents a Km value similar to those obtained for the dimeric linear and cyclic unmodified analogues, the Km value of the Phe5, Tyr12 derivative is two-fold higher than those found for the above-mentioned peptides. Moreover, as previously reported for the linear and cyclic dimeric forms of the native sequence, in the mono-tyrosine containing series of dimers the still conformationally flexible cyclic derivative shows a phosphorylation efficiency two-fold higher than those found for the linear derivatives.

Solution conformational analysis of sodium complexed [Gly6]- and [Gly9]-antamanide analogs.

To investigate the conformational flexibility of metal-complexed cyclodecapeptides, we synthesized and studied two antamanide analogs, in which the phenylalanine residue in position 6 or 9 of the sequence was substituted by Gly. Previous conformational studies on antamanide suggested that these backbone regions are affected by conformational variation. The NMR conformational study showed a high degree of flexibility for the two analogs. With sodium ions, on the other hand, [Gly9]-antamanide was able to form a fairly stable equimolar complex, whereas [Gly6]-antamanide showed a conformational heterogeneity, with one prevailing conformer. For the [Gly9]-antamanide analog, the whole NMR data, combined with extensive theoretical calculations, were consistent with the presence of 1) two beta-turns of type I, centered on Gly9-Phe10 and Ala4-Phe5, respectively; 2) a central cavity with a six-carbonyl oxygen cage, optimal for a Na+ hexacoordination; 3) strongly H-bonded amide protons for residues 1 and 6, both involved in the formation of the two type I beta-turns, which, however, exhibited some fluctuations during the molecular dynamics simulations. For the [Gly6]-antamanide-Na+ complex the prevailing conformer was consistent with a more open structure, with the partial solvent exposure of all the amide protons; that is, the Gly residue in position 6 increases the flexibility of this critical site more than does the Gly in position 9. These data in some way parallel the results of the cytotoxicity tests on B16-F10 transformed cells for the two analogs: [Gly9]-antamanide is cytotoxic after 48 h exposure, whereas [Gly6]-antamanide is almost inactive. On the contrary, both analogs are practically inactive in vivo against phalloidin.

An exploration of the effects of constraints on the phosphorylation of synthetic protein tyrosine kinase peptide substrates.

We synthesized by classical solution methods three conformational constrained analogues of EDNEYTA, a heptapeptide sequence that represents the common major autophosphorylation site of the protein tyrosine kinases (PTKs) of the Src family. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as PTK substrates was examined. The kinetic data obtained indicate that the introduction of the tyrosine-analogue constraints Tic(OH) and MeTyr, which block the ring flexibility, completely prevents the phosphorylation catalysed by the kinases Lyn and Fgr. On the other hand PTKIIB/p38syk can phosphorylate the two derivatives albeit with an efficiency lower than that found with the native sequence. A third derivative contained side chain to side chain cyclization. This analogue, in which the freedom of the phenolic moiety is not altered, can be phosphorylated by all the PTKs tested with kinetic constants comparable to the parent peptide.

Synthetic Tyr-phospho and non-hydrolyzable phosphonopeptides as PTKs and TC-PTP inhibitors.

Tyrosine-specific protein kinases and phosphatases are important signal transducing enzymes in normal cellular growth and differentiation and have been implicated in the etiology of a number of human neoplastic processes. In order to develop agents which inhibits the function of these two classes of enzymes by interfering with the binding of their substrates, we synthesized analogs derived from the peptide EDNEYTA. This sequence reproduces the main autophosphorylation site of Src tyrosine kinases. In this work we report the synthesis, by classical solution methods, of the phosphotyrosyl peptide EDNEYpTA as well as of three analogs in which the phosphotyrosine is replaced by a phosphinotyrosine and by two unnatural, non-hydrolyzable amino acids 4-phosphonomethyl-L-phenylalanine and 4-phosphono-L-phenylalanine. The Src peptide and its derivatives were tested as inhibitors of three non-receptor tyrosine kinases (Lyn, belonging to the Src family, CSK and PTK-IIB) and a non-receptor protein tyrosine phosphatase obtained from human T-cell (TC-PTP). The biomimetic analogues, which do not significantly affect the activity of CSK, PTK-IIB and TC-PTP, act as efficient inhibitors on Lyn, influencing both the exogenous phosphorylation and, especially, its autophosphorylation. In particular, the Pphe derivative may provide a basis for the design of a class of inhibitors specific for Lyn and possibly Src tyrosine kinases, capable of being used in vivo and in vitro conditions.

Linear and cyclic synthetic peptides related to the main autophosphorylation site of the Src tyrosine kinases as substrates and inhibitors of Lyn.

Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60c-src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella-Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem. 204, 1159-1163]. Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src-like tyrosine kinase activity and active site-directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N-terminal acetylated and C-terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation.

Synthesis and conformational studies on peptides corresponding to a putative autophosphorylation site of abl TPK.

The transforming gene of Abelson murine leukaemia virus (v-abl) codes for a membrane-associated tyrosine-specific protein kinase (abl TPK). Analysis of the v-abl gene has shown that both the fibroblast-transforming and tyrosine-protein kinase activities reside within a minimal region encoding a protein of 43 kDa (p43v-abl), which represents the most active, isolated form of this enzyme. Since the cellular substrates for p43v-abl are yet to be identified, we synthesized by classical solution methods the octapeptide H-Gly-Asp-Thr-Tyr-Thr-Ala-His-Ala-OH, corresponding to the structural sequence of the main putative autophosphorylation site (Tyr 515) of the abl TPK, as well as some of its analogs modified in positions -2, -1, +1 and +3. The synthetic peptides were tested as substrates for the p43v-abl. The kinetic data obtained indicate that the rates of their phosphorylation vary considerably depending on the sequence of the peptide, as expected. As a rule, no significant increment of the efficiency results from each substitution in the parent sequence. While the replacement of the two charged residues, namely Asp-2 and His-7, with neutral Ala is well tolerated, the substitution with amino acids bearing opposite charges is detrimental. The correlation between secondary structure of our synthetic octapeptides and their substrate recognition by p43v-abl was studied using CD and fluorescence spectroscopy in 5 mM Tris, in 98% TFE/Tris and in 30 mM SDS solutions. The comparison of the spectroscopic data with the kinetic parameters does not confirm a close relationship between the conformational properties of these peptides and their enzymatic role.

Specificity of T-cell protein tyrosine phosphatase toward phosphorylated synthetic peptides.

The local specificity determinants for a T-cell protein tyrosine phosphatase (TC-PTP) have been inspected with the aid of a series of synthetic peptides, either enzymically or chemically phosphorylated, derived from the phosphoacceptor sites of phosphotyrosyl proteins. The truncated form of T-cell PTP, deprived of its C-terminal down-regulatory domain, readily dephosphorylates submicromolar concentrations of eptapeptides to eicosapeptides, reproducing the C-terminal down-regulatory site of pp60c-src (Tyr527), the phosphorylated loop IV of calmodulin and the main autophosphorylation site of two protein tyrosine kinases of the src family (Tyr416 of pp60c-src and Tyr412 of p51fgr). However, phosphopeptides of similar size, derived from phosphoacceptor tyrosyl sites of the abl and epidermal-growth-factor receptor protein tyrosine kinases, the phosphorylated loop III of calmodulin, and phosphoangiotensin II undergo either very slow or undetectable dephosphorylation, even if tested up to 1 microM concentration. The replacement of either Ser-P or O-methylated phosphotyrosine for phosphotyrosine within suitable peptide substrates gives rise to totally inert derivatives. Moreover, amino acid substitutions around phosphotyrosine in the peptides src(412-418), src(414-418) and abl-(390-397) deeply influence the dephosphorylation efficiency. From these data and from a comparative analysis of efficient versus poor phosphopeptide substrates, it can be concluded that acidic residues located on the N-terminal side of phosphotyrosine, with special reference to position -3, play a crucial role in substrate recognition, while basic residues in the same positions act as negative determinants. In any event, the presence of at least two aminoacyl residues upstream of phosphotyrosine represents a necessary, albeit not sufficient, condition for detectable dephosphorylation to occur. By replacing the truncated form of TC-PTP with the full length TC-PTP, the dephosphorylation efficiencies of all peptides tested are dramatically impaired. Such an effect is invariably accounted for by a substantial increase in Km values, accompanied by a more or less pronounced decrease in Vmax values. These data support the concept that the C-terminal regulatory domain of TC-PTP exerts its function primarily by altering the affinity of the enzyme toward its phosphotyrosyl targets.

Separation of acidic peptides by reversed-phase ion-pair chromatography. Analytical application to a series of acidic substrates of casein kinases.

A series of small peptides including clusters of glutamyl residues, synthesized to study the site specificity of rat liver (L-CK2) and yeast (Y-CK2) casein kinase-2, are analytically characterized by ion-pair high-performance liquid chromatography using tetrabutylammonium as counter-ion and acetonitrile as modifier of the aqueous phase. Under these conditions peptides of slightly different acidity can be separated and the elution order parallels the hydrophobicity of the ion-pair-peptide complexes, which increases with the number of the acidic functions present in the sequence.

Conformational and binding studies on peptides related to domains I and III of calmodulin.

The conformational and ion-binding properties of two peptide fragments of 25 amino acid residues corresponding to the helix-loop sequences of domains I and III of calmodulin (CaM) were investigated by CD and Tb(3+)-mediated fluorescence spectroscopy. Both peptides exhibit very similar ion binding properties either in water or trifluoroethanol (TFE), and do not allow the differentiation of the two domains in the native protein in terms of their binding capacity. An aggregation phenomenon was observed in TFE with increase of the alpha-helical content. We suggest that the aggregation involves an interaction between the hydrophilic surfaces of amphiphilic alpha-helices in a way similar to inverse micelle formation.

Synthetic peptides including acidic clusters as substrates of yeast casein kinase-2.

The synthesis is reported of a series of glutamyl peptide analogs of the model substrate H-Ser-Glu-Glu-Glu-Glu-Glu-OH of casein kinase-2 (CK-2). A convenient HPLC method for the separation of slightly different acidic peptides is also reported. The site specificity of yeast casein kinase-2 (Y-CK2) is examined with the aid of synthesized peptide substrates.

Synthetic peptides reproducing the EGF-receptor segment homologous to the pp60v-src phosphoacceptor site. Phosphorylation by tyrosine protein kinases.

The octapeptide E-E-K-E-Y-H-A-E, corresponding to the amino acid sequence 841-845 of EGF receptor, whose tyrosine-845 is homologous to the main phosphorylation site of pp60v-src, has been synthesized together with seven shorter peptides encompassing variable segments around the tyrosine residue. The peptides have been employed as model substrates for inspecting the local structural determinants of three tyrosine protein kinases (TPKs), namely; TPK-IIB and TPK-III, isolated from lymphoid cells (Eur. J. Biochem. 172, 451-457 (1988] and the TPK encoded by the oncogene of Abelson murine leukemia virus. The phosphorylation order with the different peptide substrates is variable depending on the TPK used: in particular, the lysine residue at position -2 relative to tyrosine proved especially harmful with TPK-IIB, the peptides K-E-Y-H and K-E-Y-H-A-E being very poor substrates compared with their shorter derivatives devoid of the N-terminal lysine (E-Y-H and E-Y-H-A-E, respectively). Conversely, such a basic residue is well tolerated by the other two TPKs. The negative effect of the N-terminal lysine on TPK-IIB-catalyzed phosphorylation is accounted for by an increase of Km and can be overcome by the presence of additional glutamic acid(s) on that side. On the other hand, the C-terminal acidic doublet Ala-Glu specifically impairs the phosphorylation efficiency of abl-TPK, by lowering the Vmax value, the heptapeptide E-K-E-Y-H-A-E being much less readily phosphorylated than E-K-E-Y-H. Collectively, these results would indicate that the site specificity of tyrosine protein kinases results from the balance of positive and negative determinants whose influence on the catalytic activity of the individual enzymes can differ greatly.

Synthesis of the dodecapeptide corresponding to domain III of bovine brain calmodulin: alpha-beta shift side reactions during the synthesis by the classical method in solution.

We report details of the chemical synthesis of the dodecapeptide corresponding to the calcium binding loop III of bovine brain calmodulin (sequence 93-104) and its fragments 96-04, 93-98, and 99-104. The preparation of the peptides employed classical solution methods and a fragment-condensation strategy. The major difficulties were encountered during the synthesis of the peptides containing the N-terminal sequences -Gly-Asn-Gly- and -Asp-Lys-Asp-Gly-Ans-Gly-, in which alpha-beta shift side reactions were observed.

Conformation and ion binding properties of peptides related to calcium binding domain III of bovine brain calmodulin.

The conformational and ion binding properties of the sequences 93-104, 96-104, and 93-98 of domain III of bovine brain calmodulin (CaM) have been studied by CD and Tb3+-mediated fluorescence. In aqueous solution the interaction of all fragments with Ca2+ and Mg2+ ions is very weak and without any effect on the peptide conformation, which remains always random. In trifluoroethanol the interaction is very strong and the different fragments exhibit very distinct binding properties. In particular, the dodecapeptide fragment 93-104, and its N-terminal hexapeptide 98-104, bind calcium and magnesium with a very high binding constant (Kb greater than 10(5) M-1), undergoing a substantial conformational change. The structural rearrangement is particularly evident in the hexapeptide fragment, which tend to form a beta-bend. The C-terminal nonapeptide fragment 96-104 interacts with calcium and magnesium more weakly, and the binding process causes a decrease of ordered structure. These results suggest that, even in the entire dodecapeptide sequence corresponding to the loop of domain III of CaM, the calcium binding site is shifted toward the N-terminal hexapeptide segment. This interpretation is consistent with the results of crystallographic studies of CaM, which show that the calcium ions are located toward the amino terminal portion of the loop.

Synthetic peptide substrates for casein kinase 2. Assessment of minimum structural requirements for phosphorylation.

Unlike the peptides SAEAAA and SEEAAA which are not substrates for casein kinase 2 (CK-2) their analogs SAAEAE and SAAEAA are still significantly phosphorylated. Their Km values, however, (13.3 and 18.9 mM, respectively) are almost two orders of magnitude higher than that of SEEEEE and their Vmax values are 3- and 14-fold lower than that of SAAEEE. The peptide ESEEEEE, but not ASEEEEE, is a slightly better substrate than SEEEEE, while both RSEEEEE and SEEEKE are very poor substrates compared to ASEEEEE and SEEEAE, respectively. SAAEAE is much more responsive to polylysine stimulation and polyphosphate inhibition than is SEEEEE. Taken together these data show that a single acidic residue at the third position from the C-terminal side of the phosphorylatable amino acid represents not only a necessary, but also a sufficient condition for site recognition by CK-2. Optimal phosphorylation efficiency, however, requires an extended C-terminal cluster of several acidic residues, and can be compromised by the presence of only a basic residue either inside the acidic cluster or adjacent to the N-terminal side of the phosphoacceptor amino acid. The structure of the phosphoacceptor site can greatly influence the efficacy of substrate-directed effectors of CK-2.

Synthesis of human [15-norleucine]little-gastrin-II and des-1-tryptophan-[12-norleucine]minigastrin-II.

By the use of a newly developed procedure for the synthesis of tyrosine-O-sulfate peptides based on the direct incorporation of the suitably N alpha-protected tyrosine-O-sulfate residue along the synthetic route, the synthesis of two human gastrin-II analogues was successfully accomplished. Thereby acid labile side chain protection was applied in combination with the N alpha-benzyloxycarbonyl group in the intermediate chain elongation steps. Despite the pronounced acid-lability of the sulfate ester moiety, its hydrolysis during the final acidolytic deprotection step was significantly reduced under optimized conditions. Subsequent chromatographic purification led to the two gastrin analogues in satisfactory yields as highly pure compounds as judged by various indicative analytical assays.