Discovery of a potent class of PI3Kα inhibitors with unique binding mode via encoded library technology (ELT4)

Article abstract of DOI:10.1021/acsmedchemlett.5b00025

In the search of PI3K p110α wild type and H1047R mutant selective small molecule leads, an encoded library technology (ELT) campaign against the desired target proteins was performed which led to the discovery of a selective chemotype for PI3K isoforms from a three-cycle DNA encoded library. An X-ray crystal structure of a representative inhibitor from this chemotype demonstrated a unique binding mode in the p110α protein.

Full text of DOI:10.1021/acsmedchemlett.5b00025

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Letter
Discovery of a potent class of PI3K# inhibitors with unique
binding mode via Encoded Library Technology (ELT)
Hongfang Yang, Patricia F Medeiros, Kaushik Raha, Patricia Elkins, Kenneth E Lind, Ruth
Lehr, Nicholas D. Adams, Joelle L Burgess, Stanley J Schmidt, Steven D Knight, Kurt R Auger,
Michael D Schaber, George Joseph Franklin, Yun Ding, Jennifer L DeLorey, Paolo A Centrella,
Sibongile Mataruse, Steven R Skinner, Matthew A Clark, John W Cuozzo, and Ghotas Evindar
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00025 • Publication Date (Web): 20 Mar 2015
Downloaded from http://pubs.acs.org on March 23, 2015
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Discovery of a potent class of PI3Kα inhibitors with unique binding mode via Encoded Library
Technology (ELT)
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Hongfang Yang,a Patricia F. Medeiros,a Kaushik Raha,b Patricia Elkins,b Kenneth E. Lind,a Ruth Lehr,c
Nicholas D. Adams,d Joelle L. Burgess,d Stanley J. Schmidt,d Steven D. Knight,d Kurt R. Auger,d
Michael D. Schaber;e G. Joseph Franklin,a Yun Ding,a Jennifer L. DeLorey,a Paolo A. Centrella,a
Sibongile Mataruse,a Steven R. Skinner,a Matthew A. Clark,a John W. Cuozzo,a Ghotas Evindar a,*
a) MDR (Molecular Discovery Research) Boston, Platform Technology and Science,
GlaxoSmithKline, 830 Winter Street, Waltham, MA 02451; b) Computational and Structural
Chemistry, Platform Technology and Science, GlaxoSmithKline, 1250 South Collegeville Road,
Collegeville, Pennsylvania 19426; c) Biological Reagents and Assay Development, Platform
Technology and Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville,
Pennsylvania 19426; d) Oncology Research, GlaxoSmithKline, 1250 South Collegeville Road,
Collegeville, Pennsylvania 19426; e) Screening and Compound Profiling, Platform Technology
and Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania
19426
Email: ghotas.x.evindar@gsk.com
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ABSTRACT
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In search of PI3K p110α wildtype and H1047R mutant selective small molecule leads, an Encoded
Library Technology (ELT) campaign against the desired target proteins was performed which led to the
discovery of a selective chemotype for PI3K isoforms from a 3 cycle DNA encoded library. An Xꢀray
crystal structure of a representative inhibitor from this chemotype demonstrated a unique binding mode
in the p110α protein.
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KEYWORDS: Encoded Library Technology, ELT, PI3Kα, p110α, PI3K p110α (H1047R).
Despite advances in research and development, discovery of novel, safe and efficacious smallꢀ
molecule chemical entities as therapeutic agents remains a major challenge in the pharmaceutical
industry. It was speculated that this might have led to a decrease in the number of small molecule drugs
launched over past decade.1,2 One major contributor to low output in the drug discovery process is
limitation of suitable chemotypes or scaffolds for medicinal chemistry program initiation.3 DNAꢀ
encoded chemical libraries as a new hit identification platform have been explored for over a decade
now.4,5 Our group has recently reported on the application of Encoded Library Technology (ELT) as a
novel hit and lead discovery platform complementary to existing methods.6,7,8,9,10,11,12,13 In pursuit of an
isoform and/or mutant selective class of phosphoinositide 3ꢀkinaseꢀalpha (PI3Kα) inhibitors, ELT was
utilized to discover additional chemotypes to our inꢀhouse existing scaffolds. In this publication, we
report one class of potent and selective PI3Kα inhibitors discovered through an ELT endeavor.
A few classes of small molecule panꢀPI3K inhibitors are reported in clinical development for
oncology applications. Some of these panꢀinhibitors include ZSTKꢀ474,14 GDCꢀ0941,15 XLꢀ147,16
BKMꢀ120,17 and CHꢀ513279918. Selective inhibitors such a INKꢀ111716 and NVPꢀBYL71919 have been
reported that target PI3Kα, the most frequently mutated kinase in human cancer,20 making it a promising
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target in cancer therapy. A frequent mutation in the p110α kinase domain is H1047R.21 Recently we
described the discovery a panꢀPI3K inhibitor for clinical evaluation.22 In an effort to identify a novel
and potentially isoform and/or mutant selective class of PI3K p110α inhibitors, we performed an ELT
selection against a set of libraries. The process of affinity selection was performed against both Hisꢀ
tagged PI3Kα wildtype and the mutant H1047R. The His affinity tags allowed for the target to be
isolated by immobilization on the solid matrix, PhyNexus IMAC (immobilized metal affinity
chromatography) resin tip. Once the target was immobilized, it was exposed to the library and nonꢀ
binding library members were removed through a simple resin wash. This was repeated twice (three
rounds total) after which the binders were eluted by heat denaturation of the resin bound target,
followed by PCR and DNA sequencing. For the PI3Kα wildtype we obtained 76,457 unique sequences,
and for the PI3Kα mutant (H1047R) we obtained 47,060 unique sequences. The outcome was analyzed
to determine the binding library members that were specific to the proteins.
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Selection of a preferred scaffold was found from one of our well established libraries that was
designed around three cycles of chemistry to provide a library (DELꢀA) with a complexity of 3.5
million compounds. As described in Figure 1, the library is composed of 191 amino acids at cycle 1
(R1), 95 boronates at cycle 2 (R2), and 196 amines at cycle 3 (R3). The R1 residues were utilized as the
attachment point to the ELT headpiece DNA through their carboxylate group. The details of the library
synthesis will be the subject of a different publication in the near future.
Figure 1. Design of DELꢀA; Null = The reaction was carried out without addition of the desired BB
amino acid (R1) or boronate (R2).
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A cubic scatter plot in which each axis represents a cycle of diversity in the library was used to
analyze and visualize the selected library members for both Hisꢀtagged PI3Kα wildtype and the mutant
(H1047R). After removal of the low copyꢀnumber molecules from the analysis, the most selected and
highly enriched families were observed to be of the same scaffolds and chemotypes with copy counts
greater than 20 fold above the background (Figure 2), indicating potential for lack of mutant selective
inhibitors.6 The feature was confirmed by repeating the PI3Kα mutant (H1047R) selection against the
same library in the presence of ZSTK474,14 a known and potent ATP competitive inhibitor. The cube
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analysis of the data demonstrated that the previously selected feature (family) was competed away in the
presence of a known inhibitor, leading us to conclude that the selected feature was interacting with
PI3Kα at the ATP binding site. We then initiated offꢀDNA feature confirmation of the original PI3Kα
mutant (H1047R) selection.
Figure 2. PI3Kα wildtype selection (left), mutant (H1047R) selection (middle) and mutant selection
with ZSTK474competitor (right). Library members with a single copy removed to simplify
visualization.
The visualizations in Figure 2 show the population after removal of the sequences that occurred fewer
than 2 times to simplify data analysis. This analysis revealed the preference for one main family of
compounds over the plane within the cube represented by a 4ꢀaminoꢀNꢀ(5ꢀmethylꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
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yl)benzenesulfonamide building block (1, Scheme 1), a cycle 3 based monoꢀsynthon. Within the plane,
one observes three major lines based on cycle 2 boronate building blocks (2ꢀ4, Scheme 1). There is no
preference for cycle 1 in the selection. The possible scaffold obtained from the selection is defined by
the library backbone, building blocks 1 at cycle 3 and 2, 3, and 4 at cycle 2. Based on the feature
enrichment over No Target (NT) background noise, building block 2 at cycle 3 was potentially a
stronger outcome than 3 or 4. A representation of the active pharmacophore is illustrated in Scheme 2.
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Scheme 1. Selected building blocks at cycle 3 (1) and cycle 2 (2, 3, and 4).
Scheme 2. The inhibitor pharmacophore defined by selection analysis.
In order to confirm the feature activity, a handful of compounds needed to be synthesized offꢀDNA after
feature analysis. In offꢀDNA small molecule synthesis, a choice was made where to truncate the linker
to the encoding DNA. Since the selection analysis indicated that cycle 1 was not selected for, two
representative amines were chosen in the first round of offꢀDNA synthesis in place of the corresponding
amino acids. In addition, analogs were planned in which cycle 1 was eliminated (5aꢀf, Table 1). The
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representative set of the selected molecules were synthesized offꢀDNA following the synthetic strategy
in Scheme 3. The synthetic strategy was planned as such to establish the selected and conserved moiety
in the scaffold earlier in the synthesis to allow for testing truncated analogs and intermediates as well as
fully enumerated compounds. Reductive amination of the selected aniline with aldehyde 6 followed by
Suzuki crossꢀcoupling of the resultant aryl iodide product with an aryl boronic acid afforded 7.
Saponification of an ester in 7 gave the acid 8 which, after coupling with the desired amine, afford the
final compounds 5.
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Table 1. Proposed target compounds for offꢀDNA synthesis and structureꢀactivity relationship
exploration.
Compounds #
R1
R2
R3
H
5a
5b
H
ꢀCO2Me
ꢀCO2Me
ꢀI
H
H
5c
5d
ꢀCO2H
ꢀH
H
H
H
5e
5f
ꢀCO2H
5g
ꢀCO2H
H
5h
5i
Me
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H
5j
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Scheme 3. Synthesis of the target offꢀDNA compounds.
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The offꢀDNA synthesized compounds were assayed for their activity against PI3Kα to confirm the
activity of the selected feature. The data obtained for all compounds is reported in Table 2. Fully
enumerated compounds 5a and 5b showed excellent activity against PI3Kα wildtype and mutant
H1047R. The methyl ester 5d obtained similar activity as compounds 5a and 5b, confirming the
selection result that cycle 1 is not necessary for scaffold activity. The intermediate lacking R2 (5c)
showed no activity against PI3Kα, a strong indication that the methoxypyridine moiety is critical for
inhibitor activity. Elimination of cycle 1 and replacement of the carboxamide with a free carboxylic
acid moiety afforded compound 5e, with similar or better PI3Kα activity than the corresponding
compounds 5a and 5b. Removal of the carboxylic acid in compound 5e afforded 5f with a 10ꢀfold
decrease in PI3Kα activity, indicating a potential interaction between the carboxylic acid moiety and the
enzyme active site. As mentioned earlier, our selection data showed two additional building blocks
selected at cycle 2. These two building blocks are boronates 3 and 4. The corresponding final carboxylic
acids for these two boronates were synthesized analogous to the synthesis of 5e (compounds 5g and 5h).
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Both compounds 5g and 5h showed similar PI3Kα activity as compound 5e. PI3K isoform assay data
for the final carboxylic acids indicate 5ꢀ10 fold selectivity for alpha over beta and delta. In general, the
selected compounds showed no selectivity against mutant H1047R but they appear to be more selective
for gamma isofom.
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Table 2. Feature activity (IC50 values, reported in nM) confirmation versus the PI3K isoforms.
Compound
alpha (WT)
alpha
beta
delta
gamma
(H1047R)
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13.0
700
398
ꢀ
10
4.4
5a
5b
5c
5d
5e
5f
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11.0
4.0
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10
>25,000
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30.0
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ꢀ
8.5
149
765
125.9
501
>10,000
790
59
2.6
52
102
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44
12.6
39.8
2,000
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63.1
199
2,000
20
2.0
4.0
>10,000
25
5g
5h
5i
ꢀ
>10,000
16
5j
“ꢀ“ = Compounds were not tested in biochemical assay
To further investigate the mode of the scaffold binding to the enzyme active site, the crystal structure
of compound 5e bound to the ATP site of PI3Kα was obtained (Figure 3). As anticipated from the
selection preference for the methoxypyridine group, the methoxy group is a hinge binder with a classic
hydrogen bond interaction between the methoxy oxygen with –NH of Valꢀ851. The thiadiazole and
phenyl sulfonamide moiety bypasses the Ileꢀ848 “gatekeeper” and Tyrꢀ836 to access the “affinity
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pocket” of the active site. This mode of binding is different from a canonical type I kinase inhibitor
binding mode, which tends to maximize interactions further into the back pocket by sterically packing
against and making interactions with the residues in the vicinity of the gatekeeper Ileꢀ848.23 The unusual
binding mode of compound 5e allows extensive interactions with residues in the front of the pocket such
as Argꢀ770 and Metꢀ772 from the Pꢀloop and Glnꢀ859 from the Cꢀterminal lobe of the catalytic domain.
Since the residue composition of the Pꢀloop or the glycineꢀrich loop of protein kinases is markedly
different from lipid kinases, the ELT scaffold is likely to be selective for PI3K if these differential
interactions are maximized. The inward turn of the phenyl sulfonamide moiety, facilitated by the methyl
amine linker, places it in close proximity to the catalytic component of the enzyme, specifically the
catalytic Lysꢀ802 residue. The sulfonamide and thiadiazole group establishes two major hydrogen bonds
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+
with –NH3 of Lysꢀ802. One of these interactions is through the thiadiazole nitrogen and the second
interaction is through the oxygen atom of the sulfonamide group (Figure 2a). Additionally, pKa
calculations on compound 5e suggest that at physiological pH of 7.4, the major species (83%) carries a
negative charge on the deprotonated nitrogen atom of the sulfonamide group.24,25 The presence of a
negative charge on this moiety in the major species is likely to result in favorable charge
complimentarity between the inhibitor and the catalytic Lysꢀ802, which could contribute to the low
nanomolar potency of compound 5e in PI3Kα. Indeed, methylation of the sulfonamide nitrogen as
exemplified in 5i, resulted in significantly attenuated potency across PI3K isoforms (Table 1).
Methylation of the nitrogen is also likely to result in steric clashes with residues in this region, therefore
this result is explained as a net unfavorable steric and electrostatic effect. The xꢀray structure reveals
that the carboxylic acid group at the paraꢀposition of the central phenyl ring is involved in a network of
an ionic saltꢀbridge interaction with Argꢀ770 and a hydrogen bond with Glnꢀ859 (Figure 2b). These
strong electrostatic interactionsof the carbonyl group indicate its importance in the activity of the amide
and carboxylate analogs, although being solvent exposed the effect might be attenuated. Lastly, an
indication that the benzyl amine group picks up a hydrogen bonding interaction by acting as a Hꢀbond
donor to the backbone carbonyl oxygen of Serꢀ919 residue through its amine moiety and the methyl
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group on the thiadiazole establishes close van der Waal interactions with Tyrꢀ836 in the back pocket of
the active site.
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The selection data showed no preference for the cycle 1 synthon and, as the crystal structure reveals,
there are no major interactions between this region of the inhibitor and the protein. Therefore, cycle 1
likely only functions as a linker to attach the inhibitor to the DNA in the library. The DNA attachment
point on the scaffold provides a handle that could be used to alter the physicochemical properties of the
inhibitors.
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b)
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Figure 3. XꢀRay structure of compound 5e bound to PI3Kα; a) Key interactions of the inhibitor in the
active site depicted; b) Surface representation of the active site with the bound inhibitor demonstrates
steric packing and interactions with the residues of the Pꢀloop.
In summary, we report here the discovery of a novel and potent class of inhibitors of the p110α
catalytic subunit of PI3Kα using ELT as the lead identification technology. We have demonstrated ELT
selection for disynthons (cycles 2 and 3) allowing for truncation of the selection based on a variable
cycle 1. This demonstrates the power of the technology to establish preliminary internal SAR before
obtaining offꢀDNA activity of the compounds, and the utility of the selectionꢀbased internal data to
guide medicinal chemistry lead optimization efforts. The ELT selection output generated a scaffold
exhibiting modest isoform selectivity for PI3Kα over PI3Kβ and PI3Kδ. The xꢀray crystal structure of
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inhibitor 5e in PI3Kα demonstrated a unique binding mode in the ATP binding pocket with major
interactions with both the hinge point and the “affinity pocket” that are consistent with the selection
results and offꢀDNA compound activity.
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Notes
The authors declare no competing financial interest.
Present Addresses: † XꢀChem, Inc., Waltham, Massachusetts, USA (M.A.C., P.A.C. and J.W.C ) and
Massachusetts College of Pharmacy and Health Sciences, Worcester, Massachusetts, USA (S.M. and
J.L.S.).
ACKNOWLEDGMENT
We acknowledge Cynthia Parrish, Christopher AricoꢀMuendel, Jeff Messer, and Barry Morgan for
their support and sponsoring this ELT campaign.
SUPPORTING INFORMATION AVAILABLE
Experimental details for the synthesis of all the compounds and in vitro ADME and in vivo PK/PD data.
The coordinates of PI3Kα p110a in complex with compound 5e were deposited under PDB ID 4YKN.
This material is available free of charge via the Internet at http://pubs.acs.org.
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Discovery of a potent class of PI3Kα inhibitors with unique binding mode via Encoded Library
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Technology (ELT)
Hongfang Yang,a Patricia F. Medeiros,a Kaushik Raha,b Patricia Elkins,b Kenneth E. Lind,a Ruth Lehr,c
Nicholas D. Adams,d Joelle L. Burgess,d Stanley J. Schmidt,d Steven D. Knight,d Kurt R. Auger,d
Michael D. Schaber;e G. Joseph Franklin,a Yun Ding,a Jennifer L. DeLorey,a Paolo A. Centrella,a
Sibongile Mataruse,a Steven R. Skinner,a Matthew A. Clark,a John W. Cuozzo,a Ghotas Evindar a,*
a) MDR (Molecular Discovery Research) Boston, Platform Technology and Science,
GlaxoSmithKline, 830 Winter Street, Waltham, MA 02451; b) Computational and Structural
Chemistry, Platform Technology and Science, GlaxoSmithKline, 1250 South Collegeville Road,
Collegeville, Pennsylvania 19426; c) Biological Reagents and Assay Development, Platform
Technology and Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville,
Pennsylvania 19426; d) Oncology Research, GlaxoSmithKline, 1250 South Collegeville Road,
Collegeville, Pennsylvania 19426; e) Screening and Compound Profiling, Platform Technology
and Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania
19426
Email: ghotas.x.evindar@gsk.com
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“For Table of Contents Use Only”
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Graphic Table of Content
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Compound 5e
PI3Kķ (WT/H1047R) IC50 = 10/8.5 nM
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