Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats.

In pre-hypertension, moderate control of blood pressure (BP) can be obtained by a nutritional approach. The effects of a diet enriched with defatted larvae of the mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (TM) endowed with ACE inhibitory activity was studied in both spontaneously hypertensive rats (SHR) and in the age-matched normotensive Wistar Kyoto strain. These were fed for 4 weeks with standard laboratory rodent chow supplemented with or without TM or captopril. In SHR, the TM diet caused a significant reduction in BP, heart rate and coronary perfusion pressure, as well as an increase in red blood cell glutathione/glutathione disulphide ratio. Rat brain slices of SHR were more resistant to oxidative stress and contained lower levels of inflammatory cytokines, while vascular and liver enzyme-activities were not affected. These results suggest that TM can be considered a new functional food that can lower BP in vivo and thus control cardiovascular-associated risk factors such as hypertension.

A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1.

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.

Structure-Based Identification of HIV-1 Nucleocapsid Protein Inhibitors Active against Wild-Type and Drug-Resistant HIV-1 Strains.

HIV/AIDS is still one of the leading causes of death worldwide. Current drugs that target the canonical steps of the HIV-1 life cycle are efficient in blocking viral replication but are unable to eradicate HIV-1 from infected patients. Moreover, drug resistance (DR) is often associated with the clinical use of these molecules, thus raising the need for novel drug candidates as well as novel putative drug targets. In this respect, pharmacological inhibition of the highly conserved and multifunctional nucleocapsid protein (NC) of HIV-1 is considered a promising alternative to current drugs, particularly to overcome DR. Here, using a multidisciplinary approach combining in silico screening, fluorescence-based molecular assays, and cellular antiviral assays, we identified nordihydroguaiaretic acid (6), as a novel natural product inhibitor of NC. By using NMR, mass spectrometry, fluorescence spectroscopy, and molecular modeling, 6 was found to act through a dual mechanism of action never highlighted before for NC inhibitors (NCIs). First, the molecule recognizes and binds NC noncovalently, which results in the inhibition of the nucleic acid chaperone properties of NC. In a second step, chemical oxidation of 6 induces a potent chemical inactivation of the protein. Overall, 6 inhibits NC and the replication of wild-type and drug-resistant HIV-1 strains in the low micromolar range with moderate cytotoxicity that makes it a profitable tool compound as well as a good starting point for the development of pharmacologically relevant NCIs.

Dietary fatty acids influence the growth and fatty acid composition of the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae).

Fat is the second most abundant component of the nutrient composition of the mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) that represents also an interesting source of PUFA, especially n-6 and n-3 fatty acids, involved in prevention of cardiovascular diseases. This study investigated the possibility of modifying the fat content and the FA composition of yellow mealworms through feeding and how this would be influenced by developmental stages, pupal sex, and generation with the future aim of applying this coleopteran as a diet supplement for human health. Growth rate and cumulative mortality percentage on the different feeding substrates were also evaluated to select the optimal conditions for a mass-raising of this insect species. Despite the different fat content in the six different breeding substrates used, T. molitor larvae and pupae contained a constant fat percentage (>34% in larvae and >30% in pupae). A similar total fat content was found comparing larvae and male and female pupae of the second generation to those of the first generation. On the contrary, FA composition differed both in larvae and pupae reared on the different feeding substrates. However, the exemplars reared on the diets based on 100% bread and 100% oat flour showed SFA, PUFA percentages, and an n-6/n-3 ratio more suitable for human consumption; the diet based on beer yeast, wheat flour, and oat flour resulted in a contemporary diet that most satisfied the balance between a fat composition of high quality and favorable growth conditions.

Functional and structural characterization of 2-amino-4-phenylthiazole inhibitors of the HIV-1 nucleocapsid protein with antiviral activity.

The nucleocapsid protein (NC) is a highly conserved protein in diverse HIV-1 subtypes that plays a central role in virus replication, mainly by interacting with conserved nucleic acid sequences. NC is considered a highly profitable drug target to inhibit multiple steps in the HIV-1 life cycle with just one compound, a unique property not shown by any of the other antiretroviral classes. However, most of NC inhibitors developed so far act through an unspecific and potentially toxic mechanism (zinc ejection) and are mainly being investigated as topical microbicides. In an effort to provide specific NC inhibitors that compete for the binding of nucleic acids to NC, here we combined molecular modeling, organic synthesis, biophysical studies, NMR spectroscopy, and antiviral assays to design, synthesize, and characterize an efficient NC inhibitor endowed with antiviral activity in vitro, a desirable property for the development of efficient antiretroviral lead compounds.

Discovery and synthesis of novel benzofurazan derivatives as inhibitors of influenza A virus.

The identification of a novel hit compound inhibitor of the protein-protein interaction between the influenza RNA-polymerase PA and PB1 subunits has been accomplished by means of high-throughput screening. A small family of structurally related molecules has been synthesized and biologically evaluated with most of the compounds showing micromolar potency of inhibition against viral replication.

A combination strategy to inhibit Pim-1: synergism between noncompetitive and ATP-competitive inhibitors.

Pim-1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim-1 inhibitors, a previously identified ATP-competitive indolyl-pyrrolone scaffold was expanded to derive structure-activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP-competitive inhibitors as well as a series of 2-aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim-1. Notably, further evaluation of the 2-aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP-competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP-competitive and ATP-noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim-1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim-1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim-1 is associated with chemotherapeutic resistance.

Use of virtual screening for discovering antiretroviral compounds interacting with the HIV-1 nucleocapsid protein.

The HIV-1 nucleocapsid protein (NC) is considered as an emerging drug target for the therapy of AIDS. Several studies have highlighted the crucial role of NC within the viral replication cycle. However, although NC inhibition has provided in vitro and in vivo antiretroviral activity, drug-candidates which interfere with NC functions are still missing in the therapeutic arsenal against HIV. Based on previous studies, where the dynamic behavior of NC and its ligand binding properties have been investigated by means of computational methods, here we used a virtual screening protocol for discovering novel antiretroviral compounds which interact with NC. The antiretroviral activity of virtual hits was tested in vitro, whereas biophysical studies elucidated the direct interaction of most active compounds with NC(11-55), a peptide corresponding to the zinc finger domain of NC. Two novel antiretroviral small molecules capable of interacting with NC are presented here.

Discovery of small molecule HIV-1 integrase dimerization inhibitors.

Human immunodeficiency virus-1 integrase (HIV-1 IN) inserts the viral DNA into host cell chromatin in a multistep process. This enzyme exists in equilibrium between monomeric, dimeric, tetrameric and high order oligomeric states. However, monomers of IN are not capable of supporting its catalytic functions and the active form has been shown to be at least a dimer. As a consequence, the development of inhibitors targeting IN dimerization constitutes a promising novel antiviral strategy. In this work, we successfully combined different computational techniques in order to identify small molecule inhibitors of IN dimerization. Additionally, a novel AlphaScreen-based IN dimerization assay was used to evaluate the inhibitory activities of the selected compounds. To the best of our knowledge, this study represents the first successful virtual screening and evaluation of small molecule HIV-1 IN dimerization inhibitors, which may serve as attractive hit compounds for the development of novel anti-HIV.

Toward the discovery of novel anti-HIV drugs. Second-generation inhibitors of the cellular ATPase DDX3 with improved anti-HIV activity: synthesis, structure-activity relationship analysis, cytotoxicity studies, and target validation.

A hit optimization protocol applied to the first nonnucleoside inhibitor of the ATPase activity of human DEAD-box RNA helicase DDX3 led to the design and synthesis of second-generation rhodanine derivatives with better inhibitory activity toward cellular DDX3 and HIV-1 replication. Additional DDX3 inhibitors were identified among triazine compounds. Biological data were rationalized in terms of structure-activity relationships and docking simulations. Antiviral activity and cytotoxicity of selected DDX3 inhibitors are reported and discussed. A thorough analysis confirmed human DDX3 as a valid anti-HIV target. The compounds described herein represent a significant advance in the pursuit of novel drugs that target HIV-1 host cofactors.

A versatile and practical synthesis toward the development of novel HIV-1 integrase inhibitors.

As a continuation of our previous work, which resulted in the identification of a new hit compound as an HIV-1 integrase inhibitor, three novel series of salicylic acid derivatives were synthesized using three versatile and practical synthetic strategies and were assayed for their capacity to inhibit the catalytic activity of HIV-1 integrase. Biological evaluations revealed that some of the synthesized compounds possess good inhibitory potency in enzymatic assays and are able to inhibit viral replication in MT-4 cells at low micromolar concentrations. Finally, docking studies were conducted to analyze the binding mode of the synthesized compounds within the DNA binding site of integrase in order to refine their structure-activity relationships.

Predicting the binding mode of known NCp7 inhibitors to facilitate the design of novel modulators.

The HIV-1 nucleocapsid protein (NCp7) is an emerging target for antiretroviral therapy. Five hits have been reported to inhibit the NCp7-viral nucleic acids interaction at micromolar concentrations. We used two computationally refined structures of NCp7 as receptors to propose a reliable binding pose for these compounds, by means of computational methods. Theoretical binding modes are in agreement with available experimental data. Results lay the foundations for a rationale development of more effective NCp7 inhibitors.

Novel dual Src/Abl inhibitors for hematologic and solid malignancies.

IMPORTANCE OF THE FIELD: c-Src and Bcr-Abl are two non-receptor or cytoplasmic tyrosine kinases (TKs) that play important roles in the development of solid and hematological malignancies. Indeed, Src is overexpressed or hyperactivated in a variety of solid tumors, while Bcr-Abl is the causative agent of chronic myeloid leukemia (CML), where Src is also involved. The two enzymes share significant sequence homology and remarkable structural resemblance. AREAS COVERED IN THIS REVIEW: ATP-competitive compounds originally developed as Src inhibitors, showed to be also potent Abl inhibitors. Dasatinib, the first dual Src/Abl inhibitor approved by the US FDA in 2006 for the treatment of imatinib-resistant CML, is currently being tested in several clinical trials for the treatment of different solid tumors. SKI-606 and AZD0530 are two other important dual Src/Abl inhibitors extensively tested in animal models and in clinical trials, but not entered into therapy yet. WHAT THE READER WILL GAIN: In this review we will report the latest results regarding dasatinib, SKI-606 and AZD0530, but also the knowledge on new compounds that have appeared in the literature in the last few years, including AP24163, AP24534, XL228, DC2036. We will focus on the most recent clinical trials or on preclinical studies that are in progress on these small-molecule TK inhibitors that represent a targeted therapy with high potential against cancer. TAKE HOME MESSAGE: Molecularly targeted therapies, including the inhibition of specific TKs hyperactivated or overexpressed in many human cancers, could be less toxic than the classical non-specific cytotoxic chemotherapeutic agents; they could offer important therapeutic effects, especially if used in association with other agents such as monoclonal antibodies.

Synthesis and biological evaluation of new enantiomerically pure azole derivatives as inhibitors of Mycobacterium tuberculosis.

A series of novel enantiomerically pure azole derivatives was synthesized. The new compounds, bearing both an imidazole as well as a triazole moiety, were evaluated as antimycobacterial agents. One of them proved to have activity against Mycobaterium tuberculosis comparable to those of the classical antibacterial/antifungal drugs Econazole and Clotrimazole.

Pharmacophore modeling and molecular docking led to the discovery of inhibitors of human immunodeficiency virus-1 replication targeting the human cellular aspartic acid-glutamic acid-alanine-aspartic acid box polypeptide 3.

HIV-1 replication has been inhibited by using a compound able to target the human cellular cofactor DEAD-box ATPase DDX3, essential for HIV-1 RNA nuclear export. This compound, identified by means of a computational protocol based on pharmacophoric modeling and molecular docking calculations, represents the first small molecule with such a mechanism of action and could lay the foundations for a pioneering approach for the treatment of HIV-1 infections.

Targets looking for drugs: a multistep computational protocol for the development of structure-based pharmacophores and their applications for hit discovery.

Pharmacophoresthree-dimensional (3D) arrangements of essential features enabling a molecule to exert a particular biological effectconstitute a very useful tool in drug design both in hit discovery and hit-to-lead optimization process. Two basic approaches for pharmacophoric model generation can be used by chemists, depending on the availability or not of the target 3D structure. In view of the rapidly growing number of protein structures that are now available, receptor-based pharmacophore generation methods are becoming more and more used. Since most of them require the knowledge of the 3D structure of the ligand-target complex, they cannot be applied when no compounds targeting the binding site of interest are known. Here, a GRID-based procedure for the generation of receptor-based pharmacophores starting from the knowledge of the sole protein structure is described and successfully applied to address three different tasks in the field of medicinal chemistry.

Synthesis, in vitro, and in vivo biological evaluation and molecular docking simulations of chiral alcohol and ether derivatives of the 1,5-diarylpyrrole scaffold as novel anti-inflammatory and analgesic agents.

Following our previous research on anti-inflammatory drugs (NSAIDs), we report here the synthesis of chiral 1,5-diarylpyrroles derivatives that were characterized for their in vitro inhibitory effects toward cyclooxygenase (COX) isozymes. Analysis of enzymatic affinity and COX-2 selectivity led us to the selection of one compound (+/-)-10b that was further tested in vitro in the human whole blood (HWB) and in vivo for its anti-inflammatory activity in mice. The affinity data have been rationalized through docking simulations.

Substituted pyrazolo[3,4-b]pyridines as potent A1 adenosine antagonists: synthesis, biological evaluation, and development of an A1 bovine receptor model.

Sixty-eight new substituted pyrazolo[3,4-b]pyridine derivatives were synthesized and tested for enriching a library of active A(1) adenosine receptor (AR) antagonists belonging to the same class. These compounds were also used as an external test set to check the reliability of a 3D QSAR model recently reported by us. To investigate the binding mode of pyrazolopyridine derivatives, a model of the bovine A(1)AR (bA(1)AR) was developed by a novel homology modeling approach and used to evaluate the main interactions of the ligands with the receptor through docking studies. Results suggest important interactions of the ligands mainly with L3.33(88), T3.36(91), Q3.37(92) and H6.52(251), in agreement with mutagenesis data. The racemic mixture of the most active compound was separated into the corresponding enantiomers which showed a bA(1)AR affinity in the nanomolar range, with the R enantiomer sevenfold more active than the S enantiomer, according to results derived from calculations on the receptor model. Analysis of the bovine/human A(1)AR affinity profile of ligands supported the hypothesis that such receptors should be characterized by a different size of their binding site, responsible for the different affinity of the antagonists.

Toward novel HIV-1 integrase binding inhibitors: molecular modeling, synthesis, and biological studies.

The identification of a novel hit compound as integrase binding inhibitor has been accomplished by means of virtual screening techniques. A small family of structurally related molecules has been synthesized and biologically evaluated with one of the compounds showing an IC(50)=12 microM.

Ligand-based virtual screening, parallel solution-phase and microwave-assisted synthesis as tools to identify and synthesize new inhibitors of mycobacterium tuberculosis.

In an attempt to identify new inhibitors of the growth of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, a procedure for the generation, design, and screening of a ligand-based virtual library was applied. This used both an in silico protocol centered on a recursive partitioning (RP) model described herein, and a pharmacophoric model for antitubercular agents previously generated by our research group. Two candidates emerged from databases of commercially available compounds, both characterized by a minimum inhibitory concentration (MIC) of 25 microg mL(-1). Based on these compounds, two series of derivatives were synthesized by both parallel solution-phase and microwave-assisted synthesis, leading to enhanced antimycobacterial activity. During both the design and synthesis, attention was focused on the efficient allocation of available resources with the aim of reducing the overall costs associated with calculation and synthesis.