Mitochondrial Uncouplers: Development as Therapeutics for Metabolic Diseases

Obesity and its comorbidities have emerged as serious healthcare concerns in the western world due to increased prevalence of nutritional overabundance and decreased physical activity. Due to the significant population affected and economic burden placed on national healthcare systems, there is a demonstrated need for effective weight management therapeutics. Obesity presents clinically diverse phenotypes that increase a person's susceptibility to comorbidities that commonly result in deteriorated health (cardiovascular disease, diabetes mellitus, hypertension, etc). A comorbidity of specific relevance is non-alcoholic fatty liver disease (NAFLD) and its advanced disease state known as non-alcoholic steatohepatitis (NASH), as it has had a documented rise in prevalence parallel to that observed with obesity. Currently there are no FDA approved therapeutics for NAFLD or NASH, with the majority in clinical development aiming to mitigate the effects caused by accumulation of adipose tissue in the liver known as steatosis. An alternative therapeutic approach is to use small molecules to uncouple oxidative phosphorylation in the mitochondria by passively shuttling protons from the mitochondrial inner membrane space into the mitochondrial matrix. Mitochondrial uncoupling results in the disruption of the proton motive force leading to an upregulation of metabolism (i.e., decrease in steatosis). Small molecule mitochondrial uncouplers have recently garnered great interest for their potential in treating the advanced disease state of NASH. In this study, we report the structure-activity relationship (SAR) profiling of a 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol core, which utilizes the hydroxy moiety as the proton transporter across the mitochondrial inner membrane. We demonstrated that a wide array of substituents are tolerated with this novel scaffold that increased cellular metabolic rates in vitro using changes in oxygen consumption rate as a read-out. In particular, compound SHS4121705 (2.12i) displayed an EC50 of 4.3 M in L6 myoblast cells and excellent oral bioavailability and liver exposure in mice. In the preclinical stelic animal model (STAM) mouse model of NASH, administration of 2.12i at 25 mg kg-1day-1 resulted in decreased liver triglyceride levels and improved liver enzymes, NAFLD activity score, and fibrosis without affecting body temperature or food intake. Overall, our initial studies showcased the promise of mitochondrial uncouplers toward the treatment of NASH.
While initial results were promising, the lead compound 2.12i had reduced potency compared to the alkyl derivatives reported in the SAR, unfortunately alkyl derivatives suffered from poor physiochemical properties, possibly due to metabolism of the alkyl chain. We hypothesized that addressing metabolic liabilities of these compounds could lead to increased potency with maintained efficacy in the STAM mouse model of NASH. Herein, we detail the SAR profiling of a 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol core derivatized with 1,1'-biphenyl anilines capable of eliciting mild mitochondrial uncoupling. A wide array of substituents are tolerated, and demonstrated sustained and stable increases in ¬cellular oxygen consumption rates over a broad concentration range. In particular, compound SHS4091862 (3.9b) displayed an EC50 of 2.0 μM in L6 myoblast cells with a pharmacokinetic profile of Cmax = 46 μM and t1/2 = 4.7 h indicating excellent oral bioavailability. Administration of 3.9b at 60 mg kg-1 day-1 in the STAM mouse model of NASH decreased fibrosis, steatosis, and hepatocellular ballooning to result in a 1.9-point decrease in NAFLD activity score (NAS) compared to vehicle. No changes in food intake, body weight, alanine transaminase (ALT) or aspartate transaminase (AST) levels were observed with 3.9b. Positive control Resmetirom afforded a 1.2-point decrease in NAS score, but increased ALT levels. Cumulatively, our work demonstrates the therapeutic potential of small molecule mitochondrial uncouplers to address metabolic diseases, namely NAFLD.