Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1α interaction

Article abstract of DOI:10.1021/ja209924v

E3 ubiquitin ligases, which bind protein targets, leading to their ubiquitination and subsequent degradation, are attractive drug targets due to their exquisite substrate specificity. However, the development of small-molecule inhibitors has proven extrao

Full text of DOI:10.1021/ja209924v

Communication
pubs.acs.org/JACS
Targeting the von Hippel−Lindau E3 Ubiquitin Ligase Using Small
Molecules To Disrupt the VHL/HIF-1α Interaction
Dennis L. Buckley,† Inge Van Molle,‡ Peter C. Gareiss,§ Hyun Seop Tae,§ Julien Michel,†,⊥
Devin J. Noblin,§ William L. Jorgensen,† Alessio Ciulli,*,‡ and Craig M. Crews*,†,§,∥
†Department of Chemistry, §Department of Molecular, Cellular & Developmental Biology, and ∥Department of Pharmacology, Yale
University, New Haven, Connecticut 06511, United States
‡Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
S
* Supporting Information
subsequent VHL-mediated ubiquitination (Figure 1).13 Small-
molecule inhibition of this pathway therefore would lead to
ABSTRACT: E3 ubiquitin ligases, which bind protein
targets, leading to their ubiquitination and subsequent degrada-
tion, are attractive drug targets due to their exquisite substrate
specificity. However, the development of small-molecule
inhibitors has proven extraordinarily challenging as modulation
of E3 ligase activities requires the targeting of protein−protein
interactions. Using rational design, we have generated the first
small molecule targeting the von Hippel−Lindau protein
(VHL), the substrate recognition subunit of an E3 ligase, and
an important target in cancer, chronic anemia, and ischemia.
We have also obtained the crystal structure of VHL bound to
our most potent inhibitor, confirming that the compound
mimics the binding mode of the transcription factor HIF-1α, a
substrate of VHL. These results have the potential to guide
future development of improved lead compounds as
therapeutics for the treatment of chronic anemia and ischemia.
3 ubiquitin ligases (of which over 600 are known in humans)1
E
confer substrate specificity for ubiquitination and are more
attractive therapeutic targets than general proteasome inhibitors2,3
due to their specificity for a small number of protein substrates.
Unfortunately, the development of E3 ligase inhibitors has proven
challenging, in part due to the fact that they must disrupt protein−
protein interactions.4 These interactions are notoriously difficult to
target using small molecules due to their large contact surfaces and
the shallow grooves or flat interfaces involved. Conversely, most
small-molecule drugs bind enzymes or receptors in tight and well-
defined pockets.5 Since the discovery of nutlins, the first small-
molecule E3 ligase inhibitors,6 a few additional compounds have
been reported that target inhibitors of apoptosis proteins (IAPs),7,8
Figure 1. (A) HIF-1α accumulation leads to the transcriptional
upregulation of genes involved in the hypoxic response, such as erythro-
poietin (Epo), VEGF and others. (B) Under normoxic conditions, HIF-1α
is hydroxylated, recognized by VHL, ubiquitinated, and degraded by the
proteasome, preventing transcriptional upregulation.
increased endogenous erythropoietin production and could
supplant the current use of recombinant erythropoietin to treat
chronic anemia associated with chronic kidney disease and cancer
chemotherapy.14 To this end, PHD inhibitors are under examina-
tion in clinical trials; however, a possible alternative would be the
development of an inhibitor of the VHL/HIF-1α interaction. Such
an inhibitor may avoid the HIF-independent off-target effects ob-
served with PHD inhibitors,15 which have already proven immensely
useful as biological probes.16,17
While VHL also has HIF-1α-independent functions such as
binding to and stabilizing p53 and acting as an adaptor for the
phosphorylation of CARD9,12 these proteins likely bind VHL
differently than HIF-1α. In fact, previous work by Willam et al. has
shown that polypeptides containing the HIF-1α oxygen-depend-
ent degradation domains (ODDs) linked to the cell-permeable tat
9
10
SCFMet30
developed.
,
and SCFCdc4
;
however, the field remains under-
One E3 ubiquitin ligase with exciting therapeutic potential is the
von Hippel−Lindau (VHL) complex consisting of VHL, elongins
B and C, cullin 2, and ring box protein 1 (Rbx1).11 The primary
substrate of VHL is hypoxia-inducible factor 1α (HIF-1α), a
transcription factor that upregulates numerous genes such as the
pro-angiogenic growth factor, vascular endothelial growth factor
(VEGF), glucose transporter, GLUT1, and the red blood cell
inducing cytokine, erythropoietin, in response to low oxygen
levels.12 While HIF-1α is constitutively expressed, its intracellular
levels are kept very low under normoxic conditions via its hydroxy-
lation by prolyl hydroxylase domain (PHD) enzymes and
Received: October 21, 2011
Published: February 27, 2012
© 2012 American Chemical Society
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Communication
translocation domain stabilize HIF and induce an angiogenic
response, suggesting that competitive inhibition of VHL is capable
of producing a downstream biological response.18
We hypothesized that small-molecule inhibitors of the VHL/
HIF-1α interaction could be rationally designed using hydroxy-
proline (Hyp) as a starting point, since residue Hyp564 on
HIF-1α makes key interactions with VHL19,20 and is crucial for
HIF-1α binding.21 We used the de novo design software BOMB
to guide the selection of plausible hydroxyproline analogues.22
1 and 2 were synthesized to test a promising design featuring an
isoxazole moiety positioned to interact with a crystallographic
water observed in the structure of VHL bound to the HIF-1α
peptide (549−582)19 and a benzyl group stacked along the side
chain of Tyr98.
The compounds’ ability to bind to VHL was measured by the
competition of a fluorescent HIF-1α peptide, FAM-DEALA-
Hyp-YIPD (Kd = 560 nM).23 Fluorescence polarization (FP) of
the peptide was measured in the presence and in the absence
of the VCB complex consisting of VHL, elongin B, and elongin
C, to get the maximum and minimum values for polarization,
respectively. The polarization was then measured in the pre-
sence of VCB and serial dilutions of the small-molecule ligands in
order to observe the competition of the fluorescent peptide. These
values were then normalized to the maximum and minimum
polarization values to calculate percent inhibition of the interac-
tion, from which an IC50 was determined. Both compounds were
able to displace the fluorescent peptide albeit at high concentra-
tions (Table 1). In addition, the nonfluorescent DEALA-Hyp-YIPD
Figure 2. WaterLOGSY NMR spectroscopy shows binding of 3, but
not L-Hyp or NAc-Hyp-NMe, to VHL.
ligands (Scheme 1).26,27 These ligands were then tested for
their ability to bind VHL using the HIF-1α peptide FP
displacement assay.
Scheme 1. Solid-Phase Synthesis of VHL Ligands
Table 1. Binding of Initial Ligands to VHL
Incorporation of various halogenated benzylamines showed
that para substitution yielded the highest affinity and that
there were only slight differences of affinity between chlorides
and bromides, although the corresponding fluoride was less
potent. We also found that substitution with electron-
withdrawing groups such as esters, nitro groups, nitriles, and
ketones led to more potent ligands than substitution with
electron-donating methoxy and tert-butyl substituents. We then
considered larger heterocyclic substituents at the para posi-
tion of the benzylamine moiety and synthesized 15, which
was found to bind with a 4.1 μM IC50 value (Table 2). The
a
Average IC50 values were determined from three independent trials,
each in triplicate.
was used as a positive control and found to bind with IC50
=
0.91 μM and Kd = 180 nM by ITC. While the smaller 3 was
unable to fully displace the fluorescent peptide under the same
conditions, we observed binding to VHL through the use of
WaterLOGSY NMR spectroscopy,24 as demonstrated by the
positive ligand signals due to cross-relaxation from water in the
presence of protein (Figure 2). As no binding was observed with
smaller hydroxyproline fragments, this suggested that we identified
a minimal pharmacophore.
Encouraged by these initial results, we sought to increase the
affinity of our VHL ligands by modifying the benzylamine
moiety of 1 while maintaining the methyl-isoxazole fragment.
In order to generate analogues rapidly, we developed a solid-
phase synthesis that involved the attachment of Fmoc-Hyp-
OAllyl to Wang resin.25 Fmoc deprotection, coupling with
3-methyl-5-isoxazoleacetic acid followed by allyl ester depro-
tection, subsequent coupling with various amines, and cleavage
with trifluoroacetic acid led to the rapid generation of VHL
Kd of 15 was determined by ITC to be 5.4
0.2 μM
(Figure S3 in the Supporting Information).
The cocrystal structure of VHL bound to 15 confirmed that
it bound to the same site on VHL as HIF-1α and that the
hydroxyproline ring recapitulates the interactions seen in the
HIF-1α peptide:VHL complex (Figure 3).19,20 The hydroxyl
group makes contacts with both His115 and Ser111, and the
amide NH makes a contact with the His110 carbonyl (Figure 4).
Furthermore, the crystal structure shows that the aryl ring is
involved in a side-on interaction with the Tyr98 phenol,
possibly explaining the higher affinity of ligands containing
electron-poor aryl groups. In addition, a hydrogen bond is
formed between the oxazole CH and the Pro99 carboxyl
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Table 2. Structure−Activity Relationship of VHL Ligands
a
R
IC50 (μM)
SEM (μM)
1
3-Cl
H
117
130
149
20.5
149
32.0
>250
106
39.4
16.0
60.3
22.6
4.1
10
10
4
5
2-Cl
4-Cl
4-F
13
6
1.9
13
7
8
4-Br
4-tBu
3.6
N/A
13
9
10
11
12
13
14
15
4-OMe
4-CO2Me
4-NO2
2.2
0.6
5.3
2.0
0.4
4-CN
4-COMe
4-(5-oxazoyl)
a
Figure 4. Graphical representation showing the key interactions
between 15 and VHL.
Average IC50 values were determined from three independent trials,
each in triplicate.
ASSOCIATED CONTENT
■
S
* Supporting Information
Chemical and biochemical procedures, characterization of novel
compounds, complete refs 7 and 9, and details of the X-ray
diffraction data collection and analysis. This information is
available free of charge via the Internet at http://pubs.acs.org.
The crystal structures of pVHL-Elongin B-Elongin C and
pVHL-Elongin B-Elongin C-15 complex described in this paper
have been deposited in the Protein Data Bank (PDB codes
3ZRF and 3ZRC, respectively).
AUTHOR INFORMATION
■
Corresponding Author
Figure 3. The 2.9 Å cocrystal structure of 15 (white carbons) bound
to VHL, indicating that its binding mimics that of the HIF-1α peptide
(yellow carbons, PDB 1LM820).
craig.crews@yale.edu; ac313@cam.ac.uk
Present Address
⊥EaStCHEM School of Chemistry, Joseph Black Building, The
King’s Buildings, Edinburgh, EH9 3JJ, U.K.
oxygen. Furthermore, the side chain of Arg107 reorients slightly
(Figure S2 in the Supporting Information), and hydrogen
bonds to the oxazole nitrogen of the inhibitor. These structural
data will allow for further rational design to optimize the
potency of future inhibitors of the VHL/HIF-1α interaction.
In summary, we have described the design and synthesis of
the first small-molecule ligands for VHL, the protein
recognition subunit of an E3 ubiquitin ligase. Starting from
the minimal hydroxyproline recognition unit and using in silico
design as well as structure-guided medicinal chemistry, we were
able to improve ligand affinity for VHL to single digit micro-
molar. Furthermore, the most potent inhibitor was cocrystal-
lized with VHL, and shown to bind at the HIF-1α binding site.
These small-molecule inhibitors of the VHL/HIF-1α protein−
protein interaction have the potential to be developed into cell-
penetrant chemical probes that mimic the hypoxic response as
well as novel therapeutics to treat disease conditions such as
chronic anemia, acute ischemia, and stroke.
ACKNOWLEDGMENTS
■
This research was partially supported by the NIH AI084140,
GM032136, the HFSP STF00017/2008 (Short-Term Fellow-
ship to A.C.), the BBSRC BB/G023123/1 (David Phillips
Fellowship to A.C.), and the EU PIEF-GA-2010-275683
(Marie-Curie Intra European Fellowship to I.V.M.) and FP7-
PEOPLE-2008-IOF-234796 (Marie-Curie International Out-
going Fellowship to J.M.). D.J.N. was supported by the U.S.
Department of Defense through the National Defense Science
& Engineering Graduate Fellowship Program. We thank Ernest
So for the synthesis of NAc-Hyp-NMe and Julian Conrad for
the synthesis of FAM-DEALA-Hyp-YIPMDDDFQLRSF. We
are grateful to Dr. Dimitri Y. Chirgadze of the Crystallographic
X-ray Facility at the Department of Biochemistry, University of
Cambridge and to the technical support at Diamond and ESRF
for their assistance.
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