Characterization of Specific N-α-Acetyltransferase 50 (Naa50) Inhibitors Identified Using a DNA Encoded Library

Article abstract of DOI:10.1021/acsmedchemlett.0c00029

Two novel compounds were identified as Naa50 binders/inhibitors using DNA-encoded technology screening. Biophysical and biochemical data as well as cocrystal structures were obtained for both compounds (3a and 4a) to understand their mechanism of action. These data were also used to rationalize the binding affinity differences observed between the two compounds and a MLGP peptide-containing substrate. Cellular target engagement experiments further confirm the Naa50 binding of 4a and demonstrate its selectivity toward related enzymes (Naa10 and Naa60). Additional analogs of inhibitor 4a were also evaluated to study the binding mode observed in the cocrystal structures.

Full text of DOI:10.1021/acsmedchemlett.0c00029

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Letter
Characterization of specific N-#-acetyltransferase 50
(Naa50) inhibitors identified using a DNA encoded library
Pei-Pei Kung, Patrick Bingham, Benjamin J. Burke, Qiuxia Chen, Xuemin Cheng, Ya-Li Deng, Dengfeng
Dou, Junli Feng, Gary M. Gallego, Michael R Gehring, Stephan K Grant, Samantha Greasley, Anthony
R. Harris, Karen A. Maegley, Jordan Meier, Xiaoyun Meng, Jose L Montano, Barry A Morgan, Brigitte S.
Naughton, Prakash B Palde, Thomas A Paul, Paul Richardson, Sylvie Sakata, Alex Shaginian, William K
Sonnenburg, Chakrapani Subramanyam, Sergei Timofeevski, Jinqiao Wan, Wen Yan, and Albert E Stewart
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00029 • Publication Date (Web): 10 Apr 2020
Downloaded from pubs.acs.org on April 13, 2020
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Characterization of specific N--acetyltransferase 50 (Naa50) inhibitors
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identified using a DNA encoded library
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Pei-Pei Kung§*, Patrick Bingham§, Benjamin J. Burke§, Qiuxia ChenꞱ, Xuemin ChengꞱ, Ya-Li
Deng§, Dengfeng Dou Ʇ, Junli Feng§, Gary M. Gallego§, Michael R. Gehring§, Stephan K. Grant§,
Samantha Greasley§, Anthony R. Harris§, Karen A. Maegley§, Jordan Meierᴛ, Xiaoyun MengꞱ,
Jose L. Montanoᴛ, Barry A. Morgan†Ʇ, Brigitte S. Naughton§, Prakash B. Palde§, Thomas A.
Paul§, Paul Richardson§, Sylvie Sakata§, Alex ShaginianꞱ, William K. Sonnenburg†ǂ, Chakrapani
Subramanyam§, Sergei Timofeevski§, Jinqiao WanꞱ, Wen Yan§, and Albert E. Stewart§
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§Worldwide Research and Development, Pfizer Inc., 10770 Science Center Drive, San Diego,
California 92121, United States
ꞱHitGen Inc., Building 6, No.8, Huigu 1st East Road, Tianfu International Bio-Town, Shuangliu
District, Chengdu, Sichuan, P.R.China, 610200
†HitGen Pharmaceuticals Inc., PO Box 88240 Houston TX 77288, United States
ᴛChemical Biology Laboratory, National Cancer Institute, Frederick, Maryland 21702, United
States
ABSTRACT: Two novel compounds were identified as Naa50 binders/inhibitors using DNA-
encoded technology screening. Biophysical and biochemical data as well as co-crystal structures
were obtained for both compounds (3a and 4a) to understand their mechanism of action. These
data were also used to rationalize the binding affinity differences observed between the two
compounds and a MLGP peptide-containing substrate. Cellular target engagement experiments
further confirm the Naa50 binding of 4a and demonstrate its selectivity toward related enzymes
(Naa10 and Naa60). Additional analogs of inhibitor 4a were also evaluated to study the binding
mode observed in the co-crystal structures. Keywords: Naa50, N--acetyltransferase, acetyl-CoA,
DEL
Protein acetylation has received significant attention in the past decade due to its important
roles in regulating cell signaling. In particular, N-terminal (Nt) acetylation is known to be a highly
abundant co-translational protein modification that is controlled by a family of acetyltransferases
(NAT).1 The Nt-acetylation of a protein can affect its nuclear import and export and can also act
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as a degradation signal to control the protein’s cellular stability.1 The eukaryotic protein N-
terminal acetylation machinery is composed of six NAT protein complexes (NatA-NatF).
Knockdown studies have shown that each complex associated with the NAT machinery (i.e.,
NatA-NatF) is important for cell proliferation and normal cell cycle progression.2 The N-
terminal acetyltransferase (Naa50) enzyme is a member of the N-alpha-terminal acetyltransferase
NAT protein family. It co-exists with Naa10 and Naa15 in the NatE complex and is responsible
for the enzymatic function of the complex. Naa50 is also found to be essential for normal sister
chromatid cohesion and chromosome condensation.3-5 Therefore, an inhibitor of the Naa50
enzyme might have therapeutic applications in oncology indications.
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Recently, a potent inhibitor of Naa50 was described in the literature (compound 1, Figure
1a).6 This molecule combines the acetyl-CoA (AcCoA) co-factor that is known to associate with
the enzyme and a tetra-peptide derived from the N-terminal protein sequence common to optimal
Naa50 substrates (MLGP)7,8. Construction of inhibitor 1 was also influenced by studies of the
Naa50 biochemical mechanism which indicated formation of a ternary complex between the
AcCoA cofactor, an appropriate protein substrate, and the enzyme.9 Although compound 1 is a
potent Naa50 inhibitor, the molecule is not particularly efficient due to its large molecular weight
(ligand efficiency (LE)10 = 0.13). In addition, its high molecular weight (MW = 1223) and high
polarity (TPSA = 577 and clogP = -4.1) likely prevents facile permeability across cell membranes
and may thus compromise the use of the molecule as a robust in vitro tool compound. In this
report, we describe our initial efforts to identify a potent and selective Naa50 inhibitor with
improved physiochemical properties relative to compound 1 (i.e., reduced molecular weight and
TPSA; increased logD). As part of these studies, we were also interested in finding a more ligand
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efficient inhibitor that could serve as an appropriate starting point for future medicinal chemistry
optimizations.
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We first explored whether the pyrophosphate adenosine moiety present in compound 1 was
required for potent Naa50 inhibition. As shown in Figure 1a, a truncated analog of 1 that lacked
this functionality displayed significantly weaker anti-Naa50 activity (compound 2). Separate co-
crystal structures obtained with compounds 1 and 2 in complex with Naa50 demonstrated that, in
spite of its weaker potency, the latter molecule occupied the same region of the Naa50 active site
as the MLGP portion of 1 along with part of the co-factor binding site (Figures 1b and S1, more
details regarding the specific interactions formed between compounds 1 and 2 and the Naa50
protein are provided below). This observation afforded confidence that molecules which lacked a
pyrophosphate adenosine moiety could appropriately recognize the Naa50 active site and further
encouraged our search for new inhibitors that improved upon compounds 1 and 2.
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As one approach to the identification of new Naa50 inhibitors, we screened a 2.2 billion-
member DNA encoded library (DEL) to find novel ligands with potent affinity to the protein.11,12
Affinity selection was performed using the library with a selectively polyhistidine-tagged human
Naa50 protein construct (250 pmol enzyme concentration) in solution incubation for 60 minutes.
The polyhistidine-tagged Naa50 protein was then immobilized to magnetic beads and was washed
extensively to remove non-binding molecules. Bound library members were subsequently eluted
from the beads by heat denaturation of the protein at 75 °C. The eluent was then incubated with
fresh protein in solution to initiate a new round of affinity selection. A parallel selection against
buffer lacking the target protein was also performed to allow for the identification of molecules
that were enriched via non-target affinity mechanisms. After three rounds of such selection, the
DNA tags of the eluted molecules were PCR amplified and sequenced, and the sequences were
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translated to identify the chemical structures of putative Naa50 binders. Importantly, we also
screened the library against Naa50 in the presence of either the AcCoA cofactor or inhibitor 1.
Inclusion of AcCoA was envisioned to bias screening results toward molecules which mimicked
the MLGP tetra-peptide substrate. In contrast, screening in the presence of 1 was anticipated to
help identify compounds which bound to Naa50 outside of the catalytic site. To further increase
confidence that the identified hits were genuine Naa50 binders, we repeated the above selections
using a 5-fold lower enzyme concentration (50 pmol). It was expected that this alternative protein
concentration would help us identify the most potent binders to the Naa50 enzyme.13,14
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As shown in Figure 2, the described affinity selections afforded a large number of
enrichments associated with one specific portion of the 2.2 B-member DEL. This sub-library
(DEL951) was constructed in 3 synthesis cycles and involved derivatization of diamine-containing
core scaffolds using a variety of carboxylic acid, aldehyde, sulfonyl chloride, and isocyanate-
containing building blocks (Figure 3a, see supporting information for more details). The affinity
selection results obtained in the presence of the AcCoA co-factor indicated that three R1 building
blocks produced multiple library members that were significantly enriched via the selection
process (4 vertical rows highlighted in Figure 2, structures 2a-2c). The observed enrichments
associated with these R1 moieties were dramatically attenuated when the affinity selections were
conducted in the absence of AcCoA (supplementary information, Figure S2). This outcome
suggested that the library members derived from the 2a-2c R1 building blocks cooperatively bound
to Naa50 with AcCoA in the vicinity of the co-factor binding site and might possibly mimic the
MLGP tetra-peptide substrate. Further support for the hypothesis that the compounds bound to
the MLGP site was obtained from affinity selections performed with DEL951 in the presence of
compound 1. As shown in Figure S2, these assessments generated no significant enrichments
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associated with the DEL951 library members consistent with the blocking of the MLGP binding
site by 1 during the enrichment process.
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Encouraged by these findings, we initially selected two structurally-diverse library
members for off-DNA synthesis to confirm affinity of their non-nucleic acid portions with the
Naa50 protein (structures A and B, Figure 3b). Both of these members afforded high copy number
outcomes from the affinity-selection process using either high (250 pmol) or low (50 pmol) Naa50
concentrations (Figure 3b). The 50 pmol results provided us with increased confidence that the
molecules were genuine and relatively potent Naa50 binders. Given the structural similarity
between R1 building blocks 2b and 2c (oxazole and thiazole moieties) and the corresponding
similar affinity selection data, we chose to only conduct the off-DNA synthesis of the thiazole-
containing compound during this initial exploration phase. The off-DNA syntheses of library
members A and B and the corresponding diastereomers from Figure 3 are described in Schemes
S2-S4 (supporting information).
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As shown in Figure 4, the synthesized compound (compound 3, supporting information)
corresponding to DEL library member A increased the thermal stability of the Naa50 protein in
the presence of AcCoA using a protein thermal-shift assessment15. This result provided evidence
of direct-target engagement for the compound (Figure S3). As predicted from the affinity selection
outcomes performed with DEL951, the thermal shift outcomes were attenuated when the assay
was performed in the absence of the AcCoA (Figure 4). This observation suggested that the
compound did not bind to the Naa50 AcCoA pocket and instead bound cooperatively with the co-
factor. Biochemical testing further demonstrated the functional activity of 3 (IC50 = 2.7 M).
Therefore, both diastereomers of the compound were synthesized (3a and 3b; 3a: Scheme S2,
supporting information; 3b, supporting information) and were further evaluated in surface
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plasmon resonance (SPR, Table 1, Figure S6) and biochemical assessments (Figure 4). SPR
methodology subsequently confirmed specific association of 3a with the Naa50 protein (Kd = 1.9
M) in the presence of AcCoA, and the corresponding Naa50 biochemical inhibition activity
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closely matched the SPR binding result (BC IC50 = 2.0 M, Figure 4). In addition, a cellular
thermal shift assay (CETSA)16 conducted using A549 cell lysates demonstrated that compound 3a
could stabilize the Naa50 protein against thermal denaturation without impacting the thermal
stabilities of the related Naa10 and Naa60 enzymes (Figure S4a). Collectively, these data
demonstrated that 3a was a genuine, selective, and relatively potent Naa50 inhibitor. Similar
profiling of the 3b diastereomer demonstrated that it inhibited Naa50 in a less potent manner
relative to 3a (Kd = 7.0 M; BC IC50 = 12 M, Figure 4). Because of this weaker activity,
additional profiling of 3b was not performed.
Since the thiazole-containing library members were prepared and screened as 1:1
diastereomeric mixtures (see supporting information), we synthesized a related mixture (4,
supporting information) as well as both single diastereomers (compounds 4a (Schemes S4 and S5)
and 4b (Scheme S3) corresponding to DEL library member B for off-DNA characterization. As
shown in Figure 4, mixture 4 displayed significant thermal stabilization of Naa50 in the thermal
shift assay that was stronger than that exhibited by 3 and which was similarly weakened in the
absence of AcCoA (Figure S3). The biochemical Naa50 inhibition activity determined for 4 was
also correspondingly more potent than that associated with 3 (Figure 4). Both single diastereomers
4a and 4b displayed measurable Naa50 binding affinity and biochemical inhibitory activity with
4a having the better binding affinity and better biochemical potency relative to both 4b and 3a
(Figure 4 and Table 1). Compound 4a was also a considerably stronger binder than 3a in CETSA
testing (Figure S4a, compare results obtained at 55 °C and 61 °C for 3a and 4a, respectively,
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relative to DMSO control at 52 °C). Compound 4a maintained good selectivity for Naa50 over
Naa10 and Naa60 just like compound 3a.
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To demonstrate the ability of 4a to engage Naa50 in a complex proteome we performed
competitive chemoproteomic profiling experiments using Lys-CoA Sepharose.17 This affinity
reagent captures Naa50 through a bidentate interaction involving the substrate-binding site,18
which should be competed in the presence of 4a. Consistent with our in vitro inhibition studies,
pre-incubation of cell lysates with 4a led to potent inhibition of Naa50 capture by Lys-CoA
Sepharose, with substantial competition observed at concentrations as low as 50 nM (Figure S4b).
This closely resembles the binding constant of 4a for recombinant holo (acetyl-CoA bound) Naa50
determined by SPR. These results provide additional evidence that 4a can engage the Naa50 active
site even in cellular contexts where it may exist in multiprotein complexes, and demonstrates the
utility of chemoproteomic capture probes to provide insight into the biochemical mechanism of
acetyltransferase inhibitors in complex proteomes.
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We obtained co-crystal structures of compounds 3a and 4a with the CoA-bound Naa50 to
further understand their binding modes. CoA was utilized in these experiments to facilitate
crystallization. As demonstrated later in this work, the orientation of the bound ligand did not vary
significantly when a representative compound (4a) was co-crystallized in the presence of either
the CoA or the co-factor, AcCoA. As shown in Figure 5a and 5b, both compounds (3a and 4a)
bound to the region of the Naa50 active site adjacent to the CoA-binding pocket. Both 3a and 4a
formed H-bond interactions with the side chains of the Tyr31 and Tyr139 residues. They also
formed similar common interactions with the backbone carbonyl moieties of Met75 and His112
as well as the backbone NH of Gln114 (Figures 5a and 5b, residues depicted in green sticks). Other
H-bond interactions unique to compounds 3a and 4a include the backbone NH moieties of Arg62
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(compound 3a) and of Arg141 and Lys140 (compound 4a) (Figures 5a and 5b, residues depicted
in orange sticks). The methyl amide moiety, R1 thiazole moiety and the R2 carboxyl amide
carbonyl moiety of 4a do not form hydrogen bonds with the protein. The R2 phenyl moiety forms
hydrophobic interactions with a hydrophobic pocket created by three tyrosine residues (Y138,
Y139, and V29). The R2 tetrazole substituent of 4a forms an H-bond with the backbone NHs of
Arg141 and Lys140. It may also form a charge-charge interaction with the side chain of Arg141.
However, the precise nature of this association is uncertain as the side chain of Arg141 is
disordered in the crystal structures.
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To further investigate the binding mechanism, we performed in-depth kinetic and
thermodynamic analyses of the interactions between 4a and Naa50 in apo as well as AcCoA and
CoA complexed forms using both SPR methods (Tables 1, Figure S6).
These kinetic
measurements suggested that 4a bound to the Naa50AcCoA complex with ~4-fold greater affinity
than the apo form of enzyme. Notably, the gain in affinity for the Naa50•AcCoA complex was
associated with a decrease in the dissociation rate (koff) or increase in the dissociative half-life (t1/2)
of the complex (Table 1). To this end, significant evidence exists linking longer t1/2 to binding-
induced conformational changes leading to greater steric and electronic complementarity in
protein-ligand interactions19. Interestingly, previous NMR-based biophysical studies determined
that a peptide substrate (MLGP-RRR) displayed very weak affinity for the Naa50 protein. Our
own SPR assessments confirmed this weak binding affinity (Kd > 100 M). These results suggest
that the 4a inhibitor recognizes a pre-existing Naa50 binding pocket more efficiently than the
peptide substrate.
We also obtained a co-crystal structure of Naa50 bound to AcCoA and compound 4a
(Figure 6). This structure-enabled investigation of the differences in 4a binding modes in the
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presence of either CoA or AcCoA. In the Naa50•AcCoA crystal structure, the acetyl group of
AcCoA formed two hydrogen bonds with Naa50 including one to the backbone NH of Gln114
and one to the sidechain of Asn117 (Figure S6). In contrast, in the Naa50•AcCoA•4a complex,
4a formed a hydrogen bond with the NH of Gln114 instead of AcCoA. The acetyl group of AcCoA
repositioned to make only a long (3.1 Å) hydrogen bond with backbone NH of Leu77 of Naa50.
Interestingly, this rearrangement was not observed in co-crystal structures obtained with CoA and
Naa50 in the presence and absence of 4a. Consistent with the similar Kd values observed for 4a-
Naa50 binding with either CoA or AcCoA, the bound inhibitor conformation and protein-ligand
interactions were identical in the 4a-CoA-Naa50 and 4a-AcCoA-Naa50 crystal structures. These
observations suggested that conclusions drawn via analysis of Naa50-CoA-inhibitor complexes
(e.g. Figures 5a and 5b) could also be applied to Naa50-AcCoA-inhibitor structure.
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Since compound 4a displayed significantly improved Naa50 binding and inhibition activity
compared to 3a (with an improved LE value), we chose to further explore other hits related to 4a
from the on-DNA affinity selections as part of our follow-up activities. Accordingly, we expanded
the structure-activity relationships associated with 4a by synthesizing several closely related
DEL951 members in off-DNA form. As shown in Table 2, these molecules all afforded robust
copy numbers at both 250 pmol and 50 pmol Naa50 protein concentrations. The syntheses of
compounds 5-8 in Table 2 are described in Scheme S5-S7 (supporting information). Replacement
of the thiazole moiety in 4a with an oxazole provided a nearly equipotent Naa50 inhibitor
(compound 5). This result was consistent with the prior analysis of the DEL951 affinity selection
data which suggested that thiazole and oxazole-containing R1 building blocks produced library
members with similar Naa50 affinities (2b and 2c in Figure 2). Removal of the R2 substituent
from 4a drastically weakened Naa50 biochemical inhibition activity (6, Table 2). Deletion of the
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R2 amide carbonyl group (7) or repositioning of the tetrazole present in the R2 substituent (8) also
weakened anti-Naa50 potency but to a lesser extent than was observed for compound 6 (compound
6 is a truncated analog, not actually present in the original DEL). Interestingly, the biochemical
potencies displayed by compounds 4a and 5-8 loosely paralleled the corresponding copy numbers
(compounds with the highest copy numbers at low protein concentrations displayed the most
potent Naa50 biochemical inhibition). This observation suggested that meaningful SAR
information could be extracted from the affinity selection data generated for DEL951 prior to off-
DNA synthesis.
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In order to investigate whether the co-crystal structure of 4a could be used for further
compound optimization, several analogs of 4a were subsequently designed based on the
compound’s observed Naa50 interactions and binding mode. The syntheses of compounds 9-14
in Table 3 are described in Scheme S8-S9 (supporting information). As shown in Table 3,
replacement of the 4a carboxyl amide thiazole substituent with a methyl group afforded similar
anti-Naa50 biochemical activity (Table 3, compound 9; compare to 4a). In contrast, replacement
of the tetrazole moiety of 4a with hydrogen (10), triazole (11), N-linked triazole (12), and N-linked
tetrazole (13) all resulted in significant (>100 fold) loss of biochemical potency (Table 3).
Interestingly, the only compound from this sub-series which retained reasonable biochemical
potency relative to 4a contained a carboxylic acid moiety in the place of the tetrazole (14, Table
3). These results suggested the importance of maintaining a negative charge on the inhibitor even
if no specific charge-charge interaction is observed in the 4a-Naa50 co-crystal structure.
The co-crystal structure of 4b (the less active diastereomer of 4a) in complex with Naa50
was also solved. As shown in Figure S10, 4b formed the same H-bonds with the protein residues
as was observed in the 4a-Naa50 co-crystal structure (Figure 5b). These interactions include the
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tetrazole moiety of 4b forming hydrogen bonds with the backbone NH of Arg141 (as shown in
Figure 5b). These observations reinforce the importance of maintaining a negatively-charged
ligand fragment in close proximity to Arg141 and Lys140. Differences between the 4a and 4b
bound conformations occurred in areas where both ligands did not form H-bonds with the protein
(e.g., the thiazole methyl amide and the carboxylamide moiety connected to the phenyltetrazole).
The bound conformation of 4b exhibited conformational strain in the pendant methyl amide (about
5 kcal/mol calculated at the B3LYP-D3/DFT 6-311g**+ level in gas phase20). This strain was
needed to avoid unfavorable contacts between the ligand and the protein. It seems likely that strain
could be partly responsible for the loss of SPR and biochemical potency relative to 4a (compare
4a and 4b in Figure 4).
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As shown in Figure S5, compounds 3a and 4a occupied the same region of the Naa50
binding site as the tetra-peptide (MLGP) moiety present in 2. These binding modes were consistent
with biochemical experiments which suggested that 3a and 4a bound un-competitively to Naa50
with respect to the AcCoA co-factor and competitively to the MLGP-containing peptide substrate
analog (Figure S8 and Figure S9). In addition to making similar hydrogen bonding interactions
with Naa50 observed for the MLGP tetrapeptide, 3a and 4a also formed additional hydrophobic
interactions with protein. Therefore, stronger binding affinities were observed for both molecules
relative to the MLGP tetrapeptide. Collectively, these results suggest a biochemical inhibition
mechanism for compounds 3a and 4a in which AcCoA binding enhances the compound’s binding
affinity. Detailed comparisons among the Naa50 binding interactions observed for 3a and 4a, and
the MLGP peptide are described in the supplementary material (Figures S11 and Figure S12).
In conclusion, we utilized DEL technology to identify potent and selective Naa50 inhibitors
(compounds 3a and 4a) that bound to the substrate binding pocket. Specifically, compound 4a
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displayed potent biochemical inhibitory potency (7 nM v.s. 30 nM), reduced TPSA (224 v.s. 557),
improved LE (0.3 v.s. 0.13) and increased clogP (0.6 v.s. -4) relative to compound 1. Further
optimization using co-crystal structure information and structure-based design strategy may
provide a cell permeable tool compound that could be used to probe the effects of inhibiting Naa50
in cancer cells.
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ASSOCIATED CONTENT
Supporting Information
Details of protein purification, crystallization conditions, compound synthesis, compound
characterization, and biochemical and biophysical experiments are included in the Supporting
Information.
Accession Codes
PDB numbers:
compound 1: 6WF3
compound 2: 6WF5
compound 3a+CoA: 6WFG
compound 4a+CoA: 6WFK
compound 4a+AcCoA: 6WFN
compound 4b+AcCoA: 6WFO
AUTHOR INFORMATION
Corresponding Author
*E-mail: peipei.kung@pfizer.com. Tel: (858) 526-4867
ORCID
Pei-Pei Kung: 0000-0002-9203-7374
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Present Address
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ǂFor William K. Sonnenburg: Nurix Therapeutics, 1700 Owens Street, Suite 205, San Francisco,
CA 94158, United States
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Author Contributions
All authors have given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Simon Bergqvist, Mike Greig, Ben Bolanos, and Simon Planken for their efforts
in the early phase of this project. We also thank Sabrina Li for repeating the CETSA experiments
and Wei Wang for obtaining the 1H and 13C NMR spectra and HRMS data for compounds 3, 3a,
3b, and 14. We are grateful to Martin Edwards and Martin Wythes for their support and review
of the manuscript.
ABBREVIATIONS
NAT, N-terminus acetyltransferase; MLGP: methionine-leucine-glycine-proline; CoA: co-
enzyme A; DEL: DNA-encoded library; PCR: polymerase chain reaction.
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Figure 1a. Chemical structures of AcCoA, CoA, literature reported bi-substrate inhibitor (1), and
5
6
its truncated analog: des-A (2)
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8
9
H2N
N
H2N
N
N
N
N
N
O
O
O
O
O
O
O
O
O P O P O
OH OH
O P O P O
OH OH
S
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11
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13
14
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SH
N
N
N
N
N
N
O
O
H
H
H
H
O
OH
OH
HO
HO P O
OH
HO
HO P O
OH
O
O
AcCoA
CoA
H2N
S
N
O
O
N
O
O
O
O
H
O P O P O
OH OH
N
N
S
N
N
N
H
N
N
N
O
H
H
H
O
O
NH2
OH
O
HO
HO P O
OH
O
1
biochem. IC50: 30 nM (LE = 0.13)
TPSA: 577
S
O
O
O
O
H
N
N
S
N
H
N
H
N
H
N
H
O
OH
O
NH2
O
2
biochem. IC50: 10.5 M (LE = 0.14)
TPSA: 280
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Figure 1b. Co-crystal structure of 1 bound to Naa50 (2.3 Å, PDB ID 6WF3). Compound 1 is
drawn in orange stick representation and spans the binding pocket, from the cofactor binding site
on the left to the peptide binding site on the right. Residues that form hydrogen bonds with the
ligand (dashed lines) are drawn as green sticks. Hydrogen bonds are drawn as dashed lines.
Protein C trace is shown in grey.
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Figure 2. Cube view of DEL951 analysis
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Points represent enrichment of library member after 3 rounds of affinity selection using biotinylated Naa50 protein
(larger size = greater enrichment). Affinity selection was performed using 250 pmol Naa50 in the presence of the
AcCoA co-factor. The cube axes depict the R1, R2, and R3 building blocks employed during construction of the library.
R1 build block structures which afforded enrichment of multiple library members are also shown (2a-2c).
Note that the 2a building block afforded two vertical rows of enriched library members.
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Figure 3. (a) general construction of DEL951 and (b) specific library members (A and B) initially
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selected for off-DNA synthesis
(a)
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HN
R3
HN
R3
O
O
R1
R1
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ring
N
H
N
H
N
N
H
R2
R2
(b).
H
N
O
N
O
HN
O
R1
H
O
N
R1
N
N
H
O
N
O
S
NH HN
R3
N
O
O
R2
O
R3
R2
N
N
B
A
N
N
N
N
H
copy#
copy#
915 250 pmol Naa50+AcCoA 2161
130 50 pmol Naa50+AcCoA 1819
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Figure 4. Re-synthesis hits from DEL951 displayed binding affinity and functional activities
5
O
HN
6
7
8
HN
O
N
O
H
O
N
O
S
O
N
N
NH HN
O
H
N
O
9
N
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11
12
13
14
15
16
17
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22
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O
N
N
N
N
N
N
H
3
4
°
C
Tm (+AcCoA): +15.5
Tm (-AcCoA): -0.15
biochem. IC50: 2700 nM
C
°
C
Tm (+AcCoA):+21.4
Tm (-AcCoA): +9.6
biochem. IC50: 13 nM
LE: 0.3
C
°
°
LE: 0.23
TPSA: 125
TPSA: 224
HN
O
N
HN
O
N
O
O
H
H
O
N
O
N
N
N
H
H
O
O
N
N
N
N
3b
3a
SPR (Naa50+AcCoA) Kd: 7000 nM
biochem. IC50: 12000 nM
LE: 0.2
SPR (Naa50+AcCoA) Kd: 1900 nM
biochem. IC50: 2000 nM
LE: 0.23
TPSA: 125
TPSA: 125
O
N
O
HN
HN
N
O
N
O
S
O
N
O
S
NH HN
O
NH HN
O
N
N
O
O
N
N
N
N
N
N
N
N
H
H
4b
4a
SPR (Naa50+CoA) Kd: 471 nM
SPR (Naa50+AcCoA) Kd: 470 nM
biochem. IC50: 1600 nM
LE: 0.2
SPR (Naa50+CoA) Kd: 32 nM
SPR (Naa50+AcCoA) Kd: 27 nM
biochem. IC50: 7 nM
LE: 0.29
TPSA: 224
TPSA: 224
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Figure 5. Crystals structures of the complex of Naa50 with CoA and 3a (2.16 Å, PDB ID 6WFG)
(a) or 4a (1.87 Å, PDB ID 6WFK) (b). CoA is drawn in yellow stick representation, 3a in blue
stick representation and 4a in magenta stick representation. Residues that form hydrogen bond
interactions (dashed lines) that are common to both 3a and 4a are drawn as green sticks, while
those that are unique to each ligand as drawn as orange sticks.
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Figure 6. Crystal structure of the complex of 4a with Naa50 in the presence of AcCoA (1.07 Å,
PDB ID 6WFN). View from the top of the binding pocket. AcCoA is drawn in pale blue stick
representation while 4a is in magenta stick representation. The binding mode and hydrogen bonds
(dashed lines) between 4a and Naa50 are identical to those observed in the presence of CoA.
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Table 1. Kinetics of compound binding to Naa50_apo, Naa50AcCoA by SPR
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kon
(M-1. s-1)
koff
(s-1)
t1/2
SPR KD
Protein
Compound
(min)
(nM)
1.8 x 105
2.0 x 105
4.26 x 104
5.5 x 104
3.6 x 103
4.3 x 103
ND
3.1 x 10-3
3.2 x 10-2
4.5 x 10-3
1.4 x 10-3
1.7 x 10-3
6.0 x 10-3
ND
Naa50_apo
AcCoA
CoA
4a
4
17.6 ± 2.8
156 ± 27
105 ± 4.8
26.7 ± 7.3
470 ± 25
1010 ± 963
>10,000
Naa50_apo
0.4
2.6
8.4
8.6
3.1
ND
Naa50_apo
4a
Naa50AcCoA
Naa50AcCoA
Naa50AcCoA
Naa50AcCoA
4b
3a
3b
Measured at 10 ºC in 25 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol, 0.02% Tween 20, 2% DMSO,
pH 7.5
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Table 2. biochemical and TPSA data of analogs from DEL 951
O
HN
N
O
N
O
X
NH HN
O
N
R2
#
X
R2
Copy
number
Biochem.
IC50
TPSA
(250 pmol)
(50 pmol)
(M)
4a
5
S
2161
1819
0.007
224
208
H
O
N
N
N
N
O
1991
796
0.014
13
H
O
O
N
N
N
N
6
7
S
S
ND
169
223
338
164
0.088
H
N
N
N
N
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S
796
13
0.43
206
N
N H
N
N
O
7
8
9
10
11
12
13
14
15
16
17
18
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20
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22
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30
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43
44
45
46
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49
50
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Table 3. biochemical and TPSA data of analogs of 4a
R
N
S
NH
N
O
R1
O
R2
#
R
R1
R2
TPSA
224
Biochem.
IC50
pKa
4.0
(M)
4a
0.007
O
O
N
H
H
H
H
N
N
N
N
N
N
N
H
O
O
9
CH3
194
0.013
3.7
N
N
N
N
N
O
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CH3
CH3
CH3
CH3
CH3
H
140
21
ND
8.9
ND
ND
3.6
O
N
H
H
H
H
H
N
N
N
N
N
O
O
182
1
N
N
N
N
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
30
31
32
33
34
35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
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57
58
59
60
O
O
171
184
177
2.9
3.9
0.17
N
N
N
N
N
O
O
N
N
N
H
N
O
O
O
O
N
O
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