Discovery and Optimization of a Selective Ligand for the Switch/Sucrose Nonfermenting-Related Bromodomains of Polybromo Protein-1 by the Use of Virtual Screening and Hydration Analysis

Article abstract of DOI:10.1021/acs.jmedchem.6b00355

Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein-ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 μM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5).

Full text of DOI:10.1021/acs.jmedchem.6b00355

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Article
Discovery and optimization of a selective ligand for the Switch/
Sucrose Non-Fermenting-related bromodomains of Polybromo
protein-1 by the use of virtual screening and hydration analysis.
Vassilios Myrianthopoulos, Nicolas GABORIAUD-KOLAR, Cynthia Tallant, Michelle
Lynn Hall, Stylianos Grigoriou, Peter Moore Brownlee, Oleg Fedorov, Catherine Rogers,
David Heidenreich, Marek Wanior, Nikolaos Drosos, Nikitia Mexia, Pavel Savitsky, Tina
Bagratuni, Efstathios Kastritis, Evangelos Terpos, Panagis Filippakopoulos, Susanne Müller,
Alexios-Leandros Skaltsounis, Jessica Ann Downs, Stefan Knapp, and Emmanuel Mikros
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00355 • Publication Date (Web): 12 Sep 2016
Downloaded from http://pubs.acs.org on September 12, 2016
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Discovery and optimization of a selective ligand for the
Switch/Sucrose Non-Fermenting-related bromodomains of
Polybromo protein-1 by the use of virtual screening and
hydration analysis.
Vassilios Myrianthopoulos1, Nicolas Gaboriaud-Kolar1, Cynthia Tallant2-3, Michelle-Lynn
Hall4, Stylianos Grigoriou1, Peter Moore Brownlee5 Oleg Fedorov2-3, Catherine Rogers2-3,
,
David Heidenreich6, Marek Wanior6, Nikolaos Drosos1, Nikitia Mexia1, Pavel Savitsky2-3,
Tina Bagratuni7, Efstathios Kastritis7, Evangelos Terpos7, Panagis Filippakopoulos2-3,
Susanne Müller2,3,6, Alexios-Leandros Skaltsounis1, Jessica Ann Downs5, Stefan Knapp2,3,6*
and Emmanuel Mikros1
*
1Department of Pharmacy, University of Athens, Panepistimiopolis Zografou, GR-15771, Athens, Greece.
2Nuffield Department of Clinical Medicine, University of Oxford, Structural Genomics Consortium, Old
Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK.
3Nuffield Department of Clinical Medicine, University of Oxford, Target Discovery Institute (TDI),
Roosevelt Drive, Oxford OX3 7BN, UK.
4Schrödinger Inc., 222 Third Street, Cambridge MA 02139, United States.
5Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1
9RQ, UK.
6Johann Wolfgang Goethe-University, Institute for Pharmaceutical Chemistry and Buchmann Institute
for Life Sciences, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
7Department of Clinical Therapeutics, School of Medicine, University of Athens, Mikras Asias 75, GR-
11527, Athens, Greece.
KEYWORDS: Bromodomains, WaterMap, SZmap, pyrazoloisocoumarin, FRAP assay,
solvation mapping.
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ABSTRACT
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Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging
drug targets for development of anticancer or antiꢀinflammatory agents. In this study,
development of a selective ligand of the fifth BRD of Polybromo Proteinꢀ1 (PB1(5)) related
to Switch/Sucrose NonꢀFermenting (SWI/SNF) chromatin remodeling complexes is
presented. A compound collection was evaluated by consensus virtual screening and a hit
was identified. The biophysical study of proteinꢀligand interactions was performed using Xꢀ
ray crystallography and isothermal titration calorimetry. Collective data supported the
hypothesis that affinity improvement could be achieved by enhancing interactions of the
complex with the solvent. The derived SAR along with free energy calculations and a
consensus hydration analysis using WaterMap and SZmap algorithms guided rational design
of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 ꢁM
and an excellent selectivity profile, thus comprising a promising lead for the development of
chemical probes targeting PB1(5).
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INTRODUCTION
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Medical interventions directly targeting epigenetic functionalities are among the most
promising novel approaches for treatment of serious pathological states such as cancer,
metabolic and neurological diseases, inflammation and viral infections.1ꢀ5 The first marketed
epigenetic drug (Vorinostat) provided in 2006 the “proofꢀofꢀconcept” for targeting epigenetic
machinery by small molecules in a therapeutic manner. Since then, a multitude of epigenetic
proteins have been suggested as potential targets for pharmacotherapy, including families of
enzymes like the histone acetyltransferases (HATs), the DNA methyltransferases (DNMTs)
and the histone deacetylases and sirtuins (HDACs and SIRTs). Bromodomains (BRDs)
comprise a family of 61 epigenetic modules implicated in recognition of acetylated lysine
(Kac) residues mainly on histones.6 The BRDs can be found as components of at least 46
multidomain proteins of the human genome.7,8 The discovery of a small molecule selectively
inhibiting BRDs in bromoꢀandꢀextraꢀterminal (BET) proteins (BRD2, BRD3, BRD4, BRDT),
has provided solid evidence for BRD druggability.9 The fused triazoleꢀthienodiazepine
scaffoldꢀbased 1 ((+)ꢀJQ1) has shown antiproliferative effects in BRD4ꢀdependent human
NUT midline carcinoma (NMC) cells in vivo.10 An increasing number of studies provides a
compelling rationale for using 1 as well as a structurally related triazolobenzodiazepineꢀbased
BET inhibitor (IꢀBET762, see Figure 1A), mainly as antiproliferative or immunomodulatory
agents.11ꢀ15 The first BRD inhibitors have already entered clinical trials as chemotherapeutic
agents.16
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The establishment of the BET group of BRDs as valid therapeutic targets for the
aforementioned aggressive form of cancer, along with the increasing amount of data showing
that BRD modules critically affect several cellular functions, have led to the emergence of
BRDs as appealing targets for development of highly specific inhibitors. As all BRD are not
equally druggable, the family can be subdivided into nine subfamilies (I to IX) according to
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structural features that define druggability of each member.17 At present, an increasing
number of compounds selectively inhibiting BRDs outside the BET group (subfamily II) are
described. Among the available molecules are IꢀCBP112 and CBP30 that target subfamily III
cꢀAMP response elementꢀbinding protein binding protein (CREBBP) and E1A binding
protein p300 (EP300), compounds LP99, IꢀBRD9 and BI9564 inhibiting subfamily IV BRD7
and BRD9, compounds NI57, OFꢀ1 and PFIꢀ4 targeting subfamily IV bromodomain and
PHD finger containingꢀ1, ꢀ2 and ꢀ3 (BRPF1, BRPF2 and BRPF3), compounds GSK2801 and
BAZ2ICR targeting subfamily V bromodomain adjacent to zinc finger containingꢀdomain 2A
and 2B (BAZ2B/A) and 2 (PFIꢀ3) targeting subfamily VIII SWI/SNF related, matrix
associated, actin dependent regulator of chromatin subfamily A, member 2 and 4
(SMARCA2/4) and PB1(5), while a number of molecules targeting sets of BRDs have been
developed as well (compound structures are shown in Figure 1AꢀB, for a more complete
overview please see review references 29ꢀ30).18ꢀ30 Such compounds with single or groupꢀ
wise BRDꢀselective inhibitory properties can significantly advance the elucidation of
biological roles of targeted BRDs and may thus serve as invaluable chemical biology probes
for mechanistic studies or as possible drug candidates.31
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The BRDs of subfamily VIII have been characterized as proteins of intermediate or
difficult druggability, in contrast to highly druggable subfamilies such as I or II (the BETs) or
individual members of subfamilies III, IV and VII.17 Subfamily VIII is comprised by BRDs
that are mostly components of the chromatin remodeling complexes of SWI/SNF family.
Indeed, all SWI/SNF complexes contain a central helicase with a bromodomain
(SMARCA2/4) while the polybromoꢀassociated BRG1 or hBRMꢀassociated factor (PBAF)
complex, a subꢀtype of the SWI/SNF complex (or SWI/SNFꢀB) contains also the polybromo
protein PB1 (or BAF180) which comprises six individual BRD modules.32 Remodeling
complexes of the SWI/SNF family play a central role in development, particularly of the
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cardiac cells.33ꢀ35 Mutations in components of these chromatin modulators have been tightly
linked to development of numerous cancer types including epithelioid sarcoma and malignant
rhabdoid tumors.36ꢀ37 Especially in the case of renal cell carcinoma and pancreatic cancer,
normal expression of the PBRM1 gene encoding for PB1 protein is considered as a critical
factor of tumor progression.38ꢀ41 Moreover, truncating mutations of PB1 which have been
found in breast cancer cells suggest a possible role for PB1 as a tumor suppressor. Although
the precise mechanism for its tumor repressor activity is not yet clearly understood, it is
thought that this functionality might be partially mediated through the ability of PB1 to
regulate p21 induction.42ꢀ44 Moreover, the tumorꢀsuppressing role of PB1 has been shown to
be related to promotion of centromere cohesion and thus preservation of genomic stability in
a manner independent from its involvement in transcriptional regulation.45 An additional
aspect of its tumor suppressing role is highlighted by its activity as a repressor of
transcription in response to double strand breaks of DNA.46,47 Finally, the occurrence of six
distinct BRDs on PB1 has raised a number of questions regarding their concerted or
complementary function or even their redundancy.32 Two of PB1 BRDs lack the canonical
asparagine that coordinates the Kac carbonyl (or equivalent moieties in inhibitors).6 It is
likely that these BRDs bind therefore much weaker to inhibitors that typically hydrogen bond
directly with this asparagine residue. As PB1(5) showed strongest interaction to acetylꢀlysine
containing histone peptide arrays, it is therefore likely the most relevant BRD anchoring PB1
(and the SWI/SNF complex) to chromatin in an acetylꢀlysine dependent way. As a result,
discovery of highly specific chemotypes with inhibitory properties towards BRDs of
subfamilyꢀVIII such as PB1 and more specifically PB1(5) can be regarded as a challenging
project, which would advance the existing knowledge concerning their underlying biological
roles of these domains in health and disease. This notion is further highlighted by the finding
that, while the ATPase module seemingly surpasses SMARCA2/4 as a drug target for
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syntheticꢀlethal interventions in a number of SWI/SNFꢀrelated cancer types, targeting PB1(5)
and SMARCA2/4 does not result in toxicity but in differentiation phenotypes and thus
simultaneous inhibition of several SWI/SNF BRDs can possibly provide alternative routes for
therapeutically modulating chromatin remodeling.28, 29, 48
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In this study, the utilization of three virtual screening (VS) methodologies of high
orthogonality and their combination in a simple consensus scheme is presented. The National
Cancer Institute/Developmental Therapeutics Program (NCI/DTP) Repository was
systematically evaluated against a group of underꢀexplored BRDs with moderate druggability
and a pyrazoloisocoumarin hit showing low micromolar affinity and a promising selectivity
profile towards the fifth BRD of PB1 was identified. The attempt to optimize this ligand was
based on the integration of thermodynamic and structural data obtained by Xꢀray
crystallography and isothermal titration calorimetry (ITC) analysis with theoretical
calculations. Hydration mapping was performed by implementation of the two most robust
algorithms, namely SZmap and WaterMap which afforded a high degree of convergence.
Subsequent synthesis of rationally designed analogues where specific water molecules would
be targeted by gradual expansion of the exocyclic methyl group resulted in derivatives
demonstrating improved binding affinity and high selectivity for PB1(5) over related BRDs,
displacing the protein from chromatin in cells and reducing viability of human fibroblasts.
RESULTS AND DISCUSSION
In silico Screening Protocol and Consensus Ranking
A screening initiative was undertaken as a means to discover compounds that selectively
bind BRDs of subfamily VIII, thus providing novel and tractable scaffolds for sustaining an
inhibitor development project. The NCI/DTP compound repository comprising
approximately 260,000 entries was selected due to its openꢀaccess policy. It was reasoned
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that an inꢀsilico computational evaluation of the collection prior to experimental screening
would increase the anticipated hit recovery rate and additionally enhance the overall rational
character of the study by providing structural insight from the initial steps of the process.
Therefore, three distinct yet complementary VS methodologies were implemented in a
stepwise protocol aiming at the optimal exploitation of sampling efficacy in a timeꢀefficient
manner. These methods would directly account for binding to the protein cavity (docking and
scoring calculations featuring proteinꢀligand energy terms and implicit solvent contributions
to binding) as well as conformational likeliness of the screened compounds to a known active
compound (threeꢀdimensional similarity involving pharmacophoric sites and shape in real
space) or for the existence of hidden or nonꢀobvious molecular motifs (twoꢀdimensional
similarity of topological fingerprints representing structural features and chemical
functionality) commonly occurring between the screened collection entries and a known
active template.
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The overall workflow of the implemented integrated VS protocol is depicted in Figure 2. In
the first step, a ligandꢀbased approach would be used to evaluate the total NCI/DTP
collection and find compounds that were similar to 1. For this purpose, twoꢀdimensional
similarity screening was performed using Canvas (Schrödinger Inc.).49ꢀ51 Canvas was
additionally used in this step to create a potentially enriched subset of the initial library
containing 5,000 diverse compounds (~2% of the total). In the second step, results of the
ligandꢀbased diversity evaluation would be redirected as input to a structureꢀbased screening
approach by implementing dockingꢀscoring calculations of the enriched subset towards the
bromodomain Kacꢀbinding pocket using Glide SP (Schrödinger Inc.).52ꢀ55 In parallel, a
separate ligandꢀbased threeꢀdimensional similarity screening of the global library would be
performed using the ROCS algorithm (OpenEye Inc.).56ꢀ57 Finally, results of the different
approaches were combined by a consensus scoring scheme and the topꢀranked compounds
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would be selected for in vitro assays. The high degree of orthogonality between the two
different screening pathways was thought as a means to effectively enhance sampling
robustness and thus accuracy of the VS protocol, while their combination in a mixed serialꢀ
parallel manner was expected to optimize screening speed with the most timeꢀconsuming
structureꢀbased method being preceded by a ligandꢀbased, fast preꢀscreening of 2D similarity.
The NCI/DTP repository was independently prepared in terms of correct protonation state,
tautomerization and stereoisomerization for docking calculations and 3Dꢀsimilarity searches
using LigPrep (Schrödinger, Inc.) and Quacpac (OpenEye, Inc.), respectively. In the case of
3Dꢀsimilarity screening, Filter and Omega (OpenEye Inc.) were additionally utilized for
drugꢀlikeness filtering and conformer ensemble generation. No special preparation was
needed for 2Dꢀsimilarity searches. The query molecule used in both ligandꢀbased approaches
was 1, the only highꢀpotency BRD inhibitor known at the time of the study. The compound
does not show strong binding to any BET group BRD. However, on the basis of the highly
conserved fold and high homology between BRD4 and PB1 (sequence similarity 61.6%) as
well as in the absence of any other template, it was thought that the overall molecular
geometry of 1 would adequately outline the fundamental structural requisites for competing
Kac binding needed for hit recovery. It should also be noted that all NCI repository entries
containing the benzodiazepine (BZD) core were filtered out and excluded from the screening
study as a means to aid the discovery of novel scaffolds with BRD binding properties.
In the 2D screening approach, the structures of all NCI/DTP compounds were converted to
fingerprint (FP) strings by utilizing a variety of different algorithms for FP derivation. Then,
the structural similarity of each NCI/DTP compound with respect to the query molecule was
determined by comparing the Tanimoto distance of its FPs with the corresponding FPs
calculated for 1. Setup of the 2D screening calculations was not straightforward, as the
selection of the most appropriate combination among the wide array of FP derivation
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methods and similarity measuring metrics available in Canvas is not always trivial.51 For this
reason, a minimal training set was prepared with 20 fragments of known binding affinities for
BRD4, CREBBP and BAZ2B (SGC unpublished data) seeded into a set of 6,000 randomly
chosen entries from the NCI/DTP repository regarded as inactive decoys. Then, a total of 60
screens were performed using several recommended combinations of the Canvas 2Dꢀ
screening parameters and the efficiency of each screen to topꢀrank the 20 active fragments
among the 6,000 decoys was quantified.50 Of these 60 screens, 11 afforded high recovery
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rates of the known actives, with enrichment factor at 20% of screened library (EF20%
)
reaching 2.2ꢀ2.5 for the 11 selected FP combinations while it was 0ꢀ1.2 for the remaining
(EFmax20% is 5). These FP combinations were further selected for screening the global
NCI/DTP repository (concerning FP derivation, scaling and atomꢀtyping methods see Table
S1).
In the structureꢀbased approach, the 5,000 molecules of the enriched NCI/DTP subset
derived from 2Dꢀprescreening were sequentially docked to the BRD Kacꢀbinding pocket and
their theoretical affinity for the protein was evaluated using the GlideScore empirical scoring
function as implemented in Glide. Concerning the 3Dꢀsceening methodology, similarity
searches were performed for 200ꢀconformer ensembles of each NCI/DTP entry against 1 at
its bioactive conformation. The 3D similarity was determined in terms of overall molecular
shape overlap (shape similarity) as well as spatial similarity of pharmacophoric sites such as
positive and negative atoms, hydrogen bond donors and acceptors, hydrophobes and rings
(color similarity).
Each of the implemented screening approaches (2D similarityꢀassisted docking and scoring
screen, independent 2Dꢀ and 3Dꢀsimilarity screens) afforded a distinct list of compounds
ranked according to their potential to bind the BRD pocket and compete with acetylated
histone binding. The lists were combined by a simple consensus scoring approach accounting
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for the frequency of appearance of each compound within a given percentage threshold of the
top of the individual lists. By using this consensus scoring scheme, the 40 topꢀranked
compounds were selected for assessing their BRD binding properties in vitro using a
Differential Scanning Fluorimetry (DSF) assay.
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In Vitro Affinity Assessment of TopꢀRanked Compounds
The 40 compounds that scored higher in the consensus scheme were assayed in vitro using
the Thermal Shift DSF screening assay (Table S2). This method is based on measurement of
the thermal stabilization of a protein in the presence of a smallꢀmolecule binder, as
determined by the increase of the proteinꢀligand complex melting temperature (Tm) compared
to that of the apoꢀprotein.58 A screening panel of six BRDs was utilized for obtaining insight
into the specificity profile of the potential hits. The DSF assay was implemented with high
protein and ligand concentrations and resulted in the identification of seven compounds (3ꢀ9,
Figure 1B) with detectable temperature shifts (Table S3 for Tm curves and screening method
of selection). Out of those, five failed to bind PB1(5) but showed very weak binding towards
BRD4(2), with corresponding Tm values ranging between 0.8°C and 2.0°C. These results
were in accordance with the wellꢀestablished, favorable druggability of this BRD (and the
BET group in general).17 Only two compounds afforded a Tm value larger than 3°C.
Compound 8 (NSC76484)59 resulted in a 3.2°C stabilization towards BRD4(2). This
particular scaffold demonstrated a rather obvious structural similarity with known BRD4
inhibitors, as its tricyclic ring system contained a triazole moiety reminiscent of the
corresponding pharmacophore of 1. However, the most interesting finding was compound 9
(NSC356476)59 which showed a marked stabilization of 4.1°C towards PB1(5) and at the
same time only weak binding to BRD4(2) (Tm of 1.9°C) at 100 ꢀM compound concentration.
The results of the thermal shift assay were considered as a good indication that 9, a
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pyrazoloisocoumarin derivative of relatively low molecular weight (200Da), could constitute
a novel BRDꢀbinding scaffold and at the same time a promising hit with a highly preferable
specificity profile towards BRD related to the SWI/SNF chromatin remodeling complexes.
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Xꢀray Crystallography of LigandꢀBRD Complex
To better understand the interaction between 9 and PB1(5), the coꢀcrystal structure of the
proteinꢀligand complex was determined at a resolution of 2.02Å and the binding mode of 9
was elucidated (Table S4). The ligand bound with the benzopyrone system buried deeply in
the Kacꢀpocket, while the N3 of the pyrazole ring accepted a hydrogen bond from the amide
nitrogen of the conserved N739 (Figure 3BꢀD). With the exception of this hydrogen bond, the
ligand was stabilized mainly through hydrophobic interactions. The most important contacts
of 9 with the bromodomain are accommodated by L687 and L693 of the ZA loop, by I745 of
aCꢀhelix as well as by I683 and F684, two residues that constitute along with A682 the WPF
shelf of the Kac cavity in PB1(5). A weak edgeꢀtoꢀface πꢀπ stacking interaction could be
observed between the aromatic system of the ligand and Y738. Notably, the overall
conformation of the ZA loop in the complex of 7 and PB1(5) did not induce structural
changes when compared to the apoꢀprotein, suggesting that the unusual loop conformation
observed for PB1(5) in a previously released structure (PDB code: 3G0J) is not induced as a
result of ligand binding.
Interestingly, compound 9 displaced four of the five of the conserved buried water
molecules that are observed in the majority of the available BRDꢀligand complexes (Figure
3AꢀD). These waters form an extensive hydrogen bond network in the Kacꢀbinding cavity
which is usually retained in most of the released bromodomainꢀinhibitor coꢀcrystal structures
(Bꢀvalues of waters between 28 and 40, similar to main chain Calpha positions in the protein
backbone). In the case of 9 however, the only water molecule that was not displaced seems to
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be stably bound through a multidirectional system of four hydrogen bonds to the sidechain
hydroxyl of Y696, the sidechain amide of N734 and the backbone carbonyls of M704 and
M731 (Figure S1). This water was the most deeply buried of the aforementioned network of
conserved waters and it was located within hydrogenꢀbonding distance to both the exocyclic
carbonyl of 9 and the sidechain of the conserved Y696 of ZA loop. This water molecule is
highly conserved and forms a hydrogen bond with the hydroxyl group of Y696 in acetylꢀ
lysine containing peptides and acetylꢀlysine mimetic inhibitor complexes. An additional
water that participates in ligand binding was found in the first hydration shell on the solventꢀ
accessible side of the Kac cavity, forming a hydrogen bond bridge between N2 of 9 and the
sidechain carbonyl of N739. Finally, the good complementarity of the ligand to the acetylꢀ
lysine binding cavity was indicated by the solventꢀaccessible surface area that was buried
upon binding which was 172Å2 for the ligand (135Å2 apolar) and 188Å2 for the protein
(177Å2 apolar).
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A binding mode displacing four of the five structural water molecules similar to 9 has also
been described for 2 and salicylic acidꢀcontaining fragments.27 It was therefore explored
whether compounds containing phenolic moieties similar to 2 and salicylic acid or pyroneꢀ
related structures similar to the isocoumarin system of 9 could induce the same water
displacement motif with 9 and hence demonstrate binding affinity for PB1(5). Moreover, due
to increased stability of compounds bearing closed rings ꢀsuch as 9- against hydrolysis when
compared to 2, identifying new scaffolds interacting with SWI/SNF bromodomains using this
unique water displacing binding mode was of high interest to us. Screening of a small inꢀ
house library of natural products identified 5, 3’, 4’ꢀtrihydroxyflavone (14) and luteolin (15)
as binders of BRDs with strongest temperature shifts observed for PB1(5) (Figure 4AꢀB).
Subsequent coꢀcrystallization studies indeed confirmed the hypothesized water displacing
acetylꢀlysine binding modes for both flavones, maintaining only water W5 in the binding
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pocket (Figure 4CꢀF). The critical role this buried solvent molecule seems to have in complex
stabilization should certainly be considered when planning chemical modifications targeting
hit optimization. Yet, its net energetic contribution to binding should be carefully assessed
since there might be a considerable penalty component due to its restrained positioning. The
affinity of 15 was determined using isothermal titration calorimetry, resulting in a Kd of
12.9ꢀM.
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Isothermal Titration Calorimetry Analysis
To accurately determine the binding affinity of pyrazoloisocoumarin 9 for PB1(5), we
measured the dissociation constant (Kd) in solution using ITC. This analysis revealed a Kd
value of 11.5 ꢁM, single site binding stoichiometry and a favorable binding enthalpy change
(ꢂH of ꢀ5.6kcal/mol) were measured at 288K, affording a fairly high ligand efficiency (LE)
index of 0.46 for this hit (Table 1). The low micromolar affinity of 9 in combination with its
high LE showed that the compound could indeed be considered as a promising starting point
for rational development of analogues with improved binding affinity. The estimated
ꢂGbinding indicated that binding is dominated by enthalpic interactions, in accordance with the
structural analysis, showing the formation of polar interactions. As mentioned before, the
flavonoid hit 15 had a similar affinity (Kd of 12.9 ꢁM) (Figure 4B). However, by considering
the less pronounced originality and chemical tractability of the flavone system and, most
importantly, the poor selectivity of 15 (Figure 4A) over other BRDs and other target families
(e.g. kinases), the aforementioned scaffold was not prioritized at that stage and 9 was selected
for further activity optimization.
In an effort to devise a rational optimization strategy for the newly characterized
pyrazoloisocoumarin hit to a possible lead compound, structural and thermodynamic data
were combined and considered collectively. Inspection of the binding mode of 9 indicated
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three major extension vectors that could be utilized for hit optimization (shown in Figure 5Aꢀ
B). Nevertheless, no obvious hydrogen bonding or electrostatic interaction partner was
located within a reasonable radius around the bound ligand. Moreover, the apparent optimal
fit of 9 and PB1(5) in combination with the known overall relative rigidity of the BRD fold
set skepticism with respect to its capacity to explore more extensive models for hit expansion.
It was hypothesized that affinity of 9 could be potentially enhanced through manipulation and
fineꢀtuning of solvent effects that may affect the binding interaction. To explore this, the
computational analysis of the protein hydration pattern was undertaken using the two
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currently available algorithms, SZmap (OpenEye Inc.) and WaterMap (Schrödinger, Inc.).60ꢀ
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Computational Hydration Analysis
Computational solvation mapping of a binding site (e.g., as performed by SZmap or
WaterMap) can identify hydration sites that may confer ligand potency should that ligand for
example efficiently displace or replace water molecules of appropriate energetics. As such,
solvation mapping can suggest possible lead optimization modifications to optimize enthalpic
proteinꢀligand interactions while simultaneously decreasing unfavorable entropy that results
from localization of bulk water. The aforementioned approach can be of particular
importance in the case of BRDs. Indeed, it has been shown that BRDs accommodate solventꢀ
mediated interactions with their ligands that involve buried, conserved water molecules and
are regarded as critical for binding. This highlights the considerable impact that the
equilibrium between solvent dynamics and ligand binding can have to the druggability of
each BRD.17
The hydration analysis performed for PB1(5) was focused to three separate regions of
interest corresponding to the most promising extension vectors of the pyrazoloisocoumarin
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scaffold (Figure 5AꢀB). Those three regions were i) the inner side of the Kac cavity, ii) the
entrance of the binding cavity and iii) the periphery of the ZA channel, a narrow saddleꢀ
shaped surface created by the ZA loop in the boundary of the BRD cavity in direct contact to
the bound ligand. Those regions were extensively mapped utilizing the two abovementioned
computational approaches and inspected visually. Concerning the first region, SZmap
predicted a hydration site partially overlapping with the buried water of the Kac cavity, a site
thus needed to be efficiently replaced by a ligand polar group capable of forming equally
extensive stabilizing interactions with the protein. For the second region, SZmap predicted a
hydration site also corresponding to a highly stable water molecule that would consequently
favor binding if merely being replaced by a polar group of the ligand. However, concerning
the third region SZmap predicted a cluster of unstable waters residing at the outer boundary
of this hydration site. Those waters were seemingly trapped in a relatively hydrophobic
environment, yet in close proximity to the bulk solvent, thus they could be possibly targeted
by nonꢀpolar ligand groups (Figure 5A).
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The utilization of WaterMap was also successful in identifying the aforementioned regions
of interest concerning the protein hydration landscape (Figure 5B). Results obtained using
WaterMap showed high convergence with the corresponding SZmap data with respect to the
role of the buried water molecule (Figure S1). Both approaches predicted that this was a
highly stable water that would be rather difficult to increase binding affinity by replacing it
by the ligand. However, WaterMap predicted that the hydration site located at the entrance of
the binding site harbors a relatively unstable water, but these results were not confirmed by
SZmap. This slight discrepancy was likely due to the actual position of these waters close to
the first hydration shell of the protein and also to algorithmic differences implemented by
each methodology. However, the prediction concerning the third region of interest was in
good agreement between both methods. More specifically, at least two sites of unstable
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waters were predicted in the periphery of the ZA channel, in good overlap with the cluster of
unstable waters predicted by SZmap. Moreover, another conserved water site was identified
in fair proximity at the inner side of ZA channel. Interestingly, prediction of the latter as well
as one of the former sites was well supported by the actual presence of crystallographic water
molecules in the proteinꢀligand complex, as the unstable water was visible only in one BRD
molecule present in the asymmetric unit, while a water molecules predicted to be stable was
present in both binding sites and interacted with three hydrogen bonds to the backbone
carbonyls or amide nitrogens of residues A682, F684, L685 and R686.
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In a consensus point of view, this third site at the periphery of ZA channel was the most
promising region in terms of structural interventions targeting affinity optimization through
manipulation of the solvent component. Both approaches used for hydration analysis
suggested that this site, as occupied by unstable waters, it can be regarded as moderately
exposed to the solvent environment and, therefore, it is possibly suitable to be targeted by
nonꢀpolar ligand groups in a manner that could favor affinity by replacing those waters.
Design of Analogues and FEPꢀREST Calculations
In light of the structural, thermodynamic and hydration analysis performed so far,
inspection of the available extension vectors of 9 led to the selection of the 1ꢀmethyl group of
the pyrazole ring as the most suitable for chemical elaborations. Most substitutions on the
phenyl ring of 9 would possibly lead to steric clashes with the protein as was suggested by
the optimal fit observed for 9 in the Xꢀray structure of the complex. Efficacious replacement
of the buried structural water was deemed highly challenging given the enthalpic penalty
predicted for the elimination of this water, a notion in agreement with several lead
optimization studies available for BRDs (Figures 5 and S1). Likewise, efficient replacement
of the stable water at the inner side of the ZA channel seemed unlike due to the pronouncedly
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high free energy penalty estimated by SZmap (ꢀ12.55 kcal/mol) and WaterMap (1.97
kcal/mol) for this particular water. Moreover, any possible modifications targeting the
nitrogens of the pyrazole ring were avoided, as this part of the scaffold constitutes an
essential element of the pharmacophore that anchors 9 to the protein, as shown in Figures 3Bꢀ
D. Combined with the insufficient convergence between the two hydration mapping
methodologies concerning the region corresponding to the binding site entrance, the unstable
waters of the ZA channel were finally selected to be targeted. A conservative, proofꢀofꢀ
concept strategy was devised wherein the 1ꢀmethyl would be gradually extended to an alkyl
chain of up to four carbon atoms (i.e. from 1ꢀmethyl to 1ꢀnꢀbutyl).
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Indeed, in accordance with the predictions, the microenvironment at the outer boundary of
the ZA channel towards where the introduced alkyl chain was anticipated to be directed and
reside, is highly hydrophobic and consists of residues I683, F684, L685 and P688. Moreover,
the limited concavity and the overall shape of the region suggested that the unavoidable
conformational entropy penalty expected by restriction of the alkyl chain upon binding could
be kept minimal, thus outbalancing any unfavorable impact on ligand affinity. Yet, to check
the appropriateness of the designed modifications, Free Energy PerturbationꢀReplica
Exchange Solute Tempering (FEPꢀREST) calculations were undertaken and the predicted
changes in free energy of binding were determined. The ligand mutation module as
implemented in program Desmond (D.E. Shaw Research) was used and 5ns simulations were
performed for each transformation of the ligand either inside the protein environment or in
pure water using an explicit solvent representation. The simulation results showed that the
designed analogues demonstrated moderately higher predicted affinities compared to the
original methyl analogue. Interestingly, the most promising modification was shown to be the
introduction of the long butyl chain. While there is no accurate methodology based on FEP
calculations for distinguishing between the enthalpic and entropic components of the
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predicted free energy release, visual inspection of the structures indicated that the
displacement of the unstable waters by the ZA channel periphery by the extruding alkyl chain
was realistic. Moreover, due to the limited nature of the designed structural modifications,
FEP simulations were considered as reliable. Thus, the encouraging predictions of FEP
prompted us to proceed with synthesis of the novel analogues.
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Chemistry
Compound 9 was synthesized along with a series of new analogs as depicted in Scheme 1.
Yoon et al synthesized 4ꢀacetylꢀ1,3ꢀisochromandione from homophthalic acid and acetic
anhydride.64 Ozcan et al later achieved the synthesis of the norꢀpyrazoloisocoumarin 13
(Scheme 1) from homophthalic anhydride using a oneꢀpot reaction consisting of
Vilsmeier/1,3ꢀcyclisation.65 Taking advantage of the reactive methylene group in benzylic
position (C4) of homophthalic anhydride, a combined strategy of the two aboveꢀmentioned
literature data was developed for the generation of a first set of analogs. Introduction of
carbonyl moieties with different carbon chains lengths in C4ꢀposition of homopthalic acid is
rendered possible by nucleophilic substitution in alkaline media. The reaction of
homophthalic acid with thionyl chloride afforded homophthalic anhydride in excellent yields,
in accordance with literature data. C4ꢀdeprotonation of homophthalic anhydride in mild
conditions using anhydrous pyridine and followed by nucleophilic substitution of acetic,
propionic, butyric and valeric anhydride afforded the corresponding intermediates 9aꢀ12a in
low to moderate yields, despite numbers of efforts to optimize the conditions (time, base,
temperature), slightly improved only by the addition of anhydrous THF as solvent in the
reaction media. The 4ꢀacylꢀisochromanꢀ1,3ꢀdiones 9aꢀ12a were subjected to pyrazole
formation in presence of hydrazine monohydrate in refluxing DMF. The corresponding
isochromeno[3,4ꢀc]pyrazolꢀ5(2H)ꢀones 9ꢀ12 were obtained in low to moderate yields.
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Structural Basis of Affinity Improvement and Selectivity Assessment of New Analogues
The affinity of the novel analogues for PB1(5) was determined by ITC which demonstrated
that the synthesized derivatives were indeed stronger binders than the original methyl hit
(Figure 6B). The Kd values of 10 (ethyl), 11 (propyl) and 12 (butyl) derivatives were 3.4 ꢁM,
3.3 ꢁM and 5.1 ꢁM, respectively thus affording a 3.5 fold affinity improvement (Table 1).
Moreover, the conservative structural modifications had a minor effect on LE, as the
corresponding index was 0.48 for 10 and 0.45 for 11, while 12 afforded a slightly lower LE
of 0.41. Notably, good agreement was obtained between ꢂGbinding determined by ITC and the
corresponding values predicted by FEPꢀREST calculations for the three novel analogues with
errors within 0.6kcal/mol (Table 1). To address the structural basis of the improved affinity,
the crystallographic determination of the complexes of 10, 11 and 12 with PB1(5) was
undertaken (Table S4). As expected, the overall binding mode of the analogues carrying the
extended alkyl chain on position 1 was highly similar to the corresponding mode of the
original methyl hit, thus sustaining the basic proteinꢀligand interactions such as the hydrogen
bond of the pyrazole to N739 and the overall hydrophobic packing of the ligand aromatic
system inside the BRD cavity (Figure 7AꢀB). The alkyl chain of the novel analogues was
positioned towards the periphery of the ZA channel where it resided, most notably the butyl
moiety, in fully extended conformation stabilized by extensive contacts with P688 and the
sidechains of I683, L693 and I745. These crystallographic data were in good agreement with
the computational hydration analysis, in terms of the anticipated effect on binding affinity of
solvent reorganization and increase of buried nonꢀpolar surface area of the ligand. Indeed,
superposition of the alkyl chain binding geometries with the hydration sites predicted by
WaterMap and SZmap showed that the alkyl groups overlapped with water molecules
characterized by high free energy and thus predicted as unstable (Figure 5). Due to the lack of
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specific stabilizing interactions between those molecules and the BRD, their anticipated
release to bulk solvent would offer a fair gain in binding affinity without serious
perturbations on the overall enthalpy/entropy compensation landscape, as the ligand groups
would substitute the solvent by forming comparable contacts with the protein. Furthermore, a
binding geometry comparison between 12, 15 and 2 additionally showed that the butyl chain
of 12 matches the interactions of the aromatic decorations of both 2 and the flavonoid 15
(Figure 7CꢀD). It is thus possible that ring C of flavonoids contributes to binding affinity by
displacing unstable waters in a similar manner to that of the butyl chain of 12. In addition,
another common SAR element between the new analogues and 15 was the presence of a pair
of Hꢀbond acceptors anchoring each ligand to N739 and water W5, respectively (Figure 7Cꢀ
D).
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Notably, systematic thermodynamic and structural studies of similar systems have shown
that in many cases, increasing the length of an alkyl chain leads to gain in enthalpy that is,
hence, fully offset by an unfavorable entropy change thus affording a negative net effect on
binding affinity.66 In this study, we demonstrated the use of designing constructive
interactions with the water environment of a protein/ligand system on the basis of utilizing
available solvation mapping algorithms. In a theoretical druggability assessment of BRDs
based on the available Xꢀray crystallographic data, the degree by which different members of
the family constitute tractable drug targets was determined. In this partition, BRDs of PB1
were clustered along with 57% of total studied BRDs that were classified as of either
intermediate (PB1 BD2 and 5) or difficult (PB1 BD1, 3, 4 and 6) druggability. However, in
that study, structural water molecules of the included BRDs were left intact and thus
druggability determination was based on the assumption that those waters are not
displaceable. The results presented herein show that such an approach could be challenged by
the notion that the net contribution of solvent thermodynamics in binding cannot always be
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straightforwardly generalized across a whole family of proteins solely on the basis of
structural similarity of related proteins.
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To address the issue of selectivity and check whether the applied structural modifications
and the observed affinity improvement had any unfavorable impact to the specificity profile
of the scaffold, the affinity of 12 was determined towards a panel of 15 BRDs (Figure 6A and
Table S5) extended with a representative group of subfamilyꢀVIII members. Compound 12
showed selectivity towards PB1(5) over the closely related BRDs, with low binding (Tm not
exceeding 2°C) observed only towards PB1(3) (Table S5). Notably, no binding was observed
towards either BRD4 or, most importantly, the closely related SMARCA2, thus highlighting
the possibility of designing a high affinity chemical probe targeting exclusively PB1(5). To
further confirm these data, ITC was used confirming that 12 did not interact with SMARCA
BRDs (Table S6).
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Cellꢀbased Assays
To test the ability of 12 to bind to the bromodomain of PB1 and displace binding of PB1
from chromatin, a fluorescence recovery after photobleaching (FRAP) assay was performed.
Chromatin was hyperacetylated using the histone deacetylase inhibitor suberoylanilide
hydroxamic acid (SAHA) to increase the assay window.67 Cells were transfected with a
plasmid encoding GFP fused to either full length PB1 or to mutant PB1(2) and PB1(5)
showing reduced ability to bind to chromatin. Cells were bleached with a laser and recovery
time measured with or without compound treatment. Despite the fairly low potency in vitro
12 was able to displace PB1 from chromatin to levels of the control 2 and the mutant protein
and showed similar half recovery times of ~2s (Figure 8).28 The Kd of 2 is 48 nM for PB1(5)
measured by ITC as compared to 5.1 ꢁM for 12. This is therefore in line with the effects
observed in FRAP, where an effect on PB1(5) is seen for 2 at 1 ꢁM and for 12 at 20 ꢁM.
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FRAP experiments suggest that, at least in part, the complex can be displaced. Yet, as this
experiment has been performed using overexpressed proteins it cannot be excluded that the
endogenous SWI/SNF complex remains bound to chromatin in the presence of PB1(5)
inhibitors.
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Analogue 12 was additionally subjected to a viability assay in 1BRꢀhTERT human
fibroblast cells. The drug very slightly reduced viability of the cells above 5µM (Figure S2A)
while 2 also reduced viability to a similar level (Figure S2B). It was next determined whether
12 reduces the ability of BAF180 to bind to chromatin using chromatin fractionation and
Western blotting. Consistent with the FRAP results, treatment of 1BRꢀhTERT cells with 12
slightly reduced the levels of chromatinꢀbound PB1 (Figure S2C). Loss of PB1 sensitizes
cells to DNA damaging agents including mitomycin C (MMC).45 Whilst U2OS PB1 knockꢀ
out cells were more sensitive to MMC than control cells, 12 did not result in any significant
sensitization to this drug and this was true for experiments using 2 as well (Figure S2D). This
suggests that the remaining level of chromatinꢀbound PB1 is sufficient to perform the
function of PB1 in MMCꢀinduced DNA repair. Finally, the effect of 12 on cell proliferation
was studied using WSTꢀ1 and the multiple myeloma (MM) lines L363, H929 and JJN3. In
agreement with earlier reports, exposure of cells to 12 did not lead to significant inhibition of
proliferation on JJN3 cells.
CONCLUSION
Precise estimation of the energetics accompanying solvent dynamics and reorganization
upon ligand binding is an aspect of structureꢀbased drug design that demands attention, as it
concerns one of the less comprehensible events addressed by currently available theoretical
approaches in response to the increasing demand for highꢀquality free energy predictions.
Especially with BRDs, the available structural data seem to converge towards identifying an
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ensemble of four to five buried water molecules that are considered as an almost integral part
of the Kac binding cavity. However, whether those waters should (or could) be effectively
displaced upon compound binding, aiming at an affinity gain in a selective fashion over the
various BRD members is still debatable. Discovery of the low micromolar
pyrazoloisocoumarin ligand of PB1(5) through a consensus virtual screening approach
provided a valuable starting point for probing the importance of solvent effects in BRD hit
optimization. Although relatively low (1 molecule active towards PB1(5) out of 40 topꢀ
ranked compounds assayed in vitro), the hit rate achieved in this virtual screen was
considered satisfactory, given the serendipitous fact that the discovered hit demonstrated a
highly original binding mode involving displacement of waters in a manner not seen in most
BRDꢀligand binding events including the interaction of the template JQ1 with its target
BRD4. Rational elaboration of the scaffold was pursued by combining experimental results
obtained by ITC and Xꢀray crystallography with theoretical data derived by predictive
algorithms modeling hydration of the target protein. The effort was not focused on structural
waters occupying the BRD Kac cavity but rather on solvent trapped on the macromolecule
surface. By doing so, it successfully indicated a means to affinity gain by chemical
modifications targeting secondary contributors as an indirect and alternative route to binding
affinity optimization.
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Naïvely, it would seem that growing an alkyl chain into solvent where it cannot sustain
considerable hydrophobic interactions with the protein, would not contribute favorably to
ligand binding potency in any system.66 However, in this study it was shown that by
considering the impact of solvent dynamics in binding, one can achieve optimization of a hit
through alternative and possibly more efficient routes than those solely based on inspection
of proteinꢀligand interactions. To facilitate this, one should go beyond utilization of structureꢀ
based techniques like protein Xꢀray crystallography or docking and also look to solvent
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mapping or other methods which take into consideration solvent dynamics. The combination
of different approaches demonstrating considerable orthogonality with each other, but with
the potential to be reasonably integrated, affords a highly reliable and constructive
representation of complex systems such as those of protein/ligand/solvent regularly studied in
drug discovery. More specifically, by using innovative computational tools for solvation
mapping such as SZmap and Watermap –independently and in a consensus fashionꢀ one can
distinguish between stable (structural) and unstable (trapped) waters and thus devise
modifications targeting each category appropriately. This work shows that by releasing
unstable waters into bulk solvent, one may achieve a favorable, although small, improvement
in Kd, in contrast to the expected view that growing an alkyl chain towards water would result
to derivatives of lower affinity.
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The advances in the field of epigenetics and more specifically the discovery of compounds
interfering with BRDs in a medically relevant manner, underline the necessity for focused
screening efforts targeting proteins of the underexplored BRD subfamilies and thus enabling
elucidation of their functionalities and involvement in different pathological states. On the
other hand, rational approaches for hit identification or lead optimization can substantially
facilitate the discovery process. The integral approach presented in this study can enable the
most efficient exploitation of available structural, biophysical and biochemical data towards
rational development of hit compounds into promising leads for probe or drug discovery. The
characterization of cellꢀpermeable pyrazoloisocoumarin derivatives showing singleꢀdigit
micromolar affinity towards the fifth BRD and high selectivity over other subfamily members
such as PB1(3) and SMARCA, is anticipated to facilitate development of a chemical probe
for exploring the biological role of this protein interaction module in the epigenetic
mechanisms mediated by SWI/SNF complexes.
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Experimental Section
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Library Preparation and Virtual Screening
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Prior to calculations, the NCI/DTP Repository compounds were enumerated and prepared in
terms of correct protonation states, tautomerism and stereoisomerism using the LigPrep
routine (Schrödinger Inc.). The NCI/DTP collection was prepared independently for ROCS
screening using the enumeration and conformer generation tools provided by OpenEye Inc.
(Filter, Quacpac, Omega) with default settings. The ligandꢀbased approach was based on the
ROCS algorithm (OpenEye Inc.) with the Tanimotoꢀcombo scoring scheme and the implicit
MillsꢀDean forcefield, while 1 was utilized as the query molecule. With respect to the
structureꢀbased approach, rigid docking was performed using the Glide v. 5.7 SP sampling
algorithm and the corresponding GScore SP5 scoring scheme (Schrödinger Inc.) with the van
der Waals atom radii scaling set to 0.9 for the protein grid and to 0.8 for the screened ligands.
Protein preparation was performed by the corresponding routine as implemented in
Schrödinger 2014 suite. The three screening methodologies were combined by a simple
consensus statistical scheme on the basis of frequencies of appearance for each compound at
the top of a given percentage threshold in each of the computationally ranked lists using
Microsoft Excel 2010. For FEPꢀREST calculations, the ligand mutation module as
implemented in Desmond software (D.E. Shaw Research) was used with default settings and
5ns simulations were performed for each transformation of the ligand either inside the protein
environment or in pure water using an explicit solvent representation.
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Synthesis and Characterization of the compounds
All chemicals were purchased from Aldrich Chemical Co. Microwaveꢀassisted reactions were
performed in a single mode CEM apparatus. NMR spectra were recorded on Bruker DRX
400 and Bruker Avance 600 spectrometers (1H 400 and 600 MHz); chemical shifts are
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expressed in ppm downfield from TMS. HRMS Spectra were determined on a MSQ Orbitrap
Thermofinnigan spectrometer. Columns chromatography were conducted using flash silica
gel 60 (40ꢀ63 ꢁm) from Merck. Purity of the compounds has been determined by HPLC and
was above 95%.
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HPLC Conditions
HPLC chromatograms have been recorded on a Thermo Finnigan chain (pump P4000,
Controller SN4000, AutoSampler AS3000, UV detector UV6000LP) equipped with a
Supelco Discovery® HS C18 5ꢁm column (25cm x 4,6mm). All the solvents were degassed
in an ultrasound bath for 15 min and then filtered (nylon membrane, 0.45ꢁm) prior to use.
Samples have been diluted in HPLC grade methanol (1mg/mL) and filtered (nylon
membrane, 0.45ꢁm). Retention times (tR) are given in minutes. Two methods have been used:
Method A: H2O+0.1% CH3COOH/MeOH: 15/85 for 45min.
Method B: H2O/CH3CN: 10/90 for 30min.
Synthesis of homopthalic anhydride
Homopthalic acid (3g, 0.01665 mol) was dissolved in anhydrous CH2Cl2. Thionyl chloride
was then added (4.83mL, 0.067mol) dropwise. The mixture was then warmed at reflux for
12h. After completion of the reaction, solvent was evaporated and the crude residue dried
under high vacuum to remove remaining traces of thionyl chloride. Homopthalic was
obtained with 98% yield and used without purification.
1Η NMR (400MHz, CDCl3, δ in ppm, J in Hz): 8.25 (d, J = 7.56, 1H, Hꢀ1); 7.37 (d, J = 7.6,
1H, Hꢀ4); 7.54 (t, J = 7.56, 1H, Hꢀ2); 7.72 (t, J = 7.56, 1H, Hꢀ3); 4.17 (s, 2H, Hꢀ5).
General procedure for the synthesis of 4ꢀacylꢀ1,3ꢀisochromandione 9aꢀ12a
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Homopthalic anhydride (1eq) was dissolved in anhydrous pyridine (2eq). After 15min, the
corresponding anhydride (4eq) was added dropwise. The mixture was then stirred for 5h at
room temperature. Et2O was then added, the precipitate filtered and washed with extra
amount of Et2O to afford the desired 4ꢀacylꢀ1,3ꢀisochromandione as a white solid. The
product was then used for the next step without further purification.
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General procedure for the synthesis of isochromeno[3,4ꢀc]pyrazolꢀ5(2H)ꢀone 9ꢀ12
The desired 4ꢀacylꢀ1,3ꢀisochromandione 9a-12a (1eq) was dissolved in anhydrous DMF and
stirred for 15min. Hydrazine monohydrate was then added dropwise (4.2eq) and the mixture
warmed to reflux for 12h. After completion of the reaction, the mixture is cooled and water is
added. The precipitate is then filtered, washed with water and dried affording the
corresponding isochromeno[3,4ꢀc]pyrazolꢀ5(2H)ꢀone 9-12.
Data for 1ꢀmethylisochromeno[3,4ꢀc]pyrazolꢀ5(2H)ꢀone 9
Yield: 40%. 1Η NMR (600MHz, DMSO, δ in ppm, J in Hz): 12.39 (s, 1H, Hꢀ2); 8.20 (d, J =
8.00, 1H, Hꢀ6); 7.85 (m, 2H, Hꢀ8 and Hꢀ9); 7.49 (t, J = 8.00, 1H, Hꢀ7); 2.63 (s, 3H, 1’ꢀCH3).
13C NMR (150MHz, DMSO, δ in ppm): 161.39 (Cꢀ5); 156.97 (Cꢀ3a); 135.78 (Cꢀ5a), 135.47
(Cꢀ8), 133.58 (Cꢀ9a), 130.94 (Cꢀ6), 126.12 (Cꢀ7), 122.01 (Cꢀ9), 118.07 (Cꢀ1), 96.55 (Cꢀ9b),
11.68 (1’ꢀCH3). HRMS (ESI+) m/z = 201.0659 ([M+H]+) (Calculated Mass: 200.19). HPLC:
Method A tR =3.17min; Method B tR=2.83min.
Data for 1ꢀethylisochromeno[3,4ꢀc]pyrazolꢀ5(2H)ꢀone 10
Yield: 30%. 1Η NMR (600MHz, DMSO, δ in ppm, J in Hz): 12.95 (s, 1H, Hꢀ2); 8.21 (d, J =
7.9, 1H, Hꢀ6); 7.87 (t, J = 7.5, 1H, Hꢀ8); 7.82 (d, J = 7.5, 1H, Hꢀ9); 7.49 (t, J = 7.90, 1H, Hꢀ
7); 3.04 (q, J = 7.55, 2H, Hꢀ2’); 1.32 (t, J = 7.55, 3H, 1’ꢀCH3). 13C NMR (150 MHz, DMSO,
δ in ppm) 161.35 (Cꢀ5), 156.94 (Cꢀ3a), 141.43 (Cꢀ5a), 135.59 (Cꢀ8), 133.42 (Cꢀ9a), 131.02
(Cꢀ6), 126.13 (Cꢀ7), 122.10 (Cꢀ9), 118.16 (Cꢀ1), 95.71 (Cꢀ9b), 19.24 (Cꢀ1’), 12.61 (Cꢀ2’).
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HRMS (ESIꢀ) m/z = 213.0672 ([MꢀH]+) (Calculated Mass: 214.22). HPLC: Method A tR
=3.40min; Method B tR=2.91min.
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Data for 1ꢀpropylisochromeno[3,4ꢀc]pyrazolꢀ5(2H)ꢀone 11
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1Η NMR (600MHz, DMSO, δ in ppm, J in Hz): 12.93 (s, 1H, Hꢀ2); 8.17 (d, J = 7.91 Hz, 1H,
Hꢀ6); 7.81 (m, 2H, Hꢀ8 and Hꢀ9); 7.45 (t, J = 7.91 Hz, 1H, Hꢀ7); 2.96 (t, J = 7.41 Hz, 2H, Hꢀ
1’); 1.7 (h, J = 7.31 Hz, 2H, Hꢀ2’); 0.95 (t, J = 7.31 Hz, 3H, Hꢀ3’). 13C NMR (150 MHz,
DMSO, δ in ppm) 161.34 (Cꢀ5), 156.89 (Cꢀ3a), 140.04 (Cꢀ5a), 135.59 (Cꢀ8), 133.44 (Cꢀ9a),
131.03 (Cꢀ6), 126.15 (Cꢀ7), 122.09 (Cꢀ9), 118.20 (Cꢀ1), 96.01 (Cꢀ9b), 27.47 (Cꢀ1’), 21.30
(Cꢀ2’), 13.43 (Cꢀ3’). HRMS (ESIꢀ) m/z = 227.1287 [MꢀH]+ (Calculated Mass: 228.25).
HPLC: Method A tR=3.72min; Method B tR=3.01min.
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Data for 1ꢀbutylisochromeno[3,4ꢀc]pyrazolꢀ5(2H)ꢀone 12
1Η NMR (600 ΜΗz DMSO, δ in ppm, J in Hz) 12.95 (s, 1H, Hꢀ2), 8.21 (d, J = 7.99 Hz, 1H,
Hꢀ6), 7.87 (t, J = 7.5 Hz, 1H, Hꢀ8), 7.81 (d, J = 7.5 Hz, 1H, Hꢀ9), 7.48 (t, J = 7.99 Hz, 1H, Hꢀ
7), 3.01 (t, J = 7.50 Hz, 2H, Hꢀ1’), 1.69 (quintuplet, J = 7.52 Hz, 2H, Hꢀ2’) 1.40 (h, J = 7.52
Hz, 2H, Hꢀ3’), 0.94 (t, J = 7.39 Hz, 3H, Hꢀ4’). 13C NMR (150 MHz, DMSO, δ in ppm)
161.33 (Cꢀ5), 156.90 (Cꢀ3a), 140.21 (Cꢀ5a), 135.59 (Cꢀ8), 133.44 (Cꢀ9a), 131.04 (Cꢀ6),
126.13 (Cꢀ7), 122.05 (Cꢀ9), 118.20 (Cꢀ1), 95.94 (Cꢀ9b), 30.01 (Cꢀ1’), 25.35 (Cꢀ2’), 21.65 (Cꢀ
3’), 13.59 (Cꢀ4’). HRMS (ESIꢀ) m/z = 241.0976 (MꢀH). HPLC: tR = [MꢀH]+ (Calculated
Mass: 242.28). HPLC: Method A tR=4.29min; Method B tR=3.21min.
The activity of the compounds was validated by Xꢀray crystallography of all proteinꢀ
compound complexes and by various different binding affinity measuring methodologies
such as ITC and DSF as well as cellꢀbased assays. All active compounds are therefore
considered as not PAINS.
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Protein Expression and Purification
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cDNA encoding human the fifth BRD of PB1 (NCBI Gene ID: 55193) was obtained from the
SGC clone collection and was used as template to amplify the kinase domain region of the
protein, using the polymerase chain reaction (PCR) in the presence of Platinum® Pfx DNA
polymerase (Invitrogen™, UK). PCR products were purified (QIAquick PCR Purification
Kit, Qiagen Ltd. UK) and further subꢀcloned into a pMCSG7 derived expression vector
(pNIC28ꢀBsa4), using ligation independent cloning. This vector includes sites for ligationꢀ
independent cloning and a Tobacco Etch Virus (TEV)ꢀcleavable Nꢀterminal His6ꢀtag
(extension MHHHHHHSSGVDLGTENLYFQ*SMꢀ). After digestion with TEV protease, the
protein retains an additional serine and methionine on the Nꢀterminus. The constructs were
transformed into competent Mach1™ cells (Invitrogen™, UK) to yield the final plasmid
DNA.
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Colonies from freshly transformed plasmid DNA in competent E. coli BL21(DE3)ꢀR3ꢀ
pRARE2 cells (phageꢀresistant derivative of BL21(DE3) cell (Invitrogen™), with a pRARE
plasmid encoding rare codon tRNAs) were grown overnight at 37ºC in 30 ml of 2x Luriaꢀ
Bertani medium (LBꢀbroth, Amresco) with 50µg/ml kanamycin and 34µg/ml
chloramphenicol (startup culture). The startup culture was diluted 1:100 in terrific broth (TB,
Merck) and cell growth was allowed at 37ºC to an optical density of about 1.5 (OD600) before
the temperature was decreased to 18ºC. When the system equilibrated at 18ºC the optical
density was about 3.0 (OD600) and protein expression was induced over night at 18ºC with
0.1mM isopropylꢀβꢀDꢀthiogalactopyranoside (IPTG). The bacterial cells were harvested by
centrifugation (8,700 x g for 15min at 4ºC, FIBERLITE F9ꢀ6x1000 LEX rotor, on a Thermo
Scientific SORVALL LYNX 6000 centrifuge) and were frozen at ꢀ20ºC as pellets for storage.
Cells expressing His6 tagged protein were reꢀsuspended in lysis buffer (50mM HEPES, pH
7.5 at 25 ºC, 500mM NaCl, 20mM Imidazole, 5% glycerol, 1mM tris(2ꢀ
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