[Discovering high-affinity ligands for proteins: SAR by NMR].

Proteins of the bromodomain and extra-terminal (BET) family are epigenetics “readers” and promising therapeutic targets for cancer and other human diseases We describe herein a structure-guided design of [1,4]oxazepines as a new class of BET inhibitors and our subsequent design, synthesis, and evaluation of proteolysis-targeting chimeric (PROTAC) small-molecule BET degraders Our efforts have led to the discovery of extremely potent BET degraders, exemplified by QCA570, which effectively induces degradation of BET proteins and inhibits cell growth in human acute leukemia cell lines even at low picomolar concentrations QCA570 achieves complete and durable tumor regression in leukemia xenograft models in mice at well-tolerated dose-schedules QCA570 is the most potent and efficacious BET degrader reported to date

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Discovery of QCA570 as an Exceptionally Potent and Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of the Bromodomain and Extra-Terminal (BET) Proteins Capable of Inducing Complete and Durable Tumor Regression

The (19)F isotope is 100% naturally abundant and is the second most sensitive and stable NMR-active nucleus. Unlike the ubiquitous hydrogen atom, fluorine is nearly absent in biological systems, making it a unique bioorthogonal atom for probing molecular interactions in biology. Over 73 fluorinated proteins have been studied by (19)F NMR since the seminal studies of Hull and Sykes in 1974. With advances in cryoprobe production and fluorinated amino acid incorporation strategies, protein-based (19)F NMR offers opportunities to the medicinal chemist for characterizing and ultimately discovering new small molecule protein ligands. This review will highlight new advances using (19)F NMR for characterizing small molecule interactions with both small and large proteins as well as detailing NMR resonance assignment challenges and amino acid incorporation approaches.

Protein-Observed Fluorine NMR: A Bioorthogonal Approach for Small Molecule Discovery

Hypoxia is reported to participate in tumor progression, promote drug resistance, and immune escape within tumor microenvironment, and thus impair therapeutic effects including the chemotherapy and advanced immunotherapy. Here, a multifunctional biomimetic core-shell nanoplatform is reported for improving synergetic chemotherapy and immunotherapy. Based on the properties including good biodegradability and functionalities, the pH-sensitive zeolitic imidazolate framework 8 embedded with catalase and doxorubicin constructs the core and serves as an oxygen generator and drug reservoir. Murine melanoma cell membrane coating on the core provides tumor targeting ability and elicits an immune response due to abundance of antigens. It is demonstrated that this biomimetic core-shell nanoplatform with oxygen generation can be partial to accumulate in tumor and downregulate the expression of hypoxia-inducible factor 1α, which can further enhance the therapeutic effects of chemotherapy and reduce the expression of programmed death ligand 1 (PD-L1). Combined with immune checkpoints blockade therapy by programmed death 1 (PD-1) antibody, the dual inhibition of the PD-1/PD-L1 axis elicits significant immune response and presents a robust effect in lengthening tumor recurrent time and inhibiting tumor metastasis. Consequently, the multifunctional nanoplatform provides a potential strategy of synergetic chemotherapy and immunotherapy.

A Multifunctional Biomimetic Nanoplatform for Relieving Hypoxia to Enhance Chemotherapy and Inhibit the PD-1/PD-L1 Axis

Mesoporous materials are ideal carriers for guest molecules and they have been widely used in analytical science. The unique mesoporous structure provides special properties including large specific surface area, tunable pore size, and excellent pore connectivity. The structural properties of mesoporous materials have been largely made use of to improve the performance of analytical methods. For instance, the large specific surface area of mesoporous materials can provide abundant active sites and increase the probability of contact between analytes and active sites to produce stronger signals, thus leading to the improvement of detection sensitivity. The connections between analytical performances and the structural properties of mesoporous materials have not been discussed previously. Understanding the “structure–performance relationship” is highly important for the development of analytical methods with excellent performance based on mesoporous materials. In this review, we discuss the structural properties of mesoporous materials that can be optimized to improve the analytical performance. The discussion is divided into five sections according to the analytical performances: (i) selectivity-related structural properties, (ii) sensitivity-related structural properties, (iii) response time-related structural properties, (iv) stability-related structural properties, and (v) recovery time-related structural properties.

New insights into the structure-performance relationships of mesoporous materials in analytical science.

Labeling of proteins with fluorinated amino acids and using fluorine-19 NMR greatly simplifies the NMR spectrum. Changes in the spectrum on interaction with ligands makes protein-observed fragment-based drug screening possible. NMR spectroscopy can be used to quantify the binding affinity between proteins and low-complexity molecules, termed 'fragments'; this versatile screening approach allows researchers to assess the druggability of new protein targets. Protein-observed 19F-NMR (PrOF NMR) using 19F-labeled amino acids generates relatively simple spectra that are able to provide dynamic structural information toward understanding protein folding and function. Changes in these spectra upon the addition of fragment molecules can be observed and quantified. This protocol describes the sequence-selective labeling of three proteins (the first bromodomains of Brd4 and BrdT, and the KIX domain of the CREB-binding protein) using commercially available fluorinated aromatic amino acids and fluorinated precursors as example applications of the method developed by our research group. Fragment-screening approaches are discussed, as well as Kd determination, ligand-efficiency calculations and druggability assessment, i.e., the ability to target these proteins using small-molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment.

Protein-observed 19F-NMR for fragment screening, affinity quantification and druggability assessment

19F NMR spectroscopy of labeled proteins is a sensitive method for characterizing structure, conformational dynamics, higher-order assembly, and ligand binding. Fluorination of aromatic side chains has been suggested as a labeling strategy for small-molecule ligand discovery for protein–protein interaction interfaces. Using a model transcription factor binding domain of the CREB binding protein (CBP)/p300, KIX, we report the first full small-molecule screen using protein-observed 19F NMR spectroscopy. Screening of 508 compounds and validation by 1H–15N HSQC NMR spectroscopy led to the identification of a minimal pharmacaphore for the MLL-KIX interaction site. Hit rate analysis for the CREB-KIX and MLL-KIX sites provided a metric to assess the ligandability or “druggability” of each interface informing future medicinal chemistry efforts. The structural information from the simplified spectra and data collection speed, affords a new screening tool for analysis of protein interfaces and discovery of small molecules.

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Fragment Screening and Druggability Assessment for the CBP/p300 KIX Domain through Protein‐Observed 19F NMR Spectroscopy

Proteins are involved in nearly every biological process, which makes them of interest to a range of scientists. Previous work has shown that hand-held cameras can be used to determine the concentration of colored analytes in solution, and this paper extends the approach to reactions involving a color change in order to quantify protein concentration (e.g., green to blue). Herein, we describe the successful use of smartphone colorimetry to quantify protein concentration using two common colorimetric biochemical methods, the Bradford and biuret assays. The ease of the experimental setup makes these lab experiments accessible to a wide range of students and can be used as both high school and college level laboratory experiments.

Quantifying Protein Concentrations Using Smartphone Colorimetry: A New Method for an Established Test.

Oxygen homeostasis is important in the regulation of biological function Disease progression can be monitored by measuring oxygen levels, thus producing information for the design of therapeutic treatments Noninvasive measurements of tissue oxygenation require the development of tools with minimal adverse effects and facile detection of features of interest Fluorine magnetic resonance imaging (19F MRI) exploits the intrinsic properties of perfluorocarbon (PFC) liquids for anatomical imaging, cell tracking, and oxygen sensing However, the highly hydrophobic and lipophobic properties of perfluorocarbons require the formation of emulsions for biological studies, though stabilizing these emulsions has been challenging To enhance the stability and biological loading of perfluorocarbons, one option is to incorporate perfluorocarbon liquids into the internal space of biocompatible mesoporous silica nanoparticles Here, we developed perfluorocarbon-loaded ultraporous mesostructured silica nanoparticles (PERF

Oxygen Sensing with Perfluorocarbon-Loaded Ultraporous Mesostructured Silica Nanoparticles

Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains Structure–activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure–activity data An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement Given these findings, novel and easily synth

Clifford T. Gee

Papers

Protein-observed 19F-NMR for fragment screening, affinity quantification and druggability assessment

Fragment Screening and Druggability Assessment for the CBP/p300 KIX Domain through Protein‐Observed 19F NMR Spectroscopy

Quantifying Protein Concentrations Using Smartphone Colorimetry: A New Method for an Established Test.

Oxygen Sensing with Perfluorocarbon-Loaded Ultraporous Mesostructured Silica Nanoparticles

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen