scholarly journals Detection and quantification of GPCR mRNA: An assessment and implications of data from high-content methods

2019 ◽  
Author(s):  
Krishna Sriram ◽  
Shu Z. Wiley ◽  
Kevin Moyung ◽  
Matthew W. Gorr ◽  
Cristina Salmerón ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Splice Variants ◽  
Cardiac Fibroblasts ◽  
Cytosolic Calcium ◽  
Membrane Receptors ◽  
Pancreatic Stellate Cells ◽  
Human Pancreatic Cancer ◽  
Rna Seq ◽  
Functional Responses ◽  
Approved Drugs

AbstractG protein-coupled receptors (GPCRs) are the largest family of membrane receptors and targets for approved drugs. Analysis of GPCR expression is thus important for drug discovery and typically involves mRNA-based methods. We compared transcriptomic cDNA [Affymetrix] microarrays, RNA-seq and qPCR-based TaqMan arrays for their ability to detect and quantify expression of endoGPCRs (non-chemosensory GPCRs with endogenous agonists). In human pancreatic cancer-associated fibroblasts, RNA-seq and TaqMan arrays yielded closely correlated values for GPCR number (~100) and expression levels, as validated by independent qPCR. By contrast, the microarrays failed to identify ~30 such GPCRs and generated data poorly correlated with results from those methods. RNA-seq and TaqMan arrays also yielded comparable results for GPCRs in human cardiac fibroblasts, pancreatic stellate cells, cancer cell lines and pulmonary arterial smooth muscle cells. The magnitude of mRNA expression for several Gq/11-coupled GPCRs predicted cytosolic calcium increase and cell migration by cognate agonists. RNA-seq also revealed splice variants for endoGPCRs. Thus, RNA-seq and qPCR-based arrays are better suited than microarrays for assessing GPCR expression and can yield results predictive of functional responses--findings that have implications for GPCR biology and drug discovery.Abstract Graphic

scholarly journals Snake Venoms in Drug Discovery: Valuable Therapeutic Tools for Life Saving

Toxins ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 564 ◽  
Author(s):  
Mohamed Abd El-Aziz ◽  
Garcia Soares ◽  
Stockand
Keyword(s):  
Drug Discovery ◽  
Snake Venom ◽  
Defense Mechanisms ◽  
Membrane Receptors ◽  
Snake Venoms ◽  
Wide Range ◽  
Therapeutic Tools ◽  
Approved Drugs ◽  
Pharmacologically Active ◽  
Animal Venoms

Animal venoms are used as defense mechanisms or to immobilize and digest prey. In fact, venoms are complex mixtures of enzymatic and non-enzymatic components with specific pathophysiological functions. Peptide toxins isolated from animal venoms target mainly ion channels, membrane receptors and components of the hemostatic system with high selectivity and affinity. The present review shows an up-to-date survey on the pharmacology of snake-venom bioactive components and evaluates their therapeutic perspectives against a wide range of pathophysiological conditions. Snake venoms have also been used as medical tools for thousands of years especially in tradition Chinese medicine. Consequently, snake venoms can be considered as mini-drug libraries in which each drug is pharmacologically active. However, less than 0.01% of these toxins have been identified and characterized. For instance, Captopril® (Enalapril), Integrilin® (Eptifibatide) and Aggrastat® (Tirofiban) are drugs based on snake venoms, which have been approved by the FDA. In addition to these approved drugs, many other snake venom components are now involved in preclinical or clinical trials for a variety of therapeutic applications. These examples show that snake venoms can be a valuable source of new principle components in drug discovery.

A human airway smooth muscle cell line that retains physiological responsiveness

1989 ◽  
Vol 256 (2) ◽  
pp. C329-C335 ◽  
Author(s):  
R. A. Panettieri ◽  
R. K. Murray ◽  
L. R. DePalo ◽  
P. A. Yadvish ◽  
M. I. Kotlikoff
Keyword(s):  
Smooth Muscle ◽  
Cell Line ◽  
Airway Smooth Muscle ◽  
Cytosolic Calcium ◽  
Airway Smooth Muscle Cell ◽  
Airway Smooth Muscle Cells ◽  
Functional Coupling ◽  
Functional Responses ◽  
Human Airway Smooth Muscle ◽  
Human Airway

We report the development of a nontransformed line of human airway smooth muscle cells retaining smooth muscle-specific contractile protein expression and physiological responsiveness to agonists implicated in inflammatory airway diseases. Specific responses to histamine, leukotrienes, bradykinin, platelet-activating factor, substance P, and thromboxane analogues are demonstrated as well as functional coupling to beta-adrenergic receptors. The cell line was characterized using indirect immunofluorescence, as well as electrophoretic separation and immunoblot analysis of smooth muscle-specific actin. Functional responses were assessed by measurements of cytosolic calcium and stimulation of adenosine 3',5'-cyclic monophosphate production. The cells retain their responsiveness over many population doublings and should be a useful model to examine specific receptor-effector mechanisms, as well as the effects of neurohumoral agents on the regulation of airway smooth muscle growth and differentiation.

scholarly journals Differential Expression of BARD1 Isoforms in Melanoma

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 320
Author(s):  
Lorissa I. McDougall ◽  
Ryan M. Powell ◽  
Magdalena Ratajska ◽  
Chi F. Lynch-Sutherland ◽  
Sultana Mehbuba Hossain ◽  
...  
Keyword(s):  
Long Range ◽  
Patient Outcomes ◽  
Splice Variants ◽  
Significant Proportion ◽  
Nanopore Sequencing ◽  
Rna Seq ◽  
Splice Isoforms ◽  
Tissue Samples ◽  
Multiple Transcripts ◽  
Mrna Variants

Melanoma comprises <5% of cutaneous malignancies, yet it causes a significant proportion of skin cancer-related deaths worldwide. While new therapies for melanoma have been developed, not all patients respond well. Thus, further research is required to better predict patient outcomes. Using long-range nanopore sequencing, RT-qPCR, and RNA sequencing analyses, we examined the transcription of BARD1 splice isoforms in melanoma cell lines and patient tissue samples. Seventy-six BARD1 mRNA variants were identified in total, with several previously characterised isoforms (γ, φ, δ, ε, and η) contributing to a large proportion of the expressed transcripts. In addition, we identified four novel splice events, namely, Δ(E3_E9), ▼(i8), IVS10+131▼46, and IVS10▼176, occurring in various combinations in multiple transcripts. We found that short-read RNA-Seq analyses were limited in their ability to predict isoforms containing multiple non-contiguous splicing events, as compared to long-range nanopore sequencing. These studies suggest that further investigations into the functional significance of the identified BARD1 splice variants in melanoma are warranted.

scholarly journals A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins

2015 ◽  
Vol 90 (3) ◽  
pp. 1278-1289 ◽  
Author(s):  
Catrin Stutika ◽  
Andreas Gogol-Döring ◽  
Laura Botschen ◽  
Mario Mietzsch ◽  
Stefan Weger ◽  
...  
Keyword(s):  
Life Cycle ◽  
Fusion Protein ◽  
Rna Sequencing ◽  
Splice Variants ◽  
Productive Infection ◽  
Minus Strand ◽  
Rna Seq ◽  
Adeno Associated Virus ◽  
Divergent Transcription ◽  
Productive Replication

ABSTRACTAdeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5ˈ rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad.IMPORTANCENext-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAVin vivo, combinedin cellulaeandin silicostudies will help to forward the understanding of the unique, bipartite AAV life cycle.

Introduction to RNA-Seq and its Applications to Drug Discovery and Development

2014 ◽  
Vol 75 (5) ◽  
pp. 324-330 ◽  
Author(s):  
Zainab Khatoon ◽  
Bryan Figler ◽  
Hui Zhang ◽  
Feng Cheng
Keyword(s):  
Drug Discovery ◽  
Rna Seq ◽  
Drug Discovery And Development

scholarly journals A New Big-Data Paradigm for Target Identification and Drug Discovery

2017 ◽  
Author(s):  
Neel S. Madhukar ◽  
Prashant K. Khade ◽  
Linda Huang ◽  
Kaitlyn Gayvert ◽  
Giuseppe Galletti ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Small Molecules ◽  
Clinical Application ◽  
Small Molecule ◽  
Target Identification ◽  
Clinical Development ◽  
Data Types ◽  
Public Data ◽  
Drug Target Identification ◽  
Approved Drugs

AbstractDrug target identification is one of the most important aspects of pre-clinical development yet it is also among the most complex, labor-intensive, and costly. This represents a major issue, as lack of proper target identification can be detrimental in determining the clinical application of a bioactive small molecule. To improve target identification, we developed BANDIT, a novel paradigm that integrates multiple data types within a Bayesian machine-learning framework to predict the targets and mechanisms for small molecules with unprecedented accuracy and versatility. Using only public data BANDIT achieved an accuracy of approximately 90% over 2000 different small molecules – substantially better than any other published target identification platform. We applied BANDIT to a library of small molecules with no known targets and generated ∼4,000 novel molecule-target predictions. From this set we identified and experimentally validated a set of novel microtubule inhibitors, including three with activity on cancer cells resistant to clinically used anti-microtubule therapies. We next applied BANDIT to ONC201 – an active anti- cancer small molecule in clinical development – whose target has remained elusive since its discovery in 2009. BANDIT identified dopamine receptor 2 as the unexpected target of ONC201, a prediction that we experimentally validated. Not only does this open the door for clinical trials focused on target-based selection of patient populations, but it also represents a novel way to target GPCRs in cancer. Additionally, BANDIT identified previously undocumented connections between approved drugs with disparate indications, shedding light onto previously unexplained clinical observations and suggesting new uses of marketed drugs. Overall, BANDIT represents an efficient and highly accurate platform that can be used as a resource to accelerate drug discovery and direct the clinical application of small molecule therapeutics with improved precision.

Splice variants in the proteome: a promising and challenging field to targeted drug discovery

2015 ◽  
Vol 20 (3) ◽  
pp. 353-360 ◽  
Author(s):  
Raphael Tavares ◽  
Nicole M. Scherer ◽  
Carlos G. Ferreira ◽  
Fabricio F. Costa ◽  
Fabio Passetti
Keyword(s):  
Drug Discovery ◽  
Splice Variants ◽  
Targeted Drug ◽  
Targeted Drug Discovery

Recent Drug-Repurposing-Driven Advances in the Discovery of Novel Antibiotics

2019 ◽  
Vol 26 (28) ◽  
pp. 5363-5388 ◽  
Author(s):  
Ananda Kumar Konreddy ◽  
Grandhe Usha Rani ◽  
Kyeong Lee ◽  
Yongseok Choi
Keyword(s):  
Drug Discovery ◽  
Bacterial Infections ◽  
De Novo ◽  
Genetic Disorder ◽  
Antibacterial Properties ◽  
Drug Repurposing ◽  
Global Public Health ◽  
Approved Drugs ◽  
Fda Approved Drugs ◽  
Novel Antibiotics

: Drug repurposing is a safe and successful pathway to speed up the novel drug discovery and development processes compared with de novo drug discovery approaches. Drug repurposing uses FDA-approved drugs and drugs that failed in clinical trials, which have detailed information on potential toxicity, formulation, and pharmacology. Technical advancements in the informatics, genomics, and biological sciences account for the major success of drug repurposing in identifying secondary indications of existing drugs. Drug repurposing is playing a vital role in filling the gap in the discovery of potential antibiotics. Bacterial infections emerged as an ever-increasing global public health threat by dint of multidrug resistance to existing drugs. This raises the urgent need of development of new antibiotics that can effectively fight multidrug-resistant bacterial infections (MDRBIs). The present review describes the key role of drug repurposing in the development of antibiotics during 2016–2017 and of the details of recently FDA-approved antibiotics, pipeline antibiotics, and antibacterial properties of various FDA-approved drugs of anti-cancer, anti-fungal, anti-hyperlipidemia, antiinflammatory, anti-malarial, anti-parasitic, anti-viral, genetic disorder, immune modulator, etc. Further, in view of combination therapies with the existing antibiotics, their potential for new implications for MDRBIs is discussed. The current review may provide essential data for the development of quick, safe, effective, and novel antibiotics for current needs and suggest acuity in its effective implications for inhibiting MDRBIs by repurposing existing drugs.

Cytosolic Calcium Oscillations in Smooth Muscle Cells

Physiology ◽  
2000 ◽  
Vol 15 (1) ◽  
pp. 50-55 ◽  
Author(s):  
Jean-Pierre Savineau ◽  
Roger Marthan
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Cell Membrane ◽  
Smooth Muscle Cells ◽  
Inositol Trisphosphate ◽  
Muscle Cells ◽  
Cytosolic Calcium ◽  
Calcium Oscillations ◽  
Membrane Receptors ◽  
Cell Membrane Potential

In a variety of smooth muscle cells, agonists activating membrane receptors induce oscillations in the cytoplasmic Ca2+ concentration via an inositol trisphosphate-activated mechanism. Ca2+ oscillations participate in the control of cell membrane potential and the tone of smooth muscle. There is evidence that alterations in Ca2+ oscillations modulate smooth muscle responsiveness.

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