1,721,105 research outputs found
Next Generation Phenotypic Screening
Full text linkPhenotypic drug discovery (PDD) strategies are defined by screening and selection of hit or lead compounds based on quantifiable phenotypic endpoints without prior knowledge of the drug target. We outline the challenges associated with traditional phenotypic screening strategies and propose solutions and new opportunities to be gained by adopting modern PDD technologies. We highlight both historical and recent examples of approved drugs and new drug candidates discovered by modern phenotypic screening. Finally we offer a prospective view of a new era of PDD underpinned by a wealth of technology advances in the areas of in vitro model development, high content imaging and image-informatics, mechanism-of-action profiling and target deconvolutio
Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin-Targeting Thiazole Analogue of Bisebromoamide
No full textSystematic alanine-scanning of the linear peptide bisebromoamide (BBA), isolated from a marine cyanobacterium, is enabled by targeting the solid phase peptide synthesis of thiazole analogues. The synthetic Tz-BBA analogues have comparable cytotoxicity (nM) to bisebromoamide and cellular morphology assays indicate that they target the actin cytoskeleton. Pathway inhibition in human colon tumour (HCT-116) cells has been explored using reverse phase protein array (RPPA) analysis, which shows a dose-dependent response of IRS-1 expression. Alanine-scanning reveals a structural dependence to the cytotoxicity, actin-targeting and pathway inhibition, and allows a new readily-synthesised lead to be proposed.Johnston, Heather; Boys, Sarah; Makda, Ashraff; Carragher, Neil; Hulme, Alison. (2016). Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin-Targeting Thiazole Analogue of Bisebromoamide, [dataset]. University of Edinburgh. School of Chemistry.. http://dx.doi.org/10.7488/ds/1417
High content profiling in oesophageal adenocarcinoma to inform drug discovery and patient stratification
No full textOesophageal adenocarcinoma (OAC) is a highly heterogeneous disease, dominated
by copy number alterations and large-scale genomic rearrangements. Such
characteristics have hampered both therapeutic target discovery and the clinical
success of targeted therapies, contributing to its status as an area of unmet
therapeutic need. Phenotypic drug discovery describes the screening and selection
of hit or lead compounds based on quantifiable phenotypic endpoints from cellbased assays or model organisms without prior knowledge of the drug target.
Thus, this may prove to be a beneficial strategy for complex, heterogeneous
diseases where target biology is poorly understood and where modern, target
directed drug discovery strategies have made little impact on patient care, as
exemplified by OAC.
In the chapters that follow, I describe the development and validation of a high
content profiling assay and machine learning pipelines to identify repurposing
opportunities in OAC. Following on from this I focus on one group of compounds
which show high potency and selectivity for targeting OAC cell lines relative to
tissue-matched controls. I further explore the mechanism of action of these
compounds and potential patient stratification hypotheses to support clinical
translation.
In the first chapter I present an in-depth study of compound induced cell
morphology in the context of a heterogeneous panel of eight oesophageal cell lines.
To achieve this I profiled a small number of reference compounds and built a
library of phenotypic fingerprints for each mechanistic class. I then interrogated
the phenotypic fingerprints using a variety of supervised and unsupervised
machine learning techniques. This work demonstrates that compound induced
morphologies are reproducible across cell lines, despite underlying morphological
heterogeneity, allowing machine learning classifiers to be applied to previously
unseen cell lines. The second chapter describes the application of the high content
assay to a comprehensive small-molecule screen encompassing 19,555 small
molecules, eight cell lines, 512x384 well plates, 3.9 million images and 36 TB of data
for the identification of new drug discovery and drug repurposing opportunities
in OAC. Following on from this, in the third chapter, I focus on one particular group
of compounds identified as highly selective for OAC and explore their mechanism
of action. In the final chapter, through the integration of transcriptomic pathway
analyses we gain insight into drug selectively and the basis for future biomarkerbased clinical trials in OAC
Investigation of the impact of HNPCC gene deficiency on outcome in epithelial ovarian cancer
Full text linkHereditary non-polyposis colon cancer syndrome (HNPCC) is associated with an
increased risk of developing several types of cancer and is the most common cause
of hereditary ovarian cancer after BRCA1 and BRCA2 mutations. HNPCC results
from a germline mutation in one of the DNA mismatch repair (MMR) genes: MLH1,
MSH2, PMS1, PMS2, MSH6, MSH3 and MLH3. While there has been extensive
investigation of MMR deficiency in colorectal cancer, MMR in ovarian cancer is
relatively under-investigated. The goal of this project was to study MMR deficiency
in ovarian cancer at both the clinical and molecular level. The first aim was to
examine the frequency of MMR loss in a large patient cohort and investigate the
clinical consequences of MMR deficiency. The second aim was to describe the
molecular characteristics of MMR deficiency in ovarian cancer cell lines and
establish an in vitro cell line model of MMR deficiency in ovarian cancer. The third
aim was to identify synthetic lethal strategies for the treatment of ovarian cancer to
maximise cytotoxicity in a MMR-deficient background.
In order to characterise the clinical consequences of MMR deficiency, a large patient
cohort was studied with regard to MMR status. Three tissue microarrays consisting
of 581 ovarian tumours were constructed, and expression of the four most frequently
lost MMR proteins: MLH1, MSH2, PMS2 and MSH6 were detected by
immunohistochemistry. Afterwards, MMR status and histology subtypes were
analysed in combination with the associated clinical data. The overall incidence of
MMR deficiency (loss of any MMR protein) was 15.7%, with PMS2 being the most
frequently lost protein (9.7%). In addition, MMR deficiency tended to appear in a
grouped fashion: MLH1 with PMS2; MSH2 with MSH6. Patients with non-serous
subtypes of ovarian cancer, clear cell or mucinous especially, had higher incidence of
MMR deficiency compared to patients with serous ovarian cancer. Overall MMR
deficient patients were more likely to be diagnosed at early stages compared with
MMR proficient patients, and this is probably due to the association between MMR
deficiency and non-serous histology. However, platinum-based treatment for patients
with MMR deficiency gives no advantage over those without MMR deficiency.
Therefore better treatments for this subgroup of patients may be needed.
The features of MMR deficiency in ovarian cancer were also characterized at the
molecular level. After quantifying mRNA and protein expression of MMR genes in
19 ovarian cell lines, three cell lines (SKOV3, TOV21G and IGROV1) were found to
have a defect in MLH1 expression at both the mRNA and protein level. Interestingly,
the three cell lines also carried a defect in PMS2 expression at the protein level but
not at the mRNA level, which is consistent with our clinical data demonstrating that
MLH1 protein and PMS2 protein are paired in loss. In addition, across the 19 cell
lines, MLH1 and PMS2 showed positive correlation at both the mRNA level (R=0.53,
p=0.02) and protein level (R=0.72, p=0.0006). In order to study co-expression of
MLH1 and PMS2, a plasmid encoding the cDNA for MLH1 was transfected into the
three MLH1 deficient cell lines; and conversely siRNA targeting MLH1 was
transfected into the MMR proficient cell line A2780 and expression of MLH1
protein and PMS2 protein was quantified. The results showed that re-introduction of
MLH1 into MLH1 deficient cells resulted in increased expression of PMS2 protein,
while knocking down MLH1 in MMR proficient cells leads to decreased PMS2
protein expression. This indicates that MLH1 may play a crucial role in regulating
PMS2 protein expression. As the three MLH1 and PMS2 protein deficient cell lines
all express PMS2 mRNA, the regulation of PMS2 expression by MLH1 is likely to
be at the translational or post-translational level. However, the expression of PMS2
protein was not increased in the absence of MLH1, even when the proteasomal and
lysosomal protein degradation pathways were blocked (as seen with SKOV3 cells),
suggesting decreased PMS2 protein expression is not due to rapid degradation in the
absence of MLH1. Therefore MLH1 may play a role in regulating the synthesis of
PMS2 protein at the translational level, rather than preventing the degradation of
PMS2. Thus, to investigate the mechanism by which PMS2 protein levels are
regulated by MLH1, future work should focus on translational regulation of PMS2. In order to identify synthetic lethal strategies to target MMR deficiency in ovarian
cancer, an isogenic cell line model of MMR deficiency was established by stable
transfection of a plasmid for MLH1 and its corresponding empty vector into SKOV3
cells. The MLH1+ cell line SAC-1 and MLH1- cell line SN-5 were selected for drug
screening based on their phenotype and growth rate. The AlamarBlue assay, with z’
above 0.5, was chosen for drug screening and a kinase inhibitor library containing
362 drugs of known target was screened. Two drugs with similar structures that
targeted PLK1 showed greater growth inhibition of SN-5 compared with SAC-1.
When the two cell lines were treated with another PLK1 inhibitor, BI2536, with
different structure, a 2-fold difference in growth inhibition between SAC-1 and SN-5
was also observed, suggesting PLK1 is a potential synthetic lethal target for MLH1
deficiency in ovarian cancer.
Together these data demonstrate that clinically, MMR deficiency is associated with
non-serous subtypes of ovarian cancer and specific MMR proteins are paired in loss.
While current standard therapy offers no selective benefit to ovarian cancer patients
with MMR deficiency, inhibiting PLK1 activity may confer selective benefit
Development of an in vitro assay for high-throughput screening investigating the role of mesenchymal stem cells on castration resistant prostate cancer cell growth
Full text linkAndrogen deprivation therapy (ADT) can increase survival from prostate cancer by up to 2-3
years, but tumours invariably relapse into an ADT-unresponsive, incurable form, known as
castrate resistant prostate cancer (CRPC). CRPC is more aggressive and more likely to
metastasise to bone, worsening morbidity and mortality. Mesenchymal stem cells have
been implicated in alteration of androgen signalling within prostate cancer cells and
stimulation of metastasis and resistance to anti-tumour therapy, and thus may play an
important role in the development of castration resistance. A high throughput screen to
identify compounds that inhibit the effect of MSCs on castration resistance would thus be
valuable in development of novel chemotherapeutics against CRPC.
Clones of the human CWR22PC and murine Myc-CaP Bo prostate cancer cell lines were
characterised by their reduced growth in response to androgen deprivation, modelled using
charcoal stripped serum and the antiandrogen enzalutamide. Investigations were
performed to optimise the miniaturisation of this assay. The effect of conditioned media
from human or murine mesenchymal stem cells on this cell growth was then examined in
the presence of androgen and androgen deprivation, in a high-throughput format.
It was found that MSC-conditioned media had only a small positive effect stimulating
growth in CWR22PC cells, greatest in the enzalutamide-treated condition. In the murine
Myc-CaP Bo cell line clone 5GSH-6943#5, MSC-conditioned media significantly stimulated
castration-resistant growth in the androgen deprivation condition but not in the presence
of androgen. However, assay validation indicated that the assay developed for either cell
line was not suitable for high-throughput drug screening in its current form. Further
optimisation is thus required for use of the assays developed as a platform for high-throughput
screening to investigate the effects of various therapeutic compounds on MSC
stimulation of castration-resistant prostate cancer cell growth
The development of phenotypic approaches to advance drug discovery and development for multiple sclerosis
Full text linkMultiple Sclerosis (MS) is described as a neurodegenerative autoimmune condition causing increased disability with age. The current disease modifying therapies (DMTs), which are approved for the treatment of MS by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), are all immunomodulatory; these small molecules and monoclonal antibodies target the immune system to ameliorate the symptoms of MS. None of these therapies have the ability to halt or reverse the symptoms. In MS, immune cells are known to be autoreactive to oligodendrocytes, thus destroying the protective myelin sheath, demyelinating axons and causing neurodegeneration. Oligodendrocyte progenitor cells (OPCs) are known to differentiate into mature myelinating oligodendrocytes to naturally remyelinate demyelinated axons and this biological process can be targeted by small molecules with the hypothesis that increasing the number of mature myelinating oligodendrocytes will increase remyelination to stop the progression of, and potentially reverse, the symptoms of MS. As such, this research used high-throughput drug screening to test multiple small molecule libraries on in vitro human induced pluripotent stem cell (iPSC)-derived OPCs to identify novel compounds that promote OPC differentiation, quantified by immunocytochemistry staining for the mature oligodendrocyte protein myelin basic protein (MBP). Subsequent analysis of potential mechanisms of action (MoAs) of the hit compounds was performed using in silico techniques such as network and pathway analysis. In addition, one novel compound, SGC-CBP30, which was identified during screening, was investigated in an in vivo experimental autoimmune encephalomyelitis (EAE) model of MS in mice to show the compound’s functional remyelination capacity and reduction of MS symptoms. In total ten compounds were identified as hits that increased the differentiation of OPCs, of which five were novel structures not previously implicated in OPC differentiation and one, latanoprost, was a novel drug class that has not previously been reported in the literature within the context of MS. From the MoA profiling analysis of these compounds, eight proteins were identified as potential novel targets for promoting OPC differentiation. EP300, the target for SGC-CBP30, was also identified to be a crucial protein in several pathways associated with the other hit compound MoAs. SGC-CBP30 was found to have action in vivo via an alternative mechanism to another compound which also targets EP300 (anacardic acid); this may be due to anacardic acid’s reduced selectivity to EP300. This research has therefore been able to identify novel small molecules that promote OPC differentiation, as well as highlighting potential MoA targets for these compounds, including inhibiting EP300 with an epigenetic modulator. This research thus provides potential drug repurposing candidates for clinical trials and therapeutic targets for novel MS drug discovery and adds to the knowledge of how the compounds interact with potential protein targets and biological pathways to increase OPC differentiation and the remyelination axons
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Investigating host factors involved in human coronavirus infection in the search for antivirals
No full textIn the past two decades, three highly pathogenic human coronaviruses (hCoVs) have emerged with the most recent, SARS-CoV-2, resulting in an ongoing global pandemic. Although vaccination and virus evolution have reduced the disease burden of COVID-19, effective antivirals are still sought for vulnerable or unvaccinated populations and to prepare for future coronavirus outbreaks. As an obligate intracellular parasite, host factors are recruited and repressed by SARS-CoV-2 throughout the entire viral replication cycle. Elucidating these interactions will aid in our molecular understanding of this virus and in the identification of candidates to develop host-directed antiviral therapies. This thesis aims to identify and characterise host factors of SARS-CoV-2 in vitro in relevant cell models and demonstrate the utility of targeting host factors as an antiviral strategy to treat COVID-19 and other hCoV infections.
We take a hypothesis-driven approach to study a group of transmembrane proteins, integrins, as potential host factors of SARS-CoV-2 given the RGD-integrin binding motif presence in S. We show that integrin subunit ITGA5 is upregulated post-SARS-CoV-2 infection in Caki-1 cells and that downstream of integrins, the tyrosine kinases Src and FAK are phosphorylated with biphasic activity post-SARS-CoV-2 infection. Inhibiting Src with small molecule inhibitors reduces viral production of SARS-CoV-2 as well as replication of hCoV-229E in a dose-dependent manner with low IC50 values. Integrins and their downstream signalling pathways present attractive targets to further explore in the development of antiviral treatments against SARS-CoV-2 and other hCoVs.
We undertake, analyse, and validate a druggable-genome RNAi screen to identify multiple host factor candidate genes involved at all stages of the viral replication cycle. Direct lysis RT-qPCR quantification of viral RNA from cell supernatant following siRNA-mediated gene knockdown allowed a comprehensive analysis of the full replication cycle, including viral release. Utilising a human druggable genome siRNA assay containing ~7000 gene targets, we performed knockdowns in human kidney Caki-1 cells infected with a clinical isolate of SARS-CoV-2 EDB-2 (B.1) at readouts 24 hpi and 48 hpi. Pathway and network analysis of our hit lists showed high confidence with expected proviral and antiviral gene clusters. Moreover, meta-analysis with other
published studies of host factors in SARS-CoV-2, including CRISPR-KO screens and GWAS, demonstrated consistency and relevance of our hit lists. A cluster of proviral hits involved in vesicle-mediated transport, including multiple Rab GTPases, were validated in EDB-2 and variants Delta and Omicron. We identify Rab11a as a key host factor in SARS-CoV-2 viral release, which can be targeted using inhibitors of Rab11a cargo delivery. We demonstrate that Rab11a inhibitors have dual-inhibitory action of entry and release and is a promising antiviral for further study in targeting SARS-CoV-2.
Finally, host factor interactions and antiviral candidates are assessed using a physiologically relevant cell model of human airway epithelial cells grown at air-liquid interface (ALI). ALI cultures are differentiated and a model for infection, quantification and confocal microscopy is established for SARS-CoV-2 and hCoV-229E. We begin to optimise a protocol for siRNA knockdown in ALI cultures and assess potential host factor interactions of SARS-CoV-2 through inhibitory action of candidate host-directed antivirals.
Together, these results highlight the availability of host factors as candidate targets for new and effective antiviral therapies, and pave the way for further work to test interactions and antiviral candidates in vivo
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