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HSG4112 superseded glabridin and is a new chemical entity.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.