scholarly journals Using in Vitro Evolution and Whole Genome Analysis To Discover Next Generation Targets for Antimalarial Drug Discovery

2018 ◽  
Vol 4 (3) ◽  
pp. 301-314 ◽  
Author(s):  
Madeline R. Luth ◽  
Purva Gupta ◽  
Sabine Ottilie ◽  
Elizabeth A. Winzeler
Keyword(s):  
Drug Discovery ◽  
Genome Analysis ◽  
Antimalarial Drug ◽  
Whole Genome ◽  
Next Generation ◽  
Whole Genome Analysis ◽  
In Vitro Evolution

scholarly journals Target deconvolution using in vitro evolution in haploid human cells

2020 ◽  
Author(s):  
Juan Carlos Jado ◽  
Michelle Dow ◽  
Krypton Carolino ◽  
Gregory J. Fonseca ◽  
Trey Ideker ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Genome Analysis ◽  
Resistance Mechanisms ◽  
Human Cells ◽  
Whole Genome ◽  
Whole Genome Analysis ◽  
In Vitro Evolution ◽  
New Genes ◽  
Target Deconvolution

ABSTRACTRobust methods are needed for determining the mechanism of action and mechanism of resistance for small molecules that are active against human cells. In microbes, in vitro evolution and whole genome analysis (IVIEWGA) is the preferred method for target deconvolution and for revealing resistance mechanisms. To investigate the method’s applicability to human cells we evolved resistance to five different anticancer drugs (doxorubicin, gemcitabine, etoposide, topotecan, and paclitaxel) using a near-haploid cell line (HAP-1). Resultant clones (N=28) were compared to their isogenic parents via whole genome and whole exome sequencing (WES). High frequency alleles predicted to change protein sequence, or alleles which appeared in the same gene for multiple independent selections with the same compound were identified in only 21 genes: The set included clinically-relevant resistance genes or known drug targets (TOP1, TOP2A, DCK, WDR33, SLCO3A1), as well as new genes (SLC13A4). In addition, some lines carried structural variants that encompassed additional known resistance genes (ABCB1, WWOX and RRM1). Gene expression knockdown and knockout experiments (via shRNA and CRISPR-Cαs9 respectively) of 10 validation targets showed a high degree of specificity and accuracy in our calls and demonstrates that the same drug resistance mechanisms found in diverse clinical samples can be evolved, identified and studied in an isogenic background. Our data show that in vitro evolution and whole genome analysis is a promising method for target identification as well as for identifying resistance mechanisms.

Association of SLC15A2 genomic variation with the response to sorafenib treatment: Whole-genome analysis in patients with hepatocellular carcinoma.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 308-308
Author(s):  
Bo Hyun Kim ◽  
Yeon-Su Lee ◽  
Byung Chul Kim ◽  
Aesun Shin ◽  
Jin Sook Kim ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Functional Analysis ◽  
Genome Analysis ◽  
Whole Genome ◽  
Whole Genome Analysis ◽  
Single Nucleotide ◽  
Sorafenib Treatment ◽  
Genomic Variations ◽  
Validation Tests

308 Background: Reliable biomarkers are required to predict patient response to sorafenib. We attempted to investigate genomic variations associated with responsiveness to sorafenib treatment in patients with unresectable hepatocellular carcinoma (HCC) and their functional relevance. Methods: We obtained blood samples from 4 strong and 3 poor responders to sorafenib treatment and subjected these samples to whole-genome analysis. Next, we performed validation tests for candidate single-nucleotide polymorphisms (SNPs) in the samples of 174 HCC patients who were treated with sorafenib, followed by in vitro functional analysis and in silico analyses of candidate SNPs. Results: On average, 90 gigabases/sample was generated at ~34X sequencing depth. In total, 1813 genomic variations were perfectly matched to sorafenib responses in the clinical data; 708 were located within regions for sorafenib-target genes or drug absorption, distribution, metabolism, and excretion (ADME)-related genes—36 within the coding regions and 6 identified as non-synonymous single-nucleotide variants from 4 ADME-related genes (ABCB1, FMO3, MUSK, and SLC15A2), which potentially cause functional alterations. Validation tests of 174 patients confirmed sequencing results and revealed that patients with the C/C genotype for rs2257212 in SCL15A2 displayed higher risk for cancer progression than did patients with C/T or T/T genotypes (HR: 2.18; 95% CI, 1.15–4.15; P = 0.018). In vitro functional analysis revealed that cells harboring C/C genotype for this SNP displayed lower response to sorafenib treatment than did cells harboring the T/T genotype. Structural prediction analysis revealed change in protein phosphorylation levels, potentially affecting sorafenib-associated enzymatic activity. Conclusions: SLC15A2 could be a robust biomarker of response to sorafenib treatment in HCC patients.

scholarly journals In Vitro Whole-Genome Analysis Identifies a Susceptibility Locus for HIV-1

PLoS Biology ◽  
2008 ◽  
Vol 6 (2) ◽  
pp. e32 ◽  
Author(s):  
Corinne Loeuillet ◽  
Samuel Deutsch ◽  
Angela Ciuffi ◽  
Daniel Robyr ◽  
Patrick Taffé ◽  
...  
Keyword(s):  
Genome Analysis ◽  
Susceptibility Locus ◽  
Whole Genome ◽  
Whole Genome Analysis ◽  
Hiv 1

scholarly journals Mapping the malaria parasite druggable genome by using in vitro evolution and chemogenomics

Science ◽  
2018 ◽  
Vol 359 (6372) ◽  
pp. 191-199 ◽  
Author(s):  
Annie N. Cowell ◽  
Eva S. Istvan ◽  
Amanda K. Lukens ◽  
Maria G. Gomez-Lorenzo ◽  
Manu Vanaerschot ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Genome Analysis ◽  
Drug Targets ◽  
Malaria Parasite ◽  
Resistance Mechanisms ◽  
Whole Genome Analysis ◽  
In Vitro Evolution ◽  
A Genome ◽  
Druggable Genome

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target–inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.

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