Drug-induced resistance evolution necessitates less aggressive treatment.

Increasing body of experimental evidence suggests that anticancer and antimicrobial therapies may themselves promote the acquisition of drug resistance by increasing mutability. The successful control of evolving populations requires that such biological costs of control are identified, quantified and included to the evolutionarily informed treatment protocol. Here we identify, characterise and exploit a trade-off between decreasing the target population size and generating a surplus of treatment-induced rescue mutations. We show that the probability of cure is maximized at an intermediate dosage, below the drug concentration yielding maximal population decay, suggesting that treatment outcomes may in some cases be substantially improved by less aggressive treatment strategies. We also provide a general analytical relationship that implicitly links growth rate, pharmacodynamics and dose-dependent mutation rate to an optimal control law. Our results highlight the important, but often neglected, role of fundamental eco-evolutionary costs of control. These costs can often lead to situations, where decreasing the cumulative drug dosage may be preferable even when the objective of the treatment is elimination, and not containment. Taken together, our results thus add to the ongoing criticism of the standard practice of administering aggressive, high-dose therapies and motivate further experimental and clinical investigation of the mutagenicity and other hidden collateral costs of therapies.

Inferring the dynamics of mutated hematopoietic stem and progenitor cells induced by IFNα in myeloproliferative neoplasms.

Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose.

High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells.

Mutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.

Whole-genome analysis of drug-resistant Mycobacterium tuberculosis reveals novel mutations associated with fluoroquinolone resistance.

Multidrug-resistant and extensively drug-resistant tuberculosis (M/XDR-TB) remains a global public-health challenge. Known mutations in quinolone resistance-determination regions cannot fully explain phenotypic fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb). The aim of this study was to look for novel mutations in Mtb associated with resistance to FQ drugs using whole-genome sequencing analysis. Whole-genome sequences of 659 Mtb strains, including 214 with phenotypic FQ resistance and 445 pan-susceptible isolates, were explored for mutations associated with FQ resistance overall and with resistance to individual FQ drugs (ofloxacin, levofloxacin, moxifloxacin and gatifloxacin). Three novel genes (recC, Rv2005c and PPE59) associated with FQ resistance were identified (P < 0.00001 based on screening analysis and absence of relevant mutations in a pan-susceptible validation set of 360 strains). Nine novel single nucleotide polymorphisms (SNPs), including in gyrB (G5383A and G6773A), gyrA (G7892A), recC (G725900C and G726857T/C), Rv2005c (C2251373G, G2251420C and C2251725T) and PPE59 (C3847269T), were used for diagnostic performance analysis. Enhancing the known SNP set with five of these novel SNPs, including gyrA [G7892A (Leu247Leu)], recC [G725900C (Leu893Leu) and G726857T/C (Arg484Arg)], Rv2005c [G2251420C (Pro205Arg)] and PPE59 [C3847269T (Asn35Asn)] increased the sensitivity of detection of FQ-resistant Mtb from 83.2% (178/214) to 86.9% (186/214) while maintaining 100% specificity (360/360). No specific mutation associated with resistance to only a single drug (ofloxacin, levofloxacin, moxifloxacin or gatifloxacin) was found. In conclusion, this study reports possible additional mutations associated with FQ resistance in Mtb.

Evaluation of oral toxicity and genotoxicity of Achyranthis Radix extract.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Achyranthes bidentata Blume, Achyranthis Radix (AR), is used as a traditional medicine ingredient in East Asia. It has anti-inflammatory, anti-oxidative, and anti-diabetic activities. AIM OF THE STUDY: In the present study, we aimed to evaluate the oral toxicity and genotoxicity of single-dose and 4-week repeated-doses of AR hot water extract (ARE), under the good laboratory practice principles. MATERIALS AND METHODS: For oral toxicity studies, SD rats (n = 5 per sex and group) were administered ARE at concentrations of 500, 1000, and 2000 mg/kg/day once (single dose) or once per day for 4 weeks (repeated dose). The non-clinical genotoxicity study consisted of bacterial reverse mutation using Escherichia coli (WP2 uvrA) and Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), in vitro chromosomal aberration test with Chinese hamster lung cells (CHL/IU), and in vivo mouse bone marrow micronucleus test using bone marrow cells collected from male ICR mice (n = 5) that were orally administered ARE. RESULTS: In the single-dose oral toxicity study, mortality and treatment-related changes in body weight were not observed throughout the study, and the lethal dose was estimated to be > 2000 mg/kg in rats. In the 4-week repeated-dose oral toxicity study, ARE did not induce significant changes in body weight, organ weight, food intake, or hematological and serum biochemical parameters in any group. In the bacterial reverse mutation test, ARE did not induce gene mutations in any tested strain. In the chromosomal aberration test, ARE did not cause chromosomal aberrations. The micronucleus test showed no significant increase in the number of micronucleated polychromatic erythrocytes or the mean ratio of polychromatic to total erythrocytes. CONCLUSIONS: These results showed that ARE does not induce oral toxicity and genotoxicity in the in vivo and in vitro test systems.

Mutational spectra induced by flavonoid extracts from pepper tree (Schinus terebinthifolius, Raddi) stem bark.

Flavonoids are a diverse family of plant compounds that are involved in pigmentation, protection, and endogenous regulation. Flavonoids also have medicinal applications, suggesting that they may exert chemoprotective effects. However, some studies have shown, that some plant flavonoids have oxidative and toxic effects, including those produced by Schinus terebinthifolius. In Brazil, extracts of this plant are widely used for medical purposes. In this study, we analyzed the mutagenic potential of two flavonoid-enriched fractions from Brazilian pepper tree stem bark using Escherichia coli CC strains deficient and proficient in enzymes involved in the DNA repair of oxidative lesions. The highest mutagenic response was detected in the CC104mutMmutY strain but CC104mutY showed a higher mutation frequency than CC104mutM. The spectrum of mutations induced in plasmid DNA is composed of mutations typically caused by oxidative lesions. However, a new type of lesion must be occurred to explain the cytotoxicity, higher mutation rates in the CC104mutY strain, and the rare A:T → T:A and G:C → C:G transversions found in this work.

Modulating effect of vitamin D3 on the mutagenicity and carcinogenicity of doxorubicin in Drosophila melanogaster and in silico studies.

Vitamin D3 (VD3) deficiency increases DNA damage, while supplementation may exert a pro-oxidant activity, prevent viral infections and formation of tumors. The aim of this study was to investigate the mutagenicity and carcinogenicity of VD3 alone or in combination with doxorubicin (DXR) using the Somatic Mutation and Recombination Test and the Epithelial Tumor Test, both in Drosophila melanogaster. For better understanding of the molecular interactions of VD3 and receptors, in silico analysis were performed with molecular docking associated with molecular dynamics. Findings revealed that VD3 alone did not increase the frequency of mutant spots, but reduced the frequency of mutant spots when co-administered with DXR. In addition, VD3 did not alter the recombinogenic effect of DXR in both ST and HB crosses. VD3 alone did not increase the total frequency of tumor, but significantly reduced the total frequency of tumor when co-administered with DXR. Molecular modeling and molecular dynamics between calcitriol and Ecdysone Receptor (EcR) showed a stable interaction, indicating the possibility of signal transduction between VD3 and EcR. In conclusion, under these experimental conditions, VD3 has modulatory effects on the mutagenicity and carcinogenicity induced by DXR in somatic cells of D. melanogaster and exhibited satisfactory interactions with the EcR.

Active Compound of Pharbitis Semen (Pharbitis nil Seeds) Suppressed KRAS-Driven Colorectal Cancer and Restored Muscle Cell Function during Cancer Progression.

Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven colorectal cancer (CRC) is notorious to target with drugs and has shown ineffective treatment response. The seeds of Pharbitis nil, also known as morning glory, have been used as traditional medicine in East Asia. We focused on whether Pharbitis nil seeds have a suppressive effect on mutated KRAS-driven CRC as well as reserving muscle cell functions during CRC progression. Seeds of Pharbitis nil (Pharbitis semen) were separated by chromatography and the active compound of Pharbitis semen (PN) was purified by HPLC. The compound PN efficiently suppressed the proliferation of mutated KRAS-driven CRC cells and their clonogenic potentials in a concentration-dependent manner. It also induced apoptosis of SW480 human colon cancer cells and cell cycle arrest at the G2/M phase. The CRC related pathways, including RAS/ERK and AKT/mTOR, were assessed and PN reduced the phosphorylation of AKT and mTOR. Furthermore, PN preserved muscle cell proliferation and myotube formation in cancer conditioned media. In summary, PN significantly suppressed mutated KRAS-driven cell growth and reserved muscle cell function. Based on the current study, PN could be considered as a promising starting point for the development of a nature-derived drug against KRAS-mutated CRC progression.

Identification of a novel gene argJ involved in arginine biosynthesis critical for persister formation in Staphylococcus aureus.

Staphylococcus aureus can cause both acute and recurrent persistent infections such as peritonitis, endocarditis, abscesses, osteomyelitis, and chronic wound infections. Effective therapies to treat persistent disease are paramount. However, the mechanisms of S. aureus persistence are poorly understood. In this study, we performed a comprehensive and unbiased high-throughput mutant screen against a transposon-insertion mutant library of S. aureus USA300 and focused on the role of argJ encoding an acetyltransferase in the arginine biosynthesis pathway, whose transposon insertion caused a significant defect in persister formation using multiple drugs and stresses. Genetic complementation and arginine supplementation restored persistence in the argJ transposon insertion mutant while generation of mutations on the active site of the ArgJ protein caused a defect in persistence. Quantitative RT-PCR analysis showed that the genes encoded in the arg operon were over-expressed under drug stressed conditions and in stationary phase cultures. In addition, the argJ mutant had attenuated virulence in both mouse and C. elegans. Our studies identify a new mechanism of persistence mediated by arginine metabolism in S. aureus. These findings provide not only novel insights about the mechanisms of S. aureus persistence but also offer novel therapeutic targets that may help to develop more effective treatment of persistent S. aureus infections.

Treating the Disease, Not the Symptom: Beyond NSAIDs.

Mitigating inflammation is clearly important in cancer prevention and control. Traditionally, pharmaceuticals have taken the lead in this problem. In an attempt to 'head them off at the pass', this Forum takes a hard look at the concept of 'better living through chemicals' and limiting proinflammatory chemicals entering the body.

Simultaneous Detection of Neisseria gonorrhoeae and Fluoroquinolone Resistance Mutations to Enable Rapid Prescription of Oral Antibiotics.

BACKGROUND: Absence of rapid antimicrobial resistance testing of Neisseria gonorrhoeae (Ng) hinders personalized antibiotic treatment. To enable rapid ciprofloxacin prescription, a real-time polymerase chain reaction (PCR) for simultaneous detection of Ng and fluoroquinolone resistance-associated gyrA-S91F mutation was evaluated. METHODS: Analytical NG quantitative PCR kit (NYtor BV) performance was assessed on 50 Ng transcription-mediated amplification (TMA)-negative and 100 Ng TMA-positive samples. To assess clinical use, 200 samples were prospectively analyzed, in parallel to routine diagnostic tests. Also, 50 urine, 50 anal, 50 pharyngeal, and 50 vaginal Ng TMA-positive samples were retrospectively analyzed. To assess if patients carried strains with different ciprofloxacin sensitivity at different anatomical locations, 50 urine/anal or vaginal/anal sample pairs collected during a single visit were analyzed. RESULTS: The NG quantitative PCR kit showed 97% sensitivity and 100% specificity for Ng detection and 92% sensitivity and 99% specificity for gyrA-S91F detection. Relative to TMA results, 85% Ng detection sensitivity and 99% specificity were found. Regarding the 200 prospectively analyzed clinical samples, 13 were Ng positive, of which 10 were also tested for antibiotic susceptibility by culture. The kit showed concordance for GyrA-S91F detection in 9 of 10 samples. Ng was detected in 96% and 94% of vaginal and urine TMA-positive samples, in 84% of anal samples and only in 22% of pharyngeal samples. Discordant ciprofloxacin sensitivity was found for 2 of 26 characterized urine/anal sample pairs. CONCLUSION: The NG quantitative polymerase chain reaction (qPCR) kit can be implemented in diagnostic testing for vaginal, urine, and anal Ng TMA-positive samples to enable rapid prescription of oral ciprofloxacin.

EMS Mutagenesis of Maize Pollen.

Effective mutagenesis is critical for connecting traits of interest to specific plant genes. The development of site-directed mutagenesis and sequenced-indexed genetics resources in maize allows for targeted analysis of individual genes. These reverse genetics approaches have the potential for confirmation bias by only studying candidate genes for association with traits of interest. Genetic screens of induced, random mutations are important for identifying novel loci as well as interacting factors for known mutant loci. Chemical mutagenesis provides very high mutation rates and can be used for a variety of screen designs. This chapter provides an updated protocol for ethyl methanesulfonate (EMS) mutagenesis of maize pollen using paraffin or mineral oil. Mutagenesis occurs in mature pollen causing nonconcordant endosperm and embryo genotypes as well as sectored M1 plants. Considerations for these factors in genetic screens are discussed.

Genotoxic Assessment of the Dry Decoction of Myracrodruon urundeuva Allemão (Anacardiaceae) Leaves in Somatic Cells of Drosophila melanogaster by the Comet and SMART Assays.

Medicinal plants are worldwide used as an efficient treatment of many diseases. Myracrodruon urundeuva Allemão (Anacardiaceae) is widely used Brazilian folk medicine to treat inflammations and infections of the female genital tract, conditions of the stomach and throat, and to heal wounds on the skin and mucous membranes. Several pharmacological properties of extracts and compounds isolated from M. urundeuva are found in the literature, corroborating its uses as antiulcer and gastroprotective, anti-inflammatory and analgesic, as well as antimicrobial. Despite these many uses in traditional herbal medicine, there are few reports of its toxic-genetic effect. This work aimed to investigate the genotoxic and mutagenic potential in vivo of the dry decoction of M. urundeuva leaves on somatic cells of Drosophila melanogaster, through the Comet assay and somatic mutation and recombination test (SMART). Six concentrations (0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 mg/mL) were studied after feeding individuals for 24 hr in culture medium hydrated with extracts of M. urundeuva. In the Comet assay, all concentrations showed a genotoxic effect significantly higher than the negative control group, treated with distilled water. The two highest concentrations were also superior to the positive control group, treated with cyclophosphamide (1 mg/mL). In the SMART, there was a mutagenic effect at all concentrations tested, with a clear dose-dependent relationship. Both recombination and mutation account for these mutagenic effects. The set of results indicate that the dry decoction of M. urundeuva leaves is genotoxic and mutagenic for D. melanogaster under the experimental conditions of this study. Environ. Mol. Mutagen. 61:329-337, 2020. © 2019 Wiley Periodicals, Inc.

Genomic insights on heterogeneous resistance to vancomycin and teicoplanin in Methicillin-resistant Staphylococcus aureus: A first report from South India.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is an important clinical concern in patients, and is often associated with significant disease burden and metastatic infections. There is an increasing evidence of heterogeneous vancomycin-intermediate S. aureus (hVISA) associated treatment failure. In this study, we aim to understand the molecular mechanism of teicoplanin resistant MRSA (TR-MRSA) and hVISA. A total of 482 MRSA isolates were investigated for these phenotypes. Of the tested isolates, 1% were identified as TR-MRSA, and 12% identified as hVISA. A highly diverse amino acid substitution was observed in tcaRAB, vraSR, and graSR genes in TR-MRSA and hVISA strains. Interestingly, 65% of hVISA strains had a D148Q mutation in the graR gene. However, none of the markers were reliable in differentiating hVISA from TR-MRSA. Significant pbp2 upregulation was noted in three TR-MRSA strains, which had teicoplanin MICs of 16 or 32 µg/ml, whilst significant pbp4 downregulation was not noted in these strains. In our study, multiple mutations were identified in the candidate genes, suggesting a complex evolutionary pathway involved in the development of TR-MRSA and hVISA strains.

Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds.

Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3)1 and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington's disease, an incurable neurodegenerative disorder2. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract3. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds.

Marine Natural Products and Drug Resistance in Latent Tuberculosis.

Pyrazinamide (PZA) is the only drug for the elimination of latent Mycobacterium tuberculosis (MTB) isolates. However, due to the increased number of PZA-resistance, the chances of the success of global TB elimination seems to be more prolonged. Recently, marine natural products (MNPs) as an anti-TB agent have received much attention, where some compounds extracted from marine sponge, Haliclona sp. exhibited strong activity under aerobic and hypoxic conditions. In this study, we screened articles from 1994 to 2019 related to marine natural products (MNPs) active against latent MTB isolates. The literature was also mined for the major regulators to map them in the form of a pathway under the dormant stage. Five compounds were found to be more suitable that may be applied as an alternative to PZA for the better management of resistance under latent stage. However, the mechanism of actions behind these compounds is largely unknown. Here, we also applied synthetic biology to analyze the major regulatory pathway under latent TB that might be used for the screening of selective inhibitors among marine natural products (MNPs). We identified key regulators of MTB under latent TB through extensive literature mining and mapped them in the form of regulatory pathway, where SigH is negatively regulated by RshA. PknB, RshA, SigH, and RNA polymerase (RNA-pol) are the major regulators involved in MTB survival under latent stage. Further studies are needed to screen MNPs active against the main regulators of dormant MTB isolates. To reduce the PZA resistance burden, understanding the regulatory pathways may help in selective targets of MNPs from marine natural sources.

Helicobacter pylori Mutations Conferring Resistance to Fluoroquinolones and Clarithromycin among Dyspeptic Patients Attending a Tertiary Hospital, Tanzania.

Objectives. Helicobacter pylori (H. pylori) isolates resistant to clarithromycin and quinolones are increasing worldwide. Data regarding the magnitude of H. pylori resistance are limited in developing countries. Here, we report the prevalence of mutations conferring resistance to clarithromycin and fluoroquinolones among dyspeptic patients attending a tertiary hospital, Tanzania. Methods. Between August 2014 and August 2016, patients undergoing upper gastrointestinal endoscopy at the Bugando Medical Centre were enrolled. Biopsies were taken for polymerase chain reaction (PCR) and sequencing to detect mutations conferring resistance to clarithromycin and fluoroquinolones. Results. A total of 208 nonrepetitive biopsies were examined of which 188 (90.4%) tested positive for H. pylori specific 23S rRNA PCR. Clarithromycin resistance mutations were detected in 54/188 (28.7%) of patients tested. The most frequently detected mutation was A2143G (30) followed by A2142G (20). Out of 131 nonrepetitive biopsies tested for fluoroquinolones resistance mutations, 77/131 (58.8%) were positive, with N87I (20) mutation being the most frequently detected mutation followed by A92T mutation which was detected in 16 samples. Conclusion. A significant proportion of dyspeptic patients attending tertiary hospital in Tanzania are infected with H. pylori strains harbouring clarithromycin or fluoroquinolones resistance mutations. Detection of more than 50% of strains with fluoroquinolones resistance mutations makes the H. pylori second line treatment questionable in our setting. There is a need of surveillance of H. pylori resistance patterns in Tanzania to provide data that can guide empirical treatment to reduce associated morbidity of H. pylori infections. The correlation between A92T fluoroquinolone mutation and phenotypic resistance requires further investigations.

Mutational Processes in Hepatocellular Carcinoma: The Story of Aristolochic Acid.

Each hepatocellular carcinoma displays dozens of mutations in driver and passenger genes. The analysis of the types of substitutions and their trinucleotide context defines mutational signatures that recapitulate the endogenous and exogenous mutational processes operative in tumor cells. Aristolochic acid is present in plants from the genus Aristolochia and causes chronic nephropathy. Moreover, aristolochic acid has genotoxic properties responsible for the occurrence of urothelial carcinoma. Metabolites of aristolochic acid form DNA adducts on adenine residues leading to a specific mutational signature with almost exclusively A:T to T:A transversions, preferentially in a CTG trinucleotide context. Interestingly, this mutational fingerprint has been identified in a subset of hepatocellular carcinomas suggesting that aristolochic acid is a new risk factor for hepatocellular carcinoma. More data are warranted to capture the real impact of exposure to aristolochic acid in hepatocellular carcinoma occurrence worldwide.

Molecular analysis of glycosylphosphatidylinositol anchor deficient aerolysin resistant isolates in gulf war i veterans exposed to depleted uranium.

During the First Gulf War (1991) over 100 servicemen sustained depleted uranium (DU) exposure through wound contamination, inhalation, and shrapnel. The Department of Veterans Affairs has a surveillance program for these Veterans which has included genotoxicity assays. The frequencies of glycosylphosphatidylinositol anchor (GPIa) negative (aerolysin resistant) cells determined by cloning assays for these Veterans are reported in Albertini RJ et al. (2019: Environ Mol Mutagen). Molecular analyses of the GPIa biosynthesis class A (PIGA) gene was performed on 862 aerolysin-resistant T-lymphocyte recovered isolates. The frequencies of different types of PIGA mutations were compared between high and low DU exposure groups. Additional molecular studies were performed on mutants that produced no PIGA mRNA or with deletions of all or part of the PIGA gene to determine deletion size and breakpoint sequence. One mutant appeared to be the result of a chromothriptic event. A significant percentage (>30%) of the aerolysin resistant isolates, which varied by sample year and Veteran, had wild-type PIGA cDNA (no mutation). As described in Albertini RJ et al. (2019: Environ Mol Mutagen), TCR gene rearrangement analysis of these isolates indicated most arose from multiple T-cell progenitors (hence the inability to find a mutation). It is likely that these isolates were the result of failure of complete selection against nonmutant cells in the cloning assays. Real-time studies of GPIa resistant isolates with no PIGA mutation but with a single TCR gene rearrangement found one clone with a PIGV deletion and several others with decreased levels of GPIa pathway gene mRNAs implying mutation in other GPIa pathway genes. Environ. Mol. Mutagen. 60:470-493, 2019. © 2019 Wiley Periodicals, Inc.

Virtual screening identification of novel chemical inhibitors for aberrant interactions between pathogenic mutant SOD1 and tubulin.

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease caused by selective motor neuron death. Mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) belong to one of the four major mutation clusters responsible for pathogenesis of ALS. Toxic gain-of-function (not loss-of-function) of SOD1 mutants causes motor neuron degeneration. Aberrant protein-protein interactions (PPI) between mutant SOD1 and other proteins are involved in this toxic gain-of-function. Therefore, PPI inhibitors of mutant SOD1 not only increase understanding of ALS pathogenesis, but can also be used as novel therapeutics for ALS. Although it is challenging to identify PPI inhibitors, prior knowledge of the binding site can increase success probability. We have previously reported that tubulin interacts with N-terminal residues 1-23 of mutant SOD1. In the present study, we performed virtual screening by docking simulation of 32,791 compounds using this N-terminal binding site as prior knowledge. An established assay system for interaction inhibition between mutant SOD1-tubulin was used as an in-house model system to identify mutant SOD1 PPI inhibitors, with the goal of developing novel therapeutics for ALS. Consequently, five of six assay-executable compounds among top-ranked compounds during docking simulation inhibited the mutant SOD1-tubulin interaction in vitro. Binding mode analysis predicted that some inhibitors might bind the tubulin binding site of G85R SOD1 by pi electron interaction with the aromatic ring of the Trp32 residue of G85R SOD1. Our screening methods may contribute to the identification of lead compounds for treating ALS.