Structure-Activity Relationship Studies of Mitochondrial Uncouplers and PilB Inhibitors

Abstract

Obesity is a chronic, noncommunicable disease caused by the excessive accumulation of body fat. Rates of diagnosis have been increasing in the late 20th and early 21st centuries, with associated comorbidities also on the rise. Consequently, obesity has become a growing health epidemic globally. Development of novel pharmacological therapeutics with limited side effects is thus desirable. Chemical mitochondrial uncouplers are protonophoric, lipophilic small-molecules that transport protons from the mitochondrial intermembrane space into the matrix independent of ATP synthase, thus uncoupling nutrient oxidation from ATP production. Such a mechanism has been previously shown to be effective in mouse obesity models. Our previous work identified BAM15 (EC50 1.5 M) as a potent and efficacious mitochondrial uncoupler with potential for obesity treatment. In this study, we investigate in vitro and in vivo properties of hydroxylamine and hydrazine BAM15 derivatives and reveal the high uncoupling nature of these compounds. Our structure-activity relationship (SAR) studies demonstrated that the hydroxylamine BAM15 analogs are more potent than hydrazine ones, with particular note of hydroxylamine ester derivatives being the most efficacious of all structures assayed. For example, the most efficacious of the hydrazine series was 1.12.1 with an EC50 value of 4.6 M and 103% activity of BAM15 while compound 1.11.5 was the best among the hydroxylamine series with EC50 value of 340 nM and 118% BAM15 mitochondrial uncoupling activity in rat L6 myoblasts. Pharmacokinetic profiling of 1.12.1 and 1.11.5 indicated low exposure (2-220 nM) and short half-life (15-27 min) in mice. In a separate study, we investigated the structure-activity relationship of benserazide in the pursuit of treating drug-resistant bacterial diseases. Antimicrobial resistance is an imminent health threat worldwide due to the increasing resistances to common antibiotics of many lethal pathogens. Overuse and underdevelopment of novel antibiotics, which target bacterial cell survival, has exacerbated the problem by enabling bacteria to develop resistance mechanisms to their mechanisms of action. Development of alternative treatments for drug-resistant microbes is thus of paramount importance. Targeting virulence factors, non-vital components of pathogens that enable them to cause disease such as the type IV pilus construction enzyme PilB, is a strategy of treatment. PilB is a highly conserved hexameric ATPase responsible for pilus generation and is thus obligatorily involved in bacterial type IV pilus use. Previously, the type IV pilus has been shown to be critically important in the pathogenesis of various drug-resistant microorganisms and so presents as a suitable drug target. Recently, we reported the discovery of a potent inhibitor of PilB, the Parkinson's Disease-treating drug benserazide (IC50 3.68 μM). Herein, we report the SAR profiling of benserazide analogues and identify key moieties that enable PilB inhibition. We found that bis-hydroxyl groups on the ortho position of the aryl ring, a rigid imine, and exchange of the serine for a thiol have resulted in marked improvement in potency. Our studies identified 2.12.3 as a PilB inhibitor with an IC50 of 580 nM and selectivity for PilB over an unrelated ATPase, apyrase. These compounds provide the chemical tools to validate virulence factors as antibacterial mechanisms of action.