Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3′ untranslated region (UTR) of this β-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3′ UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3′ UTRs in the infectious virions and the minigene-transfected cells are almost identical.

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5′-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.

No abstract

Background: Citrus sinensis (L.) Osbeck peels are usually discarded as wastes; however, they are rich sources of Vitamin C, fibre, and many nutrients including phenolics and flavonoids which are also good antioxidant agents. This study aimed to examine phytochemical composition, antioxidant capabilities, cytotoxicity of C. sinensis (L.) Osbeck peel extract and and to compare the antibacterial activity with zinc nanoparticles of Citrus sinensis (L.) Osbeck peels with its extract. GC-MS analysis of the compounds present in the peels extract of Citrus sinensis (L.) Osbeck was also done. Methods: C. sinensis (L.) Osbeck fruits were collected from Sindhuli district and were taken to National Herbarium and Plant Laboratory, Godawari, Lalitpur for its identification. Extraction was done by maceration in aqueous solvent. Extract was subjected to Phytochemical screening done by color reactions with different reagents, Antioxidant activities of the peel extracts were examined via the 2,2-diphenylpicrylhydrazyl (DPPH) free radical scavenging activity. Total phenolic content and total flavonoid content of the extracts were measured via the Folin-Ciocalteau method and the aluminium chloride colorimetric method, respectively. Cytotoxic activities of the peel extracts were determined by Brine Shrimp Lethality Bioassay. Comparison of antibacterial activity of extract and zinc oxide nanoparticles prepared via green synthesis using C. sinensis (L.) Osbeck peel extracts as reducing agents. Antibacterial activity was tested by Bore well diffusion method. Result: The extractive value of C. sinensis (L.) Osbeck was found to be 8.64% in aqueous solvent. GC-MS analysis of peel extract of C. sinensis (L.) Osbeck showed the presence of 2-Methoxy-4-vinylphenol, 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, Benzoic acid, 3-Deoxy-d-mannoic lactone and 5-Hydroxymethyl-furfuralas major compounds. The qualitative phytochemical test showed the presence of tannin, alkaloid, carbohydrate, flavonoid, cardiac glycoside, terpinoid. The DPPH radical scavenging activity of C. sinensis (L.) Osbeck peel extract was 35.56 microgram/ml. TPC of C. sinensis (L.) Osbeck peel extracts was 46.07 mg GAE/g. TFC was 1.29 mg QE/g. The LD50 value of Brine Shrimp Lethality assay of the extract showed 312.5 microgram/ml which is indicative. The antibacterial activity of zinc oxide nanoparticles was found to be greater than that of the extract, but the antibacterial activity of Zn-NPs was less than that of the standard. Conclusion: Hence, the GC-MS analysis of aqueous extracts of leaves of C. sinensis (L.) Osbeck showed the presence of 20 different compounds. Phytochemicals including phenolics and flavonoids in C. sinensis (L.) Osbeck peel extracts exhibited good antioxidant properties. The extract also exhibited antibacterial activity which was 4 times less than that of the standard. The antibacterial activity of standard was 2 times greater than that of Zn-NPs. The extract also exhibited cytotoxic activity. This study indicated that C. sinensis (L.) Osbeck peels contained potential antioxidant, cytotoxic and antibacterial compounds which could be exploited as value added products.

ID 16497 Poster Board 121Regulators of G protein Signaling (RGS) proteins are negative regulators of G-protein-coupled receptors (GPCRs). They reduce the amplitude and duration of GPCR signaling by accelerating the hydrolysis of GTP to GDP through their GTPase-accelerating protein (GAP) activity. Altered RGS protein function is involved in numerous diseased states. However, the therapeutic regulation of RGS proteins is challenging as they lack binding pockets for small molecules. Therefore, identifying mechanisms that control RGS protein activity and expression could be a potential targeting strategy. A notable example where altering RGS protein activity would be beneficial is asthma, a long-term respiratory disease, characterized by inflammation of the airways, resulting in the obstruction of airflow to the lungs. Aberrant activation of Ga q and downstream signaling contribute to chronic symptoms of asthma. RGS2 is known to be selective for Ga q over other G protein subtypes and has reduced expression levels in asthmatic patients. Low RGS2 protein levels are also implicated in many other diseases such as hypertension, cancer, and heart failure. Indeed, we previously showed that pharmacologically stabilizing RGS2 protein levels inhibits Ga q -mediated signaling and are cardioprotective in a mouse model of cardiac injury, suggesting that enhanced RGS2 protein levels correlate with increased function. Therefore, targeting the mechanisms that regulate RGS2 protein levels would be a feasible therapeutic strategy.RGS2 is rapidly degraded through the ubiquitin-proteasomal system (UPS). In the UPS, ubiquitin molecules are covalently linked to the substrate through the cascade of enzymatic reactions (facilitated by E1: ubiquitin-activating enzyme; E2: ubiquitinconjugating enzyme; E3 ligase). The ubiquitinated substrate is then recognized and degraded by the 26S proteasomal complex. We recently identified the E3 ligase that recognizes RGS2. This E3 ligase complex consists of Cullin 4B (Cul4B), DNA Damage Binding Protein 1 (DDB1), and F-box Only Protein 44 (FBXO44), where FBXO44 acts as the substrate recognition site for RGS2. Therefore, we hypothesize that inhibiting the RGS2-FBXO44 interaction will lead to enhanced RGS2 levels. To identify small molecule RGS2-FBXO44 interaction inhibitors, we utilized NanoLucV R Binary Technology (NanoBiT) to detect the interaction between RGS2 and FBXO44 in a high-throughput screen (HTS). We developed a HEK-293T cell line stably expressing the RGS2-SmBit and LgBit-FBXO44 and optimized the NanoBit assay for HTS. Using this assay, we screened 1600 compounds (Life Chemicals PPI fragment library) at the Purdue Chemicals Genomics Facility. Following hit confirmation and chemical clustering, the top 20 hits that inhibited the RGS2-FBXO44 protein-protein interaction at least 50% were selected for follow-up. The concentration-dependent activity of these 20 hits was performed to only select compounds inhibiting at least 50% of the interaction and having acceptable inhibition curves (Hill slope 0...