Synergistic interaction of novel lactate dehydrogenase inhibitors with gemcitabine against pancreatic cancer cells in hypoxia.

BACKGROUND: Hypoxia is a driving force in pancreatic-ductal-adenocarcinoma (PDAC) growth, metastasis and chemoresistance. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis, ensuring supply of energy and anabolites in hypoxic-environments. Therefore, we investigated the molecular mechanisms underlying the pharmacological interaction of novel LDH-A inhibitors in combination with gemcitabine in PDAC cells. METHODS: Lactate dehydrogenase A levels were studied by quantitative RT-PCR, western blot, immunofluorescence and activity assays in 14 PDAC cells, including primary-cell-cultures and spheroids, in normoxic and hypoxic conditions. Cell proliferation, migration and key determinants of drug activity were evaluated by sulforhodamine-B-assay, wound-healing assay, PCR and LC-MS/MS. RESULTS: Lactate dehydrogenase A was significantly increased under hypoxic conditions (1% O2), where the novel LDH-A inhibitors proved to be particularly effective (e.g., with IC50 values of 0.9 vs 16.3 µM for NHI-1 in LPC006 in hypoxia vs normoxia, respectively). These compounds induced apoptosis, affected invasiveness and spheroid-growth, reducing expression of metalloproteinases and cancer-stem-like-cells markers (CD133+). Their synergistic interaction with gemcitabine, with combination index values <0.4 in hypoxia, might also be attributed to modulation of gemcitabine metabolism, overcoming the reduced synthesis of phosphorylated metabolites. CONCLUSION: Lactate dehydrogenase A is a viable target in PDAC, and novel LDH-A inhibitors display synergistic cytotoxic activity with gemcitabine, offering an innovative tool in hypoxic tumours.

QSAR models for predicting enzymatic hydrolysis of new chemical entities in 'soft-drug' design.

The work described here is aimed at developing QSAR models capable of predicting in vitro human plasma lability/stability. They were built based on a dataset comprising about 200 known compounds. 3D structures of the molecules were drawn, optimized and submitted to the calculation of molecular descriptors that enabled selecting different TR/TS set pairs, subsequently exploited to develop QSAR models. Several 'machine learning' algorithms were explored in order to obtain suitable classification models, which were then validated on the relevant TS sets. Moreover the predictive ability of the best performing models was assessed on a Prediction set (PS) comprising about 40 molecules, not strictly related, from a structural point of view, to the initial dataset, but (obviously) comprised within the validity domain of the QSAR models obtained. The study allowed selecting predictive models enabling the classification of New Chemical Entities with regard to hydrolysis rate, that may be exploited for soft-drug design.

Anticancer agents interacting with membrane glucose transporters.

The altered metabolism observed in cancer cells generally consists in increased glucose uptake and glycolytic activity. This is associated with an overexpression of glucose transporter proteins (GLUTs), which facilitate glucose uptake across the plasma membrane and play a crucial role in the survival of cancer cells. Therefore GLUTs are considered as suitable targets for the treatment of cancer. Herein we review some of the most relevant GLUT inhibitors that have been recently developed as prospective anticancer agents.

Salicylaldoxime moiety as a phenolic "A-Ring" substitute in estrogen receptor ligands.

The phenolic "A-ring" of natural and synthetic estrogen receptor (ER) ligands was effectively replaced by a planar six-member ring formed through an intramolecular hydrogen bond within a salicylaldoxime. Thus, oxime 1, a structural analogue of a triarylethylene estrogen, showed a significant binding affinity for the ER. The OH of the oxime function appears to mimic the phenolic OH present in more "classical" ER ligands because the binding reduced when the oxime OH is methylated (2) or absent (3).

Design, synthesis, and characterization of the antitumor activity of novel ceramide analogues.

A deficiency in apoptosis is one of the key events in the proliferation and resistance of malignant cells to antitumor agents; for these reasons, the search for apoptosis-inducing drugs represents a valuable approach for the development of novel anticancer therapies. In this study we report the first example of conformationally restrained analogues of ceramide (compounds 1-4), where the polar portion of the molecule has been replaced by a thiouracil (1, 3) or uracil (2, 4) ring. The evaluation of their biologic activity on CCRF-CEM human leukemia cells demonstrated that the most active was compound 1 followed by compound 2 (mean 50% inhibition of cell proliferation [IC(50)] 1.7 and 7.9 microM, respectively), while compounds 3 and 4 were inactive, as were uracil, thiouracil, and 5,6-dimethyluracil, the pyrimidine moieties of compounds 1-4. For comparison, the IC(50) of the reference substance, the cell-permeable C2-ceramide, was 31.6 microM. Compounds 1 and 2 and C2-ceramide were able to trigger apoptosis, as shown by the occurrence of DNA and nuclear fragmentation, and to release cytochrome c from treated cells. The treatment of female CD-1 nu/nu athymic mice bearing a WiDr human colon xenograft with the most active compound 1 at 2, 10, 50, and 200 mg/kg ip daily for 10 days resulted in an antitumor effect that was equivalent at 50 mg/kg or superior (200 mg/kg) to that of cyclophosphamide, 20 mg/kg ip daily, delivered on the same schedule, with markedly lower systemic toxicity. In conclusion, the present study demonstrates that the new ceramide analogues 1 and 2 are characterized by in vitro and in vivo antitumor activity and low toxicity.

Resveratrol structure and ceramide-associated growth inhibition in prostate cancer cells.

Resveratrol (3,4',5-trans-trihydroxystilbene) is a dietary polyphenol with chemopreventive properties present in grapes, red wine, peanuts and other edible products. The antiproliferative and proapoptotic effect of resveratrol in breast cancer cells can be traced to the accumulation of ceramide. In this study we demonstrate that resveratrol can also exert antiproliferative/proapoptotic effects in association with the accumulation of endogenous ceramide in the androgen receptor (AR)-negative prostate cancer cell line, PC3. Notably, resveratrol shares with other ceramide-inducing agents a phenolic moiety on its structure. For this reason we hypothesize that the phenolic moiety is critical for the ceramide-associated growth-inhibitory effects of resveratrol. We compared the ability to induce both ceramide increase and growth inhibition in PC3 cells of resveratrol and three resveratrol analogs: piceatannol (3,3',4',5-trans-tetrahydroxystilbene), with an additional hydroxyl group in the 3' position; trans-stilbene, the nonhydroxylated analog; and the semisynthetic 3,4',5-trimethoxy-trans-stilbene (TmS), with methoxyl groups in lieu of the hydroxyl groups. Of the three stilbenoids, only piceatannol (and not stilbene or TmS) produced ceramide-associated growth inhibition. These data point to the phenolic moiety of stilbenoids as a critical structural feature necessary to induce ceramide-associated growth inhibition.

Synthesis and antiviral properties of novel analogues of monophosphate and diphosphate bioactive forms of acyclovir.

New analogues (compounds 6, 7 and 9) of the mono- (8) and diphosphate (10) bioactive forms of the antiherpes drug acyclovir are described. In compound 6, the monophosphate moiety of 8 was replaced by an aminosulfonyloxy group, while in compounds 7 and 9, a phosphonoacetamidoxy and an O-ethyl phosphonoacetamidoxy moiety are, respectively present instead of the diphosphate one of 10. None of the compounds synthesized proved to possess an appreciable activity on herpes simplex virus (HSV) or human immunodeficiency virus (HIV).

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.

Identification of LDH-A as a therapeutic target for cancer cell killing via (i) p53/NAD(H)-dependent and (ii) p53-independent pathways.

Most cancer cells use aerobic glycolysis to fuel their growth. The enzyme lactate dehydrogenase-A (LDH-A) is key to cancer's glycolytic phenotype, catalysing the regeneration of nicotinamide adenine dinucleotide (NAD(+)) from reduced nicotinamide adenine dinucleotide (NADH) necessary to sustain glycolysis. As such, LDH-A is a promising target for anticancer therapy. Here we ask if the tumour suppressor p53, a major regulator of cellular metabolism, influences the response of cancer cells to LDH-A suppression. LDH-A knockdown by RNA interference (RNAi) induced cancer cell death in p53 wild-type, mutant and p53-null human cancer cell lines, indicating that endogenous LDH-A promotes cancer cell survival irrespective of cancer cell p53 status. Unexpectedly, however, we uncovered a novel role for p53 in the regulation of cancer cell NAD(+) and its reduced form NADH. Thus, LDH-A silencing by RNAi, or its inhibition using a small-molecule inhibitor, resulted in a p53-dependent increase in the cancer cell ratio of NADH:NAD(+). This effect was specific for p53(+/+) cancer cells and correlated with (i) reduced activity of NAD(+)-dependent deacetylase sirtuin 1 (SIRT1) and (ii) an increase in acetylated p53, a known target of SIRT1 deacetylation activity. In addition, activation of the redox-sensitive anticancer drug EO9 was enhanced selectively in p53(+/+) cancer cells, attributable to increased activity of NAD(P)H-dependent oxidoreductase NQO1 (NAD(P)H quinone oxidoreductase 1). Suppressing LDH-A increased EO9-induced DNA damage in p53(+/+) cancer cells, but importantly had no additive effect in non-cancer cells. Our results identify a unique strategy by which the NADH/NAD(+) cellular redox status can be modulated in a cancer-specific, p53-dependent manner and we show that this can impact upon the activity of important NAD(H)-dependent enzymes. To summarise, this work indicates two distinct mechanisms by which suppressing LDH-A could potentially be used to kill cancer cells selectively, (i) through induction of apoptosis, irrespective of cancer cell p53 status and (ii) as a part of a combinatorial approach with redox-sensitive anticancer drugs via a novel p53/NAD(H)-dependent mechanism.

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.

Study of apoptosis induction and deoxycytidine kinase/cytidine deaminase modulation in the synergistic interaction of a novel ceramide analog and gemcitabine in pancreatic cancer cells.

This study investigated the interaction between the novel ceramide analog AL6 and gemcitabine in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines, harboring different polymorphic variants of the gemcitabine catabolism enzyme cytidine deaminase (CDA). AL6 dose-dependently inhibited cell growth, induced apoptosis and synergistically enhanced the cytotoxic activity of gemcitabine. Moreover, it triggered apoptosis, which was significantly enhanced by the combination, and increased the ratio between gene expression of the activating enzyme deoxycytidine kinase (dCK) and CDA, potentially favoring gemcitabine activity. In conclusion, AL6 displays synergistic cytotoxic activity, enhances apoptosis, and favorably modulates enzymes involved in gemcitabine metabolism, supporting future investigation of this combination in pancreatic cancer.

Metabolically labile cannabinoid esters: a 'soft drug' approach for the development of cannabinoid-based therapeutic drugs.

Biphenylic ester derivatives, designed by using a 'soft-drug' approach, proved to possess good binding properties toward cannabinoid CB(1) and CB(2) receptors and, at the same time, their metabolically labile ester portion would promote a rapid systemic inactivation. This may constitute a possible solution to the psychotropic side effects encountered when cannabinoids are therapeutically employed as local analgesic or antiglaucoma agents.

Crystallographic evidence for the electronic distribution in (2,4-cyclopentadien-1-yl-idenehydrazono)triphenylphosphorane.

The title compound, C23H19N2P, can be graphically represented by several canonical forms. Its crystal structure analysis shows a clear bond alternation in the cyclopentadiene ring, which continues in the azo substructure, indicating that the resonance form containing the nonaromatic neutral cyclopentadienylidene moiety describes the actual hybrid form better than other 'inner ionic' resonance forms containing the aromatic anionic cyclopentadienylic portion. The preference for an s-transoid (E) geometry for the P1-N1-N2-C1 substructure was also confirmed over the other possible s-cisoid (Z) conformer.

Synthesis and dopaminergic properties of the two enantiomers of 3-(3,4-dimethylphenyl)-1-propylpiperidine, a potent and selective dopamine D4 receptor ligand.

The synthesis of the two enantiomers of 3-(3,4-dimethylphenyl)-1-propylpiperidine 1, a potent and selective D4 dopaminergic ligand, was performed. The 3-(3,4-dimethylphenyl)- 1-propylpiperidine with the R configuration showed an affinity for the D4 receptors 6-fold higher than the corresponding enantiomer with the S configuration. Furthermore, the (R)-1 enantiomer proved to be highly selective for D4 receptors with respect to D2-D3 receptors, with a Ki ratio higher than 25,000, while the (S)-1 enantiomer was about 100-fold less selective than the (R)-1 one.

Purification, enzymatic characterization, and inhibition of the Z-farnesyl diphosphate synthase from Mycobacterium tuberculosis.

We have recently shown that open reading frame Rv1086 of the Mycobacterium tuberculosis H37Rv genome sequence encodes a unique isoprenyl diphosphate synthase. The product of this enzyme, omega,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinomannan. We have now purified Z-farnesyl diphosphate synthase to near homogeneity using a novel mycobacterial expression system. Z-Farnesyl diphosphate synthase catalyzed the addition of isopentenyl diphosphate to omega,E-geranyl diphosphate or omega,Z-neryl diphosphate yielding omega,E,Z-farnesyl diphosphate and omega,Z,Z-farnesyl diphosphate, respectively. The enzyme has an absolute requirement for a divalent cation, an optimal pH range of 7-8, and K(m) values of 124 micrometer for isopentenyl diphosphate, 38 micrometer for geranyl diphosphate, and 16 micrometer for neryl diphosphate. Inhibitors of the Z-farnesyl diphosphate synthase were designed and chemically synthesized as stable analogs of omega,E-geranyl diphosphate in which the labile diphosphate moiety was replaced with stable moieties. Studies with these compounds revealed that the active site of Z-farnesyl diphosphate synthase differs substantially from E-farnesyl diphosphate synthase from pig brain (Sus scrofa).

7-Nitrobenzofurazan (NBD) derivatives of 5'-N-ethylcarboxamidoadenosine (NECA) as new fluorescent probes for human A(3) adenosine receptors.

New fluorescent ligands for adenosine receptors (ARs), obtained by the insertion, in the N(6) position of NECA, of NBD-moieties with linear alkyl spacers of increasing length, proved to possess a high affinity and selectivity for the A(3) subtype expressed in CHO cells. In fluorescence microscopy assays, compound 2d, the most active and selective for human A(3)-AR, permitted visualization and localization of this human receptor subtype, showing its potential suitability for internalization and trafficking studies in living cells.