Maurizio Fermeglia

Activity of dasatinib against L576P KIT mutant melanoma: Molecular, cellular, and clinical correlates

Point mutations in the KIT receptor tyrosine kinase gene have recently been identified in mucosal, acral lentiginous, and chronically sun-damaged melanomas. We have identified the first human melanoma cell line with an endogenous L576P mutation, the most common KIT mutation in melanoma (∼30-40%). In vitro testing showed that the cell viability of the L576P mutant cell line was not reduced by imatinib, nilotinib, or sorafenib small molecule KIT inhibitors effective in nonmelanoma cells with other KIT mutations. However, the viability of the mutant cells was reduced by dasatinib at concentrations as low as 10 nM (P = 0.004). Molecular modeling studies found that the L576P mutation induces structural changes in KIT that reduce the affinity for imatinib (ΔΔGbind = −2.52 kcal/mol) but not for dasatinib (ΔΔGbind = +0.32 kcal/mol). Two metastatic melanoma patients with the L576P KIT mutation were treated with dasatinib, including one patient previously treated with imatinib. Both patients had marked reduction (>50%) and elimination of tumor F18-fluorodeoxyglucose (FDG)-avidity by positron emission tomography (PET) imaging after dasatinib treatment. These data support the selective inhibitory effect of dasatinib against cells harboring the most common KIT mutation in melanoma, and thus has therapeutic implications for acrallentiginous, chronic sun-damaged, and mucosal melanomas. [Mol Cancer Ther 2009;8(8):2079–85]

Anticancer drug nanomicelles formed by self-assembling amphiphilic dendrimer to combat cancer drug resistance

Significance Nanotechnology-based drug delivery is expected to bring new hope for cancer treatment by enhancing anticancer drug efficacy, overcoming drug resistance, and reducing drug toxicity. In this respect, we developed an innovative drug delivery system based on a self-assembling amphiphilic dendrimer, which can generate supramolecular nanomicelles with large void space in their core to encapsulate anticancer drugs with high loading capacity. The resulting drug-encapsulated nanomicelles can effectively enhance drug potency and combat drug resistance by promoting cellular uptake and decreasing efflux of the anticancer drug. Moreover, this drug delivery system can significantly reduce the systemic toxicity of the free drug. The present study illustrates a successful example of how advances in dendrimer nanotechnology can be advantageously implemented to foster therapeutic perspectives. Drug resistance and toxicity constitute challenging hurdles for cancer therapy. The application of nanotechnology for anticancer drug delivery is expected to address these issues and bring new hope for cancer treatment. In this context, we established an original nanomicellar drug delivery system based on an amphiphilic dendrimer (AmDM), which could generate supramolecular micelles to effectively encapsulate the anticancer drug doxorubicin (DOX) with high drug-loading capacity (>40%), thanks to the unique dendritic structure creating large void space for drug accommodation. The resulting AmDM/DOX nanomicelles were able to enhance drug potency and combat doxorubicin resistance in breast cancer models by significantly enhancing cellular uptake while considerably decreasing efflux of the drug. In addition, the AmDM/DOX nanoparticles abolished significantly the toxicity related to the free drug. Collectively, our studies demonstrate that the drug delivery system based on nanomicelles formed with the self-assembling amphiphilic dendrimer constitutes a promising and effective drug carrier in cancer therapy.

Unifac prediction of vapor-liquid equilibria in mixed solvent-salt systems

Abstract The Sander model for correlation and prediction of salt effects on vapor—liquid equilibria (VLE) of mixed solvents combines a term of the Debye—Huckel type with a modified UNIQUAC equation with concentration dependent parameters. In this work the UNIQUAC equation has been substituted by the original UNIFAC group-contribution model with concentration independent group interaction para-meters. Group interaction parameters have been estimated between ions (Li+, Na+, K+, Ca2+, Ba2+, Sr2+, Cu2+, Ni2+, Hg2+, F−, Cl−, Br−, J−, NO3− and CH3COO−), and solvent groups (CH2, OH, CH3OH, H2O, CH3CO), while previously published group interaction parameters between solvent groups have been maintained. It is shwon that the proposed model represents VLE for solvent—water—salt mixtures with an expected average accuracy of the total pressure around 9% and of the vapor-phase mole fractions around 4%. Since it is a predictive group-contribution method it has a much broader range of applicability than the Sander model.

Sodium montmorillonite silylation: Unexpected effect of the aminosilane chain length

In this work, the silylation of sodium montmorillonite (Na-MMT) was performed in glycerol using 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and 3-[2-(2-aminoethylamino)ethylamino]-propyl-trimethoxysilane. The effects on the d-spacing of sodium montmorillonite (Na-MMT) upon reaction with three aminosilanes of different chain length were studied in details by combining experimental and computational techniques. Infrared spectroscopy was used to monitor the grafting process, while the degree of grafting was calculated using thermogravimetric analysis. X-ray diffraction experiments were carried out to evaluate the shift of the (0 0 1) basal spacing. It was found that the degree of silylation of Na-MMT increases with increasing the length of the aminosilane organic moieties, the overall aminosilane concentration, and temperature. The same beneficial effects were observed on the silicate d-spacing, as its value increases with increasing silane concentration and reaction temperature. Remarkably, however, increasing the length of the organic chains in the silane modifiers resulted in decreasing values of the Na-MMT interlayer distance. A rationale for this behavior is proposed on the basis of atomistic molecular dynamics simulation evidences.

Computer simulation of nylon-6/organoclay nanocomposites: prediction of the binding energy

Molecular mechanics/dynamics computer simulations are used to explore the atomic scale structure and to predict binding energy values for polymer/clay nanocomposites based on nylon-6, montmorillonite (MMT) and several, different quaternary ammonium salts. Our results reveal that the energy of binding between the polymeric matrix and the montmorillonite platelet shows a decreasing trend with increasing molecular volume V of the quaternary ammonium salt used as surfactant. On the other hand, both the binding energy between the polyamide and the quat, and between the quat and the montmorillonite increase with increasing V, although with a different slope. Shorter hydrocarbonic chains are more effective in producing favorable binding energies with respect to longer ones, and the substitution of hydrogen atoms with polar groups, such as –OH or –COOH on the quaternary ammonium salt generally results in a greater interaction of the quat with the polymer. Finally, under the hypothesis that the clay platelets are uniformly dispersed within the polymer matrix, the pristine clay still yields a high interfacial strength between MMT and nylon-6.

Functional analyses and molecular modeling of two c-Kit mutations responsible for imatinib secondary resistance in GIST patients

Imatinib-acquired resistance related to the presence of secondary point mutations has become a frequent event in gastrointestinal stromal tumors. Here, transient transfection experiments with plasmids carrying two different KIT-acquired point mutations were performed along with immunoprecipitation of total protein extracts, derived from imatinib-treated and untreated cells. The molecular mechanics/Poisson Boltzmann surface area computational techniques were applied to study the interactions of the wild-type and mutated receptors with imatinib at the molecular level. Biochemical analyses showed KIT phosphorylation in cells transfected with vectors carrying the specific mutant genes. Imatinib treatment demonstrated that T670I was insensitive to the drug at all the applied concentrations, whereas V654A was inhibited by 6 μM of imatinib. The modeling of the mutated receptors revealed that both substitutions affect imatinib-binding site, but to a different extent: T670I substantially modifies the binding pocket, whereas V654A induces only relatively confined structural changes. We demonstrated that T670I and V654A cause indeed imatinib-acquired resistance and that the former is more resistant to imatinib than the latter. The application of molecular simulations allowed us to quantify the interactions between the mutated receptors and imatinib, and to propose a molecular rationale for this type of drug resistance.

Mallard Blue: A High-Affinity Selective Heparin Sensor That Operates in Highly Competitive Media

We report the simple synthesis and full investigation of a novel heparin binding dye, mallard blue, an arginine-functionalized thionine. This dye binds heparin in highly competitive media, including water with high levels of competitive electrolyte, buffered aqueous solution and human serum. The dye reports on heparin levels by a significant change in its UV-vis spectroscopic profile. Molecular dynamics modeling provides detailed insight into the binding mode. Heparin binding is shown to be selective over other glycosaminoglycans, such as hyaluronic acid and chondroitin sulfate. Importantly, we demonstrate that, in the most competitive conditions, mallard blue outperforms standard dyes used for heparin sensing such as azure A.

Hydrophobically Modified Dendrons: Developing Structure-Activity Relationships for DNA Binding and Gene Transfection

This paper develops a structure-activity relationship understanding of the way in which surfactant-like dendrons with hydrophilic spermine surface groups and a variety of lipophilic units at their focal points can self-assemble and subsequently bind to DNA with high affinity. The choice of functional group at the focal point of the dendron and the high tunability of the molecular structure have a very significant impact on DNA binding. Mesoscale modeling of the mode of dendron self-assembly provides a direct insight into how the mode of self-assembly exerts its effect on the DNA binding process. In particular, the hydrophobic unit controls the number of dendrons in the self-assembled micellar structures, and hence their diameters and surface charge density. The DNA binding affinity correlates with the surface charge density of the dendron aggregates. Furthermore, these structure-activity effects can also be extended to cellular gene delivery, as surface charge density plays a role in controlling the extent of endosomal escape. It is reported that higher generation dendrons, although binding DNA less strongly than the self-assembling lower generation dendrons, are more effective for transfection. The impact of the lipophilic group at the focal point is less significant for the DNA binding ability of these larger dendrons, which is predominantly controlled by the spermine surface groups, but it does modify the levels of gene transfection. Significant synergistic effects on gene delivery were observed when employing combinations of the dendrons and polyethyleneimine (PEI, 25 kDa), with transfection becoming possible at low loading levels where the two components would not transfect individually, giving practically useful levels of gene delivery.

T315I-mutated Bcr-Abl in chronic myeloid leukemia and imatinib: insights from a computational study

The early stage of chronic myeloid leukemia is triggered by the tyrosine kinase Bcr-Abl. Imatinib mesylate, a selective inhibitor of Bcr-Abl, has been successful in chronic myeloid leukemia clinical trials, but short-lived remissions are usually observed in blast crisis patients. Sequencing of the BCR-ABL gene in relapsed patients revealed a set of mutants that mediate drug resistance. Previously reported work postulated that the missense T315I mutation both alters the three-dimensional structure of the protein binding site, thus decreasing the protein sensitivity for the drug, and does not feature a fundamental hydrogen bond that is critical for binding with imatinib. These speculations, however, were not supported by investigations at the molecular modeling level. Here, we present the results obtained from the application of molecular dynamics simulations to the study of the interactions between T315I Bcr-Abl and imatinib. For the first time, we show that, with respect to the wild-type system, the absence of the supposedly critical H-bond is not the only cause for the failure of receptor inhibition by imatinib, but also a plethora of other protein/drug interactions are drastically and unfavorably changed in the mutant protein.

Synthesis, Biological Evaluation, and Three-Dimensional in Silico Pharmacophore Model for σ1 Receptor Ligands Based on a Series of Substituted Benzo[d]oxazol-2(3H)-one Derivatives

Novel benzo[d]oxazol-2(3H)-one derivatives were designed and synthesized, and their affinities against sigma receptors were evaluated. On the basis of 31 compounds, a three-dimensional pharmacophore model for the sigma(1) receptor binding site was developed using the Catalyst 4.9 software package. The best 3D pharmacophore hypothesis, consisting of one positive ionizable, one hydrogen bond acceptor, two hydrophobic aromatic, and one hydrophobic features provided a 3D-QSAR model with a correlation coefficient of 0.89. The best hypothesis was also validated by three independent methods, i.e., the Fisher randomization test included in the CatScramble functionality of Catalyst, the leave-one-out test, and activity prediction of an additional test set. The achieved results will allow researchers to use this 3D pharmacophore model for the design and synthesis of a second generation of high affinity sigma(1) ligands, as well as to discover other lead compounds for this class of receptors.