Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without ...perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.
The discovery of protein ligands, capable of forming a reversible covalent bond with amino acid residues on a protein target of interest, may represent a general strategy for the discovery of potent ...small‐molecule inhibitors. We analyzed the ability of different aromatic aldehydes to form imines by reaction with lysine using 1H NMR techniques. 2‐Hydroxybenzaldehyde derivatives were found to efficiently form imines in the millimolar concentration range. These benzaldehyde derivatives could increase the binding affinity of protein ligands towards the cognate protein target. Affinity maturation was achieved not only by displaying ligand and aldehyde moieties on two complementary locked nucleic acid strands but also by incorporating the binding fragments in a single small‐molecule ligand. The affinity gain was only observed when lysine residues were accessible in the immediate surroundings of the ligand‐binding site and could be abrogated by quenching with a molar excess of hydroxylamine.
A lock on affinity: The simultaneous display of protein ligands and of 2‐hydroxybenzaldehyde derivatives on either two complementary LNA strands or a small‐organic molecule leads to an enhanced binding affinity by reversible covalent interaction with proximal lysine residues.
Differences in genetics and receptor expression (phenotypes) of invasive ductal breast cancer (IDC) impact on prognosis and treatment response. Immunohistochemistry (IHC), the most used technique for ...IDC phenotyping, has some limitations including its invasiveness. We explored the possibility of contrast-enhanced positron emission tomography magnetic resonance (CE-FDG PET/MR) to discriminate IDC phenotypes.
21 IDC patients with IHC assessment of oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor-2 (HER2), and antigen Ki-67 (Ki67) underwent CE-FDG PET/MR. Magnetic resonance-perfusion biomarkers, apparent diffusion coefficient (ADC), and standard uptake value (SUV) were compared with IHC markers and phenotypes, using a Student's t-test and one-way ANOVA.
ER/PR- tumours demonstrated higher Kep
and SUV
than ER or PR+ tumours. HER2- tumours displayed higher ADC
, Kep
, and SUV
than HER2+tumours. Only ADC
discriminated Ki67⩽14% tumours (lower ADC
) from Ki67>14% tumours. PET/MR biomarkers correlated with IHC phenotype in 13 out of 21 patients (62%; P=0.001).
Positron emission tomography magnetic resonance might non-invasively help discriminate IDC phenotypes, helping to optimise individual therapy options.
The aim of the present study was to evaluate the performance of whole-body diffusion-weighted imaging (WB-DWI), whole-body positron emission tomography with computed tomography (WB-PET/CT), and ...whole-body positron emission tomography with magnetic resonance imaging (WB-PET/MRI) in staging patients with untreated invasive ductal carcinoma of the breast. Fifty-one women with newly diagnosed invasive ductal carcinoma of the breast underwent WB-DWI, WB-PET/CT and WB-PET/MRI before treatment. A radiologist and a nuclear medicine physician reviewed in consensus the images from the three modalities and searched for occurrence, number and location of metastases. Final staging, according to each technique, was compared. Pathology and imaging follow-up were used as the reference. WB-DWI, WB-PET/CT and WB-PET/MRI correctly and concordantly staged 33/51 patients: stage IIA in 7 patients, stage IIB in 8 patients, stage IIIC in 4 patients and stage IV in 14 patients. WB-DWI, WB-PET/CT and WB-PET/MRI incorrectly and concordantly staged 1/51 patient as stage IV instead of IIIA. Discordant staging was reported in 17/51 patients. WB-PET/MRI resulted in improved staging when compared to WB-PET/CT (50 correctly staged on WB-PET/MRI vs. 38 correctly staged on WB-PET/CT; McNemar's test; p<0.01). Comparing the performance of WB-PET/MRI and WB-DWI (43 correct) did not reveal a statistically significant difference (McNemar test, p=0.14). WB-PET/MRI is more accurate in the initial staging of breast cancer than WB-DWI and WB-PET/CT, however, the discrepancies between WB-PET/MRI and WB-DWI were not statistically significant. When available, WB-PET/MRI should be considered for staging patient with invasive ductal breast carcinoma.
Lung cancer accounts for one in five cancer deaths worldwide and mutations in the gene encoding for the Kirsten rat sarcoma (KRAS) oncoprotein define the largest molecular subset of non-small cell ...lung cancer (NSCLC). These tumors are characterized by activated MAPK signaling, however, no targeted inhibitors of mutant KRAS or of downstream signaling molecules have yet been approved for routine clinical use.
The primary objective of this review is to critically summarize the current developmental state of MEK and ERK inhibitors in pre-clinical models and in human clinical trials for KRAS mutant lung cancer particularly in light of the newly emerging concept of immune checkpoint blockade.
We performed a Pubmed-based literature search and considered publications from the fields of basic and translational biomedicinal and biochemistry research, as well as from past and ongoing human clinical trials (www.clinicaltrials.gov).
MAPK pathway targeting agents are efficacious in pre-clinical models but their benefit is limited for patients with KRAS mutant NSCLC due to the lack of predictive factors, toxicity and the adaptive dynamic kinome reprogramming within the tumor. Overall, MEK inhibitors have advanced further in clinical development compared to ERK inhibitors. New treatment strategies as e.g. immune checkpoint blockade are currently revolutionizing the treatment paradigms and future clinical trials need to show if they replace MAPK targeting strategies or are used as add-on.
Targeted covalent small molecules have shown promise for cancers driven by KRAS G12C. Allosteric compounds that access an inducible pocket formed by movement of a dynamic structural element in KRAS, ...switch II, have been reported, but these compounds require further optimization to enable their advancement into clinical development. We demonstrate that covalent quinazoline-based switch II pocket (SIIP) compounds effectively suppress GTP loading of KRAS G12C, MAPK phosphorylation, and the growth of cancer cells harboring G12C. Notably we find that adding an amide substituent to the quinazoline scaffold allows additional interactions with KRAS G12C, and remarkably increases the labeling efficiency, potency, and selectivity of KRAS G12C inhibitors. Structural studies using X-ray crystallography reveal a new conformation of SIIP and key interactions made by substituents located at the quinazoline 2-, 4-, and 7-positions. Optimized lead compounds in the quinazoline series selectively inhibit KRAS G12C-dependent signaling and cancer cell growth at sub-micromolar concentrations.
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•Functional and structural characterization of covalent KRAS G12C inhibitors•An amino amide substituent enhances the potency and selectivity of inhibitors•Develop key assays for characterization and discovery of new KRAS inhibitors
Zeng et al. demonstrate that introduction of an amino amide substituent to the quinazoline scaffold remarkably increases the labeling efficiency and rates, potency, and selectivity of KRAS G12C inhibitors. This is supported by structural evidence that amide side chain allows additional interactions with KRAS G12C.
A versatile and Lipinski‐compliant DNA‐encoded library (DEL), comprising 366 600 glutamic acid derivatives coupled to oligonucleotides serving as amplifiable identification barcodes is designed, ...constructed, and characterized. The GB‐DEL library, constructed in single‐stranded DNA format, allows de novo identification of specific binders against several pharmaceutically relevant proteins. Moreover, hybridization of the single‐stranded DEL with a set of known protein ligands of low to medium affinity coupled to a complementary DNA strand results in self‐assembled selectable chemical structures, leading to the identification of affinity‐matured compounds.
A single‐stranded DNA‐encoded chemical library (DEL) of 366 600 compounds, based on a stereoisomeric scaffold and on a single‐stranded DNA format, allows the isolation of ligands against multiple protein targets. The library can also be hybridized to a complementary DNA strand equipped with a protein binder, in order to generate affinity‐matured ligands.
Pulmonary artery intimal sarcoma (PAIS) is a rare malignant neoplasm with imaging features that can mimic pulmonary embolism (PE). It must be recognized early because a radical resection may be ...useful to prolong survival.
A clinical case of a 57-year-old Caucasian male affected by PAIS is presented, which describes the computed tomography (CT) findings found in PAIS and the elements of overlap and differentiation with PE. The main common element is represented by the endoluminal filling defect of the pulmonary arterial vessels in contrast-enhanced CT examinations; a characteristic polypoid morphology or polylobulated contours are typical findings of PAIS. Other specific elements of the neoplasm such as wall eclipse sign, extension beyond the arterial wall, and metastasis are also explained.
The overlap of the clinical–radiological findings and the epidemiological difference between PAIS and PE cause a diagnostic delay. By knowing the differential elements, the radiologist can detect the neoplasm early to accelerate diagnosis and suggest optimal management.
Vascular compression syndromes include a group of rare changes due to extrinsic compression of veins or arteries by surrounding structures. These pathologies are often underestimated due to their ...rarity, clinicians' poor level of knowledge, and the non-specificity of their symptoms. The best known are Eagle syndrome, thoracic outlet syndrome, nutcracker syndrome, May-Thurner syndrome, Dunbar syndrome, and popliteal entrapment syndrome. This work summarizes the main ultrasonographic characteristics, symptoms, and treatments of choice for these syndromes. Knowledge of these conditions' characteristic signs is essential for the differential diagnosis. Failure to diagnose these rare diseases can expose patients to serious complications and risks to their health.
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