Butyrylcholinesterase (BChE) is recognized as a promising therapeutic target for the late stages of Alzheimer's disease (AD) due to its role in the hydrolysis of acetylcholine (ACh), while acetylcholinesterase (AChE) activity declines during disease progression. Here, we have reported an efficient chemistry procedure for the naturally occurring Amaryllidaceae alkaloid carltonine B, along with the design and synthesis of 36 novel carltonine-based analogues to determine structure-activity relationship (SAR). Most of the synthesized compounds exhibited potent and selective human BChE (hBChE) inhibition, with IC50 values ranging from low micromolar to nanomolar concentrations. The drug-like properties of the molecules were assessed by in silico tools, using the blood-brain barrier (BBB) score algorithm, and subsequently validated by in vitro permeability assessment via parallel artificial membrane permeability assay (PAMPA). The derivatives exhibited potent hBChE inhibition in the low micromolar to submicromolar range, while their cytotoxicity against human neuroblastoma (SH-SY5Y) cells was observed only at higher micromolar concentrations, indicating a favorable safety profile. The synthesized alkaloid carltonine B (37) and its N-ethyl derivative (38) emerged as the most potent and selective hBChE inhibitors, with IC50 values of 0.014 ± 0.002 μM and 0.013 ± 0.001 μM, respectively. Enzyme kinetic studies were conducted to elucidate the inhibition mechanism toward hBChE enzyme. Compound 37 demonstrated competitive inhibition with Ki value of 0.055 μM. In contrast, compound 38 showed a noncompetitive inhibition profile, with a Ki value of 0.067 μM. Molecular modeling suggested that the superior potency of compounds 37 and 38 arises from their more optimal engagement of the BChE active-site gorge compared to compound 33. For the additional safety assessment, CYP inhibition assay revealed that compounds 37 and 38 may pose a risk of CYP3A4-mediated drug-drug interactions during chronic administration.

Abstract Glioblastoma multiforme (GBM) represents the most aggressive form of primary brain tumor, characterized by rapid progression, therapeutic resistance, and near-universal recurrence. Despite multimodal treatment strategies—comprising maximal surgical resection followed by radiotherapy and temozolomide (TMZ)-based chemotherapy—median survival remains dismal, with most patients succumbing within one-year post-diagnosis. TMZ and ionizing radiation exert their cytotoxic effects primarily through the induction of DNA lesions, including single- and double-strand breaks. The cellular DNA damage response (DDR), encompassing a complex signaling network of kinases, repair enzymes, and checkpoint regulators, orchestrates the detection and repair of these lesions. Dysregulation or hyperactivation of DDR pathways—including those mediated by ATR/ATM signaling, PARP-dependent repair, and cell cycle checkpoints (e.g., WEE1, CDK1)—has been implicated in the pronounced therapeutic resistance of GBM. In this study, we pursued the rational design and synthesis of novel small molecules aimed at potentiating TMZ- and radiation-induced cytotoxicity in GBM. Over seventy compounds were generated across five focused series using structure-based design principles. Phenotypic screening was conducted in the TMZ-resistant T98G line and patient-derived GBM cultures to evaluate both intrinsic cytotoxicity and TMZ sensitization. Several lead candidates demonstrated superior chemosensitization efficacy compared to clinical DDR inhibitors. Interestingly, broad kinase profiling revealed an absence of canonical DDR kinase inhibition, indicating a potential non-classical mechanism of action. To delineate this mechanism, comprehensive quantitative proteomic analyses were performed, revealing modulation of pathways associated with DNA repair, stress response, and cell cycle control. The most promising candidates exhibited favorable pharmacokinetic and toxicological properties, along with effective blood-brain barrier penetration in vivo. This work has been supported by the grant from the Ministry of Health of the Czech Republic (NW24J-03-00005); and by the Long-term development plan Military Faculty of Medicine, Healthcare Challenges of WMD II (DZRO-VLF22-ZHN II). Citation Format: Lukas Gorecki, Lubica Muckova, Michaela Sadibolova, Ondrej Soukup. From phenotypic screening to mechanistic insight: Rational discovery of chemosensitizers for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 993.

The serotonin type 3 receptor (5-HT3R) is a ligand-gated ion channel with a unique position within the serotonergic system, yet its relevance to schizophrenia (SCZ) and its frequent psychiatric comorbidities remains underappreciated. The aim of this review is to critically synthesize preclinical and clinical evidence supporting the role of 5-HT3Rs in the pathophysiology and treatment of SCZ, with particular emphasis on negative and cognitive symptoms, as well as comorbid depressive symptoms and substance use disorders (SUDs). We summarize data demonstrating that 5-HT3R antagonists (setrons), widely used and well tolerated in non-psychiatric indications, exert circuit-level effects through modulation of mesolimbic dopaminergic signaling, cortical excitation-inhibition balance, cholinergic transmission, and neuroinflammatory and oxidative stress pathways. Clinical studies and meta-analyses suggest statistically significant but heterogeneous improvements in negative symptoms with adjunctive setron treatment in SCZ, with less consistent findings for total and cognitive outcomes and minimal or no robust effects on positive symptoms. Preclinical and clinical evidence further suggests that 5-HT3R antagonism may attenuate depressive symptomatology and reduce craving and relapse vulnerability in SUDs, highlighting its potential transdiagnostic relevance. In addition, recent advances in medicinal chemistry have yielded multifunctional ligands combining 5-HT3R antagonism with dopaminergic or other serotonergic activities, illustrating a rational polypharmacological strategy for next-generation antipsychotic development. In conclusion, we propose that 5-HT3Rs function as circuit-level modulatory hubs rather than classical antipsychotic targets. Targeting these receptors may represent a potentially well-tolerated and mechanistically distinct adjunctive strategy to address unmet therapeutic needs in SCZ, particularly negative symptoms, cognitive dysfunction, and psychiatric comorbidities.

Selective butyrylcholinesterase (BChE) inhibition is gaining renewed attention as a potential therapeutic strategy for Alzheimer's disease (AD), particularly in advanced stages marked by a shift from acetylcholinesterase (AChE) to BChE dominance. Beyond cholinergic regulation, BChE participates in metabolic, inflammatory, and affective pathways, including the enzymatic control of acyl ghrelin that influences appetite, energy balance, and mood. Preclinical and experimental evidence suggests that selective BChE inhibition may modulate cholinergic tone, enhance cognition, and exert antidepressant- and anti-anhedonic-like effects, although clinical evidence remains limited and inconclusive. Genetic polymorphisms in BChE further shape disease progression and responses to cholinesterase therapy. This review integrates advances in BChE inhibitor development with evolving insights into BChE-dependent metabolic and neuropsychiatric mechanisms, highlighting selective BChE inhibition as a multifaceted therapeutic approach in AD.

Sustained effort has been dedicated to the development of novel cholinesterase reactivators—the only causal antidotes—to counter organophosphorus (OP) intoxication. As more lethal nerve agents—such as A-agents—continue to emerge, the existing arsenal of causal antidotes remains unchanged. Approved oxime reactivators—2-PAM, HI-6, and LüH-6—are restricted by their limited efficacy spectrum, poor blood–brain barrier permeability, and suboptimal pharmacokinetics. The objective of this study is to design, synthesize, and characterize a new class of asymmetric monoquaternary bisoxime reactivators with broad-spectrum reactivation potential, favorable pharmacokinetics, and dual mechanisms of action—cholinesterase reactivation and direct OP compound degradation. In vitro and in vivo experiments identified LG-1795 as the lead candidate with the broadest OP spectrum. The averaged second-order reactivation constant (kr2) across five hAChE–OP and two hBChE-OP complexes was 16.8 mM−1 min−1, surpassing clinical standards. Notably, LG-1795 reactivated both AChE and BChE, a dual activity not previously reported for reactivators. In vivo, LG-1795 restored both cholinesterase enzymes and demonstrated prophylactic efficacy against GB, VX, and PXE following intramuscular administration, preventing symptoms in sarin-poisoned animals. These findings represent a significant advance in antidotal therapy, providing the first evidence that asymmetric monoquaternary bisoximes deliver broad-spectrum efficacy against nerve agents and pesticides while simultaneously targeting both OP-inhibited cholinesterases. The translational potential of LG-1795 supports its further preclinical development as a next-generation countermeasure for both clinical and regulatory use in chemical defense.

Chronic neuroinflammation and cholinergic dysfunction are major contributors to cognitive decline in Alzheimer's disease. Here, we report a series of pyridazine-based multitarget-directed ligands that simultaneously inhibit human butyrylcholinesterase (hBChE) and p38α mitogen-activated protein kinase (p38α MAPK). Pyridazines 8 and 21 were identified as potent dual inhibitors with submicromolar inhibitory potencies against target enzymes and high selectivity over acetylcholinesterase. X-ray crystallographic analyses revealed the experimental binding modes of 8 and 21 in hBChE and p38α MAPK, and identified conserved π-π, π-cation, and hinge region interactions that rationalize dual-target engagement. Both compounds were blood-brain barrier permeable, exhibited low cytotoxicity, and significantly attenuated lipopolysaccharide-induced proinflammatory responses and apoptosis in BV2 microglia. In vivo, compound 21 enhanced cognitive performance in mouse models of scopolamine-induced amnesia and LPS-stimulated neuroinflammatory cognitive impairment. Collectively, these results document the potential of structure-assisted design to achieve selective dual-target pharmacology within a single molecular entity.

17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) is a multifunctional mitochondrial enzyme and a potential drug target for the treatment of various pathologies, including Alzheimer's disease (AD). In this study, five new benzothiazole-derived 17β-HSD10 inhibitors were developed based on structure-activity relationship (SAR) analyses of previously published compounds. To evaluate the inhibitory effects, cytotoxicity, and therapeutic potential of these new compounds, several enzyme- and cell-based methods were employed. All prepared compounds exhibited high inhibitory potential and confirmed good biomembrane permeation. Three inhibitors (9b, 9c, and 15a) showed lower IC50 values in both enzyme- and cell-based assays than the formerly published hit compounds. The compounds were also found to reduce the pathological effects associated with 17β-HSD10 overexpression, although not the combined pathological effects of 17β-HSD10 overexpression within an amyloid-β rich environment.

Quaternary ammonium compounds (QACs) are widely used disinfectants, favored for their broad antimicrobial action and safety profile. However, viral disinfection is complex and affected by multiple variables, including the virus's envelope status, exposure time, temperature, and organic matter presence. This has created considerable ambiguity in the literature.We searched PubMed and Scopus using terms including "quaternary ammonium compounds" and "virucidal," with a focus on only registered QACs (biocides), identifying 64 studies from 419 results. These studies examined viruses from 22 families (13 enveloped, 9 non-enveloped) across different experimental conditions.QACs are highly effective against enveloped viruses, even under suboptimal conditions, and remain relevant for pandemic response. However, proper cleaning protocols are essential to ensure complete virucidal activity. Against non-enveloped viruses, QAC effectiveness is less predictable. While Parvoviridae and Picornaviridae remain highly resistant, other families like Adenoviridae, Caliciviridae, and Sedoreoviridae show variable susceptibility depending on conditions. Adding synergistic agents such as acids or alkalis can improve QAC performance and mitigate this uncertainty.

The nerve agent A-234, classified among the so-called A-series agents or "Novichoks", represents a significant challenge in the treatment of nerve agent poisoning due to its potent and irreversible inhibition of human recombinant acetylcholinesterase (HssAChE). In this study, we screened 22 structurally diverse oxime reactivators for their efficacy against A-234-inhibited HssAChE in vitro after a 10-min incubation. The initial screening was extended to GB- and VX-inhibited enzymes under the same conditions for comparison. Then, the 22 oximes were tested against A-234-inhibited HssAChE for up to 24 h. Finally, we evaluated the reactivation kinetics of the two most effective oximes over a 240-min period. While most reactivators demonstrated considerable reactivation against GB- and VX-inhibited enzymes within 10 min, none showed efficacy against A-234 in this time frame. Extended incubation over 24 h revealed that only HLö-7, MMB-4, HI-6, K027, K868, TMB-4, GM415, and LüH-6 achieved efficient reactivation (set at a 10 % threshold for a 24-h interval), with HLö-7 and methoxime (MMB-4) being the most effective. Kinetic analysis indicated that HLö-7 exhibited a superior second-order reactivation rate constant compared to MMB-4, highlighting differences in binding affinity and catalytic efficacy. In the case of A-234 poisonings, our findings indicate the most effective oxime antidotes and emphasize the need for prolonged therapy to improve clinical outcomes.

The currently approved drugs for the treatment of Alzheimer's disease (AD) fail to address its interconnected pathological processes. Inhibition of butyrylcholinesterase (BChE) and p38α mitogen-activated protein kinase (p38α MAPK) offers an innovative dual approach to mitigate two major drivers of neurodegeneration in AD: cholinergic deficit and neuroinflammation. Using structure-based drug design and a library of known p38α MAPK inhibitors, we developed first-in-class, selective dual BChE/p38α MAPK inhibitors with balanced activity against both targets. The X-ray crystal structures of the two most promising molecules bound to both enzymes were solved. Those ligands effectively reduced the production of proinflammatory markers in vitro and ex vivo in phytohemagglutinin/lipopolysaccharide neuroinflammation models. Remarkably, these compounds also significantly improved cognition in scopolamine- and lipopolysaccharide-induced models of cognitive dysfunction in mice. Because our dual-acting inhibitors target both the symptoms and the underlying neuropathology, they offer an innovative and comprehensive strategy to combat AD.

One of the traditional treatments in Alzheimer's disease (AD) is administration of memantine, the NMDA receptor antagonist. However, the molecular mechanism of the complex memantine action and the impact on the hippocampal proteome in humans is unknown. In this study, hippocampal proteins extracted from formalin-fixed paraffin-embedded post mortem tissues obtained from healthy donors (n = 15), AD patients not treated with memantine (n = 11), and AD patients treated with memantine (n = 8) were investigated using tandem mass tag (TMT)-based quantitative proteomics. Memantine medication induced subtle but distinct changes in the hippocampal proteome in AD patients. Although it did not prevent the metabolic and physiologic decline associated with AD pathology, memantine administration upregulated several mitochondrially encoded proteins and mitigated the proteomic pattern of activated phagocytes. Furthermore, memantine specifically enhanced the expression of postsynaptic glutamatergic and GABAergic receptors and components of the respective pathways without affecting presynaptic proteome. This suggests that memantine treatment in AD patients not only alleviates excitotoxic stress by inhibiting NMDA receptor activity, but also triggers broader adaptations in the synaptic signaling and plasticity.

Tuberculosis (TB) imposes a major global health challenge, aggravated by the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains. Scaffold hopping, a medicinal chemistry approach that modifies the molecular backbone of known bioactive compounds, has emerged as a promising tool in the development of novel drugs, including TB therapeutics. This perspective provides an insight into the application of scaffold hopping across varying degrees of structural modifications, highlighting successful case studies targeting key Mtb pathways, including energy metabolism, cell wall synthesis, proteasome function, and respiratory processes. Beyond traditional and in silico methods, scaffold hopping has spurred the discovery of compounds with improved pharmacological profiles, such as improved pharmacokinetics, enhanced efficacy, reduced toxicity, and resistance circumvention. The findings support scaffold hopping's potential to address the limitations of current anti-TB drugs as a versatile and innovative approach to accelerate TB drug discovery.

Dendritic cells (DCs) hijacked by intracellular bacteria contribute to pathogen dissemination and immunopathology. How bacteria achieve DC subversion remains largely unknown. Here, we describe the mechanism used by tularemia agent Francisella tularensis exploiting host mitochondrial anaplerosis. Shortly after internalization, Francisella associates with DC mitochondria, which leads to the rapid repurposing of their oxidative metabolism for production of mitochondrial reactive oxygen species (mtROS). Mitochondrial metabolic rewiring is orchestrated by the intramitochondrial signaling mediated by protein acetylation and involves switching to glutamate as the primary substrate for DC tricarboxylic acid cycle. Rather than killing the bacterium, glutamate-fueled mtROS production activates p38-dependent proinflammatory gene expression. Blocking of glutamate utilization prevents DC activation and bacterial dissemination and alleviates inflammation in vivo. Our findings underscore the importance of metabolic plasticity in antibacterial DC response and open up potential avenues for therapies targeting host metabolism.

Tuberculosis (TB) remains a major global health challenge, particularly due to the rise of multidrug-resistance and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains. In addition to Mtb, many non-tuberculous mycobacterial (NTM) strains are associated with opportunistic infections in animals and humans. In this study, we report the design, synthesis, and evaluation of novel pyrimidine derivatives as potential antimycobacterial agents. A systematic structure-activity relationship study was conducted, leading to the identification of several promising compounds. Among them, derivative 55a demonstrated the highest efficacy, exhibiting minimum inhibitory concentration (MIC99) of 8 μM against drug-susceptible Mtb, while retaining significant potency against multidrug-resistant and extensively drug-resistant clinical isolates. Additionally, compound 55a displayed excellent metabolic stability, with a half-life of 187 min and an intrinsic clearance rate of 7.41 μL/min/mg protein in human liver microsomes. Several compounds also revealed promising efficacy against M. kansasii as a representative from NTM strains. The most promising antimycobacterial agents showed no activity against Gram-positive or Gram-negative bacterial strains, indicating their selectivity towards mycobacteria. These finding highlight 55a as a promising structural motif for further optimization in the development of novel anti-TB therapeutics.

The mitochondrial enzyme 17β-hydroxysteroid dehydrogenase type 10 (HSD10) is implicated in neurodegenerative disorders, particularly Alzheimer’s disease (AD), through its interplay with the amyloid-β peptide (Aβ). However, its independent pathological role in AD remains unclear. To explore the individual effects of HSD10 and amyloid precursor protein (APP) overexpression (including the Aβ42-generating APPSwe/Ind variant), monoclonal HEK293 cell lines were developed. Cellular fitness was evaluated by measuring ATP levels, cell viability, and cytotoxicity measurements under glucose and galactose culture conditions. Mitochondrial metabolic changes were analysed using mitochondrial electron flow measurements in response to various metabolic substrates. HSD10 enzymatic activity was monitored using a fluorogenic probe, and two HSD10 inhibitors were tested for their ability to reduce cytotoxic effects. Statistical significance was determined using appropriate tests as detailed in the methods section. The overexpression of HSD10 or APPSwe/Ind led to mitochondrial dysfunction and reduced viability, particularly under glucose-deprived conditions. HSD10-driven cytotoxicity was linked to its enzymatic activity and associated with impaired TCA cycle function, reduced β-oxidation, and increased oxidative stress. In contrast, APPSwe/Ind overexpression induced Aβ42 production, glucose hypermetabolism, and enhanced β-oxidation. Aβ42 also affected HSD10 activity and further amplified its cytotoxic effects. The benzothiazole-based HSD10 inhibitor 34 restored cell viability under both HSD10 overexpression and Aβ42-rich conditions. HSD10 and Aβ42 each contribute to mitochondrial impairment via distinct metabolic pathways. These findings established HSD10 as an independent pathological factor in AD and support the potential of HSD10 inhibitors, particularly inhibitor 34, as therapeutic agents targeting mitochondrial dysfunction in AD.

Currently available antidotes against toxic organophosphorus compounds suffer from poor permeability across the blood-brain barrier (BBB) and due to this, are limited in their ability to restore the inhibited acetylcholinesterase (AChE) in the central nervous system (CNS). We designed functionalized detonation nanodiamond nanocarrier platforms to transport quaternary oxime antidotes into CNS. We showed that the nanodiamonds with covalently attached 4-oximinopyridinium moiety, cross the layer of Madin-Darby Canine Kidney (MDCK) cells, the surrogate BBB model, and demonstrate a dose-independent reactivation in vitro towards human AChE inhibited by nerve agents GB and VX, and pesticide paraoxon. Confocal microscopy visualization of tight junctions and actin cytoskeleton in MDCK and Human Umbilical Vein Endothelial Cells (HUVEC) revealed temporary disruption of tight junctions at higher nanoparticle concentrations without compromising cell viability or cytoskeletal integrity. Although reactivation was modest, the nanodiamond platform showed promise for delivering quaternary oxime to the central nervous system (CNS) in vitro. The results reveal the potential of detonation nanodiamonds as a promising delivery platform for charged therapeutic agents to CNS aimed to enhance treatment outcomes in organophosphorus poisoning.

Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder for which current treatments provide only symptomatic relief, primarily through cholinesterase (ChE) inhibition and N-methyl-d-aspartate receptor (NMDAR) antagonism. To improve therapeutic efficacy and safety, we designed and synthesized 16 novel tacrine derivatives modified at position 7 with various (hetero)aryl groups or deuterium substitution. Initially, in silico screening predicted favorable CNS permeability and oral bioavailability. Subsequent in vitro evaluations demonstrated significant inhibitory potency against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with derivatives 5i and 5m displaying particularly promising profiles. Metabolic stability assessed using human liver microsomes revealed enhanced stability for compound 5e, whereas 5i and 5m underwent rapid metabolism. Notably, compound 7 showed improved metabolic stability attributed to deuterium incorporation. The newly synthesized compounds were further tested for antagonistic activity on the GluN1/GluN2B subtype of NMDAR, with compound 5m exhibiting the most potent and voltage-independent inhibition. The ability of these compounds to permeate the blood-brain barrier (BBB) was confirmed through in vitro PAMPA assays. In preliminary hepatotoxicity screening (HepG2 cells), most derivatives exhibited higher cytotoxicity than tacrine, emphasizing the ongoing challenge in hepatotoxicity management. Based on its overall favorable profile, compound 5m advanced to in vivo pharmacokinetic studies in mice, demonstrating efficient CNS penetration, with brain concentrations exceeding plasma levels (brain-to-plasma ratio 2.36), indicating active transport across the BBB. These findings highlight compound 5m as a promising tacrine-based multi-target-directed ligand, supporting further preclinical development as a potential therapeutic candidate for AD.

Tuberculosis (TB), an infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb), was responsible for the deaths of approximately 1.3 million people in 2022. In addition, 7.5 million new cases of TB have been reported. Present-day treatments require a daily dosing of a multiple-drug regimen for a minimum of six-month, but poor adherence and other factors often lead to treatment failure. Consequently, drug-resistant TB strains have become a growing concern, leading to more complex and expensive treatments. Promising drugs such as bedaquiline, delamanid, and pretomanid have been recently released, and 19 drug candidates are currently at different phases of clinical trials, addressing the problem of drug-resistant TB. Notwithstanding recent advances, the development of effective and safe drugs with novel mechanisms of action remains a challenge due to the unique nature of Mtb. Despite the persistent need for new treatments, TB research remains underfunded, highlighting the importance of collaborations between academia and the private sector in the advancement of anti-TB drug development. This review provides a perspective on the dynamic landscape of anti-TB drug discovery in recent years, offering hope for a more effective approach to combat this persistent global health threat.