Class Of Inhibitors Research Articles

(Heteroarylmethyl)benzoic Acids as a New Class of Bacterial Cystathionine γ-Lyase Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling.

Antibiotic resistance is one of the most serious global health threats. Therefore, there is a need to develop antimicrobial agents with new mechanisms of action. Targeting of bacterial cystathionine γ-lyase (bCSE), an enzyme essential for bacterial survival, is a promising approach to overcome antibiotic resistance. Here, we described a series of (heteroarylmethyl)benzoic acid derivatives and evaluated their ability to inhibit bCSE or its human ortholog hCSE using known bCSE inhibitor NL2 as a lead compound. Derivatives bearing the 6-bromoindole group proved to be the most active, with IC50 values in the midmicromolar range, and highly selective for bCSE over hCSE. Furthermore, none of these compounds showed significant toxicity to HEK293T cells. The obtained data were rationalized by ligand-based and structure-based molecular modeling analyses. The most active compounds were also found to be an effective adjunct to several widely used antibacterial agents against clinically relevant antibiotic-resistant strains of such bacteria as Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The most potent compounds, 3h and 3i, also showed a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. Finally, compound 3i manifested potentiating activity in pneumonia, sepsis, and infected-wound in vivo models.

Bio-based ionic liquid as a corrosion inhibitor for mild steel in 5% HCl solution: Experimental and theoretical investigation

In pickling conditions, corrosion inhibitors are commonly used to mitigate the corrosive effects of the acidic environment on metal surfaces. Green corrosion inhibitors are substances that provide corrosion protection while being environmentally friendly, often derived from renewable resources or having minimal environmental impact. Ionic liquids are a class of green corrosion inhibitors that have gained attention for their potential in corrosion protection due to their unique properties. In this study, we synthesized a bio-based choline hexanoate ionic liquid (referred to as ChH) and used FT-IR and 1H NMR spectroscopy to elucidate its structure. Further the effectiveness of ChH IL was examined in preventing MS corrosion in 5% HCl at 20–80 °C. The corrosion behavior of MS was examined using weight loss, potentiodynamic polarization, and EIS measurements. Increasing inhibitor concentration and temperatures improved the inhibition performance. The highest efficiency i.e., 98.16% was observed at 3.3 × 10−3 M of ChH at 60 °C. To verify the adsorption of ChH on the MS further investigations were done using FT-IR, Raman, and XPS analysis.

Design, synthesis, molecular docking study, and α-glucosidase inhibitory evaluation of novel hydrazide–hydrazone derivatives of 3,4-dihydroxyphenylacetic acid

A series of novel Schiff base derivatives (1–28) of 3,4-dihydroxyphenylacetic acid were synthesized in a multi-step reaction. All the synthesized Schiff bases were obtained in high yields and their structures were determined by 1HNMR, 13CNMR, and HR-ESI–MS spectroscopy. Except for compounds 22, 26, 27, and 28, all derivatives show excellent to moderate α-glucosidase inhibition. Compounds 5 (IC50 = 12.84 ± 0.52 µM), 4 (IC50 = 13.64 ± 0.58 µM), 12 (IC50 = 15.73 ± 0.71 µM), 13 (IC50 = 16.62 ± 0.47 µM), 15 (IC50 = 17.40 ± 0.74 µM), 3 (IC50 = 18.45 ± 1.21 µM), 7 (IC50 = 19.68 ± 0.82 µM), and 2 (IC50 = 20.35 ± 1.27 µM) shows outstanding inhibition as compared to standard acarbose (IC50 = 873.34 ± 1.67 µM). Furthermore, a docking study was performed to find out the interaction between the enzyme and the most active compounds. With this research work, 3,4-dihydroxyphenylacetic acid Schiff base derivatives have been introduced as a potential class of α-glucosidase inhibitors that have remained elusive till now.

Synthesis of α-Hydroxy-1,2,3-Triazole-linked Sialyltransferase Inhibitors and Evaluation of Selectivity Towards ST3GAL1, ST6GAL1 and ST8SIA2.

Tumour-derived sialoglycans, bearing the charged nonulosonic sugar sialic acid at their termini, play a critical role in tumour cell adhesion and invasion, as well as evading cell death and immune surveillance. Sialyltransferases (ST), the enzymes responsible for the biosynthesis of sialylated glycans, are highly upregulated in cancer, with tumour hypersialylation strongly correlated with tumour growth, metastasis and drug resistance. As a result, desialylation of the tumour cell surface using either targeted delivery of a pan-ST inhibitor (or sialidase) or systemic delivery of a non-toxic selective ST inhibitors are being pursued as potential new anti-metastatic strategies against multiple cancers including pancreatic, ovarian, breast, melanoma and lung cancer. Herein, we have employed molecular modelling to give insights into the selectivity observed in a series of selective ST inhibitors that incorporate a uridyl ring in place of the cytidine of the natural donor (CMP-Neu5Ac) and replace the charged phosphodiester linker of classical ST inhibitors with a neutral a-hydroxy-1,2,3-triazole linker. The inhibitory activities of the nascent compounds were determined against recombinant human ST enzymes (ST3GAL1, ST6GAL1, ST8SIA2) showing promising activity and selectivity towards specific ST sub-types. Our ST inhibitors are non-toxic and show improved synthetic accessibility and drug-likeness compared to earlier nucleoside-based ST inhibitors.

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors

ROS-responsive nanoparticle delivery of ferroptosis inhibitor prodrug to facilitate mesenchymal stem cell-mediated spinal cord injury repair

Spinal cord injury (SCI) is a traumatic condition that results in impaired motor and sensory function. Ferroptosis is one of the main causes of neural cell death and loss of neurological function in the spinal cord, and ferroptosis inhibitors are effective in reducing inflammation and repairing SCI. Although human umbilical cord mesenchymal stem cells (Huc-MSCs) can ameliorate inflammatory microenvironments and promote neural regeneration in SCI, their efficacy is greatly limited by the local microenvironment after SCI. Therefore, in this study, we constructed a drug-release nanoparticle system with synergistic Huc-MSCs and ferroptosis inhibitor, in which we anchored Huc-MSCs by a Tz-A6 peptide based on the CD44-targeting sequence, and combined with the reactive oxygen species (ROS)-responsive drug nanocarrier mPEG-b-Lys-BECI-TCO at the other end for SCI repair. Meanwhile, we also modified the classic ferroptosis inhibitor Ferrostatin-1 (Fer-1) and synthesized a new prodrug Feborastatin-1 (Feb-1). The results showed that this treatment regimen significantly inhibited the ferroptosis and inflammatory response after SCI, and promoted the recovery of neurological function in rats with SCI. This study developed a combination therapy for the treatment of SCI and also provides a new strategy for the construction of a drug-coordinated cell therapy system.

Deficiency of histone deacetylases 3 in macrophage alleviates monosodium urate crystals-induced gouty inflammation in mice

BackgroundGout is caused by monosodium urate (MSU) crystals deposition to trigger immune response. A recent study suggested that inhibition of Class I Histone deacetylases (HDACs) can significantly reduce MSU crystals-induced inflammation. However, which one of HDACs members in response to MSU crystals was still unknown. Here, we investigated the roles of HDAC3 in MSU crystals-induced gouty inflammation.MethodsMacrophage specific HDAC3 knockout (KO) mice were used to investigate inflammatory profiles of gout in mouse models in vivo, including ankle arthritis, foot pad arthritis and subcutaneous air pouch model. In the in vitro experiments, bone marrow-derived macrophages (BMDMs) from mice were treated with MSU crystals to assess cytokines, potential target gene and protein.ResultsDeficiency of HDAC3 in macrophage not only reduced MSU-induced foot pad and ankle joint swelling but also decreased neutrophils trafficking and IL-1β release in air pouch models. In addition, the levels of inflammatory genes related to TLR2/4/NF-κB/IL-6/STAT3 signaling pathway were significantly decreased in BMDMs from HDAC3 KO mice after MSU treatment. Moreover, RGFP966, selective inhibitor of HDAC3, inhibited IL-6 and TNF-α production in BMDMs treated with MSU crystals. Besides, HDAC3 deficiency shifted gene expression from pro-inflammatory macrophage (M1) to anti-inflammatory macrophage (M2) in BMDMs after MSU challenge.ConclusionsDeficiency of HDAC3 in macrophage alleviates MSU crystals-induced gouty inflammation through inhibition of TLR2/4 driven IL-6/STAT3 signaling pathway, suggesting that HDAC3 could contribute to a potential therapeutic target of gout.

Empagliflozin and Risk of Incident Gout: Analysis from the EMPagliflozin Comparative Effectiveness and SafEty (EMPRISE) Cohort Study.

Hyperuricemia is frequently observed in patients with type 2 diabetes (T2D) and is associated with increased risk of gout and cardiovascular disease (CVD). Empagliflozin lowers serum urate levels by enhancing its urinary excretion. To compare initiators of empagliflozin vs dipeptidyl peptidase-4 inhibitor (DPP4i) and initiators of empagliflozin vs glucagon-like peptide-1 receptor agonist (GLP-1RA) with respect to the risk of incident gout events. Using three claims-based datasets from 08/2014 to 09/2019, we generated two cohorts (cohort 1: empagliflozin vs DPP4i; cohort 2: empagliflozin vs GLP-1RA) of adult patients with T2D and without prior history of gout or gout-specific medication dispensing separately in each dataset. To assess the risk of incident gout, we estimated hazard ratios (HR) and rate differences (RD) per 1000 person-years (PY) with their 95% confidence intervals (CI) before and after 1:1 propensity score (PS) matching adjusting for 141 baseline covariates. We identified 102,262 pairs of 1:1 propensity score-matched adults in cohort 1 and 131,216 pairs in cohort 2. Over a mean follow-up period of 8months on treatment, the risk of gout was lower in patients initiating empagliflozin compared to DPP4i (HR = 0.69: 95%CI (0.60-0.79); RD = - 2.27: 95% CI (- 3.08, 1.46)) or GLP-1RA (HR = 0.83: 95%CI (0.73-0.94); RD = - 0.99: 95%CI (- 1.66, - 0.32)). Results were consistent across subgroups (sex, age, body mass index, chronic kidney disease, heart failure, cardiovascular disease, and concurrent diuretic use) and sensitivity analyses. Among adults with T2D, the initiation of empagliflozin vs a DPP4i or GLP-1RA was associated with lower risk of incident gout, complementing results from a post hoc analysis of the EMPA-REG OUTCOME trial and previously published observational research focusing on the sodium-glucose co-transporter-2 inhibitor class in more narrowly defined study populations.

Botulinum toxin A attenuates osteoarthritis development via inhibiting chondrocyte ferroptosis through SLC7Al1/GPX4 axis

Osteoarthritis (OA) is a prevalent joint degenerative disease, resulting in a significant societal burden. However, there is currently a lack of effective treatment option available. Previous studies have suggested that Botulinum toxin A (BONT/A), a macromolecular protein extracted from Clostridium Botulinum, may improve the pain and joint function in OA patients, but the mechanism remains elusive. This study was to investigate the impact and potential mechanism of BONT/A on OA in vivo and in vitro experiment. LPS increased the levels of ROS, Fe2+and Fe3+, as well as decreased GSH levels, the ratio of GSH / GSSH and mitochondrial membrane potential. It also enhanced the degeneration of extracellular matrix (ECM) and altered the ferroptosis-related protein expression in chondrocytes. BONT/A rescued LPS-induced decrease in collagen type II (Collagen II) expression and increase in matrix metalloproteinase 13 (MMP13), mitigated LPS-induced cytotoxicity in chondrocytes, abolished the accumulation of ROS and iron, upregulated GSH and the ratio of GSH/ GSSH, improved mitochondrial function, and promoted SLC7A11/GPX4 anti-ferroptosis system activation. Additionally, intra-articular injection of BONT/A inhibited the degradation of cartilage in OA model rats. This chondroprotective effect of BONT/A was reversed by erastin (a classical ferroptosis agonist) and enhanced by liproxstatin-1 (a classic ferroptosis inhibitor). Our research confirms that BONT/A alleviates the OA development by inhibiting the ferroptosis of chondrocytes, which revealed to be a potential therapeutic mechanism for BONT/A treating the OA.

Discovery of a novel chemotype as DYRK1A inhibitors against Alzheimer's disease: Computational modeling and biological evaluation

Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) plays an essential role in Tau and Aβ pathology closely related to Alzheimer's disease (AD). Accumulative evidence has demonstrated DYRK1A inhibition is able to reduce the pathological features of AD. Nevertheless, there is no approved DYRK1A inhibitor for clinical use as anti-AD therapy. This is somewhat due to the lack of effective and safe chemotypes of DYRK1A inhibitors. To address this issue, we carried out in silico screening, in vitro assays and in vivo efficacy evaluation with the aim to discover a new class of DYRK1A inhibitors for potential treatment of AD. By in silico screening, we selected and purchased 16 potential DYRK1A inhibitors from the Specs chemical library. Among them, compound Q17 (Specs ID: AO-476/40829177) potently inhibited DYRK1A. The hydrogen bonds between compound Q17 and two amino acid residues named GLU239 and LYS188, were uncovered by molecular docking and molecular dynamics simulation. The cell-based assays showed that compound Q17 could protect the SH-SY5Y human neuroblastoma cell line from okadaic acid (OA)-induced injury by targeting DYRK1A. More importantly, compound Q17 significantly improved cognitive dysfunction of 3 × Tg-AD mice, ameliorated pathological changes, and attenuated Tau hyperphosphorylation as well as Aβ deposition. In summary, our computational modeling strategy is effective to identify novel chemotypes of DYRK1A inhibitors with great potential to treat AD, and the identified compound Q17 in this study is worthy of further study.

Abstract PO3-26-09: A novel class of RNR inhibitors that induces genomic instability and breast cancer cell death without inducing overt toxicities

Abstract Despite the development of screening techniques and therapies, nearly 30% of breast cancers progress to Stage IV metastatic disease. These advanced cancers are highly proliferative, engaging in ongoing DNA synthesis that requires ribonucleotide reductase (RNR), the rate-limiting enzyme for dNTP synthesis. RRM2, the small subunit of RNR, activates the enzyme, increases deoxynucleotide pools, and is overexpressed in many cancers, including breast cancer. Thus, RNR is an established therapeutic target in advanced breast cancer. Currently, gemcitabine is the only FDA-approved RNR inhibitor used to treat Stage IV disease. As a nucleoside analog, gemcitabine also causes severe dose-limiting toxicities (i.e. myelosuppression and hepatotoxicity) due to off-target DNA replication termination. We hypothesize that a novel, non-nucleoside, RNR inhibitor may overcome these limitations. To address this, we developed TMU27a, a derivative of naphthyl salicylic acyl hydrazone (NSAH), which was identified in an in silico screen for its high-affinity binding to the RNR catalytic site. To measure direct interactions between TMU27a and purified RNR protein, we used surface plasmon resonance, revealing on-target, tight binding (Kd = 150 nM). Unbiased docking and molecular dynamics simulations (using lowest energy calculations) predicted the binding sight of TMU27a to be the catalytic site of RNR, where it is stabilized through extensive interactions formed with conserved residues. Preliminary docking predictions also indicate multiple clash sites between TMU27a and the DNA polymerase lambda nucleotide pocket even in the lowest energy pose (unlike gemcitabine), suggesting that TMU27a inhibits RNR activity through a novel mechanism without significant off-target binding to other nucleotide binding proteins that contribute to toxicity. Demonstrating on-target inhibition, TMU27a reduces dATP levels and induces S and G2/M-phase stalling, leading to significant DNA damage and chromosomal instability. This excessive genomic damage causes growth repression, apoptosis, and senescence in several triple negative breast cancer (TNBC) cell lines. In xenograft mouse models of TNBC, TMU27a fully suppressed tumor growth without impacting mouse weight, suggesting strong drug efficacy in the absence of overt toxicity. Moreover, while short-term exposure to gemcitabine suppresses red and white blood cell populations, TMU27a does not. Together, these data indicate that TMU27a and its potential analogs are novel, non-toxic RNR inhibitors that overcome the limitations of gemcitabine for treating breast cancer. We are currently investigating how and where TMU27a binds human RNR using cryo-electron microscopy with the goal of generating a novel class of RNR inhibitors through structure-based drug design, improving binding affinity and selectivity. Citation Format: Taylor Baker, Kristen Weber-Bonk, Wei Huang, Derek Taylor, Ruth Keri. A novel class of RNR inhibitors that induces genomic instability and breast cancer cell death without inducing overt toxicities [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-09.

Abstract PO3-26-04: Discovery of novel inhibitors of the epigenetic regulatory KAT6A histone acetyl transferase and their anti-tumor activity in breast cancer cells

Abstract KAT6A is a histone acetyltransferase (HAT) which directs chromatin structural rearrangement and subsequent changes in gene transcription through acetylation of histones, primarily specific lysine residues on histone H3. KAT6A is modified in cancer, including genomic amplification, overexpression, mutation, as well as genetic rearrangement resulting in the production of fusion proteins. Dysregulation of this MYST family member and its acetyl transferase activity is known to drive gene expression and tumorigenic progression in cancers. An inhibitor of KAT6A and related MYST family member KAT6B has entered phase 1 clinical trials with durable partial responses seen in a variety of tumor types, including estrogen-receptor positive (ER+) breast cancer previously treated with a CDK4/6 inhibitor (Sommerhalder, ASCO2023). Evidence of dose dependent pharmacodynamics (H3K23Ac inhibition) was observed in peripheral blood mononuclear cells and paired tumor biopsies. Isosterix has developed 2 distinct series of KAT6A selective inhibitors which engage in the active site of KAT6A in the acetyl CoA substrate binding pocket and inhibit histone acetylation activity. The kinetics of target enzyme binding and inhibition will be discussed for both series of inhibitors. These compounds have been characterized for potency and cellular activity in a variety of assays. Both series of compounds inhibit KAT6A-mediated histone acetylation with single digit nM potency in a biochemical assay as well as significant potency in the breast cancer tumor cell line ZR-75-1, inhibiting H3K23 acetylation. Using a thermal shift assay (CETSA) and mass spectrometry-based protein quantitation, direct target engagement of KAT6A within cells has been shown. These compounds reduce cell viability of a number of breast cancer cell lines with 10 days of compound administration confirming the dependence of these tumor cell lines on KAT6A acetyl transferase activity for proliferation and cell cycle progression. Compounds are orally bioavailable with excellent pharmacokinetic (PK) properties in mice including half-lives >5 hours with oral administration. Finally, Isosterix inhibitors demonstrated anti-tumor efficacy with oral administration in a ZR-75-1 mouse xenograft tumor model. Inhibitors against this novel epigenetic target demonstrate the potential for efficacy in ER+ breast cancer as well as other tumor indications through suppression of KAT6A-directed transcriptional induction and chromatin remodeling. Table. Compound Kat6A Biochemical IC50 (µM) ZR-75-1 H3K23 Acetylation EC50 (µM) Mouse PK (p.o.) Mouse PK (i.v.) Inhibitor class %F T½ (h) Cl (mL/min/kg) SL-IST-001 0.007 0.01 100 5.2 0.47 Series 1 SL-IST-002 0.007 0.005 100 6 0.48 Series 2 Citation Format: Leslie Holsinger, Roopa RaI, Rahul R. Nagawade, Benoy Pal, Samir Pawar, Pankaj Singh, Krishna Putta, Mahaboobi Jaleel, Tove Bostrom, Jakob Karen, Qianyang Huang, Alison Hannah. Discovery of novel inhibitors of the epigenetic regulatory KAT6A histone acetyl transferase and their anti-tumor activity in breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-04.

Salvianolic Acid B Inhibits Thrombosis and Directly Blocks the Thrombin Catalytic Site

BackgroundSalvianolic acid B (SAB) is a major component of Salvia miltiorrhiza root (Danshen), widely used in East/Southeast Asia for centuries to treat cardiovascular diseases. Danshen depside salt, 85% of which is made up of SAB, is approved in China to treat chronic angina. Although clinical observations suggest that Danshen extracts inhibited arterial and venous thrombosis, the exact mechanism has not been adequately elucidated. ObjectiveTo delineate the anti-thrombotic mechanisms of SAB. MethodsWe applied platelet aggregation and coagulation assays, perfusion chambers, and intravital microscopy models. The inhibition kinetics and binding affinity of SAB to thrombin are measured by thrombin enzymatic assays, intrinsic fluorescence spectrophotometry, and isothermal titration calorimetry (ITC). We used molecular in silico docking models to predict the interactions of SAB with thrombin. ResultsSAB dose-dependently inhibited platelet activation and aggregation induced by thrombin. SAB also reduced platelet aggregation induced by ADP and collagen. SAB attenuated blood coagulation by modifying fibrin network structures and significantly decreased thrombus formation in mouse cremaster arterioles and perfusion chambers. The direct SAB-thrombin interaction was confirmed by enzymatic assays, intrinsic fluorescence spectrophotometry, and ITC. Interestingly, SAB shares key structural similarities to the trisubstituted benzimidazole class of thrombin inhibitors, such as dabigatran. Molecular docking models predicted the binding of SAB to the thrombin active site. ConclusionOur data established SAB as the first herb-derived direct thrombin catalytic site inhibitor, suppressing thrombosis through both thrombin-dependent and -independent pathways. Purified SAB may be a cost-effective agent for treating arterial and deep vein thrombosis.

PH-responsive anti-corrosion Activity of Gallic Acid-Intercalated MgAl LDH in Acidic, Neutral, and Alkaline Environments

Undoubtedly, designing pH-responsive particles is one of the most fascinating aims for corrosion experts. Local pH variations can occur through the corrosion process (chemical and electrochemical reactions), accelerating the corrosion rate. However, by designing this class of modern inhibitors, a significant part of the effects of pH variation during corrosion disappears. Herein, a novel layer double hydroxide-based (LDH) nanocomposite, which is filled by gallic acid (GA) molecules, was designed. To prove the composite inhibition proficiency, EIS tests were conducted in acid, neutral and alkaline atmospheres and then surface morphology of the protected and unprotected samples are visualized by FE-SEM images. Simultaneously, the EDS-Mapping technique was applied to predict the elemental composition of the generated inhibitive layer. The results of the EIS test indicated an increase in corrosion resistance after using nanoparticles in all three acidic, neutral and alkaline environments. In addition, the potentiodynamic polarization test confirmed the mixed-protection mechanism in these three media. Furthermore, FE-SEM approach demonstrated the formation of a protective film on metal surfaces, and the EDS-Mapping evaluation confirmed these results in line with the element recognition.

3-Epi-betulinic acid 3-O-β-D-glucopyranoside (eBAG) induces autophagy by activation of AMP-activated protein kinase in hepatocellular carcinoma

3-Epi-betulinic acid 3-O-β-D-glucopyranoside (eBAG) is a pentacyclic triterpene mainly distributed in food and medicinal plants, which exhibits various pharmacological properties. However, whether these functions are attributed to eBAG or additional components in these plants remain unknown. Herein, we report that eBAG exerted an inhibitory activity against hepatocellular carcinoma and esophageal cancer cells. EBAG induced non-apoptotic cell death in hepatocellular carcinoma cells. The eBAG-induced cell death was inhibited by knock-down of autophagy related gene (ATG) 5 and ATG7, by administration of 3-methyladenine, a selective autophagy inhibitor that suppresses phosphoinositide 3-kinase (PI3K), and by chloroquine, a classic autophagy flux inhibitor. We demonstrated that eBAG induced an autophagy-mediated cell death. Application of eBAG mimicked cellular bioenergetics depletion leading to the reduction of intracellular ATP, activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR. Co-treatment with compound C, an AMPK inhibitor, abrogated cell death induced by eBAG. We further validated the anti-tumor effect of eBAG in the murine xenograft model of hepatocellular carcinoma and found that eBAG treatment promoted the induction of autophagy and reduction of tumor growth in mice. As a functional food ingredient, eBAG is a potential therapeutic agent for the treatment of hepatocellular carcinoma and esophageal cancer.

Ethaverine and Papaverine Target Cyclin-Dependent Kinase 5 and Inhibit Lung Cancer Cell Proliferation and Migration.

CDK5 kinase plays a central role in the regulation of neuronal functions, and its hyperactivation has been associated with neurodegenerative pathologies and more recently with several human cancers, in particular lung cancer. However, ATP-competitive inhibitors targeting CDK5 are poorly selective and suffer limitations, calling for new classes of inhibitors. In a screen for allosteric modulators of CDK5, we identified ethaverine and closely related derivative papaverine and showed that they inhibit cell proliferation and migration of non small cell lung cancer cell lines. Moreover the efficacy of these compounds is significantly enhanced when combined with the ATP-competitive inhibitor roscovitine, suggesting an additive dual mechanism of inhibition targeting CDK5. These compounds do not affect CDK5 stability, but thermodenaturation studies performed with A549 cell extracts infer that they interact with CDK5 in cellulo. Furthermore, the inhibitory potentials of ethaverine and papaverine are reduced in A549 cells treated with siRNA directed against CDK5. Taken together, our results provide unexpected and novel evidence that ethaverine and papaverine constitute promising leads that can be repurposed for targeting CDK5 in lung cancer.

Substituted 4H-3,1-benzoxazine-4-one Derivatives as Inhibitors of Cathepsin G.

Cathepsin G (CatG) is a cationic serine protease with a wide substrate specificity. CatG has been reported to play a role in several pathologies, including rheumatoid arthritis, ischemic reperfusion injury, acute respiratory distress syndrome, and cystic fibrosis, among others. We aim to develop a new class of CatG inhibitors and evaluate their potency and selectivity against a series of serine proteases. We exploited chemical synthesis as well as chromogenic substrate hydrolysis assays to construct and evaluate the new inhibitors. In this communication, we report on a new class of CatG inhibitors of 4H-3,1-benzoxazin- 4-one derivatives. We constructed a small library of seven substituted 4H-3,1-benzoxazin-4-one derivatives and identified their inhibition potential against CatG. Five molecules were identified as CatG inhibitors with values of 0.84-5.5 μM. Inhibitor 2 was the most potent, with an IC50 of 0.84 ± 0.11 μM and significant selectivity over representative serine proteases of thrombin, factor XIa, factor XIIIa, and kallikrein. Thus, we propose this inhibitor as a lead molecule to guide subsequent efforts to develop clinically relevant potent and selective CatG inhibitors for use as anti-inflammatory agents.

Myosin-1C augments endothelial secretion of von Willebrand factor by linking contractile actomyosin machinery to the plasma membrane

Blood endothelial cells control the hemostatic and inflammatory response by secreting von Willebrand factor (VWF) and P-selectin from storage organelles called Weibel-Palade bodies (WPB). Actin-associated motor proteins regulate this secretory pathway at multiple points. Prior to fusion, myosin Va forms a complex that anchors WPBs to peripheral actin structures allowing maturation of content. Post-fusion, an actomyosin ring/coat is recruited and compresses the WPB to forcibly expel the largest VWF multimers. Here we provide the first evidence for the involvement of class I myosins during regulated VWF secretion. We show that the unconventional myosin-1C (Myo1c) is recruited post-fusion via its pleckstrin homology domain in an actin-independent process. This provides a link between the actin ring and phosphatidylinositol 4,5-bisphosphate (PIP2) at the membrane of the fused organelle and is necessary to ensure maximal VWF secretion. This is an active process requiring Myo1c ATPase activity as inhibition of class I myosins using the inhibitor Pentachloropseudilin or expression of an ATPase deficient Myo1c rigor mutant perturbs the expulsion of VWF and alters the kinetics of the exocytic actin ring. These data offer a novel insight into the control of an essential physiological process and provide a new way in which it can be regulated.

Site-Specifically Modified Peptide Inhibitors of Protein Tyrosine Phosphatase 1B and T-Cell Protein Tyrosine Phosphatase with Enhanced Stability and Improved In Vivo Long-Acting Activity.

Protein tyrosine phosphatase 1B (PTP1B) and TC-PTP can function in a coordinated manner to regulate diverse biological processes including insulin and leptin signaling, T-cell activation, and tumor antigen presentation, which makes them potential targets for several therapeutic applications. We have previously demonstrated that the lipidated BimBH3 peptide analogues were a new class of promising PTP1B inhibitors with once-weekly antidiabetic potency. Herein, we chemically synthesized two series of BimBH3 analogues via site-specific modification and studied their structure-activity relationship. The screened analogues S2, S6, A2-14, A2-17, A2-20, and A2-21 exhibited an improved PTP1B/TC-PTP dual inhibitory activity and achieved good stability in the plasma of mice and dogs, which indicated long-acting potential. In mouse models of type 2 diabetes mellitus (T2DM), the selected analogues S6, S7, A2-20, and A2-21 with an excellent target activity and plasma stability generated once-weekly therapeutic potency for T2DM at lower dosage (0.5 μmol/kg). In addition, evidence was provided to confirm the cell permeability and targeted enrichment of the BimBH3 analogues. In summary, we report here that site-specific modification and long fatty acid conjugation afforded cell-permeable peptidomimetic analogues of BimBH3 with enhanced stability, in vivo activity, and long-acting pharmacokinetic profile. Our findings could guide the further optimization of BimBH3 analogues and provide a proof-of-concept for PTP1B/TC-PTP targeting as a new therapeutic approach for T2DM, which may facilitate the discovery and development of alternative once-weekly anti-T2DM drug candidates.