scholarly journals Tumor immunological phenotype signature-based high-throughput screening for the discovery of combination immunotherapy compounds

2021 ◽  
Vol 7 (4) ◽  
pp. eabd7851
Author(s):  
Haiyan Wang ◽  
Shasha Li ◽  
Qianyu Wang ◽  
Zhengshuo Jin ◽  
Wei Shao ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
High Throughput Sequencing ◽  
Therapeutic Potential ◽  
Objective Response ◽  
Aurora Kinase ◽  
Gene Signature ◽  
Immune Checkpoint Blockade ◽  
Combination Immunotherapy ◽  
Immunological Phenotype

Combination immunotherapy is promising to overcome the limited objective response rates of immune checkpoint blockade (ICB) therapy. Here, a tumor immunological phenotype (TIP) gene signature and high-throughput sequencing–based high-throughput screening (HTS2) were combined to identify combination immunotherapy compounds. We firstly defined a TIP gene signature distinguishing “cold” tumors from “hot” tumors. After screening thousands of compounds, we identified that aurora kinase inhibitors (AKIs) could reprogram the expression pattern of TIP genes in triple-negative breast cancer (TNBC) cells. AKIs treatments up-regulate expression of chemokine genes CXCL10 and CXCL11 through inhibiting aurora kinase A (AURKA)–signal transducer and activator of transcription 3 (STAT3) signaling pathway, which promotes effective T cells infiltrating into tumor microenvironment and improves anti-programmed cell death 1 (PD-1) efficacy in preclinical models. Our study established a novel strategy to discover combination immunotherapy compounds and suggested the therapeutic potential of combining AKIs with ICB for the treatment of TNBC.

The Medicinal Chemistry of Therapeutic Peptides: Recent Developments in Synthesis and Design Optimizations

2019 ◽  
Vol 26 (13) ◽  
pp. 2330-2355 ◽  
Author(s):  
Anutthaman Parthasarathy ◽  
Sasikala K. Anandamma ◽  
Karunakaran A. Kalesh
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
The Past ◽  
Recombinant Production ◽  
Tremendous Progress ◽  
Recent Developments ◽  
Therapeutic Peptides ◽  
Development Processes ◽  
Delivery Strategies

Peptide therapeutics has made tremendous progress in the past decade. Many of the inherent weaknesses of peptides which hampered their development as therapeutics are now more or less effectively tackled with recent scientific and technological advancements in integrated drug discovery settings. These include recent developments in synthetic organic chemistry, high-throughput recombinant production strategies, highresolution analytical methods, high-throughput screening options, ingenious drug delivery strategies and novel formulation preparations. Here, we will briefly describe the key methodologies and strategies used in the therapeutic peptide development processes with selected examples of the most recent developments in the field. The aim of this review is to highlight the viable options a medicinal chemist may consider in order to improve a specific pharmacological property of interest in a peptide lead entity and thereby rationally assess the therapeutic potential this class of molecules possesses while they are traditionally (and incorrectly) considered ‘undruggable’.

Validation of a High-Throughput Calcium Mobilization Assay for the Human Trace Amine-Associated Receptor 1

2020 ◽  
Vol 26 (1) ◽  
pp. 140-150
Author(s):  
Ann M. Decker ◽  
Kelly M. Mathews ◽  
Bruce E. Blough ◽  
Brian P. Gilmour
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
Calcium Mobilization ◽  
Hit Rate ◽  
Wide Range ◽  
Trace Amine ◽  
The Central Nervous System ◽  
Calcium Mobilization Assay

The human trace amine-associated receptor 1 (hTAAR1) is a G protein-coupled receptor (GPCR) that is widely expressed in monoaminergic nuclei in the central nervous system and has therapeutic potential for multiple diseases, including drug addiction and schizophrenia. Thus, identification of novel hTAAR1 ligands is critical to advancing our knowledge of hTAAR1 function and to the development of therapeutics for a wide range of diseases. Herein we describe the development of a robust, 3-addition high-throughput screening (HTS) calcium mobilization assay using stable CHO-Gαq16-hTAAR1 cells, which functionally couple hTAAR1 to the promiscuous Gαq16 protein and thus allow signal transduction to occur through mobilization of internal calcium. Our previously established 96-well hTAAR1 assay was first miniaturized to the 384-well format and optimized to provide an assay with a Z′ factor of 0.84, which is indicative of a robust HTS assay. Using the 3-addition protocol, 22,000 compounds were screened and yielded a ~1% agonist hit rate and a ~0.2% antagonist hit rate. Of the antagonist hits, two confirmed hits are the most potent hTAAR1 antagonists identified to date (IC50 = 206 and 281 nM). While scientists have been studying hTAAR1 for years, the lack of suitable hTAAR1 antagonists has been a major roadblock for studying the basic pharmacology of hTAAR1. Thus, these new ligands will serve as valuable tools to study hTAAR1-mediated signaling mechanisms, therapeutic potential, and in vivo functions.

scholarly journals High-throughput sequencing screen reveals novel, transforming RAS mutations in myeloid leukemia patients

Blood ◽  
2009 ◽  
Vol 113 (8) ◽  
pp. 1749-1755 ◽  
Author(s):  
Jeffrey W. Tyner ◽  
Heidi Erickson ◽  
Michael W. N. Deininger ◽  
Stephanie G. Willis ◽  
Christopher A. Eide ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Myeloid Leukemia ◽  
High Throughput Sequencing ◽  
Point Mutations ◽  
Causative Role ◽  
Coding Region ◽  
Ras Mutations ◽  
Oncogenic Ras ◽  
Frequent Mutations

Abstract Transforming mutations in NRAS and KRAS are thought to play a causative role in the development of numerous cancers, including myeloid malignancies. Although mutations at amino acids 12, 13, or 61 account for the majority of oncogenic Ras variants, we hypothesized that less frequent mutations at alternate residues may account for disease in some patients with cancer of unexplained genetic etiology. To search for additional, novel RAS mutations, we sequenced all coding exons in NRAS, KRAS, and HRAS in 329 acute myeloid leukemia (AML) patients, 32 chronic myelomonocytic leukemia (CMML) patients, and 96 healthy individuals. We detected 4 “noncanonical” point mutations in 7 patients: N-RasG60E, K-RasV14I, K-RasT74P, and K-RasA146T. All 4 Ras mutants exhibited oncogenic properties in comparison with wild-type Ras in biochemical and functional assays. The presence of transforming RAS mutations outside of positions 12, 13, and 61 reveals that alternate mechanisms of transformation by RAS may be overlooked in screens designed to detect only the most common RAS mutations. Our results suggest that RAS mutations may play a greater role in leukemogenesis than currently believed and indicate that high-throughput screening for mutant RAS alleles in cancer should include analysis of the entire RAS coding region.

scholarly journals A Cell-Based High-Throughput Screening Assay for Posttranscriptional Utrophin Upregulation

2012 ◽  
Vol 18 (4) ◽  
pp. 400-406 ◽  
Author(s):  
Catherine Moorwood ◽  
Neha Soni ◽  
Gopal Patel ◽  
Steve D. Wilton ◽  
Tejvir S. Khurana
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Posttranscriptional Regulation ◽  
Therapeutic Potential ◽  
Dystrophin Gene ◽  
Screening Assay ◽  
Expression Screening ◽  
High Throughput Screening Assay ◽  
Promising Therapeutic Approach ◽  
Muscle Wasting Disease

Duchenne muscular dystrophy (DMD) is a devastating muscle-wasting disease caused by mutations in the dystrophin gene. Utrophin is a homologue of dystrophin that can compensate for its absence when overexpressed in DMD animal models. Utrophin upregulation is therefore a promising therapeutic approach for DMD. Utrophin is regulated at both transcriptional and posttranscriptional levels. Transcriptional regulation has been studied extensively, and assays have been described for the identification of utrophin promoter-targeting molecules. However, despite the profound impact that posttranscriptional regulation has on utrophin expression, screening assays have not yet been described that could be used to discover pharmaceuticals targeting this key phase of regulation. We describe the development and validation of a muscle cell line–based assay in which a stably expressed luciferase coding sequence is flanked by the utrophin 5′- and 3′-untranslated regions (UTRs). The assay was validated using the posttranscriptional regulation of utrophin by miR-206. The assay has a Z′ of 0.7, indicating robust performance in high-throughput format. This assay can be used to study utrophin regulatory mechanisms or to screen chemical libraries for compounds that upregulate utrophin posttranscriptionally via its UTRs. Compounds identified via this assay, used alone or in a synergistic combination with utrophin promoter-targeting molecules, would be predicted to have therapeutic potential for DMD.

scholarly journals Canvass: A Crowd-Sourced, Natural Product Screening Library for Exploring Biological Space

2018 ◽  
Author(s):  
Sara E. Kearney ◽  
Gergely Zahoránszky-Kőhalmi ◽  
Kyle R. Brimacombe ◽  
Mark J. Henderson ◽  
Caitlin Lynch ◽  
...  
Keyword(s):  
Natural Products ◽  
High Throughput ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
Structural Diversity ◽  
Biological Evaluation ◽  
Structural Class ◽  
Differential Behavior ◽  
Biological Potential ◽  
Approved Drugs

Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays. Characterization of the library in terms of physicochemical properties, structural diversity, and similarity to compounds in publicly available libraries indicates that the Canvass library contains many structural elements in common with approved drugs. The assay data generated were analyzed using a variety of quality control metrics, and the resultant assay profiles were explored using statistical methods, such as clustering and compound promiscuity analyses. Individual compounds were then sorted by structural class and activity profiles. Differential behavior based on these classifications, as well as noteworthy activities, are outlined herein. One such highlight is the activity of (–)-2(<i>S</i>)-cathafoline, which was found to stabilize calcium levels in the endoplasmic reticulum. The workflow described here illustrates a pilot effort to broadly survey the biological potential of natural products by utilizing the power of automation and high-throughput screening.

scholarly journals High-throughput screening reveals novel mutations in spinal muscular atrophy patients

2019 ◽  
Author(s):  
Jianbo Shu ◽  
Jingrui Wang ◽  
Yulian Fang ◽  
Zanmei Xu ◽  
Xiaowei Wang ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Throughput Sequencing ◽  
Muscular Atrophy ◽  
Point Mutations ◽  
Control Group ◽  
Compound Heterozygous ◽  
Single Nucleotide Variants ◽  
Illumina Hiseq

Abstract Background Some spinal muscular atrophy (SMA) cases are caused by either compound heterozygosity with a point mutation in one allele and a deletion in the other or compound heterozygous point mutations in SMN1 or other genes. Methods To explore more genes and mutations in the onset of SMA, 83 whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was firstly performed with a SALSA MLPA Kit P021 for preliminary diagnosis. Afterwards, the complete gene sequence of SMN1 gene was detected with the high-throughput sequencing platform of Illumina HiSeq-2500 to find more mutations in the 28 core families. Furthermore, 20 SMA patients were selected from the 28 prodands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. Results MLPA results showed that 22 probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. 6 SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C>T], c.[271C>T], c.[-39A>G] and g.[70240639G>C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[-41_-40insCTCT], FUT5 SNV c.[1001A>G], and MCCC2 SNV c.[-117A>G] were the 3 most frequent mutations in SMA group (95%, 85% and 75%, respectively). Conclusions We identified some mutations in both SMN1 and other genes, and c.[271C>T], c.[-41_-40insCTCT], c.[1001A>G] and c.[-117A>G] might be associated with the onset of SMA.

scholarly journals High-Throughput Screening Strategy Identifies Allosteric, Covalent Human D-Amino Acid Oxidase Inhibitor

2015 ◽  
Vol 20 (10) ◽  
pp. 1218-1231 ◽  
Author(s):  
Ryan T. Terry-Lorenzo ◽  
Keiki Masuda ◽  
Kohtaroh Sugao ◽  
Q. Kevin Fang ◽  
Michael A. Orsini ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Throughput ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
Association Studies ◽  
Therapeutic Drug ◽  
Human Populations ◽  
Acid Oxidase ◽  
Genome Wide Association Studies ◽  
Amino Acid Oxidase

Genome-wide association studies have linked polymorphisms in the gene G72 to schizophrenia risk in several human populations. Although controversial, biochemical experiments have suggested that the mechanistic link of G72 to schizophrenia is due to the G72 protein product, pLG72, exerting a regulatory effect on human D-amino acid oxidase (hDAAO) activity. In an effort to identify hDAAO inhibitors of novel mechanism of action, we designed a pLG72-directed hDAAO activity assay suitable for high-throughput screening (HTS). During assay development, we confirmed that pLG72 was an inhibitor of hDAAO. Thus, our assay employed an IC20 pLG72 concentration that was high enough to allow dynamic pLG72-hDAAO complexes to form but with sufficient remaining hDAAO activity to measure during an HTS. After conducting an approximately 150,000-compound HTS, we further characterized a class of compound hits that were less potent hDAAO inhibitors when pLG72 was present. Focusing primarily on compound 2 [2-(2,5-dimethylphenyl)-6-fluorobenzo[d]isothiazol-3(2H)-on], we demonstrated that these compounds inhibited hDAAO via an allosteric, covalent mechanism. Although there is significant interest in the therapeutic potential of compound 2 and its analogues, their sensitivity to reducing agents and their capacity to bind cysteines covalently would need to be addressed during therapeutic drug development.

scholarly journals In Vitro High Throughput Screening, What Next? Lessons from the Screening for Aurora Kinase Inhibitors

Biology ◽  
2014 ◽  
Vol 3 (1) ◽  
pp. 167-175 ◽  
Author(s):  
Thi-My-Nhung Hoang ◽  
Hong-Lien Vu ◽  
Ly-Thuy-Tram Le ◽  
Chi-Hung Nguyen ◽  
Annie Molla
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Aurora Kinase ◽  
Aurora Kinase Inhibitors

scholarly journals Functional genomics to explore cancer cell vulnerabilities

2010 ◽  
Vol 28 (1) ◽  
pp. E5 ◽  
Author(s):  
Kristopher T. Kahle ◽  
David Kozono ◽  
Kimberly Ng ◽  
Grace Hsieh ◽  
Pascal O. Zinn ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
High Throughput Sequencing ◽  
Chromosomal Abnormalities ◽  
Cost Effective ◽  
Gene Copy ◽  
Tumor Initiation ◽  
Cancer Proliferation ◽  
Synthetically Lethal

Our understanding of glioblastoma multiforme (GBM), the most common form of primary brain cancer, has been significantly advanced by recent efforts to characterize the cancer genome using unbiased high-throughput sequencing analyses. While these studies have documented hundreds of mutations, gene copy alterations, and chromosomal abnormalities, only a subset of these alterations are likely to impact tumor initiation or maintenance. Furthermore, genes that are not altered at the genomic level may play essential roles in tumor initiation and maintenance. Identification of these genes is critical for therapeutic development and investigative methodologies that afford insight into biological function. This requirement has largely been fulfilled with the emergence of RNA interference (RNAi) and high-throughput screening technology. In this article, the authors discuss the application of genome-wide, high-throughput RNAi-based genetic screening as a powerful tool for the rapid and cost-effective identification of genes essential for cancer proliferation and survival. They describe how these technologies have been used to identify genes that are themselves selectively lethal to cancer cells, or synthetically lethal with other oncogenic mutations. The article is intended to provide a platform for how RNAi libraries might contribute to uncovering glioma cell vulnerabilities and provide information that is highly complementary to the structural characterization of the glioblastoma genome. The authors emphasize that unbiased, systems-level structural and functional genetic approaches are complementary efforts that should facilitate the identification of genes involved in the pathogenesis of GBM and permit the identification of novel drug targets.

scholarly journals High-throughput screening reveals novel mutations in spinal muscular atrophy patients

2020 ◽  
Vol 46 (1) ◽  
Author(s):  
Ruiping Zhang ◽  
Chunyu Gu ◽  
Linjie Pu ◽  
Yingtao Meng ◽  
Jianbo Shu ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Throughput Sequencing ◽  
Muscular Atrophy ◽  
Hereditary Disease ◽  
Control Group ◽  
Single Nucleotide Variants ◽  
Frequent Mutations ◽  
Multiplex Ligation Probe Amplification

Abstract Background Spinal muscular atrophy (SMA) is an autosomal recessive hereditary disease associated with severe muscle atrophy and weakness in the limbs and trunk. The discovery of mutated genes is helpful in diagnosis and treatment for SMA. Methods Eighty-three whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was performed. Afterwards, the complete gene sequence of SMN1 gene was detected. Furthermore, 20 SMA patients were selected from the 28 probands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. Results Twenty-two probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. Six SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C > T], c.[271C > T], c.[−39A > G] and g.[70240639G > C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[−41_-40insCTCT], FUT5 SNV c.[1001A > G], and MCCC2 SNV c.[−117A > G] were the 3 most frequent mutations in SMA group (95, 85 and 75%, respectively). Conclusions We identified some mutations in both SMN1 and other genes, and c.[271C > T], c.[−41_-40insCTCT], c.[1001A > G] and c.[−117A > G] might be associated with the onset of SMA.

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