Elucidating Residue-Level Determinants Affecting Dimerization of Ebola Virus Matrix Protein Using High-Throughput Site Saturation Mutagenesis and Biophysical Approaches.

The Ebola virus (EBOV) is a filamentous virus that acquires its lipid envelope from the plasma membrane of the host cell it infects. EBOV assembly and budding from the host cell plasma membrane are mediated by a peripheral protein, known as the matrix protein VP40. VP40 is a 326 amino acid protein with two domains that are loosely linked. The VP40 N-terminal domain (NTD) contains a hydrophobic α-helix, which mediates VP40 dimerization. The VP40 C-terminal domain has a cationic patch, which mediates interactions with anionic lipids and a hydrophobic region that mediates VP40 dimer-dimer interactions. The VP40 dimer is necessary for trafficking to the plasma membrane inner leaflet and interactions with anionic lipids to mediate the VP40 assembly and oligomerization. Despite significant structural information available on the VP40 dimer structure, little is known on how the VP40 dimer is stabilized and how residues outside the NTD hydrophobic portion of the α-helical dimer interface contribute to dimer stability. To better understand how VP40 dimer stability is maintained, we performed computational studies using per-residue energy decomposition and site saturation mutagenesis. These studies revealed a number of novel keystone residues for VP40 dimer stability just adjacent to the α-helical dimer interface as well as distant residues in the VP40 CTD that can stabilize the VP40 dimer form. Experimental studies with representative VP40 mutants in vitro and in cells were performed to test computational predictions that reveal residues that alter VP40 dimer stability. Taken together, these studies provide important biophysical insights into VP40 dimerization and may be useful in strategies to weaken or alter the VP40 dimer structure as a means of inhibiting the EBOV assembly.

N-Alkyl Carbamoylimidazoles as Versatile Synthons for the Synthesis of Urea-Based PSMA Inhibitors.

We describe N-alkyl carbamoylimidazoles as readily available and highly versatile synthons for synthesizing urea-based prostate-specific membrane antigen (PSMA) inhibitors. Urea formation proceeded in high yields (>80%) at room temperature under aqueous conditions. All novel compounds were tested for their PSMA inhibitory potency in a cell-based radiometric binding assay. Compound 17 was identified as a novel high-affinity PSMA inhibitor (IC50 = 0.013 µM) suitable for developing an 18F-labeled radioligand for PET imaging of PSMA in prostate cancer.

[18F]ONO-8430506: A novel radioligand for PET imaging of autotaxin (ATX).

We have prepared and tested radioligand [18F]ONO-8430506 ([18F]8) as a novel ATX PET imaging agent derived from highly potent ATX inhibitor ONO-8430506. Radioligand [18F]8 could be prepared in good and reproducible radiochemical yields of 35 ± 5% (n = 6) using late-stage radiofluorination chemistry. ATX binding analysis showed that 9-benzyl tetrahydro-b-carboline 8 has about five times better inhibitory potency than clinical candidate GLPG1690 and somewhat less inhibitory potency than ATX inhibitor PRIMATX. The binding mode for compound 8 inside the catalytic pocket of ATX using computational modelling and docking protocols revealed that compound 8 resembled a comparable binding mode to that of ATX inhibitor GLPG1690. However, PET imaging studies with radioligand [18F]8 showed only relatively low tumour uptake and retention (SUV60min 0.21 ± 0.03) in the tested 8305C human thyroid tumour model reaching a tumour-to-muscle ratio of âˆ¼ 2.2 after 60 min.

Targeting the chromatin effector Pygo2 promotes cytotoxic T cell responses and overcomes immunotherapy resistance in prostate cancer.

The noninflamed microenvironment in prostate cancer represents a barrier to immunotherapy. Genetic alterations underlying cancer cell-intrinsic oncogenic signaling are increasingly appreciated for their role in shaping the immune landscape. Recently, we identified Pygopus 2 (PYGO2) as the driver oncogene for the amplicon at 1q21.3 in prostate cancer. Here, using transgenic mouse models of metastatic prostate adenocarcinoma, we found that Pygo2 deletion decelerated tumor progression, diminished metastases, and extended survival. Pygo2 loss augmented the activation and infiltration of cytotoxic T lymphocytes (CTLs) and sensitized tumor cells to T cell killing. Mechanistically, Pygo2 orchestrated a p53/Sp1/Kit/Ido1 signaling network to foster a microenvironment hostile to CTLs. Genetic or pharmacological inhibition of Pygo2 enhanced the antitumor efficacy of immunotherapies using immune checkpoint blockade (ICB), adoptive cell transfer, or agents inhibiting myeloid-derived suppressor cells. In human prostate cancer samples, Pygo2 expression was inversely correlated with the infiltration of CD8+ T cells. Analysis of the ICB clinical data showed association between high PYGO2 level and worse outcome. Together, our results highlight a potential path to improve immunotherapy using Pygo2-targeted therapy for advanced prostate cancer.

Fluorine-18 Labelled Radioligands for PET Imaging of Cyclooxygenase-2.

Molecular imaging probes enable the early and accurate detection of disease-specific biomarkers and facilitate personalized treatment of many chronic diseases, including cancer. Among current clinically used functional imaging modalities, positron emission tomography (PET) plays a significant role in cancer detection and in monitoring the response to therapeutic interventions. Several preclinical and clinical studies have demonstrated the crucial involvement of cyclooxygenase-2 (COX-2) isozyme in cancer development and progression, making COX-2 a promising cancer biomarker. A variety of COX-2-targeting PET radioligands has been developed based on anti-inflammatory drugs and selective COX-2 inhibitors. However, many of those suffer from non-specific binding and insufficient metabolic stability. This article highlights examples of COX-2-targeting PET radioligands labelled with the short-lived positron emitter 18F, including radiosynthesis and PET imaging studies published in the last decade (2012-2021).

A Narrative Review on Current Diagnostic Imaging Tools for Dentomaxillofacial Abnormalities in Children.

The current review narrates the findings and discusses the available diagnostic tools for detecting structural abnormalities. The review discusses several diagnostic tools, such as magnetic resonance imaging, cone beam computed tomography, multi detector row CT and positron emission tomography. The vital findings and comparative analysis of different diagnostic tools are presented in this review. The present review also discusses the advent of newer technologies, such as the HyperionX9 scanner with less field of view and 18F-FDG PET/CT (positron emission tomography with 2-deoxy-2-[fluorine-18] fluoro-D-glucose, integrated with computed tomography), which can give more efficient imaging of dentomaxillofacial structures. The discussion of effective comparative points enables this review to reveal the available diagnostic tools that can be used in the detection of dentomaxillofacial abnormalities in the pediatric population. The advantages and disadvantages of each tool are discussed, and the findings of past publications are also presented. Overall, this review discusses the technical details and provides a comparative analysis of updated diagnostic techniques for dentomaxillofacial diagnosis.

Synthesis, Binding Affinity Analysis, and 18 F Radiosynthesis of Small-Molecular-Weight HIF-1α-Binding Compounds.

Eleven small-molecular-weight compounds and three cyclic peptides were synthesized and evaluated for binding to hypoxia-inducible factor-1α (HIF-1α). Microscale thermophoresis analysis identified peptide [19 F]SFB-link-c-(Ppg)LLFVY 3 and small-molecule inhibitor 5 as potent HIF-1α binding compounds with KD values of 0.46±0.2 µM and 7.8±3.4 µM, respectively. Both compounds represent novel HIF-1α-targeting compounds that are predicted to interact with the PAS-B region of HIF-1α, as confirmed with molecular docking studies. Lead structures 3 and 5 were further radiolabelled with fluorine-18 for positron emission tomography (PET) imaging agents targeting HIF-1α in vivo.

Synthesis and Preclinical Evaluation of [18F]SiFA-PSMA Inhibitors in a Prostate Cancer Model.

Positron emission tomography (PET) imaging of prostate-specific membrane antigen (PSMA) with gallium-68 (68Ga) and fluorine-18 (18F) radiotracers has aroused tremendous interest over the past few years. The use of organosilicon-[18F]fluoride acceptors (SiFA) conjugated to urea-based peptidomimetic PSMA inhibitors provides a "kit-like" multidose synthesis technology. Nine novel 18F-labeled SiFA-bearing PSMA inhibitors with different linker moieties were synthesized and analyzed for their in vitro binding against [125I]I-TAAG-PSMA in LNCaP cells. IC50 values ranged from 58-570 nM. Among all compounds, [18F]SiFA-Asp2-PEG3-PSMA (IC50 = 125 nM) showed the highest tumor uptake in LNCaP tumors (SUV60min 0.73). A substantial increase in molar activity (Am) (from 7.5 ± 0.5 to 86 ± 3 GBq/µmol) led to a significant increase in LNCaP tumor uptake (SUV60min 1.18; Δ 0.45 corresponding to +62%). In vivo blocking with DCFPyL resulted in -32% uptake after 60 min. The SiFA-isotopic exchange chemistry offers a method that is readily adaptable for a "kit-type" labeling procedure and clinical translation.

Production of Proteins of the SARS-CoV-2 Proteome for Drug Discovery.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the coronavirus disease of 2019 (COVID-19). Its genome encodes two open reading frames for two large proteins, PP1a and PP1ab. Within the two polypeptide stretches, there are two proteases that process the large proteins into 15 discrete proteins essential for the assembly of the virion during its replication. We describe herein the cloning of the genes for these discrete proteins optimized for expression in Escherichia coli, production of the proteins, and their purification to homogeneity. These included all but six: NSP6, which possesses eight transmembrane regions, and five that are small proteins/peptides (E, ORF3b, ORF6, ORF7b, and ORF10). These proteins are intended for experimental validation of small-molecule binders as molecular template hits. The proof of concept was established with the ADP-ribosylhydrolase (ARH) domain of NSP3 in discovery of small-molecule templates that could serve as the basis for further optimization. The hit molecules include one submicromolar and a few low-micromolar binders to the ARH domain. Availability of these proteins in soluble forms opens up the opportunity for discoveries of novel templates with the potential for anti-COVID-19 pharmaceuticals.

Development of Fluorescence Imaging Probes for Labeling COX-1 in Live Ovarian Cancer Cells.

Recent experimental evidence demonstrated an aberrant overexpression of cyclooxygenase-1 (COX-1) in various cancers, which has stimulated the development of COX-1-selective inhibitors as promising anticancer drugs and cancer imaging agents. Herein we describe the synthesis and validation of 3-(furan-2-yl)-N-aryl 5-amino-pyrazoles as a novel class of COX-1 inhibitors, including molecular docking studies. Among all tested compounds, 4-(5-azido-3-(furan-2-yl)-1H-pyrazol-1-yl)benzoic 17 displayed a favorable COX-1 inhibition and selectivity profile (COX-1 IC50 = 0.1 µM, SI >1000 over COX-2). Compound 17 was selected as a lead structure for developing the novel COX-1-selective fluorescent probe 22. Fluorescent probe 22 was prepared via click chemistry by installing a nitro-benzoxadiazole motif as a fluorophore into the 3-(furan-2-yl)-N-aryl 5-amino-pyrazole scaffold. Fluorescence probe 22 was tested in ovarian cancer cell line OVCAR-3, confirming its usefulness for targeting and visualizing COX-1 in living cells with confocal microscopy.

Genetically Encoded Fragment-Based Discovery from Phage-Displayed Macrocyclic Libraries with Genetically Encoded Unnatural Pharmacophores.

Genetically encoded macrocyclic peptide libraries with unnatural pharmacophores are valuable sources for the discovery of ligands for many targets of interest. Traditionally, generation of such libraries employs "early stage" incorporation of unnatural building blocks into the chemically or translationally produced macrocycles. Here, we describe a divergent late-stage approach to such libraries starting from readily available starting material: genetically encoded libraries of peptides. A diketone linchpin 1,5-dichloropentane-2,4-dione converts peptide libraries displayed on phage to 1,3-diketone bearing macrocyclic peptides (DKMP): shelf-stable precursors for Knorr pyrazole synthesis. Ligation of diverse hydrazine derivatives onto DKMP libraries displayed on phage that carries silent DNA-barcodes yields macrocyclic libraries in which the amino acid sequence and the pharmacophore are encoded by DNA. Selection of this library against carbonic anhydrase enriched macrocycles with benzenesulfonamide pharmacophore and nanomolar Kd. The methodology described in this manuscript can graft diverse pharmacophores into many existing genetically encoded phage libraries and significantly increase the value of such libraries in molecular discoveries.

In Cellulo Generation of Fluorescent Probes for Live-Cell Imaging of Cylooxygenase-2.

Live-cell imaging with fluorescent probes is an essential tool in chemical biology to visualize the dynamics of biological processes in real-time. Intracellular disease biomarker imaging remains a formidable challenge due to the intrinsic limitations of conventional fluorescent probes and the complex nature of cells. This work reports the in cellulo assembly of a fluorescent probe to image cyclooxygenase-2 (COX-2). We developed celecoxib-azide derivative 14, possessing favorable biophysical properties and excellent COX-2 selectivity profile. In cellulo strain-promoted fluorogenic click chemistry of COX-2-engaged compound 14 with non/weakly-fluorescent compounds 11 and 17 formed fluorescent probes 15 and 18 for the detection of COX-2 in living cells. Competitive binding studies, biophysical, and comprehensive computational analyses were used to describe protein-ligand interactions. The reported new chemical toolbox enables precise visualization and tracking of COX-2 in live cells with superior sensitivity in the visible range.

Design, Synthesis, and Evaluation of a Luminescent Cholesterol Mimic.

The development of new chemical tools with improved properties is essential to chemical and cell biology. Of particular interest is the development of mimics of small molecules with important cellular function that allow the direct observation of their trafficking in a cell. To this end, a novel 15-azasterol has been designed and synthesized as a luminescent cholesterol mimic for the monitoring of cholesterol trafficking. The brightness of this probe, which is ∼32-times greater than the widely used dehydroergosterol probe, is combined with resistance to photobleaching in solution and in human fibroblasts and an exceptionally large Stokes-like shift of ∼150-200 nm. The photophysical properties of the probe have been studied experimentally and computationally, suggesting an intersystem crossing to the triplet excited state with subsequent phosphorescent decay. Molecular dynamics simulations show a similar binding mode of cholesterol and the azasterol probe to NPC proteins, demonstrating the structural similarity of the probe to cholesterol.

Optimization of Acetazolamide-Based Scaffold as Potent Inhibitors of Vancomycin-Resistant Enterococcus.

Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States, and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent antienterococcal agents. Through structure-activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 µg/mL (acetazolamide) to MIC = 0.007 µg/mL (22) and 1 µg/mL (26). Physicochemical properties were modified to design leads that have either high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggest the intracellular targets for the molecules are putative α-carbonic and γ-carbonic anhydrases, and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.

Assessment of correlation of periodontitis in teeth adjacent to implant and peri- implantitis.

AIMS: The present study was conducted to determine correlation between peri-implantitis and periodontitis in adjacent teeth. MATERIALS AND METHODS: The present study was conducted on 58 patients with 84 dental implants. They were divided into two groups, group I (50) was with peri-implantitis and group II (34) was without it. In all patients, probing depth (PD), gingival recession (GR), and clinical attachment loss (CAL) was calculated around implant, adjacent to implant and on contralateral side. Obtained data were statistically analyzed using statistical software IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp with one-way analysis of variance. RESULTS: Males were 30 with 52 dental implants and females were 28 with 32 dental implants. CAL was 5.82 ± 0.52 in group I and 3.62 ± 0.63 in group II (P = 0.001) around implants. PD was 4.28 ± 1.26 in group I and 2.20 ± 0.52 in group II around adjacent teeth (P = 0.002). PD around contralateral teeth was significant (P = 0.05) in group I (3.18 ± 1.01) and group II (2.71 ± 0.73). CONCLUSION: Periodontitis has negative effect on implant success. Teeth adjacent to dental implant plays an important role in deciding the success or failure of implant. Maintenance of periodontal health is of paramount importance for successful implant therapy.

Analysis of chain length, substitution patterns, and unsaturation of AM-404 derivatives as 20S proteasome stimulators.

Proteasome-mediated degradation of proteins is a vital cellular process and is performed by the ubiquitin-dependent proteasome system (UPS) and the ubiquitin-independent proteasome system (UIPS). While both systems are necessary to maintain healthy cell function, many disease states are characterized by reduced activity of the UPS, and the UIPS cannot by itself maintain proper protein levels. It has been suggested that the 20S core particle (20S CP), the isoform of the proteasome in the UIPS that can degrade proteins without a ubiquitin tag, can be stimulated with a small molecule to assist the 20S CP to accept and hydrolyze substrates more rapidly. Several small molecule stimulators of the 20S CP have since been discovered, including AM-404, an arachidonic acid derivative. AM-404 has previously been shown to inhibit fatty acid amide hydrolase activity. We wished to evaluate what structural components of AM-404 are required to stimulate the 20S CP with the long-term goal of using this information to design a stimulator with better drug-like qualities. We synthesized numerous derivatives of AM-404, varying the chain length, substitutions, and degree of unsaturation. Through this endeavor, we obtained several molecules capable of stimulating the 20S CP to various degrees. We discovered that though chain length is important, the presence of a cis-alkene in a specific location in the aliphatic chain has the greatest impact on the ability to stimulate the 20S CP. Two of the derivatives maintain modest stimulatory activity, and have improved toxicity over AM-404.

The succinct synthesis of AT13387, a clinically relevant Hsp90 inhibitor.

AT13387 is an orally bioavailable clinical candidate developed to inhibit theheat shock protein 90 (Hsp90). This article describes a modified synthetic route for the multi-gram production of AT13387 in 46% overall yield. The modified synthetic route is short, avoids stringent reaction conditions and difficult purifications, which led to increase in an overall yield.

Glutathione S-Transferase π-Activatable O2-(Sulfonylethyl Derived) Diazeniumdiolates Potently Suppress Melanoma in Vitro and in Vivo.

A group of glutathione S-transferase π (GSTπ) activatable O2-(sulfonylethyl derived) diazeniumdiolates 5-12 were designed and synthesized. These compounds could be activated by GSTπ to initiate the ß-elimination reaction, liberating an active vinyl sulfone-based GSH derivative and a diazeniumdiolate anion which subsequently releases NO in situ. The most active compound 6 released relatively high levels of NO and inhibited proliferation of melanoma B16 cells, superior to a diazeniumdiolate-based anticancer agent JS-K (1). Importantly, 6 had 8-fold less inhibitory activity against normal epithelial JB6 Cl 30-7b cells. The inhibitory activity of 6 could be diminished by an NO scavenger or GSTπ inhibitor. Furthermore, 6 induced nitration of mitochondrial tyrosine in B16 cells and inoculated B16 tumors in mice, which might be responsible for oxidation of protein leading to tumor suppression. Finally, 6 significantly retarded the B16 cells growth in a mouse xenograft model. These findings suggest that 6 may have a potential to treat melanoma.

Assessment of Root Resorption and Root Shape by Periapical and Panoramic Radiographs: A Comparative Study.

INTRODUCTION: One of the common findings encountered by the clinician at the end of orthodontic treatment is the apical root resorption. Root resorption occurs to various degrees. A severe form of root resorption is characterized by shortening of root for more than 4 mm or more than one-third of the total tooth length. A low incidence rate of resorption is observed based on radiographic findings for the diagnosis of root resorption, panoramic radiography, and periapical radiography. Hence, we evaluated the accuracy of panoramic radiographic films for assessing the root resorption in comparison with the periapical films. MATERIALS AND METHODS: This study included the assessment of all the cases in which pre- and post-treatment radiographs were available for analysis of the assessment of the amount of root resorption. Complete records of 80 patients were analyzed. Examination of a total of 900 teeth was done. Mean age of the patients in this study was 21 years ranging from 11 to 38 years. The majority of the patients in the present study were females. All the treatments were carried out by registered experienced orthodontists having minimum experience of more than 10 years. All the cases were divided into two study groups. Group I comprised panoramic radiographic findings, while group II consisted of periapical radiographic findings. For the measurement of crown portion, root portion, and the complete root length, magnification loops of over 100 powers with parallax correction with inbuilt grids were used. Assessment of the tooth length and the crown length was done by the same observers. All the results were analyzed by Statistical Package for the Social Sciences software version 6.0. RESULTS: Maximum amount of root resorption was observed in case of maxillary central incisors and canines among group I and II cases respectively. However, nonsignificant difference was obtained while comparing the mean root resorption in relation to maxillary incisors and canines among the two study groups. While comparing the overall value of root resorption among the two study groups, a significant difference was obtained. The maximum value of tooth length in both the groups was observed in cases of maxillary canines. Significant differences were observed while comparing the tooth length of various teeth among the two study groups. Among the deviated forms of root shape, dilacera-tion was the most common form of root shape detected in both the study groups. CONCLUSION: Periapical radiographs are more efficient in the assessment of the shape and resorption of the root. CLINICAL SIGNIFICANCE: Thorough evaluation of periapical radiographs is necessary for detection of even minute levels of root resorption.

Denture identification using individual national identification number of Saudi Arabia: An innovative inclusion method of casted metal.

CONTEXT: Forensic odontology is one of the branches of dentistry, which played a very important role in identification of individuals in accident, natural and mass disaster, and civil unrest and in genocide crimes. In the absence of natural teeth, marking or labeling of denture plays a vital role in the personal identification. BACKGROUND: Various types of marking or labeling methods are reported. However, many are not according to the criteria put forth by American Dental Association or other professional association. Majority of these techniques may be time consuming and expansive, may not be standardized, long lasting and do not permit the incorporation of a large amount of information. AIM: The aim of this study is to find out a denture identification technique that should be easy, less expensive, long lasting, and standardized. MATERIALS AND METHODS: This article illustrates an inclusion denture casted metal technique of the individual national identification number printed in the patient's residence number or iquama or national identity card issued by the ministry of interior, Kingdom of Saudi Arabia is used as a denture marker in the lingual surface of mandibular denture. RESULTS: The label in this method is durable and can withstand high temperature, less chances of deterioration, visible radiographically, and provide all important information about individual that is standardized, reliable, and also accessible from any remote location. CONCLUSION: Hence, the proposed technique is an easy, less expensive, long lasting, radiographically visible, and standardized method of identification.