Suite of Biochemical and Cell-Based Assays for the Characterization of Kirsten Rat Sarcoma (KRAS) Inhibitors and Degraders DOI Creative Commons

Medhanie Kidane,

Rene M. Hoffman,

Jennifer K. Wolfe-Demarco

и другие.

ACS Pharmacology & Translational Science, Год журнала: 2024, Номер 7(12), С. 3921 - 3934

Опубликована: Дек. 2, 2024

KRAS is an important oncogenic driver which mutated in numerous cancers. Recent advances the selective targeting of mutants via small molecule inhibitors and targeted protein degraders have generated increase research activity this area recent years. As such, there a need for new assay platforms to profile next generation improve on potency selectivity existing drug candidates, while evading emergence resistance. Here, we describe development panel biochemical cell-based assays evaluate binding function known chemical entities mutant KRAS. Our panels profiles quantitative interaction dissociation constants molecules against wild type, G12C, G12D, G12V KRAS, were congruent with published data. These can be leveraged additional interest beyond those described study, using both overexpressed cell-free systems endogenous levels.

Язык: Английский

Leveraging Structural and Computational Biology for Molecular Glue Discovery DOI Creative Commons
CongBao Kang, Weijun Xu

Journal of Medicinal Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Янв. 24, 2025

The discovery of molecular glues has made significant strides, unlocking new avenues for targeted protein degradation as a therapeutic strategy, thereby expanding the scope drug into territories previously considered undruggable. Pioneering molecules like thalidomide and its derivatives have paved way development small that can induce specific by hijacking cellular ubiquitin–proteasome system. Recent advancements focused on range E3 ligases target proteins be modulated glues. Structural elucidation ligase in complex with glue interest, combined computational modeling, facilitates understanding underlying mechanisms how degradation. By leveraging these tools, next generation are expected to offer unprecedented opportunities combating wide diseases, including cancer, autoimmune disorders, neurodegenerative conditions.

Язык: Английский

Процитировано

1
Luciferase complementation for cellular assays beyond protein–protein interactions DOI Open Access
Genki Kawamura,

Takeaki Ozawa

Analytical Sciences, Год журнала: 2025, Номер unknown

Опубликована: Фев. 18, 2025

Abstract Luciferase complementation assays have emerged in 2001 as a useful tool to analyze biological processes through diverse such cellular studies and vivo imaging. The assay has an advantage of wide dynamic ranges, high signal-to-noise ratios, capability for real-time monitoring events with readout bioluminescence. While it was initially harnessed detecting protein–protein interactions, biosensors based on luciferase-fragment achieved significant advancements their designs, expanding versatility applicability beyond the initial scope. This review aims provide comprehensive overview designing strategies employed split luciferase highlight bioanalytical applications. Because simple bi-molecular detection interactions by this approach is well-established, will focus introducing sensor designs using concept complementation. Graphical abstract

Язык: Английский

Процитировано

0
Rapid and high-throughput screening of proteolysis targeting chimeras using a dual-reporter system expressing fluorescence protein and luciferase DOI Creative Commons
Shuai Meng, Yuan Meng, Xuena Yang

и другие.

BMC Biology, Год журнала: 2025, Номер 23(1)

Опубликована: Фев. 21, 2025

Proteolysis targeting chimera (PROTAC), a novel drug discovery strategy, utilizes the ubiquitin-proteasome system to degrade target proteins in cells. While Western blotting, mass spectrometry, and Lumit Immunoassay have been instrumental determining protein levels, rapid screening of PROTACs continues pose challenges, necessitating development alternative methodologies. We herein reported an high-throughput method for using dual-reporter expressing Renilla luciferase (RLUC)-fused enhanced green fluorescent (EGFP). EGFP served as internal reference RLUC indicated degradation. Rapid measurement or light signals was achieved fluorescence/luminescence plate-based reader endpoint mode. The feasibility model tested ARV110, clinical trial-stage PROTAC androgen receptor (AR). In EGFP/RLUC-tAR-expressing modal cells treated with varying concentrations normalized luminescence decreased dose-dependently, confirmed via western blotting detection AR expression. Then platform used practically screen Sirtuin 2 (SIRT2) degraders from small group that we built. Normalized changes EGFP/RLUC-SIRT2 reflected degradation efficiencies PROTACs. Compounds 128 129 exhibited highest efficacies, leading dose-dependent endogenous SIRT2 MCF-7 cell line inducing growth arrest. both fluorescence chemiluminescence successfully constructed. Using this method, identified effective candidate against SIRT2. may accelerate during development.

Язык: Английский

Процитировано

0
One Tracer, Dual Platforms: Unlocking Versatility of Fluorescent Probes in TR-FRET and NanoBRET Target Engagement Assays DOI Creative Commons
Erika Y. Monroy, Xin Qi Yu, Lu Dong

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Март 27, 2025

Abstract Target engagement assays are critical for drug discovery, with Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) and Nano Bioluminescence (NanoBRET) representing two complementary approaches biochemical cellular evaluation. Traditionally, these platforms demand distinct fluorescent tracers tailored to their unique detection systems, requiring separate probe development comprehensive target characterization. Despite widespread adoption, the of platform-specific often leads increased costs experimental complexity. In this study, tracers, T2-BODIPY -FL T2-BODIPY-589, initially developed receptor-interacting protein kinase 1 (RIPK1) studies in TR-FRET NanoBRET applications respectively, were systematically evaluated performance across both under various parameters. By evaluating assay we demonstrate that can effectively bridge assays, delivering reliable measurements. its red-shifted spectral properties, exhibits superior (Z’ up 0.80) while maintaining acceptable functionality systems (Z’=0.53). contrast, provides optimal (Z’=0.57) but also demonstrates potential use 0.72), albeit reduced efficiency. Competition an unlabeled inhibitor yielded consistent binding constants all tracer-platform combinations, validating reliability quantitative Our findings highlight integrating a single tracer diverse platforms, reducing need enhancing consistency. This approach has broad implications streamlining development, improving data comparability, enables more direct comparisons between data, broader integrated discovery programs classes.

Язык: Английский

Процитировано

0
Four-color single-molecule imaging system for tracking GPCR dynamics with fluorescent HiBiT peptide DOI Creative Commons

Toshiki Yoda,

Yasushi Sako, Asuka Inoue

и другие.

Biophysics and Physicobiology, Год журнала: 2024, Номер 21(3), С. n/a - n/a

Опубликована: Янв. 1, 2024

Single-molecule imaging provides information on diffusion dynamics, oligomerization, and protein-protein interactions in living cells. To simultaneously monitor different types of proteins at the single-molecule level, orthogonal fluorescent labeling methods with photostable dyes are required. G-protein-coupled receptors (GPCRs), a major class drug targets, prototypical membrane that have been studied using techniques. Here we developed method for cell-surface GPCRs inspired by HiBiT system, which utilizes high affinity complementation between LgBiT fragments NanoLuc luciferase. We synthesized four fluorescence-labeled peptides (F-FiBiTs) color dye (Setau-488, TMR, SaraFluor 650 720). constructed multicolor total internal reflection fluorescence microscopy system allows us to track simultaneously. As proof-of-concept experiment, labeled an N-terminally LgBiT-fused GPCR (Lg-GPCR) mixture F-FiBiTs successfully tracked each within cell level. The F-FiBiT-labeled Lg-GPCRs showed agonist-dependent changes dynamics accumulation into clathrin-coated pits as observed conventional C-terminally HaloTag-fused GPCR. Taking advantage luciferase F-FiBiT Lg-GPCRs, was also applicable bioluminescence plate-reader-based assays. By combining existing such HaloTag, SNAP-tag, proteins, will be useful enhance our understanding signaling

Язык: Английский

Процитировано

2
Covalent Proximity Inducers DOI Creative Commons
Nir London

Chemical Reviews, Год журнала: 2024, Номер unknown

Опубликована: Дек. 18, 2024

Molecules that are able to induce proximity between two proteins finding ever increasing applications in chemical biology and drug discovery. The ability introduce an electrophile make such inducers covalent can offer improved properties as selectivity, potency, duration of action, reduced molecular size. This concept has been heavily explored the context targeted degradation particular for bivalent molecules, but recently, additional reported other contexts, well monovalent glues. is a comprehensive review inducers, aiming identify common trends current gaps their discovery application.

Язык: Английский

Процитировано

2
Bivalent target-binding bioPROTACs induce potent degradation of oncogenic SHP2 DOI Creative Commons
Megan T. Hoffman,

David Krum,

K. Dane Wittrup

и другие.

Journal of Biological Chemistry, Год журнала: 2024, Номер 300(9), С. 107616 - 107616

Опубликована: Июль 31, 2024

Язык: Английский

Процитировано

1
To homeostasis and beyond! Recent advances in the medicinal chemistry of heterobifunctional derivatives DOI

Diana Castagna,

Benoit Gourdet,

Roland Hjerpe

и другие.

Progress in medicinal chemistry, Год журнала: 2024, Номер unknown, С. 61 - 160

Опубликована: Янв. 1, 2024

Язык: Английский

Процитировано

1
Application of Fluorescence- and Bioluminescence-Based Biosensors in Cancer Drug Discovery DOI Creative Commons

Tynan Kelly,

Xiaolong Yang

Biosensors, Год журнала: 2024, Номер 14(12), С. 570 - 570

Опубликована: Ноя. 24, 2024

Recent advances in drug discovery have established biosensors as indispensable tools, particularly valued for their precision, sensitivity, and real-time monitoring capabilities. The review begins with a brief overview of cancer discovery, underscoring the pivotal role advancing research. Various types employed are then explored, particular emphasis on fluorescence- bioluminescence-based technologies such FRET, TR-FRET, BRET, NanoBRET, NanoBiT. These enabled breakthrough discoveries, including identification Celastrol novel YAP-TEAD inhibitor through NanoBiT-based screening, development TR-FRET assays that successfully identified Ro-31-8220 SMAD4R361H/SMAD3 interaction inducer. integration high throughput screening validation compounds is examined, highlighting successful applications LATS revealed VEGFR an upstream regulator Hippo signaling pathway. Real-time cellular responses has yielded invaluable insights into cell pathways, demonstrated by NanoBRET detecting RAF dimerization HiBiT systems protein degradation dynamics. addresses challenges linked to biosensor applications, maintaining stability complex tumor microenvironments achieving consistent sensitivity HTS applications. Emerging trends discussed, integrating artificial intelligence advanced nanomaterials enhanced performance. In conclusion, this offers comprehensive analysis dynamic field, presenting quantitative evidence impact potential revolutionize targeted treatments.

Язык: Английский

Процитировано

1
Suite of Biochemical and Cell-Based Assays for the Characterization of Kirsten Rat Sarcoma (KRAS) Inhibitors and Degraders DOI Creative Commons

Medhanie Kidane,

Rene M. Hoffman,

Jennifer K. Wolfe-Demarco

и другие.

ACS Pharmacology & Translational Science, Год журнала: 2024, Номер 7(12), С. 3921 - 3934

Опубликована: Дек. 2, 2024

KRAS is an important oncogenic driver which mutated in numerous cancers. Recent advances the selective targeting of mutants via small molecule inhibitors and targeted protein degraders have generated increase research activity this area recent years. As such, there a need for new assay platforms to profile next generation improve on potency selectivity existing drug candidates, while evading emergence resistance. Here, we describe development panel biochemical cell-based assays evaluate binding function known chemical entities mutant KRAS. Our panels profiles quantitative interaction dissociation constants molecules against wild type, G12C, G12D, G12V KRAS, were congruent with published data. These can be leveraged additional interest beyond those described study, using both overexpressed cell-free systems endogenous levels.

Язык: Английский

Процитировано

0