IR spectroscopy as a new tool for evidencing antitumor drug signatures

There is a growing interest for screening antitumor drugs for their mechanism of action on cancer cells. Yet, screening for “modes of action” presents a technical challenge that is beyond the capability of conventional methods used in cellular or molecular biology. Several studies have highlighted the advantages of using infrared spectroscopy for diagnostic purposes at the clinical level for identifying cell types. In the present work, we suggest that the Fourier Transform Infrared (FTIR) spectrum of cells exposed to anti-cancer drugs could offer a unique opportunity to obtain a fingerprint of all molecules present in the cells and to observe, with a high sensitivity, the metabolic changes induced by potential anti-cancer drugs. Ouabain is one of the most potent cardenolides, which acts by inhibiting sodium pump activity. Cardenolides represent a class of compounds that are intended to soon enter clinical trials in oncology. In order to evaluate the potential of infrared spectroscopy to yield a signature for ouabain action on cancer cells, human prostate cancer PC-3 cells were treated with 36 nM ouabain, a sub-lethal concentration. Using ouabain as a model, we have thus demonstrated the possibility of using IR spectroscopy in the assessment of the global effects of an investigational compound on the cell constituents, thus contributing to setting up a new method for screening for novel anti-cancer agents in general, and potential anti-cancer cardenolides in particular. The most spectacular data obtained strongly suggest a modification in the nature of the cell lipids.     

Development of novel membrane active lipidated peptidomimetics active against drug resistant clinical isolates

A new series of small cationic lipidated peptidomimetics have been synthesized and found to be highly active against several susceptible as well as drug resistant clinical isolates of bacteria and fungi. All lipidated peptidomimetics do not cause significant lysis of human erythrocytes (HC₅₀ > 200 μg/mL). Calcein dye leakage experiment revealed membranolytic effect of LPEP08 which was further confirmed by scanning electron microscopy (SEM). The involvement of intracellular targets as an alternate mode of action was precluded by DNA retardation assay. Additionally, LPEP08 exhibit high proteolytic stability and dose not elicit resistance against drug resistant clinical isolate of Staphylococcus aureus, even after 16 rounds of passaging. These results demonstrate the potential of lipidated peptidomimetics as biocompatible anti-infective therapeutics.     

Synthesis of a biotin-conjugate of phosmidosine O-ethyl ester as a G1 arrest antitumor drug

This paper deals with the synthesis of a stable biotin–phosmidosine conjugate molecule 3 that is required for isolation of biomolecules that bind to phosmidosine (1). It was found that introduction of a biotin residue into the 6-N position of phosmidosine could be carried out by reaction of an N⁷-Boc-7,8-dihydro-8-oxoadenosine derivative 13 with phenyl chloroformate followed by displacement with a diamine derivative 6 along with the simultaneous removal of the Boc group and one of the two phenoxycarbonyl groups and the successive condensation with an N-tritylated biotin derivative 5. The condensation of an N-prolylphosphorodiamidite derivative 4 with an appropriately protected 7,8-dihydro-8-oxoadenosine derivative 17 having the biotin residue gave the coupling product 18, which was deprotected to give the biotin–phosmidosine (O-ethyl ester) conjugate 3.     

Physiochemical characterization of substituted chromeno[4,3-b][1,5]benzodiazepine stereoisomers designed as cell membrane active antitumor agents

As an alternative to naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines (e.g., antramycin) which possess properties of DNA alkylation, we have designed several antileukemic chromeno[4,3-b][1,5]benzodiazepine derivatives with potential activity toward leukemia cell membranes and the cyclic nucleotide system. The cis and trans diastereoisomers were characterized by NMR. The absolute configurations of the enantiomers were established by X-ray diffraction and circular dichroism (CD) measurements. By means of absorption spectroscopy and determinations of fluorescence and fluorescence decay, it was found that the cancerostatically active compound (+)(6aR, 13aS)-3,4-dimethoxy-10,11-dimethyl-6,6a,7,8,13, 13a-hexahydrochromeno[4,3-b][1,5]benzodiazepine (ZIMET 54/79) and its biologically inactive (-) enantiomer (ZIMET 55/79) interact with liposomal membranes. At pH values of 6.0 and 7.3 the long-wave absorption bands of these agents showed weak bathochromic and hypochromic effects upon addition of neutral, and positively and negatively charged phosphatidylcholine and phosphatidylcholine/cholesterol liposomes. Such spectral changes are interpreted as resulting from the binding of both agents to phospholipid bilayers. Steady-state determinations using the membrane probe 1-anilino-8-naphthalenesulfonic acid (1,8-ANS) led to the observation of a small decrease in fluorescence intensity in the presence of either agent. Time-resolved measurements demonstrate that the mechanism of action of the agents occurs mainly through the partial displacement of probe molecules from regions of hydrophobic binding to areas of greater solvent accessibility. No significant differences in binding between the cancerostatically active and inactive enantiomers with liposomes (archiral systems) were detectable on the basis of spectrophotometric and fluorescence determinations. Cell membrane bound adenylate cyclase is stimulated by ZIMET 54/79, resulting in an increase of 103% in the level of cAMP in mouse L1210 leukemia cells. On examination of structure-activity relationships, it was found that the biological activity (leukemia L1210, P388, Lewis lung carcinoma, melanoma B16, increase in cAMP) is correlated with the particular configuration (6aR,13aS) and type of substituent at positions 3 and 4 of the benzo ring in the case of alkoxy groups and positions 10 and 11 for methyl groups. No activity was detected toward DNA/RNA using microbial test systems.     

In vitro binding of an orally active platinum antitumor drug, JM216 to metallothionein

The in vitro binding of an orally active anticancer drug JM216 to metallothionein is firstly investigated in this paper. It is revealed that a redox reaction following a substitution reaction from JM216 with rabbit liver Zn₇MT-II is presented. The reaction feature, the metal binding stoichiometry and the oxidation states of platinum and sulfur in the products are studied by UV-visible, chromatography and X-ray photoelectron spectroscopy methods. Parts of MT are oxidized to precipitate products with intra and intermolecular CyS-SCy disulfides linkages. Pt(IV) is reduced to its Pt(II) counterpart. And the reduced Pt(II) replace the metal ions in native MTs. Meanwhile it can also cause the dimerization of MT. Increasing the reaction ratio of JM216 to MT leads to a concomitant increase in the apparent yield of the precipitate and dimeric products and the elevation of the binding stoichiometry of Pt to the protein. Based on the experimental data, the reaction mechanism between JM216 and Zn₇MT-II in vitro are discussed.     

The monotopic membrane protein human oxidosqualene cyclase is active as monomer

The monotopic integral membrane protein 2,3-oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol the first sterol precursor of cholesterol in mammals. Therefore, it is an important target for the development of new hypocholesterolemic drugs. Here, we report the overexpression and purification of functional human OSC (hOSC) in Pichia pastoris. The obtained IC(50) for the reference inhibitor Ro 48-8071 is nearly identical for the recombinant hOSC compared to OSC from human liver microsomes. The correlation of analytical ultracentrifugation data and activity measurements showed the highest enzymatic activity for the monomeric hOSC indicating that this would be the natural form. Furthermore, these data helped us to identify the detergent for a successful crystallization of the protein. The availability of this active recombinant human membrane protein is a very important step on the way to a more detailed functional and structural characterization of OSCs.     

Cellular effects of the antitumor drug aurumacryl

The cellular effects of aurumacryl (a drug based on gold polyacrylate, which previously showed significant antitumor activity in vivo against solid tumors in mice) were studied in the model of the MCF7 stable cell line of human breast carcinoma. It was found that aurumacryl possesses cytotoxic and cytostatic effects on tumor cells. The dose-dependent cytotoxic effect of aurumacryl is expressed as the death of 60% of tumor cells after incubation with aurumacryl at a dose of 1 mg/mL for 24 h. The proliferation kinetics of the surviving fraction of tumor cells also undergoes significant changes, which is expressed in the predominant accumulation of the cells (93%) in the G₀ phase of proliferative rest and in a significant decrease in the number of proliferating cells to 7%. These data could be interpreted as evidence of the loss of the reproductive ability of the surviving cells after treatment with aurumacryl.

     

Electroporation of red blood cell membrane and its use as a drug carrier system.

Application of external electric field to cell suspension maintained at ice temperature induces pores in the cell membrane. At this stage, a drug added to the cell suspension equilibrates across the membrane. On raising the temperature to 37 degrees C, the pores appear to be sealed as the drug is retained in the cells. This method was used for encapsulation of Co-57 labelled cynocobalamin in rabbit erythrocytes. It was seen from the in vivo biokinetic study of the drug-loaded erythrocytes that the rate of elimination of the drug was considerably reduced as compared to that of the free drug, indicating that drug delivery by electroencapsulation can give a sustained release of the drug.     

Atp-bound topoisomerase ii as a target for antitumor drugs

Topoisomerase II (TOP2) poisons interfere with the breakage/reunion reaction of TOP2 resulting in DNA cleavage. In the current studies, we show that two different classes (ATP-sensitive and -insensitive) of TOP2 poisons can be identified based on their differential sensitivity to the ATP-bound conformation of TOP2. First, in the presence of 1 mm ATP or the nonhydrolyzable analog adenosine 5'-(beta,gamma-imino)triphosphate, TOP2-mediated DNA cleavage induced by ATP-sensitive TOP2 poisons (e.g. doxorubicin, etoposide, mitoxantrone, and 4'-(9-acridinylamino)methanesulfon-m-anisidide) was 30-100-fold stimulated, whereas DNA cleavage induced by ATP-insensitive TOP2 poisons (e.g. amonafide, batracylin, and menadione) was only slightly (less than 3-fold) affected. In addition, ADP was shown to strongly antagonize TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive TOP2 poisons. Second, C427A mutant human TOP2alpha, which exhibits reduced ATPase activity, was shown to exhibit cross-resistance to all ATP-sensitive but not ATP-insensitive TOP2 poisons. Third, using ciprofloxacin competition assay, TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive poisons was shown to be antagonized by ciprofloxacin. These results suggest that ATP-bound TOP2 may be the specific target of ATP-sensitive TOP2 poisons. Using Lac repressor-operator complexes as roadblocks, we show that ATP-bound TOP2 acts as a circular clamp capable of entering DNA ends and sliding on unobstructed duplex DNA.     

Aspirin as a cancer-preventing drug

Aspirin suppresses the mutator phenotype associated with hereditary nonpolyposis colorectal cancer by genetic selectionRuschoff, J. et al. (1998)Proc. Natl. Acad. Sci. U. S. A. 95, 11301–11306     

In vitro effects of the antitumor drug miltefosine on human erythrocytes and molecular models of its membrane

This study was aimed at elucidating the molecular mechanisms of the interaction of the antitumor alkylphospholipid drug miltefosine with human erythrocytes (RBC) and molecular models of its membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. X-ray results showed that the drug interacted with DMPC multilayers; however, no effects on DMPE were detected. The experimental findings obtained by differential scanning calorimetry (DSC) indicated that miltefosine altered the thermotropic behavior of both DMPC and DMPE vesicles. Fluorescence spectroscopy evidenced an increase in the fluidity of DMPC vesicles and human erythrocyte membranes. Scanning electron microscopy (SEM) observations on human erythrocytes showed that miltefosine induced morphological alterations to RBC from its normal biconcave to an echinocyte type of shape. These results confirm that miltefosine interacts with the outer moiety of the human erythrocyte membrane affecting the cell morphology.     

DNA sequence recognition by the antitumor drug ditercalinium

The antitumor drug ditercalinium is a rare example of a noncovalent DNA-binding ligand that forms bisintercalation complexes via the major groove of the double helix. Previous structural studies have revealed that the two connected pyridocarbazolium chromophores intercalate into DNA with the positively charged bis(ethylpiperidinium) linking chain oriented to the wide groove side of the helix. Although the interaction of ditercalinium with short oligonucleotides containing 4-6 contiguous GC base pairs has been examined in detail by biophysical and theoretical approaches, the sequence preference for ditercalinium binding to long DNA fragments that offer a wide variety of binding sites has been investigated only superficially. Here we have investigated both sequence preferences and possible molecular determinants of selectivity in the binding of ditercalinium to DNA, primarily using methods based upon DNase I footprinting. A range of multisite DNA substrates, including several natural restriction fragments and different PCR-generated fragments containing unconventional bases (2,6-diaminopurine, inosine, uridine, 5-fluoro- and 5-methylcytosine, 7-deazaguanine, 7-deazaadenine, and N(7)-cyanoboranoguanine), have been employed to show that ditercalinium selectively recognizes certain GC-rich sequences in DNA and to identify some of the factors which affect its DNA-binding sequence selectivity. Specifically, the footprinting data have revealed that the 2-amino group on the purines or the 5-methyl group on the pyrimidines is not essential for the formation of ditercalinium-DNA complexes whereas the major groove-oriented N(7) of guanine does appear as a key element in the molecular recognition process. The loss of N(7) at guanines but not adenines is sufficient to practically abolish sequence-selective binding of ditercalinium to DNA. Thus, as expected for a major groove binding drug, the N(7) of guanine is normally required for effective complex formation with GC base pairs, but interestingly the substitution of the N(7) with a relatively bulky cyanoborane group does not markedly affect the sequence recognition process. Therefore, the hydrogen bond accepting capability at N(7) of guanines is not sufficient to explain the GC-selective drug-DNA association, and the implications of these findings are considered.