Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen

Article abstract of DOI:10.1021/acs.jmedchem.6b01055

Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.

Full text of DOI:10.1021/acs.jmedchem.6b01055

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Article
Discovery of a Chemical Probe Bisamide (CCT251236): An
Orally Bioavailable Efficacious Pirin Ligand from a Heat
Shock Transcription Factor 1 (HSF1) Phenotypic Screen
Matthew D. Cheeseman, Nicola E. A. Chessum, Carl S Rye, A Elisa Pasqua, Michael J Tucker,
Birgit Wilding, Lindsay E Evans, Susan Lepri, Meirion Richards, Swee Y. Sharp, Salyha
Ali, Martin G. Rowlands, Lisa O'Fee, Asadh Miah, Angela Hayes, Alan T. Henley, Marissa
Powers, Robert te Poele, Emmanuel De Billy, Loredana Pellegrino, Florence I Raynaud,
Rosemary Burke, Rob L.M. van Montfort, Suzanne A. Eccles, Paul Workman, and Keith Jones
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01055 • Publication Date (Web): 23 Nov 2016
Downloaded from http://pubs.acs.org on November 25, 2016
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Therapeutics Unit
Eccles, Suzanne; The Institute of Cancer Research,
Workman, Paul; Institute of Cancer Research, Cancer Research UK Centre
for Cancer Therapeutics
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Jones, Keith; Institute for Cancer Research, CRUK Cancer Therapeutics Unit
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Discovery of a Chemical Probe Bisamide
(CCT251236): An Orally Bioavailable Efficacious
Pirin Ligand from a Heat Shock Transcription Factor
1 (HSF1) Phenotypic Screen
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Matthew D. Cheeseman,1 Nicola E. A. Chessum,1 Carl S. Rye,1 A. Elisa Pasqua,1 Michael J.
Tucker,1 Birgit Wilding,1 Lindsay E. Evans,1 Susan Lepri,1 Meirion Richards,1 Swee Y. Sharp,1
Salyha Ali,1,2 Martin Rowlands,1 Lisa O’Fee,1 Asadh Miah,1 Angela Hayes,1 Alan T. Henley,1
Marissa Powers,1 Robert te Poele,1 Emmanuel De Billy,1 Loredana Pellegrino,1 Florence
Raynaud,1 Rosemary Burke,1 Rob L. M. van Montfort,1,2 Suzanne A. Eccles,1 Paul Workman,1,*
Keith Jones1,*
1
Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London
SW7 3RP, United Kingdom.
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Division of Structural Biology at The Institute of Cancer Research, London SW7 3RP, United
Kingdom.
Key Words. HSF1, Proteomics, Pirin, Phenotypic, Bisamide.
ABSTRACT
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Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather
than measuring affinity for individual recombinant proteins, have recently attracted renewed
interest as an efficient strategy for drug discovery. In this paper, we describe the discovery of a
new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to
detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and
displays efficacy in a human ovarian carcinoma xenograft model. By developing cellꢀbased SAR
and using chemical proteomics, we identified pirin as a high affinity molecular target, which was
confirmed by SPR and crystallography.
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INTRODUCTION
Despite the recent extraordinary progress seen in cancer therapy using molecularlyꢀtargeted
drugs, the disease commonly remains stubbornly resistant to effective long term treatment. Even
when excellent responses to drugs are initially observed, resistance is almost inevitable and
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patients are then left with few treatment options as the discovery of targeted therapies in
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oncology has focused on relatively few protein families. To break this cycle and expand the
treatment options for cancer patients, new approaches are needed to discover novel druggable
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protein targets.
In phenotypic screens, small molecules undergo highꢀthroughput screening against intact cells,
rather than recombinant proteins, and discrete phenotypic changes in the cell are measured and
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quantified. Interest in phenotypic screens has increased significantly in recent years, due to their
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potential to effectively discover new drugs. Phenotypic screens have several advantages over
screens using recombinant proteins. Hits from a phenotypic screen will, by definition, be cell
permeable and have cellular activity, potentially reducing optimization cycles and timelines.
Also, because the screening approach is unbiased, established knowledge of the biology of
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molecular targets is not required. Finally, polypharmacology is often observed with small
molecules and structurally related protein families; this can be crucial for efficacy and is
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perfectly compatible with a phenotypic screening approach.
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In contrast, progressing hits from a phenotypic screen can generate a number of unique
challenges. Discovering pharmacodynamic (PD) biomarkers in vivo for use in animals can be
difficult when developing hits from phenotypic screens, as the pathways commonly need to be
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activated with an external stimulus. Cellꢀbased screens are typically more expensive and timeꢀ
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consuming, so may require a greater commitment prior to the screening campaign. Furthermore,
molecular target identification, deconvolution and validation are crucial steps if new chemical
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probes and drugs are to be discovered. These are often a bottleꢀneck in phenotypic screening.
Even with successful target deconvolution, the target discovered may not be of interest; for
example, the target may already be drugged or be a known antiꢀtarget. Finally,
polypharmacology may be a serious impediment to compound progression, because the
interaction with multiple structurally related protein targets may prove impossible to
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deconvolute. These challenges alter the balance between prioritizing druglike properties of
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compounds and an efficient target identification strategy.
To execute a successful phenotypic screening campaign, it is critical to select an appropriate
phenotype for small molecule intervention. Heatꢀshock factor protein 1 (HSF1) is a transcription
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factor and the master regulator of the ancient, canonical heatꢀshock response. A large body of
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work has verified the importance of HSF1 to tumorigenesis and cancer progression. HSF1 has
been proposed to be activated by various elements of the cancer state, potentially reprogramming
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the transcriptome in a way that is overlapping with, but distinct from, the heatꢀshock response.
Also, a strong correlation has been reported between the expression of activated HSF1 in tumors
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and adverse clinical outcomes. This evidence indicates that the inhibition of HSF1ꢀmediated
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transcription could be a viable strategy in cancer treatment. Moreover, inhibiting the HSF1
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stress pathway would represent an attempt at targeting nonꢀoncogene addiction and proteotoxic
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stress, which has been proposed to be advantageous. However, HSF1 is a ligandless
transcription factor so is unlikely to be amenable to standard drug discovery strategies and direct
inhibition with small molecules. Therefore, we proposed that an inhibitor of HSF1ꢀmediated
transcription, which antagonized the HSF1 pathway but without necessarily binding directly to
HSF1, could be discovered and developed via a cellꢀbased phenotypic screen.
RESULTS
HSF1 Phenotypic Assay. To observe HSF1ꢀmediated transcription in an in vitro setting, the
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HSF1 pathway is activated by a validated heatꢀshock protein 90 (HSP90) inhibitor, or another
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form of external stress, which initiates the heatꢀshock response. Commonly, the output of the
heatꢀshock response is quantified by measuring the induction of heatꢀshock 70 KDa protein 1
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(HSP72) expression, the stressꢀinducible HSP70 isoform. HSF1 pathway inhibitors are then
defined by their ability to block the induction of HSP72. Several HSF1ꢀmediated HSP72
induction inhibitors have been discovered via this method with different proposed molecular
,
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mechanisms of action (Figure 1).
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Figure 1. Inhibitors of HSF1ꢀmediated HSP72 induction
With the aim of discovering novel hitꢀmatter that inhibits HSF1ꢀmediated transcription, we
previously carried out a cellꢀbased highꢀthroughput phenotypic screen in U2OS human
osteosarcoma cells of ~200,000 compounds, including ~35,000 compounds from a kinaseꢀ
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focused library. The screen quantified the inhibition of HSP72 induction using the Arrayscan
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assay following treatment with the HSP90 inhibitor tanespimycin (17ꢀAAG).
The Bisamide Series. Using this screen, we identified a potent hit from the kinaseꢀfocused
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deck, bisamide 1 (CCT245232) (Figure 2). Following reꢀsynthesis, the screening hit was
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confirmed and displayed a pIC50=8.55±0.09 (IC50=2.8 nM, n=49) in our HSP72 cellꢀbased
enzymeꢀlinked immunosorbent assay (ELISA) in U2OS cells. The HSP72 cellꢀbased ELISA
assay is an alternative assay format to the Arrayscan assay for quantifying the induced
expression of HSP72 and was used as our primary phenotypic pathway assay throughout the
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study. The IC50 was defined as the concentration that inhibited the signal to 50% of the 17ꢀ
AAG (250 nM) induced HSP72 expression, relative to the control 17ꢀAAG alone (see supporting
information for details). The clear structural feature defining this chemotype was the N,N’ꢀ4ꢀ
methylꢀ1,3ꢀphenylenediamide core (Figure 2).
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Figure 2. HSF1 pathway inhibitor, bisamide 1
We had previously demonstrated that pan cyclinꢀdependent kinase (CDK) inhibitors, and
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potent CDK9 inhibitors in particular which act as transcription antagonists , can inhibit HSF1ꢀ
mediated HSP72 induction phenotype; therefore, we initially suspected a similar mechanism for
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this chemotype. However, upon biochemical screening of bisamide 1 against CDK2 and CDK9,
no inhibition was observed (<10 % inhibition at 1 ꢀM, see supporting information). Therefore,
we hypothesized that bisamide 1 was acting through a different mechanism of action and
consequently was submitted for further characterization.
Hit Characterization – Kinase Activity. HSF1 is regulated by multiple postꢀtranslational
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phosphorylations, so we hypothesized that kinase inhibition other than CDK2 and CDK9 was
causing the observed HSF1ꢀmediated HSP72 induction inhibition. Therefore, bisamide 1 was
screened against a broad kinase set using the KINOMEscan biochemical screening platform, a
binding assay which gave singleꢀpoint percentage inhibition values at 1 ꢀM (DiscoverX,
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http://www.discoverx.com). Analysis of this data set for 442 kinases revealed that bisamide 1
inhibited only 9 kinases >50% and 4 kinases >90% (see supporting information for details). To
validate the kinase hits, orthogonal functional assays were carried out on the 4 kinases that
displayed >90% inhibition. Of these, KIT, PDGFRA and PDGFRB failed to confirm in their
respective functional assays, all returning pIC50<5 (IC50>10000 nM). Bisamide 1 did display
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modest activity against BRAF, returning a pIC50=6.38 (IC50=420 nM, n=1). Although it seemed
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unlikely that the modest biochemical BRAF activity would translate into cellular activity, seven
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commercially available potent BRAF inhibitors of differing chemotypes were tested in our
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cellular HSF1ꢀmediated HSP72 induction inhibition assay for comparison and none displayed
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pIC50>5 (IC50<10000 nM). This indicated that BRAF inhibition was not an important
contributor to the observed HSF1 transcription inhibition phenotype (see supporting
information).
Hit Characterization – Cellular. To establish whether bisamide 1 displays cellular activity
without activation of the heatꢀshock response using the HSP90 inhibitor 17ꢀAAG, the growth
inhibitory effects were assayed in the U2OS cell line using the CellTiterꢀBlue (CTB) assay
(Promega). Following four days of treatment, bisamide 1 displayed a highly potent
pGI50=7.74±0.08 (GI50=18 nM, n=13), when compared to vehicle. To assess the broader single
agent cellular activity of 1, the compound was assayed for growth inhibition against a large,
geneticallyꢀdiverse panel of human cancer cell lines (Genomics of Drug Sensitivity in Cancer,
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www.cancerrxgene.org). Of the 635 cell lines assayed, 628 (99%) displayed a pGI50>6
(GI50<1000 nM), 455 (72%) a pGI50>7 (GI50<100 nM) and 18 (2.8%) a pGI50>8 (GI50<10 nM),
with no clear selectivity against any specific tissue type (see supporting information for details).
These results suggested that bisamide 1 had wideꢀranging antiꢀcancer activity and that activation
of the HSF1 pathway with an HSP90 inhibitor was not required for 1 to inhibit cancer cell
growth.
Cell-Based SAR. Given the complexity of factors underlying cellꢀbased structure activity
relationships (SAR), we focused on matched pair changes to establish which features were
crucial for the cellular activity of the bisamide chemotype. There were two primary aims to our
medicinal chemistry strategy. Firstly, because our biochemical kinase screening had failed to
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reveal any potential kinase targets, a broader target identification strategy would need to be
developed. Secondly, although bisamide 1 had excellent cellular potency, the compound had
poor aqueous solubility (kinetic solubility <2 ꢀM), which would need to be addressed to validate
HSF1 pathway inhibition as an antiꢀcancer strategy in an in vivo human tumor xenograft model.
Both goals required us to improve our understanding of the cellular SAR, particularly to identify
a vectorꢀtoꢀsolvent through which either a chemical probe linker or a solubilizing group could be
attached, without disrupting the ability of the compound to bind to the primary pharmacological
target or targets.
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To develop the cellꢀbased SAR, we switched to the human ovarian carcinoma cell line SKꢀ
OVꢀ3, which was relatively sensitive to growth inhibition and is commonly used in translational
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drug discovery both in vitro and in vivo. When the activity of bisamide 1 in the SKꢀOVꢀ3 cell
line was assayed, it gave a pIC50=7.17±0.07 (IC50=68 nM, n=4) using the HSP72 cellꢀbased
ELISA assay induced with 250 nM 17ꢀAAG. Bisamide 1 also inhibited the proliferation of SKꢀ
OVꢀ3 cells in the CTB assay with a pGI50=8.08±0.12 (GI50=8.4 nM, n=12).
Scheme 1. Synthesis of the benzodioxane bisamide replacements
Reagents and conditions: (i) Oxalyl chloride, DMF, DCM; then, pyridine, DCM. (ii) Fe, NH4Cl,
EtOH/H2O. (iii) RPhCO2H, HATU, DIPEA, DMF.
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We first focused the exploration on the benzodioxane motif (Scheme 1). The leftꢀhand ring
system analogues (Table 1, Entries 1ꢀ6) were synthesized via a threeꢀstep procedure from the
commercially available 2ꢀmethylꢀ5ꢀnitroaniline 2. Following amide bond formation, through the
reaction of 2ꢀmethylꢀ5ꢀnitroaniline 2 with 2ꢀmethylquinolineꢀ6ꢀcarboxylic acid 3, and
subsequent reduction of the nitroꢀgroup using iron, the second amide bond was generated using
the corresponding carboxylic acid in a HATUꢀmediated coupling to afford the various
benzodioxane analogues in good to moderate yields.
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Table 1. Bisamide analogues
SKꢀOVꢀ3
pIC50±SEM
(IC50, n)a,b
SKꢀOVꢀ3
pGI50±SEM
(GI50, n)a,c
Pirin SPR
pKD±SEM
(KD, n)a,d
Entry
1
Compd
Structures
7.17±0.07
(68 nM, n=4)
8.08±0.12
(8.3 nM, n=12)
7.42±0.03
(38 nM, n=3)
1
<5.00
(>10000 nM, n=2)
<6.00
(>1000 nM, n=2)
2
6
ND
<5.00
(>10000 nM, n=2)
<6.00
(>1000 nM, n=2)
<6.00
(>1000 nM, n=3)
3
4
5
6
7
8
6.35±0.07
(450 nM, n=3)
7.26±0.09
(55 nM, n=3)
7.57±0.01
(27 nM, n=3)
5.59±0.09
(2600 nM, n=4)
6.37±0.12
(430 nM, n=5)
7.74±0.03
(18 nM, n=3)
9
<5.00
(>10000 nM, n=2)
<6.00
(>1000 nM, n=2)
7.57±0.03
(27 nM, n=3)
10
<5.00
(>10000 nM, n=2)
<6.00
(>1000 nM, n=2)
7
8
11
12
13
14
ND
ND
<5.00
(>10000 nM, n=2)
<6.00
(>1000 nM, n=2)
O
<5.00
(>10000 nM, n=2)
<6.00
(>1000 nM, n=2)
<6.00
(>1000 nM, n=3)
N
NH
9
O
<5.00
(>10000 nM, n=2)
<6.00
(>1000 nM, n=2)
10
ND
7.92±0.25
(12 nM, n=5)
8.60±0.11
(8.6 nM, n=4)
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12
15
16
ND
ND
7.29±0.23
(51 nM, n=6)
8.29±0.05
(5.1 nM, n=3)
6.65±0.09
(220 nM, n=6)
8.05±0.08
(8.9 nM, n=7)
7.42±0.00
(38 nM, n=3)
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aAll results are quoted as the geometric mean±SEM. pIC50/pGI50/pKD=-log IC50/GI50/KD (M).
The number of repeats, n, are described in parentheses. ND=Not determined. bCell-based ELISA
assay for inhibition of HSP72 induction; SK-OV-3 cells were pretreated with compound at the
relevant concentration for 1 hour before the addition of 250 nM 17-AAG. HSP72 levels were
then quantified after 18 hours. cGrowth inhibition was measured after 96 hours of treatment and
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compared to vehicle control. pKD values were measured through analysis of the sensorgram at
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equilibrium where possible. Values were then fitted to a one-site specific binding model using
Graphpad Prism Version 6.
Changing the 5ꢀsubstituted dioxane isomer 1 (Table 1, entry 1) to the 6ꢀsubstituted dioxane
isomer 6 (Table 1, entry 2) resulted in a complete loss of cellular activity (pIC50<5), indicating
that the isomer of the benzodioxane was crucial. The acyclic dimethoxy analogue 7 (Table 1,
entry 3) also displayed a complete loss of cellular activity. Next, we investigated the role of the
two oxygen atoms of the dioxane ring. Removing the paraꢀoxygen of the dioxane to give the
metaꢀchroman 8 (Table 1, entry 4) resulted in a modest 5ꢀfold decrease in cellular HSF1ꢀ
mediated HSP72 induction inhibition and a 7ꢀfold decrease in growth inhibition compared to 1;
while the paraꢀchroman isomer 9 (Table 1, entry 5) displayed a further 6ꢀfold decrease in HSF1ꢀ
mediated HSP72 induction inhibition and an 8ꢀfold decrease in growth inhibition compared to 8,
which represents a 37ꢀ and 51ꢀfold decrease in cellular activity respectively compared to the
original hit, bisamide 1. Surprisingly, isochroman 10 (Table 1, entry 6) displayed no measurable
cellular activity. From these results, it was clear that the benzodioxane moiety was crucial for the
cellular activity of the bisamide series. Because the SAR surrounding the benzodioxane was
steep and complex, we decided that this region was unlikely to be solvent exposed, so would be
incompatible with linker and solubilizing group attachment.
Secondly, we investigated the role of the central ring and amide moieties in the cellular activity
of the bisamide chemotype. We hypothesized that the amide groups could play an important role
in hydrogen bonding with a potential target and in controlling the overall shape of the ligand,
while substitution at the central ring could lead to the discovery solventꢀexposed vectors. The
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synthesis of these analogues (Table 1, Entries 7ꢀ10) was similar to that described in Scheme 1.
Substitution at both the 6ꢀposition (Table 1, entry 7) and the 4ꢀposition (Table 1, entry 8) of the
central benzene ring resulted in a complete loss of cellular activity, suggesting that neither of
these vectors represented a viable route to solvent. Owing to the potential for the toluene methyl
group to affect the conformation of the rightꢀhand amide, we decided to leave this group
unchanged. NꢀMethyl substitution of the leftꢀhand amide (Table 1, entry 9) and an attempted
sulfonamide bioisosteric replacement (Table 1, entry 10) also resulted in a complete loss of
cellular activity. Again, owing to the steep SAR in this region of the chemotype, it was clearly
incompatible with linker and solubilizing group attachment.
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Finally, we examined the role of the 2ꢀmethylquinoline ring in the cellular activity of the
bisamide series. All analogues (Table 1, Entries 11ꢀ13) were synthesized via a method similar to
that described in Scheme 1. Firstly, we removed the 2ꢀmethyl group, which is both lipophilic and
has the potential to be a weak hydrogen bond donor, to give quinoline 15 (Table 1, entry 11).
Removal of this group resulted in no significant change in the cellular activity when compared to
1 (Table 1, entry 1). Next we moved the quinoline nitrogen from the 1ꢀ to the 3ꢀposition, to give
isoquinoline 16 (Table 1, entry 12). Again, no significant change in cellular activity was
observed when compared to bisamide 1. Finally, we partially reduced the quinoline ring to give
tetrahydroquinoline 17 (Table 1, entry 13). By changing to tetrahydroquinoline 17, the nitrogen
atom now acts as a hydrogen bond donor rather than an acceptor. Despite this reversal in
anticipated binding properties, only a 3.5ꢀfold decrease in HSF1ꢀmediated HSP72 induction
inhibition and no significant change in antiproliferative activity was observed when compared to
bisamide 1. The broad SAR of the quinoline region of the molecule suggested that this moiety is
solvent exposed, so could therefore be exploited for solubilizing group and linker attachment.
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Exploiting Vectors-to-Solvent – Solubilizing Group Optimization. Owing to the
complexities of the cellular SAR, attaching a solubilizing group to the solventꢀexposed region of
the ligand was considered a more expeditious strategy to generate an in vivo chemical probe
suitable for use in animal models, rather than attempting to optimize the intrinsic solubility of the
bisamide series without insight from structural information. To address this objective, we
adopted an iterative strategy to exploit the rapid synthesis of analogues that would exhibit
improved physicochemical properties and demonstrate the appropriate mouse pharmacokinetic
(PK) parameters for in vivo study (Scheme 2).
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Scheme 2. Synthesis of solubilizing group analogues
Reagents and conditions: (i) Oxalyl chloride, DMF, 1,4-benzodioxane-6-carboxylic acid DCM;
then, 4-methyl-3-nitroaniline, pyridine, DCM, RT; then Pd/C (10%), H2 (1 atm), EtOH. (ii) NaH,
RCH2OH, THF, 0 oC to reflux. (iii) n-BuLi, CO2(s), THF, -78 oC. (iv) HATU, DIPEA, DMF, RT.
The optimal route to the synthesis of these analogues would be to introduce the solubilizing
group via nucleophilic aromatic substitution on the quinoline as the final step; however, this
failed due to consistently low yields. Therefore, the solubilizing group was introduced at the start
of the synthesis. Formation of the precursor carboxylic acid 19 was achieved via lithium halogen
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exchange of the corresponding arylbromide 20. The resulting bisamides were then assayed for
cellular activity and in vitro PK parameters (Table 2).
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8
9
Table 2. Optimization of the bisamide solubilizing group
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26
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SKꢀOVꢀ3
MLM Clint
KS
Calculated
d
Entry Compd
Sol. Group
pGI50±SEM
LogD7.4
pKa (MoKa)e
(ꢀL/min/mg)b
(ꢀM)c
(GI50, n)a
8.08±0.12
(8.3 nM, n=12)
8.37±0.06
(4.3 nM, n=3)
8.94±0.10
(1.1 nM, n=4)
8.13±0.18
(7.4 nM, n=4)
1
2
3
4
35
92
1
5
2.6
3.3
2.0
2.9
NA
NA
9.5
7.7
1
21
22
23
170
76
90
7
8.87±0.09
(1.3 nM, n=8)
5
6
7
24
57
23
40
30
70
2.4
2.5
2.1
9.7
8.8
8.9
24
25
26
O
N
8.97±0.03
(1.1 nM, n=6)
8.65±0.10
(2.2 nM n=25)
apGI50=-log GI50 (M); geometric mean±SEM, n=number of biological repeats in parentheses.
For the corresponding SK-OV-3 HSP72 cell-based ELISA data see the supporting information.
bMouse liver microsome (MLM) assay was carried out at Cyprotex, arithmetic mean of n=2. In
vitro Clint is calculated from the half-life using standard procedures. Assumes the fraction
unbound in the assay is 1. cKinetic Solubility (KS) measured via an in-house HPLC method
37
from phosphate buffer at pH 7.4, all values quoted to 1 SF, the dynamic range of the assay is 1-
d
100
ꢀ
M, arithmetic mean of n=2. Measured via an in-house HPLC method, arithmetic mean of
n=2, all values quoted to 2 SF. dMoKa version 2.5.2, all values quoted to 2 SF. See supporting
information for details.
38
We first attached the oxygenꢀlinked ether chain to give glycol 21 (Table 2, entry 2). No
significant decrease in either HSF1ꢀmediated HSP72 induction inhibition (see supporting
information) or the antiproliferative activity was observed when compared to lead bisamide 1
(Table 2, entry 1). This suggested firstly, the oxygen linker had no detrimental effect on activity
and secondly, that this region of the molecule was indeed solvent exposed. To carryout multiꢀ
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parameter optimization of the solubilizing group, we used four in vitro properties to assess each
compound's potential for in vivo mouse PK: microsomal stability, kinetic solubility at pH 7.4,
lipophilicity as measured by LogD7.4 and the predicted basicity of the solubilizing group. The
glycol 21 displayed an increase in lipophilicity compared to lead bisamide 1; this increase was
reflected in the 2.4ꢀfold decrease in microsomal stability with a modest increase in kinetic
solubility. However, glycol 21 remained a low solubility compound, which is inconsistent with
good oral bioavailability. From these data, we concluded that the compound would need to be
charged to deliver the correct balance of properties. To introduce a charged moiety, we first
focused on the dimethylamine group with a 3ꢀcarbon linker. Dimethylamine 22 (Table 2, entry 3)
displayed no significant change in cellular activity, despite this analogue now being
predominately charged at physiological pH, according to the calculated pKa. Consistent with its
cationic character, dimethylamine 22 displayed a 1.4 log unit decrease in lipophilicity when
compared to 21, which was accompanied by an 18ꢀfold improvement in kinetic solubility.
Unfortunately, the decrease in lipophilicity was not reflected in an improvement in microsomal
stability. We hypothesized that this was due to oxidation of the Nꢀmethyl groups, so we moved to
cyclic amines to reduce the CYP450 mediated degradation. Morpholine 23 (Table 2, entry 4)
showed little improvement in microsomal stability or kinetic solubility when compared to 21.
Analysis of the LogD7.4 and calculated pKa for morpholine 23 suggested that the solubilizing
group was not sufficiently basic to balance the physicochemical properties of the compound.
Moving to piperidine 24 (Table 2, entry 5), we observed a better balance in physicochemical
properties, with the reduction in lipophilicity reflected in improvements in both microsomal
stability and kinetic solubility. However, piperidine 24 was predicted to be highly basic and we
were concerned that this would have a detrimental effect on permeability, and therefore on oral
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bioavailability. To reduce the basicity of the solubilizing group without increasing lipophilicity,
we reduced the linker length to give 25 (Table 2, entry 6), thereby exploiting the inductive effect
of the oxygen. Despite the 0.9 log unit decrease in predicted basicity, the kinetic solubility and
lipophilicity were unchanged. Unfortunately, 25 displayed a decrease in microsomal stability, so
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we reduced the ring size to give pyrrolidine 26 (CCT251236, Table 2, entry 7), which displayed
the desired balance of in vitro properties, while maintaining excellent cellular activity with a
pIC50=7.73±0.07 (IC50=19 nM, n=15, see Table S3 in the supporting information for details) for
inhibition of HSF1ꢀmediated HSP72 induction. The free GI50 in SKꢀOVꢀ3 cells was then
39
calculated from the free fraction in the cell assay, which gave a free GI50=1.1 nM. Western
blotting confirmed that pyrrolidine 26 blocked the HSF1ꢀmediated induction of both HSP72 and
HSP27 as representative heat shock proteins, following treatment with the HSP90 inhibitor 17ꢀ
AAG. Also, qPCR analysis demonstrated that 26 inhibited the induction of HSP72 at the mRNA
level, clearly blocking the induction of HSPA1A gene expression with a pIC50=7.40 (IC50=40
nM, n=1, see supporting information). Taking these results together, pyrrolidine 26 was selected
for in vivo study.
Mouse Pharmacokinetics (PK). To assess the potential of bisamide 26 as an in vivo chemical
tool to study HSF1ꢀmediated transcriptional activity inhibition, the compound was dosed in
BALB/c mice at 5 mg/Kg as an oral solution and iv bolus. Blood concentrations were then
measured over a 24 hour period (Table 3).
Table 3. Mouse blood PK parameters for bisamide 26
Dose
po/iv
mg/Kg
Blood Cl
ꢈꢉ
ꢈꢉ
Vss
L/Kg
Halfꢀ
life (
ꢀꢁꢂ
(
Clu
Vdu
e
ꢀꢁꢂ
(
b
ꢊ,ꢃꢄꢅꢆꢇ
ꢃꢄꢅꢆꢇ
Mouse
(
mL/min/Kg
)
%F
fub
a
d
a
c
(
)
h
)
(
mL/min/Kg
)
(
L/Kg)
h*nM)
h*nM)
a
(
)
5800
(8100ꢀ4200)
1900
(2800ꢀ1400)
9.2
(12ꢀ7.3)
4.2
(6.3ꢀ2.8)
BALB/c
Athymic
5/5
5.3
39
0.0083c
0.015d
48
29
1100
NA
510
NA
20/0
NA
NA
NA
NA
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All values are quoted to 2 SF. The 90% confidence intervals (CI) are in parentheses. NA=not
a
b
c
applicable. The geometric mean of n=3 individual mice. fub=fup/B:P, fup=0.010 (0.011-
d
0.0097), B:P@1
ꢀM=1.2:1 (1.4-1.0). fup=0.025 (0.030-0.020), B:P@1 ꢀM=1.7:1 (1.7-1.7).
cAUCu=AUC*fub. dClu=Cl/fub. eVdu=Vss/fub.
9
Analysis of the mouse PK data revealed that bisamide 26 possessed low total blood clearance
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(10% hepatic blood flow) and moderate oral bioavailability, with a halfꢀlife sufficient to allow
onceꢀaꢀday dosing. In vitro assessment of the plasma protein binding and the blood to plasma
ratio revealed that 26 was highly bound to plasma proteins (~99%); therefore, the unbound
0ꢀ
clearance was high, with a low free exposure from the 5 mg/Kg oral dose, equivalent to free Cav
24h
,
41 42
=2.0 nM.
The high unbound volume of distribution indicates that 26 readily binds to
43
tissues, consistent with the basicity of the compound.
Bisamide 26 Displays Efficacy in a Human Ovarian Carcinoma Xenograft Model. Despite
the high unbound clearance of bisamide 26 in mouse, the good oral bioavailability, halfꢀlife and
excellent in vitro cellular activity (free GI50=1.1 nM) encouraged us to test the potential efficacy
of 26 in an in vivo human tumor xenograft model. Based on the free Cav0ꢀ24h=2.0 nM observed in
nonꢀtumor bearing immunocompetent BALB/c mice following the 5 mg/kg po qd dose, a 20
mg/kg po qd dose in immunodeprived athymic mice was selected to cover ~10ꢀtimes the in vitro
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free GI50 in SKꢀOVꢀ3 cells. To our surprise, following this 20 mg/kg po dose, the free exposure
was actually a disappointing AUCu0ꢀ24h=29 h*nM, equivalent to free Cav0ꢀ24h=1.2 nM (Table 3,
see supporting information for details). Despite the lower than expected free exposure of
bisamide 26, this dose still represented coverage of the in vitro free GI50 in SKꢀOVꢀ3 cells and
45
was well tolerated in a mouse multiꢀdose tolerability study; therefore, the 20 mg/kg po qd dose
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was selected for further investigation.
SKꢀOVꢀ3 cells were injected subcutaneously into athymic mice for tumor formation. Once
tumors were established, the mice were randomized into treatment and control groups and were
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dosed orally onceꢀaꢀday with either vehicle or 20 mg/kg of bisamide 26. Tumor volumes and
mouse body weights were measured throughout and tumor weights were measured at the end of
the study, while total tumor concentrations were measured 2 and 6 hours post final dose. (Figure
3 and supporting information).
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Figure 3. Efficacy of bisamide 26 against SKꢀOVꢀ3 human ovarian carcinoma xenograft model
Blue: vehicle control n=8, Red: 26 20 mg/kg po qd n=8, (Vehicle=10% DMSO, 90% of a 25%
(2ꢀhydroxypropyl)ꢀβꢀcyclodextrin in 50 mM citrate buffer pH 5). Error bars: arithmetic
mean±SEM. Dosing breaks were carried out on days 5ꢀ12, 14, 16, 18, 20, 22, 24, 26, 29, 31
The mice were dosed intermittently throughout the study with 26 to maintain their condition,
as assessed through monitoring body weights (see supporting information). Clear therapeutic
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efficacy was observed with bisamide 26, with a Tumor Growth Inhibition (%TGI) of 70%
based on final tumor volumes. The study was terminated after 33 days and comparison of the
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control and treated arms indicated a 64 % reduction in mean tumor weights (p=0.015) with total
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tumor concentrations of 26 as high as 940 nM, consistent with the compound’s basicity and high
volume of distribution (see supporting information).
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Target Identification. Following the successful efficacy study, the potential for bisamide 26
as a chemical probe to study the effects of HSF1 transcription inhibition, both in vitro and in
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vivo, were clear. However, the variety of HSF1 transcription inhibitors in the literature suggests
there are multiple mechanisms through which this phenotype may be observed, each with
differing potential for drug discovery.
To decipher the molecular mechanism of the bisamide series, we needed to discover their
protein targets. The potency of the bisamide 26 in cellꢀbased assays suggests that only high
affinity efficacy and epistatic targets would be of interest.11 Our biochemical kinase screening
had indicated that there were no high affinity kinase targets. We therefore expanded our
biochemical screening to include other protein families. Bisamide 26 was submitted to the Cerep
Diversity Screen (Cerep, http://www.cerep.fr) comprising 98 molecular targets, including
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receptors and enzymes, measured at 10 ꢀM. Analysis of these screening data revealed that
bisamide 26 displays a good selectivity profile. Only six targets (the receptors: adenosine A2A
and A3, histamine H2 and H3, muscarinic and the enzyme acetylcholine esterase) displayed >80
% inhibition and these generally represented the highly promiscuous receptor protein targets (see
supporting information for details). No hits displayed sufficient activity to clearly relate the
molecular target to the efficacious free concentrations achieved in vitro or in vivo.
Because no clear protein families had been revealed from our biochemical screening, a
different approach was necessary. We decided to exploit a chemical proteomics strategy and
using our knowledge of the cellular SAR, we designed a protein pullꢀdown chemical probe to
identify high affinity molecular targets from a human cancer cell lysate (Figure 4).
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Figure 4. Tool compounds for target identification. All quoted cellular activities are in the SKꢀ
OVꢀ3 cell line, the numbers of repeats are in parentheses
To carry out the chemical proteomics pullꢀdown strategy, the bisamide warhead would need to
be attached via a linker to a solidꢀphase bead, without disrupting binding to the molecular target
or targets. The solubilizing group vector was the obvious choice for the linker attachment, but to
test whether cellular activity was maintained, amide 27 was designed as a cellꢀpermeable mimic
of the solidꢀphase probe. Pleasingly, amide 27 maintained excellent cellular potency (SKꢀOVꢀ3
pGI50=8.04±0.06, GI50=9.1 nM, n=4), so amine 29 was used for attachment to the bead via amide
bond formation. In addition to bisamide 26, tetrahydroquinoline 17 was selected as a second
positive control due to its distinct structural difference. These two compounds would be used to
displace specific molecular targets from the solidꢀphase probe. Also, to help distinguish false
positives from the pullꢀdown experiment, a physicochemicallyꢀmatched negative control was
designed. The dioxane isomer 6 (Table 1, entry 2) had previously been shown to lose all cellular
activity. To physicochemically match the dioxane isomer 6 to bisamide 26, the same ethoxy
pyrrolidine solubilizing group was attached to give isomer 28, which, as expected, displayed no
measurable cellular activity.
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With all four probe molecules in hand, the compounds were submitted to the stable isotope
labeling by amino acids in cell culture (SILAC) massꢀspectrometry based pullꢀdown assay
(Evotec, https:www.evotec.com) to identify and quantify molecular targets of the bisamide series
from the lysate of SKꢀOVꢀ3 cells. Every protein captured by the bead, whether specific or nonꢀ
specific, is analyzed in this methodology by quantitative mass spectrometry. By using SILAC to
quantify the relative amounts of protein captured from the lysate, this approach can, in principle,
determine the apparent affinity of every stable protein in the lysate for the surfaceꢀbound
bisamide probe, although particularly low abundance proteins may not be discovered via this
method and proteins that are unstable to the lysis conditions would not be detected.
Displacement of the captured proteins with the free active bisamides 26 and 17, then allows for
estimation of their apparent affinities for specific protein targets. Proteins that are apparently
displaced from the beadꢀbound probe by the inactive isomer 28 are considered nonꢀspecific or
irrelevant for the cellular activity and discarded. The putative protein targets can then be ranked
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based on their affinity for further investigation (Table 4, see supporting information for details).
Table 4. Molecular targets from the pullꢀdown assay using the bisamide probes in SKꢀOVꢀ3 cell
lysate
Protein EC50
(ꢀM)a
1.3
26 AC50
(ꢀM)b
1.2
26 Kiapp
(ꢀM)c
NA
17 AC50
(ꢀM)b
10
17 Kiapp
(ꢀM)c
NAd
28 AC50
(ꢀM)b
NAa
Protein
GSK3β
Pirin
1.3
1.5
0.028
10
0.19
NAa
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A: SK-OV-3 cell lysate protein binding curves for immobilized 29. B: protein displacement
curves for pyrrolidine 26. Curves are an average of two different mixing conditions. EC50 and
AC50 are determined from analysis of the curves without limits. EC50 is the apparent
a
7
8
9
immobilized bisamide probe concentration at which 50% of the available protein is bound. The
probe bisamide is assumed to be >10-fold molar excess over proteins in the lysate. AC50 is the
b
apparent free bisamide concentration at which 50% of the protein is displaced from the
immobilized bisamide probe. At least 2-fold enrichment of bound proteins when compared to the
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c
matrix. Kiapp values are estimated using the Cheng-Prusoff equation, apparent [29-
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immobilized]=67
ꢀ
M, assuming no mass transport limitation, interactions are purely
competitive and all interactions are direct. dNot Applicable (NA) – Kiapp cannot be calculated if
the interaction with the probe is indirect.
Analysis of these data from the quantified chemical proteomics pullꢀdown experiment revealed
very few molecular targets of the bisamides, consistent with the selectivity observed with our
biochemical screening. After excluding highly promiscuous proteins commonly observed in pullꢀ
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down experiments, three putative targets were identified. PDE6D displayed only weak apparent
affinity for the beadꢀbound probe and little selectivity between active (26 and 17) and inactive
(28) probes, so was not considered further. GSK3β displayed apparent high affinity for both
active analogues, with no apparent affinity for the inactive analogue. However, GSK3β had
previously been assayed in our biochemical kinase screening and both binding and functional
assays (see supporting information) had demonstrated that the bisamide series have no
measurable affinity for this kinase; we therefore concluded that GSK3β was potentially an
indirect target of the bisamide series. The final putative protein target was pirin. The binding
curve for pirin to the beadꢀbound bisamide probe was quite shallow; however, the displacement
curves for pirin, which were very similar to the displacement curves for GSK3β with both cell
active probe ligands, gave robust data with an apparent Ki=28 nM for 26 and no affinity was
observed with the inactive control. Using this method on a SKꢀOVꢀ3 cell lysate, pirin emerged as
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the only remaining putative target of the bisamide series.
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Pirin Validation. Very little is known about pirin and few papers have been published
discussing its possible function. Pirin was first identified in 1997 by Wendler et al. from a yeast
twoꢀhybrid screen to discover interactors of the transcription factor NFI/CTF1. Pirin was
described as an ironꢀbinding, metalꢀdependent protein, predominately localized within the
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nucleus, highly conserved across species and ubiquitously expressed across all tissue types.
Following this initial discovery, Scheidereit et al. reported that human pirin interacts with the
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protoꢀoncoprotein, BCL3, linking pirin with the NFκB pathway. The role of pirin in the NFκB
pathway was further demonstrated by Lui et al.; using SPR, they revealed the metalꢀdependent
formation of a pirin/p65/DNA complex and hypothesized the role of human pirin to be a redoxꢀ
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sensing transcription factor regulator. The redox activity of pirin has previously been discussed,
as the expression level of pirin is proposed to be under the control of the transcription factor
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NRF2 through changes in cellular oxidative stress. Wang et al. previously reported that human
pirin interacts with the tumor suppressor protein EAF2/U19 and that exogenous overexpression
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of pirin increased colony formation in LNCaP human prostate cancer cells. In the human
melanoma cell line WM266.4, Alcalay et al. demonstrated that shRNA knockdown of pirin could
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induce a senescence phenotype and suppress colony formation. Also in the WM266.4 cellꢀline,
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Osada et al. demonstrated that siRNA knockdown of pirin could suppress cell migration.
These results are consistent with the observation by Simizu et al. that siRNA knockdown of pirin
suppressed migration of human adenocarcinoma HeLa cells and that pirin was important for
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epithelialꢀmesenchymal transition (EMT). However, little evidence has emerged of an
antiproliferative phenotype from genetic inhibition of pirin expression and our inꢀhouse data
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using siRNA in SKꢀOVꢀ3 cells was consistent with this finding. Nonetheless, because our
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original screening paradigm was designed to discover inhibitors of transcription, the proposed
role of pirin as a redoxꢀsensitive transcription factor regulator was certainly intriguing.
To confirm pirin as a high affinity molecular target of the bisamide series, we needed to assess
the affinity of the compounds and establish SAR in an orthogonal assay format. Pirin has no
known catalytic function and no known endogenous ligand in mammalian cells, consequently we
decided to focus on surface plasmon resonance (SPR) to measure the affinity of the bisamide
series for pirin. Purified recombinant human pirin was attached to the SPR chip through a
standard amide coupling. The affinity of the bisamide ligand was assessed by equilibrium
analysis of the resulting sensorgram (Figure 5).
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Figure 5. Representative SPR sensorgram and binding isotherm of bisamide 26 bound to
recombinant pirin. The dottedꢀline represents the timeꢀpoint the equilibrium response was
measured. The binding isotherm was fitted to a oneꢀsite specific binding model using Graphpad
Prism 6
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The known pirin ligand TPh A was used as a positive control and to protect the binding site
during amine coupling to the SPR chip, but displayed only modest affinity in our hands
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(pKD=5.77±0.04, KD=1700 nM, n=4). The SPR sensorgram showed that bisamide 26 is indeed a
tightꢀbinding ligand of pirin. Equilibrium analysis revealed that bisamide 26 had a
pKD=7.36±0.01 (KD=44 nM, n=3). However, the ratio of theoretical Rmax to measured Rmax from
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the binding isotherm was 0.40, indicating apparent substoichiometric binding. This could be due
to the coupling of pirin to the SPR chip impairing protein folding or because pirin is a metalꢀ
dependent protein and the metal was leaching out into the running buffer; both effects could
negatively impact the apparent binding affinity and stoichiometry.
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To establish the SAR surrounding pirin binding, several bisamide analogues were selected for
further study. We first focused on the two other analogues used in target identification. The cell
active analogue tetrahydroquinoline 17 (Table 1, entry 13) was also found to display tightꢀ
binding affinity for pirin; in contrast, the negative control dioxane isomer 28 (pIC50<5.00,
IC50>10000 nM, n=2) displayed no measurable affinity. These data suggested that pirin SAR and
the cellular SAR were linked. Increasing the steric bulk of the solubilizing group, as exemplified
by piperidine 24 (Table 2, entry 4) again returned a high affinity pirin ligand (pKD=7.52±0.02,
KD=30 nM, n=3), consistent with the potent cellular activity. However, structural changes to the
benzodioxane amide motif proved more complex. The original hit, bisamide 1 (Table 1, entry 1),
was a tightꢀbinding pirin ligand; whereas the cell inactive analogues, methylated amide 13 (Table
1, entry 9) and dimethoxy 7 (Table 1, entry 3), displayed no affinity for pirin. However, the two
chroman isomers 8 and 9 (Table 1, Entries 4 and 5) were tightꢀbinding ligands for pirin but their
target affinity did not reflect their decrease in cellular activity when compared to bisamide 1.
Finally, the isochroman analogue 10 (Table 1, entry 6) was still a high affinity ligand for pirin,
despite displaying no cellular activity (see Table S11 supporting information for details).
Pirin is a member of the cupin super family of proteins which are so called because they all
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possess a conserved βꢀbarrel. However, due to minimal sequence homology between members
of the cupin family, crystallography is often needed to identify them. The cupins display a huge
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array of functionality, including oxidases and isomerases. To better understand pirin SAR, we
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determined the crystal structure of efficacious bisamide 26 bound to pirin (Figure 6).
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Figure 6. Xꢀray crystal structure of bisamide 26. PDB 5JCT, Pymol image of pirin (light blue
cartoon representation) in complex with bisamide 26 (cyan stick representation), 2 FoꢀFc map
contoured at 1.0 σ (blue mesh) and Pymol image of pirin (magenta, blue stick representation) in
complex with bisamide 26 (cyan stick representation), distances shown are in Ångstroms, 2 Foꢀ
Fc map contoured at 1.0 σ (blue mesh). Crystals belonged to the space group P212121 and
diffracted to resolution of 1.73 Å
Pirin is a metalꢀbinding bicupin characterized by the two βꢀbarrels that are clearly visible in
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the crystal structure. Pirin binds a single metalꢀion in one of the βꢀbarrels, which is where a
deep, small molecule binding pocket is located. The metalꢀion is clearly visible in the electron
density, although it is unclear which metalꢀion is actually bound. The metalꢀion binding site is
formed of three histidine residues (His56, His58 and His101), a glutamic acid (Glu103) and two
water molecules, showing the metalꢀion possesses octahedral coordination. Bisamide 26 forms
no direct interactions with the metalꢀion, instead a waterꢀmediated interaction is created by the
two amide functional groups, which form a hydrogen bonding pincer around this metalꢀ
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coordinated water molecule, revealing why the two amides are crucial for binding. The methylꢀ
distal amide also acts as a hydrogenꢀbond donor with an aspartic acid (Asp43) at the base of the
binding site, explaining why methylation of that group is not tolerated. The methylpyridine ring
of the quinoline substituent is clearly solvent exposed and the solubilizing group cannot be
resolved due to flexibility, consistent with the broad SAR at this position and the eventual
success of the pullꢀdown probe. The SAR around the benzodioxane ring is less clear. From the
cellular SAR, the oxygen atoms of the dioxane are crucial for activity. The narrow binding site is
consistent with the cellularlyꢀinactive dioxane isomer 28 and the cellularlyꢀinactive dimethoxy
analogue 7 having low affinity for pirin (Table 1 and Table S11), as both these groups would
cause a steric clash. However, there are no clear interactions between the protein and the dioxane
ring oxygens, so it is unsurprising that changing the positions of the oxygen atoms has little
effect on pirin affinity, such that chromans 8, 9 and isochroman 10 (Table 1, Entries 4ꢀ6) still
display high affinity for pirin, in contrast to their complex cellular SAR.
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The apparent disconnect between the pirin affinity SAR and the antiproliferative cellular SAR,
led us to hypothesize that either simply binding to pirin was not enough to recapitulate the
cellular phenotype or that binding to a second molecular target was also crucial. For example, in
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the study by Lui et al. the formation of proteinꢀprotein interactions with pirin was under the
allosteric control of the redoxꢀstate of the iron bound in the protein. Ligands bound in the metalꢀ
binding site are therefore likely to act in an allosteric manner to regulate the function of pirin. It
is commonly known that allosteric modulators display complex SAR, where small structural
changes can have little effect on protein affinity but can drastically alter the observed phenotype,
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switching ligands between positive, negative and neutral allosteric modulation.
The
alternative hypothesis consistent with the observed complex cellular SAR was that a second high
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affinity protein target was required to explain the antiproliferative activity of the bisamide 26 and
was not detected in our targetꢀID campaign due to either low abundance or poor stability in the
lysate. The dual targeting of proteins is often observed; for example, CDK4 and CDK6, where
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simultaneous inhibition of both kinases is necessary to observe an antiproliferative phenotype,
owing to the similarity of their respective binding sites, smallꢀmolecule inhibitors inevitably
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inhibit both proteins. RNAi knockdown of pirin by several groups has demonstrated very
limited effects on cell proliferation and as pirin has no known enzymatic function, discovering
and validating a cellular biomarker to investigate its role in the antiproliferative phenotype and in
vivo efficacy is an ongoing challenge.
One phenotype that has previously been associated with a smallꢀmolecule binding to pirin is
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cancer cell migration. Miyazaki et al. demonstrated that their pirin ligand, TPh A, inhibited the
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migration of the melanoma cell line, WM266.4. To investigate whether our tool compound,
bisamide 26, phenocopied this distinct pirinꢀligand chemotype, we decided to investigate its antiꢀ
migratory activity using a scratchꢀwound assay. The scratchꢀwound assay is commonly used to
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assess the effects of smallꢀmolecules on cell migration. Bisamide 26 demonstrated excellent
antiproliferative activity against the WM266.4 cell line (pGI50=7.92±0.10, GI50=12 nM, n=11).
To exclude the effect of inhibiting proliferation on migration in the scratchꢀwound assay, the
cells were plated at high confluency and the timeꢀframe of the assay was reduced to 30 h. The
relative woundꢀdensity was then measured at various concentrations and timeꢀpoints. The
negative control isomer 28 displayed no measurable antiꢀmigratory activity in stark contrast to
the potent pirin ligand bisamide 26, which was able to strongly inhibit the migration of
WM266.4 cells at 100 nM (Figure 7).
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Figure 7. The antiꢀmigratory activity of chemical probe bisamide 26. A: wound healing images
of WM266.4 cells, after 30 h the wound has almost completely healed in the control in contrast
to 100 nM 26. B: quantification of the relative wound density reveals the maximum antiꢀ
migratory activity is achieved at 100 nM 26.
DISCUSSION
Bisamide 26 displays excellent and wideꢀranging in vitro cellular potency, inhibiting both
HSF1ꢀmediated transcriptional activity (defined by the inhibition of the induced expression of
the heatꢀshock proteins HSP72 and HSP27 and HSPA1A mRNA) and cancer cell proliferation.
Also, 26 has good mouse PK, and demonstrated its anticancer effects in vivo at low free
exposures with clear tumor growth inhibition at tolerable doses. Kinase screening and broader
proteome screening, in addition to the chemical proteomics using SILAC quantified pullꢀdown
with rigorous controls and followꢀup testing, indicate that pirin was the sole specific protein
target that could be identified to date. SPR analysis of the binding of the bisamide series to pirin
confirms that these compounds are high affinity ligands, with most aspects of the pirin SAR
being consistent with the cellular SAR. However, there was an apparent disconnect surrounding
the dioxane group of the chemotype. This requires further investigation to determine whether
pirin is crucial for the in vitro antiproliferative activity and in vivo efficacy via an allosteric
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