The clinical management of malignant tumours is challenging, often leading to severe adverse effects and death. Drug resistance (DR) antagonises the effectiveness of treatments, and increasing drug dosage can worsen the therapeutic index (TI). Current efforts to overcome DR predominantly involve the use of drug combinations, including applying multiple anti-cancerous drugs, employing drug sensitisers, which are chemical agents that enhance pharmacokinetics (PK), including the targeting of cellular pathways and regulating pertinent membrane transporters.
View Article and Find Full Text PDFCyclin dependent kinase 7 (CDK7) is an important therapeutic kinase best known for its dual role in cell cycle regulation and gene transcription. Here, we describe the application of protein engineering to generate constructs leading to high resolution crystal structures of human CDK7 in both active and inactive conformations. The active state of the kinase was crystallized by incorporation of an additional surface residue mutation (W132R) onto the double phosphomimetic mutant background (S164D and T170E) that yielded the inactive kinase structure.
View Article and Find Full Text PDFSilver has long been recognized for its potent antimicrobial properties, but achieving a slow and longer-term delivery of silver ions presents significant challenges. Previous efforts to control silver ion dosages have struggled to sustain release for extended periods in biomimetic environments, especially in the presence of complex proteins. This challenge is underscored by the absence of technology for sustaining antimicrobial activity, especially in the context of orthopedic implants where long-term efficacy, extending beyond 7 days, is essential.
View Article and Find Full Text PDFThe ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2.
View Article and Find Full Text PDFIntroduction: It is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites.
View Article and Find Full Text PDFBackground: The idiopathic inflammatory myopathies (IIMs) are heterogeneous autoimmune conditions of skeletal muscle inflammation and weakness. MicroRNAs (miRNAs) are short, non-coding RNA which regulate gene expression of target mRNAs. The aim of this study was to profile miRNA and mRNA in IIM and identify miRNA-mRNA relationships which may be relevant to disease.
View Article and Find Full Text PDFWe recently introduced the Gini coefficient (GC) for assessing the expression variation of a particular gene in a dataset, as a means of selecting improved reference genes over the cohort ('housekeeping genes') typically used for normalisation in expression profiling studies. Those genes (transcripts) that we determined to be useable as reference genes differed greatly from previous suggestions based on hypothesis-driven approaches. A limitation of this initial study is that a single (albeit large) dataset was employed for both tissues and cell lines.
View Article and Find Full Text PDFBackground: It is widely believed that most xenobiotics cross biomembranes by diffusing through the phospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternative view, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptake of an individual molecular type. We recognise here that the availability of gene knockout collections allows one to assess the contributions of all potential transporters, and flow cytometry based on fluorescence provides a convenient high-throughput assay for xenobiotic uptake in individual cells.
View Article and Find Full Text PDFDysregulation of the kynurenine pathway (KP) leads to imbalances in neuroactive metabolites associated with the pathogenesis of several neurodegenerative disorders, including Huntington's disease (HD). Inhibition of the enzyme kynurenine 3-monooxygenase (KMO) in the KP normalises these metabolic imbalances and ameliorates neurodegeneration and related phenotypes in several neurodegenerative disease models. KMO is thus a promising candidate drug target for these disorders, but known inhibitors are not brain permeable.
View Article and Find Full Text PDFThe expression levels of SLC or ABC membrane transporter transcripts typically differ 100- to 10,000-fold between different tissues. The Gini coefficient characterizes such inequalities and here is used to describe the distribution of the expression of each transporter among different human tissues and cell lines. Many transporters exhibit extremely high Gini coefficients even for common substrates, indicating considerable specialization consistent with divergent evolution.
View Article and Find Full Text PDFAn authenticated U87MG clonal glioblastoma cell line was investigated to identify a sub-population of neurospheroidal (NSP) cells within the main epithelial population (U87MG). The NSP cells sorted using Fluorescence Assisted Cell Sorting (FACS) showed varied morphology, 30% lower growth rates, 40% higher IC values for temozolomide drug and could differentiate into the glial cell type (NDx). Metabolite profiling using HR-LCMS identified glucose, glutamine and serine in both populations and tryptophan only in U87MG as growth limiting substrates.
View Article and Find Full Text PDFThe transport of drug molecules is mainly determined by the distribution of influx and efflux transporters for which they are substrates. To enable tissue targeting, we sought to develop the idea that we might affect the transporter-mediated disposition of small-molecule drugs via the addition of a second small molecule that of itself had no inhibitory pharmacological effect but that influenced the expression of transporters for the primary drug. We refer to this as a "binary weapon" strategy.
View Article and Find Full Text PDFThe theory of computer science is based around universal Turing machines (UTMs): abstract machines able to execute all possible algorithms. Modern digital computers are physical embodiments of classical UTMs. For the most important class of problem in computer science, non-deterministic polynomial complete problems, non-deterministic UTMs (NUTMs) are theoretically exponentially faster than both classical UTMs and quantum mechanical UTMs (QUTMs).
View Article and Find Full Text PDFLp-PLA has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC > 1 mM).
View Article and Find Full Text PDFGene synthesis is a fundamental technology underpinning much research in the life sciences. In particular, synthetic biology and biotechnology utilize gene synthesis to assemble any desired DNA sequence, which can then be incorporated into novel parts and pathways. Here, we describe SpeedyGenes, a gene synthesis method that can assemble DNA sequences with greater fidelity (fewer errors) than existing methods, but that can also be used to encode extensive, statistically designed sequence variation at any position in the sequence to create diverse (but accurate) variant libraries.
View Article and Find Full Text PDFBackground: The idiopathic inflammatory myopathies (IIM) are autoimmune diseases characterised by acquired proximal muscle weakness, inflammatory cell infiltrates in muscle and myositis-specific/associated autoantibodies. It is unclear which pathways are involved in IIM, and the functional relationship between autoantibody targets has not been systematically explored. Protein-protein interaction and pathway analyses were conducted to identify pathways relevant to disease, using autoantibody targets and gene products of IIM-associated single nucleotide polymorphism (SNP) loci.
View Article and Find Full Text PDFSingle cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays.
View Article and Find Full Text PDFAge is well-known to be a significant factor in both disease pathology and response to treatment, yet the molecular changes that occur with age in humans remain ill-defined. Here, using transcriptome profiling of healthy human male skin, we demonstrate that there is a period of significantly elevated, transcriptome-wide expression changes occurring predominantly in middle age. Both pre and post this period, the transcriptome appears to undergo much smaller, linear changes with increasing age.
View Article and Find Full Text PDFElevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273).
View Article and Find Full Text PDFPurpose Of Review: The idiopathic inflammatory myopathies (IIMs) are a group of rare autoimmune disorders characterized by skeletal muscle weakness and inflammation. MicroRNAs (miRNAs) regulate a wide range of developmental and physiological cellular processes. New approaches to investigating the mechanisms involved in IIM, such as investigating the role of miRNAs, are vital for the development of novel therapeutics and/or better diagnostic tools.
View Article and Find Full Text PDFThe exponential development of highly advanced scientific and medical research technologies throughout the past 30 years has arrived to the point where the high number of characterized molecular agents related to pathogenesis cannot be readily integrated or processed by conventional analytical approaches. Indeed, the realization that several moieties are signatures of disease has partly led to the increment of complex diseases being characterized. Scientists and clinicians can now investigate and analyse any individual dysregulations occurring within the genomic, transcriptomic, miRnomic, proteomic, and metabolomic levels thanks to currently available advanced technologies.
View Article and Find Full Text PDFInhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis and pro-survival signaling pathways whose deregulation is often associated with tumor genesis and tumor growth. IAPs have been proposed as targets for anticancer therapy, and a number of peptidomimetic IAP antagonists have entered clinical trials. Using our fragment-based screening approach, we identified nonpeptidic fragments binding with millimolar affinities to both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP).
View Article and Find Full Text PDFThe amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions.
View Article and Find Full Text PDFThe protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists.
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