Group-Based Optimization of Potent and Cell-Active Inhibitors of the von Hippel-Lindau (VHL) E3 Ubiquitin Ligase: Structure-Activity Relationships Leading to the Chemical Probe (2S,4R)-1-((S)-2-(1-Cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH298)

Article abstract of DOI:10.1021/acs.jmedchem.7b00675

The von Hippel-Lindau tumor suppressor protein is the substrate binding subunit of the VHL E3 ubiquitin ligase, which targets hydroxylated α subunit of hypoxia inducible factors (HIFs) for ubiquitination and subsequent proteasomal degradation. VHL is a potential target for treating anemia and ischemic diseases, motivating the development of inhibitors of the VHL:HIF-α protein-protein interaction. Additionally, bifunctional proteolysis targeting chimeras (PROTACs) containing a VHL ligand can hijack the E3 ligase activity to induce degradation of target proteins. We report the structure-guided design and group-based optimization of a series of VHL inhibitors with low nanomolar potencies and improved cellular permeability. Structure-activity relationships led to the discovery of potent inhibitors 10 and chemical probe VH298, with dissociation constants <100 nM, which induced marked HIF-1α intracellular stabilization. Our study provides new chemical tools to probe the VHL-HIF pathways and new VHL ligands for next-generation PROTACs.

Full text of DOI:10.1021/acs.jmedchem.7b00675

Subscriber access provided by UNIVERSITY OF CONNECTICUT
Article
Group-based optimization of potent and cell-active inhibitors of
the von Hippel-Lindau (VHL) E3 ubiquitin ligase: structure-activity
relationships leading to the chemical probe (2S,4R)-1-((S)-2-(1-
cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH298)
Pedro Soares, Morgan S. Gadd, Julianty Frost, Carles Galdeano, Lucy
C.J. Ellis, Ola Epemolu, Sonia Rocha, Kevin D Read, and Alessio Ciulli
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00675 • Publication Date (Web): 30 Aug 2017
Downloaded from http://pubs.acs.org on August 30, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
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of their duties.

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Groupꢀbased optimization of potent and cellꢀ
active inhibitors of the von HippelꢀLindau (VHL)
E3 ubiquitin ligase: structureꢀactivity
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relationships leading to the chemical probe
(2S,4R)ꢀ1ꢀ((S)ꢀ2ꢀ(1ꢀ
cyanocyclopropanecarboxamido)ꢀ3,3ꢀ
dimethylbutanoyl)ꢀ4ꢀhydroxyꢀNꢀ(4ꢀ(4ꢀ
methylthiazolꢀ5ꢀyl)benzyl)pyrrolidineꢀ2ꢀ
carboxamide (VH298)
†
†
†,‡
†
†
†
Pedro Soares , Morgan S. Gadd , Julianty Frost , Carles Galdeano , Lucy Ellis , Ola Epemolu ,
‡
†
†*
Sonia Rocha , Kevin D. Read , Alessio Ciulli
†
Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of
Dundee, Dow Street, Dundee, DD1 5EH, Scotland, UK.
‡
Center for Gene Regulation and Expression, School of Life Sciences, University of Dundee,
Dow Street, Dundee, DD1 5EH, Scotland, UK.
*
Correspondence and requests for materials should be addressed to A.C. Eꢀmail:
a.ciulli@dundee.ac.uk
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ABSTRACT
The von Hippel–Lindau tumor suppressor protein is the substrate binding subunit of the VHL E3
ubiquitin ligase, which targets hydroxylated α subunit of hypoxia inducible factors (HIFs) for
ubiquitination and subsequent proteasomal degradation. VHL is a potential target for treating
anemia and ischemic diseases, motivating the development of inhibitors of the VHL:HIFꢀα
proteinꢀprotein interaction. Additionally, bifunctional proteolysis targeting chimeras (PROTACs)
containing a VHL ligand can hijack the E3 ligase activity to induce degradation of target
proteins. We report the structureꢀguided design and groupꢀbased optimization of a series of VHL
inhibitors with low nanomolar potencies and improved cellular permeability. Structureꢀactivity
relationships led to the discovery of potent inhibitors 10 and chemical probe VH298, with
dissociation constants <100 nM, which induced marked HIFꢀ1α intracellular stabilization. Our
study provides new chemical tools to probe the VHLꢀHIF pathways, and new VHL ligands for
nextꢀgeneration PROTACs.
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INTRODUCTION
Proteins are constantly synthesized and targeted for degradation during cellular
homeostasis. The major pathway to protein degradation is the ubiquitinꢀproteasome system
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(UPS), first reported in the early 80’s. In a highly regulated enzymatic signaling cascade,
ubiquitin is covalently attached to a substrate protein as a monomer or as a polyubiquitin chain,
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,3
promoting proteasomeꢀcatalyzed target degradation. The complexity and biological importance
of the UPS, together with implication of this pathway in a wide range of diseases, highlight its
4,5
importance as a therapeutic target. The market release of the pioneering proteasome inhibitors
bortezomib and carfilzomib, for the treatment of haematopoietic and lymphoid malignancies,
validated the UPS as a focus for therapeutic intervention and opened the door to a new series of
5ꢀ7
proteasome inhibitors. Despite their success proteasome inhibitors lack substrate specificity,
which is a significant limitation to their use as chemical probes of specific biological pathways
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,8
and leads to unwanted side effects in their use as drugs.
Specific binding of a target substrate to an E3 ubiquitin ligase is required for substrate
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,9
ubiquitination and is therefore a key step in the ubiquitination process. E3 ubiquitin ligases
determine specificity of substrate ubiquitination and thus could be regarded as more attractive
targets to center drug discovery efforts over the proteasome. However, smallꢀmolecule
modulators of E3 ligases need to target proteinꢀprotein interactions (PPIs), either directly or
10
allosterically. The typically flat and featureless nature of many PPI interfaces can be a limiting
11,12
factor in the development of potent and selective inhibitors.
To cover considerable protein
surface area that often lack wellꢀdefined pockets, most PPI inhibitors tend to be larger in size
than classical enzyme inhibitors or receptor antagonists, forcing medicinal chemists to break
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conventional drugꢀlike quality metrics.
These observations consequently led to E3 ligases
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6
being perceived as untractable target space. On the other hand, the identification of small sized
hotspots for certain PPIs, particularly those characterized by the recognition of primaryꢀtype
linear epitopes, have led to the successful observation of smallꢀmoleculeꢀsized patches on protein
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surfaces that could be adequately targeted by drugꢀlike molecules.
Today, several small
molecules that modulate PPIs have been developed as chemical probes, and many have entered
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clinical trials.
of PPI based targets, including E3 ubiquitin ligases.
The von Hippel–Lindau protein (VHL) is a member of the CullinꢀRING ligase family of
These realizations have reinvigorated drug discovery efforts for a wide range
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E3 ubiquitin ligases. The major substrate of VHL is the hypoxia inducible factor 1α (HIFꢀ1α), a
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transcription factor that drives the transcriptional program of many human genes, mainly
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involved in hypoxia adaptation.
When tissue oxygen levels are normal, HIFꢀ1α expression
levels are tightly controlled. Ironꢀ and oxygenꢀdependent hydroxylation occurs at two specific
proline residues within the HIFꢀ1α oxygenꢀdependent degradation domain (ODD), by prolyl
hydroxylase domain (PHD) enzymes. This modification leads to HIFꢀ1α specific recognition and
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ubiquitination by VHL and subsequent degradation via the UPS (Supplementary Figure 1).
The importance of this pathway has been uncovered in a wide range of diseases,
including conditions characterized by anemia, ischemia, inflammation, chronic
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neurodegeneration and more recently mitochondrial dysfunction.
Smallꢀmolecule inhibition
of this pathway could activate HIFꢀ1α expression, upregulating genes involved in the hypoxic
response, consequently providing a potential therapeutic strategy. Indeed, smallꢀmolecule PHD
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3ꢀ35
inhibitors have been developed that have shown potential in a number of pathologies.
Amongst
these,
the
candidate
FGꢀ4592
(Nꢀ[(4ꢀHydroxyꢀ1ꢀmethylꢀ7ꢀphenoxyꢀ3ꢀ
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isoquinolinyl)carbonyl]glycine, also known as roxadustat or ASP1517) has progressed to a Phase
III clinical trial to evaluate efficacy and safety in hemodialysis chronic kidney patients with
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anemia.
Despite their clinical potential, no PHD inhibitor shows selectivity between the three
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different PHD paralogs (PHD1, PHD2 and PHD3). In addition, several nonꢀHIF substrates of
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PHD enzymes have been identified.
Lack of selectivity could promote unwanted side effects
due to the different substrate specificities as well as cellular and tissue localization of PHD
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enzymes.
In contrast to PHD inhibitors, inhibitors of the VHL:HIFꢀ1α PPI would allow to block the
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pathway downstream of HIF hydroxylation by PHD enzymes. This approach could avoid HIFꢀ
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independent offꢀtarget effects and deliver new lead compounds as hypoxia mimetics. In order
to validate the chemical intervention on VHL and establish the biological consequences of
modulating its activity, small molecules must effectively penetrate cells and engage with VHL
with enough potency to productively block HIFꢀ1α binding. In a different approach, VHL
ligands can be conjugated to target ligands of interest to design VHLꢀrecruiting bivalent chimeric
43
ligands (also known as PROTACs). PROTACs can efficiently hijack the VHL ligase to induce
intracellular degradation of the desired target proteins. Previous work from our laboratory and
others have demonstrated potent and selective activities of VHLꢀrecruiting PROTACs against a
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wide range of target proteins, including Brd4,
RIPK2, and cꢀAbl kinase amongst others.
Coꢀcrystal structures of VHL bound with a hydroxylꢀproline peptide from human HIFꢀ1α
carboxyꢀterminal oxygenꢀdependent degradation (CODD) motif identified a groove on the pVHL
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surface, which accommodated the substrate peptide.
Recognizing that this primaryꢀtype PPI
could be suitably targeted by small molecules disruptors, in pioneering work, our laboratory in
collaboration with the Crews laboratory developed a firstꢀgeneration of VHL ligands with singleꢀ
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digit micromolar binding affinities, based around the central hydroxyproline core fragment.
However, these initial compounds proved inactive as PPI inhibitors in cells. Subsequent
structureꢀguided optimization led to more potent inhibitors with improved nanomolar binding
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affinities. The best inhibitor of this series, compound 1 (VH032, Figure 1A), was able to
disrupt the VHL:HIFꢀ1α PPI in cells, illustrated by its capacity to stabilize hydroxylated HIFꢀ
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1
α. Nevertheless this inhibitor showed low passive cell permeability and exhibited a large
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mismatch between in vitro potency in solution and in cells.
Figure 1. Initial optimization strategy. (A) Xꢀray crystal structure of VHL, Elongin B and
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Elongin C (VBC) protein complex with inhibitor 1 (purple carbons, PDB 4W9H) . VHL is
shown as a pale green surface and the VHL residues forming the binding pocket as orange stick
representations. (B) Designed group modifications on inhibitor 1 to improve activity.
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Motivated by a desire to drive further inhibitor optimization, analysis of the Xꢀray crystal
structure of the complex composed of VHL, Elongin B and Elongin C (VBC) with 1 suggested
an attractive pocket at the leftꢀhand side (LHS) of the VHL:HIF interface that was only partially
filled by the terminal acetamide group of 1, and could therefore provide space to further grow
and optimize the molecule (Figure 1A). Herein, we describe a systematic groupꢀbased
optimization of VHL inhibitors strating from 1, improving binding affinity, cell membrane
passive permeability and ultimately cellular activity; a campaign that led to the discovery of
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inhibitor 15 (VH298), a potent and selective probe of the hypoxia HIFꢀ1α pathway.
RESULTS AND DISCUSSION
VHL inhibitor SAR. We aimed to optimize the physicochemical properties and binding affinity
of VHL inhibitors in order to minimize the observed mismatches between biophysical and
41
cellular potency. The information gathered from the coꢀcrystal structure of inhibitor 1 bound to
VBC protein complex offered a starting point for the design of a new series of inhibitors by
adding small modifications on the LHS of the inhibitor (Figure 1A).
In the first new series of inhibitors we decided to keep the carbonyl on the LHS in order to
maintain the hydrogen bond between this group and the structural water in this pocket. Our
design strategy was to replace the three hydrogens of the acetamide methyl group, one at a time,
using alkyl groups to better fill the LHS pocket, or adding electronꢀwithdrawing groups at the
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alpha position of the LHS amide in order to lock its conformation.
It was also decided to
evaluate the effect of removing the LHS acetamide hydrogenꢀbondꢀdonor group (NH) via
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cyclization, which we hypothesized could improve cellular permeability (Figure 1B). The
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synthesized compounds were evaluated biophysically in a direct binding assay using isothermal
titration calorimetry (ITC) and for their ability to displace a highꢀaffinity HIF peptide using
fluorescence polarization (FP). In parallel, the compounds cellular activity was evaluated in
HeLa cells by monitoring protein levels of HIFꢀ1α using western blots (Table 1).
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Substitution of a single hydrogen on the acetamide group for a hydroxyl (3) maintained binding
affinity but led to a substantial loss in cellular potency. On the other hand, expansion of the alkyl
chain from methyl to ethyl (4) allowed the retention of inhibitor cellular potency despite a twoꢀ
fold loss in inhibitor binding affinity. These two observations suggested that tuning hydrogen
bond donors and lipophilicity could play as important a role as binding affinity in determining
cellular activity. Replacing all three hydrogen with fluorine (7) or chlorine groups (8) did not
impact binding affinity and cellular potency. In contrast, introduction of a bulky tertꢀbutyl group
(5) resulted in a loss in binding affinity and in cellular potency, a similar effect observed for
replacing all hydrogens for two methyl groups together with one fluorine group (9). However,
substitution of two hydrogens with a restrained cyclopropyl group (6) promoted a small
improvement in both binding affinity and cellular activity. Further replacement of the tertiary
hydrogen of 6 with a fluorine (10) led to a marked increase in both binding affinity and cellular
potency compared to 1 and 6 (Table 1). Figure 2 illustrates representative ITC and FP binding
curve for 10, the most potent inhibitor of the series, and the parent inactive epimer cisꢀ10, which
due to a change in the proline OH group orientation no longer binds to VHL.
Attempts to modify over regions of the chemical structure of 1 did not yield improvements. For
example, modification of the tert-leucine side chain for a less bulky alanine side chain (11), or
removal of the acetamide hydrogen bond donor group through cyclization into a pyrrolidinone
(12) led to significant loss of inhibitor binding affinity and cellular potency. Lastly, acetamide
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methyl group substitution for a Boc protecting group (2) was not tolerated, resulting in the
greatest loss in binding and cellular potency.
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Table 1. Chemical structure, FP K s backꢀcalculated from IC s, ITC measured K s and group
d
50
d
efficiencies (GE) [based on FP results and calculated as: GE = ∆pK /∆HA (Heavy atoms)] of
d
inhibitors 1 and 2ꢀ12.
K FP
K ITC
(nM)
GE
HIFꢀ1α Stabilization
d
d
Inhibitor
R1
R2
a
(
nM)
(kcal/mol) (Protein % levels)
O
1
2
tꢀLeu
tꢀLeu
150 ± 30
185 ± 6
ꢀ
100 ± 7
12 ± 2
N
H
6500 ± 300
ꢀ
−0.41
3
4
5
6
7
8
tꢀLeu
tꢀLeu
tꢀLeu
tꢀLeu
tꢀLeu
tꢀLeu
200 ± 30
350 ± 90
450 ± 90
170 ± 30
270 ± 50
200 ± 30
105 ± 11
−0.11
−0.36
−0.12
−0.02
−0.08
−0.04
16 ± 3
98 ± 8
65 ± 7
105 ± 8
95 ± 9
89 ± 16
ꢀ
ꢀ
132 ± 10
ꢀ
ꢀ
O
N
9
tꢀLeu
tꢀLeu
770 ± 30
90 ± 10
ꢀ
−0.24
0.08
19 ± 2
H
F
10
44 ± 9
175 ± 1
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O
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Ala
760 ± 20
ꢀ
ꢀ
ꢀ
12 ± 2
33 ± 3
N
H
1
tꢀLeu
1400 ± 100
−0.48
a
HeLa cells were treated with 50 µM of the respective inhibitor and HIFꢀ1α stabilization levels
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were measured by Western blot after 2 h treatment (protein levels normalized to HIFꢀ1α
stabilization level observed with 1).
Figure 2. Biophysical characterization of inhibitor 10 binding to VBC. (A) Competitive FP
binding assay curve, monitoring the displacement of a 20ꢀmer FAMꢀlabeled HIFꢀ1α peptide
bound from VBC by inhibitor 10 (K = 3 nM). (B) ITC titrations of 300 µM inhibitor 10 (blue)
d
or 300 µM of its cis inactive epimer (green) into 30 µM VBC protein.
To better understand the SAR of this new inhibitor series, the Xꢀray crystal structures of
inhibitors 3, 6, 10 and 11 bound to VBC were obtained. All solved structures revealed a
conserved inhibitor binding mode at the VHL:HIF interface (Figure 3), consistent with the
1
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binding of 1. The crystal structure of 3 shows two different conformations upon binding to
VBC, each with 50% of occurrence. In the first inhibitor conformation, the added OH group
points upwards forming an extra hydrogen bond with a structural water that promotes the
formation of an interaction network between Tyr112 side chain and the amide carbonyl on the
LHS of inhibitor’s hydroxyproline (Hyp) (Figure 3A). In the second conformation, the hydroxyl
group is forming a bidentate interaction with the structural water present on the far left side of
binding pocket, together with LHS amide carbonyl (Figure 3B). These favorable interactions
contribute small improvement in inhibitor binding affinity (Table 1).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
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9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Crystal structures of 6 (Figure 3C) and 10 (Figure 3D) showed the cyclopropyl group occupying
the far LHS of the VHLꢀHIF interface, maintaining the hydrogen bond interactions with the
structural water. The addition of cyclopropyl induces a conformational modification in the
residues on this side of the pocket to accommodate this moiety. The most notable conformational
change is observed on the side chain of Arg69, which adopts a bent conformation to
accommodate the new group on the LHS. From the crystal structure of inhibitor 10, the fluorine
atom at the alpha position is found anti to the amide carbonyl. This matches a known minimumꢀ
5
6,57
energy conformation for alphaꢀfluoroamides.
This effect allows the inhibitor to be preꢀ
organized in its bound conformation prior to binding, thus minimizing the entropic penalty to
binding and as a result increasing binding affinity.
Substitution of the tertꢀleucine group for a less bulky alanine in 11 was meant to more closely
mimic the sequence of the HIFꢀ1α peptide (Supplementary Figure 2). This modification however
led to a less rigid conformation on the inhibitor LHS. In the coꢀcrystal structure of 11 (Figure 3E)
the ligand bends upwards, increasing the distance between the acetamide carbonyl and the
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structural water from 2.8 ± 0.1Å to 3.8 ± 0.5Å (Supplementary Figure 3), consequently
weakening this hydrogen bond and decreasing the binding affinity.
10
11
12
13
14
15
16
17
18
19
20
21
22
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41
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Coꢀcrystal structures of firstꢀseries inhibitors. Crystal structures of VBC in complex
with 3 (A and B), 6 (C), 10 (D) and 11 (E). VHL inhibitors are shown as sticks with purple
carbons. VHL is shown as a pale green surface and the VHL residues forming the binding pocket
as orange stick representations. Inhibitorꢀbinding waters are shown as a red spheres. Hydrogen
bond interactions between inhibitors, bound waters and VHL pocket residues are shown as
dashed red lines
.
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The first inhibitor series led us to the discovery of 10, a VHL inhibitor with doubleꢀdigit
nanomolar binding affinity, good cell membrane permeability and high activity in cells (Table 1
5
9
and Figure 4A), which are some of the key parameters needed for a good chemical probe.
0
1
2
3
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5
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9
0
1
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0
1
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3
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7
8
9
0
Despite meeting these benchmarks, further investigation revealed a degree of cytotoxicity for 10,
which notably decreased cell proliferation relative to DMSO vehicle (Figure 4). The inactive
epimer cisꢀ10 still presented a degree of cytotoxicity (Figure 4B,C). Together these findings
suggested an offꢀtarget toxicity for 10 (Figure 4).
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Figure 4. Cellular activity and toxicity of 10. (A) Activity of inhibitor 10 in HeLa cells
expressing a HRE (Hypoxia Responsive Element)ꢀluciferase reporter and treated with indicated
4
1
concentrations for 24 h. Luminescence reports luciferase expression as proxy of HIFꢀα activity.
10
11
12
13
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15
16
17
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19
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21
22
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Colony formation (Figure B) and cellꢀproliferation assays (Figure C) showing toxicity in HeLa
cells after treatment with 150 µM of inhibitor 10 and its cis epimer.
The observed cytotoxicity of 10 motivated us to identify a more suitable chemical probe. Based
on the information furnished by the coꢀcrystal structure, we designed a second series of VHL
inhibitors (Figure 5).
Figure 5. Xꢀray crystal structure of VBC protein complex with inhibitor 10. (A) VHL is
shown as a pale green surface and the VHL residues forming the binding pocket as orange stick
1
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representations. (B) Designed group modifications on inhibitor structure to improve inhibitor
activity and optimize inhibitors toward a chemical probe.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
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0
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0
1
2
3
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5
6
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8
9
0
In this second series we initially studied the impact of changing the fluorine atom for other
electron withdrawing groups or promoters of intramolecular hydrogen bonds. Secondly, to
exploit the flexibility of Arg69 in the LHS pocket, we expanded the cyclopropyl ring by adding
larger and more lipophilic groups. In addition, we considered moieties that could potentially
form a hydrogen bond with the Arg69 side chain, or alternatively, together with the LHS
carbonyl amide, could form bidentate hydrogen bonds with the structural water present in the
LHS pocket. Finally, we decided to test the impact of methylating the solventꢀexposed NH group
of the LHS acetamide and of replacing the tertꢀleucine side chain for threonine to explore
53
potential hydrogen bonds at the top LHS pocket, as observed previously (Figure 5B).
Removing the hydrogen bond donor group through methylation (13) resulted in a major loss in
binding affinity and cellular potency when compared with 1, presumably due to destabilization
of the required transꢀamide bond conformation. Substitution of tertꢀleucine on 10 for a threonine
(14) side chain group also led to a loss in binding affinity and cellular activity. Conversion of the
fluorine group into an electronꢀwithdrawing group such a cyano (15) revealed a twoꢀfold
increase in binding affinity and in cellular potency compared to 1. Changing the fluorine group
for a ketone moiety (16) also improved both binding affinity and cellular activity. Modification
of the fluorine atom with an acetamide (17) retained a comparable affinity when compared with
1
but yielded a considerable loss in cellular potency, presumably as a result of poorer cell
membrane passive permeability. Expansion of the cyclopropyl ring into a cyclobutyl (18) led to a
small loss of inhibitor binding affinity but a greater cellular activity, presumably due to the
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increased lipophilicity and permeability. Conversion of the cyclopropyl group into an oxetane
(19) retained binding affinity but led to a fourꢀfold loss in cellular activity. Further conversion of
the cyclopropyl group into a cyclobutanone (20) or into an acetylated azetidine (21) led to a twoꢀ
and 20ꢀfold loss in binding affinity and a fiveꢀ and 11ꢀfold decrease in cellular potency,
respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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Table 2. Chemical structure, FP backꢀcalculated K s, ITC measured K s and Group efficiencies
d
d
(GE) [based on FP results and calculated as: GE = ∆pK /∆HA (Heavy atoms)] of inhibitors 1, 15,
d
10 and 13–21.
K FP
K ITC
(nM)
GE
(kcal/mol)
HIFꢀ1α Stabilization
(Protein % levels)
d
d
Inhibitor
1
R1
R2
(
nM)
tꢀLeu
tꢀLeu
150±30
90±10
185±6
ꢀ
100±7
200±1
O
F
N
1
0
44±9
0.08
H
O
1
1
1
1
1
3
4
5
6
7
tꢀLeu
Thr
N
5500±300
370±40
80±30
ꢀ
–1.56
–0.19
0.07
26±9
46±5
O
F
N
ꢀ
H
tꢀLeu
tꢀLeu
tꢀLeu
90±5
106±14
138±21
190±10
156±10
20±2
170±30
300±40
–0.01
–0.05
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O
O
1
1
8
9
tꢀLeu
tꢀLeu
N
H
250±50
320±20
210±12
230±20
–0.07
–0.11
138±12
34±4
N
H
O
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0
20
tꢀLeu
tꢀLeu
500±30
ꢀ
ꢀ
–0.13
–0.25
28±1
12±3
O
N
H
2
1
4900±200
N
O
a
HeLa cells were treated with 50 µM inhibitors and HIFꢀ1α stabilization levels were measured
by Western blot after 2 h treatment (protein levels normalized to HIFꢀ1α stabilization level
observed with 1).
In summary, while we were not able to improve binding affinity beyond that of inhibitor 10,
most of the designed inhibitors retained in vitro binding potencies around 100 nM, and also
retained high cellular activity, in many cases greater than starting inhibitor 1. In particular, 15
and 10 were the only compounds that showed Kd <100 nM, and positive group efficiencies
(
Table 2). Inhibitor 15 was shown to be highly cell permeable (vide infra, Table 3) and exhibited
cellular activity comparable with 10 and significantly greater than 1. Further investigation with
5 revealed no toxicity in cells (Figure 6), supporting further evaluation of it as a chemical
probe.
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Figure 6. Inhibitor 15 is not cytotoxic. Colony formation (A) and cellꢀproliferation assays (B)
showing no toxicity in HeLa cells after treatment with 150 µM of inhibitor 15 and its cis epimer.
To support the SAR results of the second inhibitor series, the Xꢀray crystal structures of 15 and
16–19 bound to VBC were obtained. These crystal structures revealed that most of the key
interactions and structural modifications observed for 10 were maintained. Described herein are
the key changes in interactions observed at the LHS from the different groups. The cyano group
on inhibitor 15 (Figure 7A) forms a hydrogen bond with a water molecule that allows the
formation of a water network similar to the one observed in the crystal structure with inhibitor 3
bound (Figure 3A). In the crystal structure of inhibitor 16 (Figure 7B) the ketone adopts an
optimal distance (2.5 ± 0.1Å) and acceptable orientation (angles formed between the C=O bond
and amide nitrogen of 90.7 ± 2.0º and between the CꢀN bond and ketone oxygen of 91.5 ± 0.8º)
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for the formation of an intramolecular hydrogen bond with the NH of inhibitor LHS amide
6
0
(
Supplementary Figure 5). The observed intramolecular interaction could stabilize the
conformation of the LHS of the inhibitor, improving affinity and cell permeability, thus
0
1
2
3
4
5
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8
9
0
1
2
3
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0
1
2
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9
0
61,62
contributing the observed high cellular activity.
The crystal structure of 17 (Figure 7C) did not show any major changes on the ligand binding
mode and on the conformation of pocket residues when compared with the crystal structure of
the fluoroꢀanalog 10 (Figure 3C). The terminal acetamide of 17 points upwards towards the
solvent, not forming any new interactions with the protein nor an intramolecular hydrogen bond
with the NH of the LHS inhibitor amide. These features, together with the extra amide Hꢀbond
donor group, likely account for the poor cellular activity of 17 (Table 2).
The structure of inhibitor 18 bound to VBC did not reveal any critical changes in either the
ligand or pocket residue conformations (Figure 7D). The cyclobutyl moiety fills the far LHS of
the pocket, inducing a conformational change to the side chain of Arg69, as previously
4
1
described. Replacement of the cyclobutyl with an oxetane group in inhibitor 19 showed that the
ether oxygen was able to replace the LHS amide in the ability to form a hydrogen bond with the
LHS structural water (Figure 7E). A hydrogen bond interaction from the oxetane oxygen (2.9 ±
0.1 Å distance) leads to an increase in the distance between the amide carbonyl oxygen and the
structural water (3.7 ± 0.2 Å) (Supplementary Figure 6). This is incompatible with the bidentate
interaction mode previously observed with inhibitor 3, a result of the increased distance between
the two oxygen atoms in 19 relative to 3, and the steric constraints of the oxetane ring.
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Figure 7. Coꢀcrystal structures of secondꢀseries inhibitors. Crystal structure of VBC in
complex with 15 (A, PDB 5LLI), 16 (B), 17 (C), 18 (D) and 19 (E) (purple carbons). VHL is
shown as a pale green surface and the VHL residues forming the binding pocket as orange stick
representations. Inhibitorꢀbinding waters are shown as a red spheres. Hydrogen bond interactions
between inhibitors, bound waters and VHL pocket residues are shown as dashed red lines
.
In summary we describe a groupꢀbased optimization of 1 that led us to the discovery of a more
potent inhibitor 10. Further optimization led us to the discovery of inhibitor 15 which showed
equivalent cellular potency when compared with 10 and was not cytotoxic in cells.
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2
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6
Chemistry. Intermediate 22, 24 and 27 were obtained following the protocol previously
4
1,55
described by us.
Intermediate 22 was used used for the synthesis of final inhibitors 4, 6ꢀ8 via
acylation or inhibitors 3, 5, 9, 10 and 16ꢀ20 by amide coupling reaction (Scheme 1). Further
treatment of intermediate 22 with 4ꢀchlorobutanoyl chloride, followed by intramolecular
cyclization, led to the synthesis of inhibitor 12 (Scheme 1). Reductive amination of 22 with
formaldehyde followed by acylation yielded inhibitor 13 (Scheme 1). An amide coupling
reaction between intermediate 22 and 1ꢀ(tertꢀbutoxycarbonyl)azetidineꢀ3ꢀcarboxylic acid led to
the intermediate 23, which after deprotection, followed by acylation, yielded inhibitor 21
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Scheme 1).
Scheme 1
HO
H
S
N
O
O
R
N
N
NH
O
i
4, 6-8
HO
HO
ii
N
O
H
S
S
H
N
N
N
O
O
N
N
N
O
H N
O
2
2
2
12
iii
HO
vi, vii
S
N
O
H
O
R
N
N
NH
O
3
, 5, 9, 10, 16-20, 23
HO
H
S
iv, v
N
O
O
N
N
N
O
HO
13
H
S
N
O
O
N
N
NH
O
21
N
O
2
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Reagents and conditions: (i) N(Et) , anhydride derivative, DCM, r.t., 2 h; (ii) 4ꢀchlorobutanoyl
3
chloride, DCM/NaOH, rt, 1 h; KOtBu, THF, 0 ºC–rt, o.n; (iii) Carboxylic acid derivative ,
HATU, DIPEA, DMF, r.t., 1 h; (iv) TFA:DCM, r.t., 30 min.; (v) N(Et) , acetic anhydride, DCM,
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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35
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39
40
41
42
43
44
45
46
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48
49
50
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57
58
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60
r.t., 2 h; (vi) Formaldehyde, DMF, r.t., 90 min.; NaBH(OAc) , r.t., 10 min.; (vii) N(Et) , acetic
3
3
anhydride, DCM, r.t., 2 h.
Intermediates 25 and 26 were obtained from intermediate 24 after deprotection with
trifluoroacetic acid followed by amide coupling reaction with Lꢀalanine or Lꢀthreonine,
respectively. Final inhibitors 11 and 14 were obtained from intermediates 25 and 26 after their
deprotection followed by acylation or amide coupling reaction, respectively (Scheme 2).
The cis epimer of inhibitor 10 was obtained from intermediate 27 following the same synthetic
pathway used for its trans conformer and, using BocꢀHyp cis epimer to obtain the desired final
inhibitor. (Scheme 3)
Scheme 2
S
S
N
N
HO
HO
H
H
iii, iv
N
N
N
N
i, ii
O
O
O
25
O
11
O
NH
NH
S
Boc
N
HO
H
N
N
24
i, v
Boc
O
S
S
N
N
HO
OH
HO
OH
H
H
iii, vi
N
N
N
N
O
O
26
O
O
O
NH
NH
Boc
14
F
2
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2
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4
4
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4
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5
5
5
5
6
Reagents and conditions: (i) TFA:DCM, r.t., 30 min.; (ii) BocꢀLꢀAla, HATU, DIPEA, DMF, r.t.,
1
h; (iii) TFA:DCM, r.t., 30 min.; (iv) N(Et) , acetic anhydride, DCM, r.t., 2 h; (v) BocꢀLꢀThr,
3
HATU, DIPEA, DMF, r.t., 1 h; (vi) 1ꢀfluorocyclopropanecarboxylic acid, HATU, DIPEA, DMF,
r.t., 1h.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3
Reagents and conditions: (i) TFA:DCM, r.t., 30 min.; (ii) 1ꢀfluorocyclopropanecarboxylic acid,
HATU, DIPEA, DMF, r.t., 1 h.
Evaluation of VHL inhibitor permeability and binding kinetics. Cell membrane passive
permeability is known to have an important role in inhibitor optimization, as it can lead to an
increase of intracellular concentration of inhibitor available to interact with the desired target,
which would be expected to increase cellular activity. Optimization of lipophilicity at
physiological pH, topological polar surface area (TPSA) and number of rotatable bonds are
63,64
known to directly impact cell membrane passive permeability.
From the SAR results it was
possible to observe that the increase in cellular potency was not only related with the increase of
inhibitor binding affinity to VBC (Figure 8A). Therefore, in order to evaluate how lipophilicity
and cell membrane passive permeability contributed to VHL inhibitors activity in cells, these
parameters were experimentally measured for a selection of the most potent inhibitors (Table 3).
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Table 3. Structures, K s measured by ITC, calculated Ligand Efficiency (LE) [LE = −ꢀG/NHA
d
6
5
65
= −RT ln K /NHA] and Ligand Lipophilic Efficiency (LLE) [LLE = pK –LogD] based on
d
d
ITC results, PAMPA measured passive permeability, chromatographic hydrophobicity index
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
logD (CHILogD ), number of hydrogen bond donors, calculated topological polar surface area
7
.4
(TPSA) and number of rotatable bonds of inhibitors 1, 15, 3, 6, 10 and 16ꢀ19.
Number of
hydrogen
LE
(kcal × mol
× NHA )
LLE
(kcal × mol
× NHA )
b
b
K ITC
ꢀ
ꢀ1
Permeability
(nm × s )
TPSA
(Å )
Rotatable
bonds
d
Inhibitor
R
ꢀ1 a
CHILogD7.4
2
(nM)
1
ꢀ1
ꢀ1
bond donor
b
groups
1
3
6
185±6
105±11
132±10
44±9
0.27
0.27
0.26
0.27
0.26
0.25
0.24
5.83
6.28
5.68
5.73
5.65
5.63
6.28
1.2
0.1
0.90
0.65
1.14
1.63
1.40
1.34
0.61
3
4
3
3
3
3
4
111.6
131.9
111.6
111.6
135.4
128.7
140.7
10
11
11
11
12
12
13
6.1
1
1
1
1
1
0
5
6
7
8
27.1
19.4
15.0
0.1
90±5
106±14
128±21
210±12
232±20
0.25
0.25
5.38
5.92
11.6
0.3
1.30
0.71
3
3
111.6
120.9
11
11
1
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Permeability of inhibitors were determined at room temperature by PAMPA, using Propanolol
(medium permeability – 62.7 nm/s) as a control.
b
Values calculated using StarDrop software.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inhibitors 10 (LE = 0.27; LLE = 5.73), 15 (LE = 0.26; LLE = 5.65) and 16 (LE = 0.25; LLE =
5.63) with permeabilities of 27.1, 19.4 and 15.0 nm/s, respectively, were found to be the most
permeable inhibitors. At the other extreme were inhibitors 3 (LE = 0.27; LLE = 6.28), 17 (LE =
0
.24; LLE = 6.28) and 19 (LE = 0.25; LLE = 5.92) with the lowest permeability values. A direct
relationship was observed between inhibitor permeability, lipophilicity and activity in cells
Figure 8B and 8C). Inhibitors 6, 10, 15, 16 and 18 were found to be more lipophilic than starting
(
inhibitor 1, contributing to their higher cellular permeability, which was thus reflected in their
reasonable intracellular free compound concentration (Supplementary Table 1) that leads to a
6
6
higher cellular potency. In contrast, analysis of TPSA, number of rotatable bonds and number
of hydrogen bond donors (HBDs) revealed no correlation with cell permeability (Table 3). Due
to limitation of our PPIꢀtargeting pharmacophore, these parameters in VHL inhibitors closely
2
64,67
approach the maximal limits for desired membrane permeability (TPSA <140 Å , HBDs <5).
Clearly, for the most active inhibitors these high values are compensated for by a higher
lipophilicity, resulting in an overall increase of inhibitors permeability and activity in cells. The
data however suggests that careful monitoring of these parameters is warranted. For example,
adding an extra HBD group (from 3 to 4) in inhibitors 3 and 17 led to marked decrease in
permeability and cellular potency compared to 1, despite the comparable CHILogD7.4 values
2
(Tables 2 and 3). Similarly, breaking beyond TPSA of 140 Å in inhibitor 17 resulted in the
lowest cell permeability and cellular activity amonst the tested inhibitors (Tables 2 and 3). These
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results argue for close monitoring of all these physicochemical properties during optimization of
a PPI inhibitor.
10
11
12
13
14
15
16
17
18
19
20
21
22
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26
27
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. Correlations between cellular activity and physicochemical parameters in vitro.
Reported values are from Tables 2 and 3, for inhibitors 1, 3, 6, 10, 15 and 16–19. (A) ITC pK vs
d
activity in cells. (B) CHILogD7.4 vs PAMPA permeability. (C) relative cellular activity vs
permeability. HIFꢀ1α protein levels measured using Western blot, as proxy of cellular activity,
and normalized to protein levels with inhibitor 10.
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1
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1
1
2
2
2
2
2
2
2
2
2
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5
6
In addition to permeability and binding affinity, binding kinetics are also key parameters that can
influence cellular activity. Longer drug residence at the target can promote the duration of
pharmacological onꢀtarget effect. In order to evaluate how the binding kinetics could influence
VHL inhibitor activity in cells, binding kinetics parameters were determined by surface plasmon
resonance for 1, 10, 15, 16 and 18, which all showed high passive membrane permeability and
high potency in cells (Table 4). The binding experiments were performed at 10 ºC and 20 ºC in
order to compare the kinetics and affinity of the inhibitors at different temperatures.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
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1
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3
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2
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0
Table 4. Structures, association (k ) and dissociation (k ) rates determined by Surface Plasmon
on
off
Resonance (SPR), K s determined by SPR (K = k /k ), calculated dissociative halfꢀtimes (t ),
d
d
off on
1/2
microsomal and plasma stability of inhibitors 1, 10, 15, 16 and 18.
Plasma
Stability
ꢀ1 ꢀ1 (1/2 life in
Microsomal
stability
mL×min ×g )
k
k
K SPR Dissociative
on
1 ꢀ1
off
ꢀ1
d
Inhibitor
1
R
a
ꢀ
(M s )
(s )
(nM)
t
1/2 (s)
(
min)
5
4
7
.5x10 ±1.0x10 (10ºC) 0.092±0.001 123 (10ºC) 7.534±0.007
6
4
0.7
>180
0
.233±0.005 107 (20ºC) 2.98±0.01
2.18x10 ±4x10 (20ºC)
O
6
5
2.4x10 ±1.0x10 (10ºC)
0.058±0.007 24 (10ºC) 11.94±0.08
0.098±0.001 16 (20ºC) 7.073±0.007
F
1
1
1
0
5
6
N
6
4
2.9
3.1
2.5
>180
>180
>180
H
5
.99x10 ±1.0x10 (20ºC)
5
4
6.6x10 ±1.1x10 (10ºC) 0.065±0.001 99 (10ºC) 10.66±0.01
5
4
0.118±0.001 52 (20ºC) 5.874±0.007
7
.8x10 ±1.0x10 (20ºC)
5
4
6.0x10 ±4x10 (10ºC)
0.069±0.004 115 (10ºC) 10.04±0.01
0.169±0.002 97 (20ºC) 4.101±0.007
5
4
8
.2x10 ±1.3x10 (20ºC)
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.17x10 ±5x10 (10ºC)
0.140±0.002 271 (10ºC) 4.95±0.01
.256±0.004 233 (20ºC) 2.71±0.01
1
8
6
4
3.0
>180
0
.10x10 ±3x10 (20ºC)
a
Dissociative halfꢀlife of inhibitorꢀVBC binary complex calculated based on inhibitor
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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dissociation rates (k ), t = ln2/k .
off
1/2
off
A close relationship was observed between dissociation rates and dissociation constants from
VBC. Inhibitors 10, 15 and 16, with higher binding affinity than 1, also showed slower
dissociation rates relative to 1 (Table 4). After comparison of the dissociative halfꢀlife (from
VBC complex) of these inhibitors, it was possible to observe higher dissociation times correlate
with higher cellular potencies. Inhibitor 10 and 15, the most active inhibitors in cells, had the
highest dissociation halfꢀlives amongst the compounds evaluated (Figure 9). It is worth pointing
out that even the slowest off rates measured for our VHL inhibitors are still relatively fast
overall. The resulting residence times are consequently all on the order of seconds, rather than
minutes, suggesting that there is much scope to optimize this parameter in future. Nevertheless,
our data suggests that even these relatively short residence times suffice for a pharmacological
6
9
effect on VHL, as shown with other drugꢀtarget interactions.
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