Ilaria Massarelli

Cytotoxic activity of polyacetylenes and polyenes isolated from roots of Echinacea pallida

The n‐hexane extracts of the roots of three medicinally used Echinacea species exhibited cytotoxic activity on human cancer cell lines, with Echinacea pallida found to be the most cytotoxic. Acetylenes are present in E. pallida lipophilic extracts but essentially absent in extracts from the other two species. In the present study, the cytotoxic effects of five compounds, two polyacetylenes (namely, 8‐hydroxy‐pentadeca‐(9E)‐ene‐11,13‐diyn‐2‐one (1) and pentadeca‐(9E)‐ene‐11,13‐diyne‐2,8‐dione (3)) and three polyenes (namely, 8‐hydroxy‐pentadeca‐(9E,13Z)‐dien‐11‐yn‐2‐one (2), pentadeca‐(9E,13Z)‐dien‐11‐yne‐2,8‐dione (4) and pentadeca‐(8Z,13Z)‐dien‐11‐yn‐2‐one (5)), isolated from the n‐hexane extract of E. pallida roots by bioassay‐guided fractionation, were investigated and the potential bioavailability of these compounds in the extract was studied.

Modulation of P-glycoprotein activity by cannabinoid molecules in HK-2 renal cells

1 Endogenous and synthetic cannabinoid molecules have been investigated as possible MDR‐1/P‐glycoprotein (P‐gp) modulators in HK‐2‐immortalized renal cells, using calcein acetoxymethylester (calcein‐AM) as a P‐gp substrate. 2 Among the endocannabinoid molecules tested, anandamide (AEA), but not 2‐arachidonoyl‐glycerol (2‐AG) or palmitoyl‐ethanolamide (PEA), increased the intracellular fluorescence emitted by calcein, a metabolic derivative of the P‐gp substrate calcein‐AM, indicative of a reduction in transport capacity. 3 All the three synthetic cannabimimetics tested, that is, R‐(+)‐methanandamide (R(+)‐MET), AM 251 and CP55,940 significantly increased calcein accumulation in the cytosol. 4 RT–PCR demonstrated that HK‐2 cells do not express CB1 or CB2 cannabinoid receptors. 5 R(+)‐MET, AM251 and CP55,940 were also evaluated as modulators of P‐gp expression, by Western blot analysis. Only AM251 weakly enhanced the protein levels (by 1.2‐fold) after a 4‐day‐long incubation with the noncytotoxic drug concentration 2 μM. 6 The present data provide the first evidence that the endocannabinoid AEA and different synthetic cannabinoids may inhibit the P‐gp activity in vitro via a cannabinoid receptor‐independent mechanism.

Development of QSAR models for predicting hepatocarcinogenic toxicity of chemicals

A dataset comprising 55 chemicals with hepatocarcinogenic potency indices was collected from the Carcinogenic Potency Database with the aim of developing QSAR models enabling prediction of the above unwanted property for New Chemical Entities. The dataset was rationally split into training and test sets by means of a sphere-exclusion type algorithm. Among the many algorithms explored to search regression models, only a Support Vector Machine (SVM) method led to a QSAR model, which was proved to pass rigorous validation criteria, in accordance with the OECD guidelines. The proposed model is capable to explain the hepatocarcinogenic toxicity and could be exploited for predicting this property for chemicals at the early stage of their development, so optimizing resources and reducing animal testing.

Synthesis and biological activities of vitamin D-like inhibitors of CYP24 hydroxylase

Selective inhibitors of CYP24A1 represent an important synthetic target in a search for novel vitamin D compounds of therapeutic value. In the present work, we show the synthesis and biological properties of two novel side chain modified 2-methylene-19-nor-1,25(OH)(2)D(3) analogs, the 22-imidazole-1-yl derivative 2 (VIMI) and the 25-N-cyclopropylamine compound 3 (CPA1), which were efficiently prepared in convergent syntheses utilizing the Lythgoe type Horner-Wittig olefination reaction. When tested in a cell-free assay, both compounds were found to be potent competitive inhibitors of CYP24A1, with the cyclopropylamine analog 3 exhibiting an 80-1 selective inhibition of CYP24A1 over CYP27B1. Addition of 3 to a mouse osteoblast culture sustained the level of 1,25(OH)(2)D(3), further demonstrating its effectiveness in CYP24A1 inhibition. Importantly, the in vitro effects on human promyeloid leukemia (HL-60) cell differentiation by 3 were nearly identical to those of 1,25(OH)(2)D(3) and in vivo the compound showed low calcemic activity. Finally, the results of preliminary theoretical studies provide useful insights to rationalize the ability of analog 3 to selectively inhibit the cytochrome P450 isoform CYP24A1.

QSAR studies on BK channel activators

QSAR studies were developed on the basis of a dataset comprising BK channel activators previously synthesized and biologically assayed in our laboratory, in order to obtain highly accurate models enabling prediction of affinity toward the channel for New Chemical Entities (NCEs). Many molecular descriptors were computed by the CODESSA software. They were initially exploited in order to rationally split the available dataset into training and test set pairs, which supplied the basis for the development of QSAR models. Models were subjected to rigorous validation analysis based on the estimate of several statistical parameters, for the seek of the most accurate and simplest model enabling prediction of BK channel affinity.

Synthesis, biological assays and QSAR studies of N-(9-benzyl-2-phenyl-8-azapurin-6-yl)-amides as ligands for A1 adenosine receptors.

2-Phenyl-9-benzyl-8-azapurines, bearing at the 6 position an amido group interposed between the 8-azapurine moiety and an alkyl or a substituted phenyl group, have been synthesised and assayed as ligands for adenosine receptors. All the compounds show high affinity for the A(1) adenosine receptor, and many of them also show a good selectivity for A(1) with respect to A(2A) and A(3) adenosine receptors. Based on the quite rich library containing such compounds and relevant biological data, QSAR models, able to rationalise the results and to give a quantitative estimate of the observed trends were also developed. The obtained models can assist in the design of new compounds selectively active on A(1) adenosine receptor.

Quantitative Structure–Activity Relationship Models for Predicting Biological Properties, Developed by Combining Structure- and Ligand-Based Approaches: An Application to the Human Ether-a-go-go-Related Gene Potassium Channel Inhibition

A strategy for developing accurate quantitative structure–activity relationship models enabling predictions of biological properties, when suitable knowledge concerning both ligands and biological target is available, was tested on a data set where molecules are characterized by high structural diversity. Such a strategy was applied to human ether‐a‐go‐go‐related gene K+ channel inhibition and consists of a combination of ligand‐ and structure‐based approaches, which can be carried out whenever the three‐dimensional structure of the target macromolecule is known or may be modeled with good accuracy. Molecular conformations of ligands were obtained by means of molecular docking, performed in a previously built theoretical model of the channel pore, so that descriptors depending upon the three‐dimensional molecular structure were properly computed. A modification of the directed sphere‐exclusion algorithm was developed and exploited to properly splitting the whole dataset into Training/Test set pairs. Molecular descriptors, computed by means of the codessa program, were used for the search of reliable quantitative structure–activity relationship models that were subsequently identified through a rigorous validation analysis. Finally, pIC50 values of a prediction set, external to the initial dataset, were predicted and the results confirmed the high predictive power of the model within a quite wide chemical space.

Design of highly specific ligands of fibrin for therapeutic applications.

The work presented here is aimed at designing high-affinity ligands for the fibrin γ(312–324) epitope. This epitope is specific for fibrin recognition, as it is exposed only on the fibrin surface, while in fibrinogen it is buried in the protein bulk. This property makes it very useful for therapeutic applications. In fact, it may be exploited in driving systems for targeted delivery of thrombolytic drugs toward the specific compartment where they are needed. It will then allow avoidance of serious unwanted side effects produced by a conventional systemic administration. The approach chosen for designing putative ligands is based on the known three-dimensional (3D) structure of the epitope. A wide virtual library made up of oligo-peptides and analogues designed by a combinatorial approach, on the basis of chemical complementarity criteria, has been screened by means of a docking/scoring approach (DOCK program). The peculiarity of the problem under study required a considerable effort in finding a method enabling the experimental validation of the design work results. In fact the selected biological target is absolutely new, so that neither a endogenous, nor a synthetic high-affinity ligand is known up to now. It does not allow for the validation of computational results by means of classical binding tests based on the use of known labeled high-affinity ligands. Preliminary binding essayes were so carried out by means of the Plasmon Surface Resonance (PSR) technique. The experimental results suggested that most of the molecules predicted to be good ligands by means of the selected computational tools, could carry the wanted affinity toward the selected target.

Optimizing QSAR models for predicting ligand binding to the drug-metabolizing cytochrome P450 isoenzyme CYP2D6.

The cytochrome P450 isozyme CYP2D6 binds a large variety of drugs, oxidizing many of them, and plays a crucial role in establishing in vivo drug levels, especially in multidrug regimens. The current study aimed to develop reliable predictive models for estimating the CYP2D6 inhibition properties of drug candidates. Quantitative structure–activity relationship (QSAR) studies utilizing 51 known CYP2D6 inhibitors were carried out. Performance achieved using models based on two‐dimensional (2D) molecular descriptors was compared with performance using models entailing additional molecular descriptors that depend upon the three‐dimensional (3D) structure of ligands. To properly compute the descriptors, all the 3D inhibitor structures were optimized such that induced‐fit binding of the ligand to the active site was accommodated. CODESSA software was used to obtain equations for correlating the structural features of the ligands to their pharmacological effects on CYP2D6 (inhibition). The predictive power of all the QSAR models obtained was estimated by applying rigorous statistical criteria. To assess the robustness and predictability of the models, predictions were carried out on an additional set of known molecules (prediction set). The results showed that only models incorporating 3D descriptors in addition to 2D molecular descriptors possessed the requisite high predictive power for CYP2D6 inhibition.

A Methylene Group on C-2 of 24,24-Difluoro-19-nor-1α,25-dihydroxyvitamin D3 Markedly Increases Bone Calcium Mobilization in Vivo

Four side chain fluorinated analogues of 1α,25-dihydroxy-19-norvitamin D have been prepared in convergent syntheses using the Wittig-Horner reaction as a key step. Structures and absolute configurations of analogues 3 and 5 were confirmed by X-ray crystallography. All analogues showed high potency in HL-60 cell differentiation and vitamin D-24-hydroxylase (24-OHase) transcription as compared to 1α,25-dihydroxyvitamin D3 (1). Most important is that all of the 20S-configured derivatives (4 and 6) had high bone mobilizing activity in vivo. However, in the 20R series, a 2-methylene group was required for high bone mobilizing activity. A change in positioning of the 20R molecule in the vitamin D receptor when the 2-methylene group is present may provide new insight into the molecular basis of bone calcium mobilization induced by vitamin D.