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Over the past decade, remarkable advances have been witnessed in the development of small-molecule probes. These molecular tools have been widely applied for interrogating proteins, pathways and drug–target interactions in preclinical research. While novel structures and designs are commonly explored in probe development, the clinical translation of small-molecule probes remains limited, primarily due to safety and regulatory considerations. Recent synergistic developments – interfacing novel chemical probes with complementary analytical technologies – have introduced and expedited diverse biomedical opportunities to molecularly characterize targeted drug interactions directly in the human body or through accessible clinical specimens (e.g., blood and ascites fluid). These integrated developments thus offer unprecedented opportunities for drug development, disease diagnostics and treatment monitoring. In this review, we discuss recent advances in the structure and design of small-molecule probes with novel functionalities and the integrated development with imaging, proteomics and other emerging technologies. We further highlight recent applications of integrated small-molecule technologies for the molecular analysis of drug–target interactions, including translational applications and emerging opportunities for whole-body imaging, tissue-based measurement and blood-based analysis. [ABSTRACT FROM AUTHOR]

Chemical probes are powerful tools for interrogating small molecule-target interactions. With additional fluorescence Turn-ON functionality, such probes might enable direct measurements of target engagement in live mammalian cells. DNS-pE (and its terminal alkyne-containing version DNS-pE2) is the first small molecule that can selectively label endogenous 3-phosphoglycerate dehydrogenase (PHGDH) from various mammalian cells. Endowed with an electrophilic vinyl sulfone moiety that possesses fluorescence-quenching properties, DNS-pE/DNS-pE2 became highly fluorescent only upon irreversible covalent modification of PHGDH. With an inhibitory property (in vitro Ki=7.4 μ m) comparable to that of known PHGDH inhibitors, our probes thus offer a promising approach to simultaneously image endogenous PHGDH activities and study its target engagement in live-cell settings. [ABSTRACT FROM AUTHOR]

BRCTs are phosphoserine-binding domains found in proteins involved in DNA repair, DNA damage response and cell cycle regulation. BRCA1 is a BRCT domain-containing, tumor-suppressing protein expressed in the cells of breast and other human tissues. Mutations in BRCA1 have been found in ca. 50 % of hereditary breast cancers. Cell-permeable, small-molecule BRCA1 inhibitors are promising anticancer agents, but are not available currently. Herein, with the assist of microarray-based platforms, we have discovered the first cell-permeable protein-protein interaction (PPI) inhibitors against BRCA1. By targeting the (BRCT)2 domain, we showed compound 15 a and its prodrug 15 b inhibited BRCA1 activities in tumor cells, sensitized these cells to ionizing radiation-induced apoptosis, and showed synergistic inhibitory effect when used in combination with Olaparib (a small-molecule inhibitor of poly-ADP-ribose polymerase) and Etoposide (a small-molecule inhibitor of topoisomerase II). Unlike previously reported peptide-based PPI inhibitors of BRCA1, our compounds are small-molecule-like and could be directly administered to tumor cells, thus making them useful for future studies of BRCA1/PARP-related pathways in DNA damage and repair response, and in cancer therapy. [ABSTRACT FROM AUTHOR]

MLN8237 is a highly potent and presumably selective inhibitor of Aurora kinase A (AKA) and has shown promising antitumor activities. Like other kinase inhibitors which target the ATP-binding site of kinases, MLN8237 might be expected to have potential cellular off-targets. Herein, we report the first photoaffinity-based, small molecule AKA probe capable of both live-cell imaging of AKA activities and in situ proteome profiling of potential off-targets of MLN8237 (including AKA-associating proteins). By using two mutually compatible, bioorthogonal reactions (copper-catalyzed azide-alkyne cycloaddition chemistry and TCO-tetrazine ligation), we demostrate small molecule-based multiplex bioimaging for simultaneous in situ monitoring of two important cell-cycle regulating kinases (AKA and CDK1). A broad range of proteins, as potential off-targets of MLN8237 and AKA's-interacting partners, is subsequently identified by affinity-based proteome profiling coupled with large-scale LC-MS/MS analysis. From these studies, we discover novel AKA interactions which were further validated by cell-based immunoprecipitation (IP) experiments. [ABSTRACT FROM AUTHOR]

Drug delivery systems (DDSs) are designed to deliver therapeutic agents to specific target sites while minimizing systemic toxicity. Recent developments in drug-loaded DDSs have demonstrated promising characteristics and paved new pathways for cancer treatment. Light, a prevalent external stimulus, is widely utilized to trigger drug release. However, conventional light sources primarily concentrate on the ultraviolet (UV) and visible light regions, which suffer from limited biological tissue penetration. This limitation hinders applications for deep-tissue tumor drug release. Given their deep tissue penetration and well-established application technology, X-rays have recently received attention for the pursuit of controlled drug release. With precise spatiotemporal and dosage controllability, X-rays stand as an ideal stimulus for achieving controlled drug release in deep-tissue cancer therapy. This article explores the recent advancements in using X-rays for stimulus-triggered drug release in DDSs and delves into their action mechanisms., (© 2023 Wiley-VCH GmbH.)

Objective: To compare the impact of permissive underfeeding versus standard enteral feeding on outcomes in critical patients requiring mechanical ventilation (MV)., Methods: A prospective randomized controlled study was conducted. Eighty-two patients requiring MV admitted to intensive care unit (ICU) of Anji People's Hospital from January 2015 to March 2017 were enrolled, and they were randomly divided into the permissive underfeeding group (n = 40, non-protein heat was 52.3-62.8 kJ×kg -1 ×d -1 , protein was 1.2-1.5 g×kg -1 ×d -1 ) and standard enteral feeding group (n = 42, non-protein heat was 104.6-125.5 kJ×kg -1 ×d -1 , protein was 1.2-1.5 g×kg -1 ×d -1 ). Permissive underfeeding group received 50% of their daily energy expenditure via enteral nutrition (EN) and standard enteral feeding group received 100% of their daily energy expenditure via EN in 24-48 hours after admitted to ICU. Nutritional status [pro-albumin (PA), serum albumin (ALB)], inflammation state [procalcitonin (PCT), hypersensitive C-reactive protein (hs-CRP)] were detected before treatment and 7 days after treatment. Duration of MV, length of ICU stay, daily insulin dosage, 28-day mortality, hospital acquired pneumonia (HAP), urinary tract infection, septic shock and other secondary infection, and the nutrition related complications were recorded., Results: Compared with before treatment, the levels of serum PA (mg/L) and ALB (g/L) were significantly increased, the levels of PCT (ng/L) and hs-CRP (mg/L) were significantly decreased at 7 days after treatment in both groups [permissive underfeeding group: PA was 127.42±65.83 vs. 80.92±60.14, ALB was 30.16±4.32 vs. 25.36±6.21, PCT was 375.8±227.2 vs. 762.3±314.5, hs-CRP was 32.19±7.53 vs. 120.48±60.24; standard enteral feeding group: PA was 132.56±61.32 vs. 86.78±47.06, ALB was 31.25±4.63 vs. 26.71±5.48, PCT was 412.1±323.4 vs. 821.7±408.6, hs-CRP was 35.86±5.69 vs. 116.38±72.16, all P < 0.05], but there was no significant difference in PA, ALB, PCT or hs-CRP at 7 days after treatment between two groups (all P > 0.05). There was no significant difference in the duration of MV, length of ICU stay, 28-day mortality or ICU-associated infection between two groups [duration of MV (hours): 162.35±20.37 vs. 153.48±18.65, length of ICU stay (days): 7.52±1.61 vs. 6.34±1.87, 28-day mortality: 17.5% vs. 19.0%, ICU-associated infection: 45.0% vs. 47.6%, all P > 0.05]. Compared with standard enteral feeding, insulin demand was significantly decreased (U/d: 13.68±10.36 vs. 26.24±18.53), and gastrointestinal intolerance was less frequent (32.5% vs. 54.8%) in the permissive underfeeding group (both P < 0.05). Kaplan-Meier survival curve analysis showed that there was no significant difference between the two groups (χ 2 = 3.216, P = 0.068)., Conclusions: The curative effect and prognosis of MV severe patients receiving permissive underfeeding are similar to those of standard enteral feeding, but it can reduce the dosage of insulin with better gastrointestinal tolerance.

Microarray screening technology has transformed the life sciences arena over the last decade. The platform is widely used in the area of mapping interaction networks, to molecular fingerprinting and small molecular inhibitor discovery. The technique has significantly impacted both basic and applied research. The microarray platform can likewise enable high-throughput screening and discovery of protein-protein interaction (PPI) inhibitors. Herein we demonstrate the application of microarray-guided PPI inhibitor discovery, using human BRCA1 as an example. Mutations in BRCA1 have been implicated in ~50 % of hereditary breast cancers. By targeting the (BRCT) 2 domain, we showed compound 15a and its prodrug 15b inhibited BRCA1 activities in tumor cells. Unlike previously reported peptide-based PPI inhibitors of BRCA1, the compounds identified could be directly administered to tumor cells, thus making them useful in targeting BRCA1/PARP-related pathways involved in DNA damage and repair response, for cancer therapy.

DOT1L is the sole protein methyltransferase that methylates histone H3 on lysine 79 (H3K79), and is a promising drug target against cancers. Small-molecule inhibitors of DOT1L such as FED1 are potential anti-cancer agents and useful tools to investigate the biological roles of DOT1L in human diseases. FED1 showed excellent in vitro inhibitory activity against DOT1L, but its cellular effect was relatively poor. In this study, we designed and synthesized photo-reactive and "clickable" affinity-based probes (AfBPs), P1 and P2, which were cell-permeable and structural mimics of FED1. The binding and inhibitory effects of these two probes against DOT1L protein were extensively investigated in vitro and in live mammalian cells (in situ). The cellular uptake and sub-cellular localization properties of the probes were subsequently studied in live-cell imaging experiments, and our results revealed that, whereas both P1 and P2 readily entered mammalian cells, most of them were not able to reach the cell nucleus where functional DOT1L resides. This offers a plausible explanation for the poor cellular activity of FED1. Finally with P1/P2, large-scale cell-based proteome profiling, followed by quantitative LC-MS/MS, was carried out to identify potential cellular off-targets of FED1. Amongst the more than 100 candidate off-targets identified, NOP2 (a putative ribosomal RNA methyltransferase) was further confirmed to be likely a genuine off-target of FED1 by preliminary validation experiments including pull-down/Western blotting (PD/WB) and cellular thermal shift assay (CETSA)., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Newly synthesized proteins constitute an important subset of the proteome involved in every cellular process, yet existing chemical tools used to study them have major shortcomings. Herein we report a suite of cell-permeable puromycin analogues capable of being metabolically incorporated into newly synthesized proteins in different mammalian cells, including neuronal cells. Subsequent labeling with suitable bioorthogonal reporters, in both fixed and live cells, enabled direct imaging and enrichment of these proteins. By taking advantage of the mutually orthogonal reactivity of these analogues, we showed multiplexed labeling of different protein populations, as well as quantitative measurements of protein dynamics by fluorescence correlation spectroscopy, could be achieved in live-cell environments., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Poly(ADP-ribose)polymerase-1 (PARP1) is a BRCT-containing enzyme (BRCT = BRCA1 C-terminus) mainly involved in DNA repair and damage response and a validated target for cancer treatment. Small-molecule inhibitors that target the PARP1 catalytic domain have been actively pursued as anticancer drugs, but are potentially problematic owing to a lack of selectivity. Compounds that are capable of disrupting protein-protein interactions of PARP1 provide an alternative by inhibiting its activities with improved selectivity profiles. Herein, by establishing a high-throughput microplate-based assay suitable for screening potential PPI inhibitors of the PARP1 BRCT domain, we have discovered that (±)-gossypol, a natural product with a number of known biological activities, possesses novel PARP1 inhibitory activity both in vitro and in cancer cells and presumably acts through disruption of protein-protein interactions. As the first known cell-permeable small-molecule PPI inhibitor of PAPR1, we further established that (-)-gossypol was likely the causative agent of PARP1 inhibition by promoting the formation of a 1:2 compound/PARP1 complex by reversible formation of a covalent imine linkage., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)