Light-activated chemical probing of nucleobase solvent accessibility inside cells.

This corrects the article DOI: 10.1038/nchembio.2548.

Light-activated chemical probing of nucleobase solvent accessibility inside cells.

The discovery of functional RNAs that are critical for normal and disease physiology continues to expand at a breakneck pace. Many RNA functions are controlled by the formation of specific structures, and an understanding of each structural component is necessary to elucidate its function. Measuring solvent accessibility intracellularly with experimental ease is an unmet need in the field. Here, we present a novel method for probing nucleobase solvent accessibility, Light Activated Structural Examination of RNA (LASER). LASER depends on light activation of a small molecule, nicotinoyl azide (NAz), to measure solvent accessibility of purine nucleobases. In vitro, this technique accurately monitors solvent accessibility and identifies rapid structural changes resulting from ligand binding in a metabolite-responsive RNA. LASER probing can further identify cellular RNA-protein interactions and unique intracellular RNA structures. Our photoactivation technique provides an adaptable framework to structurally characterize solvent accessibility of RNA in many environments.

Discovery of Novel Small-Molecule Calcium Sensitizers for Cardiac Troponin C: A Combined Virtual and Experimental Screening Approach.

Heart failure is a leading cause of death throughout the world and is triggered by a disruption of the cardiac contractile machinery. This machinery is regulated in a calcium-dependent manner by the protein complex troponin. Calcium binds to the N-terminal domain of cardiac troponin C (cNTnC) setting into motion the cascade of events leading to muscle contraction. Because of the severity and prevalence of heart failure, there is a strong need to develop small-molecule therapeutics designed to increase the calcium sensitivity of cardiac troponin in order to treat this devastating condition. Molecules that are able to stabilize an open configuration of cNTnC and additionally facilitate the binding of the cardiac troponin I (cTnI) switch peptide have the potential to enable increased calcium sensitization and strengthened cardiac function. Here, we employed a high throughput virtual screening methodology built upon the ability of computational docking to reproduce known experimental results and to accurately recognize cNTnC conformations conducive to small molecule binding using a receiver operator characteristic curve analysis. This approach combined with concurrent stopped-flow kinetic experimental verification led to the identification of a number of sensitizers, which slowed the calcium off-rate. An initial hit, compound 4, was identified with medium affinity (84 ± 30 µM). Through refinement, a calcium sensitizing agent, compound 5, with an apparent affinity of 1.45 ± 0.09 µM was discovered. This molecule is one of the highest affinity calcium sensitizers known to date.

Mechanism of Cardiac Troponin C Calcium Sensitivity Modulation by Small Molecules Illuminated by Umbrella Sampling Simulations.

Cardiac troponin C (cTnC) binds intracellular calcium and subsequently cardiac troponin I (cTnI), initiating cardiac muscle contraction. Due to its role in contraction, cTnC has been a therapeutic target in the search for small molecules to treat conditions that interfere with normal muscle contraction like the heritable cardiomyopathies. Structural studies have shown the binding location of small molecules such as bepridil, dfbp-o, 3-methyldiphenylamine (DPA), and W7 to be a hydrophobic pocket in the regulatory domain of cTnC (cNTnC) but have not shown the influence of these small molecules on the energetics of opening this domain. Here we describe an application of an umbrella sampling method used to elucidate the impact these calcium sensitivity modulators have on the free energy of cNTnC hydrophobic patch opening. We found that all these molecules lowered the free energy of opening in the absence of the cTnI, with bepridil facilitating the least endergonic transformation. In the presence of cTnI, however, we saw a stabilization of the open configuration due to DPA and dfbp-o binding, and a destabilization of the open configuration imparted by bepridil and W7. Predicted poor binding molecule NSC34337 left the hydrophobic patch in under 3 ns in conventional MD simulations suggesting that only hydrophobic patch binders stabilized the open conformation. In conclusion, this study presents a novel approach to study the impact of small molecules on hydrophobic patch opening through umbrella sampling, and it proposes mechanisms for calcium sensitivity modulation.

Cycloaddition Reactions of Azomethine Ylides and 1,3-Dienes on the C2v-Symmetrical Pentakisadduct of C60.

The reactivity of the C2v-symmetric pentakisadduct of C60 with azomethine ylides and conjugated dienes was studied experimentally and computationally. This derivative possesses four [6,6] double bonds, each with unique electrophilicity. The Diels-Alder reaction studied is a regiospecific, kinetically and thermodynamically guided [4 + 2] process producing [5:1]-hexaadducts with an octahedral addition pattern. The kinetically controlled Prato reaction gives a mixture of regioisomeric [5:1]-hexaadducts. The synthesis of geometrically well-defined supramolecular architectures may benefit from these new types of highly functionalized [5:1]-hexaadducts.

Direct Observation of an Alkylidenecarbene by Ultrafast Transient Absorption Spectroscopy.

Singlet α-methylbenzylidenecarbene has been detected and characterized for the first time by femtosecond transient absorption (fs-TA) spectroscopy. The carbene was generated by photolysis of the hydrocarbon precursor, 1-(1-phenylethylidene)-1a,9b-dihydro-1 H-cyclopropa[ l]-phenanthrene, in acetonitrile. The photolysis initially forms the singlet excited state of the precursor which then extrudes α-methylbenzylidenecarbene in 4.0 ps. The decay of α-methylbenzylidenecarbene, which was previously shown to rearrange into phenylpropyne by a 1,2-phenyl shift, occurred over 13.3 ps. Computed spectra at the TD-B3LYP/6-311+G** level of theory are consistent with the experimental observations. CASSCF(10,10)/6-311+G** calculations, using one of the carbene conformers as a model, indicated that the reference wave function is dominated by a closed-shell description.

In-silico guided design, screening, and molecular dynamic simulation studies for the identification of potential SARS-CoV-2 main protease inhibitors for the targeted treatment of COVID-19.

COVID-19, the disease responsible for the recent pandemic, is caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main protease (Mpro) of SARS-CoV-2 is an essential proteolytic enzyme that plays a number of important roles in the replication of the virus in human host cells. Blocking the function of SARS-CoV-2 Mpro offers a promising and targeted, therapeutic option for the treatment of the COVID-19 infection. Such an inhibitory strategy is currently successful in treating COVID-19 under FDA's emergency use authorization, although with limited benefit to the immunocompromised along with an unfortunate number of side effects and drug-drug interactions. Current COVID vaccines protect against severe disease and death but are mostly ineffective toward long COVID which has been seen in 5-36% of patients. SARS-CoV-2 is a rapidly mutating virus and is here to stay endemically. Hence, alternate therapeutics to treat SARS-CoV-2 infections are still needed. Moreover, because of the high degree of conservation of Mpro among different coronaviruses, any newly developed antiviral agents should better prepare us for potential future epidemics or pandemics. In this paper, we first describe the design and computational docking of a library of novel 188 first-generation peptidomimetic protease inhibitors using various electrophilic warheads with aza-peptide epoxides, α-ketoesters, and ß-diketones identified as the most effective. Second-generation designs, 192 compounds in total, focused on aza-peptide epoxides with drug-like properties, incorporating dipeptidyl backbones and heterocyclic ring motifs such as proline, indole, and pyrrole groups, yielding 8 hit candidates. These novel and specific inhibitors for SARS-CoV-2 Mpro can ultimately serve as valuable alternate and broad-spectrum antivirals against COVID-19.Communicated by Ramaswamy H. Sarma.

2-(3,5-Dinitrophenyl)-1,3-dithiane carbanion: a benzylic anion with a low energy triplet state.

Calculations at the DFT level predict that benzyl anions with strong π-electron-withdrawing groups in the meta position(s) have low energy diradical or triplet electronic states. Specifically, the 2-(3,5-dinitrophenyl)-1,3-dithiane carbanion is predicted to have nearly degenerate singlet and triplet states at the (U)B3LYP level as a free anion. Its lithium ion pair is predicted to be a ground-state triplet with a substantial (26 kcal/mol) singlet-triplet energy gap. Experiments on this anion using chemical trapping, NMR, and the Evans method strongly suggest that this anion is either a triplet or a ground-state singlet with a very low energy triplet state.

Mechanistic investigations into the cyclization and crystallization of benzobisoxazole-linked two-dimensional covalent organic frameworks.

Although many diverse covalent organic frameworks (COFs) have been synthesised over the past decade, our fundamental understanding of their nucleation and growth during the crystallization process has progressed slowly for many systems. In this work, we report the first in-depth mechanistic investigation detailing the role of nucleophilic catalysts during the formation of two distinct benzobisoxazole (BBO)-linked COFs. The BBO-COFs were constructed by reacting 1,3,5-tris(4-formylphenyl)benzene (TFPB) and 1,3,5-tris(4-formylphenyl)triazine (TFPT) C 3-symmetric monomers with a C 2-symmetric o-aminophenol substituted precursor using different nucleophiles (e.g. NaCN, NaN3, and NaSCH3). Our experimental and computational results demonstrate that the nucleophiles help initiate an oxidative dehydrogenation pathway by producing radical intermediates that are stabilized by a captodative effect. We also demonstrate that the electron deficient TFPT monomer not only aids in enhancing the crystallinity of the BBO-COFs but also participates in the delocalization of the radicals generated to help stabilize the intermediates.

Demonstration of In Vitro Resurrection of Aged Acetylcholinesterase after Exposure to Organophosphorus Chemical Nerve Agents.

After the inhibition of acetylcholinesterase (AChE) by organophosphorus (OP) nerve agents, a dealkylation reaction of the phosphylated serine, referred to as aging, can occur. When aged, known reactivators of OP-inhibited AChE are no longer effective. Realkylation of aged AChE may provide a route to reversing aging. We designed and synthesized a library of quinone methide precursors (QMPs) as proposed realkylators of aged AChE. Our lead compound (C8) from an in vitro screen successfully resurrected 32.7 and 20.4% of the activity of methylphosphonate-aged and isopropyl phosphate-aged electric-eel AChE, respectively, after 4 days. C8 displays properties of both resurrection (recovery from the aged to the native state) and reactivation (recovery from the inhibited to the native state). Resurrection of methylphosphonate-aged AChE by C8 was significantly pH-dependent, recovering 21% of activity at 4 mM and pH 9 after only 1 day. C8 is also effective against isopropyl phosphate-aged human AChE.