R. Zhang, Z. Song, X. Wang et al.
European Journal of Medicinal Chemistry 216 (2021) 113358
Fig. 1. Structures of inhibitors against PTL or NPC1L1.
Additionally, some PTL inhibitors showed significantly inhibit ac-
tivity for triglyceride elevation in mice model. Moreover, PTL in-
hibitors, orlistat and cetilistat, have been approved for the
treatment of obesity in 1998 and 2003 respectively [15,16].
Medications used for hypercholesterolemia include: cholesterol
biosynthetic inhibitors (statins), cholesterol absorption inhibitors,
proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors,
and bile acid chelating agents. The combination of cholesterol
biosynthetic inhibitors and absorption inhibitors is the first choice
for the treatment of hypercholesterolemia. Targets for cholesterol
absorption inhibitors (e.g., ezetimibe) have been identified as
Niemann-Pick C1-like 1 (NPC1L1) that is specific transporters for
cholesterol [17]. NPC1L1 has attracted more and more attention
due to its own characteristics. It is abundantly expressed in the
small intestine of humans, and responsible for transporting
cholesterol to across the apical membrane of enterocytes [18]. In
genetically NPC1L1-deficient mice, a 70% reduction in intestinal
cholesterol absorption was seen, which indicated that NPC1L1
plays an essential role in promoting intestinal cholesterol uptake
[19]. Reducing absorption of cholesterol by inhibiting NPC1L1 also
has the added advantage that there was no effect on the absorption
of fat-soluble nutrients such as fat-soluble vitamins, triglycerides,
or bile acids [20]. Thus, it has been identified as an attractive
therapeutic target to lower cholesterol blood levels. There are few
reports of NPC1L1 inhibitors at present. The most classic NPC1L1
inhibitor is ezetimibe, which has gained approval from Food and
Drug Administration in 2002 for the treatment of dyslipidemia (e.g.
hypercholesteremia) [21,22]. As a potent cholesterol absorption
inhibitor, it could effectively prevent the absorption of cholesterol,
thus lower circulating plasma cholesterol in humans by 15e20%
as we know, there were no dual-target inhibitors for PTL and
NPC1L1 up to today. In the present study, dual-target inhibitors for
PTL and NPC1L1 were designed, and dual-target effect were
demonstrated in enzyme and cell level.
2. Design and synthesis of dual-inhibitors
The design strategy of dual-inhibitors is based on the structure
alignment of classic drugs and computer molecular docking simu-
lation (Fig. 2). Orlistat and ezetimibe were selected as the starting
molecules for the dual-target inhibitor design based on the
following three reasons: (1) their structural commonality (tetra-
atomic ring); (2) the reduction of orlistat on cholesterol levels in
obese patients has been demonstrated previously [27], as well as
ezetimibe was shown to reduce the level of triglycerides in patients
with hyperlipidemia [28]; (3) proven clinical effectiveness [16,22].
In addition, it was worth noting that Saeed Alqahtani et al.’s study
has proved that orlistat could decrease dietary cholesterol ab-
sorption, which was achieved in part by inhibition of NPC1L1 [29].
Their study demonstrated that it was feasible to design NPC1L1
inhibitors based on orlistat. Considering the catalytic serine could
reach the solvent and react with substrate only when the surface
loop is open (Fig. S1), the crystal structure of PTL (PDB: 1LPB) with
open conformation was used to simulate the interaction with in-
hibitors in computer molecular docking and molecular dynamics
(MD) studies [30]. Additionally, crystal structure of NPC1L1 in
complex with an ezetimibe from Ching-Shin Huang et al.’s study
(PDB: 6V3H), revealed mechanisms of cholesterol transport and
ezetimibe inhibition, and therefore was utilized in our present
work for docking and MD studies [31].
The design process is following: (1) Orlistat is the typical
It is important to note that obese patients often exhibit high
levels of plasma cholesterol. In other word, hypercholesteremia and
obesity are often co-exist. However, as far as we know, there are no
drugs designed to treat both hypercholesteremia and obesity at the
same time. Although some studies have demonstrated that the
anti-obesity drug orlistat also showed effect of lowering cholesterol
level when compared to patients receiving placebo, orlistat is still
used as an anti-obesity drug until now, not used for the treatment
of cholesterol-lowering like statins (inhibitors of cholesterol syn-
thesis) [24e26]. As important therapeutic targets for obesity and
hypercholesteremia, PTL and NPC1L1 are of great significance for
the design and development of new therapeutic drugs. Active
compounds that target both PTL and NPC1L1 have the potential to
treat obesity and hypercholesteremia simultaneously. Thus, dual-
target inhibitors for both PTL and NPC1L1 would provide new op-
tions for obese patients with hypercholesterolemia. However, as far
representative of PTL inhibitors, which contains
b-lactone that
could form a covalent bond with the active serine residue site
(Ser152) of PTL, and thus leading to PTL inactivate. Interestingly,
ezetimibe also contains a quaternary cycloamide structure similar
to orlistat. The b-lactone has higher reactivity with the hydroxyl of
serine compared to the lactam structure, so it was used as a starting
component of the dual-target inhibitors. (2) Two alkyl chains of
orlistat fit into a hydrophobic groove of PTL and are thought to thus
mimic the interaction between the leaving fatty acid of a triglyc-
eride substrate and the PTL [30]. Alkyl chains (C-6, C-13) are also
considered as the key groups to achieve PTL inhibition. Thus, two
alkyl chains were also retained in designed dual-inhibitors. (3) For
ezetimibe, the most significant feature is the presence of three
substituted benzene rings, except for the lactam ring. In contrast,
orlistat has three distinct alkyl chains, beside
Therefore, we hypothesized that the modification with substituted
b-lactone ring.
2