Damanhour University

Faculty of Pharmacy
Pharmaceutical Chemistry Department

Drug Design – Lab. Manual

PC5107 - 5th Year
Prepared by
Dr. Shaymaa Emam Kassab
(2021-2022)
 Assignment (3)
Instructions
Deadline: Sunday 7.11.2021 at 11:00 p.m.
Material: PPT presentation of the assignment supported by
voice record for each slide of the requested 8 slides.
Any other form of the material will not be accepted!
The voice will be rechecked to verify the owner of the
material, any kind of cheating, the student will be subject to
ZERO grade on all the upcoming assignments.
 Identification of Navoximod as selective Indoleamine-2,3-dioxygenase 1
(IDO1) inhibitor

Fig. 2. The storyline of Navoximod discovery starting from the hit structure
identification to the elaboration of the clinical candidate (Navoximod).

The discovery of Navoximod (GDC-0919) started in 1989 with the

identification of 4-phenylimidazole (4-PI), having a drug-like profile with a weak
non-competitive hIDO1 inhibiting activity (IC50 = 28 µM)[24, 25]. Then,
spectroscopic measurements and the complex crystal structure of the hIDO1 with 4-
PI (PDB code: 2D0T)[26] proved the suggested profile. Besides, the X-ray image of
the complex showed that N3-4-PI [Figure 2 (1)] coordinates with Fe-heme. Based
on the above findings, NewLink Genetics (NLG) corporation group worked
diligently on 4-PI motivated by the structure-activity relationship studies disclosed
by Kumar et al. [27] tried to improve the activity against hIDO1, highlighting some
considered fundamentals in the modification axes of the hit [23]:

(i) introduction of 2`-OH to 4-PI [Figure 2 (2)] improves the activity 16 times (IC50
= 1.7 µM) the original ligand 4-PI (IC50 = 28 µM) but 2',6`-dihydroxy-4-PI gives
insignificant effect, (ii) phenyl ring accommodates small-size groups and the activity
significantly increases with the introduction of 3`-F,5`-Cl to 2`-OH-4-PI [Figure 2
(3)] to exhibit a 5-fold increase in the activity (IC50 = 0.3 µM), (iii) the short half
lifetime (t½=1h) of 2`-OH, 3`-F, 5`-Cl-4-PI [Figure 2 (3)] is attributable to the
metabolic-labile phenolic OH group, (iv) improvement of the ligand biochemical
activity, and PK profile without compromising the cellular activity is challengeable,
(v) 2`-OH, 6`-cyclohexylethoxy-4-PI [Figure 2 (4)] gives equipotent compound
(IC50 = 1.7 µM) to 2`-OH-4-PI [Figure 2 (2)] but weak cellular activity (EC50 = 80
µM).

The outcomes of the structural modification strategies forced the group to

expand the view on the chemotype modification plan to overcome the low cellular
activity and the metabolic liability. The group tried with the molecular rigidification
of 4-PI to reduce the rotatable bonds of the inhibitor that might be the reason for the
low cellular potency [28, 29]. One carbon linked the 2`-carbon with N1 of 4-PI to
generate the imidazo[5,1-a]isoindole congener [Figure 2 (5)]. The impact on the
inhibiting activity of the rigid 9-OH-imidazoisoindole inhibitor [Figure 2 (5)] (IC50
= 3.1 µM) is 4-fold the original ligand 4-PI (IC50 = 28 µM). The molecular
rigidification is favorable for improving the potency and is open to accommodate
different variables according to the molecular modeling and docking results into the
hIDO1 active site.

Identification of the freely rotating 2`-cyclohexylethoxy-4-PI [Figure 2 (4)]

having improved anti-enzymatic activity (IC50 = 1.7 µM) motivated the group to
introduce the ethylcyclohexyl group at carbon 5 of imidazoisoindole and enhanced
the activity of the racemic mixture [Figure 2 (6)] against hIDO1 (IC50 = 0.135 µM)
as well as the cellular potency (EC50 = 1.1 µM). The enhanced biochemical activity
is attributable to the deeper interactions with the B-pocket amino acid residues and
the 7-propionate-heme [30]. The docking studies into the active site of hIDO1 (PDB
code: 2D0T) revealed that the interaction with the inhibitor is stereospecific as it fits
favorably with R-enantiomer but not S-enantiomer due to the steric clash. The
preparation of the 11-hydroxy-derivative [Figure 2 (7)] allows interaction of the
introduced hydroxyl group with the 7-propionate-heme via hydrogen bond and the
hydrophobic interaction of the cyclohexyl group with Phe226. The introduction of
11-OH imparts 4-different diastereomers that are difficult to isolate. So, the
biochemical assay was applicable for the diastereomeric mixture to show a 2-fold
increase in the biochemical activity (IC50 = 0.060 µM) consistently with a 13-fold
improvement in the cellular potency (EC50 = 0.083 µM). Then, the chiral separation
of the 11-hydroxy derivative [Figure 2 (7)] identified the S,S-diastereomer as the
most potent inhibitor (hIDO1 IC50 = 0.021 µM) and emphasized the substantial
importance of C5-S-isomer for potency that dramatically abolishes the activity when
it interconverts to R-isomer (hIDO1 IC50 =18 µM). The pyridine is not a helpful
substitution to cyclohexyl because such hydrophilic radical can't accommodate near
the hydrophobic region of Phe226 in the active site. The 11-
hydroxyimidazoisoindole [Figure 2 (7)] exhibits rapid-hepatic clearance due to the
first-pass metabolism of the cyclohexyl group [31] coupled with the high potency of
the inhibitor against CYP 3A4 (IC50 = 0.17 µM). They mitigated these PK problems
by improving the polarity of the compound without compromising the biochemical
potency. Substitution of the 11-hydroxyimidazoisoindole [Figure 2 (7)] with a
fluorine atom affords the 6-fluoro derivative [Figure 2 (8)] that reduces the potential
inhibiting activity against CYP3A4 at (IC50 = 2.00 µM) 10-fold the 11-hydroxy
derivative [Figure 2 (7)] without alteration of the biochemical potency at (hIDO1
IC50 = 0.030 µM). Interestingly, the introduction of the hydrophilic 15-OH group to
the cyclohexyl ring of 6-fluoro-11-hydroxy-imidazoisoindole [Figure 2 (9)] reduces
the rate of the hepatic clearance (cLogP = 1.55) via first-pass metabolic liability and
decreases in CYP 3A4 inhibiting activity at (IC50 = 3.30 µM). The anti-enzymatic
potency maintains at (hIDO1 IC50 = 0.20 µM). The profile of the resulting derivative
[Figure 2 (9)] shows the best balance of potency, metabolic stability, and on-target
activity. Eventually, the S,R-trans-stereoisomer [Figure 2 (Navoximod)] is the best
to show potential activity at (hIDO1 IC50 = 0.028 µM), cellular potency at (IC50 =
0.075 µM), CYP 3A4 inhibiting activity at (IC50 = 5.70 µM) and hepatic clearance
is very acceptable. The kinetic study verified the reversible non-competitive
mechanism of inhibition at a high concentration of L-Trp [32]. The license of
Navoximod (NLG919) is currently under the authorization of Genentech Inc. (GDC-
0919).

The complex crystal structure of hIDO1 with Navoximod (PDB code: 6O3I)
[71] revealed the binding pattern of the inhibitor to the protein binding site. It
emphasized the occupancy of the imidazoisoindole core structure to the hydrophobic
space of the A-pocket to look coated with the hydrophobic amino acid residues of
Phe163, Phe164, Val130, and Tyr126 (Figure 3). Moreover, Cys129-SH was
proximal to 6-fluorine of Navoximod without any opportunity for fluorine-sulfur
interaction [71]. The interaction of the inhibitor extended to B-pocket via π-stacking
of the 15-hydroxycyclohexyl fragment with Phe226 and hydrogen bond with Ser235
(Figure 9). 11-OH formed H-bond with 7-propionate-Heme at a reasonable distance.

Fig. 9. Complex crystal structure of hIDO1 with Navoximod (PDB code: 6O3I). The active site amino acids-
labeled by the coded three letters/number. Sidechains of the amino acid residues of the active site-assigned
by atoms, oxygen (red), nitrogen (blue), carbon (grey), sulfur (yellow). The ligand inhibitor is pink-colored
with heteroatoms: oxygen (red), nitrogen (blue), and fluorine (green). Heme is turquoise-colored with
heteroatoms: oxygen (red) and nitrogen (blue). Hydrogen bonds are brown-dotted lines. Hydrogen bonds
are brown-dotted lines.

Phase I clinical trials of Navoximod combined with the checkpoint inhibitor,

Atezolizumab [85] on patients with advanced solid tumors, showed acceptable
safety. However, there is no clear evidence of the therapeutic benefit [91]. In another
trial, it is well-tolerated at a high dose (800 mg) and gives a stable disease response
[92].

After reading carefully the story of Navoximod identification, pick up

from the storyline the steps listed 1-8 and let us know via PPT-voice
record the action plan taken to achieve each step.