Intracellular Drug Targets In Mycobacterium Tuberculosis Revealed By A Chemo-Genetic Approach

Abstract Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is one of the most devastating infectious agents in the world. It causes chronic lung diseases to one third of the world’s population. Chemo-genetic characterization through in vitro evolution combined with whole genome sequencing analysis can identify novel drug targets and drug resistance genes in Mtb. We performed a genome analysis of 53 Mtb mutants resistant to 15 different hit compounds. We found nonsynonymous mutations/indels in 30 genes that may be associated with drug resistance acquisitions. Beyond confirming previously identified drug resistance mechanisms such as rpoB and lead targets reported in novel anti-tuberculosis drug screenings such as mmpL3, ethA, mbtA, we discovered several unrecognized candidate drug targets including prrB and TB18.5. The exploration of the M. tuberculosis chemical mutant genomes could help novel drug discovery and structural biology of compounds and asscoiated mechanisms of action relevant to tuberculosis treatment.

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

Science ◽

10.1126/science.aan4472 ◽

2018 ◽

Vol 359 (6372) ◽

pp. 191-199 ◽

Cited By ~ 84

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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Mapping the malaria parasite drug-able genome using in vitro evolution and chemogenomics

10.1101/139386 ◽

2017 ◽

Cited By ~ 1

Author(s):

Annie N. Cowell ◽

Eva S. Istvan ◽

Amanda K. Lukens ◽

Maria G. Gomez-Lorenzo ◽

Manu Vanaerschot ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Resistance Genes ◽

Genome Analysis ◽

Drug Targets ◽

Malaria Parasite ◽

Resistance Mechanisms ◽

Whole Genome

AbstractChemogenetic characterization through in vitro evolution combined with whole genome analysis is a powerful tool to discover novel antimalarial drug targets and identify drug resistance genes. Our comprehensive genome analysis of 262 Plasmodium falciparum parasites treated with 37 diverse compounds reveals how the parasite evolves to evade the action of small molecule growth inhibitors. This detailed data set revealed 159 gene amplifications and 148 nonsynonymous changes in 83 genes which developed during resistance acquisition. Using a new algorithm, we show that gene amplifications contribute to 1/3 of drug resistance acquisition events. In addition to confirming known multidrug resistance mechanisms, we discovered novel multidrug resistance genes. Furthermore, we identified promising new drug target-inhibitor pairs to advance the malaria elimination campaign, including: thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This deep exploration of the P. falciparum resistome and drug-able genome will guide future drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms of the deadliest malaria parasite.One Sentence SummaryWhole genome sequencing reveals how Plasmodium falciparum evolves resistance to diverse compounds and identifies new antimalarial drug targets.

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Drug resistance mechanisms and novel drug targets for tuberculosis therapy

Journal of Genetics and Genomics ◽

10.1016/j.jgg.2016.10.002 ◽

2017 ◽

Vol 44 (1) ◽

pp. 21-37 ◽

Cited By ~ 33

Author(s):

Md Mahmudul Islam ◽

H.M. Adnan Hameed ◽

Julius Mugweru ◽

Chiranjibi Chhotaray ◽

Changwei Wang ◽

...

Keyword(s):

Drug Resistance ◽

Drug Targets ◽

Resistance Mechanisms ◽

Tuberculosis Therapy ◽

Novel Drug ◽

Novel Drug Targets

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A genome‐scale CRISPR knock‐out screen in chronic myeloid leukemia identifies novel drug resistance mechanisms along with intrinsic apoptosis and MAPK signaling

Cancer Medicine ◽

10.1002/cam4.3231 ◽

2020 ◽

Vol 9 (18) ◽

pp. 6739-6751

Author(s):

Matthieu Lewis ◽

Valérie Prouzet‐Mauléon ◽

Florence Lichou ◽

Elodie Richard ◽

Richard Iggo ◽

...

Keyword(s):

Drug Resistance ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Resistance Mechanisms ◽

Mapk Signaling ◽

Intrinsic Apoptosis ◽

Knock Out ◽

A Genome ◽

Genome Scale ◽

Novel Drug

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Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01341-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 6873-6881 ◽

Cited By ~ 13

Author(s):

Kathryn Winglee ◽

Shichun Lun ◽

Marco Pieroni ◽

Alan Kozikowski ◽

William Bishai

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Efflux Pump ◽

Transcriptional Regulator ◽

Cross Resistance ◽

Loss Of Function ◽

Strong Activity ◽

Content Type ◽

Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

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Cross kingdom analysis of putative quadruplex-forming sequences in fungal genomes: novel antifungal targets to ameliorate fungal pathogenicity?

10.1101/2020.09.23.310581 ◽

2020 ◽

Author(s):

Emily F. Warner ◽

Natália Bohálová ◽

Václav Brázda ◽

Zoë A. E. Waller ◽

Stefan Bidula

Keyword(s):

Drug Resistance ◽

Drug Targets ◽

Protein Modification ◽

Pathogenic Fungi ◽

Significant Heterogeneity ◽

Nucleic Acid Binding ◽

Fungal Virulence ◽

Coding Regions ◽

Critical Requirement ◽

Novel Drug

AbstractFungi contribute to upwards of 1.5 million human deaths annually, are involved in the spoilage of up to a third of food crops, and have a devastating effect on plant and animal biodiversity. Moreover, this already significant issue is exacerbated by a rise in antifungal resistance and a critical requirement for novel drug targets. Quadruplexes are four-stranded secondary structures in nucleic acids which can regulate processes such as transcription, translation, replication, and recombination. They are also found in genes linked to virulence in microbes, and quadruplex-binding ligands have been demonstrated to eliminate drug resistant pathogens. Using a computational approach, we identified putative quadruplex-forming sequences (PQS) in 1362 genomes across the fungal kingdom and explored their potential involvement in virulence, drug resistance, and pathogenicity. Here we present the largest analysis of PQS in fungi and identified significant heterogeneity of these sequences throughout phyla, genera, and species. Moreover, PQS were genetically conserved. Notably, loss of PQS in cryptococci and aspergilli was associated with pathogenicity. PQS in the clinically important pathogens Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans were located within genes (particularly coding regions), mRNA, repeat regions, mobile elements, tRNA, ncRNA, rRNA, and the centromere. Genes containing PQS in these organisms were found to be primarily associated with metabolism, nucleic acid binding, transporter activity, and protein modification. Finally, PQS were found in over 100 genes associated with virulence, drug resistance, or key biological processes in these pathogenic fungi and were found in genes which were highly upregulated during germination, hypoxia, oxidative stress, iron limitation, and in biofilms. Taken together, quadruplexes in fungi could present interesting novel targets to ameliorate fungal virulence and overcome drug resistance.

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Drug Resistance Mechanisms in Mycobacterium tuberculosis

Antibiotics ◽

10.3390/antibiotics3030317 ◽

2014 ◽

Vol 3 (3) ◽

pp. 317-340 ◽

Cited By ~ 112

Author(s):

Juan Palomino ◽

Anandi Martin

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Resistance Mechanisms

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Genetic Identification and Drug-Resistance Characterization of Mycobacterium tuberculosis Using a Portable Sequencing Device. A Pilot Study

Antibiotics ◽

10.3390/antibiotics9090548 ◽

2020 ◽

Vol 9 (9) ◽

pp. 548 ◽

Cited By ~ 1

Author(s):

Jorge Cervantes ◽

Noemí Yokobori ◽

Bo-Young Hong

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Genetic Identification ◽

Sequencing Analysis ◽

Antimicrobial Resistance Genes ◽

Long Read ◽

Drug Resistance Prediction ◽

Almost All

Clinical management of tuberculosis (TB) in endemic areas is often challenged by a lack of resources including laboratories for Mycobacterium tuberculosis (Mtb) culture. Traditional phenotypic drug susceptibility testing for Mtb is costly and time consuming, while PCR-based methods are limited to selected target loci. We herein utilized a portable, USB-powered, long-read sequencing instrument (MinION), to investigate Mtb genomic DNA from clinical isolates to determine the presence of anti-TB drug-resistance conferring mutations. Data analysis platform EPI2ME and antibiotic-resistance analysis using the real time ARMA workflow, identified Mtb species as well as extensive resistance gene profiles. The approach was highly sensitive, being able to detect almost all described drug resistance conferring mutations based on previous whole genome sequencing analysis. Our findings are supportive of the practical use of this system as a suitable method for the detection of antimicrobial resistance genes, and effective in providing Mtb genomic information. Future improvements in the error rate through statistical analysis, drug resistance prediction algorithms and reference databases would make this a platform suited for the clinical setting. The small size, relatively inexpensive cost of the device, as well as its rapid and simple library preparation protocol and analysis, make it an attractive option for settings with limited laboratory infrastructure.

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A Whole Genome In Vivo Crispr Screen in Primary ALL Predicts Leukaemic Relapse

Blood ◽

10.1182/blood.v126.23.2619.2619 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2619-2619

Author(s):

Katherine Dormon ◽

Elda S Latif ◽

Matthew Bashton ◽

Deepali Pal ◽

Matthew Selby ◽

...

Keyword(s):

Drug Resistance ◽

Conflicts Of Interest ◽

Gene Set Enrichment Analysis ◽

Whole Genome ◽

Competitive Situation ◽

Nsg Mice ◽

A Genome ◽

Crispr Screen

Abstract Although paediatric acute lymphoblastic leukaemia (ALL) has a favourable prognosis, a number of cases will invariably relapse. One of the major problems associated with relapse is drug resistance, in particular to glucocorticoids, the mainstay of ALL treatment. Examining the underlying mechanisms is complicated by clonal heterogeneity within a patient and the potential impact of the leukaemic niche. To address mechanisms of drug resistance in a patient-relevant setting, we performed a genome-wide in vivo CRISPR screen in primary ALL material. To that end, we took advantage of primografted material from patient L707, who initially presented with a Dexamethasone (DEX) sensitive t(17;19) ALL, but relapsed 5 months after initial diagnosis. We transduced DEX sensitive presentation cells with the full genome GeCKOv2 CRISPR library, before transplantation into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were subsequently treated with DEX by oral gavage (15mg/kg for 5 weeks, 10mg/kg thereafter). DNA from several engrafted sites in the mouse was extracted and PCR amplified before being sequenced on the Illumina HiSeq2500. Changes in pool complexity were analysed using MaGEcK software to determine which sgRNAs were significantly enriched or depleted. By far the most significantly enriched sgRNAs were those targeting NR3C1, the gene encoding the glucocorticoid receptor. In addition, two of the top five significantly depleted sgRNAs targeted the Plexins, PLXNA1 and PLXND1. Whilst PLXNA1 is expressed at low levels, PLXND1 is highly expressed and has been linked to dexamethasone resistance. Notably, the matched relapsed material from L707 was highly DEX resistant both in tissue culture and when transplanted into NSG mice. SNP 6.0 analysis revealed a 5q deletion in the relapse, spanning 5 genes including NR3C1. Whole genome sequencing showed this was comprised of 2 deletions both targeting NR3C1, with different breakpoints for each allele. The differential gene expression between the L707 presentation and relapse established that NR3C1 was the most significant of all the genes lost at relapse, based on gene set enrichment analysis (GSEA). This contrasts with many ALL cases, where one of the downstream effectors of apoptosis is lost as opposed to NR3C1. Growth of the relapse material in vivo and in vitro was slower than the presentation in a competitive situation, but with DEX treatment the relapse phenotype began to emerge with a small percentage of cells showing a heterozygous deletion of NR3C1. These combined data strongly suggest that the NR3C1 deletion is the main driver of DEX resistance in the L707 relapse. Moreover, it proves that our in vivo CRISPR screen predicted the leukaemic relapse. These results confirm NR3C1 deletion as a driver in glucocorticoid resistance and demonstrate the power of in vivo CRISPR screens to predict mechanisms of gain of drug resistance and subsequent relapse. The parallels that can be drawn between the relapse and the CRISPR screen are striking, giving the indication that the progression from presentation to relapse may follow the same path in a patient derived xenograft setting as it did in the patient. Disclosures No relevant conflicts of interest to declare.

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Pharmacological interactions of TKIs with the P-gp drug transport protein.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.2536 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 2536-2536 ◽

Cited By ~ 1

Author(s):

Sandra Roche ◽

Kasper Pedersen ◽

Grainne Dunne ◽

Denis Collins ◽

Aoife Devery ◽

...

Keyword(s):

Drug Resistance ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Drug Transport ◽

Kinase Inhibitors ◽

Resistance Mechanisms ◽

Cancer Drug ◽

Clear Understanding ◽

Pharmacological Interactions

2536 Background: Tyrosine Kinase Inhibitors (TKIs) can interact with drug transport proteins. P-gp is a transporter with two important roles in cancer drug therapy. If overexpressed in tumour cells it can cause drug resistance. However, P-gp, expressed in tissues as part of normal drug clearance mechanisms, is also involved in termination of drug action. Hence, TKI-mediated interactions with P-gp have significant therapeutic consequences. Methods: P-gp over-expressing cancer cell lines were used to determine the inhibitor or substrate status of tyrosine kinase inhibitors (erlotinib, gefitinib, lapatinib, dasatinb, neratinib, afatinib and pazopanib). Cell proliferation assays in combination with a potent P-gp inhibitor, or P-gp substrate were also employed. Findings were augmented using LC-MS-based quantitation of cellular levels of target drugs. Results: We summarise our findings of four distinct interactions with P-gp among various TKIs. Some agents have little interaction at conventional doses; others can act as P-gp inhibitors without being substrates; substrates without being inhibitors or substrates which also prevent the actions of the transporter.Eachof the investigated TKIs has a distinct relationship with P-gp. As examples, lapatinib is an inhibitor but not a substrate, dasatinib is a substrate but not an inhibitor, while pazopanib has little interaction with P-gp. Other agents also have an effect on or are affected by P-gp to varying amounts with some of these interactions likely to be suprapharmacological. Conclusions: P-gp protein has important roles both in resistance and drug toxicology, hence, a clear understanding of the interaction of emerging drugs with this transporter is vital. Agents which are inhibitors of P-gp may have applications in drug resistance circumvention but may also greatly exacerbate the toxicity of concurrently administered P-gp substrate cytotoxics; conversely the activity of P-gp substrate TKIs may be reduced by tumour overexpression of the transporter. Hence in vitro screening of TKI-transporter interactions may identify putative TKI resistance mechanisms, help guide the development of combination schedule trials and/or reducing unwanted treatment side effects.

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