Katarzyna Pustelny

Antipsychotic drugs elicit complex cellular responses in neurons, yet the molecular mechanisms underlying their actions remain incompletely understood. Here, we employed deep proteomic profiling (DDA/DIA-MS) in LUHMES-derived dopaminergic neurons to explore the distinct proteomic profile induced by clozapine and risperidone. Pathway analysis revealed that clozapine robustly upregulated lipid metabolic pathways, particularly those involved in cholesterol and fatty acid biosynthesis. Conversely, risperidone primarily enhanced oxidative phosphorylation and modulated synaptic architecture and protein metabolic-pathways. Functional enrichment further indicated that clozapine promotes sterol metabolism, neuronal viability, and neurite outgrowth—hallmarks of neurotrophic activity – while simultaneously suppressing neurotransmission. Importantly, quantitative analysis of the nuclear subproteome enabled us to map these opposing regulatory hubs with high precision. Mechanistically, clozapine induced a transcriptionally repressive state, marked by downregulation of chromatin-opening factors CHD5 and DPF3 (BAF complex components) and HMGN1. In parallel, upregulation of the transcriptional repressor CIC reinforced this repressive signature, suggesting diminished chromatin accessibility. Furthermore, downregulation of activity-dependent regulators CRTC3 and JUNB pointed to an attenuation of excitability-driven gene expression. In contrast, risperidone upregulated TCF4, TCF7L1, FOS, HMGN1, and coactivators RREB1 and NCOA3, promoting Wnt signaling, chromatin accessibility and synaptic plasticity. Taken together, our data unveil distinct proteomic, transcriptional and epigenetic programs orchestrated by clozapine and risperidone, providing novel molecular insights into their divergent clinical actions.

The superior clinical efficacy of clozapine (CLO) in treatment‐resistant schizophrenia remains incompletely understood. To elucidate these mechanisms, we employed a multi‐platform proteomic strategy—combining data‐dependent acquisition (DDA) with fractionation, data‐independent acquisition (DIA) and targeted parallel reaction monitoring (PRM)—to analyse chronic drug‐induced proteomic changes in the prefrontal cortex (PFC) and remaining cerebral cortex (CX) of rats. This approach enabled unbiased analysis of over 6300 proteins per group in DDA and over 8700 in DIA. Consistent across all analytical methods, chronic CLO administration induced robust downregulation of the serotonin 5‐HT2A receptor (5HTR2A) in both PFC and CX, whereas risperidone (RIS) exhibited no significant effect on 5‐HTR2A levels at clinically relevant doses. Despite sharing high affinity for this receptor, CLO and RIS elicited markedly distinct signalling profiles. In the PFC, CLO inhibited serotonergic, G‐protein (Gαq and Gβγ), calcium, and cytoskeletal signalling pathways, accompanied by strong downregulation of phospholipase C delta 4 (PLCδ4), an isoform implicated in suicidal behaviour. Concurrently, CLO upregulated calcium‐independent phospholipase A2γ (iPLA2γ) and guanylyl cyclase C (GUCY2C), suggesting potential modulation of docosahexaenoic acid (DHA) signalling and cGMP‐driven synaptic plasticity. In contrast, RIS predominantly activated Gαi, Gαz, and Gβγ signalling in the CX and enhanced mitochondrial function. RIS also upregulated GUCY2C. Both drugs differentially modulated protein networks associated with potential heteromeric complexes involving 5HTR2As, including interactions with 5‐HT1A, 5‐HT2C, dopamine D2, oxytocin and cannabinoid CB1 receptors, highlighting their impact on complex serotonergic modulation. Notably, despite CLO's suppression of Gαq signalling, compensatory upregulation of Gαi was not observed, indicating a net attenuation of 5HTR2A‐mediated output rather than a simple G‐protein coupling switch. These findings advance our understanding of serotonergic, metabolic and receptor‐level adaptations induced by atypical antipsychotics and suggest that CLO's superior efficacy may derive from its unique ability to downregulate 5HTR2A and remodel signalling networks beyond classical dopaminergic mechanisms. image

In the pathogenesis of Alzheimer's disease, the overexpression of glycogen synthase kinase-3β (GSK-3β) stands out due to its multifaced nature, as it contributes to the promotion of amyloid β and tau protein accumulation, as well as neuroinflammatory processes. Therefore, in the present study, we have designed, synthesized, and evaluated a new series of GSK-3β inhibitors based on the N-(pyridin-2-yl)cyclopropanecarboxamide scaffold. We identified compound 36, demonstrating an IC₅₀ of 70 nM against GSK-3β. Subsequently, through crystallography studies and quantum mechanical analysis, we elucidated its binding mode and identified the structural features crucial for interactions with the active site of GSK-3β, thereby understanding its inhibitory potency. Compound 36 was effective in the cellular model of hyperphosphorylated tau-induced neurodegeneration, where it restored cell viability after okadaic acid treatment and showed anti-inflammatory activity in the LPS model, significantly reducing NO, IL-6, and TNF-α release. In ADME-tox in vitro studies, we confirmed the beneficial profile of 36, including high permeability in PAMPA (Pe equals 9.4) and high metabolic stability in HLMs as well as lack of significant interactions with isoforms of the CYP enzymes and lack of considerable cytotoxicity on selected cell lines (IC₅₀ > 100 μM on HT-22 cells and 89.3 μM on BV-2 cells). Based on promising pharmacological activities and favorable ADME-tox properties, compound 36 may be considered a promising candidate for in vivo research as well as constitute a reliable starting point for further studies.

A clinical casein kinase 2 inhibitor, CX-4945 (silmitasertib), shows significant affinity toward the DYRK1A and GSK3β kinases, involved in down syndrome phenotypes, Alzheimer's disease, circadian clock regulation, and diabetes. This off-target activity offers an opportunity for studying the effect of the DYRK1A/GSK3β kinase system in disease biology and possible line extension. Motivated by the dual inhibition of these kinases, we solved and analyzed the crystal structures of DYRK1A and GSK3β with CX-4945. We built a quantum-chemistry-based model to rationalize the compound affinity for CK2α, DYRK1A, and GSK3β kinases. Our calculations identified a key element for CK2α's subnanomolar affinity to CX-4945. The methodology is expandable to other kinase selectivity modeling. We show that the inhibitor limits DYRK1A- and GSK3β-mediated cyclin D1 phosphorylation and reduces kinase-mediated NFAT signaling in the cell. Given the CX-4945's clinical and pharmacological profile, this inhibitory activity makes it an interesting candidate with potential for application in additional disease areas.

The precise physiological functions and mechanisms regulating RNase Regnase‐2 (Reg‐2/ZC3H12B/MCPIP2) activity remain enigmatic. We found that Reg‐2 actively modulates neuroinflammation in nontransformed cells, including primary astrocytes. Downregulation of Reg‐2 in these cells results in increased mRNA levels of proinflammatory cytokines IL‐1β and IL‐6. In primary astrocytes, Reg‐2 also regulates the mRNA level of Regnase‐1 ( Reg‐1 /ZC3H12A/MCPIP1). Reg‐2 is expressed at high levels in the healthy brain, but its expression is reduced during neuroinflammation as well as glioblastoma progression. This process is associated with the upregulation of Reg‐1 . Conversely, overexpression of Reg‐2 is accompanied by the downregulation of Reg‐1 in glioma cells in a nucleolytic NYN/PIN domain‐dependent manner. Interestingly, low levels of Reg‐2 and high levels of Reg‐1 correlate with poor‐glioblastoma patients' prognoses. While Reg‐2 restricts the basal levels of proinflammatory cytokines in resting astrocytes, its expression is reduced in IL‐1β‐activated astrocytes. Following IL‐1β exposure, Reg‐2 is phosphorylated, ubiquitinated, and degraded by proteasomes. Simultaneously, the Reg‐2 transcript is destabilized by tristetraprolin (TTP) and Reg‐1 through the AREs elements and conservative stem‐loop structure present in its 3′UTR. Thus, the peer‐control loop, of Reg‐1 and Reg‐2 opposing each other, exists. The involvement of TTP in Reg‐2 mRNA turnover is confirmed by the observation that high TTP levels correlate with the downregulation of the Reg‐2 expression in high‐grade human gliomas. Additionally, obtained results reveal the importance of Reg‐2 in inhibiting human and mouse glioma cell proliferation. Our current studies identify Reg‐2 as a critical regulator of homeostasis in the brain.

DYRK1B has been recently recognized as a critical therapeutic target in oncology and non-alcoholic fatty liver disease. However, the lack of structural information has constrained the development of selective inhibitors for DYRK1B. Here, we employed recombinant protein production, activity assays, and crystallization to elucidate the structure of DYRK1B. We present a crystal structure of DYRK1B in complex with a known inhibitor, AZ191. For comparative analysis, we provide the crystal structure of the closely related DYRK1A kinase in complex with AZ191. Our analysis identifies the exclusiveness of the binding site in the hinge region of DYRK1B, which is pivotal for selective inhibitor design. Quantum mechanical calculations reveal a notable difference in the accessibility of the catalytic lysine between DYRK1B and DYRK1A, offering avenues for distinguishing binders to these kinases. Our findings mark a significant advancement in the quest for specific DYRK1B inhibitors, potentially offering focused efficacy compared to the current dual-specificity inhibitors targeting both DYRK1B and DYRK1A.

Background: A universal adaptor protein, MyD88, orchestrates the innate immune response by propagating signals from toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R). Receptor activation seeds MyD88 dependent formation of a signal amplifying supramolecular organizing center (SMOC)—the myddosome. Alternatively spliced variant MyD88S, lacking the intermediate domain (ID), exhibits a dominant negative effect silencing the immune response, but the mechanistic understanding is limited. Methods: Luciferase reporter assay was used to evaluate functionality of MyD88 variants and mutants. The dimerization potential of MyD88 variants and myddosome nucleation process were monitored by co-immunoprecipitation and confocal microscopy. The ID secondary structure was characterized in silico employing I-TASSER server and in vitro using nuclear magnetic resonance (NMR) and circular dichroism (CD). Results: We show that MyD88S is recruited to the nucleating SMOC and inhibits its maturation by interfering with incorporation of additional components. Biophysical analysis suggests that important functional role of ID is not supported by a well-defined secondary structure. Mutagenesis identifies Tyr116 as the only essential residue within ID required for myddosome nucleation and signal propagation (NF-κB activation). Conclusions: Our results argue that the largely unstructured ID of MyD88 is not only a linker separating toll-interleukin-1 receptor (TIR) homology domain and death domain (DD), but contributes intermolecular interactions pivotal in MyD88-dependent signaling. The dominant negative effect of MyD88S relies on quenching the myddosome nucleation and associated signal transduction.

The rising prevalence of diabetes is threatening global health. It is known not only for the occurrence of severe complications but also for the SARS-Cov-2 pandemic, which shows that it exacerbates susceptibility to infections. Current therapies focus on artificially maintaining insulin homeostasis, and a durable cure has not yet been achieved. We demonstrate that our set of small molecule inhibitors of DYRK1A kinase potently promotes β-cell proliferation, enhances long-term insulin secretion, and balances glucagon level in the organoid model of the human islets. Comparable activity is seen in INS-1E and MIN6 cells, in isolated mice islets, and human iPSC-derived β-cells. Our compounds exert a significantly more pronounced effect compared to harmine, the best-documented molecule enhancing β-cell proliferation. Using a body-like environment of the organoid, we provide a proof-of-concept that small–molecule–induced human β-cell proliferation via DYRK1A inhibition is achievable, which lends a considerable promise for regenerative medicine in T1DM and T2DM treatment.