Cytotoxicity, crosslinking and biological activity of three mitomycins.

While interstrand crosslinks (ICLs) have been considered as one type of DNA damage in the past, there is mounting evidence suggesting that these highly cytotoxic lesions are processed differently by the cellular machinery depending upon the ICL structure. In this study, we examined the crosslinking ability of three mitomycins, the structure of the ICLs they produce and the cytotoxicity of the drugs toward three different cell lines. The drugs are: mitomycin C (1), decarbamoylmitomycin C (2), and a mitomycin-conjugate (3) whose mitosane moiety is linked to a N-methylpyrrole carboxamide. We found that, overall, both MC and compound 3 show strong similarities regarding their alkylation of DNA, while DMC alkylating behavior is markedly different. To gain further insight into the mode of action of these drugs, we performed high throughput gene expression and gene ontology analysis to identify gene expression and cellular pathways most impacted by each drug treatment in MCF-7 cell lines. We observed that the novel mitomycin derivative (3) specifically causes changes in the expression of genes encoding proteins involved in cell integrity and tissue structure. Further analysis using bioinformatics (IPA) indicated that the new derivative (3) displays a stronger downregulation of major signaling networks that regulate the cell cycle, DNA damage response and cell proliferation when compared to MC and DMC. Collectively, these findings demonstrate that cytotoxic mechanisms of all three drugs are complex and are not solely related to their crosslinking abilities or the structure of the ICLs they produce.

A role of flavonoids in cytochrome c-cardiolipin interactions.

The processes preceding the detachment of cytochrome c (cyt c) from the inner mitochondrial membrane in intrinsic apoptosis involve peroxidation of cardiolipin (CL) catalyzed by cyt c-CL complex. In the present work, we studied the effect of 17 dietary flavonoids on the peroxidase activity of cyt c bound to liposomes. Specifically, we explored the relationship between peroxidase activity and flavonoids' (1) potential to modulate cyt c unfolding, (2) effect on the oxidation state of heme iron, (3) membrane permeability, (4) membrane binding energy, and (5) structure. The measurements revealed that flavones, flavonols, and flavanols were the strongest, while isoflavones were the weakest inhibitors of the oxidation. Flavonoids' peroxidase inhibition activity correlated positively with their potential to suppress Trp-59 fluorescence in cyt c as well as the number of OH groups. Hydrophilic flavonoids, such as catechin, having the lowest membrane permeability and the strongest binding with phosphocholine (PC) based on the quantum chemical calculations exhibited the strongest inhibition of Amplex Red (AR) peroxidation, suggesting a membrane-protective function of flavonoids at the surface. The results of the present research specify basic principles for the design of molecules that will control the catalytic oxidation of lipids in mitochondrial membranes. These principles take into account the number of hydroxyl groups and hydrophilicity of flavonoids.

Acetone promoted 1,4-migration of an alkoxycarbonyl group on a syn-1,2-diamine.

A 2-protected cis-amino mitosene undergoes an irreversible acetone promoted isomerization and converts to the 1-isomer. Kinetic studies and DFT calculations of the reaction are reported. An organocatalytic mechanism is proposed, involving a covalent intermediate formed by reaction of the mitosene and acetone.

Synthesis of Mitomycin C and Decarbamoylmitomycin C N(2) deoxyguanosine-adducts.

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) - a derivative of MC lacking the carbamate on C10 - are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine-mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment.

Synthesis of a major mitomycin C DNA adduct via a triaminomitosene.

We report here the synthesis of two amino precursors for the production of mitomycin C and 10-decarbamoylmitomycin C DNA adducts with opposite stereochemistry at C-1. The triamino mitosene precursors were synthesized in 5 steps from mitomycin C. In addition synthesis of the major mitomycin C-DNA adduct has been accomplished via coupling of a triaminomitosene with 2-fluoro-O(6)-(2-p-nitrophenylethyl)deoxyinosine followed by deprotection at the N(2) and O(6) positions.

Theoretical studies of the anti-tumor drug FR900482.

Hartree-Fock and density functional theory (B3LYP) calculations were applied to the study of the anti-tumor drug FR900482 and some of its analogs. Optimum geometries were obtained and it was found that the most stable conformations feature the N-H bond of the aziridine ring nitrogen "down" and the oxygen bridge and aziridine nitrogen "up". It was also found that the analog containing NH(2) (in place of the -CHO of the natural product) is the most prone to oxidation.

Theoretical studies of the reduction reaction of the anti-tumor drug FR900482.

A number of analogs of the anti-tumor drug FR900482 have been investigated with quantum chemical calculations, at the HF/6-31G(d,p) and B3LYP levels from the point of view of their energy, optimum geometry and the energetics of the reduction reaction. It was found that the parent molecule is the most prone to reduction, followed closely by fluorine-containing analogs.

An ab initio study of the guanidinium groups in saxitoxin.

Quantum chemical (Hartree-Fock) calculations were performed on neutral and protonated saxitoxin in order to obtain optimum geometries, rotational energy barriers for the guanidinium ions and proton affinities. For comparison purposes, as model compounds, guanidinium systems in five and six membered rings were also investigated. In addition, DFT (B3LYP) calculations with the 6-31G** basis set were performed and the sodium affinities of the guanidinium groups in saxitoxin were obtained. It was concluded that the inhibition of the sodium channels by the saxitoxin is due to the interaction of the guanidinium group with carboxylate groups from the wall of the channel and not to the binding of the sodium ions.

Amino acid alanine reactivity with the fingerprint reagent ninhydrin. A detailed ab initio computational study.

Ninhydrin is one of the most widely used reagents for chemical development of fingerprints on porous surfaces. The detection is based on the reaction of ninhydrin with a monoacidic component of the fingerprint to form an intensively colored compound named Ruhemann's Purple. A computational study of the mechanisms and reaction energetics of the formation of Ruhemann's Purple from ninhydrin and alanine is presented. Such a study is significant from a forensic science point of view because of the strong interest in the forensic chemistry and law enforcement communities in developing alternatives to the current generation of ninhydrin like chemicals for the detection and development of latent fingerprints. Information about the mechanism of reaction between ninhydrin and amino acids can ultimately help to design compounds with stronger chromo-fluorogenic properties in aid of detecting fingerprints at crime scenes. The three most accepted mechanisms of formation have been considered using ab initio quantum mechanical calculations. At relatively high temperature ( approximately 100 degrees C) all three mechanisms are energetically feasible. However since it is recommended that forensic analyses be performed at room temperature, a revised mechanism is proposed for the formation of Ruhemann's Purple under this condition.

An ab initio study of di- and trifluorobenzene-benzene complexes as relevant to carbonic anhydrase II-drug interactions.

Ab initio calculations at the MP2/6-31G* level have shown that variously substituted di- and trifluorobenzenes form non-covalent complexes with benzene that adopt either aromatic-aromatic or H--F binding, the choice being determined by the pattern of fluorination. The binding energies of these structures are from 3.4 to 4.5 kcal mol(-1). This range is large enough to account for observed variations in the binding affinity of a library of fluoroaromatic inhibitors of carbonic anhydrase. This enzyme has an aromatic amino acid at a central position in the active site. The diverse modes of binding of the dimers also suggest that aggregates of fluorobenzenes might adopt specified 3-dimensional shapes in the solid state.

The role of salt bridge formation in glucagon: an experimental and theoretical study of glucagon analogs and peptide fragments of glucagon.

BACKGROUND: Glucagon is a 29-residue peptide produced in the alpha cells of the pancreas that interacts with hepatic receptors to stimulate glucose production and release, via a cAMP-mediated pathway. Type 2 diabetes patients may have an excess of glucagon and, as such, glucagon antagonists might serve as diabetes drugs. The antagonists that bind to the glucagon receptor but do not exhibit activity could be analogs of glucagon. The presence of salt bridges between some residues of glucagons (such as aspartic acid) and others (such as lysine) might influence both the binding to the receptor and the activity. MATERIALS AND METHODS: Experimental-The solid phase method with 4-methylbenzilhydrilamine resin (p-MBHA resin) was used for the synthesis of glucagon analogs. Rat liver membranes were prepared from male Sprague-Dawley rats by the Neville procedure. The receptor binding essay was performed in 1% BSA, 1 mM dithiothreitol, 25 mM Tris-HCl buffer, pH 7.2. Adenyl cyclase activity was measured in an assay medium containing 1% serum albumin, 25 mM MgCl2, 2 mM dithiothreitol, 0.025 mM GTP, 5 mM ATP, 0.9 mM theophylline, 17.2 mM creatine phosphate, and 1 mg/ml creatine phosphokinase. Theoretical-Quantum chemical calculations using the Titan program with the 6-31G* basis set were performed to calculate the binding energies of salt bridges between aspartic or glutamic acids and lysine. The relative stability of cyclic conformations of glucagon segments versus the extended segments was determined. RESULTS: It was found that the cyclic Glu9-Lys12 amide compound displayed a 20-fold decrease in binding affinity. DesHis1 cyclic compounds Glu20-Lys24 amide and DesHis1Glu9 Glu20-Lys24 amide behave as glucagon antagonists. The calculations show that cyclic conformations of tetrapeptidic and pentapeptidic segments of glucagon are more stable than the extended species. CONCLUSIONS: The biological data and the theoretical calculations show that an intramolecular salt bridge might impart stability to some glucagon antagonists and, when situated at the C-terminus of glucagon, might facilitate induction of an alpha-helix upon initial hormone association with the membrane bilayer. These findings might be a useful tool for the design of new glucagon antagonists.