ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B.

The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

Cryo-EM structure of ACE2-SIT1 in complex with tiagabine.

The pharmacology of amino acid transporters in the SLC6 family is poorly developed compared to that of the neurotransmitter transporters. To identify new inhibitors of the proline transporter SIT1 (SLC6A20), its expression in Xenopus laevis oocytes was optimized. Trafficking of SIT1 was augmented by co-expression of angiotensin-converting enzyme 2 (ACE2) in oocytes but there was no strict requirement for co-expression of ACE2. A pharmacophore-guided screen identified tiagabine as a potent non-competitive inhibitor of SIT1. To understand its binding mode, we determined the cryo-electron microscopy (cryo-EM) structure of ACE2-SIT1 bound with tiagabine. The inhibitor binds close to the orthosteric proline binding site, but due to its size extends into the cytosolic vestibule. This causes the transporter to adopt an inward-open conformation, in which the intracellular gate is blocked. This study provides the first structural insight into inhibition of SIT1 and generates tools for a better understanding of the ACE2-SIT1 complex. These findings may have significance for SARS-CoV-2 binding to its receptor ACE2 in human lung alveolar cells where SIT1 and ACE2 are functionally expressed.

Small molecule MarR modulators potentiate metronidazole antibiotic activity in aerobic E. coli by inducing activation by the nitroreductase NfsA.

Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

Structure and RAF family kinase isoform selectivity of type II RAF inhibitors tovorafenib and naporafenib.

Upon activation by RAS, RAF family kinases initiate signaling through the MAP kinase cascade to control cell growth, proliferation, and differentiation. Among RAF isoforms (ARAF, BRAF, and CRAF), oncogenic mutations are by far most frequent in BRAF. The BRAFV600E mutation drives more than half of all malignant melanoma and is also found in many other cancers. Selective inhibitors of BRAFV600E (vemurafenib, dabrafenib, encorafenib) are used clinically for these indications, but they are not effective inhibitors in the context of oncogenic RAS, which drives dimerization and activation of RAF, nor for malignancies driven by aberrantly dimerized truncation/fusion variants of BRAF. By contrast, a number of "type II" RAF inhibitors have been developed as potent inhibitors of RAF dimers. Here, we compare potency of type II inhibitors tovorafenib (TAK-580) and naporafenib (LHX254) in biochemical assays against the three RAF isoforms and describe crystal structures of both compounds in complex with BRAF. We find that tovorafenib and naporafenib are most potent against CRAF but markedly less potent against ARAF. Crystal structures of both compounds with BRAFV600E or WT BRAF reveal the details of their molecular interactions, including the expected type II-binding mode, with full occupancy of both subunits of the BRAF dimer. Our findings have important clinical ramifications. Type II RAF inhibitors are generally regarded as pan-RAF inhibitors, but our studies of these two agents, together with recent work with type II inhibitors belvarafenib and naporafenib, indicate that relative sparing of ARAF may be a property of multiple drugs of this class.

Deep learning identifies explainable reasoning paths of mechanism of action for drug repurposing from multilayer biological network.

The discovery and repurposing of drugs require a deep understanding of the mechanism of drug action (MODA). Existing computational methods mainly model MODA with the protein-protein interaction (PPI) network. However, the molecular interactions of drugs in the human body are far beyond PPIs. Additionally, the lack of interpretability of these models hinders their practicability. We propose an interpretable deep learning-based path-reasoning framework (iDPath) for drug discovery and repurposing by capturing MODA on by far the most comprehensive multilayer biological network consisting of the complex high-dimensional molecular interactions between genes, proteins and chemicals. Experiments show that iDPath outperforms state-of-the-art machine learning methods on a general drug repurposing task. Further investigations demonstrate that iDPath can identify explicit critical paths that are consistent with clinical evidence. To demonstrate the practical value of iDPath, we apply it to the identification of potential drugs for treating prostate cancer and hypertension. Results show that iDPath can discover new FDA-approved drugs. This research provides a novel interpretable artificial intelligence perspective on drug discovery.

Biomolecular fingerprints of the effect of zoledronic acid on prostate cancer stem cells: Comparison of 2D and 3D cell culture models.

Revealing the potential of candidate drugs against different cancer types without disrupting normal cells depends on the drug mode of action. In the current study, the drug response of prostate cancer stem cells (PCSCs) to zoledronic acid (ZOL) grown in two-dimensional (2D) and three-dimensional (3D) culture systems was compared using Fourier transform-infrared (FT-IR) spectroscopy which is a vibrational spectroscopic technique, supporting by biochemical assays and imaging techniques. Based on our data, in 2D cell culture conditions, the ZOL treatment of PCSCs isolated according to both C133 and CD44 cell surface properties induced early/late apoptosis and suppressed migration ability. The CD133 gene expression and protein levels were altered, depending on culture systems. CD133 expression was significantly reduced in 2D cells upon ZOL treatment. FT-IR data revealed that the integrity, fluidity, and ordering/disordering states of the cell membrane and nucleic acid content were altered in both 2D and 3D cells after ZOL treatment. Regular protein structures decrease in 2D cells while glycogen and protein contents increase in 3D cells, indicating a more pronounced cytotoxic effect of ZOL for 2D cells. Untreated 3D PCSCs exhibited an even different spectral profile associated with IR signals of lipids, proteins, nucleic acids, and glycogen in comparison to untreated 2D cells. Our study revealed significant differences in the drug response and cellular constituents between 2D and 3D cells. Exploring molecular targets and/or drug-action mechanisms is significant in cancer treatment approaches; thus, FT-IR spectroscopy can be successfully applied as a novel drug-screening method in clinical research.

Drug-Target Association Kinetics in Drug Discovery.

The important role of ligand-receptor binding kinetics in drug design and discovery is increasingly recognized by the drug research community. Over the past decade, accumulating evidence has shown that optimizing the ligand's dissociation rate constant can lead to desirable duration of in vivo target occupancy and, hence, improved pharmacodynamic properties. However, the association rate constant as a pharmacological principle remains less investigated, whereas it can play an equally important role in the selection of drug candidates. This review provides a compilation and discussion of otherwise scarce and dispersed information on this topic, bringing to light the importance of drug-target association in kinetics-directed drug design and discovery.

Tafenoquine and its derivatives as inhibitors for the severe acute respiratory syndrome coronavirus 2.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely affected human lives around the world as well as the global economy. Therefore, effective treatments against COVID-19 are urgently needed. Here, we screened a library containing Food and Drug Administration (FDA)-approved compounds to identify drugs that could target the SARS-CoV-2 main protease (Mpro), which is indispensable for viral protein maturation and regard as an important therapeutic target. We identified antimalarial drug tafenoquine (TFQ), which is approved for radical cure of Plasmodium vivax and malaria prophylaxis, as a top candidate to inhibit Mpro protease activity. The crystal structure of SARS-CoV-2 Mpro in complex with TFQ revealed that TFQ noncovalently bound to and reshaped the substrate-binding pocket of Mpro by altering the loop region (residues 139-144) near the catalytic Cys145, which could block the catalysis of its peptide substrates. We also found that TFQ inhibited human transmembrane protease serine 2 (TMPRSS2). Furthermore, one TFQ derivative, compound 7, showed a better therapeutic index than TFQ on TMPRSS2 and may therefore inhibit the infectibility of SARS-CoV-2, including that of several mutant variants. These results suggest new potential strategies to block infection of SARS-CoV-2 and rising variants.

Dimethyl fumarate inhibits ZNF217 and can be beneficial in a subset of estrogen receptor positive breast cancers.

PURPOSE: The oncogenic factor ZNF217 promotes aggressive estrogen receptor (ER)+breast cancer disease suggesting that its inhibition may be useful in the clinic. Unfortunately, no direct pharmacological inhibitor is available. Dimethyl fumarate (DMF) exhibits anti-breast cancer activities, in vitro and in pre-clinical in vivo models. Its therapeutic benefits stem from covalent modification of cellular thiols such as protein cysteines, but the full profile of molecular targets mediating its anti-breast cancer effects remains to be determined. METHODS: ER+breast cancer cells were treated with DMF followed by cysteine-directed proteomics. Cells with modulated ZNF217 levels were used to probe the efficacy of DMF. RESULTS: Covalent modification of ZNF217 by DMF identified by proteomics was confirmed by using a DMF-chemical probe. Inhibition of ZNF217's transcriptional activity by DMF was evident on reported ZNF217-target genes. ZNF217 as an oncogene has been shown to enhance stem-like properties, survival, proliferation, and invasion. Consistent with ZNF217 inhibition, DMF was more effective at blocking these ZNF217-driven phenotypes in cells with elevated ZNF217 expression. Furthermore, partial knockdown of ZNF217 led to a reduction in DMF's efficacy. DMF's in vivo activity was evaluated in a xenograft model of MCF-7 HER2 cells that have elevated expression of ZNF217 and DMF treatment resulted in significant inhibition of tumor growth. CONCLUSION: These data indicate that DMF's anti-breast cancer activities in the ER+HER2+models, at least in part, are due to inhibition of ZNF217. DMF is identified as a new covalent inhibitor of ZNF217.

Cancer treatment and toxicity outlook of nanoparticles.

Diversified nanosystems with tunable physicochemical attributes have emerged as potential solution to globally devastating cancer by offering novel possibilities for improving the techniques of cancer detection, imaging, therapies, diagnosis, drug delivery and treatment. Drug delivery systems based on nanoparticles (NPs) with ability of crossing different biological barriers are becoming increasingly popular. Besides, NPs are utilized in pharmaceutical sciences to mitigate the toxicity of conventional cancer therapeutics. However, significant NPs-associated toxicity, off-targeted activities, and low biocompatibility limit their utilization for cancer theranostics and can be hazardous to cancer patients up to life-threatening conditions. NPs interact with the biomolecules and disturb their regular function by aggregating inside cells and forming a protein corona, and the formulation turns ineffective in controlling cancer cell growth. The adverse interactions between NPs and biological entities can lead to life-threatening toxicities. This review focuses on the widespread use of various NPs including zinc oxide, titanium oxide, silver, and gold, which serve as efficient nano-vehicles and demonstrate notable pharmacokinetic and pharmacodynamic advantages in cancer therapy. Subsequently, the mechanism of nanotoxicity attached with these NPs, alternate solutions and their prospect to revolutionize cancer theranostics are highlighted. This review will serve as guide for future developments associated with high-performance NPs with controlled toxicity for establishing them as modern-age nanotools to manage cancer in tailored manner.

A SERS/fluorescence dual-mode immuno-nanoprobe for investigating two anti-diabetic drugs on EGFR expressions.

A surface-enhanced Raman scattering (SERS)/fluorescence dual-mode nanoprobe was proposed to assess anti-diabetic drug actions from the expression level of the epidermal growth factor receptor (EGFR), which is a significant biomarker of breast cancers. The nanoprobe has a raspberry shape, prepared by coating a dye-doped silica nanosphere with a mass of SERS tags, which gives high gains in fluorescence imaging and SERS measurement. The in situ detection of EGFR on the cell membrane surfaces after drug actions was achieved by using this nanoprobe, and the detection results agree with the enzyme-linked immunosorbent assay (ELISA) kit. Our study suggests that rosiglitazone hydrochloride (RH) may be a potential drug for diabetic patients with breast cancer, while the anti-cancer effect of metformin hydrochloride (MH) is debatable since MH slightly promotes the EGFR expression of MCF-7 cells in this study. This sensing platform endows more feasibility for highly sensitive and accurate feedback of pesticide effects at the membrane protein level.

Genotype and Phenotype Influence the Personal Response to Prostaglandin Analogues and Beta-Blockers in Spanish Glaucoma and Ocular Hypertension Patients.

Analysis of the genotype that predicts the phenotypic characteristics of a cohort of glaucoma and ocular hypertension patients, and the correlation with their personal pharmacological response to beta-blockers (BB) and prostaglandin analogues (PGA). Prospective study that included 139 eyes from 72 patients under BB and/or PGA treatment, and in some cases other types of ocular hypotensive treatments. Five single-nucleotide polymorphisms were genotyped by real-time PCR assays: prostaglandin-F2α receptor (rs3766355, rs3753380); cytochrome-P450 2D6 (rs16947, rs769258); and beta-2-adrenergic receptor (rs1042714). Other studied variables were mean deviation (MD) of visual field, previous ocular interventions, medical treatment, baseline (bIOP), and treated intraocular pressure (tIOP). From a total of 139 eyes, 71 (51.1%) were left eyes. The main diagnosis was primary open angle glaucoma (66.2%). A total of 57 (41%) eyes were under three or more medications (PGA + BB + other) and, additionally, 57 eyes (41%) had had some kind of glaucoma surgery. The mean bIOP and tIOP were 26.55 ± 8.19 and 21.01 ± 5.54 mmHg, respectively. Significant differences in tIOP were found between heterozygous (HT) (21.07 ± 0.607 mmHg) and homozygous (HM) (20.98 ± 0.639 mmHg) rs3766355 with respect to wildtype individuals (16 ± 1.08 mmHg) (p = 0.031). The MD values presented significant differences between wildtype rs3766355 (-2 ± 2.2 dB), HT (-3.87 ± 4 dB), and HM carriers (-9.37 ± 9.51 dB) (p = 0.009). Significant differences were also observed between the MD in wildtype rs3753380 (-6.1 ± 8.67 dB), HT (-9.02 ± 8.63 dB), and HM carriers (-9.51 ± 7.44 dB) (p = 0.017). Patients carrying the variant rs3766355 in HM or HT presented clinically-significantly higher tIOP than wildtype patients. Additionally, some differences in MD were found in rs3766355 and rs3753380 carriers, and the more alleles that were affected, the worse the MD value, meaning greater severity of the glaucoma. Poor response to treatment and more visual field damage may be associated with being a carrier of these mutated alleles.

Major advances in targeted protein degradation: PROTACs, LYTACs, and MADTACs.

Of late, targeted protein degradation (TPD) has surfaced as a novel and innovative chemical tool and therapeutic modality. By co-opting protein degradation pathways, TPD facilitates complete removal of the protein molecules from within or outside the cell. While the pioneering Proteolysis-Targeting Chimera (PROTAC) technology and molecular glues hijack the ubiquitin-proteasome system, newer modalities co-opt autophagy or the endo-lysosomal pathway. Using this mechanism, TPD is posited to largely expand the druggable space far beyond small-molecule inhibitors. In this review, we discuss the major advances in TPD, highlight our current understanding, and explore outstanding questions in the field.

Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin-actin interaction.

Actin-myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin-myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure.

The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue.

Patients with cystic fibrosis (CF) harboring the P67L variant in the cystic fibrosis transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors, such as lumacaftor and tezacaftor, with rescue in model systems that far exceed what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low-temperature growth and/or intragenic suppression, which suggest a mechanism underlying P67L that (1) shares key pathogenic features with F508del, including off-pathway (non-native) folding intermediates, (2) is linked to folding stability of nucleotide-binding domains 1 and 2, and (3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the "lasso" helices 1 and 2, which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino-terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair.

Targeting primary and metastatic uveal melanoma with a G protein inhibitor.

Uveal melanoma (UM) is the most common intraocular tumor in adults. Nearly half of UM patients develop metastatic disease and often succumb within months because effective therapy is lacking. A novel therapeutic approach has been suggested by the discovery that UM cell lines driven by mutant constitutively active Gq or G11 can be targeted by FR900359 (FR) or YM-254890, which are bioavailable, selective inhibitors of the Gq/11/14 subfamily of heterotrimeric G proteins. Here, we have addressed the therapeutic potential of FR for UM. We found that FR inhibited all oncogenic Gq/11 mutants reported in UM. FR arrested growth of all Gq/11-driven UM cell lines tested, but induced apoptosis only in a few. Similarly, FR inhibited growth of, but did not efficiently kill, UM tumor cells from biopsies of primary or metastatic tumors. FR evoked melanocytic redifferentiation of UM tumor cells with low (class 1), but not high (class 2), metastatic potential. FR administered systemically below its LD50 strongly inhibited growth of PDX-derived class 1 and class 2 UM tumors in mouse xenograft models and reduced blood pressure transiently. FR did not regress xenografted UM tumors or significantly affect heart rate, liver function, hematopoiesis, or behavior. These results indicated the existence of a therapeutic window in which FR can be explored for treating UM and potentially other diseases caused by constitutively active Gq/11.

Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity.

Acetaminophen (APAP)-induced liver necrosis is a form of regulated cell death (RCD) in which APAP activates the mitogen-activated protein kinases (MAPKs) and specifically the c-Jun-N-terminal kinase (JNK) pathway, leading to necrotic cell death. Previously, we have shown that receptor interacting protein kinase-1 (RIPK1) knockdown is also protective against APAP RCD upstream of JNK. However, whether the kinase or platform function of RIPK1 is involved in APAP RCD is not known. To answer this question, we used genetic mouse models of targeted hepatocyte RIPK1 knockout (RIPK1HepCKO) or kinase dead knock-in (RIPK1D138N) and adult hepatocyte specific knockout of the cytoprotective protein A20 (A20HepCKO), known to interact with RIPK1, to study its potential involvement in MAPK signaling. We observed no difference in injury between WT and RIPK1D138N mice post APAP. However, RIPK1HepCKO was protective. We found that RIPK1HepCKO mice had attenuated pJNK activation, while A20 was simultaneously upregulated. Conversely, A20HepCKO markedly worsened liver injury from APAP. Mechanistically, we observed a significant upregulation of apoptosis signal-regulating kinase 1 (ASK1) and increased JNK activation in A20HepCKO mice compared with littermate controls. We also demonstrated that A20 coimmunoprecipitated (co-IP) with both RIPK1 and ASK1, and that in the presence of RIPK1, there was less A20-ASK1 association than in its absence. We conclude that the kinase-independent platform function of RIPK1 is involved in APAP toxicity. Adult RIPK1HepCKO mice are protected against APAP by upregulating A20 and attenuating JNK signaling through ASK1, conversely, A20HepCKO worsens injury from APAP.

Glutamate potentiates heterologously expressed homomeric acid-sensing ion channel 1a.

Acid-sensing ion channels (ASICs) participate in synaptic transmission due to the acidic content of synaptic vesicles, but their contribution to postsynaptic currents is small. This has stimulated attempts to find endogenous ASIC potentiators that could enhance ASIC-mediated currents to physiologically relevant values. Here we demonstrate that glutamate, which serves as a neurotransmitter, potentiates recombinant ASIC1a in the submillimolar concentration range. The effect of glutamate is especially interesting as ASIC's presence has been shown in glutamatergic synapses. At pH=6.5 glutamate had maximum potentiation of 87% with an EC50 value of 0.65 mM. The mechanism of potentiation is due to a shift of pH-dependent activation to less acidic values, with 0.5 mM glutamate increasing pH50 from 6.04 to 6.43. Due to this mechanism, ASIC1a in glutamatergic synapses might be intrinsically potentiated. Furthermore, this effect could compensate for the inhibition of ionotropic glutamate receptors by extracellular acidification during synaptic transmission.

P300/CBP inhibition sensitizes mantle cell lymphoma to PI3Kδ inhibitor idelalisib.

Mantle cell lymphoma (MCL) is a lymphoproliferative disorder lacking reliable therapies. PI3K pathway contributes to the pathogenesis of MCL, serving as a potential target. However, idelalisib, an FDA-approved drug targeting PI3Kδ, has shown intrinsic resistance in MCL treatment. Here we report that a p300/CBP inhibitor, A-485, could overcome resistance to idelalisib in MCL cells in vitro and in vivo. A-485 was discovered in a combinational drug screening from an epigenetic compound library containing 45 small molecule modulators. We found that A-485, the highly selective catalytic inhibitor of p300 and CBP, was the most potent compound that enhanced the sensitivity of MCL cell line Z-138 to idelalisib. Combination of A-485 and idelalisib remarkably decreased the viability of three MCL cell lines tested. Co-treatment with A-485 and idelalisib in Maver-1 and Z-138 MCL cell xenograft mice for 3 weeks dramatically suppressed the tumor growth by reversing the unsustained inhibition in PI3K downstream signaling. We further demonstrated that p300/CBP inhibition decreased histone acetylation at RTKs gene promoters and reduced transcriptional upregulation of RTKs, thereby inhibiting the downstream persistent activation of MAPK/ERK signaling, which also contributed to the pathogenesis of MCL. Therefore, additional inhibition of p300/CBP blocked MAPK/ERK signaling, which rendered maintaining activation to PI3K-mTOR downstream signals p-S6 and p-4E-BP1, thus leading to suppression of cell growth and tumor progression and eliminating the intrinsic resistance to idelalisib ultimately. Our results provide a promising combination therapy for MCL and highlight the potential use of epigenetic inhibitors targeting p300/CBP to reverse drug resistance in tumor.