Immunoproteasome and Non-Covalent Inhibition: Exploration by Advanced Molecular Dynamics and Docking Methods

The selective inhibition of immunoproteasome is a valuable strategy to treat autoimmune, inflammatory diseases, and hematologic malignancies. Recently, a new series of amide derivatives as non-covalent inhibitors of the β1i subunit with Ki values in the low/submicromolar ranges have been identified. Here, we investigated the binding mechanism of the most potent and selective inhibitor, N-benzyl-2-(2-oxopyridin-1(2H)-yl)propanamide (1), to elucidate the steps from the ligand entrance into the binding pocket to the ligand-induced conformational changes. We carried out a total of 400 ns of MD-binding analyses, followed by 200 ns of plain MD. The trajectories clustering allowed identifying three representative poses evidencing new key interactions with Phe31 and Lys33 together in a flipped orientation of a representative pose. Further, Binding Pose MetaDynamics (BPMD) studies were performed to evaluate the binding stability, comparing 1 with four other inhibitors of the β1i subunit: N-benzyl-2-(2-oxopyridin-1(2H)-yl)acetamide (2), N-cyclohexyl-3-(2-oxopyridin-1(2H)-yl)propenamide (3), N-butyl-3-(2-oxopyridin-1(2H)-yl)propanamide (4), and (S)-2-(2-oxopyridin-1(2H)-yl)-N,4-diphenylbutanamide (5). The obtained results in terms of free binding energy were consistent with the experimental values of inhibition, confirming 1 as a lead compound of this series. The adopted methods provided a full dynamic description of the binding events, and the information obtained could be exploited for the rational design of new and more active inhibitors

Virtual Screening Strategy and In Vitro Tests to Identify New Inhibitors of the Immunoproteasome

Immunoproteasome inhibition is a promising strategy for the treatment of hematological malignancies, autoimmune diseases, and inflammatory diseases. The design of non-covalent inhibitors of the immunoproteasome beta 1i/beta 5i catalytic subunits could be a novel approach to avoid the drawbacks of the known covalent inhibitors, such as toxicity due to off-target binding. In this work, we report the biological evaluation of thirty-four compounds selected from a commercially available collection. These hit compounds are the outcomes of a virtual screening strategy including a dynamic pharmacophore modeling approach onto the beta 1i subunit and a pharmacophore/docking approach onto the beta 5i subunit. The computational studies were first followed by in vitro enzymatic assays at 100 mu M. Only compounds capable of inhibiting the enzymatic activity by more than 50% were characterized in detail using Tian continuous assays, determining the dissociation constant (K-i) of the non-covalent complex where K-i is also the measure of the binding affinity. Seven out of thirty-four hits showed to inhibit beta 1i and/or beta 5i subunit. Compound 3 is the most active on the beta 1i subunit with K-i = 11.84 +/- 1.63 mu M, and compound 17 showed K-i = 12.50 +/- 0.77 mu M on the beta 5i subunit. Compound 2 showed inhibitory activity on both subunits (K-i = 12.53 +/- 0.18 and K-i = 31.95 +/- 0.81 on the beta 1i subunit and beta 5i subunit, respectively). The induced fit docking analysis revealed interactions with Thr1 and Phe31 of beta 1i subunit and that represent new key residues as reported in our previous work. Onto beta 5i subunit, it interacts with the key residues Thr1, Thr21, and Tyr169. This last hit compound identified represents an interesting starting point for further optimization of beta 1i/beta 5i dual inhibitors of the immunoproteasome

Investigating the inhibition of FTSJ1 a tryptophan tRNA-specific 2’-O-methyltransferase by NV TRIDs, as a mechanism of readthrough in nonsense mutated CFTR

Abstract: Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10% of the CFTR gene mutations are "stop" mutations, which generate a Premature Termination Codon (PTC), thus synthesizing a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, which is the ri-bosome’s capacity to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough; for some, the mechanism of action is still under debate. We in-vestigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914, and NV930, could exert their readthrough activity by in silico analysis and in vitro studies. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase

Sulfonamide moiety as "molecular chimera" in the design of new drugs

The -SO2NH- group is of great significance in modern pharmaceutical use since in sulfa-drugs it is possible to introduce easily chemical modifications, and even small changes may lead to an improved version of an already existing drug

A definitive pharmacophore modelling study on CDK2 ATP pocket binders: tracing the path of new virtual high-throughput screenings

Cyclin Dependent Kinases-2 (CDK2) are members of serine/threonine protein kinases family. They play an important role in the regulation events of the eukaryotic cell division cycle, especially during the G1 to S phase transition. Experimental evidences indicate that excessive expression of CDK2s should cause abnormal cell cycle regulation. Therefore, since long time, CDK2s have been considered potential therapeutic targets for cancer therapy. In this work, one-hundred and forty-nine complexes of inhibitors bound in the CDK2-ATP pocket were submitted to short MD simulations (10ns) and free energy calculation. Comparison with experimental data (Ki, Kd and pIC50) revealed that short simulations are exhaustive to examine the crucial ligand-protein interactions within the complexes. Information collected on MD simulations of protein-ligand complexes have been used to perform a molecular modelling approach that incorporates flexibility into structure-based pharmacophore modelling (Common Hits Approach, CHA). The high number of pharmacophore models resulting from the MD simulation was thus reduced to a few representative groups of pharmacophore models. The performance of the models have been assessed by using the ROC curves analysis. This definitive set of validated pharmacophore models could be used to screen in-house and/or commercial datasets for detection of new CDK-2 inhibitors. We provide the models to all the researchers involved in this field.Peer reviewe

Exploring the non-covalent ligand-binding mechanism on immunoproteasome by enhanced Molecular Dynamics

Selective inhibition of immunoproteasome is a valuable strategy to treat autoimmune and inflammatory diseases, and hematologic malignancies. In particular, non-covalent inhibition is strongly desirable because it is free of the drawbacks and side effects associated with covalent inhibition. Recently, a new series of amide derivatives with Ki values in the low/submicromolar ranges toward the β1i subunit have been identified as non-covalent inhibitors 1 . We investigated the binding mechanism of the most potent and selective inhibitor (1) to elucidate the steps from the ligand entrance into the binding pocket to the ligand-induced conformational changes. We carried out a total of 400ns of MD-binding analysis, followed by 200ns of plain MD. The trajectories clustering allowed identifying three representative poses evidencing new key interactions with Phe31 and Lys33 together to a flipped orientation of a representative pose. Further, Binding pose metadynamics (BPMD) studies have been performed to evaluate the binding affinity, comparing (1) with other four inhibitors of β1i subunit (2, 3, 4, and 5). Results are consistent with experimental values of inhibition, confirming (1) as a lead compound of this series. The adopted methods provided a full dynamic description of the binding events and the information obtained could be exploited for the rational design of new and more active inhibitor

Exploring the SARS-CoV-2 Proteome in the Search of Potential Inhibitors via Structure-Based Pharmacophore Modeling/Docking Approach

To date, SARS-CoV-2 infectious disease, named COVID-19 by the World Health Organization (WHO) in February 2020, has caused millions of infections and hundreds of thousands of deaths. Despite the scientific community efforts, there are currently no approved therapies for treating this coronavirus infection. The process of new drug development is expensive and time-consuming, so that drug repurposing may be the ideal solution to fight the pandemic. In this paper, we selected the proteins encoded by SARS-CoV-2 and using homology modeling we identified the high-quality model of proteins. A structure-based pharmacophore modeling study was performed to identify the pharmacophore features for each target. The pharmacophore models were then used to perform a virtual screening against the DrugBank library (investigational, approved and experimental drugs). Potential inhibitors were identified for each target using XP docking and induced fit docking. MM-GBSA was also performed to better prioritize potential inhibitors. This study will provide new important comprehension of the crucial binding hot spots usable for further studies on COVID-19. Our results can be used to guide supervised virtual screening of large commercially available libraries

Uno studio comparativo in silico sui possibili target di Ataluren e analoghi farmaci promotori di readthrough di codoni di stop prematuri

E’ noto in letteratura che Ataluren (acido 5-(fluorofenil)-1,2,4-ossadiazolil-benzoico) sia in grado di sopprimere le mutazioni non senso favorendo il readthrough dei codoni di stop prematuri, anche se il suo meccanismo di azione non risulta ancora chiaro. La probabile interazione tra Ataluren e CTFR-mRNA è stata precedentemente studiata mediante dinamica molecolare. In questo studio1, abbiamo esteso il modeling del probabile meccanismo di azione di Ataluren mediante approcci computazionali completementari, quali Induced Fit Docking (IFD), Quantum Polarized Ligand Docking (QPLD), metodi MM-GBSA e mutagenesi computazionale. Oltre a considerare il CTFR-mRNA, sono stati presi in considerazione altri target implicati nel processo di traduzione, quali la subunità 16S dell’rRNA batterico e la subunità 18S dell’rRNA eucariotico, che sono target comprovati di molti aminoglicosidi noti per la loro capacità di sopprimere l’attività di correzione svolta normalmente dal ribosoma; il fattore di rilascio eucariotico eRF1, per valutare la potenziale influenza di Ataluren sulla fine del processo di traduzione. Inoltre, è stato effettuato un confronto tra Ataluren, un suo nuovo promettente analogo NV2445 (acido 4-(5-(o-tolil)-1,3,4-ossadiazol-2-il)benzoico)2 e una serie di antibiotici aminoglicosidici. I risultati hanno confermato che mRNA è il più probabile target per Ataluren e i suoi derivati. I calcoli di energia libera di legame effettuati in seguito alla mutagenesi computazionale, hanno mostrato che il legame tra Ataluren e il codone di stop prematuro è fortemente influenzato dalla presenza di nucleotidi ausiliari nell’intorno genico

Indicaxanthin, a multi-target natural compound from Opuntia ficus-indica fruit: From its poly-pharmacological effects to biochemical mechanisms and molecular modelling studies

Over the latest years phytochemical consumption has been associated to a decreased risk of both theonset and the development of a number of pathological conditions. In this context indicaxanthin, abetalain pigment fromOpuntiaficus-indicafruit, has been the object of sound research. Explored, atfirst,for its mere antioxidant potential, Indicaxanthin is now regarded as a redox-active compound able toexert significant poly-pharmacological effects against several targets in a number of experimental con-ditions bothin vivoandin vitro. This paper aims to provide an overview on the therapeutical effects ofindicaxanthin, ranging from the anti-inflammatory to the neuro-modulatory and anti-tumoral ones andfavored by its high bioavailability. Moreover, biochemical and molecular modelling investigations areaimed to identify the pharmacological targets the compound is able to interact with and to address thechallenging development in the future researc

Fighting Antibiotic Resistance: New Pyrimidine-Clubbed Benzimidazole Derivatives as Potential DHFR Inhibitors

The present work describes the design and development of seventeen pyrimidine-clubbed benzimidazole derivatives as potential dihydrofolate reductase (DHFR) inhibitors. These compounds were filtered by using ADMET, drug-likeness characteristics calculations, and molecular docking experiments. Compounds 27, 29, 30, 33, 37, 38, and 41 were chosen for the synthesis based on the results of the in silico screening. Each of the synthesized compounds was tested for its in vitro antibacterial and antifungal activities using a variety of strains. All the compounds showed antibacterial properties against Gram-positive bacteria (Staphylococcus aureus and Staphylococcus pyogenes) as well as Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Most of the compounds either had a higher potency than chloramphenicol or an equivalent potency to ciprofloxacin. Compounds 29 and 33 were effective against all the bacterial and fungal strains. Finally, the 1,2,3,4-tetrahydropyrimidine-2-thiol derivatives with a 6-chloro-2-(chloromethyl)-1H-benzo[d]imidazole moiety are potent enough to be considered a promising lead for the discovery of an effective antibacterial agent