Marco Macchia

Inhibitors of lactate dehydrogenase isoforms and their therapeutic potentials.

In many different species, lactate dehydrogenase (LDH) constitutes a major checkpoint of anaerobic glycolysis, by catalyzing the reduction of pyruvate into lactate. This enzyme has recently received a great deal of attention since it may constitute a valid therapeutic target for diseases so different as malaria and cancer. In fact, the isoform expressed by Plasmodium falciparum (pfLDH) is a key enzyme for energy generation of malarial parasites. These species mostly depend on anaerobic glycolysis for energy production, since they lack a citric acid cycle for ATP formation. Therefore, inhibitors of pfLDH would potentially cause mortality of P. falciparum and, to this purpose, several small organic molecules have been recently designed and developed with the aim of blocking this new potential antimalarial chemotherapeutic target. Moreover, most invasive tumour phenotypes show a metabolic switch (Warburg effect) from oxidative phosphorylation to an increased anaerobic glycolysis, by promoting an upregulation of the human isoform-5 of lactate dehydrogenase (hLDH-5 or LDH-A), which is normally present in muscles and in the liver. Hence, inhibition of hLDH-5 may constitute an efficient way to interfere with tumour growth and invasiveness. This review provides an overview of the LDH inhibitors that have been developed up to now, an analysis of their possible isoform-selectivity, and their therapeutic potentials.

Estrogen receptor β ligands: Recent advances and biomedical applications

Recent work elucidating the role that the estrogen receptor β (ERβ), a member of the nuclear receptor superfamily, plays in regulating various physiological functions has highlighted the potential of this receptor subtype as a therapeutic target for several pathologies. In fact, molecules that are able to selectively activate ERβ hold promise for the treatment of certain cancers, as well as endometriosis, inflammatory diseases including rheumatoid arthritis, and cardiovascular and CNS conditions. Nevertheless, ERβ remains a challenging target because its ligand‐binding cavity is very similar to that present in ERα, and this makes it difficult to develop ligands having sufficient levels of ERβ selectivity for therapeutic use. Nevertheless, considerable advances have recently been made in developing both nonsteroidal and steroidal ERβ‐selective agonists. These molecules constitute not only important tools to probe the biological effects of the selective stimulation of ERβ, but some of them appear to be agents with considerable therapeutic potential. This study provides a detailed review of selective ERβ ligands that have been developed recently. After a brief introduction to the structure and nature of the two ERs and the biology of ERβ and its isoforms, the ligands are classified on the basis of their structures and activities. Common pharmacophore elements are highlighted throughout the description of the various chemical classes analyzed, and these elements are presented in a concluding summary overview along with a discussion of potential therapeutic applications of these agents in biomedicine.

Geranylgeraniol Overcomes the Block of Cell Proliferation by Lovastatin in C6 Glioma Cells

Abstract: It is well documented that 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitors prevent cultured mammalian cells from progressing through the cell cycle, suggesting a critical role for a mevalonate‐derived product. Recently, it has been shown that free geranylgeraniol (GG‐OH) and farnesol (F‐OH) can be utilized by C6 glioma cells for protein isoprenylation. The ability of GG‐OH and F‐OH to restore protein geranylgeranylation or farnesylation selectively has enabled us to examine the possibility that mevalonate is essential for cell proliferation because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the post‐translational modification of key regulatory proteins. In this study we report that GG‐OH, as well as mevalonate, overcomes the arrest of cell proliferation of C6 glioma cells treated with lovastatin, as assessed by increased cell numbers and a stimulation in [3H]thymidine incorporation. The increase in cell number and [3H]thymidine incorporation were significantly lower when F‐OH was added. Under these conditions [3H]mevalonate and [3H]GG‐OH are actively incorporated into a set of isoprenylated proteins in the size range of small, GTP‐binding proteins (19–27 kDa) and a polypeptide with the molecular size (46 kDa) of the smaller isoform of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase. Analysis of the proteins metabolically labeled by [3H]mevalonate and [3H]GG‐OH reveals the presence of labeled proteins containing geranylgeranylated cysteinyl residues. Consistent with geranylgeranylated proteins playing a critical role in the entry of C6 cells into the cell cycle, a (phosphonoacetamido)oxy derivative of GG‐OH, a drug previously shown to interfere with protein geranylgeranylation, prevented the increase in cell number when mevalonate or GG‐OH was added to lovastatin‐treated cells. These results strongly suggest that geranylgeranylated proteins are essential for progression of C6 cells into the S phase of the cell cycle and provide the first evidence that the “salvage” pathway for the utilization of the free isoprenols is physiologically significant in the CNS.

N6-isopentenyladenosine arrests tumor cell proliferation by inhibiting farnesyl diphosphate synthase and protein prenylation

The physiological effects of a variety of N6‐substituted adenine and adenosine derivatives called cytokinins have been documented in plants, but information on their occurrence and function in other biological system is limited. Here we investigated the anti‐proliferative effect of N6‐isopentenyladenosine (i6A), an adenosine and isoprenoid derivative, in a thyroid cell system, FRTL‐5 wild‐type, and K‐ras transformed KiMol cells. Addition of i6A to FRTL‐5 cells caused a dose‐dependent arrest of the G0‐G1 cell phase transition associated with a reduction of cells in the S phase that was much more evident in KiMol cells. I6A arrested tumor cell proliferation by inhibiting farnesyl diphosphate synthase (FPPS) and protein prenylation. Indeed the addition of farnesol reversed these effects and i6A affected protein prenylation, in particular lamin B processing. I6A effect was not mediated by the adenosine receptor but was due to a direct modulation of FPPS enzyme activity as a result of its uptake inside the cells. I6A inhibited FPPS activity more efficaciously in KiMol cells than in normal FRTL‐5. Moreover, the i6A anti‐proliferative effect was evaluated in vivo in a nude mouse xenograft model, where KiMol cells were implanted subcutaneously. Mice treated with i6A showed a drastic reduction in tumor volume. Our findings indicate that this isoprenoid end product might be used for antineoplastic therapy, an application emulating that of the lovastatin and/or farnesyltransferase inhibitors.—Laezza, C., Notarnicola, M., Caruso, M. G., Messa, C., Macchia, M., Bertini, S., Minutolo, F., Portella, G., Fiorentino, L., Stingo, S., Bifulco, M. N6‐isopentenyladenosine arrests tumor cell proliferation by inhibiting farnesyl diphosphate synthase and protein prenylation. FASEB J. 20, 412–418 (2006)

Use of β-cyclodextrin in the capillary zone electrophoretic separation of the components of clandestine heroin preparations

The present paper describes the methodological optimization and validation of a capillary zone electrophoresis method for the rapid determination of heroin, secondary products and additives present in clandestine heroin samples, by using 20 mM beta-cyclodextrins in phosphate buffer, pH 3.23. Applied potential was 15 kV and separation temperature was 24 degrees C; detection was by UV absorption at 200 nm wavelength. Heroin samples were first dissolved in CHCl3-MeOH (96:4, v/v) and injected by pressure (0.5 p.s.i., 3 s; 1 p.s.i.=6894.76 Pa) after evaporation of the organic mixture and reconstitution in aqueous buffer. Under the described conditions, phenylethylamine (internal standard), morphine, monoacetylmorphine, heroin, acetylcodeine, papaverine, codeine and narcotine were baseline resolved in less than 10 min. The limit of detection was better than 1 microg/ml for each analyte. The study of the intra-day and day-to-day precision showed, in terms of migration times, RSDs < or = 0.71% and, in terms of peak areas, RSDs < or = 3.2%. Also, the evaluation of linearity and analytical accuracy of the method provided good results for all the analytes investigated, thus allowing its application to real cases of seized controlled drug preparations.

Polyprenyl Phosphate Biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis

Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in M. tuberculosis are 10- to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in M. tuberculosis only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes omega,E,Z-farnesyl diphosphate and the other present in the cytosol that synthesizes omega,E,E,E-geranylgeranyl diphosphate. In M. smegmatis, the omega,E, Z-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the omega,E,E,E-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however, omega,E,E,E-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of M. smegmatis and M. tuberculosis can be attributed to distinct enzymatic differences between these two organisms.

Assessing the differential action on cancer cells of LDH-A inhibitors based on the N-hydroxyindole-2-carboxylate (NHI) and malonic (Mal) scaffolds

A head-to-head study of representative examples of N-hydroxyindole-2-carboxylates (NHI) and malonic derivatives (Mal) as LDH-A inhibitors was conducted, comparing the enzyme inhibition potency, cellular uptake, reduction of lactate production in cancer cells and anti-proliferative activity. Among the compounds tested, methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (2, NHI-2), a methyl ester belonging to the NHI class, displayed optimal properties in the cell-based assays, proving to be an efficient anti-glycolytic agent against cancer cells.

Structural Evolutions of Salicylaldoximes as Selective Agonists for Estrogen Receptor β

The bioisosteric replacement of the phenol ring, a signature functional group of most estrogen receptor (ER) ligands, with a hydrogen-bonded pseudocyclic ring, led to the development of a novel class of nonsteroidal ER-ligands based on a salicylaldoxime template. A series of structural modifications were applied to selected molecules belonging to the monoaryl-salicylaldoxime chemical class in an attempt to improve further their ERbeta-selective receptor affinity and agonist properties. Among several modifications, the best results were obtained by the simultaneous introduction of a meta-fluorine atom into the para-hydroxyphenyl substituent present in the 4-position of salicylaldoxime, together with the insertion of a chloro group in the 3-position of the central scaffold. The resulting compound showed the best affinity (K(i) = 7.1 nM) and selectivity for ERbeta over ERalpha. Moreover, in transcription assays, it proved to be a selective and potent ERbeta-full agonist with an EC(50) of 4.8 nM.

Synthesis and antiviral properties of 9-[(2-methyleneaminoxyethoxy)methyl]guanine derivatives as novel Acyclovir analogues.

This paper reports the synthesis and the antiviral properties of a series of 9-[(2-methyleneaminoxyethoxy)methyl]guanine derivatives, which can be viewed as analogues of the antiherpes drug Acyclovir (ACV) from which they differ in the replacement of its hydroxy group with variously substituted methyleneaminoxy moieties. Some of the newly synthesized compounds proved to possess a certain activity against HSV-1, albeit lower than that of ACV.

Dual Targeting of the Warburg Effect with a Glucose‐Conjugated Lactate Dehydrogenase Inhibitor

Effective glucose diet: We report the development and activity of glucose-conjugated LDH-A inhibitors designed for dual targeting of the Warburg effect (elevated glucose uptake and glycolysis) in cancer cells. Glycoconjugation could be applied to inhibitors of many enzymes involved in glycolysis or tumor metabolism.