Discovery of novel and potent benzhydryl-tropane trypanocides highly selective for Trypanosoma cruzi

A benzhydryl tropinone oxime that is potently toxic to Trypanosoma cruzi has been previously identified. An SAR investigation determined that no part of the original compound was superfluous and all early SAR probes led to significant drops in activity. The only alteration that could be achieved without loss of activity was replacement of the aryl chloride substituent with chloro homologues. This led to the discovery of a trifluoromethyl-containing analogue with an EC50 against T. cruzi of 30 nM and a cytotoxicity selectivity index of over 1000 relative to rat skeletal myoblast L-6 cells

Compound-mediated assay interferences in homogenous proximity assays.

Homogeneous proximity assays are widely implemented in high-throughput screening (HTS) of small-molecule libraries for drug and probe discovery. Representative technologies include amplified luminescent proximity homogeneous assays (ALPHA, which is trademarked by PerkinElmer; also informally referred to as Alpha), Förster/fluorescence resonance energy transfer (FRET), time-resolved FRET (TR-FRET) and homogeneous time-resolved fluorescence (HTRF, which is trademarked by CisBio), bioluminescence resonance energy transfer (BRET), and scintillation proximity assays (SPA). While highly useful, these assay technologies are susceptible to a variety of technology-related compound-mediated interferences, most notably signal attenuation (e.g., through quenching, inner-filter effects, light scattering), signal emission (e.g., auto-fluorescence), and disruption of affinity capture components such as affinity tags and antibodies. These assays are also susceptible to more generalized compound-mediated interferences such as nonspecific reactivity and aggregation. This chapter describes (a) the basic principles of proximity assays, (b) common sources of compound-mediated assay interference in homogenous proximity assays, and (c) counter-screens and other strategies to classify compound-mediated assay interferences in homogenous proximity assays. This information should be useful for prioritizing bioactive compounds in homogenous proximity assays for drug and chemical probe discovery

Identification of 3-aminothieno[2,3-b]pyridine-2-carboxamides and 4-aminobenzothieno[3,2-d]pyrimidines as LIMK1 inhibitors

A high throughput chemical screening campaign has led to the identification of 3-aminobenzo[b]thiophene-2-carboxamides as LIMK I inhibitors. Evolution of bicyclic hits to the tricyclic 4-aminobenzothieno[3,2-d]pyrimidine, using a traditional medicinal chemistry SAR guided approach, resulted in a significant increase in potency. Further elaboration has seen the 7-phenyl-4-aminobenzothieno[3,2-d]pyrimidine emerge as a LIMK I inhibitor lead candidat

1,2,4-Oxadiazole antimicrobials act synergistically with daptomycin and display rapid kill kinetics against MDR Enterococcus faecium

Background: Enterococcus faecium is an important nosocomial pathogen. It has a high propensity for horizontal gene transfer, which has resulted in the emergence of MDR strains that are difficult to treat. The most notorious of these, vancomycin-resistant E. faecium, are usually treated with linezolid or daptomycin. Resistance has, however, been reported, meaning that new therapeutics are urgently needed. The 1,2,4-oxadiazoles are a recently discovered family of antimicrobials that are active against Gram-positive pathogens and therefore have therapeutic potential for treating E. faecium. However, only limited data are available on the activity of these antimicrobials against E. faecium. Objectives: To determine whether the 1,2,4-oxadiazole antimicrobials are active against MDR and daptomycinnon- susceptible E. faecium. Methods: The activity of the 1,2,4-oxadiazole antimicrobials against vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium was determined using susceptibility testing, time-kill assays and synergy assays. Toxicity was also evaluated against human cells by XTT and haemolysis assays. Results: The 1,2,4-oxadiazoles are active against a range of MDR E. faecium, including isolates that display nonsusceptibility to vancomycin and daptomycin. This class of antimicrobial displays rapid bactericidal activity and demonstrates superior killing of E. faecium compared with daptomycin. Finally, the 1,2,4-oxadiazoles act synergistically with daptomycin against E. faecium, with subinhibitory concentrations reducing the MIC of daptomycin for non-susceptible isolates to a level below the clinical breakpoint. Conclusions: The 1,2,4-oxadiazoles are active against MDR and daptomycin-non-susceptible E. faecium and hold great promise as future therapeutics for treating infections caused by these difficult-to-treat isolates

Development of 1,2,4-Oxadiazole Antimicrobial Agents to Treat Enteric Pathogens within the Gastrointestinal Tract

Colonization of the gastrointestinal (GI) tract with pathogenic bacteria is an important risk factor for the development of certain potentially severe and life-threatening healthcare-associated infections, yet efforts to develop effective decolonization agents have been largely unsuccessful thus far. Herein, we report modification of the 1,2,4-oxadiazole class of antimicrobial compounds with poorly permeable functional groups in order to target bacterial pathogens within the GI tract. We have identified that the quaternary ammonium functionality of analogue 26a results in complete impermeability in Caco-2 cell monolayers while retaining activity against GI pathogens Clostridioides difficile and multidrug-resistant (MDR) Enterococcus faecium. Low compound recovery levels after oral administration in rats were observed, which suggests that the analogues may be susceptible to degradation or metabolism within the gut, highlighting a key area for optimization in future efforts. This study demonstrates that modified analogues of the 1,2,4-oxadiazole class may be potential leads for further development of colon-targeted antimicrobial agents

Nuisance compounds in cellular assays.

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity (“nuisance compounds”) are routinely encountered in cellular assays, including phenotypic and high-content screening assays. Much is known regarding compound-dependent assay interferences in cell-free assays. However, despite the essential role of cellular assays in chemical biology and drug discovery, there is considerably less known about nuisance compounds in more complex cell-based assays. In our view, a major obstacle to realizing the full potential of chemical biology will not just be difficult-to-drug targets or even the sheer number of targets, but rather nuisance compounds, due to their ability to waste significant resources and erode scientific trust. In this review, we summarize our collective academic, government, and industry experiences regarding cellular nuisance compounds. We describe assay design strategies to mitigate the impact of nuisance compounds and suggest best practices to efficiently address these compounds in complex biological settings. Nuisance compounds can waste significant resources by producing promising bioactivities that are attributable to undesirable mechanisms of action. Addressing nuisance compounds is particularly challenging in cellular assays. Dahlin et al. summarize academic, government, and industry experiences with assay design and hit triage to specifically address cellular nuisance compounds