Common genetic variation drives molecular heterogeneity in human iPSCs.

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells

Single-cell RNA-sequencing of differentiating iPS cells reveals dynamic genetic effects on gene expression

Abstract: Recent developments in stem cell biology have enabled the study of cell fate decisions in early human development that are impossible to study in vivo. However, understanding how development varies across individuals and, in particular, the influence of common genetic variants during this process has not been characterised. Here, we exploit human iPS cell lines from 125 donors, a pooled experimental design, and single-cell RNA-sequencing to study population variation of endoderm differentiation. We identify molecular markers that are predictive of differentiation efficiency of individual lines, and utilise heterogeneity in the genetic background across individuals to map hundreds of expression quantitative trait loci that influence expression dynamically during differentiation and across cellular contexts

The story of alpha-conotoxins, Vc1.1 and RgIA, on their journey to becoming therapeutics

Abstract The broad aim of this thesis is to structurally and functionally explore two α-conotoxins, from venomous sea snails, Vc1.1 and RgIA, in the hope of improving their journey to becoming analgesic therapeutics (introduction to conotoxins in Chapter 1). Vc1.1 is a two-disulfide peptide that is of interest as a potential therapeutic for the treatment of neuropathic pain. Despite investigations, limited structure-activity relationships have been conducted on this α-conotoxin. Consequently there is restricted insight into the interaction of this peptide with one of its analgesic targets, the α9α10 nicotinic acetylcholine receptor (nAChR). Late in this PhD project, the GABAB receptor was implicated as the possible target for conotoxins in neuropathic pain relief. However, there is still debate in the literature with regard to the true target of Vc1.1 and the α9α10 nAChR is still believed to be the target by some groups. This thesis predominantly focuses on the α9α10 nicotinic acetylcholine receptor. Chapter 4 of this thesis presents an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp or Lys (materials and methods described in Chapter 3) and examined using NMR spectroscopy (theory of NMR presented in Chapter 2), to determine the effects of the mutations on the structure of Vc1.1. The structural fold was found to be preserved in all peptides except where Pro was substituted. Chapter 5 explores the effect of these mutations on the blocking of acetylcholine (ACh)-evoked membrane currents at the α9α10 nAChR. Electrophysiological studies showed that the key residues for Vc1.1’s activity are Asp5-Arg7 and Asp11-Ile15, as changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. Hence, Chapter 6 describes a second generation of mutants that was synthesized, namely N9G, N9I, N9L, S4R and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4 and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of Vc1.1 or its analogs as drugs. However, despite peptides exhibiting high degrees of potency and selectivity, such as Vc1.1 and RgIA, they are potentially hindered in their development as drugs due to their stability and bioavailability limitations, leading to invasive delivery techniques. Chapter 7 presents a range of cyclic RgIA analogs, tested at their targets the α9α10 nAChR and the GABAB receptor, that retain their activity and increase their stability in human serum relative to non-cyclic RgIA. NMR spectroscopy was used to determine the structure of the non-cyclic peptide and the cyclic peptide to confirm similarities in the global fold of the peptide. Structural perturbations and reduced activities were observed for cyclic RgIA analogs cyclized via linkers composed of three and four residues. Analogs with five, six and seven residues showed no structural perturbations, but differences in their activities at the different receptors. Because cRgIA-6 showed high potency for the GABAB receptor and lower potency for the α9α10 nAChR, this study has identified a GABAB selective peptide. Additionally, because the cRgIA-7 showed high potency for the α9α10 nAChR and low potency for the GABAB receptor, a α9α10 nAChR selective analog has also been identified. With improvements in these peptides against enzymatic attack, they show great potential on their path to becoming orally available analgesics as they may be able to withstand enzymatic conditions in the stomach

Using label-free screening technology to improve efficiency in drug discovery

Introduction: Screening assays have traditionally utilized reporter labels to quantify biological responses relevant to the disease state of interest. However, there are limitations associated with the use of labels that may be overcome with temporal measurements possible with label-free. Areas covered: This review comprises general and system-specific information from literature searches using PubMed, published books and the authors' personal experience. This review highlights the label-free approaches in the context of various applications. The authors also note technical issues relevant to the development of label-free assays and their application to HTS. Expert opinion: The limitations associated with the use of transfected cell lines and the use of label-based assays are gradually being realized. As such, greater emphasis is being placed on label-free biophysical techniques using native cell lines. The introduction of 96-and 384-well plate label-free systems is helping to broker a wider acceptance of these approaches in high-throughput screening. However, potential users of the technologies remain skeptical, primarily because the physical basis of the signals generated, and their contextual relevance to cell biology and signal transduction, has not been fully elucidated. Until this is done, these new technology platforms are more likely to complement, rather than replace, traditional screening platforms

Conotoxins: natural product drug leads

Venomous marine cone snails harbour a variety of small disulfide-rich peptides called conotoxins, which target a broad range of ion channels, membrane receptors, and transporters. More than 700 species of Conus are thought to exist, each expressing a wide array of different peptides. Within this large repertoire of toxins, individual conotoxins are able to discriminate between different subtypes and isoforms of ion channels, making them valuable pharmacological probes or leads for drug design. This review gives a brief background to the discovery of conotoxins and describes their sequences, biological activities, and applications in drug design

Glycopeptide antibiotics: back to the future

Glycopeptide antibiotics have been a key weapon in the fight against bacterial infections for over half a century, with the progenitors, vancomycin (1) and teicoplanin (2), still used extensively. The increased occurrence of resistance and the effectiveness of these ‘last resort’ treatments for Gram-positive infections has led to the discovery and clinical development of second generation, semisynthetic lipoglycopeptide derivatives such as telavancin (3), dalbavancin (4) and oritavancin (5), which all possess broader spectra of activity and improved pharmacokinetic properties. Two of these new antibiotics, telavancin (3) and dalbavancin (4), were approved in the past 5 years and the third, oritavancin (5), is awaiting regulatory approval. In this review, the discovery, development and associated resistance of vancomycin (1) and teicoplanin (2), and semi-synthetic glycopeptides, telavancin (3), dalbavancin (4) and oritavancin (5), are detailed. The clinical implications of glycopeptide resistance, especially vancomycin (1), as well as the future prospects for current glycopeptide drugs and the development of new glycopeptides are discussed

C5a, but not C5a-des Arg, induces upregulation of heteromer formation between complement C5a receptors C5aR and C5L2

Receptors for C5a have an important role in innate immunity and inflammation where their expression and activation is tightly regulated. There are two known receptors for C5a: the C5a receptor (C5aR) and the C5a receptor like-2 (C5L2) receptor. Here we hypothesized that activation of C5aR might lead to heteromer formation with C5L2, as a downregulatory mechanism for C5aR signaling. To investigate this experimentally, bioluminescent resonance energy transfer (BRET) was implemented and supported by wide-field microscopy to analyze receptor localization in transfected HEK293 cells and human monocyte-derived macrophages (HMDM). BRET experiments indicated the presence of constitutive C5aR-C5L2 heteromers, where C5a, but not C5a-des Arg, was able to induce further heteromer formation, which was inhibited by a C5aR-specific antagonist. The data obtained suggest that C5aR-C5L2 can form heteromers in a process enhanced by C5a, but not by C5a-des Arg. There was also a significant difference in the levels of the anti-inflammatory cytokine IL-10 detected in HMDM following exposure to C5a compared with that seen for C5a-des Arg but no differences in the pro-inflammatory cytokines TNF alpha and IL-6. These subtle differences in C5a and C5a-des Arg induced receptor function may be of benefit in understanding the regulation of C5a in acute inflammation

Effects of cyclization on stability, structure, and activity of α-conotoxin RgIA at the α9α10 nicotinic acetylcholine receptor and GABAB receptor

α-Conotoxin RgIA is of interest as a lead in the development of drugs for neuropathic pain. It modulates the α9α10 nicotinic acetylcholine receptor (nAChR) and the GABAB receptor, both of which are implicated in antinociception. However, because of its peptidic nature, RgIA is potentially susceptible to generic problems encountered by peptide-based drugs of poor oral bioavailability, short biological half-life, and low stability. Here, we improved the biopharmaceutical properties of RgIA by backbone cyclization using 3-7 residue peptidic linkers. Cyclization with a six-residue linker does not perturb the overall structure of RgIA, improves selectivity for the GABAB receptor over the α9α10 nAChR, and improves stability in human serum. The results provide insights to further improve the therapeutic properties of RgIA and other conotoxins being considered as drug leads and confirm that cyclization is a readily applicable strategy to improve the stability of peptides with proximate N- and C-termini