A complex suite of loci and elements in eukaryotic type II topoisomerases determine selective sensitivity to distinct poisoning agents

AbstractType II topoisomerases catalyze essential DNA transactions and are proven drug targets. Drug discrimination by prokaryotic and eukaryotic topoisomerases is vital to therapeutic utility, but is poorly understood. We developed a next-generation sequencing (NGS) approach to identify drug-resistance mutations in eukaryotic topoisomerases. We show that alterations conferring resistance to poisons of human and yeast topoisomerase II derive from a rich mutational ‘landscape’ of amino acid substitutions broadly distributed throughout the entire enzyme. Both general and discriminatory drug-resistant behaviors are found to arise from different point mutations found at the same amino acid position and to occur far outside known drug-binding sites. Studies of selected resistant enzymes confirm the NGS data and further show that the anti-cancer quinolone vosaroxin acts solely as an intercalating poison, and that the antibacterial ciprofloxacin can poison yeast topoisomerase II. The innate drug-sensitivity of the DNA binding and cleavage region of human and yeast topoisomerases (particularly hTOP2β) is additionally revealed to be significantly regulated by the enzymes’ adenosine triphosphatase regions. Collectively, these studies highlight the utility of using NGS-based methods to rapidly map drug resistance landscapes and reveal that the nucleotide turnover elements of type II topoisomerases impact drug specificity.

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Plasmodium falciparum DHODH inhibitor complexes reveal flexible binding site

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314082072 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1793-C1793

Author(s):

Paul Rowland ◽

Onkar SINGH ◽

Leila Ross ◽

Francisco Gamo ◽

Maria Lafuente-Monasterio ◽

...

Keyword(s):

Drug Resistance ◽

Binding Site ◽

Crystal Structures ◽

Point Mutations ◽

Crystal Form ◽

Drug Binding ◽

Binding Modes ◽

Resistance Mutations ◽

Drug Binding Site

Malaria is a preventable and treatable disease, yet annually there are still hundreds of thousands of malaria-related deaths. The disease is caused by infection with mosquito-borne Plasmodium parasites. With hundreds of millions of cases each year there is a very high potential for drug resistance and this has compromised many existing therapies. One target under investigation is the enzyme dihydroorotate dehydrogenase (DHODH) which catalyses the rate-limiting step of pyrimidine biosynthesis and is an essential enzyme in the malaria parasite. There are currently several Plasmodium-selective DHODH inhibitors under development. To investigate the potential for drug resistance against DHODH inhibitors in vitro resistance selections were carried out using known inhibitors from different structural classes [1]. These studies identified point mutations in the drug binding site which lead to reduced sensitivity to the inhibitors, and in some cases increased sensitivity to a different inhibitor, suggesting a novel combination therapy approach to combat resistance. To help understand the significance of the inhibitor binding site mutations we determined the crystal structures of P. falciparum DHODH in complex with the inhibitors Genz-669178, IDI-6253 and IDI-6273. Co-crystallisation experiments led to a new crystal form in each case. Here we describe the crystal structures, the binding modes of the inhibitors and the great flexibility of the binding site, which is able to adjust to accommodate different inhibitor series. The structural role of the resistance mutations is also discussed.

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Importance of the Fourth Alpha-Helix within the CAP Homology Domain of Type II Topoisomerase for DNA Cleavage Site Recognition and Quinolone Action

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.9.2735-2746.2002 ◽

2002 ◽

Vol 46 (9) ◽

pp. 2735-2746 ◽

Cited By ~ 9

Author(s):

Dirk Strumberg ◽

John L. Nitiss ◽

Jiaowang Dong ◽

Jerrylaine Walker ◽

Marc C. Nicklaus ◽

...

Keyword(s):

Amino Acid ◽

Dna Cleavage ◽

Topoisomerase Ii ◽

Point Mutations ◽

Alpha Helix ◽

Single Amino Acid ◽

Type Ii ◽

Cleavage Sites ◽

Amino Acid Residues ◽

Site Recognition

ABSTRACT We report that point mutations causing alteration of the fourth alpha-helix (α4-helix) of the CAP homology domain of eukaryotic (Saccharomyces cerevisiae) type II topoisomerases (Ser740Trp, Gln743Pro, and Thr744Pro) change the selection of type II topoisomerase-mediated DNA cleavage sites promoted by Ca2+ or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, Thr744Ala substitution had minimal effect on Ca2+- and drug-stimulated DNA cleavage sites, indicating the selectivity of single amino acid substitutions within the α4-helix on type II topoisomerase-mediated DNA cleavage. The equivalent mutation in the gene for Escherichia coli gyrase causing Ser83Trp also changed the DNA cleavage pattern generated by Ca2+ or quinolones. Finally, Thr744Pro substitution in the yeast type II topoisomerase rendered the enzyme sensitive to antibacterial quinolones. This study shows that the α4-helix within the conserved CAP homology domain of type II topoisomerases is critical for selecting the sites of DNA cleavage. It also demonstrates that selective amino acid residues in the α4-helix are important in determining the activity and possibly the binding of quinolones to the topoisomerase II-DNA complexes.

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Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3470 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3470-3480 ◽

Cited By ~ 98

Author(s):

E Moran ◽

B Zerler ◽

T M Harrison ◽

M B Mathews

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Point Mutations ◽

Apparent Molecular Weight ◽

Amino Acid Position ◽

Cysteine Residue ◽

Transformation Function ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

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Expanded Spectrum of Antiretroviral-Selected Mutations in Human Immunodeficiency Virus Type 2

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa026 ◽

2020 ◽

Vol 221 (12) ◽

pp. 1962-1972 ◽

Cited By ~ 1

Author(s):

Philip L Tzou ◽

Diane Descamps ◽

Soo-Yon Rhee ◽

Dana N Raugi ◽

Charlotte Charpentier ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Drug Resistance ◽

Amino Acid ◽

Meta Analysis ◽

Multiple Comparisons ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Immunodeficiency Virus ◽

Hiv 1

Abstract Background HIV-1 and HIV-2 differ in their antiretroviral (ARV) susceptibilities and drug resistance mutations (DRMs). Methods We analyzed published HIV-2 pol sequences to identify HIV-2 treatment-selected mutations (TSMs). Mutation prevalences were determined by HIV-2 group and ARV status. Nonpolymorphic mutations were those in <1% of ARV-naive persons. TSMs were those associated with ARV therapy after multiple comparisons adjustment. Results We analyzed protease (PR) sequences from 483 PR inhibitor (PI)-naive and 232 PI-treated persons; RT sequences from 333 nucleoside RT inhibitor (NRTI)-naive and 252 NRTI-treated persons; and integrase (IN) sequences from 236 IN inhibitor (INSTI)-naive and 60 INSTI-treated persons. In PR, 12 nonpolymorphic TSMs occurred in ≥11 persons: V33I, K45R, V47A, I50V, I54M, T56V, V62A, A73G, I82F, I84V, F85L, L90M. In RT, 9 nonpolymorphic TSMs occurred in ≥10 persons: K40R, A62V, K70R, Y115F, Q151M, M184VI, S215Y. In IN, 11 nonpolymorphic TSMs occurred in ≥4 persons: Q91R, E92AQ, T97A, G140S, Y143G, Q148R, A153G, N155H, H156R, R231 5-amino acid insertions. Nine of 32 nonpolymorphic TSMs were previously unreported. Conclusions This meta-analysis confirmed the ARV association of previously reported HIV-2 DRMs and identified novel TSMs. Genotypic and phenotypic studies of HIV-2 TSMs will improve approaches to predicting HIV-2 ARV susceptibility and treating HIV-2–infected persons.

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Mutations in Plasmodium falciparumCytochrome b That Are Associated with Atovaquone Resistance Are Located at a Putative Drug-Binding Site

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.8.2100-2108.2000 ◽

2000 ◽

Vol 44 (8) ◽

pp. 2100-2108 ◽

Cited By ~ 232

Author(s):

Michael Korsinczky ◽

Nanhua Chen ◽

Barbara Kotecka ◽

Allan Saul ◽

Karl Rieckmann ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Point Mutations ◽

Drug Binding ◽

Single Amino Acid ◽

Malaria Parasites ◽

Drug Binding Site ◽

Sole Agent ◽

Amino Acid Mutations

ABSTRACT Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc 1 (CYTbc 1) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25- to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYTb.

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Interstrain Variation in the Human Cytomegalovirus DNA Polymerase Sequence and Its Effect on Genotypic Diagnosis of Antiviral Drug Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.6.1500 ◽

1999 ◽

Vol 43 (6) ◽

pp. 1500-1502 ◽

Cited By ~ 84

Author(s):

Sunwen Chou ◽

Nell S. Lurain ◽

Adriana Weinberg ◽

Guang-Yung Cai ◽

Prem L. Sharma ◽

...

Keyword(s):

Drug Resistance ◽

Amino Acid ◽

Dna Polymerase ◽

Human Cytomegalovirus ◽

Antiviral Drug ◽

Resistance Mutations ◽

Sequence Alignments ◽

Coding Sequence ◽

Antiviral Drug Resistance ◽

Almost All

ABSTRACT The polymerase (pol) coding sequence was determined for 40 independent clinical cytomegalovirus isolates sensitive to ganciclovir and foscarnet. Sequence alignments showed >98% interstrain homology and amino acid variation in only 4% of the 1,237 codons. Almost all variation occurred outside of conserved functional domains where resistance mutations have been identified.

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Acetolactate Synthase Gene Proline (197) Mutations Confer Tribenuron-Methyl Resistance in Flixweed (Descurainia sophia) Populations from China

Weed Science ◽

10.1614/ws-d-10-00099.1 ◽

2011 ◽

Vol 59 (3) ◽

pp. 376-379 ◽

Cited By ~ 28

Author(s):

Hai Lan Cui ◽

Chao Xian Zhang ◽

Shou Hui Wei ◽

Hong Jun Zhang ◽

Xiang Ju Li ◽

...

Keyword(s):

Amino Acid ◽

Molecular Basis ◽

Resistance Mechanism ◽

Point Mutations ◽

Acetolactate Synthase ◽

Amino Acid Position ◽

Resistance Level ◽

Coding Sequence ◽

Whole Plant ◽

Tribenuron Methyl

The molecular basis of resistance to tribenuron-methyl, an acetolactate synthase (ALS)–inhibiting herbicide was investigated in four resistant (R) and three susceptible (S) flixweed populations. The resistance level in the R populations was assessed in whole-plant pot experiments in a greenhouse, and resistance indices ranged from 723 to 1422. The ALS genes of the three S populations and four R populations were cloned and sequenced, and the full coding sequence of the ALS gene of flixweed was 2,004 bp. The sequences of the ALS genes of the three S populations collected from Shaanxi, Gansu, and Tianjin were identical. Comparison of the ALS gene sequences of the S and R populations withArabidopsisrevealed that proline at position 197 of the ALS gene was substituted by leucine in R population SSX-2, by alanine in R population SSX-3, and by serine in R populations TJ-2 and GS-2. In another study of two R flixweed populations from Hebei and Shaanxi, resistance was also related to mutation at position 197 of the ALS gene. Both studies confirmed tribenuron-methyl resistance in flixweed in China, with the resistance mechanism being conferred by specific ALS point mutations at amino acid position 197.

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Mutations in efflux pump Rv1258c (Tap) cause resistance to pyrazinamide and other drugs inM. tuberculosis

10.1101/249102 ◽

2018 ◽

Author(s):

Jiayun Liu ◽

Wanliang Shi ◽

Shuo Zhang ◽

Gail Cassell ◽

Dmitry A. Maslov ◽

...

Keyword(s):

Drug Resistance ◽

Drug Targets ◽

Drug Exposure ◽

Efflux Pump ◽

Active Form ◽

Point Mutations ◽

Drug Susceptibility ◽

Clinical Isolates

AbstractAlthough drug resistance inM. tuberculosisis mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in possible role of efflux in causing drug resistance. Previously, efflux genes are shown upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance inM. tuberculosisby constructing point mutations V219A, S292L in Rv1258c in the chromosome ofM. tuberculosisand assessed drug susceptibility of the constructed mutants. Interestingly, V219A, S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs inM. tuberculosis. While V219A point mutation conferred a low level resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance including PZA and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance inM. tuberculosisand address their role in affecting treatment outcome in vivo.

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Landscape of drug-resistance mutations in kinase regulatory hotspots

Briefings in Bioinformatics ◽

10.1093/bib/bbaa108 ◽

2020 ◽

Cited By ~ 3

Author(s):

Pora Kim ◽

Hanyang Li ◽

Junmei Wang ◽

Zhongming Zhao

Keyword(s):

Drug Resistance ◽

Large Scale ◽

Kinase Inhibitors ◽

Growth Factor Receptor ◽

Kinase Domain ◽

Systematic Investigation ◽

Drug Binding ◽

Data Sets ◽

Resistance Mutations ◽

Cancer Data

Abstract More than 48 kinase inhibitors (KIs) have been approved by Food and Drug Administration. However, drug-resistance (DR) eventually occurs, and secondary mutations have been found in the previously targeted primary-mutated cancer cells. Cancer and drug research communities recognize the importance of the kinase domain (KD) mutations for kinasopathies. So far, a systematic investigation of kinase mutations on DR hotspots has not been done yet. In this study, we systematically investigated four types of representative mutation hotspots (gatekeeper, G-loop, αC-helix and A-loop) associated with DR in 538 human protein kinases using large-scale cancer data sets (TCGA, ICGC, COSMIC and GDSC). Our results revealed 358 kinases harboring 3318 mutations that covered 702 drug resistance hotspot residues. Among them, 197 kinases had multiple genetic variants on each residue. We further computationally assessed and validated the epidermal growth factor receptor mutations on protein structure and drug-binding efficacy. This is the first study to provide a landscape view of DR-associated mutation hotspots in kinase’s secondary structures, and its knowledge will help the development of effective next-generation KIs for better precision medicine.

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