Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers.

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens. Despite advances in treatment with immune checkpoint inhibitors, there is an unmet need in the treatment of MSI cancers. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy.

In vivo genotoxicity testing strategies: Report from the 8th International Workshop on Genotoxicity Testing (IWGT).

The in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non-hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate "sufficient" exposure was not reached. The liver MN test using six-week-old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study.

International Workshops on Genotoxicity Testing (IWGT): Origins, achievements and ambitions.

Over the past thirty years, the International Workshops on Genotoxicity Testing (IWGT) became one of the leading groups in the field of regulatory genotoxicology, not only due to the diversity of participants with respect to geography and professional affiliation, but also due to the unique setup of recurring IWGT meetings every four years. The hallmarks of the IWGT process have been diligent initial planning approaches of the working groups, collection of data so as to stimulate data-driven discussions and debate, and striving to reach consensus recommendations. The scientific quality of the Working Groups (WGs) has been exceptional due to the selection of highly regarded experts on each topic.

Genotoxicity evaluation of a valsartan-related complex N-nitroso-impurity.

Recently, the formation of genotoxic and carcinogenic N-nitrosamines impurities during drug manufacturing of tetrazole-containing angiotensin-II blockers has been described. However, drug-related (complex) nitrosamines may also be generated under certain conditions, i.e.

Comprehensive review of genotoxicity data for diclofenac.

Diclofenac is a non-steroidal anti-inflammatory drug discovered several decades ago, which has since been used by an estimated one billion patients and has demonstrated an acceptable safety profile. In support of its marketing approval, a comprehensive set of genotoxicity studies were conducted in vitro and in vivo. Despite the fact that these studies preceded both Good Laboratory Practice (GLP) requirements and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines on genotoxicity testing, they were conducted using the best scientific principles and are considered appropriate by contemporary standards.

Pig-a gene mutation assay study design: critical assessment of 3- versus 28-day repeat-dose treatment schedules.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organisation for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93).

Intra- and inter-laboratory reproducibility of the rat blood Pig-a gene mutation assay.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organization for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93).

In vivo genotoxicity testing strategies: Report from the 7th International workshop on genotoxicity testing (IWGT).

The working group reached complete or majority agreement on many issues. Results from TGR and in vivo comet assays for 91 chemicals showed they have similar ability to detect in vivo genotoxicity per se with bacterial mutagens and Ames-positive carcinogens. TGR and comet assay results were not significantly different when compared with IARC Group 1, 2 A, and unclassified carcinogens.

X-ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan-McDermid Syndrome.

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan-McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X-ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach.

Derisking Drug-Induced Carcinogenicity for Novel Therapeutics.

Assessing the carcinogenic potential of innovative drugs spanning diverse therapeutic modalities and target biology represents a major challenge during drug development. Novel modalities, such as cell and gene therapies that involve intrinsic genetic modification of the host genome, require distinct approaches for identification of cancer hazard. We emphasize the need for customized weight-of-evidence cancer risk assessments based on mode of action that balance multiple options for preclinical identification of cancer hazard with appropriate labeling of clinical products and risk management plans.

Setting Occupational Exposure Limits for Genotoxic Substances in the Pharmaceutical Industry.

In the pharmaceutical industry, genotoxic drug substances are developed for life-threatening indications such as cancer. Healthy employees handle these substances during research, development, and manufacturing; therefore, safe handling of genotoxic substances is essential. When an adequate preclinical dataset is available, a risk-based decision related to exposure controls for manufacturing is made following a determination of safe health-based limits, such as an occupational exposure limit (OEL).

Evaluation of methyl methanesulfonate, 2,6-diaminotoluene and 5-fluorouracil: Part of the Japanese center for the validation of alternative methods (JaCVAM) international validation study of the in vivo rat alkaline comet assay.

As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined methyl methanesulfonate, 2,6-diaminotoluene, and 5-fluorouracil under coded test conditions. Rats were treated orally with the maximum tolerated dose (MTD) and two additional descending doses of the respective compounds. In the MMS treated groups liver and stomach showed significantly elevated DNA damage at each dose level and a significant dose-response relationship.

Miniaturized flow cytometry-based in vitro primary human lymphocyte micronucleus assay-validation study.

Most in vitro mammalian genotoxicity assays show a low specificity (high rate of irrelevant positive results), and therefore, lead to an increase in follow-up in vivo genotoxicity testing. One of the sources of the high rate of in vitro irrelevant positive results that find no confirmation in in vivo studies may be the characteristics of the test system used. It has been shown that cells that are p53 deficient or carry an alteration in DNA repair genes may be more prone to produce high rate of false/irrelevant positive results.

Compilation and use of genetic toxicity historical control data.

The optimal use of historical control data for the interpretation of genotoxicity results was discussed at the 2009 International Workshop on Genotoxicity Testing (IWGT) in Basel, Switzerland. The historical control working group focused mainly on negative control data although positive control data were also considered to be important. Historical control data are typically used for comparison with the concurrent control data as part of the assay acceptance criteria.

High-content micronucleus assay in genotoxicity profiling: initial-stage development and some applications in the investigative/lead-finding studies in drug discovery.

This article describes the first step toward full (that includes conditions for both absence and presence of metabolic activation) validation and drug discovery application of a 96-well, automated, high-content micronucleus (HCMN) assay. The current validation tests were performed using Chinese hamster ovary cells, in the absence of metabolic activation, against three distinct sets of drug-like compounds that represent all stages of a drug discovery pipeline. A compound categorization scheme was created based on quantitative relationships between micronucleus (MN) signals, cytotoxicity, and compound solubility.

Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat.

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints.

Strategy for genotoxicity testing--metabolic considerations.

The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens "missed" in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance.

Methylphenidate is not clastogenic in cultured human lymphocytes and in the mouse bone-marrow micronucleus test.

Methylphenidate (MPH) is one of the most frequently prescribed drugs for the treatment of attention deficit hyperactivity disorder (ADHD). A report on cytogenetic effects observed in peripheral lymphocytes from children treated for 3 months with MPH raised questions about the genetic toxicity of this compound. A critical review of this data concluded that the cytogenetic effects in treated children remain unexplained.

Structure-activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters.

Sulfonic acid esters are considered as potentially alkylating agents that may exert genotoxic effects in bacterial and mammalian cell systems. One possible source of human exposure stems from drug synthesis when the salt-forming agents methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid are used together with alcoholic solvents such as methanol, ethanol and propanol. In this study computer-assisted structural considerations and in vitro approaches (Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, and the micronucleus test using L5178Y mouse lymphoma cells) were used to assess the genotoxic properties of 19 sulfonic esters.

Induction of A:T to G:C transition mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue, in the bone marrow of Muta Mouse.

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) is a pyrimidine nucleoside analogue formerly in development for the treatment of herpes simplex virus infections. The compound proved clearly mutagenic in the mouse spot test and exhibited weak activity in the Salmonella reverse mutation test, which led to the termination of the compound's development. In another study, CEDU, administered orally to beta-galactosidase (lacZ) transgenic mice (Muta Mouse) for five days, induced a clear increase in lacZ mutant frequencies in spleen, lung, and bone marrow.

Induction of gene mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue.

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro.