Peptide-Based Strategies: Combating Alzheimer's Amyloid ß Aggregation through Ergonomic Design and Fibril Disruption.

Amyloidosis of amyloid-ß (Aß) triggers a cascade of events, leading to oxidative damage and neuronal death. Therefore, inhibiting Aß amyloidosis or disrupting the matured fibrils is the primary target to combat progressive Alzheimer's disease (AD) pathogenesis. Here, we undertake optimization strategies to improve the antiamyloid efficiency of our previously reported NF11 (NAVRWSLMRPF) peptide. Among the series of peptides tested, nontoxic and serum-stable peptide 1 or P1 containing an anthranilic acid residue shows immense potential in not only inhibiting the Aß42 amyloid formation but also disrupting the mature Aß42 fibrils into nontoxic small molecular weight soluble species. Our studies provide high-resolution characterization of the peptide's mechanism of action. With a binding affinity within the micromolar range for both the monomer and aggregated Aß42, this α/ß hybrid peptide can efficiently modulate Aß amyloidosis while facilitating the clearance of toxic aggregates and enforcing protection from apoptosis. Thus, our studies highlight that incorporating a ß-amino acid not only imparts protection from proteolytic degradation and improved stability but also functions effectively as a ß breaker, redirecting the aggregation kinetics toward off-pathway fibrillation.

Enhancing amyloid beta inhibition and disintegration by natural compounds: A study utilizing spectroscopy, microscopy and cell biology.

Amyloid proteins and peptides play a pivotal role in the etiology of various neurodegenerative diseases, including Alzheimer's disease (AD). Synthetically designed small molecules/ peptides/ peptidomimetics show promise towards inhibition of various kinds of amyloidosis. However, exploration of compounds isolated from natural extracts having such potential is lacking. Herein, we have investigated the repurposing of a traditional Indian medicine Lasunadya Ghrita (LG) in AD. LG is traditionally used to treat gut dysregulation and mental illnesses. Various extracts of LG were obtained, characterized, and analyzed for inhibition of Aß aggregation. Biophysical studies show that the water extract of LG (LGWE) is more potent in inhibiting Aß peptide aggregation and defibrillation of Aß40/Aß42 aggregates. NMR studies showed that LGWE binds to the central hydrophobic area and C-terminal residues of Aß40/Aß42, thereby modulating the aggregation, and reducing cell membrane damage. Additionally, LGWE rescues Aß toxicity in neuronal SH-SY5Y cells evident from decreases in ROS generation, membrane leakage, cellular apoptosis, and calcium dyshomeostasis. Notably, LGWE is non-toxic to neuronal cells and mouse models. Our study thus delves into the mechanistic insights of a repurposed drug LGWE with the potential to ameliorate Aß induced neuroinflammation.

Use of phosphotyrosine-containing peptides to target SH2 domains: Antagonist peptides of the Crk/CrkL-p130Cas axis.

Protein-protein interactions between SH2 domains and segments of proteins that include a post-translationally phosphorylated tyrosine residue (pY) underpin numerous signal transduction cascades that allow cells to respond to their environment. Dysregulation of the writing, erasing, and reading of these posttranslational modifications is a hallmark of human disease, notably cancer. Elucidating the precise role of the SH2 domain-containing adaptor proteins Crk and CrkL in tumor cell migration and invasion is challenging because there are no specific and potent antagonists available. Crk and CrkL SH2s interact with a region of the docking protein p130Cas containing 15 potential pY-containing tetrapeptide motifs. This chapter summarizes recent efforts toward peptide antagonists for this Crk/CrkL-p130Cas interaction. We describe our protocol for recombinant expression and purification of Crk and CrkL SH2s for functional assays and our procedure to determine the consensus binding motif from the p130Cas sequence. To develop a more potent antagonist, we employ methods often associated with structure-based drug design. Computational docking using Rosetta FlexPepDock, which accounts for peptides having a greater number of conformational degrees of freedom than small organic molecules that typically constitute libraries, provides quantitative docking metrics to prioritize candidate peptides for experimental testing. A battery of biophysical assays, including fluorescence polarization, differential scanning fluorimetry and saturation transfer difference nuclear magnetic resonance spectroscopy, were employed to assess the candidates. In parallel, GST pulldown competition assays characterized protein-protein binding in vitro. Taken together, our methodology yields peptide antagonists of the Crk/CrkL-p130Cas axis that will be used to validate targets, assess druggability, foster in vitro assay development, and potentially serve as lead compounds for therapeutic intervention.

Empowered Relief, cognitive behavioral therapy, and health education for people with chronic pain: a comparison of outcomes at 6-month Follow-up for a randomized controlled trial.

Introduction: We previously conducted a 3-arm randomized trial (263 adults with chronic low back pain) which compared group-based (1) single-session pain relief skills intervention (Empowered Relief; ER); (2) 8-session cognitive behavioral therapy (CBT) for chronic back pain; and (3) single-session health and back pain education class (HE). Results suggested non-inferiority of ER vs. CBT at 3 months post-treatment on an array of outcomes. Methods: Here, we tested the durability of treatment effects at 6 months post-treatment. We examined group differences in primary and secondary outcomes at 6 months and the degree to which outcomes eroded or improved from 3-month to 6-month within each treatment group. Results: Empowered Relief remained non-inferior to CBT on most outcomes, whereas both ER and CBT remained superior to HE on most outcomes. Outcome improvements within ER did not decrease significantly from 3-month to 6-month, and indeed ER showed additional 3- to 6-month improvements on pain catastrophizing, pain bothersomeness, and anxiety. Effects of ER at 6 months post-treatment (moderate term outcomes) kept pace with effects reported by participants who underwent 8-session CBT. Conclusions: The maintenance of these absolute levels implies strong stability of ER effects. Results extend to 6 months post-treatment previous findings documenting that ER and CBT exhibit similarly potent effects on outcomes.

Mutation of a highly conserved isoleucine residue in loop 2 of several ß-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental to infection.

All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in nonstructural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the GIF (glycine-isoleucine-phenylalanine) motif. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that led to attenuated replication and pathogenesis in cell culture and mice. In contrast, the I-A mutations had normal enzyme activity and enhanced ADP-ribose binding. Despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication.

In-silico simulation of nanoindentation on bone using a 2D cohesive finite element model.

This study proposed and validated a 2D finite element (FE) model for conducting in-silico simulations of in-situ nanoindentation tests on mineralized collagen fibrils (MCF) and the extrafibrillar matrix (EFM) within human cortical bone. Initially, a multiscale cohesive FE model was developed by adapting a previous model of bone lamellae, encompassing both MCF and EFM. Subsequently, nanoindentation tests were simulated in-silico using this model, and the resulting predictions were compared to AFM nanoindentation test data to verify the model's accuracy. The FE model accurately predicted nanoindentation results under wet conditions, closely aligning with outcomes obtained from AFM nanoindentation tests. Specifically, it successfully mirrored the traction/separation curve, nanoindentation modulus, plastic energy dissipation, and plastic energy ratio obtained from AFM nanoindentation tests. Additionally, this in-silico model demonstrated its ability to capture alterations in nanoindentation properties caused by the removal of bound water, by considering corresponding changes in mechanical properties of the collagen phase and the interfaces among bone constituents. Notably, significant changes in the elastic modulus and plastic energy dissipation were observed in both MCF and EFM compartments of bone, consistent with observations in AFM nanoindentation tests. These findings indicate that the proposed in-silico model effectively captures the influence of ultrastructural changes on bone's mechanical properties at sub-lamellar levels. Presently, no experimental methods exist to conduct parametric studies elucidating the ultrastructural origins of bone tissue fragility. The introduction of this in-silico model presents an invaluable tool to bridge this knowledge gap in the future.

New cyclophilin D inhibitor rescues mitochondrial and cognitive function in Alzheimer's disease.

Mitochondrial dysfunction is an early pathological feature of Alzheimer disease and plays a crucial role in the development and progression of Alzheimer's disease. Strategies to rescue mitochondrial function and cognition remain to be explored. Cyclophilin D (CypD), the peptidylprolyl isomerase F (PPIase), is a key component in opening the mitochondrial membrane permeability transition pore, leading to mitochondrial dysfunction and cell death. Blocking membrane permeability transition pore opening by inhibiting CypD activity is a promising therapeutic approach for Alzheimer's disease. However, there is currently no effective CypD inhibitor for Alzheimer's disease, with previous candidates demonstrating high toxicity, poor ability to cross the blood-brain barrier, compromised biocompatibility and low selectivity. Here, we report a new class of non-toxic and biocompatible CypD inhibitor, ebselen, using a conventional PPIase assay to screen a library of ∼2000 FDA-approved drugs with crystallographic analysis of the CypD-ebselen crystal structure (PDB code: 8EJX). More importantly, we assessed the effects of genetic and pharmacological blockade of CypD on Alzheimer's disease mitochondrial and glycolytic bioenergetics in Alzheimer's disease-derived mitochondrial cybrid cells, an ex vivo human sporadic Alzheimer's disease mitochondrial model, and on synaptic function, inflammatory response and learning and memory in Alzheimer's disease mouse models. Inhibition of CypD by ebselen protects against sporadic Alzheimer's disease- and amyloid-ß-induced mitochondrial and glycolytic perturbation, synaptic and cognitive dysfunction, together with suppressing neuroinflammation in the brain of Alzheimer's disease mouse models, which is linked to CypD-related membrane permeability transition pore formation. Thus, CypD inhibitors have the potential to slow the progression of neurodegenerative diseases, including Alzheimer's disease, by boosting mitochondrial bioenergetics and improving synaptic and cognitive function.

Quantification and characterization of biological activities of glansreginin A in black walnuts (Juglans nigra).

Glansreginin A has been reported to be an indicator of the quality of walnuts (Juglans spp.). However, bioactive properties of glansreginin A have not been adequately explored. In the present study, we quantified concentrations of glansreginin A in black walnuts (Juglans nigra) using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and performed an array of in vitro bioassays to characterize biological activities (e.g., antibacterial, antioxidant, anticancer capacities) of this compound. Results from HPLC-MS/MS analysis indicated that glansreginin A was presented in all 12 black cultivars examined and its contents were variable among black walnut cultivars, ranged from 6.8 mg/kg (Jackson) to 47.0 mg/kg (Hay). Glansreginin A possessed moderate antibacterial activities against Gram-positive pathogens (Staphylococcus aureus and Bacillus anthracis). This compound exhibited no antioxidant activities, did not induce the activity of antioxidant response element signaling pathways, and exerted no antiproliferative effects on tumorigenic alveolar epithelial cells and non-tumorigenic lung fibroblast cells.

Management of threatened abortion through Ayurvedic intervention: A case report.

Untreated vaginal bleeding during pregnancy can potentially give rise to various complications, with the incidence ranging from 12% to 40% of all cases. In Ayurveda, this condition is referred to as garbhashrava, which encompasses the manifestation of abortion, with raktadarshana (vaginal bleeding) serving as a key diagnostic symptom. In this present case study, 28 years old second gravida woman with amenorrhea of 3 months 08 days presented with vaginal bleeding (1 pad/day) for 15 days on and off. The case was diagnosed as first-trimester vaginal bleeding due to low-lying posterior placenta (placental cause). Pregnancy outcome depends on the severity of bleeding thus early diagnosis and proper management are the priority. Traditionally, the conventional approach involves the use of hemostatic agents and injectable hormonal support, which is an invasive method. In this present study, a local non-invasive method i.e application of gairika choorna 5 g with shatadhauta ghrita 15-20 g in the form of lepa (anointment) below the umbilicus was advised thrice a day for 1 week. This treatment resulted in complete relief from vaginal bleeding within a one-week period while maintaining the pregnancy.

Assessment of glycosaminoglycan content in bone using Raman spectroscopy.

Glycosaminoglycans (GAGs) are responsible for preserving bone tissue toughness as well as regulating collagen formation and mineralization in the extracellular matrix. However, current methods for characterization of GAGs in bone are destructive, thus unable to capture in situ changes or differences in GAGs between experimental groups. As an alternative, Raman spectroscopy is a non-destructive method and can detect concurrent changes in GAGs and other bone constituents. In this study, we hypothesized that the two most prominent Raman peaks of sulfated GAGs (at ~1066 cm-1 and at ~1378 cm-1) could be used to detect differences in GAGs content of bone. To test this hypothesis, three experimental models were utilized: an in vitro model (enzymatic removal of GAGs from human cadaver bone), an ex vivo mouse model (biglycan KO vs. WT), and an ex vivo aging model (comparing cadaveric bone samples from young and old donors). All Raman measurements were compared to Alcian blue measurements to confirm the validity of Raman spectroscopy in detecting GAGs changes in bone. Irrespective of different models, it was found that the ~1378 cm-1 peak in Raman spectra of bone was uniquely sensitive to changes of GAGs content in bone when normalized with respect to the phosphate phase (~960 cm-1); i.e., 1378 cm-1/960 cm-1 (peak intensity ratio) or 1370-1385 cm-1/930-980 cm-1 (integrated peak area ratio). In contrast, the 1070 cm-1 peak, which includes another major peak of GAGs (1066 cm-1), seemed to be compromised to detect changes of GAGs in bone due to concurrent changes of carbonate (CO3) in the similar peak range. This study validates the ability of Raman spectroscopy to detect in situ treatment-, genotype-, and age-related changes in GAG levels of bone matrix.

Resultant inward imbalanced seeding force (RIISF)-induced concave gold nanostar (CAuNS) for non-enzymatic electrocatalytic detection of serotonin and Kynurenine in human serum.

CTAC-based gold nanoseed-induced concave curvature evolution of surface boundary planes from concave gold nanocube (CAuNC) to concave gold nanostar (CAuNS) has been achieved by a novel synthetic methodology simply by controlling the extent of seed used and hence the generated 'Resultant Inward Imbalanced Seeding Force (RIISF)'. The resultant CAuNS shows an excellent enhancement in catalytic activity compared to CAuNC and other intermediates as a function of curvature-induced anisotropy. Detailed characterization evaluates the presence of an enhanced number of multiple defect sites, high energy facets, larger surface area, and roughened surface which ultimately results in an increased mechanical strain, coordinately unsaturation, and multifacet-oriented anisotropic behavior suitable for positive influence on the binding affinity of CAuNSs. While different crystalline and structural parameters improve their catalytic activity, the resultant uniform three-dimensional (3D) platform shows comparatively easy pliability and well absorptivity on the glassy carbon electrode surface for increased shelf life, a uniform structure to confine a large extent of stoichiometric systems, and long-term stability under ambient conditions for making this newly developed material a unique nonenzymatic scalable universal electrocatalytic platform. With the help of various electrochemical measurements, the ability of the platform has been established by performing highly specific and sensitive detection of the two most important human bio messengers: Serotonin (STN) and Kynurenine (KYN) which are metabolites of L-Tryptophan in the human body system. The present study mechanistically surveys the role of seed-induced RIISF-modulated anisotropy in controlling the catalytic activity which offers a universal 3D electrocatalytic sensing tenet by an electrocatalytic approach.

Doubly multivariate linear models with block exchangeable distributed errors and site-dependent covariates.

The problem of testing the intercept and slope parameters of doubly multivariate linear models with site-dependent covariates using Rao's score test (RST) is studied. The RST statistic is developed for a block exchangeable covariance structure on the error vector under the assumption of multivariate normality. We compare our developed RST statistic with the likelihood ratio test (LRT) statistic. Monte Carlo simulations indicate that the RST statistic is much more accurate than its counterpart LRT statistic and it takes significantly less computation time than the LRT statistic. The proposed method is illustrated with an example of multiple response variables measured on multiple trees in a single plot in an agricultural study.

Small-Molecule Disruptors of Mutant Huntingtin-Calmodulin Protein-Protein Interaction Attenuate Deleterious Effects of Mutant Huntingtin.

Huntington's disease is a progressive and lethal neurodegenerative disease caused by an increased CAG repeat mutation in exon 1 of the huntingtin gene (mutant huntingtin). Current drug treatments provide only limited symptomatic relief without impacting disease progression. Previous studies in our lab and others identified the abnormal binding of mutant huntingtin protein with calmodulin, a key regulator of calcium signaling. Disrupting the abnormal binding of mutant huntingtin to calmodulin reduces perturbations caused by mutant huntingtin in cell and mouse models of Huntington's disease and importantly normalizes receptor-stimulated calcium release. Using a series of high-throughput in vitro and cell-based screening assays, we identified numerous small-molecule hits that disrupt the binding of mutant huntingtin to calmodulin and demonstrate protective effects. Iterative optimization of one hit resulted in nontoxic, selective compounds that are protective against mutant huntingtin cytotoxicity and normalized receptor-stimulated intracellular calcium release in PC12 cell models of Huntington's disease. Importantly, the compounds do not work by reducing the levels of mutant huntingtin, allowing this strategy to complement future molecular approaches to reduce mutant huntingtin expression. Our novel scaffold will serve as a prototype for further drug development in Huntington's disease. These studies indicate that the development of small-molecule compounds that disrupt the binding of mutant huntingtin to calmodulin is a promising approach for the advancement of therapeutics to treat Huntington's disease.

"My Surgical Success": Feasibility and Impact of a Single-Session Digital Behavioral Pain Medicine Intervention on Pain Intensity, Pain Catastrophizing, and Time to Opioid Cessation After Orthopedic Trauma Surgery-A Randomized Trial.

BACKGROUND: Behavioral pain treatments may improve postsurgical analgesia and recovery; however, effective and scalable options are not widely available. This study tested a digital perioperative behavioral medicine intervention in orthopedic trauma surgery patients for feasibility and efficacy for reducing pain intensity, pain catastrophizing, and opioid cessation up to 3 months after surgery. METHODS: A randomized controlled clinical trial was conducted at an orthopedic trauma surgery unit at a major academic hospital to compare a digital behavioral pain management intervention ("My Surgical Success" [MSS]) to a digital general health education (HE) intervention (HE; no pain management skills). The enrolled sample included 133 patients; 84 patients were randomized (MSS, n = 37; HE, n = 47) and completed study procedures. Most patients received their assigned intervention within 3 days of surgery (85%). The sample was predominantly male (61.5%), White (61.9%), and partnered (65.5%), with at least a bachelor's degree (69.0%). Outcomes were collected at 1-3 months after intervention through self-report e-surveys and electronic medical record review; an intention-to-treat analytic framework was applied. Feasibility was dually determined by the proportion of patients engaging in their assigned treatment and an application of an 80% threshold for patient-reported acceptability. We hypothesized that MSS would result in greater reductions in pain intensity and pain catastrophizing after surgery and earlier opioid cessation compared to the digital HE control group. RESULTS: The engagement rate with assigned interventions was 63% and exceeded commonly reported rates for fully automated Internet-based e-health interventions. Feasibility was demonstrated for the MSS engagers, with >80% reporting treatment acceptability. Overall, both groups improved in the postsurgical months across all study variables. A significant interaction effect was found for treatment group over time on pain intensity, such that the MSS group evidenced greater absolute reductions in pain intensity after surgery and up to 3 months later (treatment × time fixed effects; F [215] = 5.23; P = .024). No statistically significant between-group differences were observed for time to opioid cessation or for reductions in pain catastrophizing ( F [215] = 0.20; P = .653), although the study sample notably had subclinical baseline pain catastrophizing scores (M = 14.10; 95% confidence interval, 11.70-16.49). CONCLUSIONS: Study findings revealed that a fully automated behavioral pain management skills intervention (MSS) may be useful for motivated orthopedic trauma surgery patients and reduce postsurgical pain up to 3 months. MSS was not associated with reduced time to opioid cessation compared to the HE control intervention.

Design, synthesis and evaluation of inhibitors of the SARS-CoV-2 nsp3 macrodomain.

A series of amino acid based 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low µM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization.

Discovery of small molecule inhibitors of Plasmodium falciparum apicoplast DNA polymerase.

Malaria is caused by infection with protozoan parasites of the Plasmodium genus, which is part of the phylum Apicomplexa. Most organisms in this phylum contain a relic plastid called the apicoplast. The apicoplast genome is replicated by a single DNA polymerase (apPOL), which is an attractive target for anti-malarial drugs. We screened small-molecule libraries (206,504 compounds) using a fluorescence-based high-throughput DNA polymerase assay. Dose/response analysis and counter-screening identified 186 specific apPOL inhibitors. Toxicity screening against human HepaRG human cells removed 84 compounds and the remaining were subjected to parasite killing assays using chloroquine resistant P. falciparum parasites. Nine compounds were potent inhibitors of parasite growth and may serve as lead compounds in efforts to discover novel malaria drugs.

Repurposing Avasimibe to Inhibit Bacterial Glycosyltransferases.

We are interested in identifying and characterizing small molecule inhibitors of bacterial virulence factors for their potential use as anti-virulence inhibitors. We identified from high-throughput screening assays a potential activity for avasimibe, a previously characterized acyl-coenzyme A: cholesterol acyltransferase inhibitor, in inhibiting the NleB and SseK arginine glycosyltransferases from Escherichia coli and Salmonella enterica, respectively. Avasimibe inhibited the activity of the Citrobacter rodentium NleB, E. coli NleB1, and S. enterica SseK1 enzymes, without affecting the activity of the human serine/threonine N-acetylglucosamine (O-GlcNAc) transferase. Avasimibe was not toxic to mammalian cells at up to 200 µM and was neither bacteriostatic nor bactericidal at concentrations of up to 125 µM. Doses of 10 µM avasimibe were sufficient to reduce S. enterica abundance in RAW264.7 macrophage-like cells, and intraperitoneal injection of avasimibe significantly reduced C. rodentium survival in mice, regardless of whether the avasimibe was administered pre- or post-infection. We propose that avasimibe or related derivates created using synthetic chemistry may have utility in preventing or treating bacterial infections by inhibiting arginine glycosyltransferases that are important to virulence.

Design, Synthesis and Evaluation of Inhibitors of the SARS-CoV2 nsp3 Macrodomain.

A series of amino acid based 7H -pyrrolo[2,3- d ]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp 22 and the amide backbone NH of Ile 23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe 157 and Asp 156 , part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low µM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization.

High-Throughput Screening Identifies Inhibitors for Parvovirus B19 Infection of Human Erythroid Progenitors.

Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 µM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 µM) without prominent cytotoxicity (CC50 = 71.8 µM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 µM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 µM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.

Simple and rapid high-throughput assay to identify HSV-1 ICP0 transactivation inhibitors.

Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that results in lifelong infections due to its ability to cycle between lytic replication and latency. As an obligate intracellular pathogen, HSV-1 exploits host cellular factors to replicate and aid in its life cycle. HSV-1 expresses infected cell protein 0 (ICP0), an immediate-early regulator, to stimulate the transcription of all classes of viral genes via its E3 ubiquitin ligase activity. Here we report an automated, inexpensive, and rapid high-throughput approach to examine the effects of small molecule compounds on ICP0 transactivator function in cells. Two HSV-1 reporter viruses, KOS6ß (wt) and dlx3.1-6ß (ICP0-null mutant), were used to monitor ICP0 transactivation activity through the HSV-1 ICP6 promoter:lacz expression cassette. A ≥10-fold difference in ß-galactosidase activity was observed in cells infected with KOS6ß compared to dlx3.1-6ß, demonstrating that ICP0 potently transactivates the ICP6 promoter. We established the robustness and reproducibility with a Z'-factor score of ≥0.69, an important criterium for high-throughput analyses. Approximately 19,000 structurally diverse compounds were screened and 76 potential inhibitors of the HSV-1 transactivator ICP0 were identified. We expect this assay will aid in the discovery of novel inhibitors and tools against HSV-1 ICP0. Using well-annotated compounds could identify potential novel factors and pathways that interact with ICP0 to promote HSV-1 gene expression.