Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

Energy-Based Pharmacophore and Three-Dimensional Quantitative Structure--Activity Relationship (3D-QSAR) Modeling Combined with Virtual Screening To Identify Novel Small-Molecule Inhibitors of Silent Mating-Type Information Regulation 2 Homologue 1 (SIRT1).

Silent mating-type information regulation 2 homologue 1 (SIRT1), being the homologous enzyme of silent information regulator-2 gene in yeast, has multifaceted functions. It deacetylates a wide range of histone and nonhistone proteins; hence, it has good therapeutic importance. SIRT1 was believed to be overexpressed in many cancers (prostate, colon) and inflammatory disorders (rheumatoid arthritis). Hence, designing inhibitors against SIRT1 could be considered valuable. Both structure-based and ligand-based drug design strategies were employed to design novel inhibitors utilizing high-throughput virtual screening of chemical databases. An energy-based pharmacophore was generated using the crystal structure of SIRT1 bound with a small molecule inhibitor and compared with a ligand-based pharmacophore model that showed four similar features. A three-dimensional quantitative structure-activity relationship (3D-QSAR) model was developed and validated to be employed in the virtual screening protocol. Among the designed compounds, Lead 17 emerged as a promising SIRT1 inhibitor with IC50 of 4.34 µM and, at nanomolar concentration (360 nM), attenuated the proliferation of prostate cancer cells (LnCAP). In addition, Lead 17 significantly reduced production of reactive oxygen species, thereby reducing pro inflammatory cytokines such as IL6 and TNF-α. Furthermore, the anti-inflammatory potential of the compound was ascertained using an animal paw inflammation model induced by carrageenan. Thus, the identified SIRT1 inhibitors could be considered as potent leads to treat both cancer and inflammation.

Structure-guided design and development of novel benzimidazole class of compounds targeting DNA gyraseB enzyme of Staphylococcus aureus.

The gyraseB subunit of Staphylococcus aureus DNA gyrase is a well-established and validated target though less explored for the development of novel antimicrobial agents. Starting from the available structural information in PDB (3TTZ), we identified a novel series of benzimidazole used as inhibitors of DNA gyraseB with low micromolar inhibitory activity by employing structure-based drug design strategy. Subsequently, this chemical class of DNA gyrase inhibitors was extensively investigated biologically through in vitro assays, biofilm inhibition assays, cytotoxicity, and in vivo studies. The binding affinity of the most potent inhibitor 10 was further ascertained biophysically through differential scanning fluorimetry. Further, the most potent analogues did not show any signs of cardiotoxicity in Zebra fish ether-a-go-go-related gene (zERG), a major breakthrough among the previously reported cardiotoxic gyraseB inhibitors.

Structure-based drug design of small molecule SIRT1 modulators to treat cancer and metabolic disorders.

Sirtuins comprise a family of deacetylase enzymes that catalyze the removal of an acetyl moiety from the ɛ-amino group of lysine residues within protein targets. Sirtuin 1(SIRT1), a NAD(+) dependent class III histone deacetylase is involved in a variety of human disorders such as obesity, type II diabetes, cancer and aging. Inhibition of SIRT1 could be useful for cancer treatment while activators can be useful for longevity and treating metabolic disorders. Hence we undertook an effort to design both inhibitors and activators using structure-based drug design techniques and report here the biological proof of concept. In this paper, we report diverse small molecule inhibitors with a potential to attenuate cancer growth designed based on high-throughput virtual screening and docking using the crystal structure of SIRT1. And small molecule activators with potential to suppress adipogenesis differentiation indicating their usefulness in obesity control was designed based on a homology model of SIRT1 activator domain.

Development of novel N-linked aminopiperidine-based mycobacterial DNA gyrase B inhibitors: scaffold hopping from known antibacterial leads.

DNA gyrase of Mycobacterium tuberculosis (MTB) is a type II topoisomerase that ensures the regulation of DNA topology and has been genetically demonstrated to be a bactericidal drug target. We present the discovery and optimisation of a novel series of mycobacterial DNA gyrase inhibitors with a high degree of specificity towards the mycobacterial ATPase domain. Compound 5-fluoro-1-(2-(4-(4-(trifluoromethyl)benzylamino)piperidin-1-yl)ethyl)indoline-2,3-dione (17) emerged as the most potent lead, exhibiting inhibition of MTB DNA gyrase supercoiling assay with an IC50 (50% inhibitory concentration) of 3.6 ± 0.16 µM, a Mycobacterium smegmatis GyrB IC50 of 10.6 ± 0.6 µM, and MTB minimum inhibitory concentrations of 6.95 µM and 10 µM against drug-sensitive (MTB H37Rv) and extensively drug-resistant strains, respectively. Furthermore, the compounds did not show any signs of cardiotoxicity in zebrafish ether-à-go-go-related gene (zERG), and hence constitute a major breakthrough among the otherwise cardiotoxic N-linked aminopiperidine analogues.

Can targeting SIRT-1 to treat type 2 diabetes be a good strategy? A review.

INTRODUCTION: Dysregulation of metabolic pathways, caused by imbalances in energy homeostasis, leads to type 2 diabetes characterized by high glucose concentration in the blood due to insulin resistance which is a major disorder in developed countries. AREAS COVERED: One of the recent treatment strategies is using activators of SIRT1, which has been in clinical trials. Many of the cellular processes including insulin secretion, cell cycle, and apoptosis are imperatively regulated by a family of mediators called sirtuins. First known mammalian sirtuin, SIRT1 is a positive regulator of insulin secretion, which triggers glucose uptake and utilization. Since the past decade, a major outstanding question is whether SIRT1 activation is a safe therapy for human diseases such as type 2 diabetes? This review summarizes and discusses the advances of the past decade and the challenges that will brazen out perplexity about homeostasis and metabolic pathways linked to SIRT1 and type 2 diabetes. Furthermore, we described the interlink between SIRT1 metabolic pathways of various tissues such as pancreas, skeletal muscle, adipose tissue and liver. EXPERT OPINION: However be the complexity of the pathways involved, T2DM regulated by SIRT1 affected metabolism is dropping down progressively due to profound research. In the context of interlinking all the SIRT1 pathways in T2DM we found various crucial intermediaries in metabolic tissues, which can also be targeted for future prospects.