The Worries of Out-of-Home Activities in Patients With Inflammatory Bowel Disease: A Survey Study During the COVID-19 Pandemic.

Background: Patients with inflammatory bowel disease (IBD) often experience worries related to travel due to frequent bowel movements. However, there is currently limited research focusing on the travel worries of patients with IBD. The aim of this study was to assess the level of worry regarding out-of-home activities in patients with IBD and identify factors associated with worry. Methods: This study included patients with IBD who visited the outpatient clinics between September 2020 and March 2022, during the COVID-19 pandemic. Participants completed a self-designed questionnaire, providing general clinical data and indicating their level of worry for out-of-home activities. Results: A total of 529 patients with IBD completed the questionnaire. Patients with Crohn's disease (CD) had a higher proportion of individuals under 40 years old and males compared to patients with ulcerative colitis (UC). Regarding out-of-home activities, patients with UC expressed greater worry about going out and taking buses than patients with CD. However, there were no significant differences observed between the two groups in terms of travel worries and worries about finding public washrooms. A significant majority (85.4%) of patients with clinically active IBD expressed worries about not finding public washrooms when going out, while 46.7% of patients in clinical remission had similar worries. Moreover, the worry about finding public washrooms was higher in patients with UC compared to those with CD, both during the clinical activity and remission. Conclusion: This survey conducted during the COVID-19 pandemic reported worries among patients with IBD about out-of-home activities. The patients with clinically active IBD, especially UC, expressed worries about not finding public washrooms when going out. We highlight the actual psychological and quality of life challenges faced by patients with IBD.

LC3-associated phagocytosis of neutrophils triggers tumor ferroptotic cell death in glioblastoma.

Necrosis in solid tumors is commonly associated with poor prognostic but how these lesions expand remains unclear. Studies have found that neutrophils associate with and contribute to necrosis development in glioblastoma by inducing tumor cell ferroptosis through transferring myeloperoxidase-containing granules. However, the mechanism of neutrophilic granule transfer remains elusive. We performed an unbiased small molecule screen and found that statins inhibit neutrophil-induced tumor cell death by blocking the neutrophilic granule transfer. Further, we identified a novel process wherein neutrophils are engulfed by tumor cells before releasing myeloperoxidase-containing contents into tumor cells. This neutrophil engulfment is initiated by integrin-mediated adhesion, and further mediated by LC3-associated phagocytosis (LAP), which can be blocked by inhibiting the Vps34-UVRAG-RUBCN-containing PI3K complex. Myeloperoxidase inhibition or Vps34 depletion resulted in reduced necrosis formation and prolonged mouse survival in an orthotopic glioblastoma mouse model. Thus, our study unveils a critical role for LAP-mediated neutrophil internalization in facilitating the transfer of neutrophilic granules, which in turn triggers tumor cell death and necrosis expansion. Targeting this process holds promise for improving glioblastoma prognosis.

Interneuron FGF13 regulates seizure susceptibility via a sodium channel-independent mechanism.

Developmental and Epileptic Encephalopathies (DEEs), a class of devastating neurological disorders characterized by recurrent seizures and exacerbated by disruptions to excitatory/inhibitory balance in the brain, are commonly caused by mutations in ion channels. Disruption of, or variants in, FGF13 were implicated as causal for a set of DEEs, but the underlying mechanisms were clouded because FGF13 is expressed in both excitatory and inhibitory neurons, FGF13 undergoes extensive alternative splicing producing multiple isoforms with distinct functions, and the overall roles of FGF13 in neurons are incompletely cataloged. To overcome these challenges, we generated a set of novel cell type-specific conditional knockout mice. Interneuron-targeted deletion of Fgf13 led to perinatal mortality associated with extensive seizures and impaired the hippocampal inhibitory/excitatory balance while excitatory neuron-targeted deletion of Fgf13 caused no detectable seizures and no survival deficits. While best studied as a voltage-gated sodium channel (Nav) regulator, we observed no effect of Fgf13 ablation in interneurons on Navs but rather a marked reduction in K+ channel currents. Re-expressing different Fgf13 splice isoforms could partially rescue deficits in interneuron excitability and restore K+ channel current amplitude. These results enhance our understanding of the molecular mechanisms that drive the pathogenesis of Fgf13-related seizures and expand our understanding of FGF13 functions in different neuron subsets.

Identification of membrane curvature sensing motifs essential for VPS37A phagophore recruitment and autophagosome closure.

VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.

Leukocyte immunoglobulin-like receptor B2: A promising biomarker for colorectal cancer.

According to the latest global cancer statistics, colorectal cancer (CRC) has emerged as the third most prevalent malignant tumor across the globe. In recent decades, the medical field has implemented several levels of CRC screening tests, encompassing fecal tests, endoscopic examinations, radiological examinations and blood tests. Previous studies have shown that leukocyte immunoglobulin-like receptor B2 (LILRB2) is involved in inhibiting immune cell function, immune evasion, and promoting tumor progression in acute myeloid leukemia and non-small cell lung cancer. However, its interaction with CRC has not been reported yet. Recently, a study published in the World Journal of Gastroenterology revealed that LILRB2 and its ligand, angiopoietin-like protein 2, are markedly overexpressed in CRC. This overexpression is closely linked to tumor progression and is indicative of a poor prognosis. The study highlights the potential of utilizing the concentration of LILRB2 in serum as a promising biomarker for tumors. However, there is still room for discussion regarding the data processing and analysis in this research.

ER stress elicits non-canonical CASP8 (caspase 8) activation on autophagosomal membranes to induce apoptosis.

The VPS37A gene encodes a subunit of the endosomal sorting complex required for transport (ESCRT)-I complex that is frequently lost in a wide variety of human solid cancers. We have previously demonstrated the role of VPS37A in directing the ESCRT membrane scission machinery to seal the phagophore for autophagosome completion. Here, we report that VPS37A-deficient cells exhibit an accumulation of the apoptotic initiator CASP8 (caspase 8) on the phagophore and are primed to undergo rapid apoptosis through the intracellular death-inducing signaling complex (iDISC)-mediated CASP8 activation upon exposure to endoplasmic reticulum (ER) stress. Using CRISPR-Cas9 gene editing and comparative transcriptome analysis, we identified the ATF4-mediated stress response pathway as a crucial mediator to elicit iDISC-mediated apoptosis following the inhibition of autophagosome closure. Notably, ATF4-mediated iDISC activation occurred independently of the death receptor TNFRSF10B/DR5 upregulation but required the pro-apoptotic transcriptional factor DDIT3/CHOP to enhance the mitochondrial amplification pathway for full-activation of CASP8 in VPS37A-deficient cells stimulated with ER stress inducers. Our analysis also revealed the upregulation of NFKB/NF-kB signaling as a potential mechanism responsible for restraining iDISC activation and promoting cell survival upon VPS37A depletion. These findings have important implications for the future development of new strategies to treat human cancers, especially those with VPS37A loss.Abbreviations: ATG: autophagy related; BMS: BMS-345541; CASP: caspase; CHMP: charged multivesicular body protein; DKO: double knockout; Dox: doxycycline; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; gRNA: guide RNA; GSEA: gene set enrichment analysis; GSK157: GSK2656157; iDISC: intracellular death-inducing signaling complex; IKK: inhibitor of NFKB kinase; IPA: ingenuity pathway analysis; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NFKB/NF-kB: nuclear factor kappa B; OZ: 5Z-7-oxozeaenol; RNA-seq: RNA sequencing; UPR: unfolded protein response; TFT: transcription factor target; THG: thapsigargin; TUN: tunicamycin; VPS: vacuolar protein sorting.

Alternative splice variants of the mitochondrial fission protein DNM1L/Drp1 regulate mitochondrial dynamics and tumor progression in ovarian cancer.

Aberrant mitochondrial fission/fusion dynamics have been reported in cancer cells. While post translational modifications are known regulators of the mitochondrial fission/fusion machinery, we show that alternative splice variants of the fission protein Drp1 (DNM1L) have specific and unique roles in cancer, adding to the complexity of mitochondrial fission/fusion regulation in tumor cells. Ovarian cancer specimens express an alternative splice transcript variant of Drp1 lacking exon 16 of the variable domain, and high expression of this splice variant relative to other transcripts is associated with poor patient outcome. Unlike the full-length variant, expression of Drp1 lacking exon 16 leads to decreased association of Drp1 to mitochondrial fission sites, more fused mitochondrial networks, enhanced respiration, and TCA cycle metabolites, and is associated with a more metastatic phenotype in vitro and in vivo. These pro-tumorigenic effects can also be inhibited by specific siRNA-mediated inhibition of the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the significance of the pathophysiological consequences of Drp1 alternative splicing and divergent functions of Drp1 splice variants, and strongly warrant consideration of Drp1 splicing in future studies.

What's in an E3: role of highly curved membranes in facilitating LC3-phosphatidylethanolamine conjugation during autophagy.

During autophagosome formation, ATG3, an E2-like enzyme, catalyzes the transfer of LC3-family proteins (including Atg8 in yeast and LC3- and GABARAP-subfamily members in more complex eukaryotes) from the covalent conjugated ATG3-LC3 intermediate to PE lipids in targeted membranes. A recent study has shown that the catalytically important regions of human ATG3 (hereafter referred to as ATG3), including residues 262 to 277 and 291 to 300, in cooperation with its N-terminal curvature-sensing amphipathic helix (NAH), directly interact with the membrane. These membrane interactions are functionally necessary for in vitro conjugation and in vivo cellular assays. They provide a molecular mechanism for how the membrane curvature-sensitive interaction of the NAH of ATG3 is closely coupled to its conjugase activity. Together, the data are consistent with a model in which the highly curved phagophore rims facilitate the recruitment of the ATG3-LC3 complex and promote the conjugation of LC3 to PE lipids. Mechanistically, the highly curved membranes of the phagophore rims act in much the same manner as classical E3 enzymes in the sumo/ubiquitin system, bringing substrates into proximity and rearranging the catalytic center of ATG3. Future studies will investigate how this multifaceted membrane interaction of ATG3 works with the putative E3 complex, ATG12-ATG5-ATG16L1, to promote LC3-PE conjugation.

Acid Ceramidase Inhibitor LCL-805 Antagonizes Akt Signaling and Promotes Iron-Dependent Cell Death in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell-death-promoting signaling lipid that plays a central role in therapy-induced cell death. We previously determined that acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug LCL-805 across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 µM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 µM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide.

REAP-2: An interactive quantitative tool for robust and efficient dose-response curve estimation.

REAP-2 is an interactive dose-response curve estimation tool for Robust and Efficient Assessment of drug Potency. It provides user-friendly dose-response curve estimation for in vitro studies and conducts statistical testing for model comparisons with a redesigned user interface. We also make a major update of the underlying estimation method with penalized beta regression, which demonstrates great reliability and accuracy in dose estimation and uncertainty quantification. In this note, we describe the method and implementation of REAP-2 with a highlight on potency estimation and drug comparison.

Iron inhibits glioblastoma cell migration and polarization.

Glioblastoma is one of the deadliest malignancies facing modern oncology today. The ability of glioblastoma cells to diffusely spread into neighboring healthy brain makes complete surgical resection nearly impossible and contributes to the recurrent disease faced by most patients. Although research into the impact of iron on glioblastoma has addressed proliferation, there has been little investigation into how cellular iron impacts the ability of glioblastoma cells to migrate-a key question, especially in the context of the diffuse spread observed in these tumors. Herein, we show that increasing cellular iron content results in decreased migratory capacity of human glioblastoma cells. The decrease in migratory capacity was accompanied by a decrease in cellular polarization in the direction of movement. Expression of CDC42, a Rho GTPase that is essential for both cellular migration and establishment of polarity in the direction of cell movement, was reduced upon iron treatment. We then analyzed a single-cell RNA-seq dataset of human glioblastoma samples and found that cells at the tumor periphery had a gene signature that is consistent with having lower levels of cellular iron. Altogether, our results suggest that cellular iron content is impacting glioblastoma cell migratory capacity and that cells with higher iron levels exhibit reduced motility.

Acid Ceramidase Inhibitor LCL-805 Antagonizes Akt Signaling and Promotes Iron-Dependent Cell Death in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell death-promoting signaling lipid that plays a central role in therapy-induced cell death. Acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug, LCL-805, across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 µM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 µM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide.

Extracellular vesicles from retinal pigment epithelial cells expressing R345W-Fibulin-3 induce epithelial-mesenchymal transition in recipient cells.

We have shown previously that expression of R345W-Fibulin-3 induces epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W-Fibulin-3 mutation are sufficient to induce EMT in recipient cells. ARPE-19 cells were infected with luciferase-tagged wild-type (WT)- Fibulin-3 or luciferase-tagged R345W-Fibulin-3 (R345W) using lentiviruses. EVs were isolated from the media by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC-MS/MS based proteomics. EV-associated transforming growth factor beta 1 (TGFß1) protein was measured by enzyme-linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT- or R345W-EVs. The role of EV-bound TGFß was determined by pre-incubation of EVs with a pan-TGFß blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W- EVs, while EMT drivers were enriched. Additionally, R345W-EVs had higher concentrations of TGFß1 compared to control. Critically, treatment with R345W-EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV-increased cell migration was significantly inhibited by pre-incubation of EVs with pan-TGFß-neutralising antibody. In conclusion, the expression of R345W-Fibulin-3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFß dependent manner. These results suggest that EV-bound TGFß plays a critical role in the induction of EMT in RPE cells.

Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy.

Autophagosome formation, a crucial step in macroautophagy (autophagy), requires the covalent conjugation of LC3 proteins to the amino headgroup of phosphatidylethanolamine (PE) lipids. Atg3, an E2-like enzyme, catalyzes the transfer of LC3 from LC3-Atg3 to PEs in targeted membranes. Here we show that the catalytically important C-terminal regions of human Atg3 (hAtg3) are conformationally dynamic and directly interact with the membrane, in collaboration with its N-terminal membrane curvature-sensitive helix. The functional relevance of these interactions was confirmed by in vitro conjugation and in vivo cellular assays. Therefore, highly curved phagophoric rims not only serve as a geometric cue for hAtg3 recruitment, but also their interaction with hAtg3 promotes LC3-PE conjugation by targeting its catalytic center to the membrane surface and bringing substrates into proximity. Our studies advance the notion that autophagosome biogenesis is directly guided by the spatial interactions of Atg3 with highly curved phagophoric rims.

Endogenous hydrogen sulfide inhibition suppresses tumor growth by promoting apoptosis and pyroptosis in esophageal cancer cells.

BACKGROUND: Hydrogen sulfide (H2S) has been identified as the third gaseous signaling molecule. Endogenous H2S plays a key role in the progression of various types of cancer. However, the effect of endogenous H2S on the growth of esophageal cancer (EC) remains unknown. METHODS: In this study, three kinds of H2S-producing enzymes inhibitors, DL-propargylglycine (PAG, inhibitor of cystathionine-γ-lyase), aminooxyacetic acid (AOAA, inhibitor of cystathionine-ß-synthase), and L-aspartic acid (L-Asp, inhibitor of 3-mercaptopyruvate sulfurtransferase) were used to determine the role of endogenous H2S in the growth of EC9706 and K450 human EC cells. RESULTS: The results indicated that the combination (PAG+AOAA+L-Asp) group showed higher inhibitory effects on the viability, proliferation, migration, and invasion of EC cells than PAG, AOAA, and L-Asp group. Inhibition of endogenous H2S promoted apoptosis via activation of mitogen-activated protein kinase pathway in EC cells. Endogenous H2S suppression triggered pyroptosis of EC cells by activating reactive oxygen species-mediated nuclear factor-κB signaling pathway. In addition, the combine group showed its more powerful growth-inhibitory effect on the growth of human EC xenograft tumors in nude mice without obvious toxicity. CONCLUSION: Our results indicate that inhibition of endogenous H2S production can significantly inhibit human EC cell growth via promotion of apoptosis and pyroptosis. Endogenous H2S may be a promising therapeutic target in EC cells. Novel inhibitors for H2S-producing enzymes can be designed and developed for EC treatment.

Fecal microbiota transplantation alleviates experimental colitis through the Toll-like receptor 4 signaling pathway.

BACKGROUND: Fecal microbiota transplantation (FMT) has shown promising therapeutic effects on mice with experimental colitis and patients with ulcerative colitis (UC). FMT modulates the Toll-like receptor 4 (TLR4) signaling pathway to treat some other diseases. However, it remains unknown whether this modulation is also involved in the treatment of UC. AIM: To clarify the necessity of TLR4 signaling pathway in FMT on dextran sodium sulphate (DSS)-induced mice and explain the mechanism of FMT on UC, through association analysis of gut microbiota with colon transcriptome in mice. METHODS: A mouse colitis model was constructed with wild-type (WT) and TLR4-knockout (KO) mice. Fecal microbiota was transplanted by gavage. Colon inflammation severity was measured by disease activity index (DAI) scoring and hematoxylin and eosin staining. Gut microbiota structure was analyzed through 16S ribosomal RNA sequencing. Gene expression in the mouse colon was obtained by transcriptome sequencing. RESULTS: The KO (DSS + Water) and KO (DSS + FMT) groups displayed indistinguishable body weight loss, colon length, DAI score, and histology score, which showed that FMT could not inhibit the disease in KO mice. In mice treated with FMT, the relative abundance of Akkermansia decreased, and Lactobacillus became dominant. In particular, compared with those in WT mice, the scores of DAI and colon histology were clearly decreased in the KO-DSS group. Microbiota structure showed a significant difference between KO and WT mice. Akkermansia were the dominant genus in healthy KO mice. The ineffectiveness of FMT in KO mice was related to the decreased abundance of Akkermansia. Gene Ontology enrichment analysis showed that differentially expressed genes between each group were mainly involved in cytoplasmic translation and cellular response to DNA damage stimulus. The top nine genes correlating with Akkermansia included Aqp4, Clca4a, Dpm3, Fau, Mcrip1, Meis3, Nupr1 L, Pank3, and Rps13 (|R| > 0.9, P < 0.01). CONCLUSION: FMT may ameliorate DSS-induced colitis by regulating the TLR4 signaling pathway. TLR4 modulates the composition of gut microbiota and the expression of related genes to ameliorate colitis and maintain the stability of the intestinal environment. Akkermansia bear great therapeutic potential for colitis.

Role of TPEN in Amyloid-ß25-35-Induced Neuronal Damage Correlating with Recovery of Intracellular Zn2+ and Intracellular Ca2+ Overloading.

The overproduction of neurotoxic amyloid-ß (Aß) peptides in the brain is a hallmark of Alzheimer's disease (AD). To determine the role of intracellular zinc ion (iZn2+) dysregulation in mediating Aß-related neurotoxicity, this study aimed to investigate whether N, N, N', N'­tetrakis (2­pyridylmethyl) ethylenediamine (TPEN), a Zn2+­specific chelator, could attenuate Aß25-35­induced neurotoxicity and the underlying mechanism. We used the 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay to measure the viability of primary hippocampal neurons. We also determined intracellular Zn2+ and Ca2+ concentrations, mitochondrial and lysosomal functions, and intracellular reactive oxygen species (ROS) content in hippocampal neurons using live-cell confocal imaging. We detected L-type voltage-gated calcium channel currents (L-ICa) in hippocampal neurons using the whole­cell patch­clamp technique. Furthermore, we measured the mRNA expression levels of proteins related to the iZn2+ buffer system (ZnT-3, MT-3) and voltage-gated calcium channels (Cav1.2, Cav1.3) in hippocampal neurons using RT-PCR. The results showed that TPEN attenuated Aß25-35­induced neuronal death, relieved the Aß25-35­induced increase in intracellular Zn2+ and Ca2+ concentrations; reversed the Aß25-35­induced increase in ROS content, the Aß25-35­induced increase in the L-ICa peak amplitude at different membrane potentials, the Aß25-35­induced the dysfunction of the mitochondria and lysosomes, and the Aß25-35­induced decrease in ZnT-3 and MT-3 mRNA expressions; and increased the Cav1.2 mRNA expression in the hippocampal neurons. These results suggest that TPEN, the Zn2+-specific chelator, attenuated Aß25-35­induced neuronal damage, correlating with the recovery of intracellular Zn2+ and modulation of abnormal Ca2+-related signaling pathways.

Going beyond Binary: Rapid Identification of Protein-Protein Interaction Modulators Using a Multifragment Kinetic Target-Guided Synthesis Approach.

Kinetic target-guided synthesis (KTGS) is a powerful screening approach that enables identification of small molecule modulators for biomolecules. While many KTGS variants have emerged, a majority of the examples suffer from limited throughput and a poor signal/noise ratio, hampering reliable hit detection. Herein, we present our optimized multifragment KTGS screening strategy that tackles these limitations. This approach utilizes selected reaction monitoring liquid chromatography tandem mass spectrometry for hit detection, enabling the incubation of 190 fragment combinations per screening well. Consequentially, our fragment library was expanded from 81 possible combinations to 1710, representing the largest KTGS screening library assembled to date. The expanded library was screened against Mcl-1, leading to the discovery of 24 inhibitors. This work unveils the true potential of KTGS with respect to the rapid and reliable identification of hits, further highlighting its utility as a complement to the existing repertoire of screening methods used in drug discovery.