Hemin as a Molecular Probe for Nitric Oxide Detection in Physiological Solutions: Experimental and Theoretical Assessment.

Given its pivotal role in modulating various pathological processes, precise measurement of nitric oxide (●NO) levels in physiological solutions is imperative. The key techniques include the ozone-based chemiluminescence (CL) reactions, amperometric ●NO sensing, and Griess assay, each with its advantages and drawbacks. In this study, a hemin/H2O2/luminol CL reaction was employed for accurately detecting ●NO in diverse solutions. We investigated how the luminescence kinetics was influenced by ●NO from two donors, nitrite and peroxynitrite, while also assessing the impact of culture medium components and reactive species quenchers. Furthermore, we experimentally and theoretically explored the mechanism of hemin oxidation responsible for the initiation of light generation. Although both hemin and ●NO enhanced the H2O2/luminol-based luminescence reactions with distinct kinetics, hemin's interference with ●NO/peroxynitrite- modulated their individual effects. Leveraging the propagated signal due to hemin, the ●NO levels in solution were estimated, observing parallel changes to those detected via amperometric detection in response to varying concentrations of the ●NO-donor. The examined reactions aid in comprehending the mechanism of ●NO/hemin/H2O2/luminol interactions and how these can be used for detecting ●NO in solution with minimal sample size demands. Moreover, the selectivity across different solutions can be improved by incorporating certain quenchers for reactive species into the reaction.

NIR-I emissive cyanine derived molecular probe for selective monitoring of hepatic albumin levels during hyperglycemia.

In this study, we report a small molecule optical marker BI-CyG derived from the structural engineering of a cyanine scaffold. The developed probe offers suitable advantages over existing cyanine-based albumin specific probes in terms of its excitation and emission wavelengths, which are 760 and 830-832 nm, respectively. Structural tuning of the cyanine architecture leading to extended π-conjugation and resulting in a suitable bathochromic shift in the emission wavelength of the probe is represented in this study. The probe besides emitting in the NIR region, also possesses the desirable characteristics of being a potential target selective optical marker, as established from various biophysical studies. Molecular modelling and simulation studies provided critical insights into the binding of the probe in the protein microenvironment, which was further supported by experimental studies. The probe displayed intracellular albumin selectivity and was utilized for demonstrating alteration in albumin levels in pathological states such as hyperglycemia in hepatic cells. The present study also sheds some light on using BI-CyG as an imaging probe and on the role of metformin as a suitable drug for balancing hyperglycemia-induced reduced intra-hepatic albumin levels. The study, thus, attempts to highlight the structural derivatization of cyanine to afford a potential probe for serum albumin and its deployment to image altering albumin levels in an induced pathological condition, hyperglycemia.

Preclinical evaluation of AGTR1-Targeting molecular probe for colorectal cancer imaging in orthotopic and liver metastasis mouse models.

Despite advancements in colorectal cancer (CRC) treatment, the prognosis remains unfavorable for patients with distant liver metastasis. Fluorescence molecular imaging with specific probes is increasingly used to guide CRC surgical resection in real-time and treatment planning. Here, we demonstrate the targeted imaging capacity of an MPA-PEG4-N3-Ang II probe labeled with near-infrared (NIR) fluorescent dye targeting the angiotensin II (Ang II) type 1 receptor (AGTR1) that is significantly upregulated in CRC. MPA-PEG4-N3-Ang II was highly selective and specific to in vitro tumor cells and in vivo tumors in a mouse CRC xenograft model. The favorable ex vivo imaging and in vivo biodistribution of MPA-PEG4-N3-Ang II afforded tumor-specific accumulation with low background and >10 contrast tumor-to-colorectal values in multiple subcutaneous CRC models at 8 h following injection. Biodistribution analysis confirmed the probe's high uptake in HT29 and HCT116 orthotopic and liver metastatic models of CRC with signal-to-noise ratio (SNR) values of tumor-to-colorectal and -liver fluorescence of 5.8 ± 0.6, 5.3 ± 0.7, and 2.7 ± 0.5, 2.6 ± 0.5, respectively, enabling high-contrast intraoperative tumor visualization for surgical navigation. Given its rapid tumor targeting, precise tumor boundary delineation, durable tumor retention and docking study, MPA-PEG4-N3-Ang II is a promising high-contrast imaging agent for the clinical detection of CRC.

Covalent activity-based probes for imaging of serine proteases.

Serine proteases are one of the largest mechanistic classes of proteases. They regulate a plethora of biochemical pathways inside and outside the cell. Aberrant serine protease activity leads to a wide variety of human diseases. Reagents to visualize these activities can be used to gain insight into the biological roles of serine proteases. Moreover, they may find future use for the detection of serine proteases as biomarkers. In this review, we discuss small molecule tools to image serine protease activity. Specifically, we outline different covalent activity-based probes and their selectivity against various serine protease targets. We also describe their application in several imaging methods.

Synthesis, Screening, and Evaluation of Theranostic Molecular CPCR4-Based Probe Targeting CXCR4.

Chemokines and chemokine receptors are indispensable to play a key role in the development of malignant tumors. As one of the most widely expressed chemokine receptors, chemokine (C-X-C motif) receptor 4 (CXCR4) has been a popular research focus. In most tumors, CXCR4 expression is significantly upregulated. Moreover, integrated nuclide diagnosis and therapy targeting CXCR4 show great potential. [68Ga]Ga-pentixafor, a radioligand targeting CXCR4, exhibits a strong affinity for CXCR4 both in vivo and in vitro. However, [177Lu]Lu-pentixather, the therapeutic companion of [68Ga]Ga-pentixafor, requires significant refinement to mitigate its pronounced hepatic biodistribution. The objective of this study was to synthesize theranostic molecular tracers with superior CXCR4 targeting functions. The Daudi cell line, which highly expressed CXCR4, and the MM.1S cell line, which weakly expressed CXCR4, were used in this study. Based on the pharmacophore cyclo (-d-Tyr-n-me-d-Orn-l-Arg-L-2-NAL-Gly-) (CPCR4) of pentixafor, six tracers were synthesized: [124I]I-1 ([124I]I-CPCR4), [99mTc]Tc-2 ([99mTc]Tc-HYNIC-CPCR4), [124I]I-3 ([124I]I-pentixafor), [18F]AlF-4 ([18F]AlF-NETA-CPCR4), [99mTc]Tc-5 ([99mTc]Tc-MAG3-CPCR4) and [124I]I-6 ([124I]I-pentixafor-Ga) and their radiochemical purities were all higher than 95%. After positron emission tomography (PET)/single-photon emission computed tomography (SPECT) imaging, the [124I]I-6 group exhibited the best target-nontarget ratio. At the same time, comparing the [68Ga]Ga-pentixafor group with the [124I]I-6 group, we found that the [124I]I-6 group had a better target-nontarget ratio and lower uptake in nontarget organs. Therefore, compound 6 was selected for therapeutic radionuclide (131I) labeling, and the tumor-bearing animal models were treated with [131I]I-6. The volume of the tumor site was significantly reduced in the treatment group compared with the control group, and no significant side effects were found. [124I]I-6 and [131I]I-6 showed excellent affinity for targeting CXCR4, and they showed great potential for the integrated diagnosis and treatment of tumors with high CXCR4 expression.

A rationally designed nuclei-targeting FAPI 04-based molecular probe with enhanced tumor uptake for PET/CT and fluorescence imaging.

PURPOSE: Fibroblast activation protein inhibitor (FAPI) -based probes have been widely studied in the diagnosis of various malignant tumors with positron emission tomography/computed tomography (PET/CT). However, current imaging studies of FAPI-based probes face challenges in rapid clearance rate and potential false-negative results. Furthermore, FAPI has been rarely explored in optical imaging. Considering this, further modifications are imperative to improve the properties of FAPI-based probes to address existing limitations and broaden their application scenarios. In this study, we rationally introduced methylene blue (MB) to FAPIs, thereby imparting nuclei-targeting and fluorescence imaging capabilities to the probes. Furthermore, we evaluated the added value of FAPI-based fluorescence imaging to traditional PET/CT, exploring the potential application of FAPI-based probes in intraoperative fluorescence imaging. METHODS: A new FAPI-based probe, namely NOTA-FAPI-MB, was designed for both PET/CT and fluorescence imaging by conjugation of MB. The targeting efficacy of the probe was evaluated on fibroblast activation protein (FAP)-transfected cell line and human primary cancer-associated fibroblasts (CAFs). Subsequently, PET/CT and fluorescence imaging were conducted on tumor-bearing mice. The tumor detection and boundary delineation were assessed by fluorescence imaging of tissues from hepatocellular carcinoma (HCC) patients. RESULTS: NOTA-FAPI-MB demonstrated exceptional targeting ability towards FAP-transfected cells and CAFs in comparison to NOTA-FAPI. This benefit arises from the cationic methylene blue (MB) affinity for anionic nucleic acids. PET/CT imaging of tumor-bearing mice revealed significantly higher tumor uptake of [18F]F-NOTA-FAPI-MB (standard uptake value of 2.20 ± 0.31) compared to [18F]F-FDG (standard uptake value of 1.66 ± 0.14). In vivo fluorescence imaging indicated prolonged retention at the tumor site, with retention lasting up to 24 h. In addition, the fluorescent probes enabled more precise lesion detection and tumor margin delineation than clinically used indocyanine green (ICG), achieving a 100.0% (6/6) tumor-positive rate for NOTA-FAPI-MB while 33.3% (2/6) for ICG. These findings highlighted the potential of NOTA-FAPI-MB in guiding intraoperative surgical procedures. CONCLUSIONS: The NOTA-FAPI-MB was successfully synthesized, in which FAPI and MB simultaneously contributed to the targeting effect. Notably, the nuclear delivery mechanism of the probes improved intracellular retention time and targeting efficacy, broadening the imaging time window for fluorescence imaging. In vivo PET/CT demonstrated favorable performance of NOTA-FAPI-MB compared to [18F]F-FDG. This study highlights the significance of fluorescence imaging as an adjunct technique to PET/CT. Furthermore, the encouraging results obtained from the imaging of human HCC tissues hold promise for the potential application of NOTA-FAPI-MB in intraoperative fluorescent surgery guidance within clinical settings.

A NIR-II Photoacoustic Probe for High Spatial Quantitative Imaging of Tumor Nitric Oxide in Vivo.

Nitric oxide (NO) exhibits both pro- and anti-tumor effects. Therefore, real-time in vivo imaging and quantification of tumor NO dynamics are essential for understanding the conflicting roles of NO played in pathophysiology. The current molecular probes, however, cannot provide high-resolution imaging in deep tissues, making them unsuitable for these purposes. Herein, we designed a photoacoustic probe with an absorption maximum beyond 1000 nm for high spatial quantitative imaging of in vivo tumor NO dynamics. The probe exhibits remarkable sensitivity, selective ratiometric response behavior, and good tumor-targeting abilities, facilitating ratiometric imaging of tumor NO throughout tumor progression in a micron-resolution level. Using the probe as the imaging agent, we successfully quantified NO dynamics in tumor, liver and kidney. We have pinpointed an essential concentration threshold of around 80 nmol/cm3 for NO, which plays a crucial role in the "double-edged-sword" function of NO in tumors. Furthermore, we revealed a reciprocal relationship between the NO concentration in tumors and that in the liver, providing initial insights into the possible NO-mediated communication between tumor and the liver. We believe that the probe will help resolve conflicting aspects of NO biology and guide the design of imaging agents for tumor diagnosis and anti-cancer drug screening.

Recent Advances of Aminopeptidases-Responsive Small-Molecular Probes for Bioimaging.

Aminopeptidases, enzymes with critical roles in human body, are emerging as vital biomarkers for metabolic processes and diseases. Aberrant aminopeptidase levels are often associated with diseases, particularly cancer. Small-molecule probes, such as fluorescent, fluorescent/photoacoustics, bioluminescent, and chemiluminescent probes, are essential tools in the study of aminopeptidases-related diseases. The fluorescent probes provide real-time insights into protein activities, offering high sensitivity in specific locations, and precise spatiotemporal results. Additionally, photoacoustic probes offer signals that are able to penetrate deeper tissues. Bioluminescent and chemiluminescent probes can enhance in vivo imaging abilities by reducing the background. This comprehensive review is focused on small-molecule probes that respond to four key aminopeptidases: aminopeptidase N, leucine aminopeptidase, Pyroglutamate aminopeptidase 1, and Prolyl Aminopeptidase, and their utilization in imaging tumors and afflicted regions. In this review, the design strategy of small-molecule probes, the variety of designs from previous studies, and the opportunities of future bioimaging applications are discussed, serving as a roadmap for future research, sparking innovations in aminopeptidase-responsive probe development, and enhancing our understanding of these enzymes in disease diagnostics and treatment.

Molecular Rotors: Fluorescent Sensors for Microviscosity and Conformation of Biomolecules.

The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non-canonical nucleic acid structures in live cells and other relevant biological settings.

The linkage-type and the exchange molecule affect the protein-labeling efficiency of iminoboronate probes.

Reversible bioorthogonal conjugation reactions have been exploited in the chemoproteomic field to prepare protein labeling reagents and to visualize labeled proteins. We recently demonstrated that reversible iminoboronates can be used to prepare probes from fragment libraries and that the linkage subsequently can be used to detect the labeled proteins. In this study, we determined the effect of the stability of the iminoboronate linkage on the efficiency of the labeling protocol. Our study reveals that the linkage should be stable enough to allow for efficient targeting, but should be labile enough to detect the labeled protein. Acyl hydrazides were identified as the most suitable handles for the probe synthesis step. Anthranilic hydrazides and N-hydroxy semicarbazides were found to be the most efficient read-out molecules. With these novel exchange molecules, native probe-labeled proteins could be visualized under physiological conditions.

Design, synthesis, and evaluation of a mitoxantrone probe (MXP) for biological studies.

Mitoxantrone (MX) is a robust chemotherapeutic with well-characterized applications in treating certain leukemias and advanced breast and prostate cancers. The canonical mechanism of action associated with MX is its ability to intercalate DNA and inhibit topoisomerase II, giving it the designation of a topoisomerase II poison. Years after FDA approval, investigations have unveiled novel protein-binding partners, such as methyl-CpG-binding domain protein (MBD2), PIM1 serine/threonine kinase, RAD52, and others that may contribute to the therapeutic profile of MX. Moreover, recent proteomic studies have revealed MX's ability to modulate protein expression, illuminating the complex cellular interactions of MX. Although mechanistically relevant, the differential expression across the proteome does not address the direct interaction with potential binding partners. Identification and characterization of these MX-binding cellular partners will provide the molecular basis for the alternate mechanisms that influence MX's cytotoxicity. Here, we describe the design and synthesis of a MX-biotin probe (MXP) and negative control (MXP-NC) that can be used to define MX's cellular targets and expand our understanding of the proteome-wide profile for MX. In proof of concept studies, we used MXP to successfully isolate a recently identified protein-binding partner of MX, RAD52, in a cell lysate pulldown with streptavidin beads and western blotting.

Chemical synthesis of on demand-activated SUMO-based probe by a photocaged glycine-assisted strategy.

The transiently-activated SUMO probes are conducive to understand the dynamic control of SENPs activity. Here, we developed a photocaged glycine-assisted strategy for the construction of on demand-activated SUMO-ABPs. The light-sensitive groups installed at G92 and G64 backbone of SUMO-2 can temporarily block probes activity and hamper aspartimide formation, respectively, which enabled the efficient synthesis of inert SUMO-2 propargylamide (PA). The probe could be activated to capture SENPs upon photo-irradiation not only in vitro but also in intact cells, providing opportunities to further perform intracellular time-resolved proteome-wide profiling of SUMO-related enzymes.

A molecular rotor FLIM probe reveals dynamic coupling between mitochondrial inner membrane fluidity and cellular respiration.

The inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration. The ETC relies on mobile carriers; therefore, it has long been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology. However, it is unclear if cells temporally modulate IMM fluidity upon metabolic or other stimulation. Using a photostable, red-shifted, cell-permeable molecular-rotor, Mitorotor-1, we present a multiplexed approach for quantitatively mapping IMM fluidity in living cells. This reveals IMM fluidity to be linked to cellular-respiration and responsive to stimuli. Multiple approaches combining in vitro experiments and live-cell fluorescence (FLIM) lifetime imaging microscopy (FLIM) show Mitorotor-1 to robustly report IMM 'microviscosity'/fluidity through changes in molecular free volume. Interestingly, external osmotic stimuli cause controlled swelling/compaction of mitochondria, thereby revealing a graded Mitorotor-1 response to IMM microviscosity. Lateral diffusion measurements of IMM correlate with microviscosity reported via Mitorotor-1 FLIM-lifetime, showing convergence of independent approaches for measuring IMM local-order. Mitorotor-1 FLIM reveals mitochondrial heterogeneity in IMM fluidity; between-and-within cells and across single mitochondrion. Multiplexed FLIM lifetime imaging of Mitorotor-1 and NADH autofluorescence reveals that IMM fluidity positively correlates with respiration, across individual cells. Remarkably, we find that stimulating respiration, through nutrient deprivation or chemically, also leads to increase in IMM fluidity. These data suggest that modulating IMM fluidity supports enhanced respiratory flux. Our study presents a robust method for measuring IMM fluidity and suggests a dynamic regulatory paradigm of modulating IMM local order on changing metabolic demand.

Visualize intracellular ß-galactosidase using an asymmetric near-infrared fluorescent probe with a large Stokes shift.

ß-galactosidase (ß-Gal) is an important biomarker of cell senescence and primary ovarian cancer. Therefore, it is of great significance to construct a near-infrared fluorescent probe with deep tissue penetration and a high signal-to-noise ratio for visualization of ß-galactosidase in biological systems. However, most near-infrared probes tend to have small Stokes shifts and low signal-to-noise ratios due to crosstalk between excitation and emission spectra. Using d-galactose residues as specific recognition units and near-infrared dye TJ730 as fluorophores, a near-infrared fluorescence probe SN-CR with asymmetric structure was developed for the detection of ß-Gal. The probe has a fast reaction equilibrium time (<12 min) with ß-Gal, excellent biocompatibility, near-infrared emission (738 nm), low detection limit (0.0029 U/mL), and no crosstalk between the excitation spectrum and emission spectrum (Stokes shifts 142 nm) of the probe. Cell imaging studies have shown that SN-CR can visually trace ß-Gal in different cells and distinguish ovarian cancer cells from other cells.

Development of a novel chitosan-based two-photon fluorescent nanoprobe with enhanced stability for the specific detection of endogenous H2O2.

Hydrogen peroxide (H2O2) acts as an important reactive oxygen species (ROS) and maintains the redox equilibrium in organisms. Imbalance of H2O2 concentration is associated with the development of many diseases. Traditional small molecular based fluorescent probes often show drawbacks of cytotoxicity and easily metabolic clearance. Herein, a chitosan-based two-photon fluorescent nanoprobe (DC-BI) was constructed and applied for H2O2 detection in live organisms. DC-BI was composed by chitosan nanoparticles and a two-photon fluorophore of naphthalimide analogues (BI) with H2O2-responsive property. The structure of DC-BI was characterized by NMR, FTIR, XPS, XRD, DLS and MLS analyses. As study shown, the nanoprobe DC-BI exhibited improved distribution stability and smaller cytotoxicity. In the presence of H2O2, both the absorption and emission spectra show dramatic changes, the fluorescence intensity at 580 nm obviously enhanced. Furthermore, fluorescence imaging results indicate that DC-BI is capable of imaging endogenous H2O2 in cells and zebrafish. The design and development of chitosan-based nanoprobe DC-BI has provided a general example of nanoprobe construction with excellent distribution stability, two-photon property, and biocompatibility.

An activatable near-infrared molecular reporter for fluoro-photoacoustic imaging of liver fibrosis.

Noninvasive and accurate detection of liver fibrosis is extremely significant for well-timed intervention and treatment to prevent or reverse its progression. Fluorescence imaging probes hold great potential for imaging of liver fibrosis, but they always encounter the inherent limitation of shallow penetration depth, which compromises their ability of in vivo detection. To overcome this issue, an activatable fluoro-photoacoustic bimodal imaging probe (IP) is herein developed for specific visualization of liver fibrosis. The probe IP is constructed on a near-infrared thioxanthene-hemicyanine dye that is caged with gamma-glutamyl transpeptidase (GGT) responsive substrate and linked with integrin-targeted peptide (cRGD). Such molecular design permits IP to effectively accumulate in the liver fibrosis region through specific recognition of cRGD towards integrin and activate its fluoro-photoacoustic signal after interaction with overexpressed GGT to precisely monitor the liver fibrosis. Thus, our study presents a potential strategy to design dual-target fluoro-photoacoustic imaging probes for noninvasive detection of early-stage liver fibrosis.

Synthetic Approaches of Epoxysuccinate Chemical Probes.

Synthetic chemical probes are powerful tools for investigating biological processes. They are particularly useful for proteomic studies such as activity-based protein profiling (ABPP). These chemical methods initially used mimics of natural substrates. As the techniques gained prominence, more and more elaborate chemical probes with increased specificity towards given enzyme/protein families and amenability to various reaction conditions were used. Among the chemical probes, peptidyl-epoxysuccinates represent one of the first types of compounds used to investigate the activity of the cysteine protease papain-like family of enzymes. Structurally derived from the natural substrate, a wide body of inhibitors and activity- or affinity-based probes bearing the electrophilic oxirane unit for covalent labeling of active enzymes now exists. Herein, we review the literature regarding the synthetic approaches to epoxysuccinate-based chemical probes together with their reported applications, from biological chemistry and inhibition studies to supramolecular chemistry and the formation of protein arrays.

HiBiT-SpyTag: A Minimal Tag for Covalent Protein Capture and Degrader Development.

The ability to rapidly and selectively modulate cellular protein levels using small molecules is essential for studying complex biological systems. Degradation tags, such as dTAG, allow for selective protein removal with a specific degrader molecule, but their utility is limited by the large tag size (>12 kDa) and the low efficiency of fusion product gene knock-in. Here, we describe the development of a short 24 amino acid peptide tag that enables cell-based quantification and covalent functionalization of proteins to which it is fused. The minimalistic peptide, termed HiBiT-SpyTag, incorporates the HiBiT peptide for protein level quantification and SpyTag, which forms a spontaneous isopeptide bond in the presence of the SpyCatcher protein. Transient expression of dTAG-SpyCatcher efficiently labels HiBiT-SpyTag-modified BRD4 or IRE1α in cells, and subsequent treatment with the dTAG13 degrader results in efficient protein removal without the need for full dTAG knock-in. We also demonstrate the utility of HiBiT-SpyTag for validating the degradation of the endoplasmic reticulum (ER) stress sensor IRE1α, which led to the development of the first PROTAC degrader of the protein. Our modular HiBiT-SpyTag system represents a valuable tool for the efficient development of degraders and for studying other proximity-induced pharmacology.

Development and Validation of TaqMan Chemistry Probe-Based Rapid Assay for the Detection of Echinocandin-Resistance in Candida auris.

Candida auris is a multidrug-resistant yeast pathogen causing outbreaks in health care facilities worldwide, and the emergence of echinocandin-resistant C. auris is a concern. Currently used Clinical and Laboratory Standards Institute (CLSI) and commercial antifungal susceptibility tests (AFST) are phenotype-based, slow, and not scalable, limiting their effectiveness in the surveillance of echinocandin-resistant C. auris. The urgent need for accurate and rapid methods of assessment of echinocandin resistance cannot be overstated, as this class of antifungal drugs is preferred for patient management. We report the development and validation of a TaqMan chemistry probe-based fluorescence melt curve analysis (FMCA) following asymmetric polymerase chain reaction (PCR) to assess mutations within the hot spot one (HS1) region of FKS1, the gene responsible for encoding 1,3-ß-d-glucan synthase that is a target for echinocandins. The assay correctly identified F635C, F635Y, F635del, F635S, S639F or S639Y, S639P, and D642H/R645T mutations. Of these mutations, F635S and D642H/R645T were not involved in echinocandin resistance, while the rest were, as confirmed by AFST. Of 31 clinical cases, the predominant mutation conferring echinocandin resistance was S639F/Y (20 cases) followed by S639P (4 cases), F635del (4 cases), F635Y (2 cases), and F635C (1 case). The FMCA assay was highly specific and did not cross-react with closely and distantly related Candida and other yeast and mold species. Structural modeling of the Fks1 protein, its mutants, and docked conformations of three echinocandin drugs suggest a plausible Fks1 binding orientation for echinocandins. These findings lay the groundwork for future evaluations of additional FKS1 mutations and their impact on the development of drug resistance. The TaqMan chemistry probe-based FMCA would allow rapid, high throughput, and accurate detection of FKS1 mutations conferring echinocandin resistance in C. auris.

Triubiquitin Probes for Identification of Reader and Eraser Proteins of Branched Polyubiquitin Chains.

The important roles played by branched polyubiquitin chains were recently uncovered in proteasomal protein degradation, mitotic regulation, and NF-κB signaling. With the new realization of a wide presence of branched ubiquitin chains in mammalian cells, there is an urgent need of identifying the reader and eraser proteins of the various branched ubiquitin chains. In this work, we report the generation of noncleavable branched triubiquitin probes with combinations of K11-, K48-, and K63-linkages. Through a pulldown approach using the branched triUb probes, we identified human proteins that recognize branched triubiquitin structures including ubiquitin-binding proteins and deubiquitinases (DUBs). Proteomics analysis of the identified proteins enriched by the branched triubiquitin probes points to possible roles of branched ubiquitin chains in cellular processes including DNA damage response, autophagy, and receptor endocytosis. In vitro characterization of several identified UIM-containing proteins demonstrated their binding to branch triubiquitin chains with moderate to high affinities. Availability of this new class of branched triubiquitin probes will enable future investigation into the roles of branched polyubiquitin chains through identification of specific reader and eraser proteins, and the modes of branched ubiquitin chain recognition and processing using biochemical and biophysical methods.