Contilisant+Tubastatin A Hybrids: Polyfunctionalized Indole Derivatives as New HDAC Inhibitor-Based Multitarget Small Molecules with In Vitro and In Vivo Activity in Neurodegenerative Diseases.

Herein, we describe the design, synthesis, and biological evaluation of 15 Contilisant+Tubastatin A hybrids. These ligands are polyfunctionalized indole derivatives developed by juxtaposing selected pharmacophoric moieties of Contilisant and Tubastatin A to act as multifunctional ligands. Compounds 3 and 4 were identified as potent HDAC6 inhibitors (IC50 = 0.012 µM and 0.035 µM, respectively), so they were further evaluated in Drosophila and human cell models of Parkinson's disease (PD). Both compounds attenuated PD-like phenotypes, such as motor defects, oxidative stress, and mitochondrial dysfunction in PD model flies. Ligands 3 and 4 were also studied in the transgenic Caenorhabditis elegans CL2006 model of Alzheimer's disease (AD). Both compounds were nontoxic, did not induce undesirable animal functional changes, inhibited age-related paralysis, and improved cognition in the thrashing assay. These results highlight 3 and 4 as novel multifunctional ligands that improve the features of PD and AD hallmarks in the respective animal models.

Synthesis and biochemical evaluation of new 3-amido-4-substituted monocyclic ß-lactams as inhibitors of penicillin-binding protein(s).

In the final phases of bacterial cell wall synthesis, penicillin-binding proteins (PBPs) catalyze the cross-linking of peptidoglycan. For many decades, effective and non-toxic ß-lactam antibiotics have been successfully used as mimetics of the d-Ala-d-Ala moiety of the natural substrate and employed as irreversible inhibitors of PBPs. In the years following their discovery, the emergence of resistant bacteria led to a decline in their clinical efficacy. Using Staudinger cycloaddition, we synthesized a focused library of novel monocyclic ß-lactams in which different substituents were introduced at the C4 position of the ß-lactam ring, at the C3 amino position, and at the N1 lactam nitrogen. In biochemical assays, the compounds were evaluated for their inhibitory effect on the model enzyme PBP1b from Streptococcus pneumoniae. Upon investigation of the antibacterial activity of the newly prepared compounds against ESKAPE pathogens, some compounds showed moderate inhibition. We also examined their reactivity and selectivity in a biochemical assay with other enzymes that have a catalytic serine in the active site, such as human cholinesterases, where they also showed no inhibitory activity, highlighting their specificity for bacterial targets. These compounds form the basis for further work on new monocyclic ß-lactams with improved antibacterial activity.

3,3-Dibromoflavanone, a synthetic flavonoid derivative for pain management with antidepressant-like effects and fewer side effects than those of morphine in mice.

In the pursuit of new lead compounds with fewer side effects than opioids, the novel synthetic phytochemical core, 3,3-dibromoflavanone (3,3-DBF), has emerged as a promising candidate for pain management. Acute assays demonstrated dose-dependent central and peripheral antinociceptive activity of 3,3-DBF through the µ-opioid receptor. This study aimed to explore repeated administration effects of 3,3-DBF in mice and compare them with morphine. Mice were treated with 3,3-DBF (30 mg/kg), morphine (6 mg/kg), or vehicle for 10 days, alongside single-treatment groups. Unlike morphine, 3,3-DBF demonstrated antinociceptive effects in the hot plate test without inducing tolerance. Locomotor activity and motor coordination tests (evaluated through the inverted screen and rotarod tests) revealed no significant differences between the 3,3-DBF-treated and control groups. The gastrointestinal transit assay indicated that 3,3-DBF did not induce constipation, in contrast to morphine. Furthermore, withdrawal signs assessed with the Gellert-Holtzman scale were not comparable to morphine. Additionally, 3,3-DBF exhibited antidepressant-like activity, reducing immobility time in the forced swimming and tail suspension tests, akin to imipramine. In summary, 3,3-DBF demonstrated antinociceptive effects without inducing tolerance or dependence and exhibited antidepressant properties. These findings highlight the potential of 3,3-DBF as a promising therapeutic agent for pain management and its comorbidities, offering advantages over morphine by minimizing side effects.

Probing Alzheimer's pathology: Exploring the next generation of FDDNP analogues for amyloid ß detection.

Fluorescent probes are a powerful tool for imaging amyloid ß (Aß) plaques, the hallmark of Alzheimer's disease (AD). Herein, we report the synthesis and comprehensive characterization of 21 novel probes as well as their optical properties and binding affinities to Aß fibrils. One of these dyes, 1Ae, exhibited several improvements over FDDNP, an established biomarker for Aß- and Tau-aggregates. First, 1Ae had large Stokes shifts (138-213 nm) in various solvents, thereby reducing self-absorption. With a high quantum yield ratio (φ(dichloromethane/methanol) = 104), 1Ae also ensures minimal background emission in aqueous environments and high sensitivity. In addition, compound 1Ae exhibited low micromolar binding affinity to Aß fibrils in vitro (Kd = 1.603 µM), while increasing fluorescence emission (106-fold) compared to emission in buffer alone. Importantly, the selective binding of 1Ae to Aß1-42 fibrils was confirmed by an in cellulo assay, supported by ex vivo fluorescence microscopy of 1Ae on postmortem AD brain sections, allowing unequivocal identification of Aß plaques. The intermolecular interactions of fluorophores with Aß were elucidated by docking studies and molecular dynamics simulations. Density functional theory calculations revealed the unique photophysics of these rod-shaped fluorophores, with a twisted intramolecular charge transfer (TICT) excited state. These results provide valuable insights into the future application of such probes as potential diagnostic tools for AD in vitro and ex vivo such as determination of Aß1-42 in cerebrospinal fluid or blood.

Molecular Mechanism of Labelling Functional Cysteines by Heterocyclic Thiones.

Heterocyclic thiones have recently been identified as reversible covalent warheads, consistent with their mild electrophilic nature. Little is known so far about their mechanism of action in labelling nucleophilic sidechains, especially cysteines. The vast number of tractable cysteines promotes a wide range of target proteins to examine; however, our focus was put on functional cysteines. We chose the main protease of SARS-CoV-2 harboring Cys145 at the active site that is a structurally characterized and clinically validated target of covalent inhibitors. We screened an in-house, cysteine-targeting covalent inhibitor library which resulted in several covalent fragment hits with benzoxazole, benzothiazole and benzimidazole cores. Thione derivatives and Michael acceptors were selected for further investigations with the objective of exploring the mechanism of inhibition of the thiones and using the thoroughly characterized Michael acceptors for benchmarking our studies. Classical and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were carried out that revealed a new mechanism of covalent cysteine labelling by thione derivatives, which was supported by QM and free energy calculations and by a wide range of experimental results. Our study shows that the molecular recognition step plays a crucial role in the overall binding of both sets of molecules.

Synthesis and Cholinesterase Inhibitory Activity of Selected Indole-Based Compounds.

Synthesis and anticholinesterase activity of 18 previously unpublished indole- and tryptophan-derived compounds are disclosed. These sp3-rich compounds containing an indole structural unit exhibit selective submicromolar inhibition of human butyrylcholinesterase (hBChE). The structures of the newly synthesized compounds were confirmed by 1H and 13C NMR, IR spectroscopy, and high-resolution mass spectrometry.

Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine.

The binucleophilic properties of 1,2-aminothiol and its rare occurrence in nature make it a useful reporter for tracking molecules in living systems. The 1,2-aminothiol moiety is present in cysteine, which is a substrate for a biocompatible click reaction with heteroaromatic nitriles. Despite the wide range of applications for this reaction, the scope of nitrile substrates has been explored only to a limited extent. In this study, we expand the chemical space of heteroaromatic nitriles for bioconjugation under physiologically relevant conditions. We systematically assembled a library of 116 2-cyanobenzimidazoles, 1-methyl-2-cyanobenzimidazoles, 2-cyanobenzothiazoles, and 2-cyanobenzoxazoles containing electron-donating and electron-withdrawing substituents at all positions of the benzene ring. The compounds were evaluated for their stability, reactivity, and selectivity toward the N-terminal cysteine of model oligopeptides. In comparison to the benchmark 6-hydroxy-2-cyanobenzothiazole or 6-amino-2-cyanobenzothiazole, we provide highly selective and moderately reactive nitriles as well as highly reactive yet less selective analogs with a variety of enabling attachment chemistries to aid future applications in bioconjugation, chemical biology, and nanomaterial science.

8-Hydroxyquinolylnitrones as multifunctional ligands for the therapy of neurodegenerative diseases.

We describe the development of quinolylnitrones (QNs) as multifunctional ligands inhibiting cholinesterases (ChEs: acetylcholinesterase and butyrylcholinesterase-hBChE) and monoamine oxidases (hMAO-A/B) for the therapy of neurodegenerative diseases. We identified QN 19, a simple, low molecular weight nitrone, that is readily synthesized from commercially available 8-hydroxyquinoline-2-carbaldehyde. Quinolylnitrone 19 has no typical pharmacophoric element to suggest ChE or MAO inhibition, yet unexpectedly showed potent inhibition of hBChE (IC50 = 1.06 ± 0.31 nmol/L) and hMAO-B (IC50 = 4.46 ± 0.18 µmol/L). The crystal structures of 19 with hBChE and hMAO-B provided the structural basis for potent binding, which was further studied by enzyme kinetics. Compound 19 acted as a free radical scavenger and biometal chelator, crossed the blood-brain barrier, was not cytotoxic, and showed neuroprotective properties in a 6-hydroxydopamine cell model of Parkinson's disease. In addition, in vivo studies showed the anti-amnesic effect of 19 in the scopolamine-induced mouse model of AD without adverse effects on motoric function and coordination. Importantly, chronic treatment of double transgenic APPswe-PS1δE9 mice with 19 reduced amyloid plaque load in the hippocampus and cortex of female mice, underscoring the disease-modifying effect of QN 19.

Biological Evaluation of Valeriana Extracts from Argentina with Potent Cholinesterase Inhibition for the Treatment of Neurodegenerative Disorders and Their Comorbidities-The Case of Valeriana carnosa Sm. (Caprifoliaceae) Studied in Mice.

Alzheimer's disease (AD) is a neurodegenerative disorder whose pathophysiology includes the abnormal accumulation of proteins (e.g., ß-amyloid), oxidative stress, and alterations in neurotransmitter levels, mainly acetylcholine. Here we present a comparative study of the effect of extracts obtained from endemic Argentinian species of valerians, namely V. carnosa Sm., V. clarionifolia Phil. and V. macrorhiza Poepp. ex DC from Patagonia and V. ferax (Griseb.) Höck and V. effusa Griseb., on different AD-related biological targets. Of these anxiolytic, sedative and sleep-inducing valerians, V. carnosa proved the most promising and was assayed in vivo. All valerians inhibited acetylcholinesterase (IC50 between 1.08-12.69 mg/mL) and butyrylcholinesterase (IC50 between 0.0019-1.46 mg/mL). They also inhibited the aggregation of ß-amyloid peptide, were able to chelate Fe2+ ions, and exhibited a direct relationship between antioxidant capacity and phenolic content. Moreover, V. carnosa was able to inhibit human monoamine oxidase A (IC50: 0.286 mg/mL (0.213-0.384)). A daily intake of aqueous V. carnosa extract by male Swiss mice (50 and 150 mg/kg/day) resulted in anxiolytic and antidepressant-like behavior and improved spatial memory. In addition, decreased AChE activity and oxidative stress markers were observed in treated mouse brains. Our studies contribute to the development of indigenous herbal medicines as therapeutic agents for AD.

Pseudo-irreversible butyrylcholinesterase inhibitors: Structure-activity relationships, computational and crystallographic study of the N-dialkyl O-arylcarbamate warhead.

Alongside reversible butyrylcholinesterase inhibitors, a plethora of covalent butyrylcholinesterase inhibitors have been reported in the literature, typically pseudo-irreversible carbamates. For these latter, however, most cases lack full confirmation of their covalent mode of action. Additionally, the available reports regarding the structure-activity relationships of the O-arylcarbamate warhead are incomplete. Therefore, a follow-up on a series of pseudo-irreversible covalent carbamate human butyrylcholinesterase inhibitors and the structure-activity relationships of the N-dialkyl O-arylcarbamate warhead are presented in this study. The covalent mechanism of binding was tested by IC50 time-dependency profiles, and sequentially and increasingly confirmed by kinetic analysis, whole protein LC-MS, and crystallographic analysis. Computational studies provided valuable insights into steric constraints and identified problematic, bulky carbamate warheads that cannot reach and carbamoylate the catalytic Ser198. Quantum mechanical calculations provided further evidence that steric effects appear to be a key factor in determining the covalent binding behaviour of these carbamate cholinesterase inhibitors and their duration of action. Additionally, the introduction of a clickable terminal alkyne moiety into one of the carbamate N-substituents and in situ derivatisation with azide-containing fluorophore enabled fluorescent labelling of plasma human butyrylcholinesterase. This proof-of-concept study highlights the potential of this novel approach and for these compounds to be further developed as clickable molecular probes for investigating tissue localisation and activity of cholinesterases.

Next-Generation Heterocyclic Electrophiles as Small-Molecule Covalent MurA Inhibitors.

Heterocyclic electrophiles as small covalent fragments showed promising inhibitory activity on the antibacterial target MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase, EC:2.5.1.7). Here, we report the second generation of heterocyclic electrophiles: the quaternized analogue of the heterocyclic covalent fragment library with improved reactivity and MurA inhibitory potency. Quantum chemical reaction barrier calculations, GSH (L-glutathione) reactivity assay, and thrombin counter screen were also used to demonstrate and explain the improved reactivity and selectivity of the N-methylated heterocycles and to compare the two generations of heterocyclic electrophiles.

Fragment-Sized Thiazoles in Fragment-Based Drug Discovery Campaigns: Friend or Foe?

Thiazoles exhibit a wide range of biological activities and therefore represent useful and attractive building blocks. To evaluate their usefulness and pinpoint their liabilities in fragment screening campaigns, we assembled a focused library of 49 fragment-sized thiazoles and thiadiazoles with various substituents, namely amines, bromides, carboxylic acids, and nitriles. The library was profiled in a cascade of biochemical inhibition assays, redox activity, thiol reactivity, and stability assays. Our study indicates that when thiazole derivatives are identified as screening hits, their reactivity should be carefully addressed and correlated with specific on-target engagement. Importantly, nonspecific inhibition should be excluded using experimental approaches and in silico predictions. To help with validation of hits identified in fragment screening campaigns, we can apply our high-throughput profiling workflow to focus on the most tractable compounds with a clear mechanism of action.

N-Hydroxy-N-Propargylamide Derivatives of Ferulic Acid: Inhibitors of Cholinesterases and Monoamine Oxidases.

Alzheimer's disease (AD) is a complex disorder characterized by impaired neurotransmission in cholinergic and monoaminergic neurons, which, in combination with the accumulation of misfolded proteins and increased oxidative stress, leads to the typical features of the disease at the biomolecular level. Given the limited therapeutic success of approved drugs, it is imperative to explore rationally supported therapeutic approaches to combat this disease. The search for novel scaffolds that bind to different receptors and inhibit AD disease-related enzymes could lead to new therapeutic solutions. Here, we describe N-hydroxy-N-propargylamide hybrids 1-6, which were designed by combining the structures of Contilisant-a multifunctional anti-AD ligand-and ferulic acid, a natural antioxidant with various other biological activities. Among the synthesized compounds, we identified compound 4 as a micromolar inhibitor of hAChE with a potent radical-scavenging capacity comparable to resveratrol and Trolox. In addition, compound 4 chelated copper(II) ions associated with amyloid ß pathology, mitochondrial dysfunction, and oxidative stress. The promising in vitro activity combined with favorable drug-like properties and predicted blood-brain barrier permeability make compound 4 a multifunctional ligand that merits further studies at the biochemical and cellular levels.

Cell models for Alzheimer's and Parkinson's disease: At the interface of biology and drug discovery.

Neurodegenerative diseases are severely debilitating conditions characterized primarily by progressive neuronal loss and impairment of the nervous system. Alzheimer's and Parkinson's diseases are the most common neurodegenerative disorders, and their impact is increasing as average life expectancy increases worldwide. Although the underlying mechanisms of both progressive diseases have been extensively studied, we still lack a comprehensive understanding of the molecular basis of both diseases. Current therapeutic options do not slow the progression of the diseases and only provide symptom relief. Cell models that resemble the characteristics of the disease in question are important in drug discovery projects because they provide information about the therapeutic benefits of drugs under development. Here, we review current in vitro cell models used to study the molecular basis of Alzheimer's and Parkinson's disease focusing on their potential for discovering of disease-modifying therapeutics to combat neurodegenerative diseases. We discuss phenotypic screening as an important approach for identifying novel therapeutic molecules. Advances in the development of cell-based assays for drug discovery are discussed, ranging from simple monoculture cell models to high-throughput three-dimensional cell models. Finally, we critically present the limitations of cell models and the caveats encountered in drug discovery to find effective treatment for neurodegenerative diseases.

Polyfunctionalized α-Phenyl-tert-butyl(benzyl)nitrones: Multifunctional Antioxidants for Stroke Treatment.

Nowadays, most stroke patients are treated exclusively with recombinant tissue plasminogen activator, a drug with serious side effects and limited therapeutic window. For this reason, and because of the known effects of oxidative stress on stroke, a more tolerable and efficient therapy for stroke is being sought that focuses on the control and scavenging of highly toxic reactive oxygen species by appropriate small molecules, such as nitrones with antioxidant properties. In this context, herein we report here the synthesis, antioxidant, and neuroprotective properties of twelve novel polyfunctionalized α-phenyl-tert-butyl(benzyl)nitrones. The antioxidant capacity of these nitrones was investigated by various assays, including the inhibition of lipid peroxidation induced by AAPH, hydroxyl radical scavenging assay, ABTS+-decoloration assay, DPPH scavenging assay, and inhibition of soybean lipoxygenase. The inhibitory effect on monoamine oxidases and cholinesterases and inhibition of ß-amyloid aggregation were also investigated. As a result, (Z)-N-benzyl-1-(2-(3-(piperidin-1-yl)propoxy)phenyl)methanimine oxide (5) was found to be one of the most potent antioxidants, with high ABTS+ scavenging activity (19%), and potent lipoxygenase inhibitory capacity (IC50 = 10 µM), selectively inhibiting butyrylcholinesterase (IC50 = 3.46 ± 0.27 µM), and exhibited neuroprotective profile against the neurotoxicant okadaic acid in a neuronal damage model. Overall, these results pave the way for the further in-depth analysis of the neuroprotection of nitrone 5 in in vitro and in vivo models of stroke and possibly other neurodegenerative diseases in which oxidative stress is identified as a critical player.

Screening of Big Pharma's Library against Various in-house Biological Targets.

Open innovation initiatives provide opportunities for collaboration and sharing of knowledge and experience between industry, academia, and government institutions. Through open innovation, Merck is offering a Mini Library of 80 carefully selected compounds from previous research and development projects to a broader scientific community for testing in academic drug discovery projects. These compounds are predominantly drug-like and cover a broad range of molecular targets. They could potentially interact with other enzymes, receptors, transporters, and ion channels of interest. The Mini Library was tested on seven in-house enzymes (bacterial MurA, MurC ligase, and DdlB enzyme, human MAO-A/B, human BChE, and murine AChE), and several hits were identified. A follow-up series of structural analogues provided by Merck gave a more detailed insight into the accessibility and the quality of the hit compounds. For example, sartan derivatives were moderate inhibitors of MurC, whereas bisarylureas were potent, selective, nanomolar inhibitors of hMAO-B. Importantly, 3-n-butyl-substituted indoles were identified as low nanomolar selective inhibitors of hBChE. All in all, the hit derivatives provide new starting points for the further exploration of the chemical space of high-quality enzyme inhibitors.

From tryptophan-based amides to tertiary amines: Optimization of a butyrylcholinesterase inhibitor series.

Lead optimization of a series of tryptophan-based nanomolar butyrylcholinesterase (BChE) inhibitors led to tertiary amines as highly potent, achiral, sp3-rich analogues with better synthetic accessibility and high selectivity over acetylcholinesterase (one to ten thousandfold). Taking it one step further, the introduction of a carbamate warhead on the well-explored reversible scaffold allowed conversion to pseudoirreversible inhibitors that bound covalently to BChE and prolonged the duration of inhibition (half-life of 14.8 h for compound 45a-carbamoylated enzyme). Additionally, N-hydroxyindole was discovered as a novel leaving group chemotype. The covalent mechanism of action was confirmed by time-dependency experiments, progress curve analysis, and indirectly by co-crystallization with the human recombinant enzyme. Two crystal structures of BChE-inhibitor complexes were solved and coupled with the supporting molecular dynamics simulations increased our understanding of the structure-activity relationship, while also providing the necessary structural information for future optimization of this series. Overall, this research demonstates the high versatility and potential of this series of BChE inhibitors.

Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds.

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.

ProBiS-Dock: A Hybrid Multitemplate Homology Flexible Docking Algorithm Enabled by Protein Binding Site Comparison.

The protein data bank (PDB) is a rich source of protein ligand structures, but ligands are not explicitly used in current docking algorithms. We have developed ProBiS-Dock, a docking algorithm complementary to the ProBiS-Dock Database (J. Chem. Inf. Model. 2021, 61, 4097-4107) that treats small molecules and proteins as fully flexible entities and allows conformational changes in both after ligand binding. A new scoring function is described that consists of a binding site-specific scoring function (ProBiS-Score) and a general statistical scoring function. ProBiS-Dock enables rapid docking of small molecules to proteins and has been successfully validated in silico against standard benchmarks. It enables rapid search for new active ligands by leveraging existing knowledge in the PDB. The potential of the software for drug development has been confirmed in vitro by the discovery of new inhibitors of human indoleamine 2,3-dioxygenase 1, an enzyme that is an attractive target for cancer therapy and catalyzes the first rate-determining step of l-tryptophan metabolism via the kynurenine pathway. The software is freely available to academic users at http://insilab.org/probisdock.

Upregulation of Cathepsin X in Glioblastoma: Interplay with γ-Enolase and the Effects of Selective Cathepsin X Inhibitors.

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Understanding GBM pathobiology and discovering novel therapeutic targets are critical to finding efficient treatments. Upregulation of the lysosomal cysteine carboxypeptidase cathepsin X has been linked to immune dysfunction and neurodegenerative diseases, but its role in cancer and particularly in GBM progression in patients is unknown. In this study, cathepsin X expression and activity were found to be upregulated in human GBM tissues compared to low-grade gliomas and nontumor brain tissues. Cathepsin X was localized in GBM cells as well as in tumor-associated macrophages and microglia. Subsequently, potent irreversible (AMS36) and reversible (Z7) selective cathepsin X inhibitors were tested in vitro. Selective cathepsin X inhibitors decreased the viability of patient-derived GBM cells as well as macrophages and microglia that were cultured in conditioned media of GBM cells. We next examined the expression pattern of neuron-specific enzyme γ-enolase, which is the target of cathepsin X. We found that there was a correlation between high proteolytic activity of cathepsin X and C-terminal cleavage of γ-enolase and that cathepsin X and γ-enolase were colocalized in GBM tissues, preferentially in GBM-associated macrophages and microglia. Taken together, our results on patient-derived material suggest that cathepsin X is involved in GBM progression and is a potential target for therapeutic approaches against GBM.