Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED.

Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus infection. Its structures had been elusive to the public until recently when one of the crystal forms was solved by MicroED. In this work, we report the MicroED structures of two distinct polymorphic crystal forms of paritaprevir from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropylsulfonamide and methylpyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the NS3/4A serine protease target, and can interact with the pocket and catalytic triad via hydrophobic interactions and hydrogen bonds. These results can provide further insight for optimization of the binding of acylsulfonamide inhibitors to the NS3/4A serine protease. In addition, this also demonstrate the opportunity of deriving different polymorphs and distinct macrocycle conformations from the same experiments using MicroED.

MicroED as a powerful tool for structure determination of macrocyclic drug compounds directly from their powder formulations.

Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as act as molecular chameleons and thereby reach intracellular targets. However, due to their complex structures and inherent flexibility, macrocycles are difficult to study structurally and there are limited structural data available. Herein, we use the cryo-EM method MicroED to determine the novel atomic structures of several macrocycles which have previously resisted structural determination. We show that structures of similar complexity can now be obtained rapidly from nanograms of material, and that different conformations of flexible compounds can be derived from the same experiment. These results will have impact on contemporary drug discovery as well as natural product exploration.

Improving the solubility of pseudo-hydrophobic Alzheimer's Disease medicinal chemicals through co-crystal formulation.

Natural products are ligands and potential inhibitors of Alzheimer's disease (AD) tau. Dihydromyricetin (DHM) is a CNS active natural product. Despite having signature polyphenolic character, DHM is ostensibly hydrophobic owing to intermolecular hydrogen bonds that shield hydrophilic phenols. Our research shows DHM becomes ionized at near-neutral pH allowing formulation of salts with transformed solubility. The MicroED co-crystal structure with trolamine reveals DHM salts as metastable solids with unlocked hydrogen bonding and a thermodynamic bent to solubilize in water. All salt formulations show better inhibitory activity against AD tau than the non-salt form, with efficacies correlating to enhanced solubilities. These results underscore the role of structural chemistry in guiding selection of solubilizing agents for chemical formulation. We propose DHM salts are appropriate formulations for research as dietary supplements to promote healthy aging by combating protein misfolding. Additionally, DHM is a suitable lead for medicinal chemistry and possible development of CNS pharmaceuticals.

Mask-Associated Dry Eye (MADE) in healthcare professionals working at COVID-19 pandemic clinics.

Background: Healthcare professionals working at COVID-19 pandemic clinics have to work with masks during long hours. After the widespread use of masks in the community, many mask-related side effects were reported to clinics. The increase in the number of applicants with dry eye symptoms due to mask use in ophthalmology clinics has led to the emergence of the concept of mask-associated dry eye (MADE). We think that it would be valuable to evaluate ocular surface tests with a comparative study using healthcare professionals working in pandemic clinics, which we think is the right study group to examine the effects of long-term mask use. Aims: We aimed to evaluate the mask-associated dry eye (MADE) symptoms and findings in healthcare professionals who have to work prolonged time with face masks in coronavirus disease 2019 (COVID-19) pandemic clinics. Patients and Methods: In this prospective, observational comparative clinical study, healthcare professionals who use the mask for a long time and work in COVID-19 pandemic clinics were compared with an age and sex-matched control group consisting of short-term masks users, from April 2021 to November 2021. All participants underwent the Ocular Surface Disease Index (OSDI) questionnaire, tear film break-up time (T-BUT), Oxford staining score, Schirmer's test I, and meibography with infrared transillumination. Results: The long-term mask user group consisted of 64 people, while the short-term mask user group consisted of 66 people (260 eyes, total). The OSDI score and Schirmer I measurement were not statistically different between the two groups. T-BUT was statistically significantly shorter in the long-term group (P: 0.008); lid parallel-conjunctival fold, Oxford staining score, and upper and lower lid meibography score were found to be significantly higher in the long-term group (P < 0.001, P: 0.004, P: 0.049, P: 0.044, respectively). Conclusion: Healthcare professionals with longer mask-wearing times are at greater risk of ocular surface damage. It may be considered to prevent this damage by blocking airflow to the ocular surface, such as by wearing a face mask properly or fitting it over the nose with surgical tape. Those who have to work with a mask for a long time during the COVID-19 pandemic should keep in mind the ophthalmology follow-up for eye comfort and ocular surface health.

Cervical Cordotomy for Intractable Pain: Do Postoperative Imaging Features Correlate with Pain Outcomes and Mirror Pain?

BACKGROUND AND PURPOSE: Percutaneous cervical cordotomy offers relief of unilateral intractable oncologic pain. We aimed to find anatomic and postoperative imaging features that may correlate with clinical outcomes, including pain relief and postoperative contralateral pain. MATERIALS AND METHODS: We prospectively followed 15 patients with cancer who underwent cervical cordotomy for intractable pain during 2018 and 2019 and underwent preoperative and up to 1-month postoperative cervical MR imaging. Lesion volume and diameter were measured on T2-weighted imaging and diffusion tensor imaging (DTI). Lesion mean diffusivity and fractional anisotropy values were extracted. Pain improvement up to 1 month after surgery was assessed by the Numeric Rating Scale and Brief Pain Inventory. RESULTS: All patients reported pain relief from 8 (7-10) to 0 (0-4) immediately after surgery (P = .001), and 5 patients (33%) developed contralateral pain. The minimal percentages of the cord lesion volume required for pain relief were 10.0% on T2-weighted imaging and 6.2% on DTI. Smaller lesions on DWI correlated with pain improvement on the Brief Pain Inventory scale (r = 0.705, P = .023). Mean diffusivity and fractional anisotropy were significantly lower in the ablated tissue than contralateral nonlesioned tissue (P = .003 and P = .001, respectively), compatible with acute-phase tissue changes after injury. Minimal postoperative mean diffusivity values correlated with an improvement of Brief Pain Inventory severity scores (r = -0.821, P = .004). The average lesion mean diffusivity was lower among patients with postoperative contralateral pain (P = .037). CONCLUSIONS: Although a minimal ablation size is required during cordotomy, larger lesions do not indicate better outcomes. DWI metrics changes represent tissue damage after ablation and may correlate with pain outcomes.

Structure-based inhibitors of tau aggregation.

Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.

High-Resolution Macromolecular Structure Determination by MicroED, a cryo-EM Method.

Microelectron diffraction (MicroED) is a new cryo-electron microscopy (cryo-EM) method capable of determining macromolecular structures at atomic resolution from vanishingly small 3D crystals. MicroED promises to solve atomic resolution structures from even the tiniest of crystals, less than a few hundred nanometers thick. MicroED complements frontier advances in crystallography and represents part of the rebirth of cryo-EM that is making macromolecular structure determination more accessible for all. Here we review the concept and practice of MicroED, for both the electron microscopist and crystallographer. Where other reviews have addressed specific details of the technique (Hattne et al., 2015; Shi et al., 2016; Shi, Nannenga, Iadanza, & Gonen, 2013), we aim to provide context and highlight important features that should be considered when performing a MicroED experiment.

Renal artery stenosis and abdominal aorta aneurysm in patients with pseudoexfoliation syndrome.

PURPOSE: To evaluate the renal arteries and abdominal aorta in patients with pseudoexfoliation syndrome (PEX). DESIGN: Prospective, case-control study. METHODS: The study involved 49 patients with PEX and 42 control subjects. Abdominal aorta and renal arteries were examined by Doppler ultrasonography. In both renal arteries (proximal and distal portions) and abdominal aorta, the peak systolic velocity (PSV) was measured. Renal artery stenosis (RAS) was defined as the renal artery PSV >150 cm/s or renal-to-aortic ratio (RAR) >3.0. Patients who had an abdominal aortic diameter >3 cm were recorded. Computed tomographic angiography was performed to confirm these findings in patients with RAS and/or abdominal aorta aneurysm. RESULTS: The mean PSV in the proximal renal artery was 88.3 cm/s in PEX group and 79.5 cm/s in control group (P=0.314); in distal renal artery was 91.7 cm/s in PEX group and 93.0 cm/s in control group (P=0.794); in abdominal aorta was 76.0 cm/s in PEX group and 65.2 cm/s in control group (P=0.046). RAS was observed in nine patients with PEX and in only one patient without PEX (P=0.017). Seven out of 10 patients with RAS (six patients in PEX group; one patient in control group) had hypertension. Abdominal aorta aneurysm was observed in four patients in PEX group but not in control group (P=0.061). CONCLUSIONS: Our study has demonstrated that there is a significant association between PEX and RAS. The abdominal aorta aneurysm may be seen in patients with PEX.

MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3.

BACKGROUND: Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator. RESULTS: MP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solubilized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction. CONCLUSIONS: MP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity.

Galectin-3 is associated with the plasma membrane of lens fiber cells.

PURPOSE: To discover proteins that have the potential to contribute to the tight packing of fiber cells in the lens. METHODS: Crude fiber cell membranes were isolated from ovine lens cortex. Proteins were separated by two-dimensional gel electrophoresis, and selected protein spots identified by micro-sequencing. The identification of galectin-3 was confirmed by immunoblotting with a specific antibody. The association of galectin-3 with the fiber cell plasma membrane was investigated using immunofluorescence microscopy, solubilization trials with selected reagents, and immunoprecipitation to identify candidate ligands. RESULTS: A cluster of three protein spots with an apparent molecular weight of 31,000 and isoelectric points ranging between 7 and 8.5 were resolved and identified as galectin-3. This protein was associated peripherally with the fiber cell plasma membrane and interacted with MP20, an abundant intrinsic membrane protein that had been identified previously as a component of membrane junctions between fiber cells. CONCLUSIONS: The detection of galectin-3 in the lens is a novel result and adds to the growing list of lens proteins with adhesive properties. Its location at the fiber cell membrane and its association with the junction-forming MP20 is consistent with a potential role in the development or maintenance of the tightly packed lens tissue architecture.