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•Twenty novel 2-substituted quinoline-4-carboxylic acids were synthesized.•Several compounds exhibited good hDHODH inhibitory activity.•Very low cytotoxicity against healthy HaCaT ...cell was observed.•Optimal lipophilicity was determined at physiological pH.•Molecular docking distinguished highly active from low active hDHODH inhibitors.
Twenty novel 2-substituted quinoline-4-carboxylic acids bearing amide moiety were designed and synthesized by Doebner reaction. Human dihydroorotate dehydrogenase (hDHODH) was recognized as a biological target and all compounds were screened as potential hDHODH inhibitors in an enzyme inhibition assay. The prepared heterocycles were also evaluated for their cytotoxic effects on the healthy HaCaT cell line while lipophilic properties were considered on the basis of experimentally determined logD values at physiological pH. The most promising compound 5j, with chlorine at para-position of terminal phenyl ring, showed good hDHODH inhibitory activity, low cytotoxicity, and optimal lipophilicity. The bioactive conformation of 5j on the hDHODH, determined by means of molecular docking, revealed the compound’s pharmacology and provide guidelines for further lead optimization.
Osteopetrosis is a heterogeneous group of rare hereditary diseases characterized by increased bone mass of poor quality. Autosomal-dominant osteopetrosis type II (ADOII) is most often caused by ...mutation of the
CLCN7
gene leading to impaired bone resorption. Autosomal recessive osteopetrosis (ARO) is a more severe form and is frequently accompanied by additional morbidities. We report an adult male presenting with classical clinical and radiological features of ADOII. Genetic analyses showed no amino-acid-converting mutation in
CLCN7
but an apparent haploinsufficiency and suppression of
CLCN7
mRNA levels in peripheral blood mononuclear cells. Next generation sequencing revealed low-frequency intronic homozygous variations in
CLCN7
, suggesting recessive inheritance. In silico analysis of an intronic duplication c.595-120_595-86dup revealed additional binding sites for Serine- and Arginine-rich Splicing Factors (SRSF), which is predicted to impair
CLCN7
expression. Quantitative backscattered electron imaging and histomorphometric analyses revealed bone tissue and material abnormalities. Giant osteoclasts were present and additionally to lamellar bone, and abundant woven bone and mineralized cartilage were observed, together with increased frequency and thickness of cement lines. Bone mineralization density distribution (BMDD) analysis revealed markedly increased average mineral content of the dense bone (CaMean
T
-score + 10.1) and frequency of bone with highest mineral content (CaHigh
T
-score + 19.6), suggesting continued mineral accumulation and lack of bone remodelling. Osteocyte lacunae sections (OLS) characteristics were unremarkable except for an unusually circular shape. Together, our findings suggest that the reduced expression of
CLCN7
mRNA in osteoclasts, and possibly also osteocytes, causes poorly remodelled bone with abnormal bone matrix with high mineral content. This together with the lack of adequate bone repair mechanisms makes the material brittle and prone to fracture. While the skeletal phenotype and medical history were suggestive of ADOII, genetic analysis revealed that this is a possible mild case of ARO due to deep intronic mutation.
Mutations of the endoplasmic reticulum (ER)-stress transducer OASIS (encoded by CREB3L1), cause severe recessive osteogenesis imperfecta (OI) not compatible with surviving the neonatal period, as has ...been shown in two unrelated families through a whole gene deletion vs. a qualitative alteration of OASIS. Heterozygous carriers in the described families have exhibited a mild phenotype. OASIS is a transcription factor highly expressed in osteoblasts, and OASIS−/− mice exhibit severe osteopenia and spontaneous fractures. Here, we expand the clinical spectrum by a detailed phenotypic characterization of the first case of OASIS-associated OI surviving the neonatal period, with heterozygous family members being unaffected.
All OI-associated genes were sequenced. Primary human osteoblast-like cell (hOB) and fibroblast (FB) cultures were obtained for qPCR, and steady-state collagen biochemistry. FB, hOB and skin biopsies were ultrastructurally analyzed. Bone was analyzed by μCT, histomorphometry, quantitative backscattered electron imaging (qBEI), and Raman microspectroscopy.
The proband, a boy with severe OI, had blue sclera and tooth agenesis. A homozygous CREB3L1 stop codon mutation was detected by sequencing, while several family members were heterozygotes. Markedly low levels of CREB3L1 mRNA were confirmed by qPCR in hOBs (16%) and FB (21%); however, collagen I levels were only reduced in hOBs (5–10%). Electron microscopy of hOBs showed pronounced alterations, with numerous myelin figures and diminished RER vs. normal ultrastructure of FB. Bone histomorphometry and qBEI were similar to collagen I OI, with low trabecular thickness and mineral apposition rate, and increased bone matrix mineralization. Raman microspectroscopy revealed low level of glycosaminoglycans. Clinical response to life-long bisphosphonate treatment was as expected in severe OI with steadily increasing bone mineral density, but despite this the boy suffered repeated childhood fractures.
Deficiency of OASIS can cause severe OI compatible with surviving the neonatal period. A marked decrease of collagen type I transcription was noted in bone tissue, but not in skin, and ultrastructure of hOBs was pathological. Results also suggested OASIS involvement in glycosaminoglycan secretion in bone.
•CREB3L1 stop-codon causes OI type III phenotype similar to classical collagen I OI.•Heterozygous CREB3L1 stop-codon carriers without clinical phenotype.•OASIS-associated OI clinical phenotype widened and bisphosphonate effect as expected.•Tissue-specific effects, such as affected collagen I-transcription in bone, not skin•Homozygous CREB3L1 stop-codon associates with low levels of bone glycosaminoglycans.
The design and study of efficient polymer-based drug delivery systems for the controlled release of anticancer drugs is one of the pillars of nanomedicine. The fight against metastatic and invasive ...cancers demands therapeutic candidates with increased and selective toxicity towards malignant cells, long-term activity and reduced side effects. In this sense, polyphosphazene nanocarriers were synthesized for the sustained release of the anticancer drugs camptothecin (CPT) and epirubicin (EPI). Linear poly(dichloro)phosphazene was modified with lipophilic tocopherol or testosterone glycinate, with antioxidant and antitumor activity, and with hydrophilic Jeffamine M1000 to obtain different polyphosphazene nanocarriers. It allowed us to encapsulate the lipophilic CPT and the more hydrophilic EPI. The encapsulation process was carried out via solvent exchange/precipitation, attaining a 9.2-13.6 wt% of CPT and 0.3-2.4 wt% of EPI. CPT-loaded polyphosphazenes formed 140-200 nm aggregates in simulated body physiological conditions (PBS, pH 7.4), resulting in an 80-100-fold increase of CPT solubility. EPI-loaded polyphosphazenes formed 250 nm aggregates in an aqueous medium. CPT and EPI release (PBS, pH 7.4, 37 °C) was monitored for 202 h, being almost linear during the first 8 h. The slow release of testosterone and tocopherol was also sustained for 150 h in PBS (pH 7.4 and 6.0) at 37 °C. The co-delivery of testosterone or tocopherol and the anticancer drugs from the nanocarriers was expected. Cells of the human breast cancer cell line MCF-7 demonstrated good uptake of anticancer-drug-loaded nanocarriers after 6 h. Similarly, MCF-7 spheroids showed good uptake of the anticancer-drug-loaded aggregates after 72 h. Almost all anticancer-drug-loaded polyphosphazenes exhibited similar or superior toxicity against MCF-7 cells and spheroids when compared to raw anticancer drugs. Additionally, cell-cycle arrest in the G2/M phase was increased in response to the drug-loaded nanocarriers. Almost no toxicity of anticancer-drug-loaded aggregates against primary human lung fibroblasts was observed. Furthermore, the aggregates displayed no hemolytic activity, which is in contrast to the parent anticancer drugs. Consequently, synthesized polyphosphazene-based nanocarriers might be potential nanomedicines for chemotherapy.
The direct electrochemical conversion of ethanol, a sustainable fuel, is an alternative sustainable technology of the future. In this study, membrane electrode assemblies with different electrode ...configurations for an alkaline direct ethanol fuel cell were fabricated and tested in a fuel cell device. The configurations include a catalyst-coated substrate (CCS), a catalyst-coated membrane (CCM), and a mixture of these two fabrication options. Two different anion exchange membranes were used to perform a comprehensive analysis. The fabricated CCSs and CCMs were characterized with single cell measurements, electrochemical impedance spectroscopy and scanning electron microscopy. In addition, the swelling behavior of the membranes in alkaline solution was investigated in order to obtain information for CCM production. The results of the experimental electrochemical tests show that the CCS approach provides higher power densities (42.4 mW cm-2) than the others, regardless of the membrane type.
A water-soluble hydrolysate of silk fibroin (SF) (~30 kDa) was esterified with tocopherol, ergocalciferol, and testosterone to form SF aggregates for the controlled delivery of the anticancer drug ...camptothecin (CPT). Elemental analysis and 1H NMR spectroscopy showed a degree of substitution (DS) on SF of 0.4 to 3.8 mol %. Yields of 58 to 71% on vitamins- and testosterone-grafted SF conjugates were achieved. CPT was efficiently incorporated into the lipophilic core of SF aggregates using a dialysis–precipitation method, achieving drug contents of 6.3–8.5 wt %. FTIR spectra and DSC thermograms showed that tocopherol- and testosterone-grafted SF conjugates predominantly adopted a β-sheet conformation. After the esterification of tyrosine residues on SF chains with the vitamin or testosterone, the hydrodynamic diameters almost doubled or tripled that of SF. The zeta potential values after esterification increased to about −30 mV, which favors the stability of aggregates in aqueous medium. Controlled and almost quantitative release of CPT was achieved after 6 days in PBS at 37 °C, with almost linear release during the first 8 h. MCF-7 cancer cells exhibited good uptake of CPT-loaded SF aggregates after 6 h, causing cell death and cell cycle arrest in the G2/M phase. Substantial uptake of the CPT-loaded aggregates into MCF-7 spheroids was shown after 3 days. Furthermore, all CPT-loaded SF aggregates demonstrated superior toxicity to MCF-7 spheroids compared with parent CPT. Blank SF aggregates induced no hemolysis at pH 6.2 and 7.4, while CPT-loaded SF aggregates provoked hemolysis at pH 6.2 but not at pH 7.4. In contrast, parent CPT caused hemolysis at both pH tested. Therefore, CPT-loaded SF aggregates are promising candidates for chemotherapy.
Side‐chain‐to‐side‐chain cyclization is frequently used to stabilize the α‐helical conformation of short peptides. In a previous study, we incorporated a lactam bridge between the side chains of ...Lys‐i and Asp‐i+4 in the nonapeptide 1Y, cyclo‐(2,6)‐(Ac‐VKRLQDLQY‐NH2), an artificial ligand of the inhibitor of DNA binding and cell differentiation (ID) protein with antiproliferative activity on cancer cells. Herein, we show that only the cyclized five‐residue segment adopts a helical turn whereas the C‐terminal residues remain flexible. Moreover, we present nine 1Y analogs arising from different combinations of hydrophobic residues (leucine, isoleucine, norleucine, valine, and tyrosine) at positions 1, 4, 7, and 9. All cyclopeptides except one build a lactam‐bridged helical turn; however, residue‐4 reveals less helix character than the neighboring Arg‐3 and Gln‐5, especially with residue‐4 being isoleucine, valine, and tyrosine. Surprisingly, only two cyclopeptides exhibit helix propagation until the C‐terminus, whereas the others share a remarkable outward tilting of the backbone carbonyl of the lactam‐bridged Asp‐6 (>40° deviation from the orientation parallel to the helix axis), which prevents the formation of the H‐bond between Arg‐3 CO and residue‐7 NH: As a result, the propagation of the helix beyond the lactam‐bridged sequence becomes unfavorable. We conclude that, depending on the amino‐acid sequence, the lactam bridge between Lys‐i and Asp‐i+4 can stabilize a helical turn but deviations from the ideal helix geometry are possible: Indeed, besides the outward tilting of the backbone carbonyls, the residues per turn increased from 3.6 (typical of a regular α‐helix) to 4.2, suggesting a partial helix unwinding.
The NMR solution structures of several Lysi ‐Aspi+4 lactam‐bridged short helical peptides showed a significant outward tilt of the backbone carbonyls and 4.2 residues per turn.
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