Small-molecule inhibitors of the interaction between the E3 ligase VHL and HIF1α

Article abstract of DOI:10.1002/anie.201206231

By design: Novel small-molecule inhibitors of the interaction between the von Hippel-Lindau ligase (VHL) and its molecular target HIF1α, a transcription factor involved in oxygen sensing, have been developed and studied. The most potent inhibitor binds with an IC₅₀ value of 0.9-μM and is thus the first sub-micromolar inhibitor of the VHL-HIF1α interaction. Copyright

Full text of DOI:10.1002/anie.201206231

Angewandte
Chemie
DOI: 10.1002/anie.201206231
Drug Design
Small-Molecule Inhibitors of the Interaction between the E3 Ligase
VHL and HIF1a**
Dennis L. Buckley, Jeffrey L. Gustafson, Inge Van Molle, Anke G. Roth, Hyun Seop Tae,
Peter C. Gareiss, William L. Jorgensen, Alessio Ciulli, and Craig M. Crews*
Protein–protein interactions (PPIs) are vital to most biolog-
ical processes, yet despite recent advances they remain
notoriously difficult to target due their relatively large
surfaces lacking the deep pockets of more tractable targets.[1]
While targeting these interactions with large a-helical
mimics[2–5] has been relatively successful, developing druglike
small-molecule inhibitors of PPIs remains highly challenging.
Recently, some success has resulted from the use of virtual
screening,[6] fragment-based approaches,[7] and the targeting
Figure 1. HIF1a is hydroxylated under normoxic conditions, leading to
recognition by VHL followed by ubiquitination and degradation by the
proteasome.
of “hot spots”;[8] however the hit rates for protein interfaces
remain low.[1c]
One class of PPIs with promising therapeutic potential is
that of E3 ligases with their substrates. E3 ligases bind to their
protein substrates, allowing E2 enzymes to transfer ubiquitin
subunits to the target protein. Because of their control of
widespread biological systems, E3 ligases are highly desirable
drug targets.[9] However, since the discovery of the nutlins, the
first small-molecule E3 ligase inhibitors,[10] only a handful of
E3 ligases have been successfully targeted.[11–13]
The vonHippel–Lindau protein (VHL) is a component of
a multi-subunit E3 ligase that recognizes the prolyl-hydroxy-
lated transcription factor HIF1a and tags it for degradation by
the proteasome (Figure 1).[14] However, under hypoxic con-
ditions, the prolyl hydroxylase domain enzymes (PHDs) are
unable to hydroxylate HIF1a, resulting in the accumulation
of HIF1a and subsequent upregulation of the genes involved
in the hypoxic response, including GLUT1, VEGF, and
erythropoietin. HIF1a stabilization, through the use of PHD
inhibitors,[15] is being investigated in the clinic as a possible
treatment for chronic anemia.[16] Alternatively, the inhibition
of the VHL–HIF1a interaction with peptidic inhibitors fused
to the tat translocation domain has been shown to stabilize
HIF1a,[17] illustrating that inhibition of this interaction is an
alternative or complementary strategy to PHD inhibitors for
the treatment of anemia.
Recently, we reported a series of VHL ligands, including
1 (see Table 1) capable of competitively inhibiting the binding
of a fluorescent peptide derived from HIF1a to VHL.[18]
These inhibitors contain a hydroxyproline residue, which is
crucial for binding to VHL,[19] and an isoxazolylacetamide
fragment, which was designed to interact with a water
molecule previously identified as an important part of the
hydrogen-bonding network between VHL and HIF1a.[20]
However, these molecules bound with limited potency and
only a small number of analogues were made, hindering the
ability to draw conclusions about structure–activity relation-
ships (SARs). Herein we report a detailed study of VHL
ligand SAR, including the discovery of N-terminal fragments
with an alternative binding mode, as shown by X-ray
crystallography. The optimization of both the C- and N-
terminal fragments, followed by their combination, yielded
our most potent ligand to date, which binds with an IC50 value
in the sub-micromolar range.
While optimizing the C- and N-terminal fragments for
affinity, we sought to minimize differences in ligand solubility
by testing binding affinity in a fluorescence polarization
competition assay using 10% DMSO, as opposed to the more
physiologically relevant 1% DMSO.[18] While general trends
in affinity were similar under both sets of conditions, we found
that in cases where solubility was not an issue, ligands had
lower IC50 values in 1% DMSO.
[*] D. L. Buckley, Dr. J. L. Gustafson, Dr. A. G. Roth, Dr. H. S. Tae,
Dr. P. C. Gareiss, Prof. W. L. Jorgensen, Prof. C. M. Crews
Departments of Chemistry, Molecular, Cellular & Developmental
Biology and Pharmacology
and Center for Molecular Discovery, Yale University
New Haven, CT 06511 (USA)
E-mail: craig.crews@yale.edu
Dr. I. Van Molle, Dr. A. Ciulli
Department of Chemistry, University of Cambridge
Lensfield Road, Cambridge CB2 1EW (UK)
[**] This research was supported in part by the NIH (AI084140,
GM032136), BBSRC (BB/G023123/1), and EC (PIEF-GA-2010-
275683). J.L.G. thanks the NIH for a postdoctoral fellowship
(F32GM10052101). A.G.R. is a Leopoldina-Nationale Akademie der
Wissenschaften Postdoctoral Fellow. We are grateful to the beam-
line scientists of the Proxima-1 beamline at the Soleil synchrotron
facility for their assistance.
After the discovery of 1,[18] we sought to systematically
investigate other analogues with five-membered heteroaro-
matic substituents (Table 1). After examining compounds
with various oxazolyl (1, 2, 3) and thiazolyl substituents (4, 5,
6, 7), we found that the original substitution at the 5-position
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201206231.
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Table 1: Optimization of the C-terminal fragment.
with Pro99 of VHL.. While the N-methylpyrrolyl analogues
had only moderate activity, the 4-methyloxazolyl compound 3
was slightly less potent than 1. In contrast, the 4-methylthia-
zolyl analogue 6 was not only more potent than 4 but also
more potent than the oxazolyl compound 1.
Given the apparent role of the oxazole C–H donation to
the Pro99 carbonyl group,[18] we sought to increase the
potency of the ligands by using a better H-bond donor.
Unfortunately, we were disappointed to find that the imida-
zole 11 had poor binding affinity and that the pyrazoles 12 and
13 had moderate affinity but were inferior to 1. However, the
direct comparison of pyrazole 15 and isoxazole 14, which
cannot act as an H-bond donor, was informative, showing that
the H-bond can lead to a significant increase in potency.
We simultaneously sought to optimize the N-terminal
fragment, while keeping the p-chlorobenzylamino fragment
of 16 constant because of its small size and the availability of
the starting material for its synthesis. We originally explored
aryl and heteroaryl acetamides. Disappointingly, each was
significantly worse than the isoxazolyl analogue. This
included the pyrazolyl 22 and imidazolyl compounds 19 and
21, which were designed to interact more strongly with the
structural water molecule owing to their increased capacity as
H-bond acceptors.[21] Testing more diverse fragments led to
more success (Table 2), as we found that the chlorobenzamide
derivative 24 approached the activity of the corresponding
isoxazoleacetamide 16.
Compd.
R (para)
IC50 [mm][a]
(10% DMSO)
IC50 [mm][a]
(1% DMSO)
1
2
7.0Æ0.5
11Æ1
4.1Æ0.4[b]
N.D.
3
5.1Æ0.2
12.7Æ0.7
4
5
17Æ1
14.0Æ0.5
77Æ3
119Æ2
6
3.8Æ0.3
3.2Æ0.4
7
8
9
17.0Æ0.4
16.4Æ0.6
17.8Æ0.3
19Æ1
32Æ4
33Æ9
To elucidate the mode of binding of the chlorobenzamide
fragment, a crystal structure of 24 with the pVHL-ElonginB-
ElonginC complex (VCB) was obtained (Figure 2). The
structure showed that the hydroxyproline core reorients
10
11
36Æ12
19Æ2
270Æ20
180Æ10
12
13
12.1Æ0.6
50Æ10
8.97Æ0.07
43Æ2
14
15
120Æ30
18Æ2
70Æ10
32Æ3
Figure 2. Crystal structure of VCB in complex with 24. pVHL is shown
as a pale green surface, the ligand as a gray stick representation, and
the pVHL residues forming the binding pocket as yellow stick represen-
tations.
[a] IC50 values were determined by the displacement of FAM-DEALA-Hyp-
YIPD from VCB, with the standard error of the mean (SEM) reported.
[b] Literature value.[18] N.D.=not determined.
slightly, maintaining many of the previously highlighted
interactions.[18] In particular, the hydroxy group H-bonds to
both S111 and H115, while the amide H-bonds to the H110
backbone and the Y98 side chain. Interestingly, the N-
terminal fragment clearly binds to VHL in an alternative
fashion. While the isoxazolyl moiety interacted with the
structural water previously identified[20] that binds to N67,
of the heteroaromatic substituent and at the para position of
the aryl ring was optimal.
Preliminary molecular modeling suggested that substitu-
tion of a methyl group at the N position of a pyrrole or 4-
position of an azole would increase hydrophobic interactions
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Table 2: Exploration of diverse N-terminal fragments and the discovery
Table 3: Optimization of the benzamide N-terminal fragment.
of the benzamide fragment.
Compd.
R
IC50 [mm][a]
(10%DMSO)
Compd.
R
IC50 [mm][a]
(10%DMSO)
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
2-Cl
H
52Æ2
110Æ3
24Æ2
3,4-dimethoxy
2-NH2
3-NH2,2-Me
4-NH2
4-Cl
3-F
3-Br
3-CN
3-OMe
3-OH
16
38Æ3
38Æ1
10.4Æ0.8
29Æ3
17
18
330Æ10
>1000
70Æ10
38.5Æ0.5
19.6Æ0.8
8.9Æ0.1
26.3Æ0.4
17Æ0.6
15.5Æ0.4
33Æ4
19
>1000
2-Br,4-Cl
3-Ph
isonicotinamide
32Æ1
[a] IC50 values determined by the displacement of FAM-DEALA-Hyp-YIPD
from VCB, with SEM reported.
20
21
280Æ40
>1000
cases, the methylthiazolyl analogue remained the most potent
compound. We were also pleased to find that 51 bound to
VHL with an IC50 of 0.9 mm in 1% DMSO, our most potent
compound to date, and the only small-molecule compound
thus far that inhibits the interaction between VHL and the
HIF1a peptide at concentrations below 1 mm.
22
372Æ7
We next obtained a crystal structure of the most potent
ligand, 51, in complex with VCB (Figure 3). The optimized
methylthiazolyl moiety of the C-terminal fragment binds in
a similar conformation to the oxazolyl fragment of 1 that had
been previously crystallized.[18] While the methyl group is
solvent exposed, it is possible that the larger and more
hydrophobic aromatic sulfur atom is better able to fill the
small hydrophobic pocket underneath P99 than the corre-
sponding oxygen in 1. The aryl ring of the N-terminal
23
24
300Æ50
52Æ2
[a] IC50 values determined by the displacement of FAM-DEALA-Hyp-YIPD
from VCB, with SEM reported.
R69, and H115, the benzamide instead is oriented away from
the water pocket, and lies adjacent to the side chain of W88.
We then synthesized a number of benzamide inhibitors
and found a wide variety of compounds with improved
binding potency (Table 3). In particular, we found that the 3-
amino-2-methylbenzamide compound 28, the m-cyanobenz-
amide compound 33, and the 2-bromo-4-chlorobenzamide
compound 36, were more potent than the isoxazoleacetamide
inhibitor 16. Additionally, we found that we were able to add
large substituents at the meta position while maintaining
potency.
After optimizing both outer fragments independently, we
sought to optimize potency of the overall molecule in
a combinatorial fashion (Table 4). We found that while in
some cases, such as the ethoxybenzamide class, the trend of
increasing activity of C-terminal aryl substituents from chloro
to oxazolyl to methylthiazolyl remained similar to the original
isoxazolylacetamide, in many cases the oxazolyl compound
was worse than both the chloro and methylthiazolyl. In most
Figure 3. Crystal structure of VCB with 51. pVHL is shown as a pale
green surface, the ligand as an orange stick representation, and the
pVHL residues forming the binding pocket as yellow stick representa-
tions.
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Table 4: VHL ligands with optimized C- and N-terminal fragments.
the interactions highlighted by the crystal structure
of 51 to design more drug-like inhibitors of VHL
with improved cell permeability. These ligands and
the novel interactions and binding modes highlighted
have the potential to guide the development of
future chemical probes that can target the E3 ligase
VHL or stabilize HIF1a proteins.
No. R1
R2[b] IC50 [mm][a]
IC50 [mm][a]
Ki[b]
(10% DMSO) (1% DMSO) [mm]
Received: August 3, 2012
Published online: October 12, 2012
16
1
6
Cl
37.8Æ3.1
7.0Æ0.5
3.8Æ0.3
20.5Æ1.9[c]
4.1Æ0.4[c]
3.2Æ0.4
15.2
3.0
2.4
5-oxazolyl
5-(4-methyl)thiazolyl
Keywords: drug design · E3 ubiquitin ligases ·
.
protein crystallography · protein–protein interactions ·
structure-based design
39
40
41
Cl
62.9Æ2.0
27.0Æ0.7
14.1Æ0.3
N.D.
N.D.
14.5
8.3
5-oxazolyl
5-(4-methyl)thiazolyl
19.5Æ1.0
11.2Æ0.9
32
42
43
Cl
19.6Æ0.8
39.5Æ3.7
7.7Æ0.4
N.D.
N.D.
23.3
19.9
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50
51
Cl
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