Kyriakos C. Prousis

Effect of alkylphospholipids on Candida albicans biofilm formation and maturation

OBJECTIVES The aim of this study was to evaluate miltefosine and four synthetic compounds (TCAN26, TC19, TC106 and TC117) for their in vitro inhibitory activity against Candida albicans planktonic and biofilm cells and investigate whether these compounds are able to inhibit the biofilm formation and to reduce the viability of mature C. albicans biofilm cells. METHODS The XTT reduction assay and transmission and scanning electron microscopy were employed to determine the inhibitory effects of the test compounds in comparison with amphotericin B and fluconazole against both planktonic cells and sessile cells in biofilms. RESULTS C. albicans planktonic cells were susceptible to miltefosine, TCAN26 and TC19, all alkylphospholipid compounds. Miltefosine and TCAN26 present a fungicidal activity with similar values of MIC and minimum fungicidal concentration (MFC), ranging from 2 to 8 mg/L. Cell treatment with sub-inhibitory concentrations of alkylphospholipids induced several ultrastructural alterations. In relation to biofilms, miltefosine reduced formation (38%-71%) and mature biofilms viability (32%-44%), at concentrations of 64 mg/L. TCAN26 also reduced biofilm formation (24%-30%) and mature biofilm viability (15%-20%), at concentrations of 64 mg/L. Although amphotericin B reduced biofilm formation similarly to miltefosine (51%-74%), its activity was lower on mature biofilms (24%-30%). Miltefosine antibiofilm activity was significantly higher than amphotericin B, on both formation and mature biofilms (P<0.05 and P<0.0001, respectively). Fluconazole was the least effective compound tested. CONCLUSION Promising antibiofilm activity was displayed by miltefosine and other alkylphosphocholine compounds, which could be considered a putative option for future treatment of candidaemia associated with biofilm formation, although further evaluation in in vivo systems is required.

Design and synthesis of new adamantyl-substituted antileishmanial ether phospholipids.

A series of new 2-[3-(2-alkyloxy-ethyl)-adamantan-1-yl]-ethoxy substituted ether phospholipids was synthesized and their antileishmanial activity was evaluated against Leishmania infantum amastigotes. The majority of the new analogues were significantly less cytotoxic than miltefosine while, antiparasitic activity depended on the length of the 2-alkyloxy substituent. The most potent compounds were {2-[[[3-(2-hexyloxy-ethyl)-adamant-1-yl]-ethoxy]hydroxyphosphinyloxy]ethyl}-Nu,Nu,Nu-trimethyl-ammonium inner salt (5b) and {2-[[[3-(2-octyloxy-ethyl)-adamant-1-yl]-ethoxy]hydroxyphosphinyloxy]ethyl}-Nu,Nu,Nu-trimethyl-ammonium inner salt (5c).

Steroidal Cardiac Na+/K (+) ATPase Inhibitors Exhibit Strong Anti-Cancer Potential in vitro and in Prostate and Lung Cancer Xenografts in vivo

Sodium potassium pump (Na(+)/K(+)ATPase) is a validated pharmacological target for the treatment of congestive heart failure. Recent data with inotropic drugs such as digoxin & digitoxin (digitalis) suggest a potent anti-cancer action of these drugs and promote Na(+)/K(+)ATPase as a novel therapeutic target in cancer. However, digitalis have narrow therapeutic indices, are pro-arrhythmic and are considered non-developable drugs by the pharmaceutical industry. On the contrary, a series of recently-developed steroidal inhibitors showed better pharmacological properties and clinical activities in cardiac patients. Their anti-cancer activity however, remained unknown. In this study, we synthesized seventeen steroidal cardiac inhibitors and explored for the first time their anti-cancer activity in vitro and in vivo. Our results indicate potent anti-cancer actions of steroidal cardiac inhibitors in multiple cell lines from different tumor panels including multi-drug resistant cells. Furthermore, the most potent compound identified in our studies, the 3-[(R)-3- pyrrolidinyl]oxime derivative 3, showed outstanding potencies (as measured by GI50, TGI and LC50 values) in most cells in vitro, was selectively cytotoxic in cancer versus normal cells showing a therapeutic index of 31.7 and exhibited significant tumor growth inhibition in prostate and lung xenografts in vivo. Collectively, our results suggest that previously described cardiac Na(+)/K(+)ATPase inhibitors have potent anti-cancer actions and may thus constitute strong re-purposing candidates for further cancer drug development.

FeCl3-catalysed ultrasonic-assisted, solvent-free synthesis of 4-substituted coumarins. A useful complement to the Pechmann reaction

The catalytic activity of FeCl3 for the synthesis of a variety of 4-substituted coumarins using high energy techniques has been investigated. The ultrasonic-assisted conditions provide a useful complement to the Pechmann reaction, affording the coumarin derivatives in excellent yields, under solvent-free conditions, in short reaction times using an inexpensive, mild and benign Lewis acid catalyst.

Design and synthesis of 21-alkynylaryl pregnenolone derivatives and evaluation of their anticancer activity

A series of novel C21-alkynylaryl derivatives of pregnenolone were synthesized and screened for anticancer activity against a panel of seven human cancer cell lines (LNCaP, A549, MCF7, HeLa, A431, HepG2, HT29). The data revealed that these compounds can be potential antitumour agents against the specific cell models. Compound 6f bearing a 2-trifluoromethylphenyl group displayed improved cytotoxicity towards all cancer cell lines used. A431 cells were the most sensitive with derivatives 6e-6h bearing electron withdrawing substituents exhibiting high potency with IC50 values ranging between 2.18 and 0.54μM and drastic inhibition of the prosurvival PI3K-Akt/PKB pathway.

BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

Neurotrophin receptors mediate a plethora of signals affecting neuronal survival. The p75 pan-neurotrophin receptor controls neuronal cell fate after its selective activation by immature and mature isoforms of all neurotrophins. It also exerts pleiotropic effects interacting with a variety of ligands in different neuronal or non-neuronal cells. In the present study, we explored the biophysical and functional interactions of a blood-brain-barrier (BBB) permeable, C17-spiroepoxy steroid derivative, BNN27, with p75NTR receptor. BNN27 was recently shown to bind to NGF high-affinity receptor, TrkA. We now tested the p75NTR-mediated effects of BNN27 in mouse Cerebellar Granule Neurons (CGNs), expressing p75NTR, but not TrkA receptors. Our findings show that BNN27 physically interacts with p75NTR receptors in specific amino-residues of its extracellular domain, inducing the recruitment of p75NTR receptor to its effector protein RIP2 and the simultaneous release of RhoGDI in primary neuronal cells. Activation of the p75NTR receptor by BNN27 reverses serum deprivation-induced apoptosis of CGNs resulting in the decrease of the phosphorylation of pro-apoptotic JNK kinase and of the cleavage of Caspase-3, effects completely abolished in CGNs, isolated from p75NTR null mice. In conclusion, BNN27 represents a lead molecule for the development of novel p75NTR ligands, controlling specific p75NTR-mediated signaling of neuronal cell fate, with potential applications in therapeutics of neurodegenerative diseases and brain trauma.

A Steroidal Na + /K + ATPase Inhibitor Triggers Pro-apoptotic Signaling and Induces Apoptosis in Prostate and Lung Tumor Cells

Recently we have reported potent anti-cancer actions of various steroidal Na(+)/K(+) ATPase inhibitors in multiple cell lines. Furthermore, the most powerful compound identified in this study, the 3-[(R)-3-pyrrolidinyl]oxime derivative (3-R-POD), was highly effective in various tumor cell lines in vitro, and exhibited significant tumor growth inhibition in prostate and lung xenografts in vivo. In the present study we have addressed the molecular mechanisms implicated in the anti-cancer actions of 3-R-POD. We report here that 3-R-POD induces strong apoptotic responses in A549 lung- and in DU145 prostate- cancer cells. These effects are accompanied by significant upregulation of caspase-3 activity. Focussing on A549 cells, we further demonstrate late downregulation of BCL-2- and upregulation of c-Fos- gene transcription. In addition, the steroidal Na(+)/K(+) ATPase inhibitor induced late de-phosphorylation of Focal Adhesion Kinase (FAK) and activation of p38 MAPK. Our findings suggest that the steroidal Na(+)/K(+) ATPase inhibitor 3-R-POD induces apoptosis, paralleled by altered BCL-2 and c-Fos gene transcription, inhibition of the pro-survival FAK signalling, up-regulation of the pro-apoptotic p38 MAPK pathway and stimulation of caspase-3 activity.

Solid-phase synthesis of optically active substituted 2 aminofuranones using an activated carbonate linker.

An efficient three-step solid-phase synthesis of diverse 3,5-disubstituted-2-aminofuranones has been developed. α-Hydroxy acids loaded on a nitrophenyl carbonate derivative of Wang resin are used as acylating agents for the C-acylation of active methylene compounds and the resulting intermediates provided, through a cyclative cleavage reaction, the desired product.

Functional characterization and anti-cancer action of the clinical phase II cardiac Na + /K + ATPase inhibitor istaroxime: in vitro and in vivo properties and cross talk with the membrane androgen receptor

Sodium potassium pump (Na+/K+ ATPase) is a validated pharmacological target for the treatment of various cardiac conditions. Recent published data with Na+/K+ ATPase inhibitors suggest a potent anti-cancer action of these agents in multiple indications. In the present study, we focus on istaroxime, a Na+/K+ ATPase inhibitor that has shown favorable safety and efficacy properties in cardiac phase II clinical trials. Our experiments in 22 cancer cell lines and in prostate tumors in vivo proved the strong anti-cancer action of this compound. Istaroxime induced apoptosis, affected the key proliferative and apoptotic mediators c-Myc and caspase-3 and modified actin cystoskeleton dynamics and RhoA activity in prostate cancer cells. Interestingly, istaroxime was capable of binding to mAR, a membrane receptor mediating rapid, non-genomic actions of steroids in prostate and other cells. These results support a multi-level action of Na+/K+ ATPase inhibitors in cancer cells and collectively validate istaroxime as a strong re-purposing candidate for further cancer drug development.

Three Step Solid-Phase Synthesis of Functionalized Tetronic Acids Using an Activated Carbonate Linker

Abstract An extension of our methodology on solid-phase synthesis (SPS) of five-membered heterocycles to functionalized tetronic acids is reported. The key step of our methodology is the application of a carbonate linker to the synthesis, offering us the convenience of incorporating the starting material and cleavage under mild conditions. The products were obtained in satisfactory yields and purities. The developed method can be applied to the preparation of libraries containing the tetronic acid moiety. GRAPHICAL ABSTRACT