V. Ruiz, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Figure 2. Glide screen compound ChDHFR inhibition screen. (A) ChDHFR percent activity in the presence of 500 µM Glide screen compounds. (B) Chemical structure
of compound 15, which inhibited ChDHFR by 52.2%.
Figure 3. Kinetic analysis of compound 15. (A)
Steady-state competition assay with varying con-
centrations of dihydrofolate: 5 μM, 10 μM, 25 μM,
5
0 μM, and 100 μM. Data fit to a hyperbolic equation.
(
B) Lineweaver-Burk plot for data in (A). For all
graphs, data points with circle (●) denote the reac-
tion with no inhibitor, data points with square (■)
denote the reaction with 100 μM compound 15, and
data points with diamond (◆) denote the reaction
with 250 μM compound 15. Steady-state competition
assay with respect to 100 µM NADPH at concentra-
tions of 0 µM, 100 µM and 250 µM of compound 15
indicated noncompetitive inhibition (data not
shown).
−
1
1
.20 ± 0.04 s , respectively, while the change in K
at 1.2 ± 0.2 μM, 1.3 ± 0.3 μM and 1.4 ± 0.4 μM, respectively (Fig.
A and 3B). One possible explanation for the noncompetitive me-
m
was insignificant
methylphenyl)-3-phenylthiourea moiety produced no greater than 30%
inhibition of ChDHFR (data not shown). On the other hand, several
derivatives of the 2-hydroxy-N-phenylbenzamide moiety demonstrated
significant inhibitory activity (Supplementary Table 2). In particular,
we observed that compounds containing halogen substituents para to
the hydroxyl moiety of the phenolic ring (Fig. 5A, red circle) demon-
strated greater inhibitory activity against ChDHFR. Moreover, com-
pounds containing substituents larger than a methyl group meta to the
hydroxyl moiety of the phenolic ring (Fig. 5A, blue circle) displayed
little ChDHFR inhibition. Finally, we observed that compounds con-
taining chlorine substituents meta to the amide linker of the second
aromatic ring (Fig. 5A, green circles) displayed greater levels of in-
hibition against ChDHFR, while most other substituents to this ring
resulted in compounds with little or no inhibitory activity (Fig. 5A,
Supplementary Table 2). These observations are consistent with the
structure of compounds 15D8, 15D10, 15D12, 15D13, and 15D15
(Fig. 5B), which, at 500 μM, inhibited ChDHFR by 49%, 61%, 61%,
54%, and 47%, respectively. Due to poor solubility at concentrations
greater than 500 µM, we were not able to determine IC50 values for
compounds 15D8, 15D10, 15D12, 15D13, and 15D15, however these
compounds do exhibit similar dose-dependent inhibition of ChDHFR as
observed for the parent compound 15 (Fig. S2).
3
chanism observed is that compound 15 is displacing NADPH, the co-
factor in the ChDHFR-catalyzed reduction of dihydrofolate to tetra-
hydrofolate. If this is the case, then the activity of compound 15 is
mediated by binding to the ChDHFR active site and not the proposed
binding pocket in Fig. 1C. In order to rule out binding to the active site,
we repeated the steady-state rate profile of ChDHFR at varying con-
centrations of compound 15 and NADPH, this time keeping the dihy-
drofolate concentration constant. Due to the limitations of our assay,
we were only able to determine the observed rate with respect to
1
00 µM NADPH. Compound 15 appears to display noncompetitive in-
hibition with respect to NADPH, which is evident by a decrease in kobs
,
−
1
−1
−1
1
.98 ± 0.09 s , 1.60 ± 0.06 s , and 1.33 ± 0.08 s
at 0, 100,
and 250 μM of compound 15, respectively.
Upon closer inspection of subsequent docking models of compound
1
5 with ChTS-DHFR, we observed two types of predicted binding
configurations between the compound and the non-active site pocket.
In the first configuration, the 1-(4-bromo-2-methylphenyl)-3-phe-
nylthiourea moiety of compound 15 is directed into the pocket
(
Fig. 4A), while the 2-hydroxy-N-phenylbenzamide moiety is embedded
in the pocket in the second configuration (Fig. 4B). Furthermore, each
configuration forms unique contacts with residues in the non-active site
pocket. We evaluated these observations by conducting a structure
activity relationship (SAR) study utilizing commercially available
compounds derived from the structures of the two different embedded
moieties of compound 15.
In order to derive a better understanding of the results from our SAR
study, we utilized Glide to model the binding of the SAR compounds
with the non-active site pocket of ChTS-DHFR shown in Fig. 1C. The
majority of models position the phenolic moiety of these compounds
within the non-active site pocket, while exposing a chlorine substituent
to solvent. In some models, the chlorine substituent is positioned near
residue Cys44, located at the C-terminal end of the DHFR B helix. A
At 500 µM of compound, derivatives of the 1-(4-bromo-2-
3