Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides.

Dampening functional levels of the mitochondrial deubiquitylating enzyme Ubiquitin-specific protease 30 (USP30) has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinson's Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria, which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide-containing compound, USP30inh. Activity-based protein profiling mass spectrometry confirmed target engagement, high selectivity, and potency of USP30inh for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30inh enzyme kinetics inferred slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with USP30inh, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30inh binds to this thumb-palm cleft, which guides the ubiquitin C terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

Patient satisfaction and attitudes towards immediate sequential bilateral cataract surgery.

PURPOSE: To investigate patients' first-hand experience, satisfaction and attitudes towards immediate sequential bilateral cataract surgery (ISBCS). SETTING: Royal Bournemouth Hospital, University Hospitals Dorset NHS Foundation Trust, Castle Lane East, Bournemouth, UK. DESIGN: Retrospective study using semi-structured phone interviews and qualitative content analysis. METHODS: Semi-structured telephone interviews were conducted by an independent interviewer, to explore patients' experiences of ISBCS, at least four weeks postoperatively. The open-ended responses were analysed using qualitative content analysis. Categories and meaning units were tabulated, with the number and percentage of patients contributing to each category provided. RESULTS: 25 patients were included. All patients rated their overall satisfaction of ISBCS as 'very satisfied' and would opt again to have both eyes operated on the same day. 22 patients (88%) reported a 'very good/comfortable' surgery experience. After surgery, 24 patients (96%) felt completely safe going home, with most organizing for family or friends to drive them home. None of the patients experienced any complications in the postoperative period. 24 patients (96%) experienced only minimal reduction in accomplishing daily living activities after surgery and 24 patients (96%) said they would recommend ISBCS to family and friends. CONCLUSION: The results support wider implementation of ISBCS from the patient's perspective and experience. Providing written information addressing common themes and concerns is recommended to increase patient acceptance and uptake of ISBCS preoperatively.

Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

USP28 deletion and small-molecule inhibition destabilizes c-MYC and elicits regression of squamous cell lung carcinoma.

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

Studying RNA-Protein Complexes Using X-Ray Crystallography.

A wide range of biological processes rely on complexes between ribonucleic acids (RNAs) and proteins. Determining the three-dimensional structures of RNA-protein complexes is crucial to elucidate the relationship between structure and biological function. X-ray crystallography represents the most widely used technique to characterize RNA-protein complexes at atomic resolution; however, determining their three-dimensional structures remains challenging. RNase contamination can ruin crystallization experiments by degrading RNA in complex with protein, leading to sample heterogeneity, and the conformational flexibility inherent in both protein and RNA can limit crystallizability. Furthermore, the three-dimensional structure can be difficult to accurately model at the typical diffraction limit of 2.5 Å resolution or lower for RNA-protein complex crystals. At this resolution, phosphates, which are electron dense, and bases, which are large, rigid, and planar, tend to be well resolved and easy to position in the electron density map, whereas other features, e.g., sugar atoms, can be difficult to accurately position. This chapter focuses on methods that can be used to overcome the unique problems faced when crystallizing RNA-protein complexes and determining their three-dimensional structures using X-ray crystallography.

Dupilumab-associated ocular surface disease: presentation, management and long-term sequelae.

OBJECTIVES: To determine the presenting features of ocular surface disease in patients with atopic dermatitis (AD) treated with dupilumab at a tertiary, university hospital. To establish the need for treatment of dupilumab-associated ocular surface disease and report any long-term effects on the ocular surface. METHODS: A retrospective analysis of consecutive patients treated with dupilumab for AD between January 2017 and August 2019 was undertaken. Data were collected on demographics, incidence and type of ocular disease features, natural history and treatment. RESULTS: A total of 50% (14/28) patients developed ocular symptoms with a mean time of onset of 6.75 (±6.1) weeks from starting dupilumab. Of these, 69% (9/13) were diagnosed with conjunctivitis associated with cicatrisation in two patients and periorbital skin changes in four. Of these nine, four had prior history of atopic keratoconjunctivitis. All were treated with topical steroids; two required additional ciclosporin drops. In all, 67% (6/9) patients went on to have on-going ocular inflammation requiring maintenance drops at a mean of 16 (±6.9) months of follow-up. All patients had improvement in their AD severity; only one patient discontinued dupilumab due to ocular side effects. CONCLUSION: The rate of dupilumab-associated ocular surface disease was 32%. Periorbital skin changes and conjunctival cicatrisation were noted in association with conjunctivitis. Ocular surface disease improved on topical steroids and ciclosporin but 67% of patients needed on-going treatment. Close liaison with an ophthalmologist should be considered in those patients who develop conjunctivitis or have a past history of severe ocular surface disease.

Accuracy of intraocular lens power calculation methods when targeting low myopia in monovision.

PURPOSE: To investigate the accuracy of IOL power calculation methods for refractive targets of myopia compared with emmetropia. SETTING: Lions Eye Institute, Perth, Australia. DESIGN: Retrospective analysis. METHODS: Patients undergoing bilateral, sequential cataract surgery with a plan of modest monovision were analyzed. Target refraction was plano (distance eye) and -1.25 diopters (D) (near eye). Prediction error was determined by comparing the actual postoperative refraction with the predicted postoperative refraction, calculated by the Barrett Universal II (BUII), Hill-RBF version 2.0 (Hill-RBF 2.0), Haigis, Holladay 1, SRK/T, and Hoffer Q formulas. The dataset was divided into distance and near eye subgroups. Mean and median absolute error and percentage of eyes within ±0.25, ±0.50, ±0.75, and ±1.00 D of refractive target were compared. RESULTS: The study included 88 consecutive patients. There was a consistent trend for lower refractive accuracy in the near eyes. BUII and Hill-RBF 2.0 were the most accurate overall and least affected by this phenomenon, with 1 (1.1%) and 4 (4.6%) fewer eyes, respectively, in the near subgroup achieving ±0.50 D of target. Haigis and SRK/T were most affected, with 14 (15.9%) and 11 (12.5%) fewer near eyes achieving ±0.50 D of target (P < .05). Holladay 1 and Hoffer Q occupied the middle ground, with 6 (6.8%) and 9 (10.2%) fewer near eyes achieving ±0.50 D of target. CONCLUSIONS: IOL-power calculation formulas appear to be less accurate when targeting myopia compared with emmetropia. BUII and Hill-RBF 2.0 represented good options when planning pseudophakic monovision as they were least affected by this phenomenon and can be used for both distance and near eyes.

Modified thin manual Descemet stripping endothelial keratoplasty with air-guided, non-pachymetric donor lenticule dissection: outcomes of graft thickness and complication rate.

AIM: To describe a modified technique of donor lenticule dissection for thin manual Descemet stripping endothelial keratoplasty (TM-DSEK). METHODS: Donor material was soaked in balanced salt solution (BSS) for 30min, before being mounted on an artificial anterior chamber (AAC). Rather than BSS, the AAC was filled with filtered air, resulting in a visible reflection at the corneal endothelium-air interface. This reflection served as a landmark for the depth of the dissection, facilitating the creation of a thin lenticule with low risk of perforation. Dissection was commenced at a standardized depth of 500 microns, with no initial pachymetry necessary. Totally 29 donor corneas were dissected by a novice TM-DSEK surgeon. Dissection time, central graft thickness at 2mo and complications were analysed. RESULTS: Results were similar to other endothelial keratoplasty techniques, despite the cases being performed by a novice DSEK surgeon. Mean dissection time was 7min (range 6-10). One graft perforation occurred (3.45%), but the air tamponaded the break and enabled dissection to be restarted and completed from a different location. Mean central graft thickness after at least two months follow-up was 106 microns (range 25-170). CONCLUSION: A problem with manual DSEK is the risk of graft perforation by attempting to dissect too thin a lenticule, or creating a thick graft due to fear of perforating. This modified air-guided technique addresses this problem, and is recommended for surgeons either embarking on the learning curve, or who wish to achieve more consistently thin grafts while reducing perforation rates.

Methods for Intraocular Lens Power Calculation in Cataract Surgery after Radial Keratotomy.

PURPOSE: To compare methods of calculating the required intraocular lens (IOL) power for patients undergoing cataract surgery after radial keratotomy (RK), including the 2016 update of the True K formula. DESIGN: Retrospective case series. PARTICIPANTS: A total of 52 eyes of 34 patients who had sequential RK and cataract surgery performed in the same institution by 1 of 2 surgeons. METHODS: Seven IOL calculation formulae were evaluated: True K [History], True K [Partial History], True K [No History], Double-K Holladay 1 (DK-Holladay-IOLM), Potvin-Hill, Haigis, and Haigis with a -0.50 diopter (D) offset. Biometry was obtained with the IOLMaster 500 (Carl Zeiss Meditec AG, Jena, Germany) and Pentacam (OCULUS Inc, Arlington, WA) devices. Subjective refraction was performed at 4 to 6 weeks postoperatively. The achieved spherical equivalent outcome was compared with the target outcome to calculate the absolute error for each eye with each formula. MAIN OUTCOME MEASURES: Median absolute error (MedAE) and mean absolute error (MAE), and percentage of patients within ±0.50 D, ±0.75 D, and ±1.00 D of refractive target. Mean error (ME) was also calculated to demonstrate whether a formula tended toward more myopic or hyperopic outcomes. RESULTS: Best results were achieved with the True K [History]. The MedAE was higher (0.382 vs. 0.275) with the True K [Partial History], but a similar percentage of patients (75.0%-76.6%) achieved within ±0.50 D of target. Of the methods that do not require refractive history, the True K [No History] and unadjusted Haigis were most accurate (69.2% within ±0.50 D of target), with the True K [No History] returning the lowest MedAE but also more of a tendency toward hyperopia (ME +0.269 vs. -0.006 for Haigis). The DK-Holladay-IOLM and Potvin-Hill methods were the least accurate. CONCLUSIONS: Knowledge of the refractive history significantly improves the accuracy of IOL calculations in patients undergoing cataract surgery after previous RK. The post-RK refraction appears to be the most important parameter, with inclusion of the pre-RK refraction offering a further slight improvement in MedAE. When no refractive history is available, the True K [No History] and Haigis formulae both perform well, with the added advantage of not requiring data from separate biometric devices.

Abnormal pro-gly-pro pathway and airway neutrophilia in pediatric cystic fibrosis.

BACKGROUND: Proline-glycine-proline (PGP) is a bioactive fragment of collagen generated by the action of matrix metalloproteinase-9 (MMP-9) and prolylendopeptidase (PE), and capable of eliciting neutrophil chemotaxis and epithelial remodelling. PGP is normally then degraded by leukotriene A4 hydrolase (LTA4H) to limit inflammation and remodelling. This study hypothesized that early and persistent airway neutrophilia in Cystic Fibrosis (CF) may relate to abnormalities in the PGP pathway and sought to understand underlying mechanisms. METHODS: Broncho-alveolar lavage (BAL) fluid was obtained from 38 CF (9 newborns and 29 older children) and 24 non-CF children. BAL cell differentials and levels of PGP, MMP-9, PE and LTA4H were assessed. RESULTS: Whilst PGP was present in all but one of the older CF children tested, it was absent in non-CF controls and the vast majority of CF newborns. BAL levels of MMP-9 and PE were elevated in older children with CF relative to CF newborns and non-CF controls, correlating with airway neutrophilia and supportive of PGP generation. Furthermore, despite extracellular LTA4H commonly being greatly elevated concomitantly with inflammation to promote PGP degradation, this was not the case in CF children, potentially owing to degradation by neutrophil elastase. CONCLUSIONS: A striking imbalance between PGP-generating and -degrading enzymes enables PGP accumulation in CF children from early life and potentially supports airway neutrophilia.

A Novel Pyrazolopyrimidine Ligand of Human PGK1 and Stress Sensor DJ1 Modulates the Shelterin Complex and Telomere Length Regulation.

Telomere signaling and metabolic dysfunction are hallmarks of cell aging. New agents targeting these processes might provide therapeutic opportunities, including chemoprevention strategies against cancer predisposition. We report identification and characterization of a pyrazolopyrimidine compound series identified from screens focused on cell immortality and whose targets are glycolytic kinase PGK1 and oxidative stress sensor DJ1. We performed structure-activity studies on the series to develop a photoaffinity probe to deconvolute the cellular targets. In vitro binding and structural analyses confirmed these targets, suggesting that PGK1/DJ1 interact, which we confirmed by immunoprecipitation. Glucose homeostasis and oxidative stress are linked to telomere signaling and exemplar compound CRT0063465 blocked hypoglycemic telomere shortening. Intriguingly, PGK1 and DJ1 bind to TRF2 and telomeric DNA. Compound treatment modulates these interactions and also affects Shelterin complex composition, while conferring cellular protection from cytotoxicity due to bleomycin and desferroxamine. These results demonstrate therapeutic potential of the compound series.

Using the first-eye prediction error in cataract surgery to refine the refractive outcome of the second eye.

PURPOSE: To refine the refractive outcome of the second eye after cataract surgery by deriving adjustment coefficients for intraocular lens (IOL) selection based on the prediction error (PE) of the first eye. SETTING: University Hospital Southampton, Southampton, England, and the Lions Eye Institute, Perth, Australia. DESIGN: Retrospective study of two heterogeneous datasets. METHODS: One hundred thirty-nine patients who underwent delayed sequential bilateral cataract surgery in Australia were retrospectively analyzed. The PE was determined by comparing postoperative subjective refraction with the predicted postoperative refraction (PPOR) calculated by the Barrett Universal II, Hoffer Q, Holladay I, and SRK/T formulas. Adjustment coefficients were derived for each formula and applied to the second eye's IOL calculation. Separately, patient-specific optimized IOL constants were derived from the first-eye PE and applied to the second-eye calculation. The same adjustments were applied to a dataset of 605 patients in the United Kingdom to test the validity of the Australian results. RESULTS: The study comprised data on 139 patients in Australia and 605 patients in the U.K. The Australian-derived adjustment coefficients based on PE ranged from 0.30 to 0.56 (Barrett Universal II 0.30; Hoffer Q 0.56; Holladay I 0.53; SRK/T 0.48). Applying these to the U.K. dataset led to the percentage of patients within 0.50 diopters of PPOR with their second eye improving from 70.74%, 65.29%, 69.09%, and 67.77%, with the Barrett Universal II, Hoffer Q, Holladay I, and SRK/T, respectively, to 72.73%, 68.76%, 71.57%, and 72.56%. Using patient-specific optimized IOL constants derived from the first eye had similar efficacy to formula-specific adjustment. CONCLUSION: Second-eye refinement via either formula-specific PPOR adjustment or patient-specific IOL constant adjustment improved the percentage of patients achieving the refractive target with their second eye.

A multi-centre interventional case series of 259 ab-interno Xen gel implants for glaucoma, with and without combined cataract surgery.

AIMS: To assess the efficacy of Xen in reducing intraocular pressure (IOP) in varying glaucoma subtypes. To assess the effect of combined phacoemulsification. To determine the frequency of complications and explore further bleb management needed. METHODS: Retrospective case note review of all patients undergoing Xen implantation across four centres from August 2015 to May 2017. RESULTS: In total, 259 consecutive surgeries of 226 patients were reviewed. IOP reduced from 19.3 (SD ± 6.0) mmHg preoperatively to 14.2 (SD ± 4.4) at month 12 and 13.5 (SD ± 3.3) at month 18 (p < 0.0001). Medication usage reduced from 2.6 (±1.1) preoperatively to 0.8 (±1.0) at month 12 (p < 0.0001) and 1.1 (±1.3) medications at month 18 (p < 0.0001). Simultaneous phacoemulsification did not alter outcomes as Xen IOP was 14.3 (SD ± 4.7) mmHg and Phaco-Xen was 13.8 (SD ± 2.6) mmHg at month 12 (p = 0.5367). Xen appears to be effective in previous failed filtration surgery. Adverse events included: IOP spikes of ≥30 mmHg in 33 (12.7%) cases, secondary filtration surgery required in 24 (9.3%) cases; implant exposure in 6 (2.3%) cases; persistent hypotonous maculopathy in 5 (1.9%) cases; persistent choroidal effusions in 4 (1.5%) cases; a cyclodialysis cleft secondary to implant insertion in 1 (0.5%) case; and 1 (0.5%) case of endophthalmitis post-implant bleb resuturing. In all, 40.9% of cases required postoperative bleb needling or antimetabolite injection. CONCLUSIONS: Xen reduces IOP and medications at 18 months. Adverse events are uncommon. Careful postoperative surveillance and low threshold for bleb management is needed. Xen is safe and effective in mild to moderate glaucoma.

A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors.

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.