POLARISED views and FRETting about probe modulation assays: Learning from High Throughput Screening.

Fluorescent probe modulation assays are a widely used approach to monitor displacement or stabilisation of fluorescently labelled tool ligands by test compounds. These assays allow an optical read-out of probe-receptor binding and can be used to detect compounds that compete with the labelled ligand, either directly or indirectly. Probes for both orthosteric and allosteric sites are often employed. The method can also be used to identify test compounds that may stabilise the ternary complex, offering an opportunity to discover novel molecular glues. The utility of these fluorescence-based assays within high-throughput screening has been facilitated by the use of streptavidin labelled terbium as a donor and access to a range of different acceptor fluorophores. During 2023, the High-throughput Screening group at AstraZeneca carried out 8 high-throughput screens using these approaches. In this manuscript we will present the types of assays used, an overview of the timelines for assay development and screening, the application of orthogonal artefact methods to aid hit finding and the results of the screens in terms of hit rate and the number of compounds identified with IC50 values of better than 30 µM. Learning across the development, execution and analysis of these screens will be presented.

Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly.

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

Screening for molecular glues - Challenges and opportunities.

Molecular glues are small molecules, typically smaller than PROTACs, and usually with improved physicochemical properties that aim to stabilise the interaction between two proteins. Most often this approach is used to improve or induce an interaction between the target and an E3 ligase, but other interactions which stabilise interactions to increase activity or to inhibit binding to a natural effector have also been demonstrated. This review will describe the effects of induced proximity, discuss current methods used to identify molecular glues and introduce approaches that could be adapted for molecular glue screening.

High-throughput differential scanning fluorimetry (DSF) and cellular thermal shift assays (CETSA): Shifting from manual to automated screening.

Biophysical affinity screening is increasingly being adopted as a high-throughput hit finding technique in drug discovery. Automation is highly beneficial to high-throughput screening (HTS) since a large number of compounds need to be reproducibly tested against a biological target. Herein, we describe how we have automated two biophysical affinity screening methods that rely on a thermal shift in protein melting temperature upon small molecule binding: differential scanning fluorimetry (DSF) and the cellular thermal shift assay (CETSA).

A perspective on the changing landscape of HTS.

Recently, there has been a change in the types of drug target entering early drug discovery portfolios. A significant increase in the number of challenging targets, or which would have historically been classed as intractable, has been observed. Such targets often have shallow or non-existent ligand-binding sites, can have disordered structures or domains or can be involved in protein-protein or protein-DNA interactions. The nature of the screens required to identify useful hits has, by necessity, also changed. The range of drug modalities explored has also increased and the chemistry required to design and optimise these molecules has adapted. In this review, we discuss this changing landscape and provide insights into the future requirements for small-molecule hit and lead generation.

Drug discovery for epigenetics targets.

Dysregulation of the epigenome is associated with the onset and progression of several diseases, including cancer, autoimmune, cardiovascular, and neurological disorders. Members from the three families of epigenetic proteins (readers, writers, and erasers) have been shown to be druggable using small-molecule inhibitors. Increasing knowledge of the role of epigenetics in disease and the reversibility of these modifications explain why pharmacological intervention is an attractive strategy for tackling epigenetic-based disease. In this review, we provide an overview of epigenetics drug targets, focus on approaches used for initial hit identification, and describe the subsequent role of structure-guided chemistry optimisation of initial hits to clinical candidates. We also highlight current challenges and future potential for epigenetics-based therapies.

Inhibition of Mycobacterium tuberculosis InhA: Design, synthesis and evaluation of new di-triclosan derivatives.

Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. 'Direct inhibitors' of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC50 of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC99 of 12.9 µM against M. bovis.

Molecular recognition of lipopolysaccharide by the lantibiotic nisin.

Nisin is a lanthionine antimicrobial effective against diverse Gram-positive bacteria and is used as a food preservative worldwide. Its action is mediated by pyrophosphate recognition of the bacterial cell wall receptors lipid II and undecaprenyl pyrophosphate. Nisin/receptor complexes disrupt cytoplasmic membranes, inhibit cell wall synthesis and dysregulate bacterial cell division. Gram-negative bacteria are much more tolerant to antimicrobials including nisin. In contrast to Gram-positives, Gram-negative bacteria possess an outer membrane, the major constituent of which is lipopolysaccharide (LPS). This contains surface exposed phosphate and pyrophosphate groups and hence can be targeted by nisin. Here we describe the impact of LPS on membrane stability in response to nisin and the molecular interactions occurring between nisin and membrane-embedded LPS from different Gram-negative bacteria. Dye release from liposomes shows enhanced susceptibility to nisin in the presence of LPS, particularly rough LPS chemotypes that lack an O-antigen whereas LPS from microorganisms sharing similar ecological niches with antimicrobial producers provides only modest enhancement. Increased susceptibility was observed with LPS from pathogenic Klebsiella pneumoniae compared to LPS from enteropathogenic Salmonella enterica and gut commensal Escherichia coli. LPS from Brucella melitensis, an intra-cellular pathogen which is adapted to invade professional and non-professional phagocytes, appears to be refractory to nisin. Molecular complex formation between nisin and LPS was studied by solid state MAS NMR and revealed complex formation between nisin and LPS from most organisms investigated except B. melitensis. LPS/nisin complex formation was confirmed in outer membrane extracts from E. coli.