Investigating the anti-cancer potential of pyrimethamine analogues through a modern chemical biology lens.

Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents.

Mycobacterium tuberculosis Infection of THP-1 Cells: A Model for High Content Analysis of Intracellular Growth and Drug Susceptibility.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the leading cause of death from any infectious agent worldwide, with an estimated 10 million new cases in 2019. Drug development efforts for TB have classically relied on in vitro screening campaigns without consideration for Mtb's established intracellular lifestyle, which may not reflect true drug susceptibility in vivo. Here, we introduce two intracellular screening techniques based on the detection of different fluorescent markers to enumerate bacterial burden in THP-1 monocyte derived macrophages. These techniques are able to distinguish actively growing bacteria from killed bacteria by two distinct methodologies, with the use of cell wall intercalating dye DMN-Tre or an RFP expressing Mtb. This method may also be utilised in the screening of mutant Mtb libraries to evaluate the mutations' effect on drug susceptibility and vice versa. As current high content platform technologies are able to perform a variety of functions, these techniques are broadly applicable to a multiplicity of intracellular screens. We further provide a comparison of infection techniques that may be used for drug screening (batch infection) and high content host-pathogen interaction analysis (2-day differentiation). The aim of this text is to provide the user with a solid and reproducible starting point to high content screening of intracellular Mtb, and to highlight adaptations to the protocol that may aid in future assay development.