Publications by authors named "Stefano Alcaro"

198 Publications

Selective inhibition of carbonic anhydrase IX and XII by coumarin and psoralen derivatives.

J Enzyme Inhib Med Chem 2021 Dec;36(1):685-692

Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy.

A small library of coumarin and their psoralen analogues and has been designed and synthesised to investigate the effect of structural modifications on their inhibition ability and selectivity profile towards carbonic anhydrase isoforms I, II, IX, and XII. None of the new compounds exhibited activity towards hCA I and II isozymes. Conversely, both coumarin and psoralen derivatives were active against tumour associated isoforms IX and XII in the low micromolar or nanomolar range of concentration. These data further corroborate our previous findings on analogous derivatives, confirming that both coumarins and psoralens are interesting scaffolds for the design of isozyme selective hCA inhibitors.
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http://dx.doi.org/10.1080/14756366.2021.1887171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899656PMC
December 2021

Current Updates on Naturally Occurring Compounds Recognizing SARS-CoV-2 Druggable Targets.

Molecules 2021 Jan 26;26(3). Epub 2021 Jan 26.

Dipartimento di Scienze della Salute, Università "Magna Græcia" di Catanzaro, Campus "S. Venuta", Viale Europa, 88100 Catanzaro, Italy.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified in China as the etiologic agent of the recent COVID-19 pandemic outbreak. Due to its high transmissibility, this virus quickly spread throughout the world, causing considerable health issues. The scientific community exerted noteworthy efforts to obtain therapeutic solutions for COVID-19, and new scientific networks were constituted. No certified drugs to efficiently inhibit the virus were identified, and the development of de-novo medicines requires approximately ten years of research. Therefore, the repurposing of natural products could be an effective strategy to handle SARS-CoV-2 infection. This review aims to update on current status of the natural occurring compounds recognizing SARS-CoV-2 druggable targets. Among the clinical trials actually recruited, some natural compounds are ongoing to examine their potential role to prevent and to treat the COVID-19 infection. Many natural scaffolds, including alkaloids, terpenes, flavonoids, and benzoquinones, were investigated by in-silico, in-vitro, and in-vivo approaches. Despite the large data set obtained by a computational approach, experimental evidences in most cases are not available. To fill this gap, further efforts to validate these results are required. We believe that an accurate investigation of naturally occurring compounds may provide insights for the potential treatment of COVID-19 patients.
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http://dx.doi.org/10.3390/molecules26030632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865633PMC
January 2021

4-Oxoquinolines and monoamine oxidase: When tautomerism matters.

Eur J Med Chem 2021 Mar 13;213:113183. Epub 2021 Jan 13.

CIQUP, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade Do Porto, Porto, 4169-007, Portugal. Electronic address:

4-Oxoquinoline derivatives have been often used in drug discovery programs due to their pharmacological properties. Inspired on chromone and 4-oxoquinoline chemical structure similarity, a small series of quinoline-based compounds was obtained and screened, for the first time, toward human monoamine oxidases isoforms. The data showed the N-(3,4-dichlorophenyl)-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide 10 was the most potent and selective MAO-B inhibitor (IC = 5.30 ± 0.74 nM and SI: ≥1887). The data analysis showed that prototropic tautomerism markedly influences the biological activity. The unequivocal characterisation of the quinoline tautomers was performed to understand the attained data. To our knowledge, there have been no prior reports on the characterisation of quinolone tautomers by 2D NMR techniques, namely by H-N HSQC and H-N HMBC, which are proposed as expedite tools for medicinal chemistry campaigns. Computational studies on enzyme-ligand complexes, obtained after MM-GBSA calculations and molecular dynamics simulations, supported the experimental data.
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http://dx.doi.org/10.1016/j.ejmech.2021.113183DOI Listing
March 2021

Insight on [1,3]thiazolo[4,5-e]isoindoles as tubulin polymerization inhibitors.

Eur J Med Chem 2021 Feb 24;212:113122. Epub 2020 Dec 24.

Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Università degli Studi di Palermo, Via Archirafi 32, 90123, Palermo, Italy.

A series of [1,3]thiazolo[4,5-e]isoindoles has been synthesized through a versatile and high yielding multistep sequence. Evaluation of the antiproliferative activity of the new compounds on the full NCI human tumor cell line panel highlighted several compounds that are able to inhibit tumor cell proliferation at micromolar-submicromolar concentrations. The most active derivative 11g was found to cause cell cycle arrest at the G2/M phase and induce apoptosis in HeLa cells, following the mitochondrial pathway, making it a lead compound for the discovery of new antimitotic drugs.
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http://dx.doi.org/10.1016/j.ejmech.2020.113122DOI Listing
February 2021

Joining European Scientific Forces to Face Pandemics.

Trends Microbiol 2021 02 4;29(2):92-97. Epub 2020 Dec 4.

Department of Oncology, University of Turin, IT-10126, Turin, Italy. Electronic address:

Despite the international guidelines on the containment of the coronavirus disease 2019 (COVID-19) pandemic, the European scientific community was not sufficiently prepared to coordinate scientific efforts. To improve preparedness for future pandemics, we have initiated a network of nine European-funded Cooperation in Science and Technology (COST) Actions that can help facilitate inter-, multi-, and trans-disciplinary communication and collaboration.
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http://dx.doi.org/10.1016/j.tim.2020.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716745PMC
February 2021

In Silico Food-Drug Interaction: A Case Study of Eluxadoline and Fatty Meal.

Int J Mol Sci 2020 Nov 30;21(23). Epub 2020 Nov 30.

Dipartimento di Scienze della Salute, Università "Magna Græcia" di Catanzaro, Campus "S. Venuta", Viale Europa, 88100 Catanzaro, Italy.

Food-drug interaction is an infrequently considered aspect in clinical practice. Usually, drugs are taken together with meals and what follows may adversely affect pharmacokinetic and pharmacodynamic properties, and hence, the therapeutic effects. In this study, a computational protocol was proposed to explain the different assimilations of two receptors agonists, eluxadoline and loperamide, with a peculiar pharmacokinetic profile. Compared to loperamide, eluxadoline is absorbed less after the intake of a fatty meal, and the LogP values do not explain this event. Firstly, keeping in mind the different pH in the intestinal tract, the protonation states of both compounds were calculated. Then, all structures were subjected to a conformational search by using MonteCarlo and Molecular Dynamics methods, with solvation terms mimicking the water and weak polar solvent (octanol). Both computational results showed that eluxadoline has less conformational freedom in octanol, unlike loperamide, which exhibits constant behavior in both solvents. Therefore, we hypothesize that fatty meal causes the "" of the eluxadoline molecule to prevent the exposure of the polar groups and their interaction with water, necessary for the drug absorption. Based on our results, this work could be a reasonable "case study", useful for future investigation of the drug pharmacokinetic profile.
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http://dx.doi.org/10.3390/ijms21239127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731208PMC
November 2020

Natural Products Extracted from Fungal Species as New Potential Anti-Cancer Drugs: A Structure-Based Drug Repurposing Approach Targeting HDAC7.

Molecules 2020 Nov 25;25(23). Epub 2020 Nov 25.

Dipartimento di Scienze della Salute, Università "Magna Græcia" di Catanzaro, Campus "S. Venuta", Viale Europa, 88100 Catanzaro, Italy.

Mushrooms can be considered a valuable source of natural bioactive compounds with potential polypharmacological effects due to their proven antimicrobial, antiviral, antitumor, and antioxidant activities. In order to identify new potential anticancer compounds, an chemical database of molecules extracted from both edible and non-edible fungal species was employed in a virtual screening against the isoform 7 of the Histone deacetylase (HDAC). This target is known to be implicated in different cancer processes, and in particular in both breast and ovarian tumors. In this work, we proposed the ibotenic acid as lead compound for the development of novel HDAC7 inhibitors, due to its antiproliferative activity in human breast cancer cells (MCF-7). These promising results represent the starting point for the discovery and the optimization of new HDAC7 inhibitors and highlight the interesting opportunity to apply the "drug repositioning" paradigm also to natural compounds deriving from mushrooms.
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http://dx.doi.org/10.3390/molecules25235524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728054PMC
November 2020

Key genetic elements, single and in clusters, underlying geographically dependent SARS-CoV-2 genetic adaptation and their impact on binding affinity for drugs and immune control.

J Antimicrob Chemother 2021 01;76(2):396-412

Department of Experimental Medicine, University of Rome 'Tor Vergata', Rome, Italy.

Objectives: To define key genetic elements, single or in clusters, underlying SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) evolutionary diversification across continents, and their impact on drug-binding affinity and viral antigenicity.

Methods: A total of 12 150 SARS-CoV-2 sequences (publicly available) from 69 countries were analysed. Mutational clusters were assessed by hierarchical clustering. Structure-based virtual screening (SBVS) was used to select the best inhibitors of 3-chymotrypsin-like protease (3CL-Pr) and RNA-dependent RNA polymerase (RdRp) among the FDA-approved drugs and to evaluate the impact of mutations on binding affinity of these drugs. The impact of mutations on epitope recognition was predicted following Grifoni et al. (Cell Host Microbe 2020.

27: 671-80.).

Results: Thirty-five key mutations were identified (prevalence: ≥0.5%), residing in different viral proteins. Sixteen out of 35 formed tight clusters involving multiple SARS-CoV-2 proteins, highlighting intergenic co-evolution. Some clusters (including D614GSpike + P323LRdRp + R203KN + G204RN) occurred in all continents, while others showed a geographically restricted circulation (T1198KPL-Pr + P13LN + A97VRdRp in Asia, L84SORF-8 + S197LN in Europe, Y541CHel + H504CHel + L84SORF-8 in America and Oceania). SBVS identified 20 best RdRp inhibitors and 21 best 3CL-Pr inhibitors belonging to different drug classes. Notably, mutations in RdRp or 3CL-Pr modulate, positively or negatively, the binding affinity of these drugs. Among them, P323LRdRp (prevalence: 61.9%) reduced the binding affinity of specific compounds including remdesivir while it increased the binding affinity of the purine analogues penciclovir and tenofovir, suggesting potential hypersusceptibility. Finally, specific mutations (including Y541CHel + H504CHel) strongly hampered recognition of Class I/II epitopes, while D614GSpike profoundly altered the structural stability of a recently identified B cell epitope target of neutralizing antibodies (amino acids 592-620).

Conclusions: Key genetic elements reflect geographically dependent SARS-CoV-2 genetic adaptation, and may play a potential role in modulating drug susceptibility and hampering viral antigenicity. Thus, a close monitoring of SARS-CoV-2 mutational patterns is crucial to ensure the effectiveness of treatments and vaccines worldwide.
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http://dx.doi.org/10.1093/jac/dkaa444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799093PMC
January 2021

Current status of antivirals and druggable targets of SARS CoV-2 and other human pathogenic coronaviruses.

Drug Resist Updat 2020 12 26;53:100721. Epub 2020 Aug 26.

Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy. Electronic address:

Coronaviridae is a peculiar viral family, with a very large RNA genome and characteristic appearance, endowed with remarkable tendency to transfer from animals to humans. Since the beginning of the 21st century, three highly transmissible and pathogenic coronaviruses have crossed the species barrier and caused deadly pneumonia, inflicting severe outbreaks and causing human health emergencies of inconceivable magnitude. Indeed, in the past two decades, two human coronaviruses emerged causing serious respiratory illness: severe acute respiratory syndrome coronavirus (SARS-CoV-1) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV), causing more than 10,000 cumulative cases, with mortality rates of 10 % for SARS-CoV-1 and 34.4 % for MERS-CoV. More recently, the severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) has emerged in China and has been identified as the etiological agent of the recent COVID-19 pandemic outbreak. It has rapidly spread throughout the world, causing nearly 22 million cases and ∼ 770,000 deaths worldwide, with an estimated mortality rate of ∼3.6 %, hence posing serious challenges for adequate and effective prevention and treatment. Currently, with the exception of the nucleotide analogue prodrug remdesivir, and despite several efforts, there is no known specific, proven, pharmacological treatment capable of efficiently and rapidly inducing viral containment and clearance of SARS-CoV-2 infection as well as no broad-spectrum drug for other human pathogenic coronaviruses. Another confounding factor is the paucity of molecular information regarding the tendency of coronaviruses to acquire drug resistance, a gap that should be filled in order to optimize the efficacy of antiviral drugs. In this light, the present review provides a systematic update on the current knowledge of the marked global efforts towards the development of antiviral strategies aimed at coping with the infection sustained by SARS-CoV-2 and other human pathogenic coronaviruses, displaying drug resistance profiles. The attention has been focused on antiviral drugs mainly targeting viral protease, RNA polymerase and spike glycoprotein, that have been tested in vitro and/or in clinical trials as well as on promising compounds proven to be active against coronaviruses by an in silico drug repurposing approach. In this respect, novel insights on compounds, identified by structure-based virtual screening on the DrugBank database endowed by multi-targeting profile, are also reported. We specifically identified 14 promising compounds characterized by a good in silico binding affinity towards, at least, two of the four studied targets (viral and host proteins). Among which, ceftolozane and NADH showed the best multi-targeting profile, thus potentially reducing the emergence of resistant virus strains. We also focused on potentially novel pharmacological targets for the development of compounds with anti-pan coronavirus activity. Through the analysis of a large set of viral genomic sequences, the current review provides a comprehensive and specific map of conserved regions across human coronavirus proteins which are essential for virus replication and thus with no or very limited tendency to mutate. Hence, these represent key druggable targets for novel compounds against this virus family. In this respect, the identification of highly effective and innovative pharmacological strategies is of paramount importance for the treatment and/or prophylaxis of the current pandemic but potentially also for future and unavoidable outbreaks of human pathogenic coronaviruses.
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http://dx.doi.org/10.1016/j.drup.2020.100721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448791PMC
December 2020

Pyrrolo[2',3':3,4]cyclohepta[1,2-][1,2]oxazoles, a New Class of Antimitotic Agents Active against Multiple Malignant Cell Types.

J Med Chem 2020 10 11;63(20):12023-12042. Epub 2020 Oct 11.

Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Via Archirafi 32, 90123 Palermo, Italy.

A new class of pyrrolo[2',3':3,4]cyclohepta[1,2-][1,2]oxazoles was synthesized for the treatment of hyperproliferative pathologies, including neoplasms. The new compounds were screened in the 60 human cancer cell lines of the NCI drug screen and showed potent activity with GI values reaching the nanomolar level, with mean graph midpoints of 0.08-0.41 μM. All compounds were further tested on six lymphoma cell lines, and eight showed potent growth inhibitory effects with IC values lower than 500 nM. Mechanism of action studies showed the ability of the new [1,2]oxazoles to arrest cells in the G2/M phase in a concentration dependent manner and to induce apoptosis through the mitochondrial pathway. The most active compounds inhibited tubulin polymerization, with IC values of 1.9-8.2 μM, and appeared to bind to the colchicine site. The G2/M arrest was accompanied by apoptosis, mitochondrial depolarization, generation of reactive oxygen species, and PARP cleavage.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901646PMC
October 2020

DJ-1 Proteoforms in Breast Cancer Cells: The Escape of Metabolic Epigenetic Misregulation.

Cells 2020 08 26;9(9). Epub 2020 Aug 26.

Laboratory of Proteomics, Research Center on Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Græcia Universityof Catanzaro, S Venuta University Campus, 88100 Catanzaro, Italy.

Enhanced glycolysis is a hallmark of breast cancer. In cancer cells, the high glycolytic flux induces carbonyl stress, a damaging condition in which the increase of reactive carbonyl species makes DNA, proteins, and lipids more susceptible to glycation. Together with glucose, methylglyoxal (MGO), a byproduct of glycolysis, is considered the main glycating agent. MGO is highly diffusible, enters the nucleus, and can react with easily accessible lysine- and arginine-rich tails of histones. Glycation adducts on histones undergo oxidization and further rearrange to form stable species known as advanced glycation end-products (AGEs). This modification alters nucleosomes stability and chromatin architecture deconstructing the histone code. Formation of AGEs has been associated with cancer, diabetes, and several age-related diseases. Recently, DJ-1, a cancer-associated protein that protects cells from oxidative stress, has been described as a deglycase enzyme. Although its role in cell survival results still controversial, in several human tumors, its expression, localization, oxidation, and phosphorylation were found altered. This work aimed to explore the molecular mechanism that triggers the peculiar cellular compartmentalization and the specific post-translational modifications (PTM) that, occurring in breast cancer cells, influences the DJ-1 dual role. Using a proteomic approach, we identified on DJ-1 a novel threonine phosphorylation (T125) that was found, by the in-silico tool scansite 4, as part of a putative Akt consensus. Notably, this threonine is in addition to histidine 126, a key residue involved in the formation of catalytic triade (glu18-Cys106-His126) inside the glioxalase active site of DJ. Interestingly, we found that pharmacological modulation of Akt pathway induces a functional tuning of DJ-1 proteoforms, as well as their shuttle from cytosol to nucleus, pointing out that pathway as critical in the development of DJ-1 pro-tumorigenic abilities. Deglycase activity of DJ-1 on histones proteins, investigated by coupling 2D tau gel with LC-MS/MS and 2D-TAU (Triton-Acid-Urea)-Western blot, was found correlated with its phosphorylation status that, in turn, depends from Akt activation. In normal conditions, DJ-1 acts as a redox-sensitive chaperone and as an oxidative stress sensor. In cancer cells, glycolytic rewiring, inducing increased reactive oxygen species (ROS) levels, enhances AGEs products. Alongside, the moderate increase of ROS enhances Akt signaling that induces DJ-1-phosphorylation. When phosphorylated DJ-1 increases its glyoxalase activity, the level of AGEs on histones decreases. Therefore, phospho-DJ-1 prevents glycation-induced histones misregulation and its Akt-related hyperactivity represents a way to preserve the epigenome landscape sustaining proliferation of cancer cells. Together, these results shed light on an interesting mechanism that cancer cells might execute to escape the metabolic induced epigenetic misregulation that otherwise could impair their malignant proliferative potential.
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http://dx.doi.org/10.3390/cells9091968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563694PMC
August 2020

In Silico Identification and Biological Evaluation of Antioxidant Food Components Endowed with IX and XII CA Inhibition.

Antioxidants (Basel) 2020 Aug 21;9(9). Epub 2020 Aug 21.

Dipartimento NEUROFARBA, Sezione di Scienze Farmaceutiche, Università degli Studi di Firenze, Sesto Fiorentino, 50019 Florence, Italy.

The tumor-associated isoenzymes CA IX and CA XII catalyze the hydration of carbon dioxide to bicarbonate and protons. These isoforms are highly overexpressed in many types of cancer, where they contribute to the acidification of the tumor environment, promoting tumor cell invasion and metastasis. In this work, in order to identify novel dual CA IX and XII inhibitors, virtual screening techniques and biological assays were combined. A structure-based virtual screening towards CA IX and XII was performed using a database of approximately 26,000 natural compounds. The best shared were submitted to a thermodynamic analysis and three promising best were identified and evaluated in terms of their CA IX and XII inhibitor activity. In vitro biological assays were in line with the theoretical studies and revealed that syringin, lithospermic acid, and (-)-dehydrodiconiferyl alcohol behave as good CA IX and CA XII dual inhibitors.
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http://dx.doi.org/10.3390/antiox9090775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555330PMC
August 2020

Improving the Treatment of Acute Lymphoblastic Leukemia.

Biochemistry 2020 Sep 23;59(35):3193-3200. Epub 2020 Aug 23.

School of Chemistry, Cardiff University, Park Place, Cardiff CF10 3AT, U.K.

l-Asparaginase (EC 3.5.1.1) was first used as a component of combination drug therapies to treat acute lymphoblastic leukemia (ALL), a cancer of the blood and bone marrow, almost 50 years ago. Administering this enzyme to reduce asparagine levels in the blood is a cornerstone of modern clinical protocols for ALL; indeed, this remains the only successful example of a therapy targeted against a specific metabolic weakness in any form of cancer. Three problems, however, constrain the clinical use of l-asparaginase. First, a type II bacterial variant of l-asparaginase is administered to patients, the majority of whom are children, which produces an immune response thereby limiting the time over which the enzyme can be tolerated. Second, l-asparaginase is subject to proteolytic degradation in the blood. Third, toxic side effects are observed, which may be correlated with the l-glutaminase activity of the enzyme. This Perspective will outline how asparagine depletion negatively impacts the growth of leukemic blasts, discuss the structure and mechanism of l-asparaginase, and briefly describe the clinical use of chemically modified forms of clinically useful l-asparaginases, such as Asparlas, which was recently given FDA approval for use in children (babies to young adults) as part of multidrug treatments for ALL. Finally, we review ongoing efforts to engineer l-asparaginase variants with improved therapeutic properties and briefly detail emerging, alternate strategies for the treatment of forms of ALL that are resistant to asparagine depletion.
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http://dx.doi.org/10.1021/acs.biochem.0c00354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497903PMC
September 2020

Inside Perspective of the Synthetic and Computational Toolbox of JAK Inhibitors: Recent Updates.

Molecules 2020 Jul 22;25(15). Epub 2020 Jul 22.

Dipartimento di Scienze della Salute, Università "Magna Græcia" di Catanzaro, Viale Europa, 88100 Catanzaro, Italy.

The mechanisms of inflammation and cancer are intertwined by complex networks of signaling pathways. Dysregulations in the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway underlie several pathogenic conditions related to chronic inflammatory states, autoimmune diseases and cancer. Historically, the potential application of JAK inhibition has been thoroughly explored, thus triggering an escalation of favorable results in this field. So far, five JAK inhibitors have been approved by the Food and Drug Administration (FDA) for the treatment of different diseases. Considering the complexity of JAK-depending processes and their involvement in multiple disorders, JAK inhibitors are the perfect candidates for drug repurposing and for the assessment of multitarget strategies. Herein we reviewed the recent progress concerning JAK inhibition, including the innovations provided by the release of JAKs crystal structures and the improvement of synthetic strategies aimed to simplify of the industrial scale-up.
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http://dx.doi.org/10.3390/molecules25153321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435994PMC
July 2020

Non-coding RNAs in cancer: platforms and strategies for investigating the genomic "dark matter".

J Exp Clin Cancer Res 2020 Jun 20;39(1):117. Epub 2020 Jun 20.

Laboratory of Translational Medical Oncology, Department of Experimental and Clinical Medicine, Magna Graecia University, Salvatore Venuta University Campus, 88100, Catanzaro, Italy.

The discovery of the role of non-coding RNAs (ncRNAs) in the onset and progression of malignancies is a promising frontier of cancer genetics. It is clear that ncRNAs are candidates for therapeutic intervention, since they may act as biomarkers or key regulators of cancer gene network. Recently, profiling and sequencing of ncRNAs disclosed deep deregulation in human cancers mostly due to aberrant mechanisms of ncRNAs biogenesis, such as amplification, deletion, abnormal epigenetic or transcriptional regulation. Although dysregulated ncRNAs may promote hallmarks of cancer as oncogenes or antagonize them as tumor suppressors, the mechanisms behind these events remain to be clarified. The development of new bioinformatic tools as well as novel molecular technologies is a challenging opportunity to disclose the role of the "dark matter" of the genome. In this review, we focus on currently available platforms, computational analyses and experimental strategies to investigate ncRNAs in cancer. We highlight the differences among experimental approaches aimed to dissect miRNAs and lncRNAs, which are the most studied ncRNAs. These two classes indeed need different investigation taking into account their intrinsic characteristics, such as length, structures and also the interacting molecules. Finally, we discuss the relevance of ncRNAs in clinical practice by considering promises and challenges behind the bench to bedside translation.
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http://dx.doi.org/10.1186/s13046-020-01622-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305591PMC
June 2020

BOPC1 Enantiomers Preparation and HuR Interaction Study. From Molecular Modeling to a Curious DEEP-STD NMR Application.

ACS Med Chem Lett 2020 May 28;11(5):883-888. Epub 2020 Jan 28.

Department of Drug Sciences, Medicinal Chemistry and Technology Section, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy.

The Hu family of RNA-binding proteins plays a crucial role in post-transcriptional processes; indeed, Hu-RNA complexes are involved in various dysfunctions (i.e., inflammation, neurodegeneration, and cancer) and have been recently proposed as promising therapeutic targets. Intrigued by this concept, our research efforts aim at identifying small molecules able to modulate HuR-RNA interactions, with a focus on subtype HuR, upregulated and dysregulated in several cancers. By applying structure-based design, we had already identified racemic - as promising HuR binder. In this Letter, we accomplished the enantio-resolution, the assignment of the absolute configuration, and the recognition study with HuR of enantiomerically pure -. For the first time, we apply DEEP (differential epitope mapping)-STD NMR to study the interaction of with HuR and compare its enantiomers, gaining information on ligand orientation and amino acids involved in the interaction, and thus increasing focus on the binding site model.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236254PMC
May 2020

New Dihydrothiazole Benzensulfonamides: Looking for Selectivity toward Carbonic Anhydrase Isoforms I, II, IX, and XII.

ACS Med Chem Lett 2020 May 13;11(5):852-856. Epub 2020 Feb 13.

Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy.

In the present study we investigated the structure-activity relationships of a new series of 4-[(3-ethyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides (-). All synthesized compounds, with the exception of compound , preferentially inhibit off-target hCA II isoform. Within the series, compound , bearing a 2,4-dichorophenyl substituent in position 4 of the dihydrothiazole ring, was the most potent and selective toward hCA II with an inhibitory activity in the low nanomolar range.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236253PMC
May 2020

Multi-Targeting Bioactive Compounds Extracted from Essential Oils as Kinase Inhibitors.

Molecules 2020 May 6;25(9). Epub 2020 May 6.

Dipartimento di Scienze della Salute, Università "Magna Græcia" di Catanzaro, Campus Universitario "S. Venuta", Viale Europa, Loc. Germaneto, 88100 Catanzaro, Italy.

Essential oils (EOs) are popular in aromatherapy, a branch of alternative medicine that claims their curative effects. Moreover, several studies reported EOs as potential anti-cancer agents by inducing apoptosis in different cancer cell models. In this study, we have considered EOs as a potential resource of new kinase inhibitors with a polypharmacological profile. On the other hand, computational methods offer the possibility to predict the theoretical activity profile of ligands, discovering dangerous off-targets and/or synergistic effects due to the potential multi-target action. With this aim, we performed a Structure-Based Virtual Screening (SBVS) against X-ray models of several protein kinases selected from the Protein Data Bank (PDB) by using a chemoinformatics database of EOs. By evaluating theoretical binding affinity, 13 molecules were detected among EOs as new potential kinase inhibitors with a multi-target profile. The two compounds with higher percentages in the EOs were studied more in depth by means Induced Fit Docking (IFD) protocol, in order to better predict their binding modes taking into account also structural changes in the receptor. Finally, given its good binding affinity towards five different kinases, cinnamyl cinnamate was biologically tested on different cell lines with the aim to verify the antiproliferative activity. Thus, this work represents a starting point for the optimization of the most promising EOs structure as kinase inhibitors with multi-target features.
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http://dx.doi.org/10.3390/molecules25092174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249159PMC
May 2020

New deferiprone derivatives as multi-functional cholinesterase inhibitors: design, synthesis and in vitro evaluation.

Eur J Med Chem 2020 Jul 25;198:112350. Epub 2020 Apr 25.

Department of Chimica e Tecnologia del Farmaco, Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185, Rome, Italy. Electronic address:

In order to obtain multi-functional molecules for Alzheimer's disease, a series of deferiprone derivatives has been synthesized and evaluated in vitro with the hypothesis that they can restore the cholinergic tone and attenuate the dyshomeostasis of the metals mainly involved in the pathology. These compounds were designed as dual binding site AChE inhibitors: they possess an arylalkylamine moiety connected via an alkyl chain to a 3-hydroxy-4-pyridone fragment, to allow the simultaneous interaction with catalytic active site (CAS) and peripheral anionic site (PAS) of the enzyme. Deferiprone moiety and 2-aminopyridine, 2-aminopyrimidine or 2,4-diaminopyrimidine groups have been incorporated into these compounds, in order to obtain molecules potentially able to chelate bio-metals colocalized in Aβ plaques and involved in the generation of radical species. Synthesized compounds were tested by enzymatic inhibition studies towards EeAChE and eqBChE using Ellman's method. The most potent EeAChE inhibitor is compound 5a, with a K of 788 ± 51 nM, while the most potent eqBChE inhibitors are compounds 12 and 19, with K values of 182 ± 18 nM and 258 ± 25 nM respectively. Selected compounds, among the most potent cholinesterases inhibitors, were able to form complex with iron and in some cases with copper and zinc. Moreover, these compounds were characterized by low toxicity on U-87 MG Cell Line from human brain (glioblastoma astrocytoma).
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http://dx.doi.org/10.1016/j.ejmech.2020.112350DOI Listing
July 2020

N-1,2,3-triazole-isatin derivatives for cholinesterase and β-amyloid aggregation inhibition: A comprehensive bioassay study.

Bioorg Chem 2020 05 19;98:103753. Epub 2020 Mar 19.

LAQV-REQUIMTE, University of Évora, Institute for Research and Advanced Studies, Rua Romão Ramalho, 59, 7000 Évora, Portugal; Chemistry Department, School of Science and Technology, University of Évora, Rua Romão Ramalho 59, 7000-671 Évora, Portugal. Electronic address:

Our goal was the evaluation of a series of N-1,2,3-triazole-isatin derivatives for multi-target activity which included cholinesterase (ChE) inhibition and β-amyloid (Aβ) peptide anti-aggregation. The compounds have shown considerable promise as butyrylcholinesterase (BuChE) inhibitors. Although the inhibition of eel acetylcholinesterase (eeAChE) was weak, the inhibitions against equine BuChE (eqBuChE) and human BuChE (hBuChE) were more significant with a best inhibition against eqBuChE of 0.46 μM. In some cases, these molecules gave better inhibitions for hBuChE than eqBuChE. For greater insights into their mode of action, molecular docking studies were carried out, followed by STD-NMR validation. In addition, some of these compounds showed weak Aβ anti-aggregation activity. Hepatotoxicity studies showed that they were non-hepatoxic and neurotoxicity studies using neurite outgrowth experiments led to the conclusion that these compounds are only weakly neurotoxic.
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http://dx.doi.org/10.1016/j.bioorg.2020.103753DOI Listing
May 2020

Multi-target drug discovery: An opportunity for novel and repurposed bioactive compounds.

Eur J Med Chem 2020 Apr 25;192:112188. Epub 2020 Feb 25.

Università Magna Græcia di Catanzaro and Net4Science Academic Spinoff, Campus "S. Venuta", Viale Europa, 88100, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.ejmech.2020.112188DOI Listing
April 2020

Folding intermediate states of the parallel human telomeric G-quadruplex DNA explored using Well-Tempered Metadynamics.

Sci Rep 2020 02 21;10(1):3176. Epub 2020 Feb 21.

Dipartimento di Scienze della Salute, Università "Magna Græcia" di Catanzaro, Campus Salvatore Venuta, Viale Europa, 88100, Catanzaro, Italy.

An increasingly comprehension of the folding intermediate states of DNA G-quadruplexes (G4s) is currently an important scientific challenge, especially for the human telomeric (h-tel) G4s-forming sequences, characterized by a highly polymorphic nature. Despite the G-triplex conformation was proposed as one of the possible folding intermediates for the antiparallel and hybrid h-tel G4s, for the parallel h-tel topology with an all-anti guanine orientation, a vertical strand-slippage involving the G-triplets was proposed in previous works through microseconds-long standard molecular dynamics simulations (MDs). Here, in order to get further insights into the vertical strand-slippage and the folding intermediate states of the parallel h-tel G4s, we have carried out a Well-Tempered Metadynamics simulation (WT-MetaD), which allowed us to retrieve an ensemble of six G4s having two/G-tetrad conformations derived by the G-triplets vertical slippage. The insights highlighted in this work are aimed at rationalizing the mechanistic characterisation of the parallel h-tel G4 folding process.
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http://dx.doi.org/10.1038/s41598-020-59774-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035250PMC
February 2020

Pyrazolones Activate the Proteasome by Gating Mechanisms and Protect Neuronal Cells from β-Amyloid Toxicity.

ChemMedChem 2020 02 17;15(3):302-316. Epub 2019 Dec 17.

Consiglio Nazionale delle Ricerche, Istituto di Cristallografia, Via P. Gaifami 18, 95126, Catania, Italy.

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an "open gate" mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from β-amyloid (Aβ) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aβ degradation by proteasome in a dose-dependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment.
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http://dx.doi.org/10.1002/cmdc.201900612DOI Listing
February 2020

Mediterranean products as promising source of multi-target agents in the treatment of metabolic syndrome.

Eur J Med Chem 2020 Jan 21;186:111903. Epub 2019 Nov 21.

Net4Science srl, Università "Magna Græcia", Campus Salvatore Venuta, Viale Europa, 88100, Catanzaro, Italy; Department of Experimental and Clinical Medicine, "Magna Græcia" University, Campus Salvatore Venuta, Viale Europa, 88100, Catanzaro, Italy.

Alteration of nutritional habits play an essential role on the risk of developing Metabolic Syndrome (MetS). Several epidemiological studies have shown that assuming diets rich of foods included in the Mediterranean diet (MetDiet) pattern like, such as olive oil, nuts, fruit, fiber, vegetables, wine and grain cereals has protective effects on the different risk factors characterizing the MetS. The beneficial effects of the MetDiet in the MetS are mainly due to the antioxidant and anti-inflammatory properties of the most abundant phytochemical components of such foods as polyphenols like resveratrol and oleuropein, allyl sulfides, ellagic acid, mono- and poly-unsaturated fatty acids (MUFA and PUFA), tocopherols and flavonoids like quercetin, which have shown positive results in the prevention of cardiovascular diseases (CVDs), with related risk factors, like hypertension, hypercholesterolemia and obesity. In this review, we highlighted the multi-target activities of the bioactive components contained in some foods typical of the Mediterranean area like olive oil, onion, liquorice, rosemary, oregano, hazelnut, pistachio, "Melannurca" apple, red wine, hot pepper, Citrus sp. fruits, saffron and garlic, with particular focus on their impact on health outcomes in relation to MetS main key factors, such as insulin resistance (IR) and type 2 diabetes mellitus (T2DM), endothelial dysfunctions, inflammatory response, oxidative stress and dyslipidaemic and hypercholesterolemic effects.
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http://dx.doi.org/10.1016/j.ejmech.2019.111903DOI Listing
January 2020

Molecular modelling of epitopes recognized by neoplastic B lymphocytes in Chronic Lymphocytic Leukemia.

Eur J Med Chem 2020 Jan 1;185:111838. Epub 2019 Nov 1.

Department of Health Sciences, University "Magna Græcia" of Catanzaro, Campus "S. Venuta", Viale Europa, Germaneto, 88100, Catanzaro, Italy; Net4Science srl, University "Magna Græcia", Campus Salvatore Venuta, Viale Europa, Catanzaro, 88100, Italy. Electronic address:

Identification of epitopes recognized by tumour B cells could provide insights into the molecular mechanisms of B cell tumorigenesis through aberrant B cell receptor (BCR) signalling. Here, we analysed the structure of eleven peptides binders of BCRs expressed in Chronic Lymphocytic Leukemia (CLL) patients in order to identify the chemical features required for cross-reactive binding to different CLL clonotypes. Four cross-reactive (CR) and seven no-cross-reactive (NCR) peptides were analysed by means of GRID molecular interaction fields, ligand-based pharmacophore and 3D-QSAR approaches. Based on pharmacophore model, two peptides were generated by specific amino acids substitutions of the parental NCR peptides; these new peptides resumed the common chemical features of CR peptides and bound the CLL BCR clonotypes recognized by CR peptides and parental NCR peptides. Thus, our computational approach guided the pharmacophore modelling of CR peptides. In perspective, peptide binders of CLL BCR clonotypes could represent a powerful tool for computational modelling of epitopes recognized by tumour B cells clones.
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http://dx.doi.org/10.1016/j.ejmech.2019.111838DOI Listing
January 2020

A drug repurposing screening reveals a novel epigenetic activity of hydroxychloroquine.

Eur J Med Chem 2019 Dec 17;183:111715. Epub 2019 Sep 17.

Department of Experimental and Clinical Medicine, Catanzaro, Italy.

Multiple myeloma (MM) is an incurable hematological malignancy driven by several genetic and epigenetic alterations. The hyperactivation of the Polycomb repressive complex 2 (PRC2), a multi-subunit oncogenic histone methyltransferase, has been implicated in the pathogenesis of this malignancy. Upon protein-protein interaction (PPI) between the catalytic subunit EZH2 and EED, PRC2 primarily methylates lysine 27 of histone H3 (H3K27me3), thus modulating the chromatin structure and inducing transcriptional repression. Herein, we highlight a new mechanism of action that can contribute to explain the anti-tumor activity of hydroxychloroquine (HCQ), an anti-malaric agent also known as autophagy inhibitor. By structural studies, we demonstrate that HCQ inhibits the allosteric binding of PRC2 to EED within the H3K27me3-binding pocket, thus antagonizing the PRC2 catalytic activity. In silico results are compatible with the significant reduction of the H3K27me3 levels in MM cells exerted by HCQ. Overall, these findings disclose a novel epigenetic activity of HCQ with potential implications for its clinical repositioning.
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http://dx.doi.org/10.1016/j.ejmech.2019.111715DOI Listing
December 2019

ABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron Stigmas.

Plant Cell 2019 11 23;31(11):2789-2804. Epub 2019 Sep 23.

ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, C.R. Casaccia, 00123, Rome, Italy

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in leaves.plantcell;31/11/2789/FX1F1fx1.
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http://dx.doi.org/10.1105/tpc.19.00193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881118PMC
November 2019

Review about the multi-target profile of resveratrol and its implication in the SGK1 inhibition.

Eur J Med Chem 2019 Dec 5;183:111675. Epub 2019 Sep 5.

Dipartimento di Medicina Sperimentale e Clinica, University "Magna Græcia" of Catanzaro, Viale Europa, 88100, Catanzaro, Italy. Electronic address:

Resveratrol (trans-3,4',5-trihydroxystilbene) is a polyphenolic natural product with a well-known polypharmacological profile that places it among the multi-target-directed ligands (MTDLs). Given its protective action against a wide number of chronic diseases, in this review, we introduce a general overview about the cardioprotective and antioxidant effects, the antidiabetic, neuroprotective and anti-inflammatory effects of this polyphenol. In the second part of the manuscript, we focused our attention on the anticancer activity of Resveratrol, given the alteration of many different signaling pathways, leading to suppression of tumor cell proliferation in numerous cancer types. Among the several anticancer targets involved in the mechanism of action of Resveratrol, here we introduce experimental and molecular modeling studies performed against the SGK1 protein as a novel anticancer target of Resveratrol. SGK1 inhibitors have been demonstrated to inhibit cell growth of different cancer cells. We demonstrated that resveratrol inhibits SGK1 in vitro and in intact cells, affecting proliferation and survival of HUH7 human hepatoma cells. Our findings demonstrate that resveratrol may function as a SGK1 inhibitor, suggesting possible applications in sodium retention and cancer.
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http://dx.doi.org/10.1016/j.ejmech.2019.111675DOI Listing
December 2019

Targeting multiple G-quadruplex-forming DNA sequences: Design, biophysical and biological evaluations of indolo-naphthyridine scaffold derivatives.

Eur J Med Chem 2019 Nov 16;182:111627. Epub 2019 Aug 16.

Dipartimento di Scienze della Salute, Università "Magna Græcia", Campus Salvatore Venuta, Viale Europa, 88100, Catanzaro, Italy; Net4Science srl, Università "Magna Græcia", Campus Salvatore Venuta, Viale Europa, 88100, Catanzaro, Italy.

It is well recognized that the non-canonical DNA structures known as G-quadruplexes (G4s) have a potential anticancer significance and several compounds have been discovered and evaluated as promising G4 binders with anticancer activity. Here, starting from a promising hit with an indolo-naphthyridine scaffold, a small series of five indolo-naphthyridine based derivatives have been designed and evaluated as G4-targeting compounds. FRET biophysical studies were performed on multiple DNA G4 structures, leading to the identification of a multi-target G4 stabilizer with a slight preference for the c-KIT1 and a good G4 over duplex selectivity. The good affinity of this compound against c-KIT1 G4 was also confirmed by SPR and MST experiments, while biological assays revealed its cytotoxic activity on tumour cells. Finally, Molecular Dynamics simulations helped to elucidate the stabilization effect of the selected compound against the c-KIT1 G4.
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http://dx.doi.org/10.1016/j.ejmech.2019.111627DOI Listing
November 2019

Chromenone derivatives as a versatile scaffold with dual mode of inhibition of HIV-1 reverse transcriptase-associated Ribonuclease H function and integrase activity.

Eur J Med Chem 2019 Nov 12;182:111617. Epub 2019 Aug 12.

Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria SS554, 09042, Monserrato, CA, Italy; Genetics and Biomedical Research Institute, National Research Council (CNR), Cittadella Universitaria SS554, 09042, Monserrato, CA, Italy. Electronic address:

A number of compounds targeting different processes of the Human Immunodeficiency Virus type 1 (HIV-1) life cycle have been developed in the continuing fight against AIDS. Coumarin-based molecules already proved to act as HIV-1 Protease (PR) or Integrase (IN) inhibitors and also to target HIV-1 reverse transcriptase (RT), blocking the DNA-dependent DNA-polymerase activity or the RNA-dependent DNA-polymerase activity working as common NNRTIs. In the present study, with the aim to exploit a coumarin-based scaffold to achieve the inhibition of multiple viral coded enzymatic functions, novel 4-hydroxy-2H, 5H-pyrano (3, 2-c) chromene-2, 5-dione derivatives were synthesized. The modeling studies calculated the theoretical binding affinity of the synthesized compounds on both HIV-1 IN and RT-associated Ribonuclease H (RNase H) active sites, which was confirmed by biological assays. Our results provide a basis for the identification of dual HIV-1 IN and RT RNase H inhibitors compounds.
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http://dx.doi.org/10.1016/j.ejmech.2019.111617DOI Listing
November 2019