In vitro and in vivo activity profiles of broad-spectrum bacterial ATP synthase inhibitors

Tomatidine (TO), a natural steroidal alkaloid derived from Solanaceae (e.g., tomato), is recognized for its narrow-spectrum antibiotic activity, particularly against persistent forms of Staphylococcus aureus, such as small-colony variants (SCVs). Previous studies have shown that TO exerts its effect by inhibiting S. aureus ATP synthase. In earlier work, we synthesized nearly 100 TO analogs featuring an ethylenediamine linker branched with aromatic substituents at the C3 position, and demonstrated that several of these analogs possess notable antibacterial activity against typical (non-SCV) S. aureus strains, including methicillin-resistant S. aureus (MRSA), with minimum inhibitory concentrations (MICs) ranging from 1 to 4 μg/mL. Among these, analogs incorporating an indole (TM-247) or para-substituted aryl moiety (TM-184, -I; TM-218, -Br; TM-220, -Cl; TM-303, -CF₃) emerged as lead candidates, exhibiting potent antibacterial activity in vitro against both Gram-positive and Gram-negative bacteria, including S. aureus SCVs. In the present study, we conducted a comprehensive antibacterial profiling of this compound series, including the predecessor compound TM-02, against a panel comprising 16 Gram-positive strains, 16 antibiotic-resistant Escherichia coli isolates, and two multidrug-resistant Acinetobacter baumannii strains. TM-184 emerged as the most promising candidate and was subsequently subjected to an expanded in vitro evaluation across 24 clinically relevant Gram-negative bacterial species. Furthermore, TM-184 was assessed in vivo using a neutropenic mouse thigh infection model against E. coli, demonstrating significant efficacy and resulting in a substantial reduction in bacterial burden.