Different design of enzyme-triggered CO-releasing molecules (ET-CORMs) reveals quantitative differences in biological activities in terms of toxicity and inflammation.

Acyloxydiene-Fe(CO)3 complexes can act as enzyme-triggered CO-releasing molecules (ET-CORMs). Their biological activity strongly depends on the mother compound from which they are derived, i.e. cyclohexenone or cyclohexanedione, and on the position of the ester functionality they harbour. The present study addresses if the latter characteristic affects CO release, if cytotoxicity of ET-CORMs is mediated through iron release or inhibition of cell respiration and to what extent cyclohexenone and cyclohexanedione derived ET-CORMs differ in their ability to counteract TNF-α mediated inflammation. Irrespective of the formulation (DMSO or cyclodextrin), toxicity in HUVEC was significantly higher for ET-CORMs bearing the ester functionality at the outer (rac-4), as compared to the inner (rac-1) position of the cyclohexenone moiety. This was paralleled by an increased CO release from the former ET-CORM. Toxicity was not mediated via iron as EC50 values for rac-4 were significantly lower than for FeCl2 or FeCl3 and were not influenced by iron chelation. ATP depletion preceded toxicity suggesting impaired cell respiration as putative cause for cell death. In long-term HUVEC cultures inhibition of VCAM-1 expression by rac-1 waned in time, while for the cyclohexanedione derived rac-8 inhibition seems to increase. NFκB was inhibited by both rac-1 and rac-8 independent of IκBα degradation. Both ET-CORMs activated Nrf-2 and consequently induced the expression of HO-1. This study further provides a rational framework for designing acyloxydiene-Fe(CO)3 complexes as ET-CORMs with differential CO release and biological activities. We also provide a better understanding of how these complexes affect cell-biology in mechanistic terms.

Enzyme-triggered CO-releasing molecules (ET-CORMs): evaluation of biological activity in relation to their structure.

Acyloxydiene-Fe(CO)3 complexes act as enzyme-triggered CO-releasing molecules (ET-CORMs) and can deliver CO intracellularly via esterase-mediated hydrolysis. The protective properties of structurally different ET-CORMs on hypothermic preservation damage and their ability to inhibit VCAM-1 expression were tested on cultured human umbilical vein endothelial cells (HUVEC) and renal proximal tubular epithelial cells (PTEC) using a structure-activity approach. Cytotoxicity of ET-CORMs, protection against hypothermic preservation damage, and inhibition of VCAM-1 expression were assessed. Cytotoxicity of 2-cyclohexenone and 1,3-cyclohexanedione-derived ET-CORMs was more pronounced in HUVEC compared to PTEC and was dependent on the position and type of the ester (acyloxy) substituent(s) (acetate>pivalate>palmitate). Protection against hypothermic preservation injury was only observed for 2-cyclohexenone-derived ET-CORMs and was not mediated by the ET-CORM decomposition product 2-cyclohexenone itself. Structural requirements for protection by these ET-CORMs were different for HUVEC and PTEC. Protection was affected by the nature of the ester functionality in both cell lines. VCAM-1 expression was inhibited by both 2-cyclohexenone- and 1,3-cyclohexanedione-derived ET-CORMs. 2-Cyclohexenone, but not 1,3-cyclohexanedione, also inhibited VCAM-1 expression. We demonstrate that structural alterations of ET-CORMs significantly affect their biological activity. Our data also indicate that different ET-CORMs behave differently in various cell types (epithelial vs endothelial). These findings warrant further studies not only to elucidate the structure-activity relation of ET-CORMs in mechanistic terms but also to assess if structural optimization will yield ET-CORMs with restricted cell specificity.

Lipophilic prodrugs of a triazole-containing colchicine analogue in liposomes: biological effects on human tumor cells.

Colchicine site binders--blockers of tubulin polymerization--are potential antimitotic agents for anticancer therapy. To reduce their systemic toxicity and improve biodistribution, encapsulation in nanosized liposomes may be employed. Liposomes present a convenient means for preparation of injectable formulations of hydrophobic compounds, however colchicine as such is known to leak through the lipid bilayer. In this study, newly synthesized triazole-containing analogues of colchicine and allocolchicine, and their palmitic and oleic esters (lipophilic prodrugs) were tested for anti-proliferative activity and apoptosis-inducing potential. In contrast to colchicine conjugates, whose activities ranged with those of colchicine, allocolchicine derivatives exhibited drastically lower effects and were discarded. Liposomes of about 100 nm in diameter composed of egg phosphatidylcholine--yeast phosphatidylinositol--palmitic or oleic prodrug, 8 : 1: 1, by mol, were prepared by standard extrusion technique and tested in a panel of four human tumor cell lines. Liposome formulations preserved the biological activities of the parent colchicinoid the most towards human epithelial tumor cells. Moreover, liposomal form of the oleoyl bearing colchicinoid inhibited cell proliferation more efficiently than free lipophilic prodrug. Due to substantial loading capacity of the liposomes, the dispersions contain sufficient concentration of the active agent to test wide dose range in experiments on systemic administration to animals.

Proline-rich sequence recognition domains (PRD): ligands, function and inhibition.

Low-affinity protein-protein interactions (PPI) between domains of modular proteins and short, solvent-exposed peptide sequences within their binding partners play an essential role in intracellular signaling. An important class of PPIs comprises proline-rich motifs (PRM) that are specifically recognized by PRM-binding domains (PRD). Aromatic side chains of the PRDs define the binding pockets that often recognize individual proline residues, while flanking sequences mediate specificity. Several of these PRM:PRD interactions are associated with cellular malfunction, cancer or infectious diseases. Thus, the design of PRM:PRD inhibitors by using structure-based molecular modeling as well as peptidomimetic approaches and high-throughput screening strategies is of great pharmacological interest. In this chapter we describe the molecular basis of PRM:PRD interactions, highlight their functional role in certain cellular processes and give an overview of recent strategies of inhibitor design.

Gram-scale synthesis of pinusolide and evaluation of its antileukemic potential.

Pinusolide (1), a known platelet-activating factor (PAF) receptor binding antagonist, was synthesized from lambertianic acid (2), a labdane-type diterpene readily accessible in multigram quantities from the Siberian pine tree. It was shown that 1 not only decreases the proliferation activity of tumor cells at relatively low concentrations but specifically induces apoptosis at 100 microM via the mitochondrial pathway in the Burkitt lymphoma cell line BJAB. Also, using primary lymphoblasts and leukemic cells from children with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), a significant DNA fragmentation in pinusolide-treated cells could be detected in an ex vivo apoptosis assay.

Piceatannol, a hydroxylated analog of the chemopreventive agent resveratrol, is a potent inducer of apoptosis in the lymphoma cell line BJAB and in primary, leukemic lymphoblasts.

The stilbene phytochemicals resveratrol and piceatannol have been reported to possess substantial antitumorigenic and antileukemic activities, respectively. Although recent experimental data revealed the proapoptotic potency of resveratrol, the molecular mechanisms underlying the antileukemic activity have not yet been studied in detail. In the present study, we show that resveratrol, as well as the hydroxylated analog piceatannol, are potent inducers of apoptotic cell death in BJAB Burkitt-like lymphoma cells with an ED50 concentration of 25 microM. Further experiments revealed that treatment of BJAB cells with both substances led to a concentration-dependent activation of caspase-3 and mitochondrial permeability transition. Using BJAB cells overexpressing a dominant-negative mutant of the Fas-associated death domain (FADD) adaptor protein to block death receptor-mediated apoptosis, we demonstrate that resveratrol- and piceatannol-induced cell death in these cells is independent of the CD95/Fas signaling pathway. To explore the antileukemic properties of both compounds in more detail, we extended our study to primary, leukemic lymphoblasts. Interestingly, piceatannol but not resveratrol is a very efficient inducer of apoptosis in this ex vivo assay with leukemic lymphoblasts of 21 patients suffering from childhood lymphoblastic leukemia (ALL).

Unexpected endo selectivity of conjugate nucleophilic addition to an arene-Cr(CO)3 complex: enantioselective synthesis of the diterpene 11-epi-helioporin B.

[reaction: see text]. Starting from a nonracemic planar-chiral arene tricarbonyl chromium complex, an epimer of the bioactive marine diterpene helioporin B was synthesized in a highly stereoselective fashion. The stereostructure of the product (11-epi-helioporin B) corresponds to that of other serrulatane-type natural products. In a key step of the synthesis, 2-lithioacetonitrile undergoes conjugate nucleophilic addition to an unsaturated complex. Remarkably, this addition occurs in an endo mode, i.e., from the complexed face of the pi-ligand.

A catalytic-enantioselective entry to planar chiral pi-complexes: enantioselective methoxycarbonylation of 1,2-dichlorobenzene-Cr(CO)(3).

The palladium-catalyzed mono-methoxycarbonylation of 1,2-dichlorobenzene tricarbonylchromium(0) has been achieved with up to 95% ee in the presence of the chiral ferrocene ligand (R,S)-PPF-pyrrolidine. It was found that the enantioselectivity strongly depends on the reaction time (conversion). Obviously, the initial enantioselectivity is enhanced by a subsequent kinetic resolution connected to the formation of the bis-methoxycarbonylated byproduct.

Density functional investigation of reactive intermediates derived from alkyne-Co2(CO)6 complexes.

By applying the hybrid density functional method B3LYP and a flexible all-electron basis set, structures and energies of reactive intermediates derived from the 1-butyne complex of Co2(CO), (1) were calculated. In particular, the geometry, electronic distribution, and configurational stability of the cationic, radical, and anionic Co2(CO)6-complexed propargylic species were studied. The calculations revealed that the configurational barrier, that is, the racemization barrier for the antarafacial migration of the CHCH3 group, is low (7.6 kcalmol(-1)) for the radical and is similar to the experimental value for the corresponding cation (ca. 10 kcalmol(-1)). However, a high racemization barrier (23.7 kcalmol(-1)) for the anionic intermediate suggests the possibility of stereospecific reactions involving Co2(CO)6-complexed propargylic anions.