Exploring Chemical Substructures Essential for hERG K+ Channel Blockade by Synthesis and Biological Evaluation of Dofetilide Analogues

In this study we followed a new approach to analyze molecular substructures required for hERG channel blockade. We designed and synthesized 40 analogues of dofetilide ( 1 ), a potent hERG potassium channel blocker, and established structure–activity relationships (SAR) for their interaction with this important cardiotoxicity‐related off‐target. Structural modifications to dofetilide were made by diversifying the substituents on the phenyl rings and the protonated nitrogen and by varying the carbon chain length. The analogues were evaluated in a radioligand binding assay and SAR data were derived with the aim to specify structural features that give rise to hERG toxicity.     

A Comprehensive Survey of Prospective Structure-Based Virtual Screening for Early Drug Discovery in the Past Fifteen Years

Structure-based virtual screening (SBVS), also known as molecular docking, has been increasingly applied to discover small-molecule ligands based on the protein structures in the early stage of drug discovery. In this review, we comprehensively surveyed the prospective applications of molecular docking judged by solid experimental validations in the literature over the past fifteen years. Herein, we systematically analyzed the novelty of the targets and the docking hits, practical protocols of docking screening, and the following experimental validations. Among the 419 case studies we reviewed, most virtual screenings were carried out on widely studied targets, and only 22% were on less-explored new targets. Regarding docking software, GLIDE is the most popular one used in molecular docking, while the DOCK 3 series showed a strong capacity for large-scale virtual screening. Besides, the majority of identified hits are promising in structural novelty and one-quarter of the hits showed better potency than 1 μM, indicating that the primary advantage of SBVS is to discover new chemotypes rather than highly potent compounds. Furthermore, in most studies, only in vitro bioassays were carried out to validate the docking hits, which might limit the further characterization and development of the identified active compounds. Finally, several successful stories of SBVS with extensive experimental validations have been highlighted, which provide unique insights into future SBVS drug discovery campaigns.     

Toward Quantitative Models in Safety Assessment: A Case Study to Show Impact of Dose–Response Inference on hERG Inhibition Models

Due to challenges with historical data and the diversity of assay formats, in silico models for safety-related endpoints are often based on discretized data instead of the data on a natural continuous scale. Models for discretized endpoints have limitations in usage and interpretation that can impact compound design. Here, we present a consistent data inference approach, exemplified on two data sets of Ether-à-go-go-Related Gene (hERG) K+ inhibition data, for dose–response and screening experiments that are generally applicable for in vitro assays. hERG inhibition has been associated with severe cardiac effects and is one of the more prominent safety targets assessed in drug development, using a wide array of in vitro and in silico screening methods. In this study, the IC₅₀ for hERG inhibition is estimated from diverse historical proprietary data. The IC₅₀ derived from a two-point proprietary screening data set demonstrated high correlation (R = 0.98, MAE = 0.08) with IC_50s derived from six-point dose–response curves. Similar IC₅₀ estimation accuracy was obtained on a public thallium flux assay data set (R = 0.90, MAE = 0.2). The IC₅₀ data were used to develop a robust quantitative model. The model’s MAE (0.47) and R² (0.46) were on par with literature statistics and approached assay reproducibility. Using a continuous model has high value for pharmaceutical projects, as it enables rank ordering of compounds and evaluation of compounds against project-specific inhibition thresholds. This data inference approach can be widely applicable to assays with quantitative readouts and has the potential to impact experimental design and improve model performance, interpretation, and acceptance across many standard safety endpoints.     

Understanding of molecular substructures that contribute to hERG K+ channel blockade: synthesis and biological evaluation of E-4031 analogues

Cardiotoxicity is a common side effect of a large variety of drugs that is often caused by off-target human ether-à-go-go-related gene (hERG) potassium channel blockade. In this study, we designed and synthesized a series of derivatives of the class III antiarrhythmic agent E-4031. These compounds where evaluated in a radioligand binding assay and automated patch clamp assay to establish structure-activity relationships (SAR) for their inhibition of the hERG K(+) channel. Structural modifications of E-4031 were made by altering the peripheral aromatic moieties with a series of distinct substituents. Additionally, we synthesized several derivatives with a quaternary nitrogen and modified the center of the molecule by introduction of an additional nitrogen and deletion of the carbonyl oxygen. Some modifications caused a great increase in affinity for the hERG K(+) channel, while other seemingly minor changes led to a strongly diminished affinity. Structures with quaternary amines carrying an additional aromatic moiety were found to be highly active in radioligand binding assay. A decrease in affinity was achieved by introducing an amide functionality in the central scaffold without directly interfering with the pK(a) of the essential basic amine. The knowledge gained from this study could be used in early stages of drug discovery and drug development to avoid or circumvent hERG K(+) channel blockade, thereby reducing the risk of cardiotoxicity, related arrhythmias and sudden death.     

In Silico Drug Repositioning to Target the SARS-CoV-2 Main Protease as Covalent Inhibitors Employing a Combined Structure-Based Virtual Screening Strategy of Pharmacophore Models and Covalent Docking

The epidemic caused by the SARS-CoV-2 coronavirus, which has spread rapidly throughout the world, requires urgent and effective treatments considering that the appearance of viral variants limits the efficacy of vaccines. The main protease of SARS-CoV-2 (M^pro) is a highly conserved cysteine proteinase, fundamental for the replication of the coronavirus and with a specific cleavage mechanism that positions it as an attractive therapeutic target for the proposal of irreversible inhibitors. A structure-based strategy combining 3D pharmacophoric modeling, virtual screening, and covalent docking was employed to identify the interactions required for molecular recognition, as well as the spatial orientation of the electrophilic warhead, of various drugs, to achieve a covalent interaction with Cys145 of M^pro. The virtual screening on the structure-based pharmacophoric map of the SARS-CoV-2 M^pro in complex with an inhibitor N3 (reference compound) provided high efficiency by identifying 53 drugs (FDA and DrugBank databases) with probabilities of covalent binding, including N3 (Michael acceptor) and others with a variety of electrophilic warheads. Adding the energy contributions of affinity for non-covalent and covalent docking, 16 promising drugs were obtained. Our findings suggest that the FDA-approved drugs Vaborbactam, Cimetidine, Ixazomib, Scopolamine, and Bicalutamide, as well as the other investigational peptide-like drugs (DB04234, DB03456, DB07224, DB7252, and CMX-2043) are potential covalent inhibitors of SARS-CoV-2 M^pro.     

Host-Assisted Delivery of a Model Drug to Genomic DNA: Key Information from Ultrafast Spectroscopy and in silico Study

Drug delivery to a target without adverse effects is one of the major criteria for clinical use. Herein, we have made an attempt to explore the delivery efficacy of SDS surfactant in a monomer and micellar stage during the delivery of the model drug, Toluidine Blue (TB) from the micellar cavity to DNA. Molecular recognition of pre-micellar SDS encapsulated TB with DNA occurs at a rate constant of k₁ ∼652 s⁻¹. However, no significant release of encapsulated TB at micellar concentration was observed within the experimental time frame. This originated from the higher binding affinity of TB towards the nano-cavity of SDS at micellar concentration which does not allow the delivery of TB from the nano-cavity of SDS micelles to DNA. Thus, molecular recognition controls the extent of DNA recognition by TB which in turn modulates the rate of delivery of TB from SDS in a concentration-dependent manner.     

A comparative reverse docking strategy to identify potential antineoplastic targets of tea functional components and binding mode

The main functional components of green tea, such as epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC), are found to have a broad antineoplastic activity. The discovery of their targets plays an important role in revealing the antineoplastic mechanism. Therefore, to identify potential target proteins for tea polyphenols, we have taken a comparative virtual screening approach using two reverse docking systems, one based on Autodock software and the other on Tarfisdock. Two separate in silico workflows were implemented to derive a set of target proteins related to human diseases and ranked by the binding energy score. Several conventional clinically important proteins with anti-tumor effects are screened out from the PDTD protein database as the potential receptors by both procedures. To further analyze the validity of docking results, we study the binding mode of EGCG and the potential target protein Leukotriene A4 hydrolase in detail. We indicate that interactions mediated by electrostatic and hydrogen bond play a key role in ligand binding. EGCG binds to the enzyme with certain orientation and conformation that is suitable for nucleophilic attacks by several electrical residues inside the enzyme's activity cavity. This study provides useful information for studying the antitumor mechanism of tea's functional components. The comparative reverse docking strategy presented generates a tractable set of antineoplastic proteins for future experimental validation as drug targets against tumors.     

Homology Model Adjustment and Ligand Screening with a Pseudoreceptor of the Human Histamine H4 Receptor

A new pseudoreceptor modeling method (PRPS) was applied to the refinement of a homology model of the human histamine H₄ receptor (H₄R), the prediction of a ligand binding site, and virtual screening. Retrieval of two new H₄R ligands demonstrates the biological relevance of the pseudoreceptor model and provides a means for finding new hits and leads in the early phases of drug discovery.

     

Discovery and Validation of Lmj_04_BRCT Domain,a Novel Therapeutic Target: Identification of Candidate Drugs for Leishmaniasis

Since many of the currently available antileishmanial treatments exhibit toxicity, low effectiveness, and resistance, search and validation of new therapeutic targets allowing the development of innovative drugs have become a worldwide priority. This work presents a structure-based drug discovery strategy to validate the Lmj_04_BRCT domain as a novel therapeutic target in Leishmania spp. The structure of this domain was explored using homology modeling, virtual screening, and molecular dynamics studies. Candidate compounds were validated in vitro using promastigotes of Leishmania major, L. amazonensis, and L. infantum, as well as primary mouse macrophages infected with L. major. The novel inhibitor CPE2 emerged as the most active of a group of compounds against Leishmania, being able to significantly reduce the viability of promastigotes. CPE2 was also active against the intracellular forms of the parasites and significantly reduced parasite burden in murine macrophages without exhibiting toxicity in host cells. Furthermore, L. major promastigotes treated with CPE2 showed significant lower expression levels of several genes (α-tubulin, Cyclin CYCA, and Yip1) related to proliferation and treatment resistance. Our in silico and in vitro studies suggest that the Lmj_04_BRCT domain and its here disclosed inhibitors are new potential therapeutic options against leishmaniasis.     

Applications of Biomaterials in 3D Cell Culture and Contributions of 3D Cell Culture to Drug Development and Basic Biomedical Research

The process of evaluating the efficacy and toxicity of drugs is important in the production of new drugs to treat diseases. Testing in humans is the most accurate method, but there are technical and ethical limitations. To overcome these limitations, various models have been developed in which responses to various external stimuli can be observed to help guide future trials. In particular, three-dimensional (3D) cell culture has a great advantage in simulating the physical and biological functions of tissues in the human body. This article reviews the biomaterials currently used to improve cellular functions in 3D culture and the contributions of 3D culture to cancer research, stem cell culture and drug and toxicity screening.     

In Vitro and In Vivo Pharmacological Characterization of a Novel TRPM8 Inhibitor Chemotype Identified by Small-Scale Preclinical Screening

Transient receptor potential melastatin type 8 (TRPM8) is a target for the treatment of different physio-pathological processes. While TRPM8 antagonists are reported as potential drugs for pain, cancer, and inflammation, to date only a limited number of chemotypes have been investigated and thus a limited number of compounds have reached clinical trials. Hence there is high value in searching for new TRPM8 antagonistic to broaden clues to structure-activity relationships, improve pharmacological properties and explore underlying molecular mechanisms. To address this, the EDASA Scientific in-house molecular library has been screened in silico, leading to identifying twenty-one potentially antagonist compounds of TRPM8. Calcium fluorometric assays were used to validate the in-silico hypothesis and assess compound selectivity. Four compounds were identified as selective TRPM8 antagonists, of which two were dual-acting TRPM8/TRPV1 modulators. The most potent TRPM8 antagonists (BB 0322703 and BB 0322720) underwent molecular modelling studies to highlight key structural features responsible for drug–protein interaction. The two compounds were also investigated by patch-clamp assays, confirming low micromolar potencies. The most potent compound (BB 0322703, IC₅₀ 1.25 ± 0.26 μM) was then profiled in vivo in a cold allodinya model, showing pharmacological efficacy at 30 μM dose. The new chemotypes identified showed remarkable pharmacological properties paving the way to further investigations for drug discovery and pharmacological purposes.     

Comprehensive Analysis of Neonatal versus Adult Unilateral Decortication in a Mouse Model Using Behavioral,Neuroanatomical, and DNA Microarray Approaches

Previously, studying the development, especially of corticospinal neurons, it was concluded that the main compensatory mechanism after unilateral brain injury in rat at the neonatal stage was due in part to non-lesioned ipsilateral corticospinal neurons that escaped selection by axonal elimination or neuronal apoptosis. However, previous results suggesting compensatory mechanism in neonate brain were not correlated with high functional recovery. Therefore, what is the difference among neonate and adult in the context of functional recovery and potential mechanism(s) therein? Here, we utilized a brain unilateral decortication mouse model and compared motor functional recovery mechanism post-neonatal brain hemisuction (NBH) with adult brain hemisuction (ABH). Three analyses were performed: (1) Quantitative behavioral analysis of forelimb movements using ladder walking test; (2) neuroanatomical retrograde tracing analysis of unlesioned side corticospinal neurons; and (3) differential global gene expressions profiling in unlesioned-side neocortex (rostral from bregma) in NBH and ABH on a 8 × 60 K mouse whole genome Agilent DNA chip. Behavioral data confirmed higher recovery ability in NBH over ABH is related to non-lesional frontal neocortex including rostral caudal forelimb area. A first inventory of differentially expressed genes genome-wide in the NBH and ABH mouse model is provided as a resource for the scientific community.     

Identification of a potent inhibitor of human dual-specific phosphatase, VHR, from computer-aided and NMR-based screening to cellular effects

Human vaccinia H1-related phosphatase (VHR) is a dual-specific phosphatase (DSPs) that plays an important role in the mitogen-activated protein (MAP) kinase cascade regulation. It is also a potential drug target for diseases that are related to immune response. By combining a virtual and NMR-based ligand-screening strategy, we successfully identified four VHR inhibitors, of which GATPT ((glucosamine-aminoethoxy)triphenyltin) can bind to VHR with a K(i) value of 2.54 muM. The putative binding mode of GATPT was constructed by a molecular docking simulation to provide structural insights into the ligand-binding mechanism. Furthermore, we found that this compound can significantly inhibit the dephosphorylation of the extracellular regulated kinases (ERKs), and c-Jun N-terminal kinases (JNKs) and block the G(1)-S phase transition in the cell cycle. Therefore, GATPT is a promising lead structure for designing more effective inhibitors of VHR.     

New approaches to determine the interface height of fire smoke based on a three-layer zone model and its verification in large confined space

Large confined space has high incidence of fires, which seriously threatens the safety of people working there. Understanding the distribution of smoke in such large space is critical to fire development prediction and smoke control. Three improved methods for the stratification interface prediction of fire smoke are developed, including of improved intra-variance, integral ratio and N-percentage methods. In these methods, the interface height is determined by the vertical temperature distribution based on a three-layer smoke zone model, which is an improvement of a two-layer zone model. Thereafter, the three improved methods are applied to several typical fire cases simulated CFD to predict the smoke interface, and their applicability and reliability are verified by comparison of the smoke stratification results with the filed simulation results. Results show that the three improved methods can effectively determine the location of the three-layer zone model's interface, and they have the ability to predict smoke interface for fires with different fire source types and ventilation conditions.     

A New Approach to the Dissolution Tests Management to Obtain Kinetic and Thermodynamic Data: Release of a Model Drug from Glyceryl Behenate Matrix Tablets

This study is focused on the use of glyceryl behenate as a lipophilic excipient of matrix tablets providing controlled drug release. The aim of this study is to evaluate activation energy (EA) and changes of the thermodynamic parameters (ΔH, ΔS, ΔG) of a dissolution process. These values, which have not yet been published, can lead to better understanding of a drug release mechanism and can extend the use options of glyceryl behenate in the pharmaceutical industry. Values of ΔH, ΔS, ΔG and E_A, providing an overall thermodynamic view on the studied matrix tablets, are evaluated based on the temperature-dependences of the release rate constant of a model drug (temperature range 25 - 45 °C). The studied lipophilic matrix tablets contain 10% to 50% of glyceryl behenate. Dissolution testing is carried out in an aqueous solution of HCl with addition of NaCl (pH1.2). Positive values of ΔH in the range of 3.83 to 56.13 kJ mol^-1 and positive values of ΔG indicate that the dissolution of the studied glyceryl behenate matrix tablets is an endothermic process which does not proceed spontaneously (in a temperature range of 25 - 45 °C). The negative slope of the linear curves of enthalpy-entropy compensation confirms the entropy-driven dissolution. Practical Applications : A better understanding of the dissolution process is an important aspect, e.g., in the field of drug formulation strategy. In this study, it is confirmed that the influence of temperature on the model drug release rate is negligible for tablets containing more than 40% of glyceryl behenate. It is an important result for drug design due to the reduction of risk of a possible dose dumping effect induced by temperature and the prevention of in vivo therapeutic failure.