Diethyl fumarate exhibits potent activity against differentially culturable Mycobacterium tuberculosis persisters

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of death from a single infectious agent. During infection, Mtb forms drug-tolerant persisters, including differentially culturable (DC) cells that fail to grow on solid media and contribute to prolonged TB treatment and disease relapse. DC Mtb formation is driven by host inflammatory responses, particularly nitric oxide (NO), suggesting that anti-inflammatory agents may limit persister generation. Here, we developed a workflow to evaluate the activity of drug candidates against DC Mtb persisters. This approach was validated using dimethyl fumarate (DMF) derivatives, as DMF has previously been shown to eradicate DC Mtb but is limited by instability and poor solubility. We assessed DMF and nine DMF derivatives for antimycobacterial activity. The workflow included antimicrobial susceptibility testing against Mycobacterium tuberculosis (Mtb) and Mycobacterium abscessus (Mabs), followed by cytotoxicity evaluation and measurement of NO release in lipopolysaccharide (LPS)-stimulated macrophages. Selected DMF derivatives were further tested against DC Mtb persisters generated in vitro using a NO donor as well as persisters recovered from interferon γ-stimulated macrophages. DMF and diethyl fumarate (DEF) inhibited Mtb growth, while ethyl-3-benzoylacrylate (EBL) and methyl-trans-4-oxo-2-pentenoate (MTOP) exhibited bactericidal activity against both Mtb and Mabs. DEF effectively eliminated DC Mtb in infected macrophages, with activity comparable to DMF. This anti-DC Mtb persister effect was associated with reduced NO production, highlighting the importance of anti-inflammatory activity in targeting DC Mtb persisters. DMF and its more soluble and stable derivative DEF represent promising candidates for the development of therapies targeting DC Mtb persisters. Their dual anti-inflammatory and anti-mycobacterial properties support their potential as a novel therapeutic strategy against persistent tuberculosis infections.