A pan-respiratory antiviral chemotype targeting a transient host multi-protein complex.

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

Simultaneous recordings of pupil size variation and locus coeruleus activity in mice.

An extensive literature describes how pupil size reflects neuromodulatory activity, including the noradrenergic system. Here, we present a protocol for the simultaneous recording of optogenetically identified locus coeruleus (LC) units and pupil diameter in mice under different conditions. We describe steps for building an optrode, performing surgery to implant the optrode and headpost, searching for opto-tagged LC units, and performing dual LC-pupil recording. We then detail procedures for data processing and analysis. For complete details on the use and execution of this protocol, please refer to Megemont et al.1.

Kinetics of the Simplest Criegee Intermediate Reaction with Water Vapor: Revisit and Isotope Effect.

The kinetics of the simplest Criegee intermediate (CH2OO) reaction with water vapor was revisited. By improving the signal-to-noise ratio and the precision of water concentration, we found that the kinetics of CH2OO involves not only two water molecules but also one and three water molecules. Our experimental results suggest that the decay of CH2OO can be described as d[CH2OO]/dt = -kobs[CH2OO]; kobs = k0 + k1[water] + k2[water]2 + k3[water]3; k1 = (4.22 ± 0.48) × 10-16 cm3 s-1, k2 = (10.66 ± 0.83) × 10-33 cm6 s-1, k3 = (1.48 ± 0.17) × 10-50 cm9 s-1 at 298 K and 300 Torr with the respective Arrhenius activation energies of Ea1 = 1.8 ± 1.1 kcal mol-1, Ea2 = -11.1 ± 2.1 kcal mol-1, Ea3 = -17.4 ± 3.9 kcal mol-1. The contribution of the k3[water]3 term becomes less significant at higher temperatures around 345 K, but it is not ignorable at 298 K and lower temperatures. By quantifying the concentrations of H2O and D2O with a Coriolis-type direct mass flow sensor, the kinetic isotope effect (KIE) was investigated at 298 K and 300 Torr and KIE(k1) = k1(H2O)/k1(D2O) = 1.30 ± 0.32; similarly, KIE(k2) = 2.25 ± 0.44 and KIE(k3) = 0.99 ± 0.13. These mild KIE values are consistent with theoretical calculations based on the variational transition state theory, confirming that the title reaction has a broad and low barrier, and the reaction coordinate involves not only the motion of a hydrogen atom but also that of an oxygen atom. Comparing the results recorded under 300 Torr (N2 buffer gas) with those under 600 Torr, a weak pressure effect of k3 was found. From quantum chemistry calculations, we found that the CH2OO + 3H2O reaction is dominated by the reaction pathways involving a ring structure consisting of two water molecules, which facilitate the hydrogen atom transfer, while the third water molecule is hydrogen-bonded outside the ring. Furthermore, analysis based on dipole capture rates showed that the CH2OO(H2O) + (H2O)2 and CH2OO(H2O)2 + H2O pathways will dominate in the three water reaction.

The locus coeruleus mediates behavioral flexibility.

Behavioral flexibility is the ability to adjust behavioral strategies in response to changing environmental contingencies. A major hypothesis in the field posits that the activity of neurons in the locus coeruleus (LC) plays an important role in mediating behavioral flexibility. To test this hypothesis, we developed a tactile-based rule-shift detection task in which mice responded to left and right whisker deflections in a context-dependent manner and exhibited varying degrees of switching behavior. Recording spiking activity from optogenetically tagged neurons in the LC at millisecond precision during task performance revealed a prominent graded correlation between baseline LC activity and behavioral flexibility, where higher baseline activity following a rule change was associated with faster behavioral switching to the new rule. Increasing baseline LC activity with optogenetic activation accelerated task switching and improved task performance. Overall, our study provides important evidence to reveal the link between LC activity and behavioral flexibility.

Reaction Kinetics of Criegee Intermediates with Nitric Acid.

This work investigated the reaction kinetics of HNO3 with four Criegee intermediates (CIs): CH2OO, (CH3)2COO, methyl vinyl ketone oxide (MVKO), and methacrolein oxide (MACRO). Our results show that these reactions are extremely fast with rate coefficients of (1.51 ± 0.45) × 10-10, (3.54 ± 1.06) × 10-10, (3.93 ± 1.18) × 10-10, and (3.0 ± 1.0) × 10-10 cm3 s-1 for reactions of HNO3 with CH2OO, (CH3)2COO, syn-MVKO, and anti-MACRO, respectively. This is consistent with previous results for the reactions between CIs and carboxylic acids, but the rate coefficient of CH2OO + HNO3 in the literature [Foreman Angew. Chem. 2016, 128, 10575] was found to be overestimated by a factor of 3.6. In addition, we did not observe any significant pressure dependence in the HNO3 reactions with CH2OO and (CH3)2COO under 100-400 Torr. Our results indicate that in a dry area with severe NOx pollution, the reactions of CIs with HNO3 and their products may be worthy of attention, but these reactions may be insignificant under high-humidity conditions. However, CI reactions with HNO3 may not play an important role in the atmospheric removal processes of HNO3 because of the low concentrations of CIs.

Isolation and Annotation of the Genome Sequences of Bacteriophages InvictusManeo (Subcluster K5) and Netyap (Subcluster L2).

The mycobacteriophages InvictusManeo (K5 subcluster) and Netyap (L2 subcluster) were isolated from soils in Cullowhee Creek, Cullowhee, North Carolina. Both exhibit Siphoviridae morphology and infect Mycobacterium smegmatis mc2155. The InvictusManeo genome is 61,147 bp and contains 96 predicted protein-coding genes, whereas the Netyap genome is 76,366 bp with 131 predicted protein-coding genes.

Absolute photodissociation cross sections of thermalized methyl vinyl ketone oxide and methacrolein oxide.

Methyl vinyl ketone oxide (MVKO) and methacrolein oxide (MACRO) are resonance-stabilized Criegee intermediates which are formed in the ozonolysis reaction of isoprene, the most abundant unsaturated hydrocarbon in the atmosphere. The absolute photodissociation cross sections of MVKO and MACRO were determined by measuring their laser depletion fraction at 352 nm, which was deduced from their time-resolved UV-visible absorption spectra. After calibrating the 352 nm laser fluence with the photodissociation of NO2, for which the absorption cross section and photodissociation quantum yield are well known, the photodissociation cross sections of thermalized (299 K) MVKO and MACRO at 352 nm were determined to be (3.02 ± 0.60) × 10-17 cm2 and (1.53 ± 0.29) × 10-17 cm2, respectively. Using their reported spectra and photodissociation quantum yields, their peak absorption cross sections were deduced to be (3.70 ± 0.74) × 10-17 cm2 (at 371 nm, MVKO) and (3.04 ± 0.58) × 10-17 cm2 (at 397 nm, MACRO). These values agree fairly with our theoretical predictions and are substantially larger than those of smaller, alkyl-substituted Criegee intermediates (CH2OO, syn-CH3CHOO, (CH3)2COO), revealing the effect of extended conjugation. With their cross sections, we also quantified the synthesis yields of MVKO and MACRO in the present experiment to be 0.22 ± 0.10 (at 299 K and 30-700 torr) and 0.043 ± 0.019 (at 299 K and 500 torr), respectively, relative to their photolyzed precursors. The lower yield of MACRO can be related to the high endothermicity of its formation channel.

Pupil diameter is not an accurate real-time readout of locus coeruleus activity.

Pupil diameter is often treated as a noninvasive readout of activity in the locus coeruleus (LC). However, how accurately it can be used to index LC activity is not known. To address this question, we established a graded relationship between pupil size changes and LC spiking activity in mice, where pupil dilation increased monotonically with the number of LC spikes. However, this relationship exists with substantial variability such that pupil diameter can only be used to accurately predict a small fraction of LC activity on a moment-by-moment basis. In addition, pupil exhibited large session-to-session fluctuations in response to identical optical stimulation in the LC. The variations in the pupil-LC relationship were strongly correlated with decision bias-related behavioral variables. Together, our data show that substantial variability exists in an overall graded relationship between pupil diameter and LC activity, and further suggest that the pupil-LC relationship is dynamically modulated by brain states, supporting and extending our previous findings (Yang et al., 2021).

A Pan-respiratory Antiviral Chemotype Targeting a Transient Host Multiprotein Complex.

We present a small molecule chemotype, identified by an orthogonal drug screen, exhibiting nanomolar activity against members of all the six viral families causing most human respiratory viral disease, with a demonstrated barrier to resistance development. Antiviral activity is shown in mammalian cells, including human primary bronchial epithelial cells cultured to an air-liquid interface and infected with SARS-CoV-2. In animals, efficacy of early compounds in the lead series is shown by survival (for a coronavirus) and viral load (for a paramyxovirus). The drug target is shown to include a subset of the protein 14-3-3 within a transient host multi-protein complex containing components implicated in viral lifecycles and in innate immunity. This multi-protein complex is modified upon viral infection and largely restored by drug treatment. Our findings suggest a new clinical therapeutic strategy for early treatment upon upper respiratory viral infection to prevent progression to lower respiratory tract or systemic disease. One Sentence Summary: A host-targeted drug to treat all respiratory viruses without viral resistance development.

Substituent Effect in the Reactions between Criegee Intermediates and 3-Aminopropanol.

Via intramolecular H atom transfer, 3-aminopropanol is more reactive toward Criegee intermediates, in comparison with amines or alcohols. Here we accessed the substituent effect of Criegee intermediates in their reactions with 3-aminopropanol. Through real-time monitoring the concentrations of two Criegee intermediates with their strong UV absorption at 340 nm, the experimental rate coefficients at 298 K (100-300 Torr) were determined to be (1.52 ± 0.08) × 10-11 and (1.44 ± 0.22) × 10-13 cm3 s-1 for the reactions of 3-aminopropanol with (CH3)2COO (acetone oxide) and CH2CHC(CH3)OO (methyl vinyl ketone oxide), respectively. Compared to our previous experimental value for the reaction with syn-CH3CHOO, (1.24 ± 0.13) × 10-11 cm3 s-1, we can see that the methyl substitution at the anti position has little effect on the reactivity while the vinyl substitution causes a drastic decrease in the reactivity. Our theoretical calculations based on CCSD(T)-F12 energies reproduce this 2-order-of-magnitude decrease in the rate coefficient caused by the vinyl substitution. Using the activation strain model, we found that the interaction of Criegee intermediates with 3-aminopropanol is weaker for the case of vinyl substitution. This effect can be further rationalized by the delocalization of the lowest unoccupied molecular orbital for the vinyl-substituted Criegee intermediates. These results would help us better estimate the impact of similar reactions like the reactions of Criegee intermediates with water vapor, some of which could be difficult to measure experimentally but can be important in the atmosphere. We also found that the B2PLYP-D3BJ/aug-cc-pVTZ calculation can reproduce the CCSD(T)-F12 reaction barrier energies within ca. 1 kcal mol-1, indicating that the use of the B2PLYP-D3BJ method is promising for future predictions of the reactions of larger Criegee intermediates.

A rapid degradation of calponin 2 is required for cytokinesis.

Calponin 2 is an actin cytoskeleton-associated protein and plays a role in regulating cell motility-related functions such as phagocytosis, migration, and division. We previously reported that overexpression of calponin 2 inhibits the rate of cell proliferation. To investigate the underlying mechanism, our present study found that the levels of endogenous calponin 2 in NIH3T3 and HEK293 cells rapidly decreased before cell division characterized by an absence at the actin contractile ring. In cells lacking endogenous calponin 2, transfective expression of GFP-fusion calponin 2 inhibited cell proliferation similar to that of nonfusion calponin 2. Fluorescent imaging studies of mitotic cells indicated that a proper level of calponin 2 expression and effective degradation during cytokinesis are necessary for normal cell division. Computer-assisted dynamic image analysis of dividing cells revealed that overexpression of calponin 2 significantly affects motility and shape behaviors of cells only on the interval from the start of anaphase to the start of cytokinesis, i.e., the pre-cytokinesis phase, but not on the interval from the start of cytokinesis to 50% completion of cytokinesis. The pre-cytokinesis degradation of calponin 2 was attenuated by MG132 inhibition of the ubiquitin proteasome and inhibitor of protein kinase C (PKC), suggesting that PKC phosphorylation-triggered degradation of calponin 2 could determine the rate of cytokinesis. The novel role of calponin 2 in regulating the rate of cytokinesis may be targeted for therapeutic applications such as in an inhibition of malignant tumor growth.

Surprisingly long lifetime of methacrolein oxide, an isoprene derived Criegee intermediate, under humid conditions.

Ozonolysis of isoprene, the most abundant alkene, produces three distinct Criegee intermediates (CIs): CH2OO, methyl vinyl ketone oxide (MVKO) and methacrolein oxide (MACRO). The oxidation of SO2 by CIs is a potential source of H2SO4, an important precursor of aerosols. Here we investigated the UV-visible spectroscopy and reaction kinetics of thermalized MACRO. An extremely fast reaction of anti-MACRO with SO2 has been found, kSO2 = (1.5 ± 0.4) × 10-10 cm3 s-1 (±1σ, σ is the standard deviation of the data) at 298 K (150 - 500 Torr), which is ca. 4 times the value for syn-MVKO. However, the reaction of anti-MACRO with water vapor has been observed to be quite slow with an effective rate coefficient of (9 ± 5) × 10-17 cm3 s-1 (±1σ) at 298 K (300 to 500 Torr), which is smaller than current literature values by 1 or 2 orders of magnitude. Our results indicate that anti-MACRO has an atmospheric lifetime (best estimate ca. 18 ms at 298 K and RH = 70%) much longer than previously thought (ca. 0.3 or 3 ms), resulting in a much higher steady-state concentration. Owing to larger reaction rate coefficient, the impact of anti-MACRO on the oxidation of atmospheric SO2 would be substantial, even more than that of syn-MVKO.

Kinetics of Unimolecular Decay of Methyl Vinyl Ketone Oxide, an Isoprene-Derived Criegee Intermediate, under Atmospherically Relevant Conditions.

Isoprene is the most abundant unsaturated hydrocarbon in the atmosphere. Ozonolysis of isoprene produces methyl vinyl ketone oxide (MVKO), which may react with atmospheric SO2, formic acid, and other important species at substantial levels. In this study, we utilized ultraviolet absorption to monitor the unimolecular decay kinetics of syn-MVKO in real time at 278-319 K and 100-503 Torr. After removing the contributions of radical reactions and wall loss, the unimolecular decay rate coefficient of syn-MVKO was measured to be kuni = 70 ± 15 s-1 (1σ uncertainty) at 298 K with negligible pressure dependence. In addition, kuni increases from ca. 30 s-1 at 278 K to ca. 175 s-1 at 319 K with an effective Arrhenius activation energy of 8.3 ± 2.5 kcal mol-1, kuni(T) = (9.3 × 107)exp(-4200/T) s-1. Our results indicate that unimolecular decay is the major sink of MVKO in the troposphere. The data would improve the estimation for the steady-state concentrations of MVKO and thus its oxidizing ability.

Intercalated disc protein Xinß is required for Hippo-YAP signaling in the heart.

Intercalated discs (ICD), specific cell-to-cell contacts that connect adjacent cardiomyocytes, ensure mechanical and electrochemical coupling during contraction of the heart. Mutations in genes encoding ICD components are linked to cardiovascular diseases. Here, we show that loss of Xinß, a newly-identified component of ICDs, results in cardiomyocyte proliferation defects and cardiomyopathy. We uncovered a role for Xinß in signaling via the Hippo-YAP pathway by recruiting NF2 to the ICD to modulate cardiac function. In Xinß mutant hearts levels of phosphorylated NF2 are substantially reduced, suggesting an impairment of Hippo-YAP signaling. Cardiac-specific overexpression of YAP rescues cardiac defects in Xinß knock-out mice-indicating a functional and genetic interaction between Xinß and YAP. Our study reveals a molecular mechanism by which cardiac-expressed intercalated disc protein Xinß modulates Hippo-YAP signaling to control heart development and cardiac function in a tissue specific manner. Consequently, this pathway may represent a therapeutic target for the treatment of cardiovascular diseases.

Reactions of Criegee Intermediates are Enhanced by Hydrogen-Atom Relay Through Molecular Design.

We report a type of highly efficient double hydrogen atom transfer (DHAT) reaction. The reactivities of 3-aminopropanol and 2-aminoethanol towards Criegee intermediates (syn- and anti-CH3 CHOO) were found to be much higher than those of n-propanol and propylamine. Quantum chemistry calculation has confirmed that the main mechanism of these very rapid reactions is DHAT, in which the nucleophilic attack of the NH2 group is catalyzed by the OH group which acts as a bridge of HAT. Typical gas-phase DHAT reactions are termolecular reactions involving two hydrogen bonding molecules; these reactions are typically slow due to the substantial entropy reduction of bringing three molecules together. Putting the reactive and catalytic groups in one molecule circumvents the problem of entropy reduction and allows us to observe the DHAT reactions even at low reactant concentrations. This idea can be applied to improve theoretical predictions for atmospherically relevant DHAT reactions.

The role of the iodine-atom adduct in the synthesis and kinetics of methyl vinyl ketone oxide-a resonance-stabilized Criegee intermediate.

Isoprene is the most abundant alkene in the atmosphere. Ozonolysis of isoprene produces three very reactive carbonyl oxides (Criegee intermediates), including formaldehyde oxide, methyl vinyl ketone oxide (MVKO, CH3(C2H3)COO), and methacrolein oxide. The latter two Criegee intermediates are resonance-stabilized due to the vinyl group. Recently, the electronic spectrum of thermalized MVKO has been reported [Caravan, et al., Proc. Natl. Acad. Sci. U. S. A., 2020, 117, 9733]. In this work, we utilized this strong UV/visible absorption to investigate the reaction kinetics of MVKO with SO2 under a wide pressure range of 4 to 700 Torr. We followed a new method [Barber, et al., J. Am. Chem. Soc., 2018, 140, 10866], in which MVKO is produced through the reaction of a resonance-stabilized iodoalkene radical with O2. The experimental data are consistent with a kinetic model that the reaction goes through an adduct of CH3(C2H3)CIOO, similar to the cases of H/alkyl substituted Criegee intermediates. However, different from the H/alkyl adducts, which are stable over the time scales of typical kinetic experiments, this vinyl adduct CH3(C2H3)CIOO is less stable and decomposes to MVKO + I at a time scale of 10-3 s (faster at higher temperature), consistent with the results of quantum chemistry calculations and the fact that the resonance stabilization is disrupted at the adduct structure. The adduct decomposition is the major pathway that forms MVKO for pressures higher than 50 Torr. In addition, temperature dependence has been investigated for 278-319 K. The experimental activation energy of the adduct decomposition was measured to be 12.7 ± 0.3 kcal mol-1, consistent with the calculated dissociation energy of the adduct to MVKO + I (14 kcal mol-1). Furthermore, the temperature dependent rate coefficient of MVKO + SO2 reaction has been measured to be kSO2 = (4.0 ± 0.6) × 10-11 cm3 s-1 at 4-700 Torr and 298 K with a negative activation energy of -3.7 ± 0.4 kcal mol-1.

Bidirectional pharmacological perturbations of the noradrenergic system differentially affect tactile detection.

The brain neuromodulatory systems heavily influence behavioral and cognitive processes. Previous work has shown that norepinephrine (NE), a classic neuromodulator mainly derived from the locus coeruleus (LC), enhances neuronal responses to sensory stimuli. However, the role of the LC-NE system in modulating perceptual task performance is not well understood. In addition, systemic perturbation of NE signaling has often been proposed to specifically target the LC in functional studies, yet the assumption that localized (specific) and systemic (nonspecific) perturbations of LC-NE have the same behavioral impact remains largely untested. In this study, we trained mice to perform a head-fixed, quantitative tactile detection task, and administered an α2 adrenergic receptor agonist or antagonist to pharmacologically down- or up-regulate LC-NE activity, respectively. We addressed the outstanding question of how bidirectional perturbations of LC-NE activity affect tactile detection, and tested whether localized and systemic drug treatments exert the same behavioral effects. We found that both localized and systemic suppression of LC-NE impaired tactile detection by reducing motivation. Surprisingly, while locally activating LC-NE enabled mice to perform in a near-optimal regime, systemic activation impaired behavior by promoting impulsivity. Our results demonstrate that localized silencing and activation of LC-NE differentially affect tactile detection, and that localized and systemic NE activation induce distinct behavioral changes.

Direct kinetic measurements and theoretical predictions of an isoprene-derived Criegee intermediate.

Isoprene has the highest emission into Earth's atmosphere of any nonmethane hydrocarbon. Atmospheric processing of alkenes, including isoprene, via ozonolysis leads to the formation of zwitterionic reactive intermediates, known as Criegee intermediates (CIs). Direct studies have revealed that reactions involving simple CIs can significantly impact the tropospheric oxidizing capacity, enhance particulate formation, and degrade local air quality. Methyl vinyl ketone oxide (MVK-oxide) is a four-carbon, asymmetric, resonance-stabilized CI, produced with 21 to 23% yield from isoprene ozonolysis, yet its reactivity has not been directly studied. We present direct kinetic measurements of MVK-oxide reactions with key atmospheric species using absorption spectroscopy. Direct UV-Vis absorption spectra from two independent flow cell experiments overlap with the molecular beam UV-Vis-depletion spectra reported recently [M. F. Vansco, B. Marchetti, M. I. Lester, J. Chem. Phys. 149, 44309 (2018)] but suggest different conformer distributions under jet-cooled and thermal conditions. Comparison of the experimental lifetime herein with theory indicates only the syn-conformers are observed; anti-conformers are calculated to be removed much more rapidly via unimolecular decay. We observe experimentally and predict theoretically fast reaction of syn-MVK-oxide with SO2 and formic acid, similar to smaller alkyl-substituted CIs, and by contrast, slow removal in the presence of water. We determine products through complementary multiplexed photoionization mass spectrometry, observing SO3 and identifying organic hydroperoxide formation from reaction with SO2 and formic acid, respectively. The tropospheric implications of these reactions are evaluated using a global chemistry and transport model.

Unimolecular decomposition rates of a methyl-substituted Criegee intermediate syn-CH3CHOO.

Criegee intermediates play important roles in atmospheric chemistry. Methyl Criegee intermediate, CH3CHOO, has two conformers, syn- and anti-conformers. Syn-CH3CHOO would undergo fast unimolecular decomposition to form OH radical via 1,4 H-atom transfer. In this work, unimolecular decomposition of syn-CH3CHOO was probed in real time with UV absorption spectroscopy at 278-318 K and 100-700 torr. We used water vapor as the scavenger of anti-CH3CHOO to distinguish the absorption signals of the two conformers. After removing the contributions from reactions with radical byproducts, reaction with water vapor and wall loss, we obtained the unimolecular reaction rate coefficient of syn-CH3CHOO (at 300 torr), which increases from (67 ± 15) s-1 at 278 K, (146 ± 31) s-1 at 298 K, to (288 ± 81) s-1 at 318 K with an Arrhenius activation energy of ca. 6.4 kcal mol-1 and a weak pressure dependence for 100-700 torr. Compared to previous studies, this work provides temperature dependent unimolecular rates of syn-CH3CHOO at higher pressures, which are more relevant to atmospheric conditions.