Impurity study of tecovirimat.

This article delineates the systematic identification, synthesis, and impurity control methods used during the manufacturing process development of tecovirimat, an antiviral drug that treats monkeypox. Critical impurities were synthesized, and their chemical structure was confirmed through NMR analysis, GC, and HPLC mass spectrometry. The results established a thorough approach to identify, address, and control impurities to produce high-quality tecovirimat drug substance in accordance with International Conference on Harmonization (ICH)-compliant standards. This study is the first of its kind to evaluate both process and genotoxic impurities in tecovirimat, demonstrating effective control measures during commercial sample investigations and scaling up to a 60-kg batch size. The findings highlight the importance of critical impurity characterization and control in pharmaceutical development and production to ensure the safety and efficacy of the final product.

Microplastics in marine-derived traditional Chinese medicine, potential threat to patients.

Microplastics (MPs) contamination is widely found in marine organisms. Marine traditional Chinese medicines (MTCM) are derived from marine organisms, but there are no relevant reports on detecting MPs in MTCM. This study selected samples of MTCM from two representative pharmaceutical companies, Brand F and Brand Z, including mother-of-pearl, stone cassia, seaweed, pumice, oyster, kombu, calcined Concha Arcae, cuttlebone, and clam shell to detect and analyze the presence of MPs. The abundance, type, color, size, and composition of MPs were investigated. Varying degrees of MPs contamination was present in all MTCM. The abundance of MPs in different MTCM ranged from 0.07 to 9.53 items/g. Their type, color, and size are similar, mainly fiber, transparent and size <2 mm. The composition of MPs is primarily made of cotton, cellulose and rayon. This study contributes to the first record of MPs in MTCM. Our results show that microplastic pollution is common in MTCM, which may cause potential risk to patients consuming MTCM.

Total Synthesis of Pseudouridimycin.

Pseudouridimycin (1), a potent antibiotic against both Gram-positive and Gram-negative bacteria including multi-drug-resistant strains with a new mode of action isolated from Streptomyces sp., was synthesized by a convergent strategy from 5'-amino-pseudouridine 5 and N-hydroxy-dipeptide 26 in 23% total yield. The key intermediate 26 was synthesized by hydroxylaminolysis of the nitrone derived from glutamine and subsequent glycylation with glycine chloride. The synthetic method provides an efficient and practical way for the synthesis of N-hydroxylated peptidyl nucleoside.

Switching Effect of p-CuO Nanotube/n-In2S3 Nanosheet Heterostructures for High-Performance Room-Temperature H2S Sensing.

High operating temperature and low response restrict the application of H2S sensors. Due to the strong chemical affinity of CuO to H2S and the large band gap and high stability of ß-In2S3, CuO nanotube/In2S3 nanosheet p/n heterostructures have been delicately designed for binder-free gas sensors by a facile method consisting of sputtering, chemical etching, and annealing. A switching effect of H2S concentration on the response of CuO/In2S3 gas sensors has been observed. When exposed to low-concentration H2S (1-10 ppm), the response is less than 0.10 and dominated by the surface-type adsorption-desorption process between CuO and H2S. When exposed to high-concentration H2S, the sensor exhibits a superior response of 3511 toward 50 ppm H2S, considerable selectivity, and long-term stability at room temperature. This dramatically enhanced response can be explained by the transformed junction from the CuO/In2S3 heterojunction to the CuS/In2S3 Schottky junction. These results suggest that the binder-free ceramic tube-type CuO/In2S3 gas sensor with considerable performance will have promising potential for H2S gas detection. Moreover, this method provides an effective strategy to fabricate other binder-free gas sensors.

A Neural Network with Convolutional Module and Residual Structure for Radar Target Recognition Based on High-Resolution Range Profile.

In the conventional neural network, deep depth is required to achieve high accuracy of recognition. Additionally, the problem of saturation may be caused, wherein the recognition accuracy is down-regulated with the increase in the number of network layers. To tackle the mentioned problem, a neural network model is proposed incorporating a micro convolutional module and residual structure. Such a model exhibits few hyper-parameters, and can extended flexibly. In the meantime, to further enhance the separability of features, a novel loss function is proposed, integrating boundary constraints and center clustering. According to the experimental results with a simulated dataset of HRRP signals obtained from thirteen 3D CAD object models, the presented model is capable of achieving higher recognition accuracy and robustness than other common network structures.

Discovery of (2E)-3-{2-butyl-1-[2-(diethylamino)ethyl]-1H-benzimidazol-5-yl}-N-hydroxyacrylamide (SB939), an orally active histone deacetylase inhibitor with a superior preclinical profile.

A series of 3-(1,2-disubstituted-1H-benzimidazol-5-yl)-N-hydroxyacrylamides (1) were designed and synthesized as HDAC inhibitors. Extensive SARs have been established for in vitro potency (HDAC1 enzyme and COLO 205 cellular IC(50)), liver microsomal stability (t(1/2)), cytochrome P450 inhibitory (3A4 IC(50)), and clogP, among others. These parameters were fine-tuned by carefully adjusting the substituents at positions 1 and 2 of the benzimidazole ring. After comprehensive in vitro and in vivo profiling of the selected compounds, SB939 (3) was identified as a preclinical development candidate. 3 is a potent pan-HDAC inhibitor with excellent druglike properties, is highly efficacious in in vivo tumor models (HCT-116, PC-3, A2780, MV4-11, Ramos), and has high and dose-proportional oral exposures and very good ADME, safety, and pharmaceutical properties. When orally dosed to tumor-bearing mice, 3 is enriched in tumor tissue which may contribute to its potent antitumor activity and prolonged duration of action. 3 is currently being tested in phase I and phase II clinical trials.

Acylurea connected straight chain hydroxamates as novel histone deacetylase inhibitors: Synthesis, SAR, and in vivo antitumor activity.

Thirty-six novel acylurea connected straight chain hydroxamates were designed and synthesized. Structure-activity relationships (SAR) were established for the length of linear chain linker and substitutions on the benzoylurea group. Compounds 5g, 5i, 5n, and 19 showed 10-20-fold enhanced HDAC1 potency compared to SAHA. In general, the cellular potency pIC(50) (COLO205) correlates with enzymatic potency pIC(50) (HDAC1). Compound 5b (SB207), a structurally simple and close analogue to SAHA, is more potent against HDAC1 and HDAC6 compared to the latter. As a representative example of this series, good in vitro enzymatic and cellular potency plus an excellent pharmacokinetic profile has translated into better efficacy than SAHA in both prostate cancer (PC3) and colon cancer (HCT116) xenograft models.

N-Hydroxy-1,2-disubstituted-1H-benzimidazol-5-yl acrylamides as novel histone deacetylase inhibitors: design, synthesis, SAR studies, and in vivo antitumor activity.

A series of N-hydroxy-1,2-disubstituted-1H-benzimidazol-5-yl acrylamides were designed and synthesized as novel HDAC inhibitors. General SAR has been established for the substituents at positions 1 and 2, as well as the importance of the ethylene group and its attachment to position 5. Optimized compounds are much more potent than SAHA in both enzymatic and cellular assays. A representative compound, 23 (SB639), has demonstrated antitumor activity in a colon cancer xenograft model.

Function of the SIRT1 protein deacetylase in cancer.

The NAD(+)-dependent protein deacetylase SIRT1 is linked to cellular survival pathways by virtue of keeping the tumor suppressor gene p53 and members of the forkhead transcription factor family deacetylated. To validate SIRT1 as a therapeutic anti-cancer target, we performed immunohistochemistry experiments to study the in vivo expression of SIRT1 in cancer specimens. We show that human SIRT1 is highly expressed in cancer cell lines as well as in tissue samples from colon carcinoma patients. Interestingly, there is a strong cytosolic component in the SIRT1 expression pattern. We further characterized SIRT1 in p53-wild-type and -mutant cell lines and show that SIRT1 mRNA-knockdown leads to a p53-independent decrease of cell proliferation and induction of apoptosis. In addition, SIRT1 expression has been found to be inducible upon DNA damage. A previously discovered small molecule SIRT1 inhibitor with nanomolar in vitro activity has been tested in cancer relevant assays. The SIRT1 inhibitory compound showed no potent anti-proliferative activity despite hitting its molecular target within tumor cells. From these studies we conclude that it may not be sufficient to block the catalytic function of SIRT1, and that its survival effects may be mainly brought about by means other then the deacetylase function. The increased cytosolic expression of SIRT1 in cancer cells could be an indicator of such novel functions.

SIRT1 modulating compounds from high-throughput screening as anti-inflammatory and insulin-sensitizing agents.

The nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase SIRT1 has been linked to fatty acid metabolism via suppression of peroxysome proliferator-activated receptor gamma (PPAR-gamma) and to inflammatory processes by deacetylating the transcription factor NF-kappaB. First, modulation of SIRT1 activity affects lipid accumulation in adipocytes, which has an impact on the etiology of a variety of human metabolic diseases such as obesity and insulin-resistant diabetes. Second, activation of SIRT1 suppresses inflammation via regulation of cytokine expression. Using high-throughput screening, the authors identified compounds with SIRT1 activating and inhibiting potential. The biological activity of these SIRT1-modulating compounds was confirmed in cell-based assays using mouse adipocytes, as well as human THP-1 monocytes. SIRT1 activators were found to be potent lipolytic agents, reducing the overall lipid content of fully differentiated NIH L1 adipocytes. In addition, the same compounds have anti-inflammatory properties, as became evident by the reduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). In contrast, a SIRT1 inhibitory compound showed a stimulatory activity on the differentiation of adipocytes, a feature often linked to insulin sensitization.

Zebrafish atonal homologue zath3 is expressed during neurogenesis in embryonic development.

Basic helix-loop-helix (bHLH) transcriptional activators function in development of various cell lineages, including the central nervous system. One of the bHLH proteins, Math3/Xath3/NeuroM, was suggested to act as a late proneural gene in the mouse, Xenopus, and chick. Here, we isolated a zebrafish homologue, named zath3, and analyzed its expression pattern in zebrafish embryos. In the neural plate, zath3 is expressed first in the primordia of the tegmentum and trigeminal ganglia and three classes of primary neurons: sensory neurons, interneurons, and motor neurons. During later development, zath3 transcripts were localized along the boundaries of the optic tectum in the midbrain and rhombomeres of the hindbrain. Analyses of zath3 expression in three mid-hindbrain boundary mutants, acerebellar, no isthmus, and spiel-ohne-grensen, indicated that distribution of zath3 mRNAs in the midbrain and hindbrain was dramatically disturbed. In addition, these mutants also affect expression of zath3 in the neuroretina.

Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter.

A 1,934-bp muscle-specific promoter from the zebrafish mylz2 gene was isolated and characterized by transgenic analysis. By using a series of 5' promoter deletions linked to the green fluorescent protein (gfp) reporter gene, transient transgenic analysis indicated that the strength of promoter activity appeared to correlate to the number of muscle cis-elements in the promoter and that a minimal -77-bp region was sufficient for a relatively strong promoter activity in muscle cells. Stable transgenic lines were obtained from several mylz2-gfp constructs. GFP expression in the 1,934-bp promoter transgenic lines mimicked well the expression pattern of endogenous mylz2 mRNA in both somitic muscle and nonsomitic muscles, including fin, eye, jaw, and gill muscles. An identical pattern of GFP expression, although at a much lower level, was observed from a transgenic line with a shorter 871-bp promoter. Our observation indicates that there is no distinct cis-element for activation of mylz2 in different skeletal muscles. Furthermore, RNA encoding a dominant negative form of cAMP-dependent protein kinase A was injected into mylz2-gfp transgenic embryos and GFP expression was significantly reduced due to an expanded slow muscle development at the expense of GFP-expressing fast muscle. The mylz2-gfp transgene was also transferred into two zebrafish mutants, spadetail and chordino, and several novel phenotypes in muscle development in these mutants were discovered.

The functional specificity of NeuroD is defined by a single amino acid residue (N11) in the basic domain.

In zebrafish, the basic helix-loop-helix (bHLH) gene neuroD specifies distinct neurons in the spinal cord. A preliminary experiment indicated that a related bHLH gene, ndr1a, normally expressed only in the olfactory organ in late embryos, also functions as neuroD to induce ectopic formation of spinal cord neurons in early embryos after introduction of its mRNA into early embryos. To define the functional specificity of these bHLH proteins, several mutant forms with selected point mutations in the basic domain were constructed and tested for inducing sensory neurons in the spinal cord. Our data indicate that the functional specificity of NeuroD to define sensory neurons is mainly due to a single residue (asparagine 11) in its basic domain.

Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.

A zebrafish cDNA encoding a novel keratin protein was characterized and named keratin8, or krt8. krt8 expression was initiated at 4.5 hr postfertilization, immediately after the time of zygotic genome activation. The expression is limited to a single layer of envelope cells on the surface of embryos and, in later stages, it also appears in the innermost epithelial layer of the anterior- and posteriormost portions of the digestive tract. In adult, its expression was limited to the surface layer of stratified epithelial tissues, including skin epidermis and epithelia of mouth, pharynx, esophagus, and rectum but not in the gastral and intestinal epithelia. By using a 2.2-kb promoter from krt8, several stable green fluorescent protein (gfp) transgenic zebrafish lines were established. All of these transgenic lines displayed GFP expression in tissues mentioned above except for the rectum; therefore, the pattern of transgenic GFP expression is essentially identical to that of the endogenous krt8 mRNAs. krt8-GFP fusion protein was also expressed in zebrafish embryos under a ubiquitous promoter, and the fusion protein was capable of assembling into intermediate filaments only in the epithelia that normally expressed krt8 mRNAs, indicating the specificity of keratin assembly in vivo.