ACS Infectious Diseases
Article
cleft floor and with the hydrophobic portion of the L68 and
E81 side chains. The 3-hydroxyl-5-chloro phenyl ring, which is
nearly parallel with the cleft floor, enables the 3-hydroxyl group
to interact with the E81 carbonyl oxygen via H-bonding, and
the Cl atom to interact with the side chains of L63 and L68
(Figure 3A,B). The phenyl ring, at its closest point, is also 3.60
Å from the M31 CE atom, a favorable interaction that is also
probable to hinder rotation of the ring toward a perpendicular
position relative to the cleft floor (Figure 3C,D). It is also
interesting to note that the 3-hydroxyl-5-chloro phenyl ring is
positioned to form a pseudo edge-to-face interaction with the
indole ring of W35; the phenyl ring, at its closest position, is
3.77 Å from the indole ring CZ2 atom, the distance between the
centroids of the phenyl and the indole 6-member ring is 5.92
Å, and the angle between the planes of these rings is 67.4°,
suggesting a weak edge-to-face interaction.
Despite significant effort, we could not obtain good
diffraction data from BfrB crystals soaked in solutions
containing KM-5-25. However, we were able to obtain the
structure of the fluorinated analogue JAG-5-7 (Figure 4).
Strong electron density consistent with JAG-5-7 was observed
in 8 of the 12 subunits. The shorter −(CH2)− linker places the
2-hydroxyl-5-fluoro phenyl ring well within the cleft formed by
the side chains of L68 and E81, with the ring nearly
perpendicular to the cleft floor. The electron density from
the 2-hydroxyl-5-fluoro phenyl ring is consistent with the ring
experiencing one orientation in certain subunits (Figure 4A,B)
and with the ring in two conformations in other subunits
(Figure 4C,D). In one of these orientations the hydroxyl group
forms a H-bond with the carbonyl oxygen of G80, whereas the
fluorine atom packs against the side chain of L68. The
structure of KM-5-50 (Figure S2), also places the 3-hydroxyl-
5-chloro phenyl ring within the cleft formed by L68 and E81,
in a similar nearly perpendicular conformation relative to the
cleft floor, except that no H-bonding was observed for the 3-
hydroxyl group.
Taken together, the structures of the 4-aminoisonindoline-
1,3-dione derivatives reported here and those reported
previously25 provide important insights. The linkers and the
phenyl rings of all the compounds containing a −(CH2)3−
linker adopt nearly identical binding modes, with the phenyl
rings oriented parallel to the cleft floor (Figure S3) and
positioned to engage the 6 member ring of the indole in W35.
Superposing the structures of analogue 16 and KM-5-66
(Figure S4A) shows that the phenyl ring in 16 is notably
pitched relative to the phenyl ring in KM-5-66 (the angle
between the mean planes of both rings is 22.6°), and the 3-
hydroxyl group is not engaged in H-bonding interactions. In
comparison, the Cl atom at position 5 in KM-5-66 appears to
induce a nearly parallel orientation of the ring relative to the
cleft floor and a conformation that places the 3-hydroxyl within
H-bonding distance of the carbonyl oxygen of E81. Together,
the H-bonding engagement of the 3-hydroxyl group, the
packing of the Cl atom with the side chains of L63 and L68,
and the more extensive packing of the phenyl ring against the
cleft floor residues are probably responsible for the higher
affinity of KM-5-66 for BfrB relative to 16. It is also interesting
to note that in the structures of all the compounds with a
shorter −(CH2)− linker the phenyl ring is nearly perpendic-
ular to the cleft floor. This is illustrated by superposing the
structures of KM-5-66 and JAG-5-7 (Figure S4B), which
shows that the phenyl rings of both compounds adopt a nearly
perpendicular angle (74°) relative to one another. The
structural information currently available suggests that the
shorter linker and the relatively less efficient packing of the
phenyl ring against the hydrophobic portions of the L68 and
E81 side chains may contribute to the higher Kd values of these
compounds relative to those with a −(CH2)3− linker.
Planktonic P. aeruginosa Cells Treated with 4-
Aminoisoindoline-1,3-dione Derivatives Overproduce
Pyoverdine. Previous studies directed at evaluating the
repercussions of blocking the BfrB-Bfd complex in P. aeruginosa
cells relied on deleting the bfd gene (Δbfd). These
investigations showed that blockade of the BfrB-Bfd complex
in planktonic Δbfd cells causes an irreversible accumulation of
iron in BfrB and iron deficiency in the cytosol. The resultant
phenotype is hyperproduction of pyoverdine relative to the
wild type cells.23 Pyoverdine is a siderophore produced by
P. aeruginosa when the cells experience iron limitation.39
A
similar pyoverdine overproduction phenotype was observed
when wild type P. aeruginosa cells were treated with small
molecule inhibitors of the BfrB-Bfd complex (11 and 16).25
Therefore, to determine that compounds KM-5-25 and KM-5-
66 inhibit iron mobilization from BfrB in the P. aeruginosa
cytosol, we investigated whether cells treated with these
compounds express the characteristic pyoverdine hyper-
production phenotype. To this end, planktonic cells were
cultured in the presence of KM-5-25 (70 μM) or KM-5-66
(50 μM) for 27 h in M63 media and the content of the
secreted pyoverdine in the cell-free spent media was analyzed
by measuring the fluorescence intensity at 460 nm. Normal-
izing the intensity of pyoverdine fluorescence to CFU/mL
shows that as expected, cells treated with KM-5-25 or KM-5-
66 secrete ∼5-fold more pyoverdine than the untreated control
(Figure S5), an overproduction level similar to that observed
with the Δbfd mutant.23 These observations indicate that both
analogues bind BfrB in the P. aeruginosa cytosol, block the
BfrB-Bfd interaction and inhibit iron mobilization from BfrB,
resulting in cytosolic iron limitation that is manifested in a
pyoverdine hyperproduction phenotype. The cytosolic iron
limitation caused by treating planktonic cultures with KM-5-25
or KM-5-66 exerts a bacteriostatic effect on the cells, as
indicated by the IC50 values in Table 1. In stark contrast, when
the same compounds are used to treat P. aeruginosa biofilms, a
bactericidal effect is observed. The results from these
experiments are discussed below.
4-Aminoisoindoline-1,3-dione Derivatives Kill P. aer-
uginosa Cells in Mature Biofilms. A characteristic of
biofilms is their high tolerance to antimicrobial agents.
Tolerance is a physiological condition which does not involve
mutation and enables bacteria to survive in the presence of
antibiotics.40−43 The persistent biofilm phenotype is thought
to arise from several factors, including restricted penetration of
antibiotic molecules due to interactions with components of
the biofilm matrix, slow cell metabolism in the biofilm,
differential expression of specific genes, and the presence of
persister cells. In addition, biofilms are composed of distinct
subpopulations that exhibit different physiological activity;
cells in the biofilm interior exhibit low metabolic activity,
distinct from the high metabolism of cells near the sur-
face.40,44,45 The dissimilar metabolic activity is thought to
result from a concentration gradient of O2 and nutrients, which
are high at the biofilm surface and low in the deeper layers of
the biofilm.45,46 Commercial antibiotics that interfere with cell
replication (e.g., ciprofloxacin), or protein translation (e.g.,
tobramycin), preferentially kill the metabolically active bacteria
E
ACS Infect. Dis. XXXX, XXX, XXX−XXX
Soldano, Anabel
