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1.
Proc Natl Acad Sci U S A ; 120(11): e2219523120, 2023 03 14.
Article in English | MEDLINE | ID: mdl-36893269

ABSTRACT

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.


Subject(s)
COVID-19 , Humans , Animals , Mice , RNA, Small Interfering/genetics , COVID-19/therapy , SARS-CoV-2/genetics , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Oligonucleotides , Lung
2.
Chembiochem ; 24(22): e202300544, 2023 11 16.
Article in English | MEDLINE | ID: mdl-37666794

ABSTRACT

Given the importance of mRNA with 5'-cap, easy access to RNA substrates with different 7m G caps, of high quality and in large quantities is essential to elucidate the roles of RNA and the regulation of underlying processes. In addition to existing synthetic routes to 5'-cap RNA based on enzymatic, chemical or chemo-enzymatic methods, we present here an all-chemical method for synthetic RNA capping. The novelty of this study lies in the fact that the capping reaction is performed on solid-support after automated RNA assembly using commercial 2'-O-propionyloxymethyl ribonucleoside phosphoramidites, which enable final RNA deprotection under mild conditions while preserving both 7m G-cap and RNA integrity. The capping reaction is efficiently carried out between a 5'-phosphoroimidazolide RNA anchored on the support and 7m GDP in DMF in the presence of zinc chloride. Substantial amounts of 7m G-cap RNA (from 1 to 28 nucleotides in length and of any sequence with or without internal methylations) containing various cap structures (7m GpppA, 7m GpppAm , 7m Gpppm6 A, 7m Gpppm6 Am , 7m GpppG, 7m GpppGm ) were obtained with high purity after IEX-HPLC purification. This capping method using solid-phase chemistry is convenient to perform and provides access to valuable RNA substrates as useful research tools to unravel specific issues regarding cap-related processes.


Subject(s)
Methyltransferases , Ribonucleosides , Methyltransferases/metabolism , RNA Caps , Methylation , RNA, Messenger
3.
Molecules ; 24(9)2019 May 10.
Article in English | MEDLINE | ID: mdl-31083395

ABSTRACT

The United States is currently experiencing an opioid crisis, with more than 47,000 deaths in 2017 due to opioid overdoses. Current approaches for opioid identification and quantification in body fluids include immunoassays and chromatographic methods (e.g., LC-MS, GC-MS), which require expensive instrumentation and extensive sample preparation. Our aim was to develop a portable point-of-care device that can be used for the instant detection of opioids in body fluids. Here, we reported the development of a morphine-sensitive fluorescence-based sensor chip to sensitively detect morphine in the blood using a homogeneous immunoassay without any washing steps. Morphine-sensitive illuminating peptides were identified using a high throughput one-bead one-compound (OBOC) combinatorial peptide library approach. The OBOC libraries contain a large number of random peptides with a molecular rotor dye, malachite green (MG), that are coupled to the amino group on the side chain of lysine at different positions of the peptides. The OBOC libraries were then screened for fluorescent activation under a confocal microscope, using an anti-morphine monoclonal antibody as the screening probe, in the presence and absence of free morphine. Using this novel three-step fluorescent screening assay, we were able to identify the peptide-beads that fluoresce in the presence of an anti-morphine antibody, but lost fluorescence when the free morphine was present. After the positive beads were decoded using automatic Edman microsequencing, the morphine-sensitive illuminating peptides were then synthesized in soluble form, functionalized with an azido group, and immobilized onto microfabricated PEG-array spots on a glass slide. The sensor chip was then evaluated for the detection of morphine in plasma. We demonstrated that this proof-of-concept platform can be used to develop fluorescence-based sensors against morphine. More importantly, this technology can also be applied to the discovery of other novel illuminating peptidic sensors for the detection of illicit drugs and cancer biomarkers in body fluids.


Subject(s)
Analgesics, Opioid/analysis , Analgesics, Opioid/blood , Body Fluids/chemistry , Combinatorial Chemistry Techniques/methods , Morphine/analysis , Morphine/blood , Peptides/chemistry , Chromatography, Liquid , High-Throughput Screening Assays , Humans , Peptide Library
4.
Anal Chem ; 89(13): 7000-7008, 2017 07 05.
Article in English | MEDLINE | ID: mdl-28530391

ABSTRACT

We report an analytical method to determine peptide loading of "one-bead one-compound" (OBOC) combinatorial peptide libraries at single-bead level. The quantification is based on a linear relationship between the amount of N-terminal amino groups on individual peptide beads and the intensity of Raman signal obtained from a specifically designed reporter labeled on amino groups. Confocal Raman spectroscopy was employed to characterize peptide loading of beads with defined peptide sequences and from OBOC combinatorial peptide libraries. Although amine loading of blank TentaGel beads was found to be uniform, peptide loading among beads of OBOC peptide libraries varied substantially, particularly for those libraries with long sequences. Construction of OBOC libraries can be monitored with this novel analytical technique so that synthetic conditions can be optimized for the preparation of high-quality OBOC peptide libraries. As the variability of peptide loading of individual library beads can significantly influence the screening results, quantitative information obtained by this method will allow us to gain insight into the complexity and challenge of OBOC library synthesis and screening.

5.
Proc Natl Acad Sci U S A ; 110(22): 8858-63, 2013 May 28.
Article in English | MEDLINE | ID: mdl-23610407

ABSTRACT

The initiation of mRNA degradation often requires deprotection of its 5' end. In eukaryotes, the 5'-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5'-monophosphorylated RNA that is a substrate for 5' exoribonucleases. In bacteria, the 5'-triphosphate group of primary transcripts is also converted to a 5' monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo- and 5' exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNA targets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base-mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria.


Subject(s)
Bacillus subtilis/enzymology , Guanosine/metabolism , Models, Molecular , Pyrophosphatases/chemistry , Pyrophosphatases/metabolism , RNA Caps/metabolism , RNA Stability/physiology , Chromatography, High Pressure Liquid , Crystallization , Guanosine Triphosphate/metabolism , Magnetic Resonance Spectroscopy , Oligonucleotides/genetics , Polymerase Chain Reaction , Protein Conformation , RNA Stability/genetics , Species Specificity , Substrate Specificity , Nudix Hydrolases
6.
RNA ; 18(4): 856-68, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22334760

ABSTRACT

The 5' end of eukaryotic mRNA carries a N(7)-methylguanosine residue linked by a 5'-5' triphosphate bond. This cap moiety ((7m)GpppN) is an essential RNA structural modification allowing its efficient translation, limiting its degradation by cellular 5' exonucleases and avoiding its recognition as "nonself" by the innate immunity machinery. In vitro synthesis of capped RNA is an important bottleneck for many biological studies. Moreover, the lack of methods allowing the synthesis of large amounts of RNA starting with a specific 5'-end sequence have hampered biological and structural studies of proteins recognizing the cap structure or involved in the capping pathway. Due to the chemical nature of N(7)-methylguanosine, the synthesis of RNAs possessing a cap structure at the 5' end is still a significant challenge. In the present work, we combined a chemical synthesis method and an enzymatic methylation assay in order to produce large amounts of RNA oligonucleotides carrying a cap-0 or cap-1. Short RNAs were synthesized on solid support by the phosphoramidite 2'-O-pivaloyloxymethyl chemistry. The cap structure was then coupled by the addition of GDP after phosphorylation of the terminal 5'-OH and activation by imidazole. After deprotection and release from the support, GpppN-RNAs or GpppN(2'-Om)-RNAs were purified before the N(7)-methyl group was added by enzymatic means using the human (guanine-N(7))-methyl transferase to yield (7m)GpppN-RNAs (cap-0) or (7m)GpppN(2'-Om)-RNAs (cap-1). The RNAs carrying different cap structures (cap, cap-0 or, cap-1) act as bona fide substrates mimicking cellular capped RNAs and can be used for biochemical and structural studies.


Subject(s)
Methyltransferases/metabolism , RNA Caps , Humans , Methylation
7.
PLoS Pathog ; 8(9): e1002912, 2012 Sep.
Article in English | MEDLINE | ID: mdl-23028313

ABSTRACT

The dengue virus (DV) is an important human pathogen from the Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5Pol(DV) of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5Pol(DV) contains a specific priming site for adenosine 5'-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn(2+) is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5Pol(DV) ensures the conservation of the 5'-adenosine by strongly discriminating against viral templates containing an erroneous 3'-end nucleotide in the presence of Mg(2+). In the presence of Mn(2+), NS5Pol(DV) is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo.


Subject(s)
Dengue Virus/enzymology , Dengue Virus/genetics , Genome, Viral , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/metabolism , Adenosine Triphosphate/metabolism , DNA Primers , Manganese/chemistry , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Templates, Genetic , Viral Nonstructural Proteins/genetics
8.
Org Biomol Chem ; 12(41): 8325-35, 2014 Nov 07.
Article in English | MEDLINE | ID: mdl-25212512

ABSTRACT

Strategies to couple non-steroidal anti-inflammatory drugs (NSAIDs) to a glucosamine hydrochloride salt via an amino acid linker are investigated and a series of novel NSAID-glucosamine bioconjugates have been prepared.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemistry , Glucosamine/chemistry , Amino Acids/chemistry , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Molecular Structure
9.
Bioorg Med Chem ; 21(17): 5461-9, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-23810677

ABSTRACT

5'-Triphosphate 2'-5'-oligoadenylate (2-5A) is the central player in the 2-5A system that is an innate immunity pathway in response to the presence of infectious agents. Intracellular endoribonuclease RNase L activated by 2-5A cleaves viral and cellular RNA resulting in apoptosis. The major limitations of 2-5A for therapeutic applications is the short biological half-life and poor cellular uptake. Modification of 2-5A with biolabile and lipophilic groups that facilitate its uptake, increase its in vivo stability and release the parent 2-5A drug in an intact form offer an alternative approach to therapeutic use of 2-5A. Here we have synthesized the trimeric and tetrameric 2-5A species bearing hydrophobic and enzymolabile pivaloyloxymethyl groups at 3'-positions and a triphosphate at the 5'-end. Both analogs were able to activate RNase L and the production of the trimer 2-5A (the most active) was scaled up to the milligram scale for antiviral evaluation in cells infected by influenza virus or respiratory syncytial virus. The trimer analog demonstrated some significant antiviral activity.


Subject(s)
Adenine Nucleotides/chemistry , Antiviral Agents/chemical synthesis , Endoribonucleases/chemistry , Oligoribonucleotides/chemistry , Adenine Nucleotides/chemical synthesis , Adenine Nucleotides/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Cell Line, Tumor , Endoribonucleases/metabolism , Fluorescence Resonance Energy Transfer , Humans , Influenza A virus/drug effects , Oligoribonucleotides/chemical synthesis , Oligoribonucleotides/pharmacology , Polymerization , Solid-Phase Synthesis Techniques
10.
Chem Biol Drug Des ; 98(1): 102-113, 2021 07.
Article in English | MEDLINE | ID: mdl-33955172

ABSTRACT

Non-steroidal anti-inflammatory drugs (NSAIDs) are a powerful class of inhibitors targeting two isoforms of the family of cyclooxygenase enzymes (COX-1 and COX-2). While NSAIDs are widely used in the management of pain, in particular as a treatment for osteo- and rheumatoid arthritis, their long-term use has been associated with numerous on- and off-target effects. As the carboxylic acid moiety present in common NSAIDs is responsible for some of their adverse effects, but is not required for their anti-inflammatory activity, we sought to mask this group through direct coupling to glucosamine, which is thought to prevent cartilage degradation. We report herein the conjugation of commonly prescribed NSAIDs to glucosamine hydrochloride and the use of molecular docking to show that addition of the carbohydrate moiety to the parent NSAID can enhance binding in the active site of COX-2. In a preliminary, in vitro screening assay, the diclofenac-glucosamine bioconjugate exhibited 10-fold greater activity toward COX-2, making it an ideal candidate for future in vivo studies. Furthermore, in an intriguing result, we observed that the mefenamic acid-glucosamine bioconjugate displayed enhanced activity toward COX-1 rather than COX-2.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemistry , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/metabolism , Cyclooxygenase Inhibitors/chemistry , Glucosamine/chemistry , Glycoconjugates/chemistry , Mefenamic Acid/chemistry , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Catalytic Domain , Cyclooxygenase Inhibitors/adverse effects , Diclofenac/chemistry , Drug Design , Glycoconjugates/adverse effects , Mefenamic Acid/adverse effects , Molecular Docking Simulation , Protein Binding , Protein Conformation , Stomach , Structure-Activity Relationship
11.
ACS Comb Sci ; 19(5): 299-307, 2017 05 08.
Article in English | MEDLINE | ID: mdl-28378995

ABSTRACT

Membrane active peptides (MAPs) represent a class of short biomolecules that have shown great promise in facilitating intracellular delivery without disrupting cellular plasma membranes. Yet their clinical application has been stalled by numerous factors: off-target delivery, a requirement for high local concentration near cells of interest, degradation en route to the target site, and in the case of cell-penetrating peptides, eventual entrapment in endolysosomal compartments. The current method of deriving MAPs from naturally occurring proteins has restricted the discovery of new peptides that may overcome these limitations. Here, we describe a new branch of assays featuring high-throughput functional screening capable of discovering new peptides with tailored cell uptake and endosomal escape capabilities. The one-bead-one-compound (OBOC) combinatorial method is used to screen libraries containing millions of potential MAPs for binding to synthetic liposomes, which can be adapted to mimic various aspects of limiting membranes. By incorporating unnatural and d-amino acids in the library, in addition to varying buffer conditions and liposome compositions, we have identified several new highly potent MAPs that improve on current standards and introduce motifs that were previously unknown or considered unsuitable. Since small variations in pH and lipid composition can be controlled during screening, peptides discovered using this methodology could aid researchers building drug delivery platforms with unique requirements, such as targeted intracellular localization.


Subject(s)
Cell-Penetrating Peptides/chemistry , Liposomes/chemistry , Peptide Library , Amino Acids/chemistry , Cell Line , Cell-Penetrating Peptides/metabolism , Cell-Penetrating Peptides/toxicity , Combinatorial Chemistry Techniques , Fluorescent Dyes/chemistry , High-Throughput Screening Assays , Humans , Hydrogen-Ion Concentration , Microspheres , Rhodamines/chemistry , Surface Properties
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