Novel multifunctional tacrine-donepezil hybrids against Alzheimer's disease: Design synthesis and bioactivity studies.

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.

Coumarins-lipophilic cations conjugates: Efficient mitocans targeting carbonic anhydrases.

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.

Neurodegeneration: can metabolites from Eremurus persicus help?

The number of patients affected by neurodegenerative diseases is increasing worldwide, and no effective treatments have been developed yet. Although precision medicine could represent a powerful tool, it remains a challenge due to the high variability among patients. To identify molecules acting with innovative mechanisms of action, we performed a computational investigation using SAFAN technology, focusing specifically on HuD. This target belongs to the human embryonic lethal abnormal visual-like (ELAV) proteins and plays a key role in neuronal plasticity and differentiation. The results highlighted that the molecule able to bind the selected target was (R)-aloesaponol-III-8-methyl ether [(R)-ASME], a metabolite extracted from Eremurus persicus. Notably, this molecule is a TNF-α inhibitor, a cytokine involved in neuroinflammation. To obtain a suitable amount of (R)-ASME to confirm its activity on HuD, we optimized the extraction procedure. Together with ASME, another related metabolite, germichrysone, was isolated. Both ASME and germichrysone underwent biological investigation, but only ASME confirmed its ability to bind HuD. Given the multifactorial nature of neurodegenerative diseases, we decided to investigate ASME as a proteasome activator, being molecules endowed with this kind of activity potentially able to counteract aggregations of dysregulated proteins. ASME was able to activate the considered target both in enzymatic and cellular assays. Therefore, ASME may be considered a promising hit in the fight against neurodegenerative diseases.

New Insights for Polyphenolic Compounds as Naturally Inspired Proteasome Inhibitors.

Polyphenols, an important class of natural products, are widely distributed in plant-based foods. These compounds are endowed with several biological activities and exert protective effects in various physiopathological contexts, including cancer. We herein investigated novel potential mechanisms of action of polyphenols, focusing on the proteasome, which has emerged as an attractive therapeutic target in cancers such as multiple myeloma. We carried out a structure-based virtual screening study using the DrugBank database as a repository of FDA-approved polyphenolic molecules. Starting from 86 polyphenolic compounds, based on the theoretical binding affinity and the interactions established with key residues of the chymotrypsin binding site, we selected 2 promising candidates, namely Hesperidin and Diosmin. The further assessment of the biologic activity highlighted, for the first time, the capability of these two molecules to inhibit the ß5-proteasome activity and to exert anti-tumor activity against proteasome inhibitor-sensitive or resistant multiple myeloma cell lines.

An Update on Recent Studies Focusing on the Antioxidant Properties of Salvia Species.

Nutrition has crucial effects and a significant role in disease prevention. Recently, nutraceuticals have attracted much attention in scientific research due to their pleiotropic effects and relatively non-toxic behavior. Among the biological effects displayed by plants belonging to the Lamiaceae family, such as antibacterial, anticancer, anti-inflammatory, and anticholinesterase, sage is well known for its antioxidant properties and is a rich source of numerous compounds that are biologically active, amongst them polyphenols, with more than 160 types identified. In this review we summarized some of the significant studies published in the last decade reporting the most employed extraction methods and the different assays that are useful for establishing the antioxidant properties of some sage species. Even though the scientific literature contains plenty of data regarding the antioxidant properties of many sage species, further studies are needed in order to gain a deeper understanding of the mechanism of action and the compounds responsible for their antioxidant activity. Finally, it should be taken into account that the data on the antioxidant properties of sage extracts are often difficult to compare with each other, since a series of variables in the extraction procedures, the type of assay used, and standardization may affect the final result.

Identification of HuR-RNA Interfering Compounds by Dynamic Combinatorial Chemistry and Fluorescence Polarization.

The RNA binding protein HuR regulates the post-transcriptional process of different oncogenes and tumor suppressor genes, and its dysregulation is linked with cancer. Thus, modulating the complex HuR-RNA represents a promising anticancer strategy. To search for novel HuR ligands able to interfere with the HuR-RNA complex, the protein-templated dynamic combinatorial chemistry (pt-DCC) method was utilized. The recombinant RRM1+2 protein construct, which contains essential domains for ligand-HuR binding and exhibits enhanced solubility and stability compared to the native protein, was used for pt-DCC. Seven acylhydrazones with over 80% amplification were identified. The binding of the fragments to HuR extracted from DCC was validated using STD-NMR, and molecular modeling studies revealed the ability of the compounds to bind HuR at the mRNA binding pocket. Notably, three compounds effectively interfered with HuR-RNA binding in fluorescence polarization studies, suggesting their potential as foundational compounds for developing anticancer HuR-RNA interfering agents.

Targeting non-coding RNAs: Perspectives and challenges of in-silico approaches.

The growing information currently available on the central role of non-coding RNAs (ncRNAs) including microRNAs (miRNAS) and long non-coding RNAs (lncRNAs) for chronic and degenerative human diseases makes them attractive therapeutic targets. RNAs carry out different functional roles in human biology and are deeply deregulated in several diseases. So far, different attempts to therapeutically target the 3D RNA structures with small molecules have been reported. In this scenario, the development of computational tools suitable for describing RNA structures and their potential interactions with small molecules is gaining more and more interest. Here, we describe the most suitable strategies to study ncRNAs through computational tools. We focus on methods capable of predicting 2D and 3D ncRNA structures. Furthermore, we describe computational tools to identify, design and optimize small molecule ncRNA binders. This review aims to outline the state of the art and perspectives of computational methods for ncRNAs over the past decade.

2H-chromene and 7H-furo-chromene derivatives selectively inhibit tumour associated human carbonic anhydrase IX and XII isoforms.

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.

Discovery of first novel sigma/HDACi dual-ligands with a potent in vitro antiproliferative activity.

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.

Molecular and Structural Aspects of Clinically Relevant Mutations of SARS-CoV-2 RNA-Dependent RNA Polymerase in Remdesivir-Treated Patients.

(1) Background: SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target to fight COVID-19, and many RdRp inhibitors nucleotide/nucleoside analogs, such as remdesivir, have been identified or are in clinical studies. However, the appearance of resistant mutations could reduce their efficacy. In the present work, we structurally evaluated the impact of RdRp mutations found at baseline in 39 patients treated with remdesivir and associated with a different degree of antiviral response in vivo. (2) Methods: A refined bioinformatics approach was applied to assign SARS-CoV-2 clade and lineage, and to define RdRp mutational profiles. In line with such a method, the same mutations were built and analyzed by combining docking and thermodynamics evaluations with both molecular dynamics and representative pharmacophore models. (3) Results: Clinical studies revealed that patients bearing the most prevalent triple mutant P323L+671S+M899I, which was present in 41% of patients, or the more complex mutational profile P323L+G671S+L838I+D738Y+K91E, which was found with a prevalence of 2.6%, showed a delayed reduced response to remdesivir, as confirmed by the increase in SARS-CoV-2 viral load and by a reduced theoretical binding affinity versus RdRp (ΔGbindWT = -122.70 kcal/mol; ΔGbindP323L+671S+M899I = -84.78 kcal/mol; ΔGbindP323L+G671S+L838I+D738Y+K91E = -96.74 kcal/mol). Combined computational approaches helped to rationalize such clinical observations, offering a mechanistic understanding of the allosteric effects of mutants on the global motions of the viral RNA synthesis machine and in the changes of the interactions patterns of remdesivir during its binding.

Discovery of AD258 as a Sigma Receptor Ligand with Potent Antiallodynic Activity.

The design and synthesis of a series of 2,7-diazaspiro[4.4]nonane derivatives as potent sigma receptor (SR) ligands, associated with analgesic activity, are the focus of this work. In this study, affinities at S1R and S2R were measured, and molecular modeling studies were performed to investigate the binding pose characteristics. The most promising compounds were subjected to in vitro toxicity testing and subsequently screened for in vivo analgesic properties. Compound 9d (AD258) exhibited negligible in vitro cellular toxicity and a high binding affinity to both SRs (KiS1R = 3.5 nM, KiS2R = 2.6 nM), but not for other pain-related targets, and exerted high potency in a model of capsaicin-induced allodynia, reaching the maximum antiallodynic effect at very low doses (0.6-1.25 mg/kg). Functional activity experiments showed that S1R antagonism is needed for the effects of 9d and that it did not induce motor impairment. In addition, 9d exhibited a favorable pharmacokinetic profile.

Cryo-EM and Molecular Dynamics Simulations Reveal Hidden Conformational Dynamics Controlling Ammonia Transport in Human Asparagine Synthetase.

How dynamical motions in enzymes might be linked to catalytic function is of significant general interest, although almost all relevant experimental data, to date, has been obtained for enzymes with a single active site. Recent advances in X-ray crystallography and cryogenic electron microscopy offer the promise of elucidating dynamical motions for proteins that are not amenable to study using solution-phase NMR methods. Here we use 3D variability analysis (3DVA) of an EM structure for human asparagine synthetase (ASNS) in combination with atomistic molecular dynamics (MD) simulations to detail how dynamic motions of a single side chain mediates interconversion of the open and closed forms of a catalytically relevant intramolecular tunnel, thereby regulating catalytic function. Our 3DVA results are consistent with those obtained independently from MD simulations, which further suggest that formation of a key reaction intermediate acts to stabilize the open form of the tunnel in ASNS to permit ammonia translocation and asparagine formation. This conformational selection mechanism for regulating ammonia transfer in human ASNS contrasts sharply with those employed in other glutamine-dependent amidotransferases that possess a homologous glutaminase domain. Our work illustrates the power of cryo-EM to identify localized conformational changes and hence dissect the conformational landscape of large proteins. When combined with MD simulations, 3DVA is a powerful approach to understanding how conformational dynamics regulate function in metabolic enzymes with multiple active sites.

Hit identification of novel small molecules interfering with MALAT1 triplex by a structure-based virtual screening.

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.

Synthesis, Computational Insights, and Evaluation of Novel Sigma Receptors Ligands.

The development of diazabicyclo[4.3.0]nonane and 2,7-diazaspiro[3.5]nonane derivatives as sigma receptors (SRs) ligands is reported. The compounds were evaluated in S1R and S2R binding assays, and modeling studies were carried out to analyze the binding mode. The most notable compounds, 4b (AD186, KiS1R = 2.7 nM, KiS2R = 27 nM), 5b (AB21, KiS1R = 13 nM, KiS2R = 102 nM), and 8f (AB10, KiS1R = 10 nM, KiS2R = 165 nM), have been screened for analgesic effects in vivo, and their functional profile was determined through in vivo and in vitro models. Compounds 5b and 8f reached the maximum antiallodynic effect at 20 mg/kg. The selective S1R agonist PRE-084 completely reversed their action, indicating that the effects are entirely dependent on the S1R antagonism. Conversely, compound 4b sharing the 2,7-diazaspiro[3.5]nonane core as 5b was completely devoid of antiallodynic effect. Interestingly, compound 4b fully reversed the antiallodynic effect of BD-1063, indicating that 4b induces an S1R agonistic in vivo effect. The functional profiles were confirmed by the phenytoin assay. Our study might establish the importance of 2,7-diazaspiro[3.5]nonane core for the development of S1R compounds with specific agonist or antagonist profile and the role of the diazabicyclo[4.3.0]nonane in the development of novel SR ligands.

Targeting SARS-CoV-2 Main Protease: A Successful Story Guided by an In Silico Drug Repurposing Approach.

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.

Uncovering Novel Capsaicin Inhibitory Activity towards Human Carbonic Anhydrase Isoforms IX and XII by Combining In Silico and In Vitro Studies.

Hot pepper (Capsicum annuum) represents one of the most widespread functional foods of the Mediterranean diet, and is associated with a reduced risk of developing cardiovascular disease, cancer, and mental disorders. In particular, its bioactive spicy molecules, named Capsaicinoids, exhibit polypharmacological properties. Among them, Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is the most studied and reported in variegated scientific contributions for its beneficial effects, often linked to mechanisms of action unrelated to the activation of Transient Receptor Potential Vanilloid 1 (TRPV1). In this study, we present the application of in silico methods to Capsaicin for evaluating its inhibitory activity against the tumor-associated human (h) expressed CA IX and XII. In vitro assays confirmed Capsaicin inhibitory activity towards the most relevant tumor-related hCA isoforms. In particular, the hCAs IX and XII showed an experimental KI value of 0.28 µM and 0.064 µM, respectively. Then, an A549 model of non-small cell lung cancer, typically characterized by an elevated expression of hCA IX and XII, was employed to test the inhibitory effects of Capsaicin in vitro under both normoxic and hypoxic conditions. Finally, the migration assay revealed that Capsaicin [10 µM] inhibits cells from moving in the A549 cells model.

In Silico-Guided Rational Drug Design and Synthesis of Novel 4-(Thiophen-2-yl)butanamides as Potent and Selective TRPV1 Agonists.

We describe an in silico-guided rational drug design and the synthesis of the suggested ligands, aimed at improving the TRPV1-ligand binding properties and the potency of N-(4-hydroxy-3-methoxybenzyl)-4-(thiophen-2-yl) butanamide I, a previously identified TRPV1 agonist. The docking experiments followed by molecular dynamics simulations and thermodynamic analysis led the drug design toward both the introduction of a lipophilic iodine and a flat pyridine/benzene at position 5 of the thiophene nucleus. Most of the synthesized compounds showed high TRPV1 efficacy and potency as well as selectivity. The molecular modeling analysis highlighted crucial hydrophobic interactions between Leu547 and the iodo-thiophene nucleus, as in amide 2a, or between Phe543 and the pyridinyl moiety, as in 3a. In the biological evaluation, both compounds showed protective properties against oxidative stress-induced ROS formation in human keratinocytes. Additionally, while 2a showed neuroprotective effects in both neurons and rat brain slices, 3a exhibited potent antinociceptive effect in vivo..

Identification of pyrrolo[3',4':3,4]cyclohepta[1,2-d][1,2]oxazoles as promising new candidates for the treatment of lymphomas.

Unsatisfactory outcomes for relapsed/refractory lymphoma patients prompt continuing efforts to develop new therapeutic strategies. Our previous studies on pyrrole-based anti-lymphoma agents led us to synthesize a new series of twenty-six pyrrolo[3',4':3,4]cyclohepta[1,2-d] [1,2]oxazole derivatives and study their antiproliferative effects against a panel of four non-Hodgkin lymphoma cell lines. Several candidates showed significant anti-proliferative effects, with IC50's reaching the sub-micromolar range in at least one cell line, with compound 3z demonstrating sub-micromolar growth inhibitory effects towards the entire panel. The VL51 cell line was the most sensitive, with an IC50 value of 0.10 µM for 3z. Our earlier studies had shown that tubulin was a prominent target of many of our oxazole derivatives. We therefore examined their effects on tubulin assembly and colchicine binding. While 3u and 3z did not appear to target tubulin, good activity was observed with 3d and 3p. Molecular docking and molecular dynamics simulations allowed us to rationalize the binding mode of the synthesized compounds toward tubulin. All ligands exhibited a better affinity for the colchicine site, confirming their specificity for this binding pocket. In particular, a better affinity and free energy of binding was observed for 3d and 3p. This result was confirmed by experimental data, indicating that, although both 3d and 3p significantly affected tubulin assembly, only 3d showed activity comparable to that of combretastatin A-4, while 3p was about 4-fold less active. Cell cycle analysis showed that compounds 3u and especially 3z induced a block in G2/M, a strong decrease in S phase even at low compound concentrations and apoptosis through the mitochondrial pathway. Thus, the mechanism of action of 3u and 3z remains to be elucidated. Very high selectivity toward cancer cells and low toxicity in human peripheral blood lymphocytes were observed, highlighting the good potential of these agents in cancer therapy and encouraging further exploration of this compound class to obtain new small molecules as effective lymphoma treatments.

HSV-1 Glycoprotein D and Its Surface Receptors: Evaluation of Protein-Protein Interaction and Targeting by Triazole-Based Compounds through In Silico Approaches.

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.

In Silico and In Vitro Study of Antioxidant Potential of Urolithins.

In this work, quantum chemical calculations based on density functional theory (DFT) were performed to predict the antioxidant potential of four bioactive gut microbiota metabolites of the natural polyphenols ellagitannins (ETs) and ellagic acid (EA), also known as urolithins (UROs). In order to evaluate their ability to counter the effect of oxidative stress caused by reactive oxygen species (ROS), such as the hydroperoxyl radical (•OOH), different reaction mechanisms were investigated, considering water and lipid-like environments. Through our in silico results, it emerged that at physiological pH, the scavenging activity of all urolithins, except urolithin B, are higher than that of trolox and other potent antioxidants existing in nature, such as EA, α-mangostin, allicin, caffeine and melatonin. These findings were confirmed by experimental assays.