RG-7916 is a first-in-class drug candidate for the treatment of spinal muscular atrophy (SMA) that functions by modulating pre-mRNA splicing of the SMN2 gene, resulting in a 2.5-fold increase in ...survival of motor neuron (SMN) protein level, a key protein lacking in SMA patients. RG-7916 is currently in three interventional phase 2 clinical trials for various types of SMA. In this report, we show that SMN-C2 and -C3, close analogs of RG-7916, act as selective RNA-binding ligands that modulate pre-mRNA splicing. Chemical proteomic and genomic techniques reveal that SMN-C2 directly binds to the AGGAAG motif on exon 7 of the SMN2 pre-mRNA, and promotes a conformational change in two to three unpaired nucleotides at the junction of intron 6 and exon 7 in both in vitro and in-cell models. This change creates a new functional binding surface that increases binding of the splicing modulators, far upstream element binding protein 1 (FUBP1) and its homolog, KH-type splicing regulatory protein (KHSRP), to the SMN-C2/C3–SMN2 pre-mRNA complex and enhances SMN2 splicing. These findings underscore the potential of small-molecule drugs to selectively bind RNA and modulate pre-mRNA splicing as an approach to the treatment of human disease.
Decarboxylative borylation Li, Chao; Wang, Jie; Barton, Lisa M. ...
Science,
06/2017, Letnik:
356, Številka:
6342
Journal Article
Recenzirano
Odprti dostop
The widespread use of alkyl boronic acids and esters is frequently hampered by the challenges associated with their preparation. We describe a simple and practical method to rapidly access densely ...functionalized alkyl boronate esters from abundant carboxylic substituents. This broad-scope nickel-catalyzed reaction uses the same activating principle as amide bond formation to replace a carboxylic acid moiety with a boronate ester. Application to peptides allowed expedient preparations of α-amino boronic acids, often with high stereoselectivity, thereby facilitating synthesis of the alkyl boronic acid drugs Velcade and Ninlaro as well as a boronic acid version of the iconic antibiotic vancomycin. The reaction also enabled the discovery and extensive biological characterization of potent human neutrophil elastase inhibitors, which offer reversible covalent binding properties.
Cholesterol is a major component of the plasma membranes (PMs) of animal cells, comprising 35-40mol% of total PM lipids. Recent studies using cholesterol-binding bacterial toxins such as domain 4 of ...Anthrolysin O (ALOD4) and fungal toxins such as Ostreolysin A (OlyA) have revealed new insights into the organization of PM cholesterol. These studies have defined three distinct pools of PM cholesterol-a fixed pool that is essential for membrane integrity, a sphingomyelin (SM)-sequestered pool that can be detected by OlyA, and a third pool that is accessible and can be detected by ALOD4. Accessible cholesterol is available to interact with proteins and transport to the endoplasmic reticulum (ER), and controls many cellular signaling processes including cholesterol homeostasis, Hedgehog signaling, and bacterial and viral infection. Here, we provide detailed descriptions for the use of ALOD4 and OlyA, both of which are soluble and non-lytic proteins, to study cholesterol organization in the PMs of animal cells. Furthermore, we describe two new versions of ALOD4 that we have developed to increase the versatility of this probe in cellular studies. One is a dual His
and FLAG epitope-tagged version and the other is a fluorescent version where ALOD4 is fused to Neon, a monomeric fluorescent protein. These new forms of ALOD4 together with previously described OlyA provide an expanded collection of tools to sense, visualize, and modulate levels of accessible and SM-sequestered cholesterol on PMs and study the role of these cholesterol pools in diverse membrane signaling events.
Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential ...for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8
T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.
Fluorescence lifetime ophthalmoscopy (FLIO) is an emerging clinical modality that could provide biomarkers of retinal health beyond fluorescence intensity. Adaptive optics (AO) ophthalmoscopy ...provides the confocality to measure fluorescence lifetime (FL) primarily from the retinal pigment epithelium (RPE) whereas clinical FLIO has greater influence from fluorophores in the inner retina and lens. Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) measures of FL in vivo could provide insight into RPE health at different stages of disease. In this study, we assess changes in pentosan polysulfate sodium (PPS) toxicity, a recently described toxicity that has clinical findings similar to advanced age-related macular degeneration.
AOFLIO was performed on three subjects with PPS toxicity (57-67 years old) and six age-matched controls (50-64 years old). FL was analyzed with a double exponential decay curve fit and with phasor analysis. Regions of interest (ROIs) were subcategorized based on retinal features on optical coherence tomography (OCT) and compared to age-matched controls.
Twelve ROIs from PPS toxicity subjects met the threshold for analysis by curve fitting and 15 ROIs met the threshold for phasor analysis. Subjects with PPS toxicity had prolonged FL compared to age-matched controls. ROIs of RPE degeneration had the longest FLs, with individual pixels extending longer than 900 ps.
Our study shows evidence that AOFLIO can provide meaningful information in outer retinal disease beyond what is obtainable from fluorescence intensity alone. More studies are needed to determine the prognostic value of AOFLIO.
Recent studies using two cholesterol-binding bacterial toxin proteins, perfringolysin O (PFO) and domain 4 of anthrolysin O (ALOD4), have shown that cholesterol in the plasma membranes (PMs) of ...animal cells resides in three distinct pools. The first pool comprises mobile cholesterol, accessible to both PFO and ALOD4, that is rapidly transported to the endoplasmic reticulum (ER) to signal cholesterol excess and maintain cholesterol homeostasis. The second is a sphingomyelin (SM)-sequestered pool inaccessible to PFO and ALOD4 but that becomes accessible by treatment with SM-degrading sphingomyelinase (SMase). The third is an essential pool also inaccessible to PFO and ALOD4 that cannot be liberated by SMase treatment. The accessible cholesterol pool can be trapped on PMs of live cells by nonlytic ALOD4, blocking its transport to the ER. However, studies of the two other pools have been hampered by a lack of available tools. Here, we used ostreolysin A (OlyA), which specifically binds SM/cholesterol complexes in membranes, to study the SM-sequestered cholesterol pool. Binding of nonlytic OlyA to SM/cholesterol complexes in PMs of live cells depleted the accessible PM cholesterol pool detectable by ALOD4. Consequently, transport of accessible cholesterol from PM to ER ceased, thereby activating SREBP transcription factors and increasing cholesterol synthesis. Thus, OlyA and ALOD4 both control movement of PM cholesterol, but through different lipid-binding mechanisms. We also found that PM-bound OlyA was rapidly internalized into cells, whereas PM-bound ALOD4 remained on the cell surface. Our findings establish OlyA and ALOD4 as complementary tools to investigate cellular cholesterol transport.
Novel coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome virus (SARS‐CoV‐2) has become a global health care crisis. The Centers for Disease Control and Prevention (CDC) ...lists immunocompromised patients, including those requiring immunosuppression following renal transplantation, as high risk for severe disease from SARS‐CoV‐2. Treatment for other viral infections in renal transplant recipients often includes a reduction in immunosuppression; however, no current guidelines are available recommending the optimal approach to managing immunosuppression in the patients who are infected with SARS‐CoV‐2. It is currently advised to avoid corticosteroids in the treatment of SARS‐CoV‐2 outside of critically ill patients. Recently published cases describing inpatient care of COVID‐19 in renal transplant recipients differ widely in disease severity, time from transplantation, baseline immunosuppressive therapy, and the modifications made to immunosuppression during COVID‐19 treatment. This review summarizes and compares inpatient immunosuppressant management strategies of recently published reports in the renal transplant population infected with SARS‐CoV‐2 and discusses the limitations of corticosteroids in managing immunosuppression in this patient population.
Kinetic analysis of surface reactions at the single molecule level is important for understanding the influence of the substrate and solvent on reaction dynamics and mechanisms, but it is difficult ...with current methods. Here we present a stochastic kinetic analysis of the oxygenation of cobalt octaethylporphyrin (CoOEP) at the solution/solid interface by monitoring fluctuations from equilibrium using scanning tunneling microscopy (STM) imaging. Image movies were used to monitor the oxygenated and deoxygenated state dwell times. The rate constants for CoOEP oxygenation are k a = 4.9 × 10–6 s–1·Torr–1 and k d = 0.018 s–1. This is the first use of stochastic dwell time analysis with STM to study a chemical reaction, and the results suggest that it has great potential for application to a wide range of surface reactions. Expanding these stochastic studies to further systems is key to unlocking kinetic information for surface-confined reactions at the molecular level, especially at the solution/solid interface.
Scanning tunneling microscopy (STM) was used to observe and quantify single-molecule diffusion at the solution/solid interface and at the argon/solid interface. This work investigates the influence ...of the temperature, solvent, and STM tip on isolated molecular surface diffusion through analysis of the molecular trajectories in sequential STM images. The surface diffusion of YC6S-Pc2 in phenyloctane was found to be thermally activated with almost no motion observed at 5 °C, whereas, above 30 °C molecular motion and/or adsorption/desorption are so rapid that it becomes difficult to track single molecules. The surface diffusion of molecules also depended on solvents; solvents with greater dipole moments (and presumably greater interaction with Au(111)) reduced diffusivity, while the absence of a solvent (i.e., argon/solid interface) increased diffusivity. At room temperature, the influence of the STM tip was quantified by varying the sample bias voltage, with the diffusion coefficient varying between 0.6 × 10–17 and 16 × 10–17 cm2/s. This is the first quantitative study of single-molecule (as opposed to vacancy) diffusion at the solution/solid interface. An important implication of this study is that even in the case of very strong adsorbate–substrate interactions, the STM tip can significantly mobilize surface molecules and thereby enhance the formation of self-assembled films. Moreover, because the tip-induced displacements are not unidirectional, one cannot diagnose tip-induced motion by analyzing the displacements at one set-point and scan rate. Particular care must be taken in any STM-based studies of self-assembly kinetics at the solution–solid interface.
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