Sequence-specific DNA cleavage by dipeptides disubstituted with chlorambucil and 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid.

Two dipeptides, each containing a lysyl residue, were disubstituted with chlorambucil (CLB) and 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid (DMQ-MA): DMQ-MA-Lys(CLB)-Gly-NH2 (DM-KCG) and DMQ-MA-beta-Ala-Lys(CLB)-NH2 (DM-BKC). These peptide-drug conjugates were designed to investigate sequence-specificity of DNA cleavage directed by the proximity effect of the DNA cleavage chromophore (DMQ-MA) situated close to the alkylating agent (CLB) inside a dipeptide moiety. Agarose electrophoresis studies showed that DM-KCG and DM-BKC possess significant DNA nicking activity toward supercoiled DNA whereas CLB and its dipeptide conjugate Boc-Lys(CLB)-Gly-NH2 display little DNA nicking activity. ESR studies of DMQ-MA and DM-KCG both showed five hyperfine signals centered at g = 2.0052 and are assigned to four radical forms at equilibrium, which may give rise to a semiquinone radical responsible for DNA cleavage. Thermal cleavage studies at 90 degrees C on a 265-mer test DNA fragment showed that besides alkylation and cleavage at G residues, reactions with DM-KCG and DM-BKC show a preference for A residues with the sequence pattern: 5'-G-(A)n-Pur-3' > 5'-Pyr-(A)n-Pyr-3' (where n = 2-4). By contrast, DNA alkylation and cleavage by CLB occurs at most G and A residues with less sequence selectivity than seen with DM-KCG and DM-BKC. Thermal cleavage studies using N7-deazaG and N7-deazaA-substituted DNA showed that strong alkylation and cleavage at A residues by DM-KCG and DM-BKC is usually flanked on the 3' side by a G residue whereas strong cleavage at G residues is flanked by at least one purine residue on either the 5' or 3' side. At 65 degrees C, it is notable that the preferred DNA cleavage by DM-KCG and DM-BKC at A residues is significantly more marked than for G residues in the 265-mer DNA; the strongest sites of A-specific reaction occur within the sequences 5'-Pyr-(A)n-Pyr-3'; 5'-Pur-(A)n-G-3' and 5'-Pyr-(A)n-G-3'. In pG4 DNA, cleavage by DM-KCG and DM-BKC is much greater than that by CLB at room temperature and at 65 degrees C. It was also observed that DM-KCG and DM-BKC cleaved at certain pyrimidine residues: C40, T66, C32, T34, and C36. These cleavages were also sequence selective since the susceptible pyrimidine residues were flanked by two purine residues on both the 5' and 3' sides or by a guanine residue on the 5' side. These findings strongly support the proposal that once the drug molecule is positioned so as to permit alkylation by the CLB moiety, the DMQ-MA moiety is held close to the alkylation site, resulting in markedly enhanced sequence-specific cleavage.

Synthesis and DNA nicking studies of a novel cyclic peptide: cyclo[Lys-Trp-Lys-Ahx-].

Two novel cyclic tetrapeptides: cyclo[Lys-Tyr-Lys-Ahx-] 7a and cyclo[Lys-Trp-Lys-Ahx-] 7b were synthesized by coupling protected amino acid in solution and the subsequent cyclization effected by the pentafluorophenyl ester method as described in previous papers. These cyclic peptides were designed and synthesized to study their interaction with DNA, based on previous reports that linear peptides Lys-Tyr-Lys and Lys-Trp-Lys could bind to various forms of DNA and cleaved supercoiled DNA at apurinic sites. Ethidium bromide displacement assay showed that the apparent DNA binding constant of linear Lys-Tyr-Lys and cyclic peptide 7a are far below 1 x 10(3) M(-1), whereas those of cyclic peptide 7b and linear Lys-Trp-Lys are 1.9 x 10(4) M(-1) and 9.5 x 10(3) M(-1), respectively. Kinetic studies using agarose gel electrophoresis showed that cyclic peptide 7b and Lys-Trp-Lys possessed DNA nicking activity on natural supercoiled phi X174 DNA with nicking rate of 50.7 and 75.6 pM min(-1) at 65 degrees C, respectively, whereas cyclic peptide 7a and linear Lys-Tyr-Lys were devoid of the corresponding activity. The DNA nicking rate increased significantly with increase in reaction temperature. At reaction temperatures lower than 65 degrees C, the DNA nicking rate of cyclic peptide 7b exceeded that of linear Lys-Trp-Lys. The addition of 1 microM ferrous ion did not give significant enhancement effect on the DNA nicking rate by the peptides. UV irradiation gave a marked rate enhancement on the DNA nicking rate of linear Lys-Trp-Lys and a moderate enhancement on the DNA nicking rate of cyclic peptide 7b.

Synthesis, DNA cleavage and cytotoxicity of some novel cyclic peptide-2,6-dimethoxyhydroquinone-3-mercaptoacetic acid conjugates containing D-amino acids.

This paper reports an ongoing study of the use of small-ring-size cyclic peptides as carriers of a potential antitumor agent: 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid (DMQ-MA). Three new cyclic tripeptide-DMQ-MA conjugates--cyclo[D-Val-Lys(DMQ-MA)-gamma-aminobutyric acid (GABA)-], cyclo[Val-Lys(DMQ-MA)-GABA-] and cyclo[D-Val-D-Lys(DMQ-MA)-GABA-]--were synthesized. The isomeric cyclic tripeptide-DMQ-MA conjugates were designed and synthesized to study the effect of stereoisomerism of the conjugates on cytotoxicity. The cyclic peptides were synthesized by coupling protected amino acids in solution and the final cyclization performed by the pentafluorophenyl ester method as described previously. After removing the lysyl-Z protecting group of the cyclic peptides the conjugation was achieved by reacting with the pentafluorophenyl ester of DMQ-MA. Electron spin resonance (ESR) studies of these three cyclic tripeptide-DMQ-MA conjugates showed that hydroxyl radicals were generated as a non-linear function of L-ascorbic acid (AH2) concentration. The IC50 of the cyclic tripeptide-DMQ-MA conjugates against a human pulmonary carcinoma cell line (PC-9 cells) under the synergistic activation of AH2 ranges from 0.4 to 1.6 microM, which is significantly lower than the parent compound DMQ-MA (6.1 microM). Agarose gel electrophoresis showed that DMQ-MA and these cyclic peptide-DMQ-MA conjugates are capable of cleaving supercoiled plasmid DNA to open circular and linear forms, even in the absence of AH2. The effects of enantiomeric and diastereomeric variations of these cyclic tripeptide-DMQ-MA conjugates on the cytotoxicity against PC-9 cells were discussed.

Synthesis, DNA binding, and sequence specificity of DNA alkylation by some novel cyclic peptide-chlorambucil conjugates.

In an effort to investigate the potential of cyclic peptides as carriers for cytotoxic agents, we synthesized four cyclic peptide-chlorambucil conjugates: cyclo[Lys(CHB)-Lys(CHB)-Gaba-] (peptide Y), cyclo[Lys(CHB)-Gly-Lys(CHB)-Gaba-] (peptide A), cyclo[Lys(CHB)-beta-Ala-Lys(CHB)-Gaba-] (peptide B) and cyclo[Lys(CHB)-Gaba-Lys(CHB)-Gaba-] (peptide C). The cyclic peptides were synthesized by coupling protected amino acid residues in solution and the subsequent cyclization was performed by the pentafluorophenyl ester method as described previously (Sheh et al., 1990, 1993a,b). After deblocking the lysyl-carbobenzyloxy protecting group (Z), the conjugation was achieved by reaction with the pentafluorophenyl ester of chlorambucil (CHB). These cyclic peptides differ from one another in ring size and are disubstituted with CHB via the epsilon-amino group of the lysyl residue. The various conjugates were designed to study the effect of ring size on the mode of DNA binding and alkylation. A DNA-binding assay using lambda-DNA with ethidium bromide showed that whereas peptide Y and CHB have no observable binding affinity, the apparent binding constants for peptide A, peptide B and peptide C on lambda-DNA were determined to be 2.36 x 10(5), 1.27 x 10(5) and 3.50 x 10(5), respectively. Thus, it is suggested that cyclic peptides bearing aliphatic side chains attached to a ring larger in size than 14 members would be more favourable as regards augmenting the binding affinity. DNase I footprinting showed that no footprinting patterns were observed for the 253-mer fragment and 117-mer fragment with peptide A, but two new bands corresponding to G69 and G80 were observed for the 117-mer fragment. DNA alkylation studies using a piperidine cleavage assay on the 117-mer DNA fragment showed that the sequence selectivity, judged by reaction intensity observed with peptide A, peptide B and peptide C, was similar to that seen with CHB alone. The selectivity of alkylation for both CHB and its peptide derivatives appears to be: 3'-Pur-G-Pyr-5' > 3'-Pyr-G-Pyr-5' > 3'-Pyr-G-Pur-5' = 3'Pur-G-Pur-5'. However, there are apparent differences in the intensity of alkylation by peptides A, B, C and CHB at certain guanine residues.

Studies of the synthesis, immunology, and cytotoxicity of a cyclic octapeptide corresponding to TNF-alpha-(59-66).

In an attempt to investigate possible binding domains of the tumor necrosis factors (TNF), we have previously synthesized a cyclic hexapeptide corresponding to murine TNF-(127-132) (cTNF-1). In this report, we describe the synthesis and biological activity of another cyclic octapeptide corresponding to human TNF-alpha-(59-66) (cTNF-2). The design of these cyclic peptides is based on their high sequence homology with corresponding fragments of human TNF-alpha or TNF-beta. Similar to cTNF-1, the cyclic octapeptide cTNF-2 displayed low in vitro cytotoxicity against human HeLa and HEP-2 cell lines. The cyclic peptides cTNF-2 and cTNF-1 were then tested for the induction of interleukin-1 (IL-1) production from human peripheral blood mononuclear cells and monocytes in vitro. At low concentrations, the IL-1 levels induced by these cyclic peptides were similar to that of recombinant TNF-alpha. However, the IL-1 production by cTNF-2 stimulation was dose-dependently increased and reached that of a lipopolysaccharide (LPS; 0.1 micrograms/mL) level. These findings suggest that the fragments corresponding to human TNF-alpha-(59-66) and murine TNF-(127-132) may represent certain binding domains of the tumor necrosis factors that elicit IL-1 production.

Synthesis and cytotoxicity studies of novel cyclic peptide-2,6-dimethoxyhydroquinone-3-mercaptoacetic acid conjugates.

In an effort to investigate the use of small-ring-size cyclic peptides as carriers of new antitumor agents, we synthesized three cyclic tripeptide-cytotoxic agent conjugates. The cytotoxic agent conjugated to the epsilon-amino group of the lysyl residue of the cyclic peptides is 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid (DMQ-MA), (Sheh et al., 1992). The cyclic peptides were synthesized by coupling protected amino acid residues in solution and the subsequent cyclization performed by the pentafluorophenyl ester method as described previously (Sheh et al., 1985, 1987, 1990). After deblocking the lysyl-Z group of the peptides, the conjugation was achieved by reaction with the pentafluorophenyl ester of DMQ-MA. The three cyclic peptides exhibited potent cytotoxicity against two solid tumor cell lines (KB and PC-9) under the synergistic activation of L-ascorbic acid. Electron spin resonance (ESR) studies of DMQ-MA and two conjugates showed that massive hydroxyl radicals were generated as a non-linear function of L-ascorbic acid concentration. These studies indicate that the hydroxyl radical is a possible mediator of cytotoxicity for these conjugates and that small-ring-size cyclic peptides are potentially useful carriers of cytotoxic agents.

Cytotoxicity studies on some novel 2,6-dimethoxyhydroquinone derivatives.

Six synthetic 2,6-dimethoxyhydroquinone derivatives were shown to have different degrees of cytotoxicity to two human tumor cell lines (KB and PC-9) under the synergistic activation of L-ascorbic acid. Two representative compounds displayed very low time-schedule-independent index, showing that the cytotoxic action is independent of time of drug treatment. The addition of catalase produced a significant inhibitory effect on the cytotoxicity of two representative compounds, indicating that the cytotoxic action is mediated by the generation of H2O2, which may yield hydroxyl radicals via various mechanisms. ESR studies employing the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) showed that massive hydroxyl radicals were generated from four of these drugs as a non-linear function of L-ascorbic acid concentration. The results indicate the possible involvement of hydroxyl radicals in the cytotoxic action of these novel drugs.

Synthesis of a cyclic hexapeptide with sequence corresponding to murine tumor necrosis factor-(127-132) as a novel potential antitumor agent.

A cyclic hexapeptide cyclo(Lys-Gly-Asp-Gln-Leu-Ser-) 10 was synthesized stepwise in solution by acylation of peptide ester trifluoroacetates directly with preactivated Boc-amino acids using the DCC/HOBt method; the final cyclization reaction was performed using the pentafluorophenyl ester method in solution (1-4). This peptide is a cyclic derivative of murine tumor necrosis factor-(127-132) and is designed as a potential antitumor agent. The cyclic peptide 10 displayed weak cytotoxic activity on three of the four human tumor cell lines tested.

Synthesis of cyclic peptide homologs of glutathione as potential antitumor agents.

Two cyclic tripeptide homologs, cyclo(Glu[Cys-beta-Ala-]-OH) 8a, and cyclo(Glu[Cys-Gaba-]-OH) 8b, were synthesized by the pentafluorophenyl ester method in solution. These cyclic peptides are cyclo homologs of glutathione and are designed as potential antitumor agents. The 1H- and 13C-n.m.r. spectral parameters of cyclo(Glu[Cys(Bzl)-beta-Ala-]-OH) 7a were measured in DMSO-d6 and a possible conformation has been proposed. The cyclic peptide 8a showed low cytotoxic activities against three human tumor cell lines: KB, HeLa, and Colo 205.

Design, synthesis, and testing of potential antisickling agents. 9. Cyclic tetrapeptide homologs as mimics of the mutation site of hemoglobin S.

As part of our continuing search for new agents which might be useful for the treatment of sickle-cell anemia, we have synthesized two cyclic tetrapeptide homologs, cyclo(-Val-Glu[-Thr-Pro-]-OH) (1a) and cyclo(-Phe-Glu[-Thr-Pro-]-OH (1b), and a tetrapeptide lactone homolog cyclo(H-Thr-Pro-Val-Glu-OH) (2). The intent was that these peptides would mimic a tetrapeptide region around the mutation site of HbS and thus be able to bind at the acceptor site of HbS and thereby inhibit polymerization. The synthesis of the linear peptides was accomplished in solution using both the polymeric reagent (PHBT) and DCC/HOBT methods; cyclization was accomplished by an improved method. 13C-n.m.r. studies were performed which allowed us to assign the conformation about the Thr-Pro bond in 1a and 2 as trans. The cyclic peptides were tested for their ability to increase the solubility of HbS under deoxygenating conditions, but only 1a had any antigelling activity, albeit low.

High-resolution proton nuclear magnetic resonance studies of the glucocerebrosidase activator protein from Gaucher spleen.

A heat-stable protein factor (HSF) obtained from the spleen of a patient with Gaucher's disease that activates glucocerebrosidase was studied by 600-MHz proton NMR spectroscopy. Assignments for a number of aromatic and aliphatic resonances were made on the basis of spin-decoupling, pH-titration, and resolution-enhancement experiments. The upfield ring current shifted aliphatic region and the downfield aromatic region were examined by nuclear Overhauser effect (NOE) methods using both pulsed Fourier-transform spectroscopy and correlation spectroscopy. It was found that a number of upfield-shifted methyl groups and certain methylene groups of specific aliphatic amino acid residues are in proximity relationships with several aromatic residues, forming a compact hydrophobic clustering site. Of special interest, tyrosine A, phenylalanine A, tryptophan B1, and tryptophan B2 were found to be located close to a cluster of aliphatic residues, indicating that the hydrophobic site of the HSF is conformationally rigid and its tertiary structure very compact. A two-dimensional structural model of the hydrophobic site of HSF is proposed.

Design, synthesis, and testing of antisickling agents. 2. Proline derivatives designed for the donor site.

We have used a three-dimensional model of deoxyhemoglobin to design potential antisickling agents with an intended binding site in the vicinity of the beta-6 mutation (donor site). Two proline derivatives, (4S)-1-butyryl-4-[(carboxymethyl)amino]-L-proline (9a) and its 1-benzoyl analogue (9b), were designed to interact, via ionic or hydrogen bonds, with polar residues beta His-2, beta Thr-4, and beta Lys-132 of hemoglobin S (HbS). Two other proline derivatives containing a salicylate leaving group, (4S)-1-butyryl-4-[(carboxymethyl)methylamino]-L-proline, 2-ester with salicyclic acid (14a), and its 1-benzoyl analogue (14b), were designed to bind covalently to beta Lys-132, as well as to interact with beta His-2 and beta Thr-4 via ionic and hydrogen bonds. This paper describes the synthesis of these agents, beginning with natural L-hydroxyproline methyl ester, and the testing of their ability to increase or decrease the solubility of dHbS by using a standard solubility assay. The covalent derivatives 14a,b were found to be inactive, while the noncovalent compounds 9a,b showed weak antigelling activity, below that observed for phenylalanine. The presence of only weak activity does not invalidate this approach, since only one structural parameter has been investigated.