Search

LETTER REPLY TO MA AND WANG: Reliabilityofvariousinvitroactivityassayson SARS-CoV-2mainproteaseinhibitors Zhe Li a,1 , Yuxi Lin b,c,1, Yi-You Huang a,1, Runduo Liu a , Chang-Guo Zhan d,e,2 , Xin Wang b,f,2 , and Hai-Bin Luo a,g,2  Ma and Wang (1) tested our recently reported severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) main protease (M pro) noncovalent inhibitors (2) using their in vitro assays, and they obtained negligible or much lower inhibitory activities compared to ours. Notably,thefluorescence resonanceenergytransfer(FRET)-basedenzymaticassay used by Ma and Wang (1) is different from our FRETbased enzymatic assay in the M pro protein (discussed below) and the FRET substrate [longer than the more popularly used substrate (3, 4) utilized in our assay]. Ma and Wang (1) also used native mass spectrometry (MS) and thermal shift assays (TSA) to detect the protein-ligand binding. [Extracted from the article]

Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates associated with protein misfolding diseases. Although they differ in morphology, the two forms are often treated indiscriminately, β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concentration at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersaturated solution, analogous to crystallization. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradiation, and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Solubility and supersaturation will be key factors for further understanding the aberrant aggregation of proteins. [ABSTRACT FROM AUTHOR]

The aggregation of amyloid-β (Aβ) on lipid bilayers has been implicated as a mechanism by which Aβ exerts its toxicity in Alzheimer's disease (AD). Lipid bilayer thinning has been observed during both oxidative stress and protein aggregation in AD, but whether these pathological modifications of the bilayer correlate with Aβ misfolding is unclear. Here, we studied peptide-lipid interactions in synthetic bilayers of the shortchain lipid dilauroyl phosphatidylcholine (DLPC) as a simplified model for diseased bilayers to determine their impact on Aβ aggregate, protofibril, and fibril formation. Aβ aggregation and fibril formation in membranes composed of dioleoyl phosphatidylcholine (DOPC) or 1-palmitoyl-2-oleoyl phosphatidylcholine mimicking normal bilayers served as controls. Differences in aggregate formation and stability were monitored by a combination of thioflavin-T fluorescence, circular dichroism, atomic force microscopy, transmission electron microscopy, and NMR. Despite the ability of all three lipid bilayers to catalyze aggregation, DLPC accelerates aggregation at much lower concentrations and prevents the fibrillation of Aβ at low micromolar concentrations. DLPC stabilized globular, membrane-associated oligomers, which could disrupt the bilayer integrity. DLPC bilayers also remodeled preformed amyloid fibrils into a pseudo-unfolded, molten globule state, which resembled on-pathway, protofibrillar aggregates. Whereas the stabilized, membrane-associated oligomers were found to be nontoxic, the remodeled species displayed toxicity similar to that of conventionally prepared aggregates. These results provide mechanistic insights into the roles that pathologically thin bilayers may play in Aβ aggregation on neuronal bilayers, and pathological lipid oxidation may contribute to Aβ misfolding. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global crisis. There is no therapeutic treatment specific for COVID-19. It is highly desirable to identify potential antiviral agents against SARS-CoV-2 from existing drugs available for other diseases and thus repurpose them for treatment of COVID-19. In general, a drug repurposing effort for treatment of a new disease, such as COVID-19, usually starts from a virtual screening of existing drugs, followed by experimental validation, but the actual hit rate is generally rather low with traditional computational methods. Here we report a virtual screening approach with accelerated free energy perturbation-based absolute binding free energy (FEP-ABFE) predictions and its use in identifying drugs targeting SARS-CoV-2 main protease (Mpro). The accurate FEP-ABFE predictions were based on the use of a restraint energy distribution (RED) function, making the practical FEP-ABFE-based virtual screening of the existing drug library possible. As a result, out of 25 drugs predicted, 15 were confirmed as potent inhibitors of SARS-CoV-2 Mpro. The most potent one is dipyridamole (inhibitory constant Ki = 0.04 µM)which has shown promising therapeutic effects in subsequently conducted clinical studies for treatment of patients with COVID-19. Additionally, hydroxychloroquine (Ki = 0.36 µM) and chloroquine (Ki = 0.56 µM) were also found to potently inhibit SARS-CoV-2 Mpro. We anticipate that the FEP-ABFE prediction-based virtual screening approach will be useful in many other drug repurposing or discovery efforts. [ABSTRACT FROM AUTHOR]

Tamoxifen, a widely used modulator of the estrogen receptor (ER), targets ER-positive breast cancer preferentially. We used a powerful validation-based insertion mutagenesis method to find that expression of a dominant-negative, truncated form of the histone deacetylase ZIP led to resistance to tamoxifen. Consistently, increased expression of full-length ZIP gives the opposite phenotype, inhibiting the expression of genes whose products mediate resistance. An important example is JAK2. By binding to two specific sequences in the promoter, ZIP suppresses JAK2 expression. Increased expression and activation of JAK2 when ZIP is inhibited lead to increased STAT3 phosphorylation and increased resistance to tamoxifen, both in cell culture experiments and in a mouse xenograft model. Furthermore, data from human tumors are consistent with the conclusion that decreased expression of ZIP leads to resistance to tamoxifen in ERpositive breast cancer. [ABSTRACT FROM AUTHOR]

As a central feature of neuroinflammation, microglial dysfunction has been increasingly considered a causative factor of neurodegeneration implicating an intertwined pathology with amyloidogenic proteins. Herein, we report the smallest synthetic molecule (N,N′- diacetyl-p-phenylenediamine [DAPPD]), simply composed of a benzene ring with 2 acetamide groups at the para position, known to date as a chemical reagent that is able to promote the phagocytic aptitude ofmicroglia and subsequently ameliorate cognitive defects. Based on our mechanistic investigations in vitro and in vivo, 1) the capability of DAPPD to restore microglial phagocytosis is responsible for diminishing the accumulation of amyloid-β (Aβ) species and significantly improving cognitive function in the brains of 2 types of Alzheimer’s disease (AD) transgenic mice, and 2) the rectification of microglial function by DAPPD is a result of its ability to suppress the expression of NLRP3 inflammasome-associated proteins through its impact on the NF-κB pathway. Overall, our in vitro and in vivo investigations on efficacies andmolecular-level mechanisms demonstrate the ability of DAPPD to regulate microglial function, suppress neuroinflammation, foster cerebral Aβ clearance, and attenuate cognitive deficits in AD transgenic mouse models. Discovery of such antineuroinflammatory compounds signifies the potential in discovering effective therapeutic molecules against AD-associated neurodegeneration. [ABSTRACT FROM AUTHOR]

Amyloid fibrils form in supersaturated solutions via a nucle-ation and growth mechanism. We proposed that ultrasonication may be an effective agitation to trigger nucleation that would otherwise not occur under the persistent metastability of super-saturation. However, the roles of supersaturation and effects of ultrasonication have not been elucidated in detail except for limited cases. Insulin is an amyloidogenic protein that is useful for investigating the mechanisms underlying amyloid fibrillation with biological relevance. We studied the alcohol-induced amyloid fibrillation of insulin using various concentrations of 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoro-2-propanol at pH 2.0 and 4.8. Ultrasonic irradiation effectively triggered fibrillation under conditions in which insulin retained persistent supersaturation. Structural analyses by circular dichroism, Fourier transform infrared spectroscopy, transmission electron microscopy, and atomic force microscopy revealed that the dominant structures of fibrils varied between parallel and antiparallel β-sheets depending on the solvent conditions. pH and alcohol concentration-dependent phase diagrams showed a marked difference before and after the ultrasonic treatment, which indicated that the persistent metastability of supersaturation determined the conformations of insulin. These results indicate the importance of an alternative view of amyloid fibrils as supersaturation-limited crystal-like aggregates formed above the solubility limit. [ABSTRACT FROM AUTHOR]