The biochemically defined super relaxed state of myosin-A paradox.

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.

Depressive-like behavior accompanies neuroinflammation in an animal model of bipolar disorder symptoms induced by ouabain.

INTRODUCTION: A previous study from our Laboratory showed no alteration in inflammatory parameters seven days after ouabain (OUA) administration, a Na+K+ATPase inhibitor, which was previously considered only a mania model. However, the administration of OUA in rats was recently validated as a model of bipolar disorder (BD) symptoms, demonstrating that 14 days after single intracerebroventricular (ICV) administration, OUA also induces depressive-like behavior. Therefore, it is important to investigate the long-term effect of OUA on inflammatory parameters since this mechanism seems to play a key role in BD physiopathology. METHODS: Adult male Wistar rats received a single ICV administration of OUA or artificial cerebrospinal fluid (aCSF). From the fourth day after the ICV infusion, the rats received saline or Lithium (Li) for 14 days. The open-field test was performed on the 7th day after OUA. On the 14th day, locomotion was re-evaluated, and the forced swimming test (FST) was used to evaluate depressive-like behavior. Inflammatory parameters were assessed in the frontal cortex and hippocampus. RESULTS: OUA increased the locomotion of rats after seven days, considered a mania-like behavior. In the FST, OUA increased the time of immobility on the 14th day, considered a depressive-like behavior. Li reversed the mania-like behavior and partially reversed the depressive-like behavior. Furthermore, OUA increased the levels of interleukin (IL)-1ß, IL-6, IL-10, TNF-α, and CINC-1 in the frontal cortex and hippocampus. Li treatment reverses all these inflammatory alterations. CONCLUSION: This study suggests that the long-term Na+K+ATPase inhibition effects induce depressive-like behavior, which was accompanied by inflammation in the BD symptoms model.

Fluorescence Lifetime Imaging of pH along the Secretory Pathway.

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein, pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. Therefore, we applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block endoplasmic reticulum (ER)-Golgi trafficking. Finally, we followed the pH changes of newly synthesized molecules of the inflammatory cytokine tumor necrosis factor-α while they were in transit from the ER via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution and can be used to assess organellar pH in disease models.

The C-terminal segment of Leishmania major HslU: Toward potential inhibitors of LmHslVU activity.

It is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. We explore the possibility to target the parasite mitochondrial HslVU protease, which is essential for growth and has no analogue in the human host. For this, we develop compounds potentially inhibiting the complex assembly by mimicking the C-terminal (C-ter) segment of the ATPase HslU. We previously showed that a dodecapeptide derived from Leishmania major HslU C-ter segment (LmC12-U2, Cpd 1) was able to bind to and activate the digestion of a fluorogenic substrate by LmHslV. Here, we present the study of its structure-activity relationships. By replacing each essential residue with related non-proteinogenic residues, we obtained more potent analogues. In particular, a cyclohexylglycine residue at position 11 (cpd 24) allowed a more than three-fold gain in potency while reducing the size of compound 24 from twelve to six residues (cpd 50) without significant loss of potency, opening the way toward short HslU C-ter peptidomimetics as potential inhibitors of HslV proteolytic function. Finally, conjugates constituted of LmC6-U2 analogues and a mitochondrial penetrating peptide were found to penetrate into the promastigote form of L. infantum and to inhibit the parasite growth without showing toxicity toward human THP-1 cells at the same concentration (i.e. 30 µM).

A new porphyrin as selective substrate-based inhibitor of breast cancer resistance protein (BCRP/ABCG2).

The ABCG2 transporter plays a pivotal role in multidrug resistance, however, no clinical trial using specific ABCG2 inhibitors have been successful. Although ABC transporters actively extrude a wide variety of substrates, photodynamic therapeutic agents with porphyrinic scaffolds are exclusively transported by ABCG2. In this work, we describe for the first time a porphyrin derivative (4B) inhibitor of ABCG2 and capable to overcome multidrug resistance in vitro. The inhibition was time-dependent and 4B was not itself transported by ABCG2. Independently of the substrate, the porphyrin 4B showed an IC50 value of 1.6 µM and a mixed type of inhibition. This compound inhibited the ATPase activity and increased the binding of the conformational-sensitive antibody 5D3. A thermostability assay confirmed allosteric protein changes triggered by the porphyrin. Long-timescale molecular dynamics simulations revealed a different behavior between the ABCG2 porphyrinic substrate pheophorbide a and the porphyrin 4B. Pheophorbide a was able to bind in three different protein sites but 4B showed one binding conformation with a strong ionic interaction with GLU446. The inhibition was selective toward ABCG2, since no inhibition was observed for P-glycoprotein and MRP1. Finally, this compound successfully chemosensitized cells that overexpress ABCG2. These findings reinforce that substrates may be a privileged source of chemical scaffolds for identification of new inhibitors of multidrug resistance-linked ABC transporters.

Synthesis, characterization and biological evaluation of thiadiazole amide derivatives as nucleoside triphosphate diphosphohydrolases (NTPDases) inhibitors.

Importance of extracellular nucleotides is widely understood. These nucleotides act as ligand for P2X and P2Y receptors and modulate a variety of biological functions. However, their extracellular concentration is maintained by a chain of enzymes termed as ecto-nucleotidases. Amongst them, nucleoside triphosphate diphosphohydrolases (NTPDases) is an important enzyme family responsible for the dephosphorylation of these nucleotides. Overexpression of NTPDases leads to many pathological conditions such as cancer and thrombosis. So far, only a few NTPDase inhibitors have been reported. Considering this scarcity of (NTPDase) inhibitors, a number of thiadiazole amide derivatives were synthesized and screened against human (h)-NTPDases. Several compounds showed promising inhibitory activity; compound 5a (IC50 (µM); 0.05 ± 0.008) and 5g (IC50 (µM); 0.04 ± 0.006) appeared to be the most distinguished molecules corresponding to h-NTPDase1 and -2. However, h-NTPDase3 was the least susceptible isozyme and only three compounds (5d, 5e, 5j) strongly inhibited h-NTPDase3. Interestingly, compound 5e was recognized as the most active compound that showed dual inhibition against h-NTPDase3 as well as against h-NTPDase8. For better comprehension of binding mode of these inhibitors, most potent inhibitors were docked with their respective isozyme.

Targeting RIOK2 ATPase activity leads to decreased protein synthesis and cell death in acute myeloid leukemia.

Novel therapies for the treatment of acute myeloid leukemia (AML) are urgently needed, because current treatments do not cure most patients with AML. We report a domain-focused, kinome-wide CRISPR-Cas9 screening that identified protein kinase targets for the treatment of AML, which led to the identification of Rio-kinase 2 (RIOK2) as a potential novel target. Loss of RIOK2 led to a decrease in protein synthesis and to ribosomal instability followed by apoptosis in leukemic cells, but not in fibroblasts. Moreover, the ATPase function of RIOK2 was necessary for cell survival. When a small-molecule inhibitor was used, pharmacological inhibition of RIOK2 similarly led to loss of protein synthesis and apoptosis and affected leukemic cell growth in vivo. Our results provide proof of concept for targeting RIOK2 as a potential treatment of patients with AML.

Identification of Influenza PAN Endonuclease Inhibitors via 3D-QSAR Modeling and Docking-Based Virtual Screening.

Structural and biochemical studies elucidate that PAN may contribute to the host protein shutdown observed during influenza A infection. Thus, inhibition of the endonuclease activity of viral RdRP is an attractive approach for novel antiviral therapy. In order to envisage structurally diverse novel compounds with better efficacy as PAN endonuclease inhibitors, a ligand-based-pharmacophore model was developed using 3D-QSAR pharmacophore generation (HypoGen algorithm) methodology in Discovery Studio. As the training set, 25 compounds were taken to generate a significant pharmacophore model. The selected pharmacophore Hypo1 was further validated by 12 compounds in the test set and was used as a query model for further screening of 1916 compounds containing 71 HIV-1 integrase inhibitors, 37 antibacterial inhibitors, 131 antiviral inhibitors and other 1677 approved drugs by the FDA. Then, six compounds (Hit01-Hit06) with estimated activity values less than 10 µM were subjected to ADMET study and toxicity assessment. Only one potential inhibitory 'hit' molecule (Hit01, raltegravir's derivative) was further scrutinized by molecular docking analysis on the active site of PAN endonuclease (PDB ID: 6E6W). Hit01 was utilized for designing novel potential PAN endonuclease inhibitors through lead optimization, and then compounds were screened by pharmacophore Hypo1 and docking studies. Six raltegravir's derivatives with significant estimated activity values and docking scores were obtained. Further, these results certainly do not confirm or indicate the seven compounds (Hit01, Hit07, Hit08, Hit09, Hit10, Hit11 and Hit12) have antiviral activity, and extensive wet-laboratory experimentation is needed to transmute these compounds into clinical drugs.

Myristic Acid Inhibits the Activity of the Bacterial ABC Transporter BmrA.

ATP-binding cassette (ABC) transporters are conserved in all kingdoms of life, where they transport substrates against a concentration gradient across membranes. Some ABC transporters are known to cause multidrug resistances in humans and are able to transport chemotherapeutics across cellular membranes. Similarly, BmrA, the ABC transporter of Bacillus subtilis, is involved in excretion of certain antibiotics out of bacterial cells. Screening of extract libraries isolated from fungi revealed that the C14 fatty acid myristic acid has an inhibitory effect on the BmrA ATPase as well as the transport activity. Thus, a natural membrane constituent inhibits the BmrA activity, a finding with physiological consequences as to the activity and regulation of ABC transporter activities in biological membranes.

Cinnamophilin overcomes cancer multi-drug resistance via allosterically modulating human P-glycoprotein on both drug binding sites and ATPase binding sites.

Cancer multi-drug resistance (MDR) caused by P-glycoprotein (P-gp) efflux is a critical unresolved clinical concern. The present study analyzed the effect of cinnamophilin on P-gp inhibition and MDR reversion. The effect of cinnamophilin on P-gp was investigated through drug efflux assay, ATPase assay, MDR1 shift assay, and molecular docking. The cancer MDR-reversing ability and mechanisms were analyzed through cytotoxicity and combination index (CI), cell cycle, and apoptosis experiments. P-gp efflux function was significantly inhibited by cinnamophilin without influencing the drug's expression or conformation. Cinnamophilin uncompetitively inhibited the efflux of doxorubicin and rhodamine 123 and exhibited a distinct binding behavior compared with verapamil, the P-gp standard inhibitor. The half maximal inhibitory concentration of cinnamophilin for doxorubicin and rhodamine 123 efflux was 12.47 and 11.59 µM, respectively. In regard to P-gp energy consumption, verapamil-stimulated ATPase activity was further enhanced by cinnamophilin at concentrations of 0.1, 1, 10, and 20 µM. In terms of MDR reversion, cinnamophilin demonstrated synergistic cytotoxic effects when combined with docetaxel, vincristine, or paclitaxel. The CI was < 0.7 in all experimental combination treatments. The present study showed that cinnamophilin possesses P-gp-modulating effects and cancer MDR resensitizing ability.

DisA Restrains the Processing and Cleavage of Reversed Replication Forks by the RuvAB-RecU Resolvasome.

DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. Bacillus subtilis RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.

Discovery of an eIF4A Inhibitor with a Novel Mechanism of Action.

Increased protein synthesis is a requirement for malignant growth, and as a result, translation has become a pharmaceutical target for cancer. The initiation of cap-dependent translation is enzymatically driven by the eukaryotic initiation factor (eIF)4A, an ATP-powered DEAD-box RNA-helicase that unwinds the messenger RNA secondary structure upstream of the start codon, enabling translation of downstream genes. A screen for inhibitors of eIF4A ATPase activity produced an intriguing hit that, surprisingly, was not ATP-competitive. A medicinal chemistry campaign produced the novel eIF4A inhibitor 28, which decreased BJAB Burkitt lymphoma cell viability. Biochemical and cellular studies, molecular docking, and functional assays uncovered that 28 is an RNA-competitive, ATP-uncompetitive inhibitor that engages a novel pocket in the RNA groove of eIF4A and inhibits unwinding activity by interfering with proper RNA binding and suppressing ATP hydrolysis. Inhibition of eIF4A through this unique mechanism may offer new strategies for targeting this promising intersection point of many oncogenic pathways.

High mutations in fatty acid metabolism contribute to a better prognosis of small-cell lung cancer patients treated with chemotherapy.

BACKGROUND: The majority of patients with small-cell lung cancer (SCLC) show a good response in the early stages of treatment, but more than 90% of patients will develop drug resistance. Therefore, biomarkers are urgently needed to identify patients who can benefit from systemic treatment. METHODS: We prospectively enrolled 52 extensive-stage SCLC patients before treatment from a local hospital to identify mutations related to patient prognosis, and verified them in the published Jiang's cohort and George's cohort. RESULTS: We found that patients with high mutations (mut-high) in the fatty acid (FA) metabolism pathway had a longer progression-free survival (PFS) in the local hospital cohort (HR = 0.446, 95% CI, 0.207-0.959, p = 0.0387) and a longer overall survival (OS) in Jiang's cohort (HR = 0.549, 95% CI, 0.314-0.960, p = 0.0351) than patients with low mutations (mut-low). Multivariate analysis suggested that mut-high status was an independent prognostic factor in both cohorts. George's cohort verified that mut-high status was associated with a longer OS than mut-low status (HR = 0.730, 95% CI 0.440-1.220, p = 0.2277). The possible mechanisms were as follows: the frequency of mutated FA synthase (FASN) in the mut-high group was greater than that in the mut-low group, and pathways related to the cell cycle, DNA repair, and oxidative phosphorylation were enriched in the mut-high group. CONCLUSIONS: The prognosis of SCLC patients treated with chemotherapy was better among patients with more mutations in the FA metabolism pathway, and the underlying mechanisms could be found at the genome and transcriptome levels.

Multi-Tissue Transcriptomic-Informed In Silico Investigation of Drugs for the Treatment of Dengue Fever Disease.

Transcriptomics, proteomics and pathogen-host interactomics data are being explored for the in silico-informed selection of drugs, prior to their functional evaluation. The effectiveness of this kind of strategy has been put to the test in the current COVID-19 pandemic, and it has been paying off, leading to a few drugs being rapidly repurposed as treatment against SARS-CoV-2 infection. Several neglected tropical diseases, for which treatment remains unavailable, would benefit from informed in silico investigations of drugs, as performed in this work for Dengue fever disease. We analyzed transcriptomic data in the key tissues of liver, spleen and blood profiles and verified that despite transcriptomic differences due to tissue specialization, the common mechanisms of action, "Adrenergic receptor antagonist", "ATPase inhibitor", "NF-kB pathway inhibitor" and "Serotonin receptor antagonist", were identified as druggable (e.g., oxprenolol, digoxin, auranofin and palonosetron, respectively) to oppose the effects of severe Dengue infection in these tissues. These are good candidates for future functional evaluation and clinical trials.

Divergent synthesis and elaboration of structure activity relationship for quinoline derivatives as highly selective NTPDase inhibitor.

Quinoline derivatives have interesting biological profile. In continuation for the comprehensive evaluations of substituted quinoline derivatives against human nucleoside triphosphate diphosphohydrolases (h-NTPDases) a series of substituted quinoline derivatives (2a-g, 3a-f, 4, 5a-c, 6) was synthesized. The inhibitory activities of the synthesized compounds were evaluated against four isoenzymes of human nucleoside triphosphate diphosphohydrolases (h-NTPDases). These quinoline derivatives had IC50 (µM) values in the range of 0.20-1.75, 0.77-2.20, 0.36-5.50 and 0.90-1.82 for NTPDase1, NTPDase2, NTPDase3 and NTPDase8, respectively. The derivative 3f was the most active compound against NTPDase1 (IC50, 0.20 ± 0.02 µM) that also possessed selectivity towards NTPDase1. Similarly, derivative 3b (IC50, 0.77 ± 0.06), 2h (IC50, 0.36 ± 0.01) and 2c (IC50, 0.90 ± 0.08) displayed excellent activity corresponding to NTPDase2, NTPDase3 and NTPdase8. The compound 5c emerged as a selective inhibitor of NTPDase8. The most active compounds were then investigated to determine their mode of inhibition and finally binding interactions of the active compounds were analyzed through molecular docking studies. The obtained results strongly support the quinoline scaffold's potential as potent and selective NTPDase inhibitor.

Kolaflavanone, a biflavonoid derived from medicinal plant Garcinia, is an inhibitor of mitotic kinesin Eg5.

Mitotic kinesin Eg5 remains a validated target in antimitotic therapy because of its essential role in the formation and maintenance of bipolar mitotic spindles. Although numerous Eg5 inhibitors of synthetic origin are known, only a few inhibitors derived from natural products have been reported. In our study, we focused on identifying novel Eg5 inhibitors from medicinal plants, particularly Garcinia species. Herein, we report the inhibitory effect of kolaflavanone (KLF), a Garcinia biflavonoid, on the ATPase and microtubule-gliding activities of mitotic kinesin Eg5. Additionally, we showed the interaction mechanism between Eg5 and KLF via in vitro and in silico analyses. The results revealed that KLF inhibited both the basal and microtubule-activated ATPase activities of Eg5. The inhibitory mechanism is allosteric, without a direct competition with adenosine-5'-diphosphate for the nucleotide-binding site. KLF also suppressed the microtubule gliding of Eg5 in vitro. The Eg5-KLF model obtained from molecular docking showed that the biflavonoid exists within the α2/α3/L5 (α2: Lys111-Glu116 and Ile135-Asp149, α3: Asn206-Thr226; L5: Gly117-Gly134) pocket, with a binding pose comparable to known Eg5 inhibitors. Overall, our data suggest that KLF is a novel allosteric inhibitor of mitotic kinesin Eg5.

Synthesis and evaluation of diphyllin ß-hydroxyl amino derivatives as novel V-ATPase inhibitors.

Natural diphyllin glycosides were identified as potent vacuolar H+ -ATPase (V-ATPase) inhibitors. A series of diphyllin ß-hydroxyl amino derivatives were designed and synthesized as novel diphyllin derivatives. Most of these derivatives displayed potent cytotoxicity against six cancer cell lines with IC50 values in the submicromolar to nanomolar concentration range. Compounds 2b, 2c, 2l, 2m, and 2n showed similar V-ATPase inhibitory potency to Bafilomycin A1. Compound 2l exhibited potent activity of modulation of lysosomal pH and cytoplasmic pH.

Structure-activity relationship and mechanism of flavonoids on the inhibitory activity of P-glycoprotein (P-gp)-mediated transport of rhodamine123 and daunorubicin in P-gp overexpressed human mouth epidermal carcinoma (KB/MDR) cells.

This study was aimed to investigate the inhibitory activity of flavonoids on P-glycoprotein (P-gp). Effects of 39 flavonoids on the cellular uptake (CU) of rhodamine123 (Rho) and daunomycin (DNR) were investigated in both parental KB and P-gp overexpressed KB/MDR cells. The inhibition mechanism of selected flavonoids was further investigated by measuring the ATPase activity and expression level of P-gp. Twelve flavonoids improved the uptake of Rho (↑RhoF) and nineteen flavonoids increased the uptake of DNR (↑DNRF) in KB/MDR cells with nine flavonoids overlapped. Structure-activity relationship (SAR) indicated that 8-OCH3, and 2'-OH have a negative effect on Rho and DNR transport. Whereas 5-OH, 5-OCH3, 6-OH, 7-OCH3, 3'-OH, and 4'-OH, are essential for inhibition of flavonoids on P-gp and reversing the resistance of Rho and DNR. Eleven selected flavonoids significantly induced the basal P-gp-ATPase activity but much lower than that induced by verapamil. Tangeretin, galangin, kaempferol, quercetin, and morin significantly reversed the ATPase activity stimulated by verapamil. Six of eleven flavonoids significantly decreased P-gp expression, whereas three flavonoids slightly increased P-gp expression. These results provide valuable information that flavonoids can effectively reverse multidrug resistance of P-gp-mediated transport of nutraceutical and drugs by co-administration.

Inhibition of heat shock proteins increases autophagosome formation, and reduces the expression of APP, Tau, SOD1 G93A and TDP-43.

Aberrant expression and denaturation of Tau, amyloid-beta and TDP-43 can lead to cell death and is a major component of pathologies such as Alzheimer's Disease (AD). AD neurons exhibit a reduced ability to form autophagosomes and degrade proteins via autophagy. Using genetically manipulated colon cancer cells we determined whether drugs that directly inhibit the chaperone ATPase activity or cause chaperone degradation and endoplasmic reticulum stress signaling leading to macroautophagy could reduce the levels of these proteins. The antiviral chaperone ATPase inhibitor AR12 reduced the ATPase activities and total expression of GRP78, HSP90, and HSP70, and of Tau, Tau 301L, APP, APP692, APP715, SOD1 G93A and TDP-43. In parallel, it increased the phosphorylation of ATG13 S318 and eIF2A S51 and caused eIF2A-dependent autophagosome formation and autophagic flux. Knock down of Beclin1 or ATG5 prevented chaperone, APP and Tau degradation. Neratinib, used to treat HER2+ breast cancer, reduced chaperone levels and expression of Tau and APP via macroautophagy, and neratinib interacted with AR12 to cause further reductions in protein levels. The autophagy-regulatory protein ATG16L1 is expressed as two isoforms, T300 or A300: Africans trend to express T300 and Europeans A300. We observed higher basal expression of Tau in T300 cells when compared to isogenic A300 cells. ATG16L1 isoform expression did not alter basal levels of HSP90, HSP70 or HSP27, however, basal levels of GRP78 were reduced in A300 cells. The abilities of both AR12 and neratinib to stimulate ATG13 S318 and eIF2A S51 phosphorylation and autophagic flux was also reduced in A300 cells. Our data support further evaluation of AR12 and neratinib in neuronal cells as repurposed treatments for AD.