G. Ma et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115902
Fig. 5. Active site of NDM-1 bound with compounds with extra phenyl ring. A) Superposition of the active sites of NDM-1 bound with compound 3 (6-membered
ring, green) and compound 11 (6-membered ring connected with extra phenyl ring, blue). The residues, zinc ions and water molecules are shown as same as
described in Fig. 2. B) Stereo view of the active site of NDM-1 in complex with a racemate dimer of compound 11. The racemate compound was connected by a
disulfide bond. The half corresponding to compound 11 is colored with light green carbon atoms and the racemate half is colored with dark green carbon atoms. The
carboxylate group coordinates to Zn2 and H-bonds to residue K211. A hydroxide bridges the two zinc ions. Extensive hydrophobic interactions formed between the
compound and residues in L3 loop (M67, F70, V73) and L10 loop (L218) and W93. Pi-pi stacking formed among the ring structures of residue F70 and the two rings in
the racemate dimer. Residue N220 in L10 loop swings away from the compound.
loop, which contributes the major inhibition potency for D-captopril. We
wondered if the 5-membered proline ring can be replaced with a 6- or
even 7-membered ring thus two new compounds, 3 and 4, were syn-
thesized corresponding to the 6- and 7-membered ring derivatives,
respectively. Enzymatic assays showed that the 7-membered ring de-
rivative (compound 4) retained similar inhibition potency (IC50 = 20.4
µM) with that of D-captopril (21.3 µM in our assay condition and 20.1
µM in reference, 7.9 µM in reference ) while the 6-membered ring
derivative (compound 3) inhibited the activity of NDM-1 more effec-
tively with IC50 of 6.8 µM. Structural studies showed that the 6-
membered ring formed more hydrophobic interaction with the side
chains of residues M67 and F70 (Fig. 4A and Fig. S1A&C) and the stable
H-bond in D-captopril was kept unchanged. In contrast, for the 7-
membered ring derivative, the hydrophobic interaction was weakened
possibly due to the larger ring size and the farther distance from residue
F70 (Fig. 4A and Fig. S1B&D). And a H-bond formed between the
carboxylate group in the 7-membered ring derivative and a different
water molecule that was not stabilized by residues L218 and H189
further tested if replacing the carboxylate group with an amide group
(compound 7) would affect its inhibition potency. Compared to the para-
carboxylate (compound 6), the hydrophobic interaction with residue
F70 is retained for compound 7 while the H-bonds with water molecules
were weakened either by longer distance or due to lack of stabilization
by nearby residues (Fig. 4B and Fig. S2C&D). And hence, the IC50 for
compound 7 is 11.1 µM, a little bit higher than that of compound 6.
Residue K211 plays an important role in the hydrolysis of β-lactams
by NDM-1 through salt-bridging with the β-lactam carboxylate
group.9 Recently, a reaction compound was designed to modify res-
2
6
7
,29
3
0
idue K211 effectively inhibited the activity of NDM-1. The MBL sub-
class B1 enzymes BlaB, IMP-1, VIM-2 and BCII2
6,31,32
share a similar
lysine or arginine residue functioning like K211 in NDM-1. In our
modelling based on compound 6 (para-carboxylate), it was possible to
extend the para-carboxylate group to form a direct salt-bridge with the
terminal amine of residue K211, which might further stabilize the
compound at the active site. We then synthesized two compounds, one
with carboxylate group (compound 8) and the other with amide (com-
pound 9). However, enzymatic inhibition assays showed that their in-
hibition potency did not increase as expected (IC50 of 19.8 and 27.9 µM
for compound 8 and 9, respectively). Structural analysis revealed similar
hydrophobic interaction with residue F70 compared to that for com-
pound 7 (Fig. 4C and Fig. S3). Similar to that for compound 6, one stable
H-bond formed with a water molecule that was stabilized by the main
chain carbonyl oxygen of residue H250. And the other H-bond formed
with an unstable water molecule.
(
Fig. 4A). Up to now, the 6-membered ring derivative (compound 3) is
the one with the highest inhibition potency and we will further develop
compounds based on this 6-membered ring structure.
The carboxylate groups in both D-captopril and compound 3 form H-
bonds with the main chain amine of residue N220 and also a stable water
molecule (Fig. 2B and Fig. 4A). Based on our compound modelling, it is
possible to further enhance the H-bonding interaction with main chain
amine or carbonyl groups of residues in L10 loop. We then synthesized
several compounds to evaluate their inhibition effects. The carboxylate
group was moved to the meta- or para-position of the 6-membered ring
to yield compound 5 and compound 6, respectively. The inhibition po-
tency of compound 5 (IC50 = 28.4 µM) is lower than that of compound 3
The hydrophobic interaction between different compounds and
residues in L3 loop plays essential role in the tight binding of com-
pounds, which was evidenced by substitution of the proline ring with the
6-membered ring in compound 3. Further, we synthesized compounds
with an extra phenyl ring aimed to enhance the hydrophobic interaction.
In one design, the extra phenyl ring was added as in compound 10.
Enzymatic assays showed very weak inhibition activity for compound 10
with IC50 more than 300 µM. Compound docking suggests that the extra
phenyl ring might be too near to residues L73 in L3 loop and H250 at the
active site. Next, another phenyl ring was added as in compound 11.
Structural determination revealed that the bulky phenyl ring pushed the
side chain of residue F70 outward but still formed strong hydrophobic
interaction with residue M67 and F70 (Fig. 5A). Enzymatic assays also
supported its higher inhibition potency with IC50 of 4.6 µM.
(
carboxylate at the ortho-position) and is comparable to that of D-
captopril. In contrast, movement of the carboxylate group to the para-
position (compound 6) increases its inhibition potency to IC50 of 4.9 µM.
Again, we solved the high-resolution structures of both compounds with
NDM-1 and studied the interactions between the active site of NDM-1
and the compounds (Fig. 4B an Fig. S2). Structural analysis showed
that compared to the ortho-carboxylate, meta-carboxylate showed less
hydrophobic interaction with residue F70 and the H-bond with the main
chain amine of residue N220 was also weakened (Fig. 4B and
Fig. S2A&B). In contrast, the hydrophobic interaction is strengthened in
the para-carboxylate compared to the ortho-carboxylate (Fig. S2C&D),
where it forms two H-bonds with two water molecules that are stabilized
by residues N220, K211 and one active site coordination residue H250
The structure for NDM-1 in complex with compound 11 (with an
extra phenyl ring) was obtained by soaking compound 11 into pre-
formed NDM-1 crystals for a period of minutes to hours. The structure
is similar to that of NDM-1/D-captopril where one NDM-1 molecule
(
Fig. 4B and Fig. S2C&D). For the para-carboxylate compound, we
5