We present a method for the synthesis of aryl-substituted squaramides through the Liebeskind-Srogl cross-coupling reaction performed on solid support. This approach offers a unique application for the cross-coupling reaction, allowing for the rapid and efficient production of a diverse range of substituted analogs within a combinatorial framework. Through our technique, we successfully synthesized derivatives that were previously unattainable. Additionally, the optimized conditions have been effectively applied in a scale-up reaction. The derivatives show potential for the treatment of drug-resistant tuberculosis.
- Publication type
- Journal Article MeSH
Developing methodological approaches for discovering novel pathways is a key challenge in the life science research. Biological pathways are regulated-in higher eukaryotes-by a vast diversity of linear peptide motifs that mediate combinatorial specificity in signal transduction pathways. The E3 ubiquitin ligase component (MDM2) is such a protein that interacts with target proteins containing linear motifs such as p53. Drug leads, such as Nutlin-3, that bind to the MDM2 hydrophobic pocket mimic p53 and can release p53 from MDM2 control and this can lead to cell death. However, these drug leads act allosterically, having agonist effects on MDM2's functions and there are other proteins whose steady state levels can be altered by Nutlin-3. As cell density can alter the proliferation state of cell populations, we examined the impact of Nutlin-3 on levels of newly synthesized proteins using pulse-SILAC mass spectrometry. The data demonstrate that at differing cell densities or population-wide proliferation rates, different newly synthesized proteins dominate the proteome landscape in a Nutlin-3 dependent manner. These data further confirm that the cell state in a population of cells can in turn impact on the MDM2 signalling landscape. This methodology forms a blueprint for biomarker discovery using clinical samples that can detect changes in the synthesis rate of proteins in cell populations treated with specific agents. Broader implications highlight tools that can be used to study allosteric regulation of protein-drug combinations.
- Keywords
- MDM2, P53, biomarker discovery, mass spectrometry, pulse‐SILAC,
- MeSH
- Imidazoles * pharmacology MeSH
- Humans MeSH
- Tumor Suppressor Protein p53 metabolism MeSH
- Piperazines * pharmacology MeSH
- Cell Proliferation drug effects MeSH
- Proteome * metabolism MeSH
- Proteomics methods MeSH
- Proto-Oncogene Proteins c-mdm2 metabolism MeSH
- Signal Transduction drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Imidazoles * MeSH
- MDM2 protein, human MeSH Browser
- Tumor Suppressor Protein p53 MeSH
- nutlin 3 MeSH Browser
- Piperazines * MeSH
- Proteome * MeSH
- Proto-Oncogene Proteins c-mdm2 MeSH
The evolution of proteins is primarily driven by the combinatorial assembly of a limited set of pre-existing modules known as protein domains. This modular architecture not only supports the diversity of natural proteins but also provides a robust strategy for protein engineering, enabling the design of artificial proteins with enhanced or novel functions for various industrial applications. Among these functions, oligomerization plays a crucial role in enhancing protein activity, such as by increasing the binding capacity of antibodies. To investigate the potential of engineering oligomerization, we examined the transferability of the sequence domain encoded by exon 5 (Ex5), which was originally responsible for the oligomerization of ameloblastin (AMBN). We designed a two-domain protein composed of Ex5 in combination with a monomeric, globular, and highly stable protein, specifically calmodulin (CaM). CaM represents the opposite protein character to AMBN, which is highly disordered and has a dynamic character. This engineered protein, termed eCaM, successfully acquired an oligomeric function, inducing self-assembly under specific conditions. Biochemical and biophysical analyses revealed that the oligomerization of eCaM is both concentration- and time-dependent, with the process being reversible upon dilution. Furthermore, mutating a key oligomerization residue within Ex5 abolished the self-assembly of eCaM, confirming the essential role of the Ex5 motif in driving oligomerization. Our findings demonstrate that the oligomerization properties encoded by Ex5 can be effectively transferred to a new protein context, though the positioning of Ex5 within the protein structure is critical. This work highlights the potential of enhancing monomeric proteins with oligomeric functions, paving the way for industrial applications and the development of proteins with tailored properties.
- Publication type
- Journal Article MeSH
PURPOSE: Patients with high-grade serous ovarian carcinoma (HGSOC) are virtually insensitive to immune checkpoint inhibitors (ICI) employed as standalone therapeutics, at least in part reflecting microenvironmental immunosuppression. Thus, conventional chemotherapeutics and targeted anticancer agents that not only mediate cytotoxic effects but also promote the recruitment of immune effector cells to the HGSOC microenvironment stand out as promising combinatorial partners for ICIs in this oncological indication. EXPERIMENTAL DESIGN: We harnessed a variety of transcriptomic, spatial, and functional assays to characterize the differential impact of neoadjuvant paclitaxel-carboplatin on the immunological configuration of paired primary and metastatic HGSOC biopsies as compared to neoadjuvant chemotherapy (NACT)-naïve HGSOC samples from five independent patient cohorts. RESULTS: We found NACT-driven endoplasmic reticulum stress and calreticulin exposure in metastatic HGSOC lesions culminates with the establishment of a dense immune infiltrate including follicular T cells (TFH cells), a prerequisite for mature tertiary lymphoid structure (TLS) formation. In this context, TLS maturation was associated with an increased intratumoral density of ICI-sensitive TCF1+PD1+ CD8+ T cells over their ICI-insensitive TIM-3+PD1+ counterparts. Consistent with this notion, chemotherapy coupled with a PD1-targeting ICI provided a significant survival benefit over either therapeutic approach in syngeneic models of HGSOC bearing high (but not low) tumor mutational burden. CONCLUSIONS: Altogether, our findings suggest that NACT promotes TLS formation and maturation in HGSOC lesions, de facto preserving an intratumoral ICI-sensitive T-cell phenotype. These observations emphasize the role of rational design, especially relative to the administration schedule, for clinical trials testing chemotherapy plus ICIs in patients with HGSOC. See related commentary by Bravo Melgar and Laoui, p. 10.
- MeSH
- CD8-Positive T-Lymphocytes * immunology drug effects MeSH
- Tertiary Lymphoid Structures * immunology pathology MeSH
- Hepatocyte Nuclear Factor 1-alpha * genetics metabolism MeSH
- Immune Checkpoint Inhibitors * therapeutic use pharmacology MeSH
- Carboplatin administration & dosage pharmacology therapeutic use MeSH
- Humans MeSH
- Tumor Microenvironment * immunology drug effects MeSH
- Ovarian Neoplasms * drug therapy immunology pathology MeSH
- Neoadjuvant Therapy methods MeSH
- Paclitaxel administration & dosage therapeutic use pharmacology MeSH
- Antineoplastic Combined Chemotherapy Protocols therapeutic use pharmacology MeSH
- Cystadenocarcinoma, Serous drug therapy pathology immunology MeSH
- Endoplasmic Reticulum Stress drug effects immunology MeSH
- Lymphocytes, Tumor-Infiltrating immunology drug effects metabolism MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Hepatocyte Nuclear Factor 1-alpha * MeSH
- Immune Checkpoint Inhibitors * MeSH
- Carboplatin MeSH
- Paclitaxel MeSH
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are intriguing compounds with potential pharmacological applications. While many RiPPs are known as antimicrobial agents, a limited number of RiPPs with anti-proliferative effects in cancer cells are available. Here we report the discovery of nostatin A (NosA), a highly modified RiPP belonging among nitrile hydratase-like leader peptide RiPPs (proteusins), isolated from a terrestrial cyanobacterium Nostoc sp. Its structure was established based on the core peptide sequence encoded in the biosynthetic gene cluster recovered from the producing strain and subsequent detailed nuclear magnetic resonance and high-resolution mass spectrometry analyses. NosA, composed of a 30 amino-acid peptide core, features a unique combination of moieties previously not reported in RiPPs: the simultaneous presence of oxazole/thiazole heterocycles, dehydrobutyrine/dehydroalanine residues, and a sactionine bond. NosA includes an isobutyl-modified proline residue, highly unusual in natural products. NosA inhibits proliferation of multiple cancer cell lines at low nanomolar concentration while showing no hemolysis. It induces cell cycle arrest in S-phase followed by mitochondrial apoptosis employing a mechanism different from known tubulin binding and DNA damaging compounds. NosA also inhibits Staphylococcus strains while it exhibits no effect in other tested bacteria or yeasts. Due to its novel structure and selective bioactivity, NosA represents an excellent candidate for combinatorial chemistry approaches leading to development of novel NosA-based lead compounds.
- MeSH
- Apoptosis * drug effects MeSH
- Cytostatic Agents pharmacology chemistry isolation & purification chemical synthesis MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Nostoc chemistry metabolism MeSH
- Drug Discovery MeSH
- Cell Proliferation * drug effects MeSH
- Antineoplastic Agents * pharmacology chemistry isolation & purification chemical synthesis MeSH
- Drug Screening Assays, Antitumor MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Cytostatic Agents MeSH
- Antineoplastic Agents * MeSH
Colorectal cancer (CRC) has the highest mortality rate among cancer types, emphasizing the need for auxiliaries to 5-fluorouracil (5-FU) due to resistance and side effects. Metabolites produced by probiotic bacteria exhibit promising anticancer properties against CRC. In the current study, the anticancer effects of cell extract of three potential probiotic lactobacilli strains isolated from camel milk, Lactobacillus helveticus, Lactobacillus gallinarum, and Lactiplantibacillus plantarum, as well as that of the standard probiotic strain Lacticaseibacillus rhamnosus GG (LGG), on the human colon cancer cell line (HT-29) and the normal HEK293 cell line separately or in combination with 5-FU, were evaluated. This study isolated strains from camel milk and compared their probiotic properties to those of LGG. The cell viability, cell apoptosis, and Th17 cytokine production were assessed using the MTT assay, acridine orange/ethidium bromide (AO/EB) staining, and flow cytometry techniques, respectively. The cell extracts of lactobacilli strains combined with 5-FU reduced HT-29 cell viability effectively and increased cell apoptosis. Nevertheless, the cell extracts of lactobacilli strains combined with 5-FU controlled the cytotoxic impact of 5-FU on HEK-293 cell viability and reduced cell apoptosis. No significant differences were observed among the strains. Moreover, the cell extracts from the strains combined with 5-FU increased the levels of cytokines IFN-γ, TNF-α, and IL-17A, all of which contribute to immunity against tumors. The performance of the studied strains was similar to that of the standard probiotic strain (LGG). The investigation revealed that cell extracts from lactobacilli strains may serve as a promising complementary anticancer treatment.
- Keywords
- 5-Fluorouracil, Anticancer activity, Colorectal cancer, Cytokine, Lactobacilli, Probiotic,
- Publication type
- Journal Article MeSH
Musculoskeletal infections (MIs) are among the most difficult-to-treat staphylococcal diseases due to antibiotic resistance. This has encouraged the development of innovative strategies, such as combination therapy, to combat MI. The aim of this study was to investigate the in vitro antistaphylococcal activity of anti-inflammatory drugs and the combined antimicrobial effect of celecoxib and oxacillin. The minimum inhibitory concentrations (MICs) of 17 anti-inflammatory drugs against standard strains and clinical isolates of S. aureus, including methicillin-resistant strains (MRSAs), were determined using the broth microdilution method. The fractional inhibitory concentration indices (FICIs) were evaluated using checkerboard assays. Celecoxib produced the most potent antistaphylococcal effect against all tested strains (MICs ranging from 32 to 64 mg/L), followed by that of diacerein against MRSA3 and MRSA ATCC 33592 (MIC 64 mg/L). Several synergistic effects were observed against the tested S. aureus strains, including MRSA (FICI ranging from 0.087 to 0.471). The strongest synergistic interaction (FICI 0.087) was against MRSA ATCC 33592 at a celecoxib concentration of 2 mg/L, with a 19-fold oxacillin MIC reduction (from 512 to 26.888 mg/L). This is the first report on the combined antistaphylococcal effect of celecoxib and oxacillin. These findings suggest celecoxib and its combination with oxacillin as perspective agents for research focused on the development of novel therapies for MI caused by S. aureus. This study further indicates that celecoxib could resensitize certain MRSA strains, in some cases, to be susceptible to β-lactams (e.g., oxacillin) that were not previously tested. It is essential to mention that the in vitro concentrations of anti-inflammatory drugs are higher than those typically obtained in patients. Therefore, an alternative option for its administration could be the use of a drug delivery system for the controlled slow release from an implant at the infection site.
- Keywords
- antibacterial activity, antistaphylococcal synergistic effect, methicillin-resistant S. aureus, musculoskeletal infections, non-steroidal anti-inflammatory drugs,
- MeSH
- Anti-Bacterial Agents * pharmacology MeSH
- Anti-Inflammatory Agents * pharmacology MeSH
- Celecoxib * pharmacology MeSH
- Humans MeSH
- Methicillin-Resistant Staphylococcus aureus * drug effects MeSH
- Microbial Sensitivity Tests * MeSH
- Oxacillin * pharmacology MeSH
- Staphylococcal Infections drug therapy microbiology MeSH
- Staphylococcus aureus * drug effects MeSH
- Drug Synergism * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Anti-Bacterial Agents * MeSH
- Anti-Inflammatory Agents * MeSH
- Celecoxib * MeSH
- Oxacillin * MeSH
Telemedicine is an emerging development in the healthcare domain, where the Internet of Things (IoT) fiber optics technology assists telemedicine applications to improve overall digital healthcare performances for society. Telemedicine applications are bowel disease monitoring based on fiber optics laser endoscopy, gastrointestinal disease fiber optics lights, remote doctor-patient communication, and remote surgeries. However, many existing systems are not effective and their approaches based on deep reinforcement learning have not obtained optimal results. This paper presents the fiber optics IoT healthcare system based on deep reinforcement learning combinatorial constraint scheduling for hybrid telemedicine applications. In the proposed system, we propose the adaptive security deep q-learning network (ASDQN) algorithm methodology to execute all telemedicine applications under their given quality of services (deadline, latency, security, and resources) constraints. For the problem solution, we have exploited different fiber optics endoscopy datasets with images, video, and numeric data for telemedicine applications. The objective is to minimize the overall latency of telemedicine applications (e.g., local, communication, and edge nodes) and maximize the overall rewards during offloading and scheduling on different nodes. The simulation results show that ASDQN outperforms all telemedicine applications with their QoS and objectives compared to existing state action reward state (SARSA) and deep q-learning network (DQN) policy during execution and scheduling on different nodes.
- Keywords
- Adaptive security deep q-learning network, Deep reinforcement learning, Fiber-optics, Internet of things, Telemedicine applications,
- MeSH
- Algorithms MeSH
- Deep Learning * MeSH
- Internet of Things * MeSH
- Humans MeSH
- Fiber Optic Technology MeSH
- Telemedicine * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The emergence of biofilm-induced drug tolerance poses a critical challenge to public healthcare management. Pseudomonas aeruginosa, a gram-negative opportunistic bacterium, is involved in various biofilm-associated infections in human hosts. Towards this direction, in the present study, a combinatorial approach has been explored as it is a demonstrably effective strategy for managing microbial infections. Thus, P. aeruginosa has been treated with cuminaldehyde (a naturally occurring phytochemical) and gentamicin (an aminoglycoside antibiotic) in connection to the effective management of the biofilm challenges. It was also observed that the test molecules could show increased antimicrobial activity against P. aeruginosa. A fractional inhibitory concentration index (FICI) of 0.65 suggested an additive interaction between cuminaldehyde and gentamicin. Besides, a series of experiments such as crystal violet assay, estimation of extracellular polymeric substance (EPS), and microscopic images indicated that an enhanced antibiofilm activity was obtained when the selected compounds were applied together on P. aeruginosa. Furthermore, the combination of the selected compounds was found to reduce the secretion of virulence factors from P. aeruginosa. Taken together, this study suggested that the combinatorial application of cuminaldehyde and gentamicin could be considered an effective approach towards the control of biofilm-linked infections caused by P. aeruginosa.
- Keywords
- Pseudomonas aeruginosa, Antibiofilm, Antimicrobial, Cuminaldehyde, Gentamicin,
- MeSH
- Anti-Bacterial Agents * pharmacology MeSH
- Benzaldehydes * pharmacology MeSH
- Biofilms * drug effects MeSH
- Cymenes pharmacology MeSH
- Virulence Factors MeSH
- Gentamicins * pharmacology MeSH
- Humans MeSH
- Microbial Sensitivity Tests * MeSH
- Pseudomonas aeruginosa * drug effects physiology MeSH
- Drug Synergism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Anti-Bacterial Agents * MeSH
- Benzaldehydes * MeSH
- cuminaldehyde MeSH Browser
- Cymenes MeSH
- Virulence Factors MeSH
- Gentamicins * MeSH
Fixation of atmospheric N2 by free-living diazotrophs accounts for an important proportion of nitrogen naturally introduced to temperate grasslands. The effect of plants or fertilization on the general microbial community has been extensively studied, yet an understanding of the potential combinatorial effects on the community structure and activity of free-living diazotrophs is lacking. In this study we provide a multilevel assessment of the single and interactive effects of different long-term fertilization treatments, plant species and vicinity to roots on the free-living diazotroph community in relation to the general microbial community in grassland soils. We sequenced the dinitrogenase reductase (nifH) and the 16S rRNA genes of bulk soil and root-associated compartments (rhizosphere soil, rhizoplane and root) of two grass species (Arrhenatherum elatius and Anthoxanthum odoratum) and two herb species (Galium album and Plantago lanceolata) growing in Austrian grassland soils treated with different fertilizers (N, P, NPK) since 1960. Overall, fertilization has the strongest effect on the diazotroph and general microbial community structure, however with vicinity to the root, the plant effect increases. Despite the long-term fertilization, plants strongly influence the diazotroph communities emphasizing the complexity of soil microbial communities' responses to changing nutrient conditions in temperate grasslands.
- MeSH
- Nitrogen Fixation MeSH
- Plant Roots * microbiology MeSH
- Poaceae MeSH
- Oxidoreductases genetics metabolism MeSH
- Grassland * MeSH
- Fertilizers * analysis MeSH
- Soil chemistry MeSH
- Soil Microbiology * MeSH
- Rhizosphere MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Oxidoreductases MeSH
- Fertilizers * MeSH
- Soil MeSH
- RNA, Ribosomal, 16S MeSH
