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.

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.

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.

Overcoming Depression with 5-HT2A Receptor Ligands.

Depression is a multifactorial disorder that affects millions of people worldwide, and none of the currently available therapeutics can completely cure it. Thus, there is a need for developing novel, potent, and safer agents. Recent medicinal chemistry findings on the structure and function of the serotonin 2A (5-HT2A) receptor facilitated design and discovery of novel compounds with antidepressant action. Eligible papers highlighting the importance of 5-HT2A receptors in the pathomechanism of the disorder were identified in the content-screening performed on the popular databases (PubMed, Google Scholar). Articles were critically assessed based on their titles and abstracts. The most accurate papers were chosen to be read and presented in the manuscript. The review summarizes current knowledge on the applicability of 5-HT2A receptor signaling modulators in the treatment of depression. It provides an insight into the structural and physiological features of this receptor. Moreover, it presents an overview of recently conducted virtual screening campaigns aiming to identify novel, potent 5-HT2A receptor ligands and additional data on currently synthesized ligands acting through this protein.

Docking-Based 3D-QSAR Studies for 1,3,4-oxadiazol-2-one Derivatives as FAAH Inhibitors.

This work aimed to construct 3D-QSAR CoMFA and CoMSIA models for a series of 31 FAAH inhibitors, containing the 1,3,4-oxadiazol-2-one moiety. The obtained models were characterized by good statistical parameters: CoMFA Q2 = 0.61, R2 = 0.98; CoMSIA Q2 = 0.64, R2 = 0.93. The CoMFA model field contributions were 54.1% and 45.9% for steric and electrostatic fields, respectively. In the CoMSIA model, electrostatic, steric, hydrogen bond donor, and hydrogen acceptor properties were equal to 34.6%, 23.9%, 23.4%, and 18.0%, respectively. These models were validated by applying the leave-one-out technique, the seven-element test set (CoMFA r2test-set = 0.91; CoMSIA r2test-set = 0.91), a progressive scrambling test, and external validation criteria developed by Golbraikh and Tropsha (CoMFA r20 = 0.98, k = 0.95; CoMSIA r20 = 0.98, k = 0.89). As the statistical significance of the obtained model was confirmed, the results of the CoMFA and CoMSIA field calculation were mapped onto the enzyme binding site. It gave us the opportunity to discuss the structure-activity relationship based on the ligand-enzyme interactions. In particular, examination of the electrostatic properties of the established CoMFA model revealed fields that correspond to the regions where electropositive substituents are not desired, e.g., in the neighborhood of the 1,3,4-oxadiazol-2-one moiety. This highlights the importance of heterocycle, a highly electronegative moiety in this area of each ligand. Examination of hydrogen bond donor and acceptor properties contour maps revealed several spots where the implementation of another hydrogen-bond-donating moiety will positively impact molecules' binding affinity, e.g., in the neighborhood of the 1,3,4-oxadiazol-2-one ring. On the other hand, there is a large isopleth that refers to the favorable H-bond properties close to the terminal phenoxy group of a ligand, which means that, generally speaking, H-bond acceptors are desired in this area.

The Universal 3D QSAR Model for Dopamine D2 Receptor Antagonists.

In order to search for novel antipsychotics acting through the D2 receptor, it is necessary to know the structure-activity relationships for dopamine D2 receptor antagonists. In this context, we constructed the universal three-dimensional quantitative structure-activity relationship (3D- QSAR) model for competitive dopamine D2 receptor antagonists. We took 176 compounds from chemically different groups characterized by the half maximal inhibitory concentration (IC50)from the CHEMBL database and docked them to the X-ray structure of the human D2 receptor in the inactive state. Selected docking poses were applied for Comparative Molecular Field Analysis (CoMFA) alignment. The obtained CoMFA model is characterized by a cross-validated coefficient Q2 of 0.76 with an optimal component of 5, R2 of 0.92, and an F value of 338.9. The steric and electrostatic field contributions are 67.4% and 32.6%, respectively. The statistics obtained prove that the CoMFA model is significant. Next, the IC50 of the 16 compounds from the test set was predicted with R2 of 0.95. Finally, a progressive scrambling test was carried out for additional validation. The CoMFA fields were mapped onto the dopamine D2 receptor binding site, which enabled a discussion of the structure-activity relationship based on ligand-receptor interactions. In particular, it was found that one of the desired steric interactions covers the area of a putative common allosteric pocket suggested for some other G protein-coupled receptors (GPCRs), which would suggest that some of the known dopamine receptor antagonists are bitopic in their essence. The CoMFA model can be applied to predict the potential activity of novel dopamine D2 receptor antagonists.

In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation.

Antibodies are important tools in anatomical pathology and research, but the quality of in situ protein detection by immunohistochemistry greatly depends on the choice of antibodies and the abundance of the targeted proteins. Many antibodies used in scientific research do not meet requirements for specificity and sensitivity. Accordingly, methods that improve antibody performance and produce quantitative data can greatly advance both scientific investigations and clinical diagnostics based on protein expression and in situ localization. We demonstrate here protocols for antibody labeling that allow specific protein detection in tissues via bright-field in situ proximity ligation assays, where each protein molecule must be recognized by two antibodies. We further demonstrate that single polyclonal antibodies or purified serum preparations can be used for these dual recognition assays. The requirement for protein recognition by pairs of antibody conjugates can significantly improve specificity of protein detection over single-binder assays.

Single Chain Antibodies as Tools to Study transforming growth factor-ß-Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens.

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-ß signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-ß signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-ß signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.

Parallel visualization of multiple protein complexes in individual cells in tumor tissue.

Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.

Intercellular variation in signaling through the TGF-ß pathway and its relation to cell density and cell cycle phase.

Fundamental open questions in signal transduction remain concerning the sequence and distribution of molecular signaling events among individual cells. In this work, we have characterized the intercellular variability of transforming growth factor ß-induced Smad interactions, providing essential information about TGF-ß signaling and its dependence on the density of cell populations and the cell cycle phase. By employing the recently developed in situ proximity ligation assay, we investigated the dynamics of interactions and modifications of Smad proteins and their partners under native and physiological conditions. We analyzed the kinetics of assembly of Smad complexes and the influence of cellular environment and relation to mitosis. We report rapid kinetics of formation of Smad complexes, including native Smad2-Smad3-Smad4 trimeric complexes, in a manner influenced by the rate of proteasomal degradation of these proteins, and we found a striking cell to cell variation of signaling complexes. The single-cell analysis of TGF-ß signaling in genetically unmodified cells revealed previously unknown aspects of regulation of this pathway, and it provided a basis for analysis of these signaling events to diagnose pathological perturbations in patient samples and to evaluate their susceptibility to drug treatment.

Sampling and Scoring in Protein-Protein Docking.

Protein-protein docking is considered one of the most important techniques supporting experimental proteomics. Recent developments in the field of computer science helped to improve this computational technique so that it better handles the complexity of protein nature. Sampling algorithms are responsible for the generation of numerous protein-protein ensembles. Unfortunately, a primary docking output comprises a set of both near-native poses and decoys. Application of the efficient scoring function helps to differentiate poses with the most favorable properties from those that are very unlikely to represent a natural state of the complex. This chapter explains the importance of sampling and scoring in the process of protein-protein docking. Moreover, it summarizes advances in the field.

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.

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.

Transcriptional profiling of human glioblastoma vessels indicates a key role of VEGF-A and TGFß2 in vascular abnormalization.

Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFß2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFß signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFß2 may represent a new target for vascular normalization therapy.

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.

What are the challenges with multi-targeted drug design for complex diseases?

INTRODUCTION: Current findings on multifactorial diseases with a complex pathomechanism confirm that multi-target drugs are more efficient ways in treating them as opposed to single-target drugs. However, to design multi-target ligands, a number of factors and challenges must be taken into account. AREAS COVERED: In this perspective, we summarize the concept of application of multi-target drugs for the treatment of complex diseases such as neurodegenerative diseases, schizophrenia, diabetes, and cancer. We discuss the aspects of target selection for multifunctional ligands and the application of in silico methods in their design and optimization. Furthermore, we highlight other challenges such as balancing affinities to different targets and drug-likeness of obtained compounds. Finally, we present success stories in the design of multi-target ligands for the treatment of common complex diseases. EXPERT OPINION: Despite numerous challenges resulting from the design of multi-target ligands, these efforts are worth making. Appropriate target selection, activity balancing, and ligand drug-likeness belong to key aspects in the design of ligands acting on multiple targets. It should be emphasized that in silico methods, in particular inverse docking, pharmacophore modeling, machine learning methods and approaches derived from network pharmacology are valuable tools for the design of multi-target drugs.

Multitarget Derivatives of D2AAK1 as Potential Antipsychotics: The Effect of Substitution in the Indole Moiety.

Schizophrenia is a complex disease which is best treated with multitarget drugs, such as atypical antipsychotics. Previously, using structure-based virtual screening, we found a virtual hit, D2AAK1, with nanomolar affinity for dopamine and serotonin receptors important in schizophrenia pharmacotherapy. As a part of an optimization campaign of D2AAK1, we obtained 17 derivatives that also display a multitarget profile. Selected compounds were tested against off-targets in schizophrenia, i. e., histamine H1 receptor and muscarinic M1 receptor, and these did not display considerable affinity for these receptors. The two most promising compounds were subjected to behavioral studies. These compounds decreased amphetamine-induced hyperactivity in mice which indicates their antipsychotic potential. The compounds did not interfere with the memory consolidation in mice, as determined in the passive avoidance test. The favorable pharmacological profile of these compounds was rationalized using molecular modeling.

Current Approaches and Tools Used in Drug Development against Parkinson's Disease.

Parkinson's disease is a progressive neurodegenerative disorder characterized by the death of nerve cells in the substantia nigra of the brain. The treatment options for this disease are very limited as currently the treatment is mainly symptomatic, and the available drugs are not able to completely stop the progression of the disease but only to slow it down. There is still a need to search for new compounds with the most optimal pharmacological profile that would stop the rapidly progressing disease. An increasing understanding of Parkinson's pathogenesis and the discovery of new molecular targets pave the way to develop new therapeutic agents. The use and selection of appropriate cell and animal models that better reflect pathogenic changes in the brain is a key aspect of the research. In addition, computer-assisted drug design methods are a promising approach to developing effective compounds with potential therapeutic effects. In light of the above, in this review, we present current approaches for developing new drugs for Parkinson's disease.

A protein interaction node at the neurotransmitter release site: domains of Aczonin/Piccolo, Bassoon, CAST, and rim converge on the N-terminal domain of Munc13-1.

Multidomain scaffolding proteins organize the molecular machinery of neurotransmitter vesicle dynamics during synaptogenesis and synaptic activity. We find that domains of five active zone proteins converge on an interaction node that centers on the N-terminal region of Munc13-1 and includes the zinc-finger domain of Rim1, the C-terminal region of Bassoon, a segment of CAST1/ELKS2, and the third coiled-coil domain (CC3) of either Aczonin/Piccolo or Bassoon. This multidomain complex may constitute a center for the physical and functional integration of the protein machinery at the active zone. An additional connection between Aczonin and Bassoon is mediated by the second coiled-coil domain of Aczonin. Recombinant Aczonin-CC3, expressed in cultured neurons as a green fluorescent protein fusion protein, is targeted to synapses and suppresses vesicle turnover, suggesting involvements in synaptic assembly as well as activity. Our findings show that Aczonin, Bassoon, CAST1, Munc13, and Rim are closely and multiply interconnected, they indicate that Aczonin-CC3 can actively participate in neurotransmitter vesicle dynamics, and they highlight the N-terminal region of Munc13-1 as a hub of protein interactions by adding three new binding partners to its mechanistic potential in the control of synaptic vesicle priming.