Slipknot or Crystallographic Error: A Computational Analysis of the Plasmodium falciparum DHFR Structural Folds.

The presence of protein structures with atypical folds in the Protein Data Bank (PDB) is rare and may result from naturally occurring knots or crystallographic errors. Proper characterisation of such folds is imperative to understanding the basis of naturally existing knots and correcting crystallographic errors. If left uncorrected, such errors can frustrate downstream experiments that depend on the structures containing them. An atypical fold has been identified in P. falciparum dihydrofolate reductase (PfDHFR) between residues 20-51 (loop 1) and residues 191-205 (loop 2). This enzyme is key to drug discovery efforts in the parasite, necessitating a thorough characterisation of these folds. Using multiple sequence alignments (MSA), a unique insert was identified in loop 1 that exacerbates the appearance of the atypical fold-giving it a slipknot-like topology. However, PfDHFR has not been deposited in the knotted proteins database, and processing its structure failed to identify any knots within its folds. The application of protein homology modelling and molecular dynamics simulations on the DHFR domain of P. falciparum and those of two other organisms (E. coli and M. tuberculosis) that were used as molecular replacement templates in solving the PfDHFR structure revealed plausible unentangled or open conformations of these loops. These results will serve as guides for crystallographic experiments to provide further insights into the atypical folds identified

Intravenous Formulation of HET0016 Decreased Human Glioblastoma Growth and Implicated Survival Benefit in Rat Xenograft Models

Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at 60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered vascular kinetics in early and late treatment groups (p \u3c 0.05). Similar growth inhibition was observed in syngeneic GL261 GBM (p \u3c 0.05). Survival studies using patient derived xenografts of GBM811, showed prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016 and TMZ (p \u3c 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA), in addition to inflammation and angiogenesis markers in the treatment group (p \u3c 0.05). Our results indicate that HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016 significantly prolonged survival in PDX GBM811 model

The Molecular Organization of Human cGMP Specific Phosphodiesterase 6 (PDE6): Structural Implications of Somatic Mutations in Cancer and Retinitis Pigmentosa.

In the cyclic guanosine monophosphate (cGMP) signaling pathway, phosphodiesterase 6 (PDE6) maintains a critical balance of the intracellular concentration of cGMP by catalyzing it to 5' guanosine monophosphate (5'-GMP). To gain insight into the mechanistic impacts of the PDE6 somatic mutations that are implicated in cancer and retinitis pigmentosa, we first defined the structure and organization of the human PDE6 heterodimer using computational comparative modelling. Each subunit of PDE6αβ possesses three domains connected through long α-helices. The heterodimer model indicates that the two chains are likely related by a pseudo two-fold axis. The N-terminal region of each subunit is comprised of two allosteric cGMP-binding domains (Gaf-A & Gaf-B), oriented in the same way and interacting with the catalytic domain present at the C-terminal in a way that would allow the allosteric cGMP-binding domains to influence catalytic activity. Subsequently, we applied an integrated knowledge-driven in silico mutation analysis approach to understand the structural and functional implications of experimentally identified mutations that cause various cancers and retinitis pigmentosa, as well as computational saturation mutagenesis of the dimer interface and cGMP-binding residues of both Gaf-A, and the catalytic domains. We studied the impact of mutations on the stability of PDE6αβ structure, subunit-interfaces and Gaf-cGMP interactions. Further, we discussed the changes in interatomic interactions of mutations that are destabilizing in Gaf-A (R93L, V141 M, F162 L), catalytic domain (D600N, F742 L, F776 L) and at the dimer interface (F426A, F248G, F424 N). This study establishes a possible link of change in PDE6αβ structural stability to the experimentally observed disease phenotypes

Medication errors in the Middle East countries: a systematic review of the literature

Background: Medication errors are a significant global concern and can cause serious medical consequences for patients. Little is known about medication errors in Middle Eastern countries. The objectives of this systematic review were to review studies of the incidence and types of medication errors in Middle Eastern countries and to identify the main contributory factors involved. Methods: A systematic review of the literature related to medication errors in Middle Eastern countries was conducted in October 2011 using the following databases: Embase, Medline, Pubmed, the British Nursing Index and the Cumulative Index to Nursing & Allied Health Literature. The search strategy included all ages and languages. Inclusion criteria were that the studies assessed or discussed the incidence of medication errors and contributory factors to medication errors during the medication treatment process in adults or in children. Results: Forty-five studies from 10 of the 15 Middle Eastern countries met the inclusion criteria. Nine (20%) studies focused on medication errors in paediatric patients. Twenty-one focused on prescribing errors, 11 measured administration errors, 12 were interventional studies and one assessed transcribing errors. Dispensing and documentation errors were inadequately evaluated. Error rates varied from 7.1% to 90.5% for prescribing and from 9.4% to 80% for administration. The most common types of prescribing errors reported were incorrect dose (with an incidence rate from 0.15% to 34.8% of prescriptions), wrong frequency and wrong strength. Computerised physician rder entry and clinical pharmacist input were the main interventions evaluated. Poor knowledge of medicines was identified as a contributory factor for errors by both doctors (prescribers) and nurses (when administering drugs). Most studies did not assess the clinical severity of the medication errors. Conclusion: Studies related to medication errors in the Middle Eastern countries were relatively few in number and of poor quality. Educational programmes on drug therapy for doctors and nurses are urgently needed

Slipknot or Crystallographic Error: A Computational Analysis of the Plasmodium falciparum DHFR Structural Folds

The presence of protein structures with atypical folds in the Protein Data Bank (PDB) is rare and may result from naturally occurring knots or crystallographic errors. Proper characterisation of such folds is imperative to understanding the basis of naturally existing knots and correcting crystallographic errors. If left uncorrected, such errors can frustrate downstream experiments that depend on the structures containing them. An atypical fold has been identified in P. falciparum dihydrofolate reductase (PfDHFR) between residues 20–51 (loop 1) and residues 191–205 (loop 2). This enzyme is key to drug discovery efforts in the parasite, necessitating a thorough characterisation of these folds. Using multiple sequence alignments (MSA), a unique insert was identified in loop 1 that exacerbates the appearance of the atypical fold-giving it a slipknot-like topology. However, PfDHFR has not been deposited in the knotted proteins database, and processing its structure failed to identify any knots within its folds. The application of protein homology modelling and molecular dynamics simulations on the DHFR domain of P. falciparum and those of two other organisms (E. coli and M. tuberculosis) that were used as molecular replacement templates in solving the PfDHFR structure revealed plausible unentangled or open conformations of these loops. These results will serve as guides for crystallographic experiments to provide further insights into the atypical folds identified

Understanding the impacts of missense mutations on structures and functions of human cancer-related genes: A preliminary computational analysis of the COSMIC Cancer Gene Census.

Genomics and genome screening are proving central to the study of cancer. However, a good appreciation of the protein structures coded by cancer genes is also invaluable, especially for the understanding of functions, for assessing ligandability of potential targets, and for designing new drugs. To complement the wealth of information on the genetics of cancer in COSMIC, the most comprehensive database for cancer somatic mutations available, structural information obtained experimentally has been brought together recently in COSMIC-3D. Even where structural information is available for a gene in the Cancer Gene Census, a list of genes in COSMIC with substantial evidence supporting their impacts in cancer, this information is quite often for a single domain in a larger protein or for a single protomer in a multiprotein assembly. Here, we show that over 60% of the genes included in the Cancer Gene Census are predicted to possess multiple domains. Many are also multicomponent and membrane-associated molecular assemblies, with mutations recorded in COSMIC affecting such assemblies. However, only 469 of the gene products have a structure represented in the PDB, and of these only 87 structures have 90-100% coverage over the sequence and 69 have less than 10% coverage. As a first step to bridging gaps in our knowledge in the many cases where individual protein structures and domains are lacking, we discuss our attempts of protein structure modelling using our pipeline and investigating the effects of mutations using two of our in-house methods (SDM2 and mCSM) and identifying potential driver mutations. This allows us to begin to understand the effects of mutations not only on protein stability but also on protein-protein, protein-ligand and protein-nucleic acid interactions. In addition, we consider ways to combine the structural information with the wealth of mutation data available in COSMIC. We discuss the impacts of COSMIC missense mutations on protein structure in order to identify and assess the molecular consequences of cancer-driving mutations.TLB acknowledges support through his Wellcome Trust Investigator Award 200814/Z/16/

COSMIC Cancer Gene Census 3D database: understanding the impacts of mutations on cancer targets.

Mutations in hallmark genes are believed to be the main drivers of cancer progression. These mutations are reported in the Catalogue of Somatic Mutations in Cancer (COSMIC). Structural appreciation of where these mutations appear, in protein-protein interfaces, active sites or deoxyribonucleic acid (DNA) interfaces, and predicting the impacts of these mutations using a variety of computational tools are crucial for successful drug discovery and development. Currently, there are 723 genes presented in the COSMIC Cancer Gene Census. Due to the complexity of the gene products, structures of only 87 genes have been solved experimentally with structural coverage between 90% and 100%. Here, we present a comprehensive, user-friendly, web interface (https://cancer-3d.com/) of 714 modelled cancer-related genes, including homo-oligomers, hetero-oligomers, transmembrane proteins and complexes with DNA, ribonucleic acid, ligands and co-factors. Using SDM and mCSM software, we have predicted the impacts of reported mutations on protein stability, protein-protein interfaces affinity and protein-nucleic acid complexes affinity. Furthermore, we also predicted intrinsically disordered regions using DISOPRED3.Wellcome Trust Investigator Award, PHZJ/489 RG83114 (2016–21)

Predicted structural mimicry of spike receptor-binding motifs from highly pathogenic human coronaviruses.

Viruses often encode proteins that mimic host proteins in order to facilitate infection. Little work has been done to understand the potential mimicry of the SARS-CoV-2, SARS-CoV, and MERS-CoV spike proteins, particularly the receptor-binding motifs, which could be important in determining tropism and druggability of the virus. Peptide and epitope motifs have been detected on coronavirus spike proteins using sequence homology approaches; however, comparing the three-dimensional shape of the protein has been shown as more informative in predicting mimicry than sequence-based comparisons. Here, we use structural bioinformatics software to characterize potential mimicry of the three coronavirus spike protein receptor-binding motifs. We utilize sequence-independent alignment tools to compare structurally known protein models with the receptor-binding motifs and verify potential mimicked interactions with protein docking simulations. Both human and non-human proteins were returned for all three receptor-binding motifs. For example, all three were similar to several proteins containing EGF-like domains: some of which are endogenous to humans, such as thrombomodulin, and others exogenous, such as Plasmodium falciparum MSP-1. Similarity to human proteins may reveal which pathways the spike protein is co-opting, while analogous non-human proteins may indicate shared host interaction partners and overlapping antibody cross-reactivity. These findings can help guide experimental efforts to further understand potential interactions between human and coronavirus proteins

Whole Exome Sequencing reveals NOTCH1 mutations in anaplastic large cell lymphoma and points to Notch both as a key pathway and a potential therapeutic target.

Patients diagnosed with Anaplastic Large Cell Lymphoma (ALCL) are still treated with toxic multi-agent chemotherapy and as many as 25-50% of patients relapse. To understand disease pathology and to uncover novel targets for therapy, Whole-Exome Sequencing (WES) of Anaplastic Lymphoma Kinase (ALK)+ ALCL was performed as well as Gene-Set Enrichment Analysis. This revealed that the T-cell receptor (TCR) and Notch pathways were the most enriched in mutations. In particular, variant T349P of NOTCH1, which confers a growth advantage to cells in which it is expressed, was detected in 12% of ALK+ and ALK- ALCL patient samples. Furthermore, we demonstrate that NPM-ALK promotes NOTCH1 expression through binding of STAT3 upstream of NOTCH1. Moreover, inhibition of NOTCH1 with γ-secretase inhibitors (GSIs) or silencing by shRNA leads to apoptosis; co-treatment in vitro with the ALK inhibitor Crizotinib led to additive/synergistic anti-tumour activity suggesting this may be an appropriate combination therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, Crizotinib-resistant and sensitive ALCL were equally sensitive to GSIs. In conclusion, we show a variant in the extracellular domain of NOTCH1 that provides a growth advantage to cells and confirm the suitability of the Notch pathway as a second-line druggable target in ALK+ ALCL.This work was supported by grants from the Ministry of Science, Kingdom of Saudi Arabia to SDT, AI and SM (grant number 74497) and Bloodwise to SDT (grant number 12065). HL is supported by a Department of Pathology, University of Cambridge Pathology Centenary Fund PhD studentship. SDT, LK, OM, SK, NP, SPD, CGP, WW, CDW and CL are in receipt of funding from a European Union Horizon 2020 Marie Sklodowska-Curie Innovative Training Network (ITN-ETN) Grant, Award No.: 675712. CL is supported by Czech Science Foundation Research Grant No. 19-23424Y and by research infrastructures EATRIS-CZ (LM2015064) and the NCMG (LM2015091) funded by MEYS CR. WK is supported by the KinderkrebsInitiative Buchholz, Holm-Seppensen