scholarly journals A combined computational and experimental strategy identifies mutations conferring resistance to drugs targeting the BCR-ABL fusion protein

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Jinxin Liu ◽  
Jianfeng Pei ◽  
Luhua Lai
Keyword(s):  
Drug Resistance ◽  
In Vitro Selection ◽  
Binding Constants ◽  
Computational Prediction ◽  
Resistance Mutations ◽  
Experimental Strategy ◽  
Drug Resistance Mutations ◽  
Suggested Strategy

AbstractDrug resistance is of increasing concern, especially during the treatments of infectious diseases and cancer. To accelerate the drug discovery process in combating issues of drug resistance, here we developed a computational and experimental strategy to predict drug resistance mutations. Using BCR-ABL as a case study, we successfully recaptured the clinically observed mutations that confer resistance imatinib, nilotinib, dasatinib, bosutinib, and ponatinib. We then experimentally tested the predicted mutants in vitro. We found that although all mutants showed weakened binding strength as expected, the binding constants alone were not a good indicator of drug resistance. Instead, the half-maximal inhibitory concentration (IC50) was shown to be a good indicator of the incidence of the predicted mutations, together with change in catalytic efficacy. Our suggested strategy for predicting drug-resistance mutations includes the computational prediction and in vitro selection of mutants with increased IC50 values beyond the drug safety window.

scholarly journals Dramatic Activation of Antibiotic Production in Streptomyces coelicolor by Cumulative Drug Resistance Mutations

2008 ◽  
Vol 74 (9) ◽  
pp. 2834-2840 ◽  
Author(s):  
Guojun Wang ◽  
Takeshi Hosaka ◽  
Kozo Ochi
Keyword(s):  
Drug Resistance ◽  
Multiple Drug Resistance ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Strain Improvement ◽  
Multiple Drug ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Order Of Magnitude

ABSTRACT We recently described a new method to activate antibiotic production in bacteria by introducing a mutation conferring resistance to a drug such as streptomycin, rifampin, paromomycin, or gentamicin. This method, however, enhanced antibiotic production by only up to an order of magnitude. Working with Streptomyces coelicolor A3(2), we established a method for the dramatic activation of antibiotic production by the sequential introduction of multiple drug resistance mutations. Septuple and octuple mutants, C7 and C8, thus obtained by screening for resistance to seven or eight drugs, produced huge amounts (1.63 g/liter) of the polyketide antibiotic actinorhodin, 180-fold higher than the level produced by the wild type. This dramatic overproduction was due to the acquisition of mutant ribosomes, with aberrant protein and ppGpp synthesis activity, as demonstrated by in vitro protein synthesis assays and by the abolition of antibiotic overproduction with relA disruption. This new approach, called “ribosome engineering,” requires less time, cost, and labor than other methods and may be widely utilized for bacterial strain improvement.

scholarly journals Coevolution analysis of amino-acids reveals diversified drug-resistance solutions in viral sequences: a case study of hepatitis B virus

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Elin Teppa ◽  
Francesca Nadalin ◽  
Christophe Combet ◽  
Diego Javier Zea ◽  
Laurent David ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Coevolution Analysis ◽  
B Virus ◽  
Compensatory Mutations ◽  
Viral Sequences

Abstract The study of mutational landscapes of viral proteins is fundamental for the understanding of the mechanisms of cross-resistance to drugs and the design of effective therapeutic strategies based on several drugs. Antiviral therapy with nucleos(t)ide analogues targeting the hepatitis B virus (HBV) polymerase protein (Pol) can inhibit disease progression by suppression of HBV replication and makes it an important case study. In HBV, treatment may fail due to the emergence of drug-resistant mutants. Primary and compensatory mutations have been associated with lamivudine resistance, whereas more complex mutational patterns are responsible for resistance to other HBV antiviral drugs. So far, all known drug-resistance mutations are located in one of the four Pol domains, called reverse transcriptase. We demonstrate that sequence covariation identifies drug-resistance mutations in viral sequences. A new algorithmic strategy, BIS2TreeAnalyzer, is designed to apply the coevolution analysis method BIS2, successfully used in the past on small sets of conserved sequences, to large sets of evolutionary related sequences. When applied to HBV, BIS2TreeAnalyzer highlights diversified viral solutions by discovering thirty-seven positions coevolving with residues known to be associated with drug resistance and located on the four Pol domains. These results suggest a sequential mechanism of emergence for some mutational patterns. They reveal complex combinations of positions involved in HBV drug resistance and contribute with new information to the landscape of HBV evolutionary solutions. The computational approach is general and can be applied to other viral sequences when compensatory mutations are presumed.

scholarly journals In VitroResistance Profile of the Candidate HIV-1 Microbicide Drug Dapivirine

2011 ◽  
Vol 56 (2) ◽  
pp. 751-756 ◽  
Author(s):  
Susan M. Schader ◽  
Maureen Oliveira ◽  
Ruxandra-Ilinca Ibanescu ◽  
Daniela Moisi ◽  
Susan P. Colby-Germinario ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Reverse Transcriptase ◽  
Sexual Transmission ◽  
Transcriptase Inhibitor ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Development Of Resistance ◽  
Sexual Transmission Of Hiv ◽  
Hiv 1

ABSTRACTAntiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cellsin vitroin the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries.

scholarly journals Development of FF-10101, a Novel Irreversible FLT3 Inhibitor, Which Overcomes Drug Resistance Mutations

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1353-1353 ◽  
Author(s):  
Toshiyuki Nakatani ◽  
Ken Uda ◽  
Takeshi Yamaura ◽  
Masaru Takasaki ◽  
Akimi Akashi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Research Funding ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Growth Inhibitory ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Ic50 Values

Abstract Background: FLT3 is one of the most frequently mutated genes in acute myeloid leukemia (AML). Internal tandem duplication (ITD) of juxtamembrane domain sequence and missense point mutations at D835 residue within kinase domain are major mutations of FLT3 in AML. These mutations induce constitutive activation of FLT3 and its downstream pathway, resulting in aberrant cell proliferation of AML cells. FLT3 is, therefore, believed to be an attractive drug target for AML. Several FLT3 inhibitors were evaluated in clinical trials and they demonstrated clinical efficacy; however, drug-resistant secondary mutations such as F691L and D835 mutations with FLT3-ITD were often appeared. Therefore development of novel FLT3 inhibitors is required to overcome resistance to current FLT3 inhibitors. Here we report that a novel irreversible FLT3 inhibitor, FF-10101 is a promising agent for AML therapy. Methods: Bone marrow samples from patients with AML were subjected to Ficoll-Hypaque density gradient centrifugation. Informed consent was obtained from all patients, and approval was obtained from the ethics committee of our institute. In vitro growth inhibitory assay was performed with leukemia cell lines and 32D transfectants in liquid culture and primary AML cells in semisolid culture. Cell viability was determined by MTS assay or ATP quantification assay. For in vivo efficacy study, leukemia xenograft mouse model was prepared by tale vein injection of MOLM-13 cells or primary AML cells. Efficacies of tested compounds were evaluated by detection of human CD45-positive cells in bone marrow cells obtained from femurs at the end of studies. Results: Kinase profiling assay with 216 human recombinant kinases revealed that FF-10101 selectively and potently inhibited kinase activities of wtFLT3 and FLT3 D835Y with IC50 values of 0.20 nM and 0.16 nM, respectively. In MV4-11 cells, FF-10101 treatment decreased phosphorylation of FLT3 and its downstream molecules in a dose-dependent manner. FF-10101 treatment for 2 days demonstrated growth inhibitory effect on FLT3-dependent human cell lines, MV4-11, MOLM-13, MOLM-14 and 32D transfectants expressing FLT3-ITD with equal to or greater potency than Quizartinib, a highly potent FLT3 inhibitor currently under clinical development for AML (FF-10101 IC50=0.83 nM-2.4 nM, Quizartinib IC50=0.95 nM-4.5 nM). Cell cycle arrest was observed followed by increased sub-G1 population in MV4-11 cells treated with 1 nM FF-10101. Importantly, FF-10101 retained growth inhibitory activities against 32D transfectants expressing drug resistance mutations such as FLT3-ITD/D835Y, FLT3-ITD/Y842C or FLT3-ITD/Y842H with IC50 values of 0.66-3.1 nM, although Quizartinib demonstrated weak inhibitory effects with IC50 values of 85-150nM. In mice xenografted with MOLM-13, oral administration of 5 mg/kg FF-10101 once daily for 8 days significantly decreased MOLM-13 cells in bone marrow as compared to vehicle administration (p<0.001). Next, anti-leukemic effect of FF-10101 was assessed by using primary AML cells. In vitro cell growth assay, 1 week treatment of FF-10101 significantly reduced primary AML cells harboring FLT3-ITD. Growth inhibitory effect was also observed in primary AML cells harboring FLT3 D835H mutation, although Quizartinib had little effect. When 10 mg/kg FF-10101 was orally administrated twice daily to mice xenografted with primary AML cells with FLT3-ITD, AML cells in bone marrow were significantly reduced with comparable efficacy of Quizartinib. Furthermore, FF-10101 demonstrated more potent efficacy than Quizartinib in mice xenografted with primary AML cells harboring FLT3 D835H mutation. FF-10101 also retained its efficacy against mice xenografted with residual AML cells in Quizartinib-treated mice inoculated with primary AML cells harboring FLT3 D835H. Conclusions: We have developed a novel irreversible FLT3 inhibitor, FF-10101. FF-10101 showed potent anti-leukemic effect on cell lines and primary AML cells by selective inhibition of FLT3 both in vitro and in vivo. Notably, FF-10101 also has potency against drug resistance mutations. These results strongly indicate that FF-10101 is a promising agent for AML patients with FLT3 mutations. Phase I study of FF-10101 for AML patients is planned for 2016. Disclosures Nakatani: FUJIFILM Corporation: Employment. Uda:FUJIFILM Corporation: Employment. Yamaura:FUJIFILM Corporation: Employment. Takasaki:FUJIFILM Corporation: Employment. Ishikawa:GlaxoSmithKline K.K.: Research Funding. Hagiwara:FUJIFILM Corporation: Employment. Kiyoi:Eisai Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; Toyama Chemical CO., LTD.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding.

scholarly journals Fitness Cost of Chromosomal Drug Resistance-Conferring Mutations

2002 ◽  
Vol 46 (5) ◽  
pp. 1204-1211 ◽  
Author(s):  
Peter Sander ◽  
Burkhard Springer ◽  
Therdsak Prammananan ◽  
Antje Sturmfels ◽  
Martin Kappler ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Low Cost ◽  
Antibiotic Consumption ◽  
Fitness Cost ◽  
Resistance Mutations ◽  
Competition Assay ◽  
Drug Resistance Mutations ◽  
Compensatory Mutations ◽  
Bacterial Protein Synthesis

ABSTRACT To study the cost of chromosomal drug resistance mutations to bacteria, we investigated the fitness cost of mutations that confer resistance to different classes of antibiotics affecting bacterial protein synthesis (aminocyclitols, 2-deoxystreptamines, macrolides). We used a model system based on an in vitro competition assay with defined Mycobacterium smegmatis laboratory mutants; selected mutations were introduced by genetic techniques to address the possibility that compensatory mutations ameliorate the resistance cost. We found that the chromosomal drug resistance mutations studied often had only a small fitness cost; compensatory mutations were not involved in low-cost or no-cost resistance mutations. When drug resistance mutations found in clinical isolates were considered, selection of those mutations that have little or no fitness cost in the in vitro competition assay seems to occur. These results argue against expectations that link decreased levels of antibiotic consumption with the decline in the level of resistance.

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