Protein mimetic 2D FAST rescues alpha synuclein aggregation mediated early and post disease Parkinson's phenotypes.

Abberent protein-protein interactions potentiate many diseases and one example is the toxic, self-assembly of α-Synuclein in the dopaminergic neurons of patients with Parkinson's disease; therefore, a potential therapeutic strategy is the small molecule modulation of α-Synuclein aggregation. In this work, we develop an Oligopyridylamide based 2-dimensional Fragment-Assisted Structure-based Technique to identify antagonists of α-Synuclein aggregation. The technique utilizes a fragment-based screening of an extensive array of non-proteinogenic side chains in Oligopyridylamides, leading to the identification of NS132 as an antagonist of the multiple facets of α-Synuclein aggregation. We further identify a more cell permeable analog (NS163) without sacrificing activity. Oligopyridylamides rescue α-Synuclein aggregation mediated Parkinson's disease phenotypes in dopaminergic neurons in early and post disease Caenorhabditis elegans models. We forsee tremendous potential in our technique to identify lead therapeutics for Parkinson's disease and other diseases as it is expandable to other oligoamide scaffolds and a larger array of side chains.

Process Robustness in Lipid Nanoparticle Production: A Comparison of Microfluidic and Turbulent Jet Mixing.

The recent clinical and commercial success of lipid nanoparticles (LNPs) for nucleic acid delivery has incentivized the development of new technologies to manufacture LNPs. As new technologies emerge, researchers must determine which technologies to assess and how to perform comparative evaluations. In this article, we use a quality-by-design approach to systematically investigate how the mixer technology used to form LNPs influences LNPstructure. Specifically, a coaxial turbulent jet mixer and a staggered herringbone microfluidic mixer were systematically compared via matched formulation and process conditions. A full-factorial design-of-experiments study with three factors and three levels was executed for each mixer to compare process robustness in the production of antisense oligonucleotide (ASO) LNPs. ASO-LNPs generated with the coaxial turbulent jet mixer were consistently smaller, had a narrower particle size distribution, and had a higher ASO encapsulation as compared to the microfluidic mixer, but had a greater variation in internal structure with less ordered cores. A subset of the study was replicated for mRNA-LNPs with comparable trends in particle size and encapsulation, but more frequent bleb features for LNPs produced by the coaxial turbulent jet mixer. The study design used here provides a road map for how researchers may compare different mixer technologies (or process changes more broadly) and how such studies can inform process robustness and manufacturing control strategies.

"Common-Precursor" Protein Mimetic Approach to Rescue Aß Aggregation-Mediated Alzheimer's Phenotypes.

Abberent protein-protein interactions (aPPIs) are associated with an array of pathological conditions, which make them important therapeutic targets. The aPPIs are mediated via specific chemical interactions that spread over a large and hydrophobic surface. Therefore, ligands that can complement the surface topography and chemical fingerprints could manipulate aPPIs. Oligopyridylamides (OPs) are synthetic protein mimetics that have been shown to manipulate aPPIs. However, the previous OP library used to disrupt these aPPIs was moderate in number (∼30 OPs) with very limited chemical diversity. The onus is on the laborious and time-consuming synthetic pathways with multiple chromatography steps. We have developed a novel chromatography-free technique to synthesize a highly diverse chemical library of OPs using a "common-precursor" approach. We significantly expanded the chemical diversity of OPs using a chromatography-free high-yielding method. To validate our novel approach, we have synthesized an OP with identical chemical diversity to a pre-existing OP-based potent inhibitor of Aß aggregation, a process central to Alzheimer's disease (AD). The newly synthesized OP ligand (RD242) was very potent in inhibiting Aß aggregation and rescuing AD phenotypes in an in vivo model. Moreover, RD242 was very effective in rescuing AD phenotypes in a post-disease onset AD model. We envision that our "common-precursor" synthetic approach will have tremendous potential as it is expandable for other oligoamide scaffolds to enhance affinity for disease-relevant targets.

Stable H-bond networks are crucial for selective CDK4 inhibition revealed from comprehensive in silico investigation.

CDK1 and CDK4 are highly similar isoforms but with apparently diverse cellular functions, which makes it fundamental to discover selective CDK4 inhibitors that could accurately control the process of cell cycle of the specific organization so as to restore normal physiological state. In current research, interaction modes of CDK1 and CDK4 inhibitors were investigated through combined in silico strategies to elucidate the selectivity mechanism against CDK4 over CDK1, revealing that H-bond networks formed with key amino acids such as LYS33 and LEU83 of CDK1 and VAL93 of CDK4 are crucial for CDK4 selective inhibition, which would provide a theoretical basis for the design of selective CDK4 inhibitors.

Foldamers reveal and validate therapeutic targets associated with toxic α-synuclein self-assembly.

Parkinson's disease (PD) is a progressive neurodegenerative disorder for which there is no successful prevention or intervention. The pathological hallmark for PD involves the self-assembly of functional Alpha-Synuclein (αS) into non-functional amyloid structures. One of the potential therapeutic interventions against PD is the effective inhibition of αS aggregation. However, the bottleneck towards achieving this goal is the identification of αS domains/sequences that are essential for aggregation. Using a protein mimetic approach, we have identified αS sequences-based targets that are essential for aggregation and will have significant therapeutic implications. An extensive array of in vitro, ex vivo, and in vivo assays is utilized to validate αS sequences and their structural characteristics that are essential for aggregation and propagation of PD phenotypes. The study aids in developing significant mechanistic and therapeutic insights into various facets of αS aggregation, which will pave the way for effective treatments for PD.

Revealing the interaction modes of 5-HT2A receptor antagonists and the Structure-Based virtual screening from FDA and TCMNP database.

5-hydroxytryptamine 2A (5-HT2A) receptor is emerging as an important target for numerous psychoactive drugs due to its imperative roles in psychological diseases. In fact, multiple 5-HT2A receptor antagonists were developed to treat numerous psychiatric disorders, however, their clinical outcome was far from ideal probably due to a blurry information of the exact interaction modes between the receptor and its antagonists. Impressively, with a recent release of its crystal structure, we carefully analyzed the receptor-ligand interactions with Protein Contacts Atlas, structure-based pharmacophore models, and molecular dynamics (MD) simulations to sum up the chemical features for antagonists interacting with 5-HT2A receptor. Moreover, the molecular docking-based virtual screening was applied to discover potential 5-HT2A receptor antagonists from FDA and TCMNP databases. Intriguingly, after a systematic assessment of the docking scores, binding modes and free energies, as well as their MD simulations performances, three compounds in TCMNP database were highlighted to be potential 5-HT2A receptor antagonists. Fascinatedly, these three hits also exhibited highly binding affinities with dopamine D2 receptor (D2R) due to the similarity of the ligand binding pockets of the receptors, indicating them to be promising dual target molecules that are of great benefit for anti-psychotic-drug research and development. In addition, ADME/Tox predictions were conducted for a primary evaluation of their developing potential. Together, this study not only revealed the exact interaction modes between 5-HT2A receptor and its antagonists, which shed a light on a better access for developing its novel antagonists, but also provided promising dual D2 and 5-HT2A receptor antagonists.Communicated by Ramaswamy H. Sarma.

Discovery of small molecule inhibitors through pharmacophore modeling, molecular docking, molecular dynamics simulation and experimental validation against myeloid cell leukemia-1 (Mcl-1).

Myeloid cell leukemia-1 (Mcl-1) protein is a family of Bcl-2 (B cell lymphoma 2) rich proteases of the most common increase threshold for genetic aberrations observed in human cancer, including lung, breast, pancreatic, cervical, and ovarian cancers as well as leukemia and lymphoma. Mcl-1 is recognized as an attractive drug target in number of diseases, including cancer. In the present study we surveyed and collected queries compounds from PDB database of Mcl-1 protein and generated pharmacophore-based models adapted to screen the drug-like compounds from FDA approved database. The 206 best lead molecules from pharmacophore-screening were further evaluated by molecular docking, molecular dynamics simulation, MM-GBSA calculation, as well as experimental validation. Two hits, ZINC00601272 and ZINC00002166, showed the best docking scores, which showed a tendency to inhibit cell viability of HL60 and K562 leukemia cells with Mcl-1 expressions. Conclusively, the present study provides structural information of Mcl-1 inhibitors for next generations of cancer therapeutics through computational and experimental validation approach.Communicated by Ramaswamy H. Sarma.

Refined pharmacophore features for virtual screening of human thromboxane A2 receptor antagonists.

For a long time, the structural basis of TXA2 receptor is limited due to the lack of crystal structure information, till the release of the crystal structure of TXA2 receptor, which deepens our understanding about ligand recognition and selectivity mechanisms of this physiologically important receptor. In this research, we report the successful implementation in the discovery of an optimal pharmacophore model of human TXA2 receptor antagonists through virtual screening. Structure-based pharmacophore models were generated based on two crystal structures of human TXA2 receptor (PDB entry 6IIU and 6IIV). Docking simulation revealed interaction modes of the virtual screening hits against TXA2 receptor, which was validated through molecular dynamics simulation and binding free energy calculation. ADMET properties were also analyzed to evaluate the toxicity and physio-chemical characteristics of the hits. The research would provide valuable insight into the binding mechanisms of TXA2 receptor antagonists and thus be helpful for designing novel antagonists.

The diagnostic performance of current tumour markers in surveillance for recurrent testicular cancer: A diagnostic test accuracy systematic review.

In this diagnostic test accuracy systematic review we summarise the evidence on the diagnostic accuracy of blood α-fetoprotein (AFP), human chorionic gonadotropin (HCG) and lactate dehydrogenase (LDH) in surveillance for testicular cancer recurrence in adults. We searched four electronic databases for studies that reported the diagnostic accuracy of HCG, AFP, and/or LDH in sufficient detail for sensitivity and specificity to be calculated by extracting a 2 × 2 table comparing biomarker positivity with testicular cancer recurrence. Screening, data extraction and QUADAS-2 quality assessment were completed by two independent reviewers. From 2406 studies, nine met our inclusion criteria. Eight reported data at the per-patient level. Sample sizes were small (range 5 to 449 patients) and clinical heterogeneity precluded meta-analysis. In most studies the specificity for recurrence with AFP and HCG was high (90-100%) but sensitivity was often relatively low, suggesting that many recurrences would not be detected by tumour markers alone. The diagnostic performance of LDH appears poorer. Studies were methodologically weak, with probable selection, incorporation and partial verification bias, and many studies were excluded for not reporting on recurrence-free patients. Limitations including small sample sizes, high heterogeneity, and inconsistent and incomplete reporting mean these results must be interpreted with caution. Despite inclusion of biomarkers in international surveillance guidance, there remains a lack of high quality evidence about their accuracy, optimal thresholds, and the most effective surveillance strategy in relation to contemporary investigative modalities. Higher quality research using data from modern-day follow-up cohorts is necessary to identify opportunities to reduce unnecessary testing.

Deep learning for detecting tumour-infiltrating lymphocytes in testicular germ cell tumours.

AIMS: To evaluate if a deep learning algorithm can be trained to identify tumour-infiltrating lymphocytes (TILs) in tissue samples of testicular germ cell tumours and to assess whether the TIL counts correlate with relapse status of the patient. METHODS: TILs were manually annotated in 259 tumour regions from 28 whole-slide images (WSIs) of H&E-stained tissue samples. A deep learning algorithm was trained on half of the regions and tested on the other half. The algorithm was further applied to larger areas of tumour WSIs from 89 patients and correlated with clinicopathological data. RESULTS: A correlation coefficient of 0.89 was achieved when comparing the algorithm with the manual TIL count in the test set of images in which TILs were present (n=47). In the WSI regions from the 89 patient samples, the median TIL density was 1009/mm2. In seminomas, none of the relapsed patients belonged to the highest TIL density tertile (>2011/mm2). TIL quantifications performed visually by three pathologists on the same tumours were not significantly associated with outcome. The average interobserver agreement between the pathologists when assigning a patient into TIL tertiles was 0.32 (Kappa test) compared with 0.35 between the algorithm and the experts, respectively. A higher TIL density was associated with a lower clinical tumour stage, seminoma histology and lack of lymphovascular invasion. CONCLUSIONS: Deep learning-based image analysis can be used for detecting TILs in testicular germ cell cancer more objectively and it has potential for use as a prognostic marker for disease relapse.

Development of a best-practice clinical guideline for the use of bleomycin in the treatment of germ cell tumours in the UK.

Bleomycin, a cytotoxic chemotherapy agent, forms a key component of curative regimens for lymphoma and germ cell tumours. It can be associated with severe toxicity, long-term complications and even death in extreme cases. There is a lack of evidence or consensus on how to prevent and monitor bleomycin toxicity. We surveyed 63 germ cell cancer physicians from 32 cancer centres across the UK to understand their approach to using bleomycin. Subsequent guideline development was based upon current practice, best available published evidence and expert consensus. We observed heterogeneity in practice in the following areas: monitoring; route of administration; contraindications to use; baseline and follow-up investigations performed, and advice given to patients. A best-practice clinical guideline for the use of bleomycin in the treatment of germ cell tumours has been developed and includes recommendations regarding baseline investigations, the use of pulmonary function tests, route of administration, monitoring and patient advice. It is likely that existing heterogeneity in clinical practice of bleomycin prescribing has significant economic, safety and patient experience implications. The development of an evidence-based consensus guideline was supported by 93% of survey participants and aims to address these issues and homogenise practice across the UK.

Changing Practice Evaluation-Stage 1 Seminoma: Outcomes With Adjuvant Treatment Versus Surveillance: Risk Factors for Recurrence and Optimizing Follow-up Protocols-Experience From a Supraregional Center.

BACKGROUND: Stage 1 seminoma is frequently cured by radical orchiectomy; however, the management strategies after this diagnosis vary in terms of the use of adjuvant treatment and the nature of the follow-up protocols. We analyzed stage 1 seminomas treated in the Thames Valley Cancer Network for outcomes to determine whether any factors are predictive of recurrence. We also studied relapses to determine the optimal follow-up schedule and protocol. MATERIALS AND METHODS: Data were obtained from centers within the Thames Valley Cancer Network for a 12-year period from 2004 to 2016. We identified 501 patients with stage 1 seminoma. RESULTS: Relapses occurred in 6.2% of the patients receiving adjuvant treatment and 6.1% of those who did not. The only statistically significant predictive factor identified for relapse was rete testis invasion, and the risk was greater when only stromal rete invasion was included, rather than pagetoid as well. A trend was seen toward an increased risk with increased tumor size, but the difference was not statistically significant. Recurrences developed within the first 2 years after surgery in nearly 75% of cases and were identified through surveillance computed tomography scans in 54.8% of the patients. All relapses were treated curatively. CONCLUSION: Active surveillance leads to excellent outcomes for stage 1 seminoma; however, adjuvant treatment should be reserved for those with high-risk disease. Follow-up schedules should include computed tomography imaging during the first 3 years, long-term measurement of tumor markers, and mechanisms for patients to be seen promptly should symptoms of tumor recurrence occur.

Incidentally detected testicular lesions <10 mm in diameter: can orchidectomy be avoided?

OBJECTIVE: To investigate the pathology of excised testicular lesions <10 mm in size. PATIENTS AND METHODS: The pathological reports of 2 681 patients with testicular lesions from Barts Health NHS Trust and Oxford University Hospitals NHS Foundation Trust were reviewed as part of a service evaluation audit from January 2003 to May 2016. Cases in which the lesion had a maximum diameter of <10 mm were selected. Clinical features were also accessed, where available, to examine patient demographics, prediagnostic levels of serum markers, ultrasonographic findings and clinical details. RESULTS: A total of 81 patients with a lesion size <10 mm on histology were identified and, of these, 16 (20%) had a lesion diameter <5 mm. Of the 81 patients, 56 (69%) had benign lesions. Of 16 patients with a benign lesion <5 mm in diameter, 15 underwent orchidectomy and just one underwent partial orchidectomy. Preoperative tumour markers were available in 47/81 patients. None of the 16 malignant tumours in these 47 patients were associated with raised tumour markers, while seven of 31 remaining patients with benign lesions had raised α-fetoprotein and lactate dehydrogenase levels. In total there were 25/81 malignant cases (31%), which were all germ cell tumours (GCTs): 15 seminomas (60%) and 10 non-seminomatous GCTs (40%). Only one GCT had a diameter of <5 mm, and this was a regressed tumour within an 18-mm area of granulomatous inflammation. Only one GCT relapsed: a clinical stage I, embryonal carcinoma of 6 mm in maximum diameter. The 56 'benign' cases included 34 sex cord stromal tumours, including 23 Leydig cell tumours (41%), eight Sertoli cell tumours (14%) and three mixed sex cord stromal tumours (5%). None showed any malignant features. The remaining 22/56 lesions (40%) were lesions with no further follow-up. Benign lesions seemed to be associated with a small diameter, and we found <5 mm to be the best threshold for predicting benign vs malignant lesions (P = 0.002). CONCLUSION: The majority of testicular lesions <10 mm, identified by radiology, were benign, although approxmiately one-third were malignant. In the present study, 100% of lesions <5 mm in diameter were benign. Tumour markers appear to be unhelpful in the distinction of these small tumours. We suggest that regular ultrasound surveillance be more widely used for testicular lesions of this size. Testicular tumours now have a very high cure rate and changes in size of lesions may be monitored prospectively with minimal risk of increased morbidity. Patients who undergo an orchidectomy for lesions <5 mm are 'victims of modern imaging technology'. If surgery is undertaken in lesions 5-10 mm, patients should be counselled that two-thirds of cases are benign.