Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or p53.

Purpose: Over the last few years, covalent fragment-based drug discovery has gained significant importance. Thus, striving for more warhead diversity, we conceived a library consisting of 20 covalently reacting compounds. Our covalent fragment library (CovLib) contains four different warhead classes, including five α-cyanoacacrylamides/acrylates (CA), three epoxides (EO), four vinyl sulfones (VS), and eight electron-deficient heteroarenes with a leaving group (SNAr/SN). Methods: After predicting the theoretical solubility of the fragments by LogP and LogS during the selection process, we determined their experimental solubility using a turbidimetric solubility assay. The reactivities of the different compounds were measured in a high-throughput 5,5'-dithiobis-(2-nitrobenzoic acid) DTNB assay, followed by a (glutathione) GSH stability assay. We employed the CovLib in a (differential scanning fluorimetry) DSF-based screening against different targets: c-Jun N-terminal kinase 3 (JNK3), ubiquitin-specific protease 7 (USP7), and the tumor suppressor p53. Finally, the covalent binding was confirmed by intact protein mass spectrometry (MS). Results: In general, the purchased fragments turned out to be sufficiently soluble. Additionally, they covered a broad spectrum of reactivity. All investigated α-cyanoacrylamides/acrylates and all structurally confirmed epoxides turned out to be less reactive compounds, possibly due to steric hindrance and reversibility (for α-cyanoacrylamides/acrylates). The SNAr and vinyl sulfone fragments are either highly reactive or stable. DSF measurements with the different targets JNK3, USP7, and p53 identified reactive fragment hits causing a shift in the melting temperatures of the proteins. MS confirmed the covalent binding mode of all these fragments to USP7 and p53, while additionally identifying the SNAr-type electrophile SN002 as a mildly reactive covalent hit for p53. Conclusion: The screening and target evaluation of the CovLib revealed first interesting hits. The highly cysteine-reactive fragments VS004, SN001, SN006, and SN007 covalently modify several target proteins and showed distinct shifts in the melting temperatures up to +5.1 °C and -9.1 °C.

Fluacrypyrim Protects Hematopoietic Stem and Progenitor Cells against Irradiation via Apoptosis Prevention.

Ionizing radiation (IR)-induced hematopoietic injury has become a global concern in the past decade. The underlying cause of this condition is a compromised hematopoietic reserve, and this kind of hematopoietic injury could result in infection or bleeding, in addition to lethal mishaps. Therefore, developing an effective treatment for this condition is imperative. Fluacrypyrim (FAPM) is a recognized effective inhibitor of STAT3, which exhibits anti-inflammation and anti-tumor effects in hematopoietic disorders. In this context, the present study aimed to determine whether FAPM could serve as a curative agent in hematopoietic-acute radiation syndrome (H-ARS) after total body irradiation (TBI). The results revealed that the peritoneally injection of FAPM could effectively promote mice survival after lethal dose irradiation. In addition, promising recovery of peripheral blood, bone marrow (BM) cell counts, hematopoietic stem cell (HSC) cellularity, BM colony-forming ability, and HSC reconstituting ability upon FAPM treatment after sublethal dose irradiation was noted. Furthermore, FAPM could reduce IR-induced apoptosis in hematopoietic stem and progenitor cells (HSPCs) both in vitro and in vivo. Specifically, FAPM could downregulate the expressions of p53-PUMA pathway target genes, such as Puma, Bax, and Noxa. These results suggested that FAPM played a protective role in IR-induced hematopoietic damage and that the possible underlying mechanism was the modulation of apoptotic activities in HSCs.

Class I Biocompatible DLP-Printed Acrylate Impairs Adhesion and Proliferation of Human Mesenchymal Stromal Cells in Indirect Cytotoxicity Assay.

The popular method of digital light processing 3D printing (DLP) for complex and individual laboratory equipment requires materials that are as inert as possible for use in contact with cells for subsequent investigations. However, the per se incomplete curing of acrylate resins by UV light leaves residuals that are not suitable for cell culture application. Therefore, we evaluated the cytotoxicity of four commercially available acrylate resins with bone marrow-derived human mesenchymal stromal cells (BM-hMSC) in an indirect cytotoxicity test. This involved incubating the printed cylinders in Transwell™ inserts for 7 days. While the degree of crosslinking did not increase significantly between freshly printed and stored samples (3 weeks in ambient conditions), the storage improved the material's performance in terms of cytocompatibility. The DNA amount and LDH activity showed a direct influence of the resin residuals on cell adhesion. The class I acrylate Surgical Guide™ left no adherent cells after 7 days, regardless of previous storage. In comparison, the Basic Ivory™ resin after storage allowed same amount of adherent cells after 7 days as the polystyrene reference. We conclude that resin residuals of certain materials are released, which allows the use of the resins in indirect contact with cells thereafter.

Development and evaluation of a drug-in-adhesive transdermal delivery system for delivery of olanzapine.

OBJECTIVES: Olanzapine (OZP) is a safe and effective atypical antipsychotic drug used in treating schizophrenia and bipolar disorders. The dosage forms currently on the market for OZP are administered via oral or intramuscular routes. However, there are many problems associated with oral and intramuscular routes of drug administration. Thus, our aim was to develop a drug-in-adhesive transdermal delivery system (TDS) that can deliver OZP for 3 days. METHODS: We determined passive permeation, effect of oleic acid as chemical enhancer, and delivery of OZP across different skin types. Based on preliminary studies and saturation solubility of OZP in different pressure-sensitive adhesives (PSAs), we formulated and characterized solution-based TDS in acrylate PSA and suspension-based TDS in silicone and PIB PSA, with oleic acid as chemical enhancer. RESULTS: Acrylate solution-based TDS, silicone, and PIB suspension-based TDS delivered 58.97 ± 6.59 µg/sq.cm, 129.34 ± 16.59 µg/sq.cm, and 245.00 ± 2.51 µg/sq.cm, respectively, using in vitro permeation testing. PIB PSA suspension-based TDS met the 3 days desired target delivery. Skin irritation testing using In vitro EpiDermTM skin irritation test (EPI-200-SIT) kit found PIB TDS to be nonirritant. CONCLUSION: The PIB PSA suspension-based TDS could serve as a potentially effective transdermal delivery system for olanzapine.

Permanent, Antimicrobial Coating to Rapidly Kill and Prevent Transmission of Bacteria, Fungi, Influenza, and SARS-CoV-2.

Microbial adhesion and contamination on shared surfaces can lead to life-threatening infections with serious impacts on public health, economy, and clinical practices. The traditional use of chemical disinfectants for sanitization of surfaces, however, comes with its share of health risks, such as hazardous effects on the eyes, skin, and respiratory tract, carcinogenicity, as well as environmental toxicity. To address this, we have developed a nonleaching quaternary small molecule (QSM)-based sprayable coating which can be fabricated on a wide range of surfaces such as nylon, polyethylene, surgical mask, paper, acrylate, and rubber in a one-step, photocuring technique. This contact-active coating killed pathogenic bacteria and fungi including drug-resistant strains of Staphylococcus aureus and Candida albicans within 15-30 min of contact. QSM coatings withstood multiple washes, highlighting their durability. Interestingly, the coated surfaces exhibited rapid killing of pathogens, leading to the prevention of their transmission upon contact. The coating showed membrane disruption of bacterial cells in fluorescence and electron microscopic investigations. Along with bacteria and fungi, QSM-coated surfaces also showed the complete killing of high loads of influenza (H1N1) and SARS-CoV-2 viruses within 30 min of exposure. To our knowledge, this is the first report of a coating for multipurpose materials applied in high-touch public places, hospital equipment, and clinical consumables, rapidly killing drug-resistant bacteria, fungi, influenza virus, and SARS-CoV-2.

The Cysteine Desulfurase IscS Is a Significant Target of 2-Aminoacrylate Damage in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes eight members of the Rid protein superfamily. PA5339, a member of the RidA subfamily, is required for full growth and motility of P. aeruginosa. Our understanding of RidA integration into the metabolic network of P. aeruginosa is at an early stage, with analyses largely guided by the well-established RidA paradigm in Salmonella enterica. A P. aeruginosa strain lacking RidA has a growth and motility defect in a minimal glucose medium, both of which are exacerbated by exogenous serine. All described ridA mutant phenotypes are rescued by supplementation with isoleucine, indicating the primary generator of the reactive metabolite 2-aminoacrylate (2AA) in ridA mutants is a threonine/serine dehydratase. However, the critical (i.e., phenotype determining) targets of 2AA leading to growth and motility defects in P. aeruginosa remained undefined. This study was initiated to probe the effects of 2AA stress on the metabolic network of P. aeruginosa by defining the target(s) of 2AA that contribute to physiological defects of a ridA mutant. Suppressor mutations that restored growth to a P. aeruginosa ridA mutant were isolated, including an allele of iscS (encoding cysteine desulfurase). Damage to IscS was identified as a significant cause of growth defects of P. aeruginosa during enamine stress. A suppressing allele encoded an IscS variant that was less sensitive to damage by 2AA, resulting in a novel mechanism of phenotypic suppression of a ridA mutant. IMPORTANCE 2-aminoacrylate (2AA) is a reactive metabolite formed as an intermediate in various enzymatic reactions. In the absence of RidA, this metabolite can persist in vivo where it attacks and inactivates specific PLP-dependent enzymes, causing metabolic defects and organism-specific phenotypes. This work identifies the cysteine desulfurase IscS as the critical target of 2AA in Pseudomonas aeruginosa. A single substitution in IscS decreased sensitivity to 2AA and suppressed growth phenotypes of a ridA mutant. Here, we provide the first report of suppression of a ridA mutant phenotype by altering the sensitivity of a target enzyme to 2AA.

Zwitterionic Amino Acid-Derived Polyacrylates as Smart Materials Exhibiting Cellular Specificity and Therapeutic Activity.

The synthesis of new amino acid-containing, cell-specific, therapeutically active polymers is presented. Amino acids served as starting material for the preparation of tailored polymers with different amino acids in the side chain. The reversible addition-fragmentation chain-transfer (RAFT) polymerization of acrylate monomers yielded polymers of narrow size distribution (D ≤ 1.3). In particular, glutamate (Glu)-functionalized, zwitterionic polymers revealed a high degree of cytocompatibility and cellular specificity, i.e., showing association to different cancer cell lines, but not with nontumor fibroblasts. Energy-dependent uptake mechanisms were confirmed by means of temperature-dependent cellular uptake experiments as well as localization of the polymers in cellular lysosomes determined by confocal laser scanning microscopy (CLSM). The amino acid receptor antagonist O-benzyl-l-serine (BzlSer) was chosen as an active ingredient for the design of therapeutic copolymers. RAFT copolymerization of Glu acrylate and BzlSer acrylate resulted in tailored macromolecules with distinct monomer ratios. The targeted, cytotoxic activity of copolymers was demonstrated by means of multiday in vitro cell viability assays. To this end, polymers with 25 mol % BzlSer content showed cytotoxicity against cancer cells, while leaving fibroblasts unaffected over a period of 3 days. Our results emphasize the importance of biologically derived materials to be included in synthetic polymers and the potential of zwitterionic, amino acid-derived materials for cellular targeting. Furthermore, it highlights that the fine balance between cellular specificity and unspecific cytotoxicity can be tailored by monomer ratios within a copolymer.

Protective and therapeutic role of mango pulp and eprosartan drug and their anti-synergistic effects against thioacetamide-induced hepatotoxicity in male rats.

The present study was done to evaluate the protective and therapeutic role of mango pulp (M), eprosartan drug (E), and their co-administration (EM) against hepatotoxicity induced by thioacetamide (T). Seven groups of rats were prepared as follows: the control (C) group (normal rats), T group (the rats were injected with T), T-M group (the rats were injected with T, and then treated with M), T-E group (the rats were injected with T, and then treated with E), T-EM group (the rats were injected with T, and then treated with E and M), M-TM-M group (the rats were administered with M before, during, and after T injection), and M group (the healthy rats were administered with M only). Firstly, the characterizations of M were determined. Also, the markers of hepatic oxidative stress [malondialdehyde (MDA) and glutathione (GSH) levels and the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GSR)], inflammation and fibrosis [(tumor necrosis factor-α (TNF-α) and platelet-derived growth factor-BB (PDGF-BB) levels and gene expression of transforming growth factor-beta1(TGF-ß1)], and liver functions and microscopic examination were evaluated. The present results revealed that M contains 419 ± 1.04 µg total phenolics as gallic acid equivalent and 6.8 ± 0.05 µg total flavonoids as quercetin equivalent. The analysis of phenolics and flavonoids showed the presence of chlorogenic, caffeic, 2,5-dihydroxy benzoic, 3,5-dicaffeoylquinic, 4,5-dicaffeoylquinic, tannic, cinnamic acidS, and catechin, phloridzin, and quercetin with different concentrations. Also, M contains various minerals with different concentrations involving potassium, calcium, magnesium, sodium, iron, copper, zinc, and manganese. The current results showed that the total antioxidant capacity of 1 g of M was 117.2 ± 1.16 as µg ascorbic acid equivalent. Our biochemical studies showed that all treatments significantly reduced T-induced hepatotoxicity and liver injuries, as the oxidative stress and inflammatory and fibrotic markers were diminished where MDA level and the activities of GST, GSSG, and GR were decreased when compared with T group. In contrast, GSH level and the activities of SOD and GPx and GSH/GSSG ratio were increased. In addition, TNF-α and PDGF-BB levels were reduced, and the gene expression of TGF-ß1 was down-regulated. Consequently, the liver functions were significantly improved. In conclusion, each E, M, and EM has a therapeutic effect against T-induced hepatotoxicity via the reduction of the OS, inflammation, and fibrosis. Unfortunately, treatment with M and E simultaneously revealed the less effectiveness than the treatment with M or E demonstrates the presence of anti-synergistic effect between them. Additionally, M-TM-M treatment showed a better effect than T-M treatment against T-induced hepatotoxicity revealing the prophylactic role of M. The administration of healthy rats with M for 12 weeks has no side effect.

In Vitro Antitumor Evaluation of Acrylic Acid Derivatives Bearing Quinolinone Moiety as Novel Anticancer Agents.

BACKGROUND: Due to the emergence of resistance to available anticancer agents, the demand for new cytotoxic agents has grown. OBJECTIVE: This study aims at synthesis and cytotoxic evaluation of new acrylic acid derivatives bearing quinolinone and halogenated quinolinone derivatives against three cancer cell lines. METHODS: New acrylic acid derivatives bearing quinolinone and halogenated quinolinone moieties were synthesized and screened for their cytotoxic activity against breast MCF-7, liver HepG2, and colon HCT-116 cancer cell lines. RESULTS: Molecules 3 and 8 showed the most potent cytotoxic activity against HCT-116. DNA flow cytometry assay showed cell cycle arrest at the G1 phase and cellular apoptosis. Moreover, molecules 3 and 8 showed cyclin-dependent kinase 2 (CDK2) inhibitory activity compared to the untreated control sample. CONCLUSION: Acrylic acid derivatives bearing quinolinone and halogenated quinolinone moieties represent an important core and could be used as a lead for further development of drug compounds in order to achieve promising therapeutic results.

Eprosartan: A closer insight into its neuroprotective activity in rats with focal cerebral ischemia-reperfusion injury.

Eprosartan (EPRO), an angiotensin receptor type-1 (AT-1) blocker, exhibited neuroprotective activities in ischemic stroke resulting from focal cerebral ischemia in rats. The current study aimed to clarify the neuroprotective role of EPRO in middle carotid artery occlusion (MCAO)-induced ischemic stroke in rats. Fifty-six male Wistar rats were divided into four groups (n = 14 per group): sham-operated group, sham receiving EPRO (60 mg/kg/day, po) group, ischemia-reperfusion (IR) group, and IR receiving EPRO (60 mg/kg/day, po) group. MCAO led to a remarkable impairment in motor function together with stimulation of inflammatory and apoptotic pathways in the hippocampus of rats. After MCAO, the AT1 receptor in the brain was stimulated, resulting in activation of Janus kinase 2/signal transducers and activators of transcription 3 signaling generating more neuroinflammatory milieu and destructive actions on the hippocampus. Augmentation of caspase-3 level by MCAO enhanced neuronal apoptosis synchronized with neurodegenerative effects of oxidative stress biomarkers. Pretreatment with EPRO opposed motor impairment and decreased oxidative and apoptotic mediators in the hippocampus of rats. The anti-inflammatory activity of EPRO was revealed by downregulation of nuclear factor-kappa B and tumor necrosis factor-ß levels and (C-X-C motif) ligand 1 messenger RNA (mRNA) expression. Moreover, the study confirmed the role of EPRO against a unique pathway of hypoxia-inducible factor-1α and its subsequent inflammatory mediators. Furthermore, upregulation of caveolin-1 mRNA level was also observed along with decreased oxidative stress marker levels and brain edema. Therefore, EPRO showed neuroprotective effects in MCAO-induced cerebral ischemia in rats via attenuation of oxidative, apoptotic, and inflammatory pathways.

Heterodimeric GW7604 Derivatives: Modification of the Pharmacological Profile by Additional Interactions at the Coactivator Binding Site.

(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a derivative of (Z)-4-hydroxytamoxifen (4-OHT) was linked by diaminoalkane spacers to molecules that are known binders to the coactivator binding site (benzimidazole or thioxo-quinazolinone scaffolds). With this modification, an optimization of the pharmacological profile was achieved. The most active thioxo-quinazolinone derivative 16 showed extraordinarily high affinity to the estrogen receptor (ER) ß (RBA = 110%), inhibited effectively the coactivator recruitment (IC50 = 20.88 nM (ERα) and 28.34 nM (ERß)), acted as a pure estradiol (E2) antagonist in a transactivation assay (IC50 = 18.5 nM (ERα) and 7.5 nM (ERß)), and downregulated the ERα content in MCF-7 cells with an efficacy of 60% at 1 µM. The cytotoxicity was restricted to hormone-dependent MCF-7 (IC50 = 4.2 nM) and tamoxifen-resistant MCF-7TamR cells (IC50 = 476.6 nM). The compounds bearing a thioxo-quinazolinone moiety can therefore be assigned as pure E2-antagonistic selective ER degraders/downregulators. By contrast, the benzimidazole derivatives acted solely as pure antagonists without degradation of the ER.

Donor acceptor fluorophores: synthesis, optical properties, TD-DFT and cytotoxicity studies.

Donor-π-acceptor (D-π-A) fluorophores consisting of a donor unit, a π linker, and an acceptor moiety have attracted attention in the last decade. In this study, we report the synthesis, characterization, optical properties, TD-DFT, and cytotoxicity studies of 17 near infrared (NIR) D-π-A analogs which have not been reported so far to the best of our knowledge. These fluorophores have chloroacrylic acid as the acceptor unit and various donor units such as indole, benzothiazole, benzo[e]indole, and quinoline. The fluorophores showed strong absorption in the NIR (700-970 nm) region due to their enhanced intramolecular charge transfer (ICT) between chloroacrylic acid and the donor moieties connected with the Vilsmeier-Haack linker. The emission wavelength maxima of the fluorophores were in between 798 and 870 nm. Compound 20 with a 4-quinoline donor moiety showed an emission wavelength above 1000 nm in the NIR II window. The synthesized fluorophores were characterized by 1H NMR and 13C NMR, and their optical properties were studied. Time dependent density functional theory (TD-DFT) calculations showed that the charge transfer occurs from the donor groups (indole, benzothiazole, benzo[e]indole, and quinoline) to the acceptor chloroacrylic acid moiety. Fluorophores with [HOMO] to [LUMO+1] transitions were shown to possess a charge separation character. The cytotoxicity of selected fluorophores, 4, 7, 10 and 12 was investigated against breast cancer cell lines and they showed better activity than the anti-cancer agent docetaxel.

Molecular and Genetic Bases of Inhibition of Tick-Borne Encephalitis Virus Replication by Eprosartan and Ribavirin.

The antiviral activity of eprosartan (compound selected in silico) towards highly and low-virulent strains of tick-borne encephalitis virus was compared in vitro with activity of ribavirin. Study of the cytopathogenic activity of the virus on SPEV cells by ELISA, IFAT, and PCR showed similar results: both substances (eprosartan and ribavirin) promoted elimination of tick-borne encephalitis virus. Ribavirin exhibited intracellular inhibition towards both strains: the selectivity index for highly virulent Dal'negorsk strain was 160, for low-virulent Primorye-437 strain - 113. Eprosartan inhibited intracellular replication of Dal'negorsk strain (13.7) and less so that of Primorye-437 strain (2.9). The efficiency of virtual screening of the ligand (eprosartan) was demonstrated for highly virulent, but not low virulent tick-borne encephalitis strain.

Cardioprotective Effect of (Z)-2-Acetoxy-3-(3,4-Dihydroxyphenyl) Acrylic Acid: Inhibition of Apoptosis in Cardiomyocytes.

BACKGROUND: Although many studies have been performed to elucidate the molecular mechanisms of heart failure, an effective pharmacological therapy to protect cardiac tissues from severe loss of contractile function associated with heart failure after acute myocardial infarction (MI) has yet to be developed. METHODS: We examined the cardioprotective effects of (Z)-2-acetoxy-3-(3,4-dihydroxyphenyl) acrylic acid, a new compound with potent antioxidant and antiapoptotic activities in a rat model of heart failure. (Z)-2-Acetoxy-3-(3,4-dihydroxyphenyl) acrylic acid was systemically delivered to rats 6 weeks after MI at different doses (15, 30, and 60 mg/kg). Cardiac function was assessed by hemodynamic measurements. The expression of proinflammatory cytokines, apoptosis-related molecules, and markers of adverse ventricular remodeling was measured using RT-PCR and Western blot. RESULTS: Treatment with (Z)-2-acetoxy-3-(3,4-dihydroxyphenyl) acrylic acid significantly improved cardiac function, in particular by increasing dP/dt. Simultaneously, the expression of the proinflammatory cytokines TNF-α and IL-1ß was markedly reduced in the treatment group compared with the MI group. In addition, (Z)-2-acetoxy-3-(3,4-dihydroxyphenyl) acrylic acid-treated tissues displayed decreased expression of Bax, caspase-3, and caspase-9 and increased expression of Bcl-2, which was in part due to the promotion of Akt phosphorylation. CONCLUSION: These data demonstrated that (Z)-2-acetoxy-3-(3,4-dihydroxyphenyl) acrylic acid possesses potent cardioprotective effects against cardiac injury in a rat model of heart failure, which is mediated, at least in part, by suppression of the inflammatory and cell apoptosis responses.

A masked initiation region in retinoblastoma protein regulates its proteasomal degradation.

Retinoblastoma protein (Rb) is a tumor suppressor that binds and represses E2F transcription factors. In cervical cancer cells, human papilloma virus (HPV) protein E7 binds to Rb, releasing it from E2F to promote cell cycle progression, and inducing ubiquitination of Rb. E7-mediated proteasomal degradation of Rb requires action by another protease, calpain, which cleaves Rb after Lys 810. However, it is not clear why cleavage is required for Rb degradation. Here, we report that the proteasome cannot initiate degradation efficiently on full-length Rb. Calpain cleavage exposes a region that is recognized by the proteasome, leading to rapid proteolysis of Rb. These findings identify a mechanism for regulating protein stability by controlling initiation and provide a better understanding of the molecular mechanism underlying transformation by HPV.

Antimalarial 9-Methoxystrobilurins, Oudemansins, and Related Polyketides from Cultures of Basidiomycete Favolaschia Species.

Fourteen new compounds, oudemansins 1-4, oudemansinols 5-7, favolasins 8-10, favolasinin (12), polyketides 13-15, and (R,E)-2,4-dimethyl-5-phenyl-4-pentene-2,3-diol (16), together with nine known compounds were isolated from the basidiomycete fungus Favolaschia sp. BCC 18686. Two new compounds, favolasin E (11) and 9-oxostrobilurin E (17), were isolated from the closely related organism Favolaschia calocera BCC 36684 along with nine ß-methoxyacrylate-type derivatives. Compounds in the class of oudemansins and strobilurins exhibited moderate to strong antimalarial activity with relatively low cytotoxicity against Vero cells (African green monkey kidney fibroblasts). Potent antimalarial activity was demonstrated for 9-methoxystrobilurins G, K, and E (IC50 values 0.061, 0.089, and 0.14 µM, respectively). The structure-activity relationships (SAR) for antimalarial activity is proposed on the basis of the activity of the new and several known ß-methoxyacrylate derivatives in combination with the data from previously isolated compounds. Furthermore, several compounds showed specific cytotoxicity against NCI-187 cells (human small-cell lung cancer), although the SAR was different from that for antimalarial activity.

Inhibition of the ERK1/2-ubiquitous calpains pathway attenuates experimental pulmonary fibrosis in vivo and in vitro.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease with poor prognosis. Epithelial-mesenchymal transition (EMT) has been reported to play an important role in IPF. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) cascade, which regulates EMT and oncogenesis, has been implicated in the pathogenesis of IPF. Calpains, Ca2+-dependent cysteine proteinases that mediate controlled proteolysis of many specific substrates including epithelial cell marker E-cadherin, participate in organ fibrosis. Calpain-1 and calpain-2 of calpain family are ubiquitous calpains. ERK1/2 signaling stimulates the ubiquitous calpains activity in cancer development, but whether ERK1/2 signaling mediates the ubiquitous calpains activity in pulmonary fibrosis is unknown. Here we investigated whether inhibition of ERK1/2 signaling and the ubiquitous calpains attenuated experimental pulmonary fibrosis and examined the potential mechanism. Our results showed that inhibition of ERK1/2 signaling and the ubiquitous calpains both attenuated bleomycin (BLM)-induced lung fibrosis in mice. Inhibition of ERK1/2 signaling downregulated the expression of calpain-1 and calpain-2 in vivo and in vitro. We detected decreased E-cadherin expression and increased calpain-1 expression in IPF patients. Inhibition of ERK1/2 signaling and the ubiquitous calpains both suppressed the development of EMT in vivo and in vitro. Our study indicated that inhibition of the ERK1/2-ubiquitous calpains pathway protected pulmonary fibrosis from BLM, possibly via inhibition of EMT. Therefore, targeting ubiquitous calpains may be a potential strategy to attenuate IPF.

Cellular Uptake of Gold Nanoparticles and Their Movement in 3D Multicellular Tumor Spheroids: Effect of Molecular Weight and Grafting Density of Poly(2-hydroxyl ethyl acrylate).

It is known that the size of gold nanoparticles (GNPs) is not the only determining factor in the uptake by cells such as cancer cells. The surface functionalization plays a crucial role, in particular the nature of the ligand as well as the molecular weight and the grafting density. Here, poly(2-hydroxy ethyl) acrylate (pHEA) with molecular weights ranging from 10, 20 to 39 g mol-1 via reversible addition-fragmentation chain transfer polymerization is synthesized. These polymers are used directly to coat GNPs with sizes of 20, 40, and 70 nm as the trithiocarbonate functionality can strongly bind to the gold surface. The library of nine GNP is found to be nontoxic against lung carcinoma cells A549 and has negligible albumin protein absorption as determined by quartz crystal microbalance. Laser scanning confocal microscopy and flow cytometry reveal that GNP coated with medium length pHEA displays the highest cellular uptake while the effect of the size is not statistically significant. In contrast, multicellular tumor spheroids, which is a 3D model that simulates the tissue, enable the penetration of GNP coated with the longest pHEA chain while it also appears that smaller GNPs have now a clear advantage.

Design, synthesis and biological evaluation of benzoylacrylic acid shikonin ester derivatives as irreversible dual inhibitors of tubulin and EGFR.

In this study, a series of shikonin derivatives combined with benzoylacrylic had been designed and synthesized, which showed an inhibitory effect on both tubulin and the epidermal growth factor receptor (EGFR). In vitro EGFR and cell growth inhibition assay demonstrated that compound PMMB-317 exhibited the most potent anti-EGFR (IC50 = 22.7 nM) and anti-proliferation activity (IC50 = 4.37 µM) against A549 cell line, which was comparable to that of Afatinib (EGFR, IC50 = 15.4 nM; A549, IC50 = 6.32 µM). Our results on mechanism research suggested that, PMMB-317 could induce the apoptosis of A549 cells in a dose- and time-dependent manner, along with decrease in mitochondrial membrane potential (MMP), production of ROS and alterations in apoptosis-related protein levels. Also, PMMB-317 could arrest cell cycle at G2/M phase to induce cell apoptosis, and inhibit the EGFR activity through blocking the signal transduction downstream of the mitogen-activated protein MAPK pathway and the anti-apoptotic kinase AKT pathway; typically, such results were comparable to those of afatinib. In addition, PMMB-317 could suppress A549 cell migration through the Wnt/ß-catenin signaling pathway in a dose-dependent manner. Additionally, molecular docking simulation revealed that, PMMB-317 could simultaneously combine with EGFR protein (5HG8) and tubulin (1SA0) through various forces. Moreover, 3D-QSAR study was also carried out, which could optimize our compound through the structure-activity relationship analysis. Furthermore, the in vitro and in vivo results had collectively confirmed that PMMB-317 might serve as a promising lead compound to further develop the potential therapeutic anticancer agents.

Anti-chlamydial activities of cell-permeable hydrophobic dipeptide-containing derivatives.

The obligate intracellular bacteria chlamydia is major human pathogen that causes millions of trachoma, sexually transmitted infections and pneumonia worldwide. We serendipitously found that both calpain inhibitors z-Val-Phe-CHO and z-Leu-Nle-CHO showed marked inhibitory activity against chlamydial growth in human epithelial HeLa cells, whereas other calpain inhibitors not. These peptidomimetic inhibitors consist of N-benzyloxycarbonyl group and hydrophobic dipeptide derivatives. Both compounds strongly restrict the chlamydial growth even addition at the 12 h post infection. Notably, inhibitors-mediated growth inhibition of chlamydia was independent on host calpain activity. Electron microscopic analysis revealed that z-Val-Phe-CHO inhibited chlamydial growth by arresting bacterial cell division and RB-EB re-transition, but not by changing into persistent state. We searched and found that z-Leu-Leu-CHO and z-Phe-Ala-FMK also inhibited chlamydial growth. Neither biotin-hydrophobic dipeptide nor morpholinoureidyl-hydrophobic dipeptide shows inhibitory effects on chlamydial intracellular growth. Our results suggested the possibility of some chemical derivatives based on z-hydrophobic dipeptide group for future therapeutic usage to the chlamydial infectious disease.