Structural basis of human monocarboxylate transporter 1 inhibition by anti-cancer drug candidates.

Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer treatment because of their enhanced expression in various tumors. Here, we report five cryo-EM structures, at resolutions of 3.0-3.3 Å, of human MCT1 bound to lactate or inhibitors in the presence of Basigin-2, a single transmembrane segment (TM)-containing chaperon. MCT1 exhibits similar outward-open conformations when complexed with lactate or the inhibitors BAY-8002 and AZD3965. In the presence of the inhibitor 7ACC2 or with the neutralization of the proton-coupling residue Asp309 by Asn, similar inward-open structures were captured. Complemented by structural-guided biochemical analyses, our studies reveal the substrate binding and transport mechanism of MCTs, elucidate the mode of action of three anti-cancer drug candidates, and identify the determinants for subtype-specific sensitivities to AZD3965 by MCT1 and MCT4. These findings lay out an important framework for structure-guided drug discovery targeting MCTs.

Energy status dictates PD-L1 protein abundance and anti-tumor immunity to enable checkpoint blockade.

Aberrant energy status contributes to multiple metabolic diseases, including obesity, diabetes, and cancer, but the underlying mechanism remains elusive. Here, we report that ketogenic-diet-induced changes in energy status enhance the efficacy of anti-CTLA-4 immunotherapy by decreasing PD-L1 protein levels and increasing expression of type-I interferon (IFN) and antigen presentation genes. Mechanistically, energy deprivation activates AMP-activated protein kinase (AMPK), which in turn, phosphorylates PD-L1 on Ser283, thereby disrupting its interaction with CMTM4 and subsequently triggering PD-L1 degradation. In addition, AMPK phosphorylates EZH2, which disrupts PRC2 function, leading to enhanced IFNs and antigen presentation gene expression. Through these mechanisms, AMPK agonists or ketogenic diets enhance the efficacy of anti-CTLA-4 immunotherapy and improve the overall survival rate in syngeneic mouse tumor models. Our findings reveal a pivotal role for AMPK in regulating the immune response to immune-checkpoint blockade and advocate for combining ketogenic diets or AMPK agonists with anti-CTLA4 immunotherapy to combat cancer.

Inhibition of retrograde transport protects mice from lethal ricin challenge.

Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.

PIEZO1 targeting in macrophages boosts phagocytic activity and foam cell apoptosis in atherosclerosis.

The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE-/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis.

Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques.

HDLs carry sphingosine-1-phosphate (S1P) and stimulate signaling pathways in different cells including macrophages and endothelial cells, involved in atherosclerotic plaque development. HDL signaling via S1P relies on the HDL receptor scavenger receptor class B, type I (SR-B1) and the sphingosine-1-phosphate receptor 1 (S1PR1), which interact when both are heterologously overexpressed in the HEK293 cell line. In this study, we set out to test if SR-B1 and S1PR1 interacted in primary murine macrophages in culture and atherosclerotic plaques. We used knock-in mice that endogenously expressed S1PR1 tagged with eGFP-(S1pr1eGFP/eGFP mice), combined with proximity ligation analysis to demonstrate that HDL stimulates the physical interaction between SR-B1 and S1PR1 in primary macrophages, that this is dependent on HDL-associated S1P and can be blocked by an inhibitor of SR-B1's lipid transfer activity or an antagonist of S1PR1. We also demonstrate that a synthetic S1PR1-selective agonist, SEW2871, stimulates the interaction between SR-B1 and S1PR1 and that this was also blocked by an inhibitor of SR-B1's lipid transport activity. Furthermore, we detected abundant SR-B1/S1PR1 complexes in atherosclerotic plaques of S1pr1eGFP/eGFP mice that also lacked apolipoprotein E. Treatment of mice with the S1PR1 antagonist, Ex26, for 12 h disrupted the SR-B1-S1PR1 interaction in atherosclerotic plaques. These findings demonstrate that SR-B1 and S1PR1 form ligand-dependent complexes both in cultured primary macrophages and within atherosclerotic plaques in mice and provide mechanistic insight into how SR-B1 and S1PR1 participate in mediating HDL signaling to activate atheroprotective responses in macrophages.

Effects of the Thiol Methyltransferase Inhibitor (±)-2,3-Dichloro-α-Methylbenzylamine (DCMB) on the Pharmacokinetics and Metabolism of Vicagrel in Rats.

P2Y12 receptor inhibitors are commonly used in clinical antiplatelet therapy, typically alongside other medications. Vicagrel, a promising P2Y12 receptor inhibitor, has submitted a new drug marketing application to the United States Food and Drug Administration. Its primary metabolites and some metabolic pathways are identical to those of clopidogrel. The aim of this study was to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine (DCMB) on the metabolism and pharmacokinetics of vicagrel. In vitro incubation with human and rat liver microsomes revealed that DCMB significantly inhibited the methylation of vicagrel's thiol metabolite M15-1. Rats were orally administered 6 mg/kg [14C]vicagrel (100 µCi/kg) 1 hour after peritoneal injection with or without DCMB (80 mg/kg). Compared with the control group, the plasma of DCMB-pretreated rats exhibited maximum plasma concentration (C max) decrease and time to reach C max (T max) delay for all vicagrel-related substances, the methylation product of the thiol metabolite (M9-2), and the derivatization product of the active thiol metabolite (MP-M15-2). However, no significant changes in area under the curve (AUC) or half-life (t 1/2) were observed. DCMB had negligible effect on the total radiological recovery of vicagrel within 72 hours, although the rate of vicagrel excretion slowed down within 48 hours. DCMB had a negligible impact on the metabolic pathway of vicagrel. Overall, the present study found that DCMB did not significantly affect the total exposure, metabolic pathways, metabolite profiles, or total excretion rates of vicagrel-related metabolites in rats, but led to C max decrease, T max delay, and slower excretion rate within 48 hours. SIGNIFICANCE STATEMENT: This study used liquid chromatography-tandem mass spectrometry combined with radiolabeling technology to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine on the absorption, metabolism, and excretion of vicagrel in rats. This work helps to better understand the in vivo metabolism of active thiol metabolites of P2Y12 inhibitors such as clopidogrel, vicagrel, etc.

REV-ERBα agonist SR10067 attenuates Th2 cytokine-mediated barrier dysfunction in human bronchial epithelial cells.

Allergens and Th2 cytokines affect the homeostatic environment in the airways, leading to increased mucus production by goblet cells associated with altered adherens junctional complex (AJC) and tight junction (TJ) proteins responsible for maintaining epithelial barrier function. Circadian clock-dependent regulatory mechanisms such as inflammation and epithelial barrier function are gaining more attention due to their therapeutic potential against allergic inflammatory lung diseases. Currently, there are no studies to support whether REV-ERBα activation can attenuate Th2 cytokine-induced epithelial barrier dysfunction in human bronchial epithelial cells. We hypothesized that Th2 cytokine-induced epithelial barrier dysfunction may be protected by activating REV-ERBα. Treatment with Th2 cytokines or HDM significantly reduced the cell impedance, as confirmed by transepithelial electrical resistance (TEER). However, pre-treatment with SR10067 attenuated Th2 cytokine-induced barrier dysfunction, such as decreased permeability, improved TEER, localization of AJC and TJ proteins, and mRNA and protein levels of selected epithelial barrier and circadian clock targets. Overall, we showed for the first time that REV-ERBα activation regulates altered epithelial barrier function that may have direct implications for the treatment of asthma and other allergic diseases.

Comparative Efficacy of Dopamine Partial Agonists by Doses for Treatment-Resistant Depression: A Systematic Review and Dose-Response Model-Based Network Meta-analysis.

BACKGROUND: The augmentative antidepressant effects of dopamine partial agonists (aripiprazole, brexpiprazole, and cariprazine) for treatment-resistant depression have been compared in a previous network meta-analysis. However, the comparative efficacy of the dose-responses of these drugs remains unclear. Therefore, we aimed to estimate the dose-response relationships and compare the effects of each dopamine partial agonist doses. METHODS: We conducted a systematic review of the Cochrane Library, PubMed, CINHAL, and ClinicalTrials.gov databases until January 1, 2023. Double-blind, randomized, placebo-controlled trials evaluating aripiprazole, brexpiprazole, and cariprazine for treatment-resistant depression were included. A random-effect dose-response model-based network meta-analysis was conducted. This study was registered in PROSPERO (CRD42023393035). RESULTS: The maximum effective doses were 5.5 mg for aripiprazole, 1.6 mg for brexpiprazole, and 1.5 mg for cariprazine, respectively. Although all doses of the 3 drugs were significantly more effective than placebo, aripiprazole ranging from 5.5 to 12.5 mg was significantly more effective than brexpiprazole 0.5 mg and cariprazine ranging from 0.5 to 1 mg. Moreover, aripiprazole ranging from 7.5 to 12.5 mg was significantly more effective than all doses of cariprazine. In addition, brexpiprazole ranging from 1 to 3 mg was significantly more effective than cariprazine 0.5 mg and brexpiprazole ranging from 1.6 to 2.5 mg was significantly superior to cariprazine 1 mg. There were no doses at which brexpiprazole overcame aripiprazole, and cariprazine overcame aripiprazole or brexpiprazole. CONCLUSIONS: Aripiprazole, brexpiprazole, and cariprazine may be effective in treatment-resistant depression in that order, with the maximum effective doses at 5.5 mg, 1.6 mg, and 1.5 mg, respectively.

Domino Reaction Protocol to Synthesize Benzothiophene-Derived Chemical Entities as Potential Therapeutic Agents.

An efficient synthesis of 3-amino-2-formyl-functionalized benzothiophenes by a domino reaction protocol and their use to synthesize a library of novel scaffolds have been reported. Reactions of ketones and 1,3-diones with these amino aldehyde derivatives formed a series of benzothieno[3,2-b]pyridine and 3,4-dihydro-2H-benzothiopheno[3,2-b]quinolin-1-one, respectively. A plausible mechanism for the formation of fused pyridine derivatives by the Friedlander reaction has been elucidated by density functional theory (DFT) calculations. Furthermore, hydrazones were obtained by reacting the aldehyde functional group of benzothiophenes with different hydrazine derivatives. Preliminary screening of these compounds against several bacterial strains and cancer cell lines led to the discovery of several hit molecules. Hydrazone and benzothieno[3,2-b]pyridine derivatives are potent cytotoxic and antibacterial agents, respectively. One of the potent compounds effected ∼97% growth inhibition of the LOX IMVI cell line at 10 µM concentration.

Discovery of 5-phenyl-3-ureidothiophene-2-carboxamides as protective agents for ALS patient iPSC-derived motor neurons.

We discovered novel neuroprotective compounds by phenotypic screening using SOD1-mutant amyotrophic lateral sclerosis (ALS) patient induced pluripotent stem cell (iPSC)-derived motor neurons. Mechanistic analysis showed that the protective effect of initial hit compound 1 was likely due to the inhibition of MAP4Ks, including MAP4K4, a member of the MAP4K kinase family. Structural transformation led to compound 15f, which showed improved MAP4K4 inhibitory activity and superior neuroprotective effects compared to 1 in motor neurons. The results suggest that structural optimization based on MAP4K4 inhibitory activity might improve the neuroprotective effect of this series of compounds.

Discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for treating cancers with microsatellite instability.

Microsatellite instability (MSI) is a hypermutable condition caused by DNA mismatch repair system defects, contributing to the development of various cancer types. Recent research has identified Werner syndrome ATP-dependent helicase (WRN) as a promising synthetic lethal target for MSI cancers. Herein, we report the first discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for MSI cancer therapy. Initial computational analysis and biological evaluation identified a new scaffold for a WRN inhibitor. Subsequent SAR study led to the discovery of a highly potent WRN inhibitor. Furthermore, we demonstrated that the optimal compound induced DNA damage and apoptotic cell death in MSI cancer cells by inhibiting WRN. This study provides a new pharmacophore for WRN inhibitors, emphasizing their therapeutic potential for MSI cancers.

Regulation of exercise ability and glycolipid metabolism by synthetic SR9009 analogues as new REV-ERB-α agonists.

SR9009 is an activator of REV-ERBs with diverse biological activities, including improving exercise tolerance and controlling skeletal muscle mass. To optimise the carbamate motif of SR9009, analogues of SR9009 were synthesised and evaluated. All of them showed REV-ERB-α agonist activities. Among them, 5a, 5f, 5 g, 5m, and 5p showed potencies equivalent to or slightly higher than the potency of SR9009 in vitro. These data indicate that the halogenated benzyl group is an indispensable active group in these compounds. 5m, 5p and SR9009 improved exercise tolerance in normal mice in vivo. Additionally, in hyperlipidemic mice, 5m and 5p not only improved exercise tolerance but also lowered blood lipid levels. 5m and 5p displayed stronger hypoglycaemic activity than SR9009.

Synthesis and biological studies of 2-aminothiophene derivatives as positive allosteric modulators of glucagon-like peptide 1 receptor.

As a step toward the development of novel small-molecule positive allosteric modulators (PAMs) of glucagon-like peptide 1 receptor (GLP-1R) for the treatment of type 2 diabetes, obesity, and heart diseases, we discovered a novel 2-amino-thiophene (2-AT) based lead compound bearing an ethyl 3-carboxylate appendage. In this work, we report the syntheses and biological studies of more than forty 2-AT analogs, that have revealed a 2-aminothiophene-3-arylketone analogue 7 (MW 299) showing approximately a 2-fold increase in insulin secretion at 5 µM when combined with the GLP-1 peptide at 10 nM. In vivo studies using CD1 mice at a dose of 10 mg/kg, clearly demonstrated that the blood plasma glucose level was lowered by 50% after 60 min. Co-treatment of 7 with sitagliptin, an inhibitor of GLP-1 degrading enzyme Dipeptidyl Peptidase IV, further confirmed 7 to be an effective PAM of GLP-1R. The small molecular weight and demonstrated allosteric modulating properties of these compound series, show the potential of these scaffolds for future drug development.

Synthesis and photodynamic activity of novel thieno[3,2-b]thiophene fused BODIPYs with good bio-solubility and anti-aggregation effect.

To discover new photosensitizers with long wavelength UV-visible absorption, high efficiency, and low side effects for photodynamic therapy, here, a series of novel thieno[3,2-b]thiophene-fused BODIPY derivatives were designed, synthesized and characterized. These compounds had a distinct absorption band at 640-680 nm, fluorescence emission at 650-760 nm, and good solubility with anti-aggregation effects. These new compounds possessed obvious singlet oxygen generation ability and photodynamic anti-Eca-109 cancer cells activities in vitro. Among them, compound II4 could be well uptaked by Eca-109 cells, and result in the apoptosis after laser irradiation, and have outstanding photodynamic efficiency both in vitro and in vivo. Therefore, II4 could be considered as a potential photosensitizer drug candidate for PDT and photo-imaging.

New thiophene-1,3,4-oxadiazole-thiazolidine-2,4-dione hybrids: Synthesis, MCF-7 inhibition and binding studies.

Two synthetic methods were proposed for the preparation of a new series of thiophene-1,3,4-oxadiazole-thiazolidine-2,4-dione hybrids (TOT-1 to 15) and their structures were elucidated based on spectral data. Studies on cytotoxicity, ROS, cellular uptake and interactions of TOT-14 with calf thymus DNA were carried out. Anticancer activity of compounds, TOT-1 to 15 on breast cancer (MCF-7) cell lines was investigated. The IC50 values for the standard, epirubicin hydrochloride and TOT-12, 13, 14 and 15 were found to be 6.78, 5.52, 6.53, 4.83 and 5.57 µg/mL, respectively. Notably, TOT-14 exhibited a remarkable antiproliferative activity with a strikingly selective inhibitory effect compared to standard. This specific selectivity could be attributed to the synergistic effect of increased cellular uptake and generation of higher ROS in cancer cells after irradiation. The binding constant of 4.25 x 103 M-1 indicated the moderate interaction between TOT-14 and ct-DNA. The docking score of TOT derivativeswas substantially identical to the docking score of epirubicin hydrochloride. The designed molecules complied with the requirements for drug-likeness and ADME.

Blocking potential metabolic sites on NAT to improve its safety profile while retaining the pharmacological profile.

The number of opioid-related overdose deaths and individuals that have suffered from opioid use disorders have significantly increased over the last 30 years. FDA approved maintenance therapies to treat opioid use disorder may successfully curb drug craving and prevent relapse but harbor adverse effects that reduce patient compliance. This has created a need for new chemical entities with improved patient experience. Previously our group reported a novel lead compound, NAT, a mu-opioid receptor antagonist that potently antagonized the antinociception of morphine and showed significant blood-brain barrier permeability. However, NAT belongs to thiophene containing compounds which are known structural alerts for potential oxidative metabolism. To overcome this, 15 NAT derivatives with various substituents at the 5'-position of the thiophene ring were designed and their structure-activity relationships were studied. These derivatives were characterized for their binding affinity, selectivity, and functional activity at the mu opioid receptor and assessed for their ability to antagonize the antinociceptive effects of morphine in vivo. Compound 12 showed retention of the basic pharmacological attributes of NAT while improving the withdrawal effects that were experienced in opioid-dependent mice. Further studies will be conducted to fully characterize compound 12 to examine whether it would serve as a new lead for opioid use disorder treatment and management.

Identification of novel ureido benzothiophenes as dual VEGFR-2/EGFR anticancer agents.

Presently, dual-targeting by a single small molecule stands out as a fruitful cancer-fighting strategy. Joining the global effort to fight cancer, a leading cause of death worldwide, we report in this study a novel set for benzothiophene-based aryl urea derivatives as potential anti-proliferative candidates endowed with dual VEGFR-2/EGFR inhibitory activities. The prepared ureido benzothiophenes 6a-r have been evaluated for their anticancer action on a panel of tumor cell lines, namely PanC-1, MCF-7, and HepG2 cells. Most newly synthesized benzo[b]thiophene ureas disclosed effective cytotoxic activities against the examined cancer cell lines. In particular, compound 6q, with an appended 4-trifluoromethoxy group on the terminal phenyl ring, exhibited the most significant cytotoxic activity in MCF-7 with IC50 3.86 ± 0.72 ug/mL; IC50 of 3.65 ± 0.18 ug/ml in PanC-1 cell line and an IC50 of 4.78 ± 0.06 ug/ml in HepG2. After that, derivatives that exhibited the most potent cytotoxic activities (6g, 6j, 6q, and 6r) were further evaluated as VEGFR-2 and EGFR inhibitors. Fortunately, they displayed low nanomolar IC50 values against both enzymes, where compound 6q emerged to possess superior inhibitory effects towards both EGFR and VEGFR-2 with IC50 46.6 nM and 11.3 nM simultaneously compared to the reference medications Erlotinib and Sorafenib, respectively. The docked structure of 6q within the catalytic region of VEGFR-2 and EGFR kinases was acquired and studied so that we could investigate potential binding mechanisms for the target ureido benzothiophenes. Hence, the benzothiophene-based aryl urea scaffold has great potential for advancing the development of highly effective dual inhibitors targeting both EGFR and VEGFR-2, which can serve as effective candidates for anticancer therapy.

Discovery of a potent Gilteritinib-based FLT3-PROTAC degrader for the treatment of Acute myeloid leukemia.

Fms-like tyrosine receptor kinase 3 (FLT3) proteolysis targeting chimeras (PROTACs) emerge as a promising approach to overcome the limitations of FLT3 inhibitors, while the development of orally bioavailable FLT3-PROTACs faces great challenges. Here, we report the rational design and evaluation of a series of Gilteritinib-based FLT3-PROTACs. Among them, B3-2 exhibited the strongest antiproliferative activity against FLT3-ITD mutant AML cells, and significantly induced FLT3-ITD protein degradation. Mechanistic investigations demonstrated that B3-2 induced FLT3-ITD degradation in a ubiquitin-proteasome-dependent manner. More importantly, B3-2 exhibited an oral bioavailability of 5.65%, and oral administration of B3-2 showed good antitumor activity in MV-4-11 xenograft models. Furthermore, B3-2 showed strong antiproliferative activity against FLT3 resistant mutations, highlighting its potential in overcoming drug resistance.

Discovery of novel thiophene-3-carboxamide derivatives as potential VEGFR-2 inhibitors with anti-angiogenic properties.

VEGFR-2 is an attractive target for the development of anti-tumor drugs and plays a crucial role in tumor angiogenesis. This study reports a series of novel thiophene-3-carboxamide derivatives based on PAN-90806 as VEGFR-2 inhibitors, among which compound 14d exhibits excellent anti-proliferative activity against HCT116, MCF7, PC3, and A549 cell lines, and has effective VEGFR-2 inhibitory activity with an IC50 value of 191.1 nM. Additionally, CETSA results indicated that VEGFR-2 was a relevant target of compound 14d in the cell lines, and compound 14d could also inhibit VEGFR-2 protein phosphorylation in A549 cell line. Furthermore, compound 14d inhibited colony formation, cell migration, and HUVECs tube formation in a dose-dependent manner. The mechanism by which 14d induced cancer cell death involves blocking the cell cycle, increasing ROS production, inducing apoptosis, and dose-dependently reducing the levels of phosphorylated ERK and MEK. Molecular docking and molecular dynamics simulations had shown that compound 14d could stably bind to the active site of VEGFR-2. These results confirmed that compound 14d might be a promising lead compound for anti-angiogenesis.

Synthesis, biological evaluation, and molecular docking studies of novel N-substituted piperazine-tethered thiophene-3-carboxamide selenides as potent antiproliferative agents with EGFR kinase inhibitory activity.

In the context of structural investigation and optimization of various potential EGFR inhibitors, a novel series of asymmetrical piperazine-tethered trisubstituted thiophene-3-carboxamide selenide derivatives were synthesized and evaluated for their antiproliferative potential against selected human cancer cell lines. These derivatives, built based on a previously identified hit molecule, were synthesized via multiple-step reactions, including optimization of the C-Se cross-coupling reaction. Two compounds, 17i and 18i, displayed significant cytotoxicity (IC50 value: 4.82 ± 0.80 µM and 1.43 ± 0.08 µM) against HCT116 and A549 cancer cell lines, respectively. Quantitative analysis of apoptotic stages using Annexin V-FITC/PI double staining validated their apoptotic potential. Further, compound 18i demonstrated a remarkable inhibition of EGFR kinase, with an IC50 concentration of 42.3 nM. The lead compound 18i, with remarkable in vitro cytotoxicity, apoptosis induction capability, and EGFR inhibition, emerges as a promising candidate for anticancer therapy.