Discovery and in†vitro Evaluation of Novel Serotonin 5-HT2A Receptor Ligands Identified Through Virtual Screening.

The 5-HT2A receptor is a molecular target of high pharmacological importance. Ligands of this protein, particularly atypical antipsychotics, are useful in the treatment of numerous mental disorders, including schizophrenia and major depressive disorder. Structure-based virtual screening using a 5-HT2A receptor complex was performed to identify novel ligands for the 5-HT2A receptor, serving as potential antidepressants. From the Enamine screening library, containing over 4 million compounds, 48 molecules were selected for subsequent experimental validation. These compounds were tested against the 5-HT2A receptor in radioligand binding assays. From the tested batch, six molecules were identified as ligands of the main molecular target and were forwarded to a more detailed in vitro profiling. This included radioligand binding assays at 5-HT1A, 5-HT7, and D2 receptors and functional studies at 5-HT2A receptors. These compounds were confirmed to show a binding affinity for at least one of the targets tested in vitro. The success rate for the inactive template-based screening reached 17 %, while it was 9 % for the active template-based screening. Similarity and fragment analysis indicated the structural novelty of the identified compounds. Pharmacokinetics for these molecules was determined using in silico approaches.

Vibrational Optical Activity of Amyloid Fibrils.

Amyloid fibrils are supramolecular systems showing distinct chirality at different levels of their complex multilayered architectures. Due to the regular long-range chiral organization, amyloid fibrils exhibit the most intense Vibrational Optical Activity (VOA) signal observed up to now, making VOA techniques: Vibrational Circular Dichroism (VCD) and Raman Optical Activity (ROA) very promising tools to explore their structures, handedness and intricate polymorphism. This concept article reviews up-to-date experimental studies on VOA applications to investigate amyloid fibrils highlighting its future potential in analyzing of these unique supramolecular systems, in particular in the context of biomedicine and nanotechnology.

Efficient delivery of carotenoids to adipocytes with albumin.

Carotenoids are very effectively delivered by albumin to adipocytes. The uptake of carotenoids to the cells occurs in the form of self-aggregates that localize in the vicinity of the adipocyte membrane, as shown by high spatial resolution Raman spectroscopy. The binding of carotenoids to albumin and the mechanism of their transport were elucidated with the help of chiroptical spectroscopies, in tandem with molecular docking and molecular dynamics simulations. In particular, apart from the recognized high affinity pocket of albumin that binds a carotenoid monomer in domain I, we have identified a hydrophobic periphery area in domain IIIB that loosely bounds the self-aggregated carotenoid in aqueous media and enables its easy detachment in hydrophobic environments. This explains the effectiveness of albumins as nanocarriers of carotenoids to adipocytes in vitro.

Direct oral and fiber-derived butyrate supplementation as an anti-obesity treatment via different targets.

BACKGROUND & AIMS: Butyric (one of the short-chain fatty acids), a major byproduct of the fermentation of non-digestible carbohydrates (e.g. fiber), is supposed to have anti-obesity and anti-inflammatory properties. However, butyrate's potential and mechanism in preventing obesity and the efficient form of administration remain to be clarified. METHODS: Hence, we studied the effect of oral supplementation with 5% (w/w) sodium butyrate and 4% (w/w) ß-glucan (fiber) on young male mice (C57BL/6J) with high-fat diet-induced obesity (HFD: 60 kcal% of fat + 1% of cholesterol). Six weeks old mice were fed diets based on HFD or control (AIN-93G) diet with/without supplements for 4 weeks. The unique, interdisciplinary approach combining several Raman-based techniques (including Raman microscopy and fiber optic Raman spectroscopy) and next-generation sequencing was used to ex vivo analyze various depots of the adipose tissue (white, brown, perivascular) and gut microbiome, respectively. RESULTS: The findings demonstrate that sodium butyrate more effectively prevent the pathological increase in body weight caused by elevated saturated fatty acids influx linked to a HFD in comparison to ß-glucan, thereby entirely inhibiting diet-induced obesity. Moreover, butyrate significantly affects the white adipose tissue (WAT) reducing the epididymal WAT mass in comparison to HFD without supplements, and decreasing lipid saturation in the epididymal WAT and perivascular adipose tissue of the thoracic aorta. Contrarily, ß-glucan significantly changes the composition and diversity of the gut microbiome, reversing the HFD effect, but shows no effect on the epididymal WAT mass and therefore the weight gain inhibition is not as effective as with sodium butyrate. CONCLUSIONS: Here, oral supplementation with sodium butyrate and ß-glucan (fiber) has been proven to have an anti-obesity effect through two different targets. Administration-dependent effects that butyrate imposes on the adipose tissue (oral administration) and microbiome (fiber-derived) make it a promising candidate for the personalized treatment of obesity.

A comprehensive assessment of palmatine as anticonvulsant agent - In vivo and in silico studies.

Previously, we demonstrated that palmatine (PALM) - an isoquinoline alkaloid from Berberis sibrica radix, exerted antiseizure activity in the pentylenetetrazole (PTZ)-induced seizure assay in larval zebrafish. The aim of the present study was to more precisely characterize PALM as a potential anticonvulsant drug candidate. A range of zebrafish and mouse seizure/epilepsy models were applied in the investigation. Immunostaining analysis was conducted to assess the changes in mouse brains, while in silico molecular modelling was performed to determine potential targets for PALM. Accordingly, PALM had anticonvulsant effect in ethyl 2-ketopent-4-enoate (EKP)-induced seizure assay in zebrafish larvae as well as in the 6 Hz-induced psychomotor seizure threshold and timed infusion PTZ tests in mice. The protective effect in the EKP-induced seizure assay was confirmed in the local field potential recordings. PALM did not affect seizures in the gabra1a knockout line of zebrafish larvae. In the scn1Lab-/- zebrafish line, pretreatment with PALM potentiated seizure-like behaviour of larvae. Repetitive treatment with PALM, however, did not reduce development of PTZ-induced seizure activity nor prevent the loss of parvalbumin-interneurons in the hippocampus of the PTZ kindled mice. In silico molecular modelling revealed that the noted anticonvulsant effect of PALM in EKP-induced seizure assay might result from its interactions with glutamic acid decarboxylase and/or via AMPA receptor non-competitive antagonism. Our study has demonstrated the anticonvulsant activity of PALM in some experimental models of seizures, including a model of pharmacoresistant seizures induced by EKP. These results indicate that PALM might be a suitable new drug candidate but the precise mechanism of its anticonvulsant activity has to be determined.

Organization of Carotenoid Aggregates in Membranes Studied Selectively using Resonance Raman Optical Activity.

In living organisms, carotenoids are incorporated in biomembranes, remarkably modulating their mechanical characteristics, fluidity, and permeability. Significant resonance enhancement of Raman optical activity (ROA) signals of carotenoid chiral aggregates makes resonance ROA (RROA), a highly selective tool to study exclusively carotenoid assemblies in model membranes. Hence, RROA is combined with electronic circular dichroism (ECD), dynamic light scattering (DLS), molecular dynamics, and quantum-chemical calculations to shed new light on the carotenoid aggregation in dipalmitoylphosphatidylcholine (DPPC) liposomes. Using representative members of the carotenoid family: apolar α-carotene and more polar fucoxanthin and zeaxanthin, the authors demonstrate that the stability of carotenoid aggregates is directly linked with their orientation in membranes and the monomer structures inside the assemblies. In particular, polyene chain distortion of α-carotene molecules is an important feature of J-aggregates that show increased orientational freedom and stability inside liposomes compared to H-assemblies of more polar xanthophylls. In light of these results, RROA emerges as a new tool to study active compounds and drugs embedded in membranes.

The application of WaterMap-guided structure-based virtual screening in novel drug discovery.

INTRODUCTION: Nowadays, it is widely accepted that water molecules play a key role in binding a ligand to a molecular target. Neglecting water molecules in the process of molecular recognition was the result of several failures of the structure-based drug discovery campaigns. The application of WaterMap, in particular WaterMap-guided molecular docking, enables the reasonably accurate and quick description of the location and energetics of water molecules at the ligand-protein interface. AREAS COVERED: In this review, the authors shortly discuss the importance of water in drug design and discovery and provide a brief overview of the computational approaches used to predict the solvent-related effects for the purposes of presenting WaterMap in the context of other available techniques and tools. A concise description of WaterMap concept is followed by the presentation of WaterMap-assisted virtual screening literature published between 2013 and 2023. EXPERT OPINION: In recent years, WaterMap software has been extensively used to support structure-based drug design, in particular structure-based virtual screening. Indeed, it is a useful tool to rescore docking results considering water molecules in the binding pocket. Although WaterMap allows for the consideration of the dynamic behavior of water molecules in the binding site, for best accuracy, its application in conjunction with other techniques such as molecular mechanics-generalized Born surface area of FEP (Free Energy Perturbation) is recommended.

Caspases in Alzheimer's Disease: Mechanism of Activation, Role, and Potential Treatment.

With the aging of the population, treatment of conditions emerging in old age, such as neurodegenerative disorders, has become a major medical challenge. Of these, Alzheimer's disease, leading to cognitive dysfunction, is of particular interest. Neuronal loss plays an important role in the pathophysiology of this condition, and over the years, a great effort has been made to determine the role of various factors in this process. Unfortunately, until now, the exact pathomechanism of this condition remains unknown. However, the most popular theories associate AD with abnormalities in the Tau and ß-amyloid (Aß) proteins, which lead to their deposition and result in neuronal death. Neurons, like all cells, die in a variety of ways, among which pyroptosis, apoptosis, and necroptosis are associated with the activation of various caspases. It is worth mentioning that Tau and Aß proteins are considered to be one of the caspase activators, leading to cell death. Moreover, the protease activity of caspases influences both of the previously mentioned proteins, Tau and Aß, converting them into more toxic derivatives. Due to the variety of ways caspases impact the development of AD, drugs targeting caspases could potentially be useful in the treatment of this condition. Therefore, there is a constant need to search for novel caspase inhibitors and evaluate them in preclinical and clinical trials.

In Silico Analysis, Anticonvulsant Activity, and Toxicity Evaluation of Schisandrin B in Zebrafish Larvae and Mice.

The aim of this study is to evaluate the anticonvulsant potential of schisandrin B, a main ingredient of Schisandra chinensis extracts. Schisandrin B showed anticonvulsant activity in the zebrafish larva pentylenetetrazole acute seizure assay but did not alter seizure thresholds in the intravenous pentylenetetrazole test in mice. Schisandrin B crosses the blood-brain barrier, which we confirmed in our in silico and in vivo analyses; however, the low level of its unbound fraction in the mouse brain tissue may explain the observed lack of anticonvulsant activity. Molecular docking revealed that the anticonvulsant activity of the compound in larval zebrafish might have been due to its binding to a benzodiazepine site within the GABAA receptor and/or the inhibition of the glutamate NMDA receptor. Although schisandrin B showed a beneficial anticonvulsant effect, toxicological studies revealed that it caused serious developmental impairment in zebrafish larvae, underscoring its teratogenic properties. Further detailed studies are needed to precisely identify the properties, pharmacological effects, and safety of schisandrin B.

Exceptionally enhanced Raman optical activity (ROA) of amyloid fibrils and their prefibrillar states.

Amyloid fibrils form remarkable, multi-layered chiral supramolecular architectures. The proximity of interacting oscillators in the chiral fibril supramolecules is responsible for the unusual sensitivity of vibrational circular dichroism (VCD) for fibril formation. Surprisingly, up to now, such characteristics have not been shown for ROA, although it displays the same vibrational markers of fibrils as VCD, including the amide I band. Here, we report an exceptionally large enhancement of the ROA signal detected for mature amyloid fibrils and their prefibrillar states. Remarkably, the same ROA signal has been obtained for fibrils of homologous lysozymes and the dissimilar protein, insulin, indicating a possible common enhanced ROA spectrum, analogous to that for VCD for all amyloid fibrils investigated to date. The ROA signal is observed at earlier stages of fibril formation than VCD and provides access to a considerably broader range of vibrations. Further studies are necessary to verify the applicability of ROA for the analysis of amyloid fibrils.

Chirality induction and amplification in supramolecular systems exhibiting vibrational optical activity.

Vibrational optical activity (VOA) refers to two vibrational techniques: vibrational circular dichroism (VCD) and Raman optical activity (ROA) that are sensitive both to the chirality and the molecular structure, typically surpassing electronic optical activity (EOA) in recognition of fine structural details. However, the measurement of VOA is inherently obstructed as the intensity of the VOA signal is typically 10-4-10-5 of the intensity of the parent IR or Raman signals. This feature considerably limits the practical applications of VOA and is a fundamental reason why various strategies are currently developed to enhance the VOA intensity. This perspective review discusses up-to-date studies focusing on applications of VOA to analyse supramolecular, mostly biogenic, systems showing induction and amplification of chirality. Most attention is devoted to two types of biogenic supramolecular assemblies providing unique enhancement of VOA: amyloid fibrils showing enormous VCD and carotenoid aggregates exhibiting resonantly enhanced ROA.

Novel multi-target ligands of dopamine and serotonin receptors for the treatment of schizophrenia based on indazole and piperazine scaffolds-synthesis, biological activity, and structural evaluation.

Schizophrenia is a chronic mental disorder that is not satisfactorily treated with available antipsychotics. The presented study focuses on the search for new antipsychotics by optimising the compound D2AAK3, a multi-target ligand of G-protein-coupled receptors (GPCRs), in particular D2, 5-HT1A, and 5-HT2A receptors. Such receptor profile may be beneficial for the treatment of schizophrenia. Compounds 1-16 were designed, synthesised, and subjected to further evaluation. Their affinities for the above-mentioned receptors were assessed in radioligand binding assays and efficacy towards them in functional assays. Compounds 1 and 10, selected based on their receptor profile, were subjected to in vivo tests to evaluate their antipsychotic activity, and effect on memory and anxiety processes. Molecular modelling was performed to investigate the interactions of the studied compounds with D2, 5-HT1A, and 5-HT2A receptors on the molecular level. Finally, X-ray study was conducted for compound 1, which revealed its stable conformation in the solid state.

Development and Characterization of Novel Selective, Non-Basic Dopamine D2 Receptor Antagonists for the Treatment of Schizophrenia.

The dopamine D2 receptor, which belongs to the family of G protein-coupled receptors (GPCR), is an important and well-validated drug target in the field of medicinal chemistry due to its wide distribution, particularly in the central nervous system, and involvement in the pathomechanism of many disorders thereof. Schizophrenia is one of the most frequent diseases associated with disorders in dopaminergic neurotransmission, and in which the D2 receptor is the main target for the drugs used. In this work, we aimed at discovering new selective D2 receptor antagonists with potential antipsychotic activity. Twenty-three compounds were synthesized, based on the scaffold represented by the D2AAK2 compound, which was discovered by our group. This compound is an interesting example of a D2 receptor ligand because of its non-classical binding to this target. Radioligand binding assays and SAR analysis indicated structural modifications of D2AAK2 that are possible to maintain its activity. These findings were further rationalized using molecular modeling. Three active derivatives were identified as D2 receptor antagonists in cAMP signaling assays, and the selected most active compound 17 was subjected to X-ray studies to investigate its stable conformation in the solid state. Finally, effects of 17 assessed in animal models confirmed its antipsychotic activity in vivo.

Discovery of novel arylpiperazine-based DA/5-HT modulators as potential antipsychotic agents - Design, synthesis, structural studies and pharmacological profiling.

Schizophrenia is a mental disorder with a complex pathomechanism involving many neurotransmitter systems. Among the currently used antipsychotics, classical drugs acting as dopamine D2 receptor antagonists, and drugs of a newer generation, the so-called atypical antipsychotics, can be distinguished. The latter are characterized by a multi-target profile of action, affecting, apart from the D2 receptor, also serotonin receptors, in particular 5-HT2A and 5-HT1A. Such profile of action is considered superior in terms of both efficacy in treating symptoms and safety. In the search for new potential antipsychotics of such atypical receptor profile, an attempt was made to optimize the arylpiperazine based virtual hit, D2AAK3, which in previous studies displayed an affinity for D2, 5-HT1A and 5-HT2A receptors, and showed antipsychotic activity in vivo. In this work, we present the design of D2AAK3 derivatives (1-17), their synthesis, and structural and pharmacological evaluation. The obtained compounds show affinities for the receptors of interest and their efficacy as antagonists/agonists towards them was confirmed in functional assays. For the selected compound 11, detailed structural studies were carried out using molecular modeling and X-ray methods. Additionally, ADMET parameters and in vivo antipsychotic activity, as well as influence on memory and anxiety processes were evaluated in mice, which indicated good therapeutic potential and safety profile of the studied compound.

Chiral and Structural Polymorphism of Fibril Architectures of Homologous Lysozymes.

Amyloid fibrils are fascinating and complex structures with the multilayered chiral organization. Using the multimodal methodology, including VCD, ECD, cryo-EM, and TEM, we characterized in detail different levels of organization (secondary structure/protofilament/mesoscopic structure) of amyloid fibrils prepared from proteins highly homologous in the structure (hen egg white and human lysozymes). Our results demonstrate that small changes in the native protein structure or preparation conditions translate into significant differences in the handedness and architecture of the formed fibrils at various levels of their complexity. In particular, fibrils of hen egg white and human lysozymes obtained in vitro at the same preparation conditions, possess different secondary structure, protofilament twist and ultrastructure. Yet, formed fibrils adopted a relatively similar mesoscopic structure, as observed in high-resolution 3D cryo-EM, scarcely used up to now for fibrils obtained in vitro in denaturing condition. Our results add to other puzzling experiments implicating the indeterministic nature of fibril formation.

Illuminating the "Twilight Zone": Advances in Difficult Protein Modeling.

Homology modeling was long considered a method of choice in tertiary protein structure prediction. However, it used to provide models of acceptable quality only when templates with appreciable sequence identity with a target could be found. The threshold value was long assumed to be around 20-30%. Below this level, obtained sequence identity was getting dangerously close to values that can be obtained by chance, after aligning any random, unrelated sequences. In these cases, other approaches, including ab initio folding simulations or fragment assembly, were usually employed. The most recent editions of the CASP and CAMEO community-wide modeling methods assessment have brought some surprising outcomes, proving that much more clues can be inferred from protein sequence analyses than previously thought. In this chapter, we focus on recent advances in the field of difficult protein modeling, pushing the threshold deep into the "twilight zone", with particular attention devoted to improvements in applications of machine learning and model evaluation.

The Role of Genetics in the Development and Pharmacotherapy of Depression and Its Impact on Drug Discovery.

Complex disorders, such as depression, remain a mystery for scientists. Although genetic factors are considered important for the prediction of one's vulnerability, it is hard to estimate the exact risk for a patient to develop depression, based only on one category of vulnerability criteria. Genetic factors also regulate drug metabolism, and when they are identified in a specific combination, may result in increased drug resistance. A proper understanding of the genetic basis of depression assists in the development of novel promising medications and effective disorder management schemes. This review aims to analyze the recent literature focusing on the correlation between specific genes and the occurrence of depression. Moreover, certain aspects targeting a high drug resistance identified among patients suffering from major depressive disorder were highlighted in this manuscript. An expected direction of future drug discovery campaigns was also discussed.

Synthesis, Structural and Behavioral Studies of Indole Derivatives D2AAK5, D2AAK6 and D2AAK7 as Serotonin 5-HT1A and 5-HT2A Receptor Ligands.

Serotonin receptors are involved in a number of physiological functions and regulate aggression, anxiety, appetite, cognition, learning, memory, mood, nausea, sleep, and thermoregulation. Here we report synthesis and detailed structural and behavioral studies of three indole derivatives: D2AAK5, D2AAK6, and D2AAK7 as serotonin 5-HT1A and 5-HT2A receptor ligands. X-ray studies revealed that the D2AAK5 compound crystallizes in centrosymmetric triclinic space group with one molecule in the asymmetric unit. The main interaction between the ligands and the receptors is the salt bridge between the protonatable nitrogen atom of the ligands and the conserved Asp (3.32) of the receptors. The complexes were stable in the molecular dynamic simulations. MD revealed that the studied ligands are relatively stable in their binding sites, with the exception of D2AAK7 in the serotonin 5-HT1A receptor. D2AAK7 exerts anxiolytic activity in the EPM test, while D2AAK5 has a beneficial effect on the memory processes in the PA test.

Funnel metadynamics and behavioral studies reveal complex effect of D2AAK1 ligand on anxiety-like processes.

Anxiety is a troublesome symptom for many patients, especially those suffering from schizophrenia. Its regulation involves serotonin receptors, targeted e.g. by antipsychotics or psychedelics such as LSD. 5-HT2A receptors are known for an extremely long LSD residence time, enabling minute doses to exert a long-lasting effect. In this work, we explore the changes in anxiety-like processes induced by the previously reported antipsychotic, D2AAK1. In vivo studies revealed that the effect of D2AAK1 on the anxiety is mediated through serotonin 5-HT1A and 5-HT2A receptors, and that it is time-dependent (anxiogenic after 30 min, anxiolytic after 60 min) and dose-dependent. The funnel metadynamics simulations suggest complicated ligand-5HT2AR interactions, involving an allosteric site located under the third extracellular loop, which is a possible explanation of the time-dependency. The binding of D2AAK1 at the allosteric site results in a broader opening of the extracellular receptor entry, possibly altering the binding kinetics of orthosteric ligands.