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Multiple Myeloma (MM) is an incurable malignancy characterised by altered expression of coding and non-coding genes promoting tumour growth and drug resistance. Although the crucial role of long non-coding RNAs (lncRNAs) in MM is clearly established, the function of the non-coding RNAome, which might allow the design of novel therapeutics, is largely unknown. We performed an unbiased CRISPR-Cas9 loss-of-function screen of 671 lncRNAs in MM cells and their Bortezomib (BZB)-resistant derivative. To rank functionally and clinically relevant candidates, we designed and used a bioinformatic prioritisation pipeline combining functional data from cellular screens with prognostic and transcriptional data from MM patients. With this approach, we unveiled and prioritised 8 onco-lncRNAs essential for MM cell fitness, associated with high expression and poor prognosis in MM patients. The previously uncharacterised RP11-350G8.5 emerged as the most promising target, irrespective of BZB resistance. We i) demonstrated the anti-tumoral effect obtained by RP11-350G8.5 inhibition in vitro and in vivo; ii) highlighted a modulation of the unfolded protein response and the induction of immunogenic cell death triggered by the RP11-350G8.5 knock-out, via RNA-sequencing and molecular studies; iii) characterised its cytoplasmic homing through RNA-FISH; iv) predicted its 2D structure and identified 2 G-quadruplex and 3 hairpin-forming regions by biophysical assays, including Thioflavin T, 1H-NMR and circular dichroism to pave the way to the development of novel targeted therapeutics. Overall we provided innovative insights about unexplored lncRNAs in MM and identified RP11-350G8.5 as an oncogenic target for treatment-naïve and BZB-resistant MM patients.
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Hematological cancers are among the most common cancers in adults and children. Despite significant improvements in therapies, many patients still succumb to the disease. Therefore, novel therapies are needed. The Wiskott-Aldrich syndrome protein (WASp) family regulates actin assembly in conjunction with the Arp2/3 complex, a ubiquitous nucleation factor. WASp is expressed exclusively in hematopoietic cells and exists in two allosteric conformations: autoinhibited or activated. Here, we describe the development of EG-011, a first-in-class small molecule activator of the WASp auto-inhibited form. EG-011 possesses in vitro and in vivo anti-tumor activity as a single agent in lymphoma, leukemia, and multiple myeloma, including models of secondary resistance to PI3K, BTK, and proteasome inhibitors. The in vitro activity was confirmed in a lymphoma xenograft. Actin polymerization and WASp binding was demonstrated using multiple techniques. Transcriptome analysis highlighted homology with drugs-inducing actin polymerization.
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Sigma-1 receptor (S1R) is involved in a large array of biological functions due to its ability to interact with various proteins and ion channels. Crystal structures of human S1R revealed the trimeric organization for which each protomer comprises the ligand binding pocket. This study applied a multistep computational procedure to develop a pharmacophore model obtained from molecular dynamics simulations of available cocrystal structures of well-known S1R ligands. Apart from the well-established positive ionizable and hydrophobic features, the obtained model included an additional specific hydrophobic feature and different excluded volumes, thus increasing the selectivity of the model as well as a more detailed determination of the distance between two essential features. The obtained pharmacophore model passed the validation test by receiver operating characteristic (ROC) curve analysis of active and inactive S1R ligands. Finally, the pharmacophoric performance was experimentally investigated through the synthesis and binding assay of new 4-phenylpiperazine-based compounds. The most active new ligand 2-(3-methyl-1-piperidyl)-1-(4-phenylpiperazin-1-yl)ethanone (3) showed an S1R affinity close to the reference compound haloperidol (Ki values of 4.8 and 2.6 nM, respectively). The proposed pharmacophore model can represent a useful tool to design and discover new potent S1R ligands.
Assuntos
Simulação de Dinâmica Molecular , Receptores sigma , Receptor Sigma-1 , Receptores sigma/metabolismo , Receptores sigma/química , Ligantes , Humanos , Piperazinas/química , Piperazinas/metabolismo , Ligação Proteica , Sítios de Ligação , Conformação ProteicaRESUMO
Being aware of the need to develop more efficient therapies against cancer, herein we disclose an innovative approach for the design of selective antiproliferative agents. We have accomplished the conjugation of a coumarin fragment with lipophilic cations (triphenylphosphonium salts, guanidinium) for providing mitochondriotropic agents that simultaneously target also carbonic anhydrases IX and XII, involved in the development and progression of cancer. The new compounds prepared herein turned out to be strong inhibitors of carbonic anhydrases IX and XII of human origin (low-to-mid nM range), also endowed with high selectivity, exhibiting negligible activity towards cytosolic CA isoforms. Key interactions with the enzyme were analysed using docking and molecular dynamics simulations. Regarding their in vitro antiproliferative activities, an increase of the tether length connecting both pharmacophores led to a clear improvement in potency, reaching the submicromolar range for the lead compounds, and an outstanding selectivity towards tumour cell lines (S.I. up to >357). Cytotoxic effects were also analysed on MDR cell lines under hypoxic and normoxic conditions. Chemoresistance exhibited by phosphonium salts, and not by guanidines, against MDR cells was based on the fact that the former were found to be substrates of P-glycoprotein (P-gp), the pump responsible for extruding foreign chemicals; this situation was reversed by administrating tariquidar, a third generation P-gp inhibitor. Moreover, phosphonium salts provoked a profound depolarization of mitochondria membranes from tumour cells, thus probably compromising their oxidative metabolism. To gain insight into the mode of action of title compounds, continuous live cell microscopy was employed; interestingly, this technique revealed two different antiproliferative mechanisms for both families of mitocans. Whereas phosphonium salts had a cytostatic effect, blocking cell division, guanidines led to cell death via apoptosis.
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Antineoplásicos , Anidrases Carbônicas , Compostos Organofosforados , Humanos , Anidrases Carbônicas/metabolismo , Sais , Relação Estrutura-Atividade , Antígenos de Neoplasias/metabolismo , Antineoplásicos/química , Cumarínicos/química , Guanidinas , Inibidores da Anidrase Carbônica/química , Estrutura MolecularRESUMO
A series of tacrine-donepezil hybrids were synthesized as potential multifunctional anti-Alzheimer's disease (AD) compounds. For this purpose, tacrine and the benzylpiperidine moiety of donepezil were fused with a hydrazone group to achieve a small library of tacrine-donepezil hybrids. In agreement with the design, all compounds showed inhibitory activity toward both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with IC50 values in the low micromolar range. Kinetic studies on the most potent cholinesterase (ChE) inhibitors within the series showed a mixed-type inhibition mechanism on both enzymes. Also, the docking studies indicated that the compounds inhibit ChEs by dual binding site (DBS) interactions. Notably, tacrine-donepezil hybrids also exhibited significant neuroprotection against H2O2-induced cell death in a differentiated human neuroblastoma (SH-SY5Y) cell line at concentrations close to their IC50 values on ChEs and showed high to medium blood-brain barrier (BBB) permeability on human cerebral microvascular endothelial cells (HBEC-5i). Besides, the compounds do not cause remarkable toxicity in a human hepatocellular carcinoma cell line (HepG2) and SH-SY5Y cells. Additionally, the compounds were predicted to also have good bioavailability. Among the tested compounds, H4, H16, H17, and H24 stand out with their biological profile. Taken together, the proposed novel tacrine-donepezil scaffold represents a promising starting point for the development of novel anti-ChE multifunctional agents against AD.
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Acetilcolinesterase , Doença de Alzheimer , Barreira Hematoencefálica , Butirilcolinesterase , Inibidores da Colinesterase , Donepezila , Desenho de Fármacos , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores , Tacrina , Tacrina/farmacologia , Tacrina/química , Humanos , Donepezila/farmacologia , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Doença de Alzheimer/tratamento farmacológico , Butirilcolinesterase/metabolismo , Relação Estrutura-Atividade , Acetilcolinesterase/metabolismo , Barreira Hematoencefálica/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Estrutura Molecular , Relação Dose-Resposta a Droga , Células Hep G2 , Linhagem Celular TumoralRESUMO
The SARS-CoV-2 main protease (Mpro) is a crucial enzyme for viral replication and has been considered an attractive drug target for the treatment of COVID-19. In this study, virtual screening techniques and in vitro assays were combined to identify novel Mpro inhibitors starting from around 8000 FDA-approved drugs. The docking analysis highlighted 17 promising best hits, biologically characterized in terms of their Mpro inhibitory activity. Among them, 7 cephalosporins and the oral anticoagulant betrixaban were able to block the enzyme activity in the micromolar range with no cytotoxic effect at the highest concentration tested. After the evaluation of the degree of conservation of Mpro residues involved in the binding with the studied ligands, the ligands' activity on SARS-CoV-2 replication was assessed. The ability of betrixaban to affect SARS-CoV-2 replication associated to its antithrombotic effect could pave the way for its possible use in the treatment of hospitalized COVID-19 patients.
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COVID-19 , Humanos , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/química , Reposicionamento de Medicamentos , Ligantes , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica MolecularRESUMO
In this work, new steroidal aromatase inhibitors (AIs) were designed, synthesized, and tested. In one approach, C-ring substituted steroids namely those functionalized at C-11 position with an α or ß hydroxyl group or with a carbonyl group as well as C-9/C-11 steroidal olefins and epoxides were studied. It was found that the carbonyl group at C-11 is more beneficial for aromatase inhibition than the hydroxyl group, and that the C-ring epoxides were more potent than the C-ring olefins, leading to the discovery of a very strong AI, compound 7, with an IC50 of 0.011 µM, better than Exemestane, the steroidal AI in clinical use, which presents an IC50 of 0.050 µM. In another approach, we explored the biological activity of A-ring C-1/C-2 steroidal olefins and epoxides in relation to aromatase inhibition and compared it with the biological activity of C-ring C-9/C-11 steroidal olefins and epoxides. On the contrary to what was observed for the C-ring olefins and epoxides, the A-ring epoxides were less potent than A-ring olefins. Finally, the effect of 7ß-methyl substitution on aromatase inhibition was compared with 7α-methyl substitution, showing that 7ß-methyl is better than 7α-methyl substitution. Molecular modelling studies showed that the 7ß-methyl on C-7 seems to protrude into the opening to the access channel of aromatase in comparison to the 7α-methyl. This comparison led to find the best steroidal AI (12a) of this work with IC50 of 0.0058 µM. Compound 12a showed higher aromatase inhibition capacity than two of the three AIs currently in clinical use.
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Inibidores da Aromatase , Aromatase , Inibidores da Aromatase/farmacologia , Aromatase/metabolismo , Esteroides/farmacologia , Relação Estrutura-Atividade , Compostos de EpóxiRESUMO
Designing and discovering compounds for dual-target inhibitors is challenging to synthesize new, safer, and more efficient drugs than single-target drugs, especially to treat multifactorial diseases such as cancer. The simultaneous regulation of multiple targets might represent an alternative synthetic approach to optimize patient compliance and tolerance, minimizing the risk of target-based drug resistance due to the modulation of a few targets. To this end, we conceived for the first time the design and synthesis of dual-ligands σR/HDACi to evaluate possible employment as innovative candidates to address this complex disease. Among all synthesized compounds screened for several tumoral cell lines, compound 6 (Kiσ1R = 38 ± 3.7; Kiσ2R = 2917 ± 769 and HDACs IC50 = 0.59 µM) is the most promising candidate as an antiproliferative agent with an IC50 of 0.9 µM on the HCT116 cell line and no significant toxicity to normal cells. Studies of molecular docking, which confirmed the affinity over σ1R and a pan-HDACs inhibitory behavior, support a possible balanced affinity and activity between both targets.
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Sistemas de Liberação de Medicamentos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Linhagem Celular Tumoral , Células HCT116RESUMO
Tumour associated carbonic anhydrases (CAs) IX and XII have been recognised as potential targets for the treatment of hypoxic tumours. Therefore, considering the high pharmacological potential of the chromene scaffold as selective ligand of the IX and XII isoforms, two libraries of compounds, namely 2H-chromene and 7H-furo-chromene derivatives, with diverse substitution patterns were designed and synthesised. The structure of the newly synthesised compounds was characterised and their inhibitory potency and selectivity towards human CA off target isoforms I, II and cancer-associated CA isoforms IX and XII were evaluated. Most of the compounds inhibit CA isoforms IX and XII with no activity against the I and II isozymes. Thus, while the potency was influenced by the substitution pattern along the chromene scaffold, the selectivity was conserved along the series, confirming the high potential of both 2H-chromene and 7H-furo-chromene scaffolds for the design of isozyme selective inhibitors.
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Anidrases Carbônicas , Neoplasias , Humanos , Anidrase Carbônica IX , Anidrases Carbônicas/metabolismo , Anidrase Carbônica I , Anidrase Carbônica II , Relação Estrutura-Atividade , Inibidores da Anidrase Carbônica/farmacologia , Inibidores da Anidrase Carbônica/química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Antígenos de Neoplasias/química , Benzopiranos/farmacologia , Isoenzimas/metabolismo , Estrutura MolecularRESUMO
Nowadays, RNA is an attractive target for the design of new small molecules with different pharmacological activities. Among several RNA molecules, long noncoding RNAs (lncRNAs) are extensively reported to be involved in cancer pathogenesis. In particular, the overexpression of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of multiple myeloma (MM). Starting from the crystallographic structure of the triple-helical stability element at the 3'-end of MALAT1, we performed a structure-based virtual screening of a large commercial database, previously filtered according to the drug-like properties. After a thermodynamic analysis, we selected five compounds for the in vitro assays. Compound M5, characterized by a diazaindene scaffold, emerged as the most promising molecule enabling the destabilization of the MALAT1 triplex structure and antiproliferative activity on in vitro models of MM. M5 is proposed as a lead compound to be further optimized for improving its affinity toward MALAT1.
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RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/química , Relação Estrutura-AtividadeRESUMO
Protein-protein interactions (PPI) represent attractive targets for drug design. Thus, aiming at a deeper insight into the HSV-1 envelope glycoprotein D (gD), protein-protein docking and dynamic simulations of gD-HVEM and gD-Nectin-1 complexes were performed. The most stable complexes and the pivotal key residues useful for gD to anchor human receptors were identified and used as starting points for a structure-based virtual screening on a library of both synthetic and designed 1,2,3-triazole-based compounds. Their binding properties versus gD interface with HVEM and Nectin-1 along with their structure-activity relationships (SARs) were evaluated. Four [1,2,3]triazolo[4,5-b]pyridines were identified as potential HSV-1 gD inhibitors, for their good theoretical affinity towards all conformations of HSV-1 gD. Overall, this study suggests promising basis for the design of new antiviral agents targeting gD as a valuable strategy to prevent viral attachment and penetration into the host cell.
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Herpesvirus Humano 1 , Humanos , Nectinas/metabolismo , Herpesvirus Humano 1/metabolismo , Moléculas de Adesão Celular/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/químicaRESUMO
Multiple myeloma (MM) is an aggressive and incurable disease for most patients, characterized by periods of treatment, remission and relapse. The introduction of new classes of drugs, such as proteasome inhibitors (PIs), has improved survival outcomes in these patient populations. The proteasome is the core of the ubiquitin-proteasome system (UPS), a complex and conserved pathway involved in the control of multiple cellular processes, including cell cycle control, transcription, DNA damage repair, protein quality control and antigen presentation. To date, PIs represent the gold standard for the treatment of MM. Bortezomib was the first PI approved by the FDA, followed by next generation of PIs, namely carfilzomib and ixazomib. Natural agents play an important role in anti-tumor drug discovery, and many of them have recently been reported to inhibit the proteasome, thus representing a new potential source of anti-MM drugs. Based on the pivotal biological role of the proteasome and on PIs' significance in the management of MM, in this review we aim to briefly summarize recent evidence on natural compounds capable of inhibiting the proteasome, thus triggering anti-MM activity.
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Antineoplásicos , Mieloma Múltiplo , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Complexo de Endopeptidases do Proteassoma , Antineoplásicos/efeitos adversos , Bortezomib/uso terapêuticoRESUMO
BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.
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DNA Ligase Dependente de ATP , Reparo do DNA , Flavonoides , Mieloma Múltiplo , Animais , Camundongos , Anexina A5/genética , Anexina A5/metabolismo , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligases/química , DNA Ligases/genética , DNA Ligases/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Fenóis , Proteínas Recombinantes/metabolismoRESUMO
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 (Saccharomyces cerevisiae), 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 Crocus sativus 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 C. sativus 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 Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.
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Transportadores de Cassetes de Ligação de ATP/metabolismo , Carotenoides/metabolismo , Crocus/metabolismo , Proteínas de Plantas/metabolismo , Vacúolos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Vias Biossintéticas , Clonagem Molecular , Crocus/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Cinética , Extratos Vegetais , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Distribuição Tecidual/fisiologia , Nicotiana/genética , Nicotiana/metabolismoRESUMO
The chromane ring system is widely distributed in nature and has proven to be a highly potent pharmacophore in medicinal chemistry, which includes the area of Alzheimer's and Parkinson's diseases. We report on the development of a gem-dimethylchroman-4-ol family that was shown to give good inhibition of equine serum butyrylcholinesterase (eqBuChE) (in the range 2.9 - 7.3 µM) and in the same range of currently used drugs. We also synthesized a small library of gem-dimethylchroman-4-amine compounds, via a simple reductive amination of the corresponding chromanone precursor, that were also selective for eqBuChE presenting inhibitions in the range 7.6 - 67 µM. Kinetic studies revealed that they were mixed inhibitors. Insights into their mechanism of action were obtained through molecular docking and STD-NMR experiments, and the most active examples showed excellent drug-likeness and pharmacological properties predicted using Swiss-ADME. We also prepared a set of propargyl gem-dimethylchromanamines, for monoamine oxidase (MAO) inhibition but they were only moderately active (the best being 28% inhibition at 1 µM on MAO-B). Overall, our compounds were found to be best suited as inhibitors for BuChE.
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Doença de Alzheimer , Doença de Alzheimer/tratamento farmacológico , Animais , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Cavalos , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/farmacologia , Relação Estrutura-AtividadeRESUMO
The insurgence of drug resistance in treating Multiple Myeloma (MM) still represents a major hamper in finding effective treatments, although over the past decades new classes of drugs, such as proteasome inhibitors and immunomodulatory drugs, have been discovered. Recently, our research team, within a Nature-Aided Drug Discovery project, isolated from Hibiscus Sabdariffa L. calyces the secondary metabolite called Hib-ester which possesses antiproliferative properties against human multiple myeloma RPMI 8226 cells, reduces migration and cell invasion and inhibits proteasome without neurotoxic effects. In the present study, we explored the chemical spaces of the hit compound Hib-ester. We explored the structure-activity relationships (SAR), and we optimized the scaffold through sequentially modifying Hib-ester subunits. Compound screening was performed based on cytotoxicity against the RPMI 8226 cells to assess the potential efficacy toward human MM. The ability of the most effective molecules to inhibit the proteasome was evaluated and the binding mode of the most promising compounds in the proteasome chymotrypsin binding pocket was deciphered through molecular modeling simulations. Compounds 13 and 14 are more potent than Hib-ester, demonstrating that our strategy was suitable for the identification of a novel chemotype for developing possible drug candidates and hopefully widening the drug armamentarium against MM.
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Antineoplásicos , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular Tumoral , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Ésteres , Antineoplásicos/uso terapêuticoRESUMO
Microtubule-targeting agents (MTAs) are effective drugs for cancer treatment. A novel diaryl [1,2]oxazole class of compounds binding the colchicine site was synthesized as cis-restricted-combretastatin-A-4-analogue and then chemically modified to have improved solubility and a wider therapeutic index as compared to vinca alkaloids and taxanes. On these bases, a new class of tricyclic compounds, containing the [1,2]oxazole ring and an isoindole moiety, has been synthetized, among which SIX2G emerged as improved MTA. Several findings highlighted the ability of some chemotherapeutics to induce immunogenic cell death (ICD), which is defined by the cell surface translocation of Calreticulin (CALR) via dissociation of the PP1/GADD34 complex. In this regard, we computationally predicted the ability of SIX2G to induce CALR exposure by interacting with the PP1 RVxF domain. We then assessed both the potential cytotoxic and immunogenic activity of SIX2G on in vitro models of multiple myeloma (MM), which is an incurable hematological malignancy characterized by an immunosuppressive milieu. We found that the treatment with SIX2G inhibited cell viability by inducing G2/M phase cell cycle arrest and apoptosis. Moreover, we observed the increase of hallmarks of ICD such as CALR exposure, ATP release and phospho-eIF2α protein level. Through co-culture experiments with immune cells, we demonstrated the increase of (i) CD86 maturation marker on dendritic cells, (ii) CD69 activation marker on cytotoxic T cells, and (iii) phagocytosis of tumor cells following treatment with SIX2G, confirming the onset of an immunogenic cascade. In conclusion, our findings provide a framework for further development of SIX2G as a new potential anti-MM agent.
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Antineoplásicos , Mieloma Múltiplo , Alcaloides de Vinca , Humanos , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Calreticulina/metabolismo , Linhagem Celular Tumoral , Colchicina/farmacologia , Morte Celular Imunogênica , Isoindóis/farmacologia , Microtúbulos/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Oxazóis/farmacologia , Taxoides/farmacologia , Alcaloides de Vinca/farmacologia , Pemetrexede/farmacologia , Pemetrexede/uso terapêuticoRESUMO
Human monoamine oxidases (hMAOs) are well-established targets for the treatment of neurological disorders such as depression, Parkinson's disease and Alzheimer's disease. Despite the efforts carried out over the years, few selective and reversible MAO inhibitors are on the market. Thus, a continuous search for new compounds is needed. Herein, MAO inhibitors were searched among the non-chiral constituents of Bergamot Essential Oil (BEO) with the aid of computational tools. Accordingly, molecular modeling simulations were carried out on both hMAO-A and hMAO-B for the selected constituents. The theoretically predicted target recognition was then used to select the most promising compounds. Among the screened compounds, Bergamottin, a furocoumarin, showed selective hMAO-B inhibitory activity, fitting its active site well. Molecular dynamics simulations were used to deeply analyze the target recognition and to rationalize the selectivity preference. In agreement with the computational results, experimental studies confirmed both the hMAO inhibition properties of Bergamottin and its preference for the isoform B.
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Monarda , Óleos Voláteis , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/farmacologia , Óleos Voláteis/farmacologia , Relação Estrutura-AtividadeRESUMO
The SARS-CoV-2 non-structural protein 13 (nsp13) helicase is an essential enzyme for viral replication and has been identified as an attractive target for the development of new antiviral drugs. In detail, the helicase catalyzes the unwinding of double-stranded DNA or RNA in a 5' to 3' direction and acts in concert with the replication-transcription complex (nsp7/nsp8/nsp12). In this work, bioinformatics and computational tools allowed us to perform a detailed conservation analysis of the SARS-CoV-2 helicase genome and to further predict the druggable enzyme's binding pockets. Thus, a structure-based virtual screening was used to identify valuable compounds that are capable of recognizing multiple nsp13 pockets. Starting from a database of around 4000 drugs already approved by the Food and Drug Administration (FDA), we chose 14 shared compounds capable of recognizing three out of four sites. Finally, by means of visual inspection analysis and based on their commercial availability, five promising compounds were submitted to in vitro assays. Among them, PF-03715455 was able to block both the unwinding and NTPase activities of nsp13 in a micromolar range.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Humanos , Reposicionamento de Medicamentos , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , DNA Helicases/metabolismo , Antivirais/farmacologiaRESUMO
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.