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Benzolactams have unique biological activity and high utility in the synthesis of valuable compounds with direct applicability to oxindole alkaloids and antibacterial agents. Despite recent advances in organic chemistry and the growing number of reported methods for synthesizing benzolactams, their preparation still requires a multistep process. C-H amination reactions can convert aromatic C(sp

Small cell lung cancer (SCLC) is frequently found disseminated at first presentation and holds a poor prognosis due to emerging resistance to first-line platinum-based and second-line topotecan chemotherapy. The present investigation tested the antitumor activity of rovalpituzumab tesirine (ROVA-T), a cytotoxic anti-DLL3 drug conjugate, against two SCLC and a corresponding SCLC CTC cell line established from a ROVA-T-resistant patient to characterize the mechanism of recurrence. Two cell lines were established from an SCLC patient progressing under ROVA-T therapy and characterized with respect to chemosensitivity against this drug as well as against currently applied chemotherapeutics and for their delta-like 3 (DLL3) expression. The chemosensitivity assays demonstrate that most SCLC lines show IC50 values exceeding the ROVA-T in-vivo concentrations and that slow-growing cells and lines showing spheroidal growth or proliferation as corresponding circulating tumor cells (CTCs) exhibit higher resistance. Chemosensitivity of the cell lines is not correlated with DLL3 protein expression possibly due to toxicity of the free payload in tissue culture. The clinical trials and experimental results demonstrate that refractoriness to ROVA-T is linked to a low initial tumor expression of DLL3, loss of DLL3 expression, higher chemoresistance to ROVA-T and the putative formation of resistant spheroids by the SCLC cells.

Due to the neuroprotective role of the Na+/Ca2+ exchanger (NCX) isoforms NCX1 and NCX3, we synthesized novel benzodiazepinone derivatives of the unique NCX activator Neurounina-1, named compounds 1-19. The derivatives are characterized by a benzodiazepinonic nucleus linked to five- or six-membered cyclic amines via a methylene, ethylene, or acetyl spacer. The compounds have been screened on NCX1/NCX3 isoform activities by a high-throughput screening approach, and the most promising were characterized by patch-clamp electrophysiology and Fura-2AM video imaging. We identified two novel modulators of NCX: compound 4, inhibiting NCX1 reverse mode, and compound 14, enhancing NCX1 and NCX3 activity. Compound 1 displayed neuroprotection in two preclinical models of brain ischemia. The analysis of the conformational and steric features led to the identification of the molecular volume required for selective NCX1 activation for mixed NCX1/NCX3 activation or for NCX1 inhibition, providing the first prototypal model for the design of optimized isoform modulators.

The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. P2X4 activity is associated with neuropathic pain, vasodilation, and pulmonary secretion and is therefore of therapeutic interest. The structure-activity relationship of P2X4 antagonists is poorly understood. Here we elucidate the structure-activity of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) at human P2X4 by combining pharmacology, electrophysiology, molecular modeling, and medicinal chemistry. 5-BDBD antagonized P2X4 in a noncompetitive manner but lacked effect at human P2X2. Molecular modeling and site-directed mutagenesis suggested an allosteric binding site for 5-BDBD located between two subunits in the body region of P2X4, with M109, F178, Y300, and I312 on one subunit and R301 on the neighboring subunit as key residues involved in antagonist binding. The bromine group of 5-BDBD was redundant for the antagonist activity of 5-BDBD, although an interaction between the carbonyl group of 5-BDBD and R301 in P2X4 was associated with 5-BDBD activity. 5-BDBD could inhibit the closed channel but poorly inhibited the channel in the open/desensitizing state. We hypothesize that this is due to constriction of the allosteric site after transition from closed to open channel state. We propose that M109, F178, Y300, R301, and I312 are key residues for 5-BDBD binding; provide a structural explanation of how they contribute to 5-BDBD antagonism; and highlight that the limited action of 5-BDBD on open versus closed channels is due to a conformational change in the allosteric site. SIGNIFICANCE STATEMENT: Activity of P2X4 receptor is associated with neuropathic pain, inflammation, and vasodilatation. Molecular information regarding small-molecule interaction with P2X4 is very limited. Here, this study provides a structural explanation for the action of the small-molecule antagonist 5-BDBD at the human P2X4 receptor.

Introduction This open-label, phase 1–2 study evaluated the safety and efficacy of rovalpituzumab tesirine (Rova-T), an antibody-drug conjugate targeting DLL3, plus immune checkpoint inhibitors nivolumab plus or minus ipilimumab in previously treated extensive-stage SCLC (ES SCLC). Methods Patients with histologically or cytologically confirmed, previously treated (two or more lines of therapy) ES SCLC were enrolled into two cohorts. Cohort 1 received 0.3 mg/kg Rova-T (once every 6 wk for two cycles) plus 360 mg nivolumab (two 3-wk cycles beginning on week 4). Cohort 2 received the same dosage of Rova-T as cohort 1 plus 1 mg/kg nivolumab (four 3-wk cycles) and 1 mg/kg ipilimumab (beginning week 4). Both cohorts received 480 mg nivolumab every 4 weeks starting at week 10. Key objectives were to evaluate safety and tolerability and efficacy (per Response Evaluation Criteria in Solid Tumors version 1.1). The response-related results are based on centrally read data. Results A total of 42 patients received therapy: cohort 1, n = 30; cohort 2, n = 12. Overall, 43% received two or more previous lines of therapy. All patients experienced one or more treatment-emergent adverse event (TEAE); 41 patients reported AEs considered related to the study drug by the investigator. The most frequent TEAE was pleural effusion (n = 20, 48%); most common grade greater than or equal to 3 was anemia (n = 9, 21%). Three grade 5 TEAEs considered related to the study drug were reported (cohort 1): pneumonitis (n = 2), acute kidney injury (n = 1). The objective response rate was 30% (12 of 40): cohort 1, 27.6% (8 of 29); cohort 2, 36.4% (4 of 11); all partial responses. Conclusions Despite encouraging antitumor activity in previously treated ES SCLC, combination therapy with Rova-T and nivolumab plus or minus ipilimumab was not well tolerated at the dose levels and administration schedules evaluated.

Delta‐like canonical Notch ligand 3 (DLL3) is a member of the Delta/Serrate/Lag2 (DSL) Notch receptor ligand family and plays a crucial role in Notch signaling, which influences various cellular processes including differentiation, proliferation, survival, and apoptosis. DLL3 is expressed throughout the presomitic mesoderm and is localized to the rostral somatic compartments; mutations in DLL3 induce skeletal abnormalities such as spondylocostal dysostosis. Recently, DLL3 has attracted interest as a novel molecular target due to its high expression in neuroendocrine carcinoma of the lung. Moreover, a DLL3‐targeting Ab‐drug conjugate, rovalpituzumab tesirine (ROVA‐T), has been developed as a new treatment with proven antitumor activity. However, the development of ROVA‐T was suspended because of shorter overall survival compared to topotecan, the second‐line standard treatment. Thus, several studies on the mechanism and function of DLL3 in several malignancies are underway to find a new strategy for targeting DLL3. In this review, we discuss the roles of DLL3 in various malignancies and the future perspectives of DLL3‐related research, especially as a therapeutic target., Delta‐like canonical Notch ligand 3 (DLL3) plays a pivotal role in maintaining malignant growth and is related to poor prognosis, especially in relatively rare neuroendocrine subtypes.

The peripheral benzodiazepine receptor (TSPO/PBR) is highly conserved among different species but with perplexing biochemical functions. Multiple ligands of TSPO show commendable regulatory activities in lots of biological functions, such as neuro-protection, cholesterol transport, and so on. These researches support that TSPO may be a potential target for disease treatment and drug development. Previous studies have shown that its ligands benzodiazepines show a satisfactory effect on melanogenic promotion. However, the potential application of TSPO in drug development for pigmentary disorder needs further investigation. In this study, we confirmed the melanogenesis induction of TSPO ligand, Ro5-4864 in mouse melanoma cell lines, human skin tissue, and zebrafish embryos by inducing melanin synthesis and melanosome transport. Molecular genetics and pharmacological studies showed that TSPO deficiency did not affect melanin production in B16F10 cells and zebrafish embryos, nor did it affect the melanin promotion effect of Ro5-4864. Whether or not TSPO exists, the expression of lots of melanogenesis-related proteins, such as TYR, TRP-1, DCT, Mlph, and Rab27 was upregulated with the Ro5-4864 administration. These results indicated that Ro5-4864 induces melanogenesis in a TSPO-independent manner, which is inconsistent with previous research. This research is a reminder that we need to be very careful during target validation in drug development.

Ischemic stroke is the most serious disease that harms human beings. In principle, its treatment is to restore blood flow supply as soon as possible. However, after the blood flow is restored, it will lead to secondary brain injury, that is, ischemia-reperfusion injury. The mechanism of ischemia-reperfusion injury is very complicated. This study showed that P2X4 receptors in the pyramidal neurons of rat hippocampus were significantly upregulated in the early stage of ischemia-reperfusion injury. Neurons with high expression of P2X4 receptors are neurons that are undergoing apoptosis. Intraventricular injection of the P2X4 receptor antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) and PSB-12062 can partially block neuronal apoptosis, to promote the survival of neurons, indicating that ATP through P2X4 receptors is involved in the process of cerebral ischemia-reperfusion injury. Therefore, identifying the mechanism of neuronal degeneration induced by extracellular ATP via P2X4 receptors after ischemia-reperfusion will likely find new targets for the treatment of ischemia-reperfusion injury, and will provide a useful theoretical basis for the treatment of ischemia-reperfusion injury.

Cinazepam C19H14BrClN2O5, (“LevanaⓇ ІC”) a partial GABAA receptor agonist, and its active metabolite 3-hydroxyphenazepam C15H10BrClN2O2 were comparatively assessed in vitro using nerve terminals isolated from rat cortex (synaptosomes). At the presynaptic site, cinazepam (100 and 200 µM) facilitated synaptosomal transporter-mediated [3H]GABA uptake by enhancing both the initial rate and accumulation, and decreased the ambient level and transporter-mediated release of [3H]GABA. Whereas, 3-hydroxyphenazepam decreased the uptake and did not change the ambient synaptosomal level and transporter-mediated release of [3H]GABA. To exclude GABA transporter influence, NO-711, the transporter blocker, was applied and it was found that exocytotic release of [3H]GABA decreased, whereas tonic release of [3H]GABA was not changed in the presence of both cinazepam or 3-hydroxyphenazepam after treatment of synaptosomes with NO-711. In fluorimetric studies using potential- and pH-sensitive dyes rhodamine 6G and acridine orange, respectively, it was found that cinazepam hyperpolarized the synaptosomal plasma membrane, and increased synaptic vesicle acidification, whereas, 3-hydroxyphenazepam demonstrated opposite effects on these parameters. Therefore, action of cinazepam and its active metabolite 3-hydroxyphenazepam on GABAergic neurotransmission was different. Therapeutic effects of cinazepam can be associated with its ability to hyperpolarize the plasma membrane, to increase synaptic vesicle acidification and capacity of its active metabolite 3-hydroxyphenazepam to inhibit GABA transporter functioning.

Small cell lung cancer (SCLC) is a leading cause of cancer death worldwide, with few treatment options. Rovalpituzumab tesirine (Rova-T) is an antibody-drug conjugate that targets delta-like 3 on SCLC cells to deliver a cytotoxic payload directly to tumor cells. In this study, the cardiac safety profile of Rova-T was assessed by evaluating changes in QT interval, electrocardiogram (ECG) waveform, heart rate, and proarrhythmic adverse events (AEs) after treatment with Rova-T in patients with previously treated extensive-stage SCLC. Patients underwent ECG monitoring for 2 weeks after each of 2 i.v. infusions of 0.3 mg/kg Rova-T over 30 minutes, administered 6 weeks apart. Forty-six patients received at least one dose of Rova-T. At the geometric mean Rova-T maximum serum concentration of 7,940 ng/mL, ECG monitoring showed no significant changes in the Fridericia-corrected QT (QTcF) interval; the upper limit of the 2-sided 90% confidence interval did not exceed 10 msec for any time point. There were no clinically significant changes in QRS or PR intervals, ECG waveforms, or heart rate after Rova-T administration. All patients experienced a treatment-emergent AE (TEAE); 78% had a grade ≥ 3 TEAE, 59% had a serious TEAE, and 41% had a cardiac-related TEAE. The TEAEs that might signal proarrhythmia tendencies were uncommon. Confirmed partial responses were observed in 24% of patients. Based on the evaluation of ECG data collected in this study from patients treated with Rova-T at 0.3 mg/kg i.v. administered every 6 weeks, a QTcF effect of clinical concern can be excluded.

Protein arginine methyltransferase 6 (PRMT6) catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes, and is associated with multiple cancers. To date, a highly selective PRMT6 inhibitor has not been reported. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, (R)-2 (SGC6870). (R)-2 is a potent PRMT6 inhibitor (IC(50) = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and nonepigenetic targets. Notably, the crystal structure of the PRMT6−(R)-2 complex and kinetic studies revealed (R)-2 binds a unique, induced allosteric pocket. Additionally, (R)-2 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, (R)-2’s enantiomer, (S)-2 (SGC6870N), is inactive against PRMT6 and can be utilized as a negative control. Collectively, (R)-2 is a well-characterized PRMT6 chemical probe and a valuable tool for further investigating PRMT6 functions in health and disease.

DNA‐encoded chemical libraries (DECLs) are collections of chemical moieties individually coupled to distinctive DNA barcodes. Compounds can be displayed either at the end of a single DNA strand (i. e., single‐pharmacophore libraries) or at the extremities of two complementary DNA strands (i. e., dual‐pharmacophore libraries). In this work, we describe the use of a dual‐pharmacophore encoded self‐assembly chemical (ESAC) library for the affinity maturation of a known 4,5‐dihydrobenzodiazepinone ring (THBD) acetyl‐lysine (KAc) mimic for the cyclic‐AMP response element binding protein (CREB) binding protein (CREBBP or CBP) bromodomain. The new pair of fragments discovered from library selection showed a sub‐micromolar affinity for the CREBBP bromodomain in fluorescence polarization and ELISA assays, and selectivity against BRD4(1). © 2020 Wiley-VCH GmbH. ISSN:1860-7179 ISSN:1860-7187

Background and purpose Resurgence in the use of chloroquine as a potential treatment for COVID-19 has seen recent cases of fatal toxicity due to unintentional overdoses. Protocols for the management of poisoning recommend diazepam, although there are uncertainties in its pharmacology and efficacy in this context. The aim was to assess the effects of diazepam in experimental models of chloroquine cardiotoxicity. Experimental approach In vitro experiments involved cardiac tissues isolated from rats and incubated with chloroquine alone or in combination with diazepam. In vivo models of toxicity involved chloroquine administered intravenously to pentobarbitone-anaesthetised rats and rabbits. Randomised, controlled treatment studies in rats assessed diazepam, clonazepam and Ro5-4864 administered: (i) prior, (ii) during and (iii) after chloroquine and the effects of diazepam: (iv) at high dose, (v) in urethane-anaesthetised rats and (vi) co-administered with adrenaline. Key results Chloroquine decreased the developed tension of left atria, prolonged the effective refractory period of atria, ventricular tissue and right papillary muscles, and caused dose-dependent impairment of haemodynamic and electrocardiographic parameters. Cardiac arrhythmias indicated impairment of atrioventricular conduction. Studies (i), (ii) and (v) showed no differences between treatments and control. Diazepam increased heart rate in study (iv) and as with clonazepam also prolonged the QTc interval in study (iii). Combined administration of diazepam and adrenaline in study (vi) improved cardiac contractility but caused hypokalaemia. Conclusion and implications Neither diazepam nor other ligands for benzodiazepine binding sites protect against or attenuate chloroquine cardiotoxicity. However, diazepam may augment the effects of positive inotropes in reducing chloroquine cardiotoxicity. Linked articles This article is part of a themed issue on The Pharmacology of COVID-19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc.

In the course of screening lipopolysaccharide (LPS)-induced nitric oxide (NO) production inhibitors, two related benzodiazepine derivatives, cyclopenol and cyclopenin, were isolated from the extract of a deep marine-derived fungal strain, Aspergillus sp. SCSIOW2. Cyclopenol and cyclopenin inhibited the LPS-induced formation of NO and secretion of IL-6 in RAW264.7 cells at nontoxic concentrations. In terms of the mechanism underlying these effects, cyclopenol and cyclopenin were found to inhibit the upstream signal of NF-κB activation. These compounds also inhibited the expression of IL-1β, IL-6, and inducible nitric oxide synthase (iNOS) in mouse microglia cells, macrophages in the brain. In relation to the cause of Alzheimer's disease, amyloid-β-peptide is known to induce inflammation in the brain. Therefore, the present study investigated the ameliorative effects of these inhibitors on an in vivo Alzheimer's model using flies. Learning deficits were induced by the overexpression of amyloid-β42 in flies, and cyclopenin but not cyclopenol was found to rescue learning impairment. Therefore, novel anti-inflammatory activities of cyclopenin were identified, which may be useful as a candidate of anti-inflammatory agents for neurodegenerative diseases.

The effects of CHK1 inhibitor on cell cycle kinetics have not been fully investigated yet. In this study, we closely analyzed this kinetics using a CHK1 inhibitor (PF00477736) in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). This system allowed us to visualize cell cycle progression following CHK1 inhibitor treatment in real-time. FACS analysis showed that high levels of DNA damage as determined by γH2AX immunostaining was induced in S phase and that polyploid cells harboring the same levels of DNA damage appeared thereafter. Surprisingly, time-lapse imaging of Fucci fluorescence revealed that many cells entered M phase at once and exhibited prolonged mitosis; eventually progressing to G1 phase not accompanied by cytokinesis; this is an endomitosis-like event. Most of these cells then underwent S/G2 phases at least once, which corroborated the appearance of polyploid cells. However, a small fraction of cells with 2 N DNA content still remained 24 h after the treatment. When co-treated with MAD2 inhibitor, a core factor constituting spindle checkpoint, the 2 N DNA cell fraction disappeared and almost all cells exhibited endomitosis, leading to enhanced sensitivity. Detailed cell cycle analysis revealed that induction of an endomitosis-like event might be associated with CHK1 inhibitor-induced cell death in HeLa cells.

Prostate cancer (PCa) remains a leading cause of cancer-related deaths in American men and treatment options for metastatic PCa are limited. There is a critical need to identify new mechanisms that contribute to PCa progression, that distinguish benign from lethal disease, and that have potential for therapeutic targeting. P2X4 belongs to the P2 purinergic receptor family that is commonly upregulated in cancer and is associated with poorer outcomes. We observed P2X4 protein expression primarily in epithelial cells of the prostate, a subset of CD66+ neutrophils, and most CD68+ macrophages. Our analysis of tissue microarrays representing 491 PCa cases demonstrated significantly elevated P2X4 expression in cancer- compared with benign-tissue spots, in prostatic intraepithelial neoplasia, and in PCa with ERG positivity or with PTEN loss. High-level P2X4 expression in benign tissues was likewise associated with the development of metastasis after radical prostatectomy. Treatment with the P2X4-specific agonist cytidine 5'-triphosphate (CTP) increased Transwell migration and invasion of PC3, DU145, and CWR22Rv1 PCa cells. The P2X4 antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) resulted in a dose-dependent decrease in viability of PC3, DU145, LNCaP, CWR22Rv1, TRAMP-C2, Myc-CaP, BMPC1, and BMPC2 cells and decreased DU145 cell migration and invasion. Knockdown of P2X4 attenuated growth, migration, and invasion of PCa cells. Finally, knockdown of P2X4 in Myc-CaP cells resulted in significantly attenuated subcutaneous allograft growth in FVB/NJ mice. Collectively, these data strongly support a role for the P2X4 purinergic receptor in PCa aggressiveness and identify P2X4 as a candidate for therapeutic targeting. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Objective To investigate the histopathological effects of a peripheral benzodiazepine receptor agonist (Ro5-4864) on epidural fibrosis (EF) in an experimental study model (post-laminectomy) in rats. Methods A total of 32 albino Wistar rats were randomly divided into four equal groups (n = 8). In Group 1, no treatment was applied after laminectomy (control group). In Group 2, hemostasis was achieved after Laminectomy, and the surgical procedure was terminated by placing a 2-mm absorbable gelatin sponge dipped in saline into the epidural space. In Group 3, low-dose (4 mg/kg) Ro5-4864 was administered 30 minutes before the surgery. In Group 4, high-dose (8 mg/kg) Ro5-4864 was administered 30 minutes before the surgery. A histopathological examination was performed to evaluate arachnoidal invasion and EF. Results Our data revealed the EF was significantly reduced in rats treated with high-dose Ro5-4864 (Group 4) compared to the control and saline-soaked Spongostan groups (p = 0.000 and p = 0.006, respectively). There was no significant difference between the groups treated with high- and low-dose Ro5-4864. Arachnoidal invasion was not seen in any of the rats in the high-dose R05-4864 group. However, the arachnoidal invasion results did not significantly differ between the study groups (p = 0.052 = 0.05). Conclusions Our study showed that Ro5-4864 could be effective in reducing EF in rats after.

Small cell lung cancer (SCLC) is characterised by high relapse rates. Tumour-initiating cells (TICs) are responsible for drug resistance and recurrence of cancer. Rovalpituzumab tesirine (Rova-T), a potent humanised antibody–drug conjugate, selectively targets delta-like protein 3, which is highly expressed in SCLC TICs. The experimental drug CBL0137 (CBL) inhibits the histone chaperone FACT (facilitates chromatin transcription), which is required for the expression of transcription factors that are essential for TIC maintenance. Rova-T and CBL each target SCLC TICs as single agents. However, acquired or intrinsic resistance to single agents is a major problem in cancer. Therefore, we investigated the potential effect of combining Rova-T and CBL in SCLC to eradicate TICs more effectively. Our preclinical studies report a novel and highly translatable therapeutic strategy of dual targeting TICs using Rova-T in combination with CBL to potentially increase survival of SCLC patients.

The failure of highly active antiretroviral therapy (HAART) has been largely responsible for the existence of latent human immunodeficiency virus type 1 (HIV-1) reservoirs. The “shock and kill” strategy was confirmed to reactivate HIV-1 latent reservoirs by latency-reversing agents (LRAs) for accelerated HIV-1 clearance. However, a single LRA might be insufficient to induce HIV-1 reactivation from latency due to the complexity of the multiple signaling regulatory pathways that establish the HIV-1 latent reservoir. Therefore, combinations of LRAs or dual-mechanism LRAs are urgently needed to purge the latent reservoirs. We demonstrate here for the first time that a dual-target inhibitor with a specific suppressive effect on both BRD4 and TIP60, CPI-637, could reactivate latent HIV-1 in vitro by permitting Tat to bind positive transcription elongation factor b (P-TEFb) and assembling Tat-super-elongation complex (SEC) formation. In addition, CPI-637-mediated TIP60 downregulation further stimulated BRD4 dissociation from the HIV-1 long terminal repeat (LTR) promoter, allowing Tat to more effectively bind P-TEFb compared to BRD4 inhibition alone. Much more importantly, CPI-637 exerted a potent synergistic effect but alleviated global T cell activation and blocked viral spread to uninfected bystander CD4+ T cells with minimal cytotoxicity. Our results indicate that CPI-637 opens up the prospect of novel dual-target inhibitors for antagonizing HIV-1 latency and deserves further investigation for development as a promising LRA with a “shock and kill” strategy.

Cholecystokinin A receptor (CCKAR) belongs to family A G-protein-coupled receptors and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with a sulfated ligand and to couple with divergent G-protein subtypes, including Gs, Gi and Gq. However, the basis for G-protein coupling promiscuity and ligand recognition by CCKAR remains unknown. Here, we present three cryo-electron microscopy structures of sulfated CCK-8-activated CCKAR in complex with Gs, Gi and Gq heterotrimers, respectively. CCKAR presents a similar conformation in the three structures, whereas conformational differences in the ‘wavy hook’ of the Gα subunits and ICL3 of the receptor serve as determinants in G-protein coupling selectivity. Our findings provide a framework for understanding G-protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8., Cryo-EM structures of sulfated cholecystokinin 8 bound to the cholecystokinin A receptor in complex with Gs, Gi and Gq heterotrimers reveal structural determinants for G-protein coupling selectivity.

CREBBP (CBP/KAT3A) and its paralogue EP300 (KAT3B) are lysine acetyltransferases (KATs) that are essential for human development. They each comprise 10 domains through which they interact with >400 proteins, making them important transcriptional co-activators and key nodes in the human protein-protein interactome. The bromodomains of CREBBP and EP300 enable the binding of acetylated lysine residues from histones and a number of other important proteins, including p53, p73, E2F, and GATA1. Here, we report a work to develop a high-affinity, small-molecule ligand for the CREBBP and EP300 bromodomains [(-)-OXFBD05] that shows >100-fold selectivity over a representative member of the BET bromodomains, BRD4(1). Cellular studies using this ligand demonstrate that the inhibition of the CREBBP/EP300 bromodomain in HCT116 colon cancer cells results in lowered levels of c-Myc and a reduction in H3K18 and H3K27 acetylation. In hypoxia (

Background White matter injury (WMI) is a major neuropathological event associated with intracerebral hemorrhage (ICH). P2X purinoreceptor 4 (P2X4R) is a member of the P2X purine receptor family, which plays a crucial role in regulating WMI and neuroinflammation in central nervous system (CNS) diseases. Our study investigated the role of P2X4R in the WMI and the inflammatory response in mice, as well as the possible mechanism of action after ICH. Methods ICH was induced in mice via collagenase injection. Mice were treated with 5-BDBD and ANA-12 to inhibit P2X4R and tropomyosin-related kinase receptor B (TrkB), respectively. Immunostaining and quantitative polymerase chain reaction (qPCR) were performed to detect microglial phenotypes after the inhibition of P2X4R. Western blots (WB) and immunostaining were used to examine WMI and the underlying molecular mechanisms. Cylinder, corner turn, wire hanging, and forelimb placement tests were conducted to evaluate neurobehavioral function. Results After ICH, the protein levels of P2X4R were upregulated, especially on day 7 after ICH, and were mainly located in the microglia. The inhibition of P2X4R via 5-BDBD promoted neurofunctional recovery after ICH as well as the transformation of the pro-inflammatory microglia induced by ICH into an anti-inflammatory phenotype, and attenuated ICH-induced WMI. Furthermore, we found that TrkB blockage can reverse the protective effects of WMI as well as neuroprotection after 5-BDBD treatment. This result indicates that P2X4R plays a crucial role in regulating WMI and neuroinflammation and that P2X4R inhibition may benefit patients with ICH. Conclusions Our results demonstrated that P2X4R contributes to WMI by polarizing microglia into a pro-inflammatory phenotype after ICH. Furthermore, the inhibition of P2X4R promoted pro-inflammatory microglia polarization into an anti-inflammatory phenotype, enhanced brain-derived neurotrophic factor (BDNF) production, and through the BDNF/TrkB pathway, attenuated WMI and improved neurological function. Therefore, the regulation of P2X4R activation may be beneficial for the reducing of ICH-induced brain injury.

Purinergic signaling plays a major role in T cell activation leading to IL-2 production and proliferation. However, it is unclear whether purinergic signaling contributes to the differentiation and activation of effector T cells. In this study, we found that the purinergic receptor P2X4 was associated with human Th17 cells but not with Th1 cells. Inhibition of P2X4 receptor with the specific antagonist 5-BDBD and small interfering RNA inhibited the development of Th17 cells and the production of IL-17 by effector Th17 cells stimulated via the CD3/CD28 pathway. Our results showed that P2X4 was required for the expression of retinoic acid-related orphan receptor C, which is the master regulator of Th17 cells. In contrast, inhibition of P2X4 receptor had no effect on Th1 cells and on the production of IFN-γ and it did not affect the expression of the transcription factor T-bet (T-box transcription factor). Furthermore, inhibition of P2X4 receptor reduced the production of IL-17 but not of IFN-γ by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis. In contrast to P2X4, inhibition of P2X7 and P2Y11 receptors had no effects on Th17 and Th1 cell activation. Finally, treatment with the P2X4 receptor antagonist 5-BDBD reduced the severity of collagen-induced arthritis in mice by inhibiting Th17 cell expansion and activation. Our findings provide novel insights into the role of purinergic signaling in T cell activation and identify a critical role for the purinergic receptor P2X4 in Th17 activation and in autoimmune arthritis.

The discovery and optimization of a novel series of PTHR1 antagonists are described. Starting from known PTHR1 antagonists, we identified more potent 1,4-benzodiazepin-2-one derivatives by means of a scaffold-hopping approach. The representative compound 23 (DS08210767) exhibited nanomolar-level PTHR1 antagonist activity and potential oral bioavailability in a pharmacokinetic study.

Abnormal growth of the intimal layer of blood vessels (neointima formation) contributes to the progression of atherosclerosis and in-stent restenosis. Recent evidence shows that the 18-kDa translocator protein (TSPO), a mitochondrial membrane protein, is involved in diverse cardiovascular diseases. In this study we investigated the role of endogenous TSPO in neointima formation after angioplasty in vitro and in vivo. We established a vascular injury model in vitro by using platelet-derived growth factor-BB (PDGF-BB) to stimulate rat thoracic aortic smooth muscle cells (A10 cells). We found that treatment with PDGF-BB (1–20 ng/mL) dose-dependently increased TSPO expression in A10 cells, which was blocked in the presence of PKC inhibitor or MAPK inhibitor. Overexpression of TSPO significantly promoted the proliferation and migration in A10 cells, whereas downregulation of TSPO expression by siRNA or treatment with TSPO ligands PK11195 or Ro5-4864 (10 4 nM) produced the opposite effects. Furthermore, we found that PK11195 (10−10 4 nM) dose-dependently activated AMPK in A10 cells. PK11195-induced inhibition on the proliferation and migration of PDGF-BB-treated A10 cells were abolished by compound C (an AMPK-specific inhibitor, 10 3 nM). In rats with balloon-injured carotid arteries, TSPO expression was markedly upregulated in the carotid arteries. Administration of PK11195 (3 mg/kg every 3 days, ip), starting from the initial balloon injury and lasting for 2 weeks, greatly attenuated carotid neointima formation by suppressing balloon injury-induced phenotype switching of VSMCs (increased α-SMA expression). These results suggest that TSPO is a vascular injury-response molecule that promotes VSMC proliferation and migration and is responsible for the neointima formation after vascular injury, which provides a novel therapeutic target for various cardiovascular diseases including atherosclerosis and restenosis.

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.

Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers. Despite high rates of response to first-line chemotherapy and radiotherapy, patients with extensive-stage disease eventually relapse, and very few patients survive more than 5 years from diagnosis. Treatment options for recurrent or refractory disease are limited, and the treatments that do exist are associated with significant treatment-related toxicities. Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand that is highly expressed in SCLC and other neuroendocrine tumors but minimally expressed in normal tissues. It is therefore being explored as a potential therapeutic target in SCLC. Here, we review the preclinical and clinical evidence for targeting DLL3 in SCLC and discuss several DLL3-specific therapies being developed for the treatment of SCLC: the antibody-drug conjugate rovalpituzumab tesirine, the bispecific T cell engager immuno-oncology therapy AMG 757, and the chimeric antigen receptor T cell therapy AMG 119.

P2X receptors (P2XRs) are a family of ATP-gated ionic channels that are expressed in numerous excitable and non-excitable cells. Despite the great advance on the structure and function of these receptors in the last decades, there is still lack of specific and potent antagonists for P2XRs subtypes, especially for the P2X4R. Here, we studied in detail the effect of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) on ATP-induced currents mediated by the rat P2X4R and compared its specificity among another rat P2XRs. We found that 5-BDBD is a potent P2X4R antagonist, with an IC50 of 0.75 μM when applied for 2 min prior and during ATP stimulation. Moreover, at 10 μM concentration, 5-BDBD did not affect the ATP-induced P2X2aR, P2X2bR, and P2X7R current amplitude or the pattern of receptor desensitization. However, at 10 μM concentration but not 0.75 μM 5-BDBD inhibited the P2X1R and P2X3R-gated currents by 13 and 35% respectively. Moreover, we studied the effects of 5-BDBD in long-term potentiation experiments performed in rat hippocampal slices, finding this antagonist can partially decrease LTP, a response that is believed to be mediated in part by endogenous P2X4Rs. These results indicate that 5-BDBD could be used to study the endogenous effects of the P2X4R in the central nervous system and this antagonist can discriminate between P2X4R and other P2XRs, when they are co-expressed in the same tissue.

Latency-reversing agents (LRAs) are considered a potential strategy for curing cells of HIV-1 infection. Certain protein kinase C (PKC) activators have been previously reported to be LRAs because they can reverse HIV latency. In the present study, we examined the activities of a panel of benzolactam derivatives against cells latently infected with HIV. Using determination of p24 antigen in cell supernatants or altered intracellular GFP expression to measure HIV reactivation from latently infected cells along with a cytotoxicity assay, we found that some of the compounds exhibited latency-reversing activity, which was followed by enhanced release of HIV particles from the cells. One derivative, BL-V8-310, displayed activity in ACH-2 and J-Lat cells latently infected with HIV at a concentration of 10 nm or higher, which was superior to the activity of another highly active PKC activator, prostratin. These results were confirmed with peripheral blood cells from HIV-infected patients. We also found that these drugs up-regulate the expression of caspase 3 and enhance apoptosis specifically in latently HIV-infected cells. Moreover, combining BL-V8-310 with a bromodomain-containing 4 (BRD4) inhibitor, JQ1, not only enhanced HIV latency-reversing activity, but also reduced the effect on cytotoxic cytokine secretion from CD4+ T-cells induced by BL-V8-310 alone. Our results suggest that BL-V8-310 and its related benzolactam derivatives are potential LRA lead compounds that are effective in reversing HIV latency and reducing viral reservoirs in HIV-positive individuals with few adverse effects.