María C. Núñez

Choline kinase inhibitors as a novel approach for antiproliferative drug design

Recent progress in deciphering the molecular basis of carcinogenesis is of utmost importance to the development of new anticancer strategies. To this end, it is essential to understand the regulation of both normal cell proliferation and its alterations in cancer cells. We have previously demonstrated that in ras-transformed cells there is an increased level of phosphorylcholine (PCho) resulting from a constitutive activation on choiline kinase (ChoK). The importance of ChoK for the regulation of cell proliferation has also been proposed since an inhibitor for this enzyme, hemicholinium-3 (HC-3), drastically reduces entry into the S phase after stimulation with growth factors. Here we report the synthesis of several new compounds which are highly specific inhibitors for ChoK, with up to 1000-fold or 600-fold increased inhibitory activity, compared to HC-3 under ex vivo or in vitro conditions respectively. These novel compounds also drastically reduce entry into the S phase after stimulation with specific growth factors. A more profound inhibition of cell proliferation was observed in ras-, src- and mos-transformed cells in the presence of ChoK inhibitors, compared to their parental, untransformed NIH3T3 cells. By contrast, this effect was not observed in fos-transformed cells. While ras, src and mos transformation is associated with elevated levels of ChoK activity, fos-induced transformation does not affect ChoK activity. The inhibitory effect on proliferation of the new compounds correlates with their ability to inhibit the production of phosphorylcholine in whole cells, a proposed novel second messenger for cell proliferation. These results strongly support a critical role of choline kinase in the regulation of cell growth and makes this enzyme a novel target for the design of new antiproliferative and anticancer drugs.

Inhibition of choline kinase as a specific cytotoxic strategy in oncogene-transformed cells.

Cancer treatment is in the need of selective drugs that can interfere specifically with signalling pathways affected during the carcinogenic process. Identification of new potential molecular targets is the key event in the design of new anticancer strategies. Once identified, attempts for the generation of specific molecules to regulate their function can be achieved. The relevance of deregulation of choline kinase (ChoK, E.C. 2.7.1.32) in oncogene-driven cell transformation has been previously demonstrated. Here we provide strong evidence that MN58b, a selective inhibitor of ChoK, is rather specific to this enzyme, with no effect on a variety of oncogene-activated signalling pathways involved in the regulation of cell proliferation. MN58b does not affect MAPKs, PI3K, and other enzymes involved in the regulation of phospholipid metabolism such as phospholipases C, D, and A2, CTP:phosphocholine cytidylyltransferase, or diacylglycerol choline-phosphotransferase. Consistent with this specificity, ectopic expression of ChoK resulted in resistance to its inhibitor. Finally, nontransformed cells were able to resume cell proliferation after removal of the drug, while transformed cells were irreversibly affected. These results indicate that inhibition of ChoK is a rather specific strategy for the cytotoxic treatment of transformed cells.

Homochiral Drugs: A Demanding Tendency of the Pharmaceutical Industry

The issue of drug chirality is now a major theme in the design and development of new drugs, underpinned by a new understanding of the role of molecular recognition in many pharmacologically relevant events. In general, three methods are utilized for the production of a chiral drug: the chiral pool, separation of racemates, and asymmetric synthesis. Although the use of chiral drugs predates modern medicine, only since the 1980s has there been a significant increase in the development of chiral pharmaceutical drugs. An important commercial reason is that as patents on racemic drugs expire, pharmaceutical companies have the opportunity to extend patent coverage through development of the chiral switch enantiomers with desired bioactivity. Stimulated by the new policy statements issued by the regulatory agencies, the pharmaceutical industry has systematically begun to develop chiral drugs in enantiometrically enriched pure forms. This new trend has caused a tremendous change in the industrial small- and large-scale production to enantiomerically pure drugs, leading to the revisiting and updating of old technologies, and to the development of new methodologies of their large-scale preparation (as the use of stereoselective syntheses and biocatalyzed reactions). The final decision whether a given chiral drug will be marketed in an enantiomerically pure form, or as a racemic mixture of both enantiomers, will be made weighing all the medical, financial and social proficiencies of one or other form. The kinetic, pharmacological and toxicological properties of individual enantiomers need to be characterized, independently of a final decision.

Novel Substituted Quinazolines for Potent EGFR Tyrosine Kinase Inhibitors

The type I receptor tyrosine kinases (RTKs) are involved in various aspects of cell growth, survival, and differentiation. Among the known RTKs, the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2) are two widely studied proteins that are prototypic members of the ErbB family which also includes ErbB-3 (Her-3) and ErbB-4 (Her-4). Overexpression of ErbB-2 and EGFR has been associated with aggressive disease and poor patient prognosis in a range of human tumour types (e.g. breast, lung, ovarian, prostate, and squamous carcinoma of head and neck). Disruption of signal transduction of these kinases has been shown to have an antiproliferative effect. Various approaches have been developed to target the ErbB signalling pathways including monoclonal antibodies (trastuzumab/Herceptin™ and cetuximab/Erbitux™) directed against the receptor, and synthetic tyrosine kinase inhibitors (gefitinib/Iressa™ and erlotinib/Tarceva™). Since many tumours overexpress ErbB receptors, simultaneous targeting of multiple ErbB receptors therefore becomes a promising approach to cancer treatment. Lapatinib (Tykerb™), a potent dual EGFR/ErbB-2 inhibitor, was approved for the treatment of ErbB-2-positive breast cancer. Despite years of intensive research on EGFR inhibitors, there is a surprising dearth of chemically distinct small inhibitors with a high degree of selectivity. There is also a need for new scaffolds due to the recent finding of EGFR mutations which render the kinase resistant to gefinitib and erlotinib. The structures under study will be quinazolines with different substituents. The structure-activity relationships and biological evaluation of compounds published during the last four years will be reviewed herein.

Homodimeric bis-quaternary heterocyclic ammonium salts as potent acetyl- and butyrylcholinesterase inhibitors: a systematic investigation of the influence of linker and cationic heads over affinity and selectivity.

A molecular library of quaternary ammonium salts (QASs), mainly composed of symmetrical bis-quaternary heterocyclic bromides exhibiting choline kinase (ChoK) inhibitory activity, were evaluated for their ability to inhibit acetyl- and butyrylcholinesterase (AChE and BChE, respectively). The molecular framework of QASs consisted of two positively charged heteroaromatic (pyridinium or quinolinium) or sterically hindered aliphatic (quinuclidinium) nitrogen rings kept at an appropriate distance by lipophilic rigid or semirigid linkers. Many homodimeric QASs showed AChE and BChE inhibitory potency in the nanomolar range along with a low enzymatic selectivity. Computational studies on AChE, BChE, and ChoK allowed identification of the key molecular determinants for high affinity and selectivity over either one of the three enzymes and guided the design of a hybrid bis-QAS (56) exhibiting the highest AChE affinity (IC(50) = 15 nM) and selectivity over BChE and ChoK (SI = 50 and 562, respectively) and a promising pharmacological potential in myasthenia gravis and neuromuscular blockade.

QSAR of 1,1'-(1,2-ethylenebisbenzyl)bis(4-substitutedpyridinium) dibromides as choline kinase inhibitors: a different approach for antiproliferative drug design.

Ten new structures of a series of the title compounds were synthesized and screened for their activity to inhibit choline kinase under ex vivo conditions. Their inhibitory potency correlates with the 13C chemical shifts (in CD3OD) of the methylene group bearing the positively charged nitrogen. The inhibitory effect on proliferation against the HT-29 cell line is strongly dependent on its ability to inhibit the production of phosphorylcholine.

Synthesis and anticancer activity of (R,S)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purines.

A series of eleven 2‐ and 6‐substituted (R,S)‐9‐(2,3‐dihydro‐1,4‐benzoxathiin‐3‐ylmethyl)‐9H‐purine derivatives was obtained by applying a standard Mitsunobu protocol that led to a six‐membered ring contraction from (R,S)‐3,4‐dihydro‐2H‐1,5‐benzoxathiepin‐3‐ol via an episulfonium intermediate. The signal ∼δ=151 ppm, which corresponds to the C4′ carbon atom, is unequivocal proof of the N9′ regioisomer. The potential of the target molecules as anticancer agents is reflected in their activity against the MCF‐7 cancer cell line. The most active compounds have IC50 values of (6.18±1.70) and (8.97±0.83) μM. The results indicate that the anticancer activity for the most active compounds is correlated with their capacity to induce apoptosis.

Design, synthesis and anticancer activity against the MCF-7 cell line of benzo-fused 1,4-dihetero seven- and six-membered tethered pyrimidines and purines.

Having previously reported the synthesis and anticancer activities of cyclic 5-fluorouracil (5-FU) O,N-acetalic compounds, the decision was made to change 5-FU for uracil (U), with the prospect of finding an antiproliferative agent endowed with a new mechanism of action. The use of a reverse transcription-PCR-based assay decreased cyclin D1 mRNA, suggesting that this cyclic U O,N-acetalic compound exerts its regulatory action on cyclin D1 at the level of transcription. Following the ongoing Anticancer Drug Programme we planned the synthesis of compounds bearing a natural pyrimidine base and also, the oxygen atom at position 1 of the seven-membered cycle was replaced by its isosteric sulfur atom, and its oxidized states. Next, the pyrimidine base was substituted for the purine one, with the objective of increasing both the lipophilicity and the structural diversity of the target molecules. If the previously described compounds were not prodrugs, it would not be necessary to maintain the O,N-acetalic characteristic. Therefore, molecules were designed in which both structural entities (such as the benzoheterocyclic ring and the purine base) were linked by a heteroatom-C-C-N bond. A series of (RS)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purine derivatives was obtained and the anticancer activity for the most active compounds was correlated with their capability to induce apoptosis. Finally, completing a SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H- or 9H-purines was prepared. The studies by microarray technology showed that the main molecular targets of some of these compounds are pro-apoptotic genes with protein kinase activity such as GP132, ERN1 or RAC1, which prevent the metastatic progression.

Enantiospecific Synthesis of Heterocycles Linked to Purines: Different Apoptosis Modulation of Enantiomers in Breast Cancer Cells

The issue of chiral drug is now a major theme in the design, discovery and development of new drugs. It has been shown for many pharmaceuticals that only one enantiomer contains the desired activity, and the synthesis of such drug molecules in their optically pure form is becoming increasingly important. Mitsunobu reaction was carried out between (R)- and (S)-3,4-dihydro-2H-1,5-benzoxathiepin-3-ol and purines under microwave irradiation. A contraction into a six-membered ring takes place with concomitant inversion at the stereocentre with excellent enatiomeric excesses giving rise to the homochiral 9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purines. The anti-tumour activity of all enantiomers is reported against the caspase-3-deficient MCF-7 and the wild type SKBR-3 human breast cancer cells. The most active homochiral compound displays an IC50 of 1.85 μM and induces inhibition of the translation initiation factor eIF2α. All homochiral compounds included in this study show different apoptotic effects between both enantiomers with levels up to 99%. We have analyzed caspase-mediated apoptotic pathways on enantiomers and racemates. We have found a homochiral derivative that activates the canonical intrinsic caspase-8/caspase-3 apoptotic pathway on the MCF-7 cells, and a racemic compound that induces caspase-2 activation. Moreover, we demonstrate the involvement of caspase activation during cell death induced by these compounds in SKBR-3 cells.

Distamycin A as Stem of DNA Minor Groove Alkylating Agents

Analogues of naturally occurring antitumor agents, such as distamycin A, which bind in the minor groove of DNA, represent a new class of anticancer compounds currently under investigation. Distamycin A has driven researchers attention not only for their biological activity, but also for its non intercalative binding to the minor groove of double-stranded B-DNA, where it forms strong reversible complex preferentially at the nucleotide sequences consisting of 4-5 adjacent AT base pairs. The pyrrole-amide skeleton of distamycin A has been also used as DNA sequence selective vehicles for the delivery of alkylating functions to DNA targets, leading to a sharp increase of its cytotoxicity, in comparison to that, very weak, of distamycin itself. In the last few years, several hybrid compounds, in which known antitumor derivatives or simple active moieties of known antitumor agents have been tethered to distamycin frames, have been designed, synthesized and tested. Several efforts have been made to modify DNA sequence selectivity and stability of the distamycin and the structural modifications have been based on replacement of pyrrole by other heterocycles and/or benzoheterocycles obtaining a novel class of minor groove binding molecules called lexitropsins. The role of the amidino moiety, by means of the substitution with various groups, which includes ionizable, acid or basic, and non-ionizable groups, has been also studied. The synthesis of a hybrid deriving among the combination of the distamycin A and naturally occurring alkylating agent has been also reported. Several classes of distamycin derivatives that have been reported in the published literature have been described in this review article.