Discussion:
In this study, three main findings related to HSG4112 arose. First, four structural modifications to glabridin – 3”,4” double bond hydrogenation, C-2’ hydroxy group preservation, C-4’ etherification, and enantiomerization – enhanced efficacy and stability. Second, HSG4112 had robust effect on HFD-mice, in adiposity, fatty liver, and transcriptome. Third, HSG4112 increased energy expenditure in mice and glucose uptake in muscle cell.
HSG4112 superseded glabridin and is a new chemical entity. Some evidence exists in literature to explain its superiority. Dehydrogenation at C-3”,4” double bond could have increased activity because of more structural flexibility that allows the resorcinol group to interact with the target more effectively (Jirawattanapong, Saifah and Patarapanich, 2009). Ethoxylation at 4’ carbon, as Bae et al . (2020) suggests, may have improved drug bioavailability and stability within the body, because metabolic clearance likely happens through glucuronidation of C-4 hydroxy group in glabridin (Guo et al. , 2015), which is absent in HSG4112. Still, the exact biochemical mechanisms of the four modified components are unknown. A greater number of animals per group – not simply for screening purpose – and modifications made directly to glabridin would benefit arguments on SAR of glabridin. Examining the unanswered questions of our SAR study may provide additional drug compounds and aid in understanding of the compound’s molecular mechanism.
Because HSG4112 showed robust in vivo efficacy, NASH and T2DM are ideal secondary target indications. Liver histology results and normalization of parameters related to insulin resistance and glucose imbalance (Paz-Filho et al. , 2012) support HSG4112’s development for the above two diseases, respectively. Furthermore, weight loss itself is immensely beneficial to NASH (Vilar-Gomez et al. , 2015) and T2DM (Wilding, 2014). Because the body weight of HSG4112 group fully normalized at the end of 6-week terminal sacrifice, it is unclear whether all the beneficial effects are primary or secondary to weight loss; therefore, future investigation into primary target and mechanism of action is needed. Hepatocyte ballooning and fibrosis were absent in HFD-mice model, and liver biopsy was not done pre-to-post to account for variability of NASH histology (Jensen et al. , 2020); additional liver fibrosis animal model (Castro and Diehl, 2018) or liver-specific fat accumulation model (Boland et al. , 2019) may be more appropriate.
Increased energy expenditure is the main mode of action of HSG4112, evidenced by transcriptomic and metabolic analysis. Because qRT-PCR was performed on selected 68 genes, unbiased RNAseq at an early timepoint before weight reduction could further benefit transcriptomic analysis and interpretation of primary or causal signals induced by HSG4112. Furthermore, while phosphorylated hypothalamic AMPK levels were tested, AMPK levels in peripheral tissues – specifically liver, muscle, and fat – and in respective cells should be measured for further confirmation of activation of AMPK signaling. Still, within our current approach, we observed consistent gene regulation pattern in the direction towards enhanced energy expenditure: upregulation of fatty acid oxidation, lipid metabolism, and glucose metabolism. This is consistent with signals induced by exercise; the observed increase in PDK4 andPGC-1ɑ levels in muscle tissue similarly occurs after exercising (Ookawara et al. , 2002; Wang and Sahlin, 2012). BecausePGC-1ɑ regulates mitochondrial biogenesis and AMPK is known to mediate mitochondrial fission (Toyama et al. , 2016), HSG4112 may act to improve mitochondrial function or dynamics; this hypothesis is yet to be tested. On the other hand, robust evidence of unaffected UCP1 mRNA and protein level, as well as unchanged body temperature of animals in various toxicology studies, demonstrate that HSG4112 does not increase thermogenesis. It remains unknown whether the above genes are primary or secondary, and such investigation merits attention.
In vitro glucose uptake assay shows that glucose is being transported to muscle cell, indicating enhanced need or use of energy in muscle cell. It is possible that cell-specific glucose transporter is directly affected, or that HSG4112 increases need of energy in muscle; in the latter case, mitochondria is again a promising target organelle of HSG4112 because of its dominant presence in muscle and few presences in WAT. Surprisingly, HSG4112’s effect here is synergistic with insulin at a level markedly greater than that of metformin; this is helpful in surmising the drug’s potential combinatorial effect in obese diabetic patients, who are injected with insulin regularly.
Overall, our results strongly suggest that the main mode of action of HSG4112 is enhanced energy expenditure. The implication of such effect is that HSG4112 presents an innovative method of pharmacological weight-loss that is comparable to ‘exercise’ portion of lifestyle modification, instead of ‘diet’ portion. This is significant in two part: (i) HSG4112 can be used in combination with other anti-obesity drugs, which focus only on ‘diet’ portion; (ii) yo-yo effect is expectedly marginal in HSG4112 administration, because muscle activity and mass are presumably maintained. A key factor that enabled this discovery is in vivo phenotypic screening, which takes into account the holistic aspect of biological pathways, is more human-translatable, and applicable towards other relevant indications. Because of this approach, the exact mechanism of HSG4112 remains unknown and potentially novel; investigation into such mechanism will provide meaningful understanding of the energy metabolism and enable further development of drugs targeting metabolic diseases. Currently, HSG4112 is in phase 1 clinical trial for obesity. Human translation and proof of concept (POC) will in future demonstrate the potent effect of HSG4112 as energy expenditure enhancer treating metabolic diseases.