GS-441524-Diphosphate-Ribose Derivatives as Nanomolar Binders and Fluorescence Polarization Tracers for SARS-CoV-2 and Other Viral Macrodomains.

Viral macrodomains that can bind to or hydrolyze protein adenosine diphosphate ribosylation (ADP-ribosylation) have emerged as promising targets for antiviral drug development. Many inhibitor development efforts have been directed against the severe acute respiratory syndrome coronavirus 2 macrodomain 1 (SARS-CoV-2 Mac1). However, potent inhibitors for viral macrodomains are still lacking, with the best inhibitors still in the micromolar range. Based on GS-441524, a remdesivir precursor, and our previous studies, we have designed and synthesized potent binders of SARS-CoV-2 Mac1 and other viral macrodomains including those of Middle East respiratory syndrome coronavirus (MERS-CoV), Venezuelan equine encephalitis virus (VEEV), and Chikungunya virus (CHIKV). We show that the 1'-CN group of GS-441524 promotes binding to all four viral macrodomains tested while capping the 1″-OH of GS-441524-diphosphate-ribose with a simple phenyl ring further contributes to binding. Incorporating these two structural features, the best binders show 20- to 6000-fold increases in binding affinity over ADP-ribose for SARS-CoV-2, MERS-CoV, VEEV, and CHIKV macrodomains. Moreover, building on these potent binders, we have developed two highly sensitive fluorescence polarization tracers that only require nanomolar proteins and can effectively resolve the binding affinities of nanomolar inhibitors. Our findings and probes described here will facilitate future development of more potent viral macrodomain inhibitors.

The violent collapse of vapor bubbles in cryogenic liquids.

Vapor bubbles in cryogenic fluids may collapse violently under subcooled and pressurized conditions. Despite important implications for engineering applications such as cavitation erosion in liquid propellant rocket engines, these intense phenomena are still largely unexplored. In this paper, we systematically investigate the ambient conditions leading to the occurrence of violent collapses in liquid nitrogen and analyze their thermodynamic characteristics. Using Brenner's time ratio χ, the regime of violent collapse is identified in the ambient pressure-temperature parameter space. Complete numerical simulations further refine the prediction and illustrate two classes of collapses. At 1 < χ < 10, the collapse is impacted by significant thermal effects and attains only moderate wall velocity. Only when χ > 10 does the collapse show more inertial features. A mechanism analysis pinpoints a critical time when the surrounding liquid enters supercritical state. The ultimate collapse intensity is shown to be closely associated with the dynamics at this moment. Our study provides a fresh perspective to the treatment of cavitation in cryogenic fluids. The findings can be instrumental in engineering design to mitigate adverse effects arising from intense cavitational activities.

Iso-ADP-Ribose Fluorescence Polarization Probe for the Screening of RNF146 WWE Domain Inhibitors.

Poly-ADP-ribosylation is an important protein post-translational modification with diverse biological consequences. After binding poly-ADP-ribose on axis inhibition protein 1 (AXIN1) through its WWE domain, RING finger protein 146 (RNF146) can ubiquitinate AXIN1 and promote its proteasomal degradation and thus the oncogenic WNT signaling. Therefore, inhibiting the RNF146 WWE domain is a potential antitumor strategy. However, due to a lack of suitable screening methods, no inhibitors for this domain have been reported. Here, we developed a fluorescence polarization (FP)-based competition assay for the screening of RNF146 WWE inhibitors. This assay relies on a fluorescently tagged iso-ADP-ribose tracer compound, TAMRA-isoADPr. We report the design and synthesis of this tracer compound and show that it is a high-affinity tracer for the RNF146 WWE domain. This provides a convenient assay and will facilitate the development of small-molecule inhibitors for the RNF146 WWE domain.

A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain.

Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening (HTS) techniques for macrodomain inhibitors is of great interest. Herein, using a novel tracer TAMRA-ADPr, an ADP-ribose compound conjugated with tetramethylrhodamine, we developed a robust fluorescence polarization assay for various viral and human macrodomains including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1, MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC50 6.4 µM) and GS441524 (IC50 15.2 µM), two literature-reported small-molecule inhibitors of SARS-CoV-2 Macro1. Our data suggest that GS441524 is highly selective for SARS-CoV-2 Macro1 over other human and viral macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p-nitrophenol, IC50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC50 590 nM) as the most potent SARS-CoV-2 Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2 Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes to the binding affinity. Our data demonstrate that this fluorescence polarization assay is a useful addition to the HTS methods for the identification of macrodomain inhibitors.

Penetration of hydroxyl radicals in the aqueous phase surrounding a cavitation bubble.

In the sonochemical degradation of nonvolatile compounds, the free radicals must be delivered into the aqueous solution from the cavitation bubble to initiate reduction-oxidation reactions. The penetration depth in the liquid becomes an important parameter that influences the radical delivery efficiency and eventual treatment performance. However, the transport of radicals in the liquid phase is not well understood yet. In this paper, we focus on the most reactive OH radical and numerically simulate its penetration behavior. This is realized by solving the coupled equations of bubble dynamics, intracavity chemistry, and radical dispersion in the aqueous phase. The results present both the local and global penetration patterns for the OH radicals. By performing simulations over a wide range of acoustic parameters, we find an undesirable phenomenon that the penetration can be adversely suppressed when strengthening the radical production. A mechanistic analysis attributes this to the excessively vigorous recombination reactions associated with high radical concentrations near the bubble interface. In this circumstance, the radicals are massively consumed and converted into molecular species before they can appreciably diffuse away. Our study sheds light on the interplay between radical production inside the bubble and dispersion in the outside liquid. The derived conclusions provide guides for sonochemical applications from a new perspective.

Production and dispersion of free radicals from transient cavitation Bubbles: An integrated numerical scheme and applications.

As an advanced oxidation process with a wide range of applications, sonochemistry relies on acoustic cavitation to induce free radicals for degrading chemical contaminants. The complete process includes two critical steps: the radical production inside the cavitation bubble, and the ensuing dispersion of these radicals into the bulk solution. To grasp the physicochemical details in this process, we developed an integrated numerical scheme with the ability to quantitatively describe the radical production-dispersion behavior. It employs coupled simulations of bubble dynamics, intracavity chemical reactions, and diffusion-reaction-dominated mass transport in aqueous solutions. Applying this method to the typical case of argon and oxygen bubbles, the production mechanism for the main radicals is revealed. Moreover, the temporal-spatial distribution of the radicals in the liquid phase is presented. The results demonstrate that the enhanced radical production observed in oxygen bubbles can be traced to the initiation reaction O2 + H2O â†’ OH+HO2, which requires relatively low activation energy. In the outside liquid region, the dispersion of radicals is limited by robust recombination reactions. The simulated penetration depth of OH is around 0.2 µm and agrees with reported experimental measurements. The proposed numerical approach can be employed to better capture the radical activity and is instrumental in optimizing the engineering application of sonochemistry.

Discovery of the First Potent IDO1/IDO2 Dual Inhibitors: A Promising Strategy for Cancer Immunotherapy.

Indoleamine 2,3-dioxygenase-1 (IDO1) plays an important role in tumor immune escape. However, unsatisfactory clinical efficacies of selective IDO1 inhibitors have impeded their further development, suggesting that they do not exert sufficient antitumor effects by selectively inhibiting IDO1. IDO2, an isoenzyme of IDO1, is overexpressed in some human tumors, and emerging evidence suggests that concomitant inhibition of IDO1/2 may have synergistic effects in cancer treatment, revealing a promising cancer immunotherapeutic strategy. Herein, we describe the discovery of compound 4t, the first inhibitor targeting both IDO1/2 that has excellent in vitro inhibitory activity (IDO1 IC50 = 28 nM and IDO2 IC50 = 144 nM). Notably, 4t (TGI = 69.7%) exhibited significantly stronger in vivo antitumor potency than epacadostat (TGI = 49.4%) in CT26 xenograft mouse models, highlighting the advantages of IDO1/2 dual inhibitors for tumor immunotherapy. Preliminary mechanistic studies in vivo further identified that 4t exerts its antitumor effect by inhibiting IDO1/2.

Curcumin Complex Analogues as Near-Infrared Fluorescent Probes for Monitoring all Aß Species in the Early Alzheimer's Disease Model.

Aggregation of amyloid ß-peptide (Aß) is closely related to the pathology of Alzheimer's disease (AD). In this pathology, the beginning stage is characterized by excessive accumulation of Aß monomers due to imbalanced Aß in the process of clearance. The Aß peptide exists in many forms such as soluble and insoluble Aß species, both of which coexist during the progression of AD and contribute to AD pathology. Thus, probes capable of monitoring all Aß species are highly desirable. While there are several fluorescent probes for detecting insoluble Aß, it is still challenging to monitor all Aß forms by using probes. Here, we describe a near-infrared fluorescent chemical probe, termed AD-1, developed through complexation of curcumin analogues with a stabilizer, which has good photophysical properties and shows high binding to all Aß species in solution tests. Furthermore, AD-1 exhibited good blood-brain barrier penetrating ability and low cytotoxicity. More importantly, it was successfully applied to 4-month-young APP/PS1 mice imaging noninvasively.

Photothermal and Photodynamic Therapies via NIR-Activated Nanoagents in Combating Alzheimer's Disease.

It is well established that the polymerization of amyloid-ß peptides into fibrils/plaques is a critical step during the development of Alzheimer's disease (AD). Phototherapy, which includes photodynamic therapy and photothermal therapy, is a highly attractive strategy in AD treatment due to its merits of operational flexibility, noninvasiveness, and high spatiotemporal resolution. Distinct from traditional chemotherapies or immunotherapies, phototherapies capitalize on the interaction between photosensitizers or photothermal transduction agents and light to trigger photochemical reactions to generate either reactive oxygen species or heat effects to modulate Aß aggregation, ultimately restoring nerve damage and ameliorating memory deficits. In this Review, we provide an overview of the recent advances in the development of near-infrared-activated nanoagents for AD phototherapies and discuss the potential challenges of and perspectives on this emerging field with a special focus on how to improve the efficiency and utility of such treatment. We hope that this Review will spur preclinical research and the clinical translation of AD treatment through phototherapy.

An inverse method to fast-track the calculation of phase diagrams for sonoluminescing bubbles.

A sound driven air bubble can be transformed into an argon bubble emitting light pulses stably. The very foundation to investigate the sonoluminescing bubble is to accurately determine the ambient radius and gas composition in the interior. The conventional approach is to model the air-to-argon transformation process through a large number of bubble dynamics simulations to obtain the physical parameters of the ultimate argon bubble. In this paper, we propose a highly efficient method to pinpoint this information in a phase diagram. The method is based on the diffusive equilibrium for each species inside the bubble and derives the ambient radius and composition inversely. To calculate the former parameter, the bisection algorithm is employed to consecutively narrow down the searching range until the equilibria is approached. Afterward, several cycles of full dynamics simulations are conducted to refine the composition. The method is validated using published experimental data. The calculated ambient radii deviate from the test results by less than 1 µm, which falls within the margin of measurement error. The advantages of this method over the semi-analytical approach reported by Hilgenfeldt et al. [J. Fluid Mech. 365 (1998)] are also discussed. Our study provides a standard procedure to calculate the ambient radius and composition and is beneficial for the numerical simulation of sonoluminescing bubbles.

Time-efficient and contrast-free magnetic resonance imaging approach to the diagnosis of deep vein thrombosis on black-blood gradient-echo sequence: a pilot study.

BACKGROUND: Black-blood thrombus imaging (BTI) has shown to be advantageous for the diagnosis of deep vein thrombosis (DVT). However, previous techniques using fast spin echo have a high specific absorption rate. As DANTE (delay alternating with nutation for tailored excitation) black-blood preparation can suppress blood flows over a broad range of velocities, we hypothesized that a DANTE black-blood preparation combined with a fast low-angle shot (FLASH) gradient-echo readout-DANTE-FLASH could be used to diagnose DVT. METHODS: Eleven healthy volunteers and 30 suspected DVT patients were recruited to undergo DANTE-FLASH and magnetic resonance direct thrombus imaging (MRDTI). The suspected DVT patients were also examined by ultrasound (US). For the segment level, a total of 1,066 venous vessel segments were analyzed. Using US and MRDTI as the references, the sensitivity (SE), specificity (SP), positive and negative predictive values (PPV and NPV), and accuracy (ACC) of DANTE-FLASH were calculated. To quantitatively compare image quality between DANTE-FLASH and MRDTI, image signal-to-noise ratio (SNR), apparent contrast-to-noise ratio (CNR) between muscle and the venous lumen, and the apparent CNR between the thrombus and venous lumen were measured. Additionally, diagnostic confidence, image quality, and clot burden were also evaluated. RESULTS: Using the consensus results of US and MRDTI as a standard reference, the diagnostic SE, SP, PPV, NPV, and ACC of DANTE-FLASH for the 2 readers were 97.0% and 93.2%, 99.0% and 98.2%, 93.4% and 87.9%, 99.6% and 99.0%, and 98.8% and 97.6%, respectively. According to the image quantitative analysis results, DANTE-FLASH demonstrated higher image SNR and CNR than MRDTI. The image quality and diagnostic confidence scores of DANTE-FLASH were higher than MRDTI (3.66±0.44 vs. 3.52±0.52, P<0.001, and 3.84±0.36 vs. 3.76±0.41, P<0.001). There was excellent agreement between DANTE-FLASH and MRDTI on clot burden evaluation. CONCLUSIONS: DANTE-FLASH provided better image quality than MRDTI and accurately detected thrombi. It may, therefore, serve as a safe and convenient alternative for the diagnosis of DVT.

Interpreting the influence of liquid temperature on cavitation collapse intensity through bubble dynamic analysis.

The violent collapse of inertial bubbles generates high temperature inside and emits strong impulsive pressure. Previous tests on sonoluminescence and cavitation erosion showed that the influence of liquid temperature on these two parameters is different. In this paper, we conducted a bubble dynamic analysis to explore the mechanism of the temperature effect and account for the above difference. The results show that the increase of vapor at higher liquid temperatures changes both the external compression pressure and the internal cushion and is responsible for the variation of bubble collapse intensity. The different trends of the collapsing temperature and emitted sound pressure are caused by the energy distribution during the bubble collapse. Moreover, a series of simulations are conducted to establish the distribution map of the optimum liquid temperature where the collapse intensity is maximized. The relationship between the collapse intensity and the radial dynamics of the bubble is discussed and the reliable indicator is identified. This study provides a clear picture of how the thermodynamic process changes cavitation aggressiveness and enriches the understanding of this complex thermal-hydrodynamic phenomenon.

Versatile near-infrared fluorescent probe for in vivo detection of Aß oligomers.

Amyloid-ß oligomers (AßOs) enrichment in brain is highly related to Alzheimer's pathogenesis, but tracing them in the brain by imaging technique is still a great challenge due to their heterogeneity and metastability. Herein, a new near-infrared (NIR) fluorescent probe, namely, PTO-41, was designed and synthesized to specifically target AßOs. PTO-41 possesses excellent functional properties including optimal fluorescent properties (emission maxima at 680 nm upon interacting with AßOs), high affinity (Kd = 349 nM), low cell toxicity, desirable lipophilicity (log P = 2.24), and fast wash out from the brain (brain2 min/brain60 min = 5.0). Furthermore, PTO-41 exhibits a high sensitivity toward AßOs in vitro phantom imaging experiments. More importantly, PTO-41 shows great capacity to differentiate between 4-month-old APP/PS1 model mice from age-matched control mice using in vivo imaging. In summary, PTO-41 almost meets all the requirements as a versatile NIR fluorescent probe for the detection of AßOs both in vitro and in vivo.

Design, synthesis and biological evaluation of N-hydroxy-aminobenzyloxyarylamide analogues as novel selective κ opioid receptor antagonists.

Aminobenzyloxyarylamide derivatives 1a-i and 2a-t were designed and synthesized as novel selective κ opioid receptor (KOR) antagonists. The benzoyl amide moiety of LY2456302 was changed into N-hydroxybenzamide and benzisoxazole-3(2H)-one to investigate whether it could increase the binding affinity or selectivity for KOR. All target compounds were evaluated in radioligand binding assays for opioid receptor binding affinity. These efforts led to the identification of compound 1c (κ Ki = 179.9 nM), which exhibited high affinity for KOR. Moreover, the selectivity of KOR over MOR and DOR increased nearly 2-fold and 7-fold, respectively, compared with (±)LY2456302.

Discovery of Novel Dual Poly(ADP-ribose)polymerase and Phosphoinositide 3-Kinase Inhibitors as a Promising Strategy for Cancer Therapy.

Concomitant inhibition of PARP and PI3K pathways has been recognized as a promising strategy for cancer therapy, which may expand the clinical utility of PARP inhibitors. Herein, we report the discovery of dual PARP/PI3K inhibitors that merge the pharmacophores of PARP and PI3K inhibitors. Among them, compound 15 stands out as the most promising candidate with potent inhibitory activities against both PARP-1/2 and PI3Kα/δ with pIC50 values greater than 8. Compound 15 displayed superior antiproliferative profiles against both BRCA-deficient and BRCA-proficient cancer cells in cellular assays. The prominent synergistic effects produced by the concomitant inhibition of the two targets were elucidated by comprehensive biochemical and cellular mechanistic studies. In vivo, 15 showed more efficacious antitumor activity than the corresponding drug combination (Olaparib + BKM120) in the MDA-MB-468 xenograft model with a tumor growth inhibitory rate of 73.4% without causing observable toxic effects. All of the results indicate that 15, a first potent dual PARP/PI3K inhibitor, is a highly effective anticancer compound.

Development of Near-Infrared Fluorescent Probes for Use in Alzheimer's Disease Diagnosis.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder. Currently, there are no available treatments that can effectively stop or reverse the progression of the disease, and existing therapeutics can only alleviate the symptoms. Thus, it remains urgent to develop effective early-stage AD diagnostic methods. In recent years, the search for near-infrared fluorescent (NIRF) probes of AD hallmarks has become a promising research field. In this Review, we will focus on small-molecule NIRF probes used to detect ß-amyloid, tau proteins, and reactive oxygen species in vivo during the past 4 years. We believe that some new directions we raise herein will benefit the future development of NIRF probes in the field of AD research.

Discovery of novel nonpeptide small-molecule NRP1 antagonists: Virtual screening, molecular simulation and structural modification.

Multifaceted roles of vascular endothelial growth factor (VEGF)-neuropilin-1 (NRP1) interaction have been implicated in cancer, but reports on small-molecule inhibitors of VEGF-NRP1 interaction are scarce. Herein, we describe the identification of 1, a novel nonpeptide small-molecule NRP1 antagonist with moderate activity via structure-based virtual screening. Ensemble docking and molecular dynamics (MD) simulations of 1 were carried out and an interesting binding model was obtained. We found that the "aromatic box" enclosed by Tyr297, Trp301 and Tyr353 of NRP1 is critical for NRP1-1 binding. Further structure modification of 1 based on the binding model derived from MD simulations resulted in the identification of 12a with significantly improved activity.

Targeting VEGF-neuropilin interactions: a promising antitumor strategy.

Inhibition of vascular endothelial growth factor (VEGF) or its corresponding receptor (VEGFR) has been validated as an efficacious antiangiogenetic approach for cancer treatment. More recently, neuropilins (NRPs), the essential coreceptors for VEGF, have also been shown to have a significant role in VEGF signaling. Given the multifaceted effects of VEGF-NRP interactions on tumor initiation and progression, the exploration of new chemical entities that selectively block these interactions has recently attracted considerable interest as a novel antitumor strategy. Here, we summarize the biological functions of VEGF-NRP interactions in tumor biology, analyze the structural basis for these interactions, and present a detailed discussion of the development of the NRP antagonists reported so far.

Altered Gray Matter Volume in Stable Chronic Obstructive Pulmonary Disease with Subclinical Cognitive Impairment: an Exploratory Study.

Gray matter volume deficits have been identified in cognitively impaired patients with chronic obstructive pulmonary disease (COPD). However, it remains unknown whether the gray matter volume is altered in COPD patients with subclinical cognitive impairment. To determine whether any gray matter abnormalities are present in these patients, neuropsychological tests and structural MRI data were analyzed from 60 patients with COPD and 60 age-, gender-, education-, and handedness-matched normal controls (NCs). The COPD patients had similar Mini-Mental State Examination (MMSE) scores compared with the NCs. However, they had reduced Montreal Cognitive Assessment (MoCA) scores for visuospatial and executive and naming and memory functions (P < 0.001). Voxel-based morphometry (VBM) analysis revealed that the COPD patients had significantly lowered gray matter volumes in several brain regions, including the left precuneus (PrCU), bilateral calcarine (CAL), right superior temporal gyrus/middle temporal gyrus (STG/MTG), bilateral fusiform gyrus (FG), and right inferior parietal lobule (IPL) (P < 0.01, corrected). Importantly, the forced vital capacity (FVC) was found to be associated with the gray matter volume in the calcarine. The present study confirmed that brain structural changes were present in stable COPD patients with subclinical cognitive impairment. These findings may provide new insights into the pathogenesis of COPD.