An in situ combinatorial methodology to synthesize and screen chemical probes

Article abstract of DOI:10.1039/c8cc06991c

Chemical probes that label proteins of interest in the context of complex biological samples are useful research tools. The reactive group that forms the covalent bond with the target protein has a large effect on the selectivity and selecting the appropriate group determines the success of a probe. We here report the development of a combinatorial methodology based on imine chemistry that enables straightforward in situ synthesis and screening of different reactive groups and thereby simplifies identification of probe leads. Using our methodology, we found chemical probes targeting BirA and chloramphenicol acetyl transferase, two proteins associated with antibacterial activity and resistance.

Full text of DOI:10.1039/c8cc06991c

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An in situ combinatorial methodology to
synthesize and screen chemical probes†
Cite this:DOI: 10.1039/c8cc06991c
Antonie J. van der Zouwen, ‡ Jonas Lohse, ‡ Lianne H. E. Wieske,
Received 28th August 2018,
Accepted 4th December 2018
Katharina F. Hohmann, Ramon van der Vlag
and Martin D. Witte
*
DOI: 10.1039/c8cc06991c
rsc.li/chemcomm
Chemical probes that label proteins of interest in the context of (bio)chemical analysis of these probes are time consuming and
complex biological samples are useful research tools. The reactive form a serious bottleneck in probe development. Combinatorial
group that forms the covalent bond with the target protein has a approaches and fragment-based screening have been explored to
large effect on the selectivity and selecting the appropriate group speed-up synthesis of chemical probes.6 However, in the former
determines the success of a probe. We here report the development approach the individual probes still need to be purified and
of a combinatorial methodology based on imine chemistry that analyzed, while the latter is more suitable for the serendipitous
enables straightforward in situ synthesis and screening of different identification of leads. The field of drug discovery has tackled
reactive groups and thereby simplifies identification of probe leads. similar challenges by developing combinatorial chemistries that
Using our methodology, we found chemical probes targeting BirA combine preparing and screening of compound libraries in one
and chloramphenicol acetyl transferase, two proteins associated simple operation.7 Imine chemistry has proven to be suitable for
with antibacterial activity and resistance.
this purpose, since it allows straightforward linking of fragments
by mixing reaction partners, with only water as the by-product.
Inspired by these in situ approaches and encouraged by earlier
Chemical probes that covalently introduce a bioorthogonal
handle or reporter group onto a protein of interest (POI) have reports of imine-containing probes,8 we set out to develop a similar
become fundamental research tools.1 They enable functional combinatorial strategy for the synthesis and screening of chemical
studies in biologically relevant settings and have been applied probes. We here report the first proof-of-concept study of how
to identify the targets of biologically active compounds.2 To acylhydrazone and oxime formation can be used to tether ligands
prepare chemical probes for a POI, often ligands or inhibitors and inhibitors to reactive groups in situ (Fig. 1). With this method,
are functionalized with a reactive group that covalently modifies we functionalized several a-nucleophiles with commonly used
an amino acid within the proximity of the binding pocket.3 reactive groups. The in situ generated probes label their targets
A variety of reactive groups has been employed for this purpose bovine carbonic anhydrase II (CA-II), streptavidin (Strp), avidin (AVI)
and selecting the appropriate ligand-reactive group combination and BirA. The selectivity and specificity of a particular ligand-reactive
is key to the success of the probe.4 Not only may the reactive group combination is easily assessed by gel-based screening of
group alter the biological activity, but it also determines to the reaction mixtures. We believe that our modular approach can
a large extent if proteins get labeled at all.5 Probes will only dramatically simplify the identification of new leads for probes.
label their target efficiently if the reactive group is positioned
correctly.4k Molecular modeling can address this issue in part,
but often knowledge of the targets is lacking and a major
challenge therefore remains in finding the optimal ligand-
reactive group combination. As a consequence, sets of probes
are synthesized that vary in the position and the chemotype
of the reactive group.4e,k The synthesis, purification and
Fig. 1 Schematic representation of the combinatorial probe synthesis
approach. a-Nucleophile ligands (L) that bind to the protein of interest
(POI) are mixed with carbonyl-containing reactive groups (R). The resulting
probes modify the POI with a TAG (bioorthogonal handle or fluorophore).
By comparing the TAG signal of the targeted probe with a non-targeted
control (red cross), the efficiency of the probe can be determined.
Chemical Biology II, Stratingh Institute for Chemistry, University of Groningen,
9747AG Groningen, The Netherlands. E-mail: M.D.Witte@rug.nl
† Electronic supplementary information (ESI) available: Supplementary figures
and tables, experimental procedures and compound characterization. See DOI:
10.1039/c8cc06991c
‡ These authors contributed equally.
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To study if combinatorial chemistry can be employed to screen For the proof-of-concept studies, we followed previously reported
ligand-reactive group combinations, we decided to synthesize labeling protocols.4a,b,k We added the resulting probes to the protein
aldehyde and ketone derivatives of acyl imidazoles,4a diazotransfer mixture in HEPES buffer (pH 7.4) and incubated for 2 hours.
reagents,4b Michael acceptors,4c alkylating agents,4d sulfonyl Subsequently, the bioorthogonal handle introduced by the probe
fluorides,4e–g sulfonate esters,4h Woodward’s reagent K4i and was reacted with a fluorophore reporter group using either copper
photocrosslinkers4j–l (Fig. 2). These reactive groups are commonly catalyzed or strain-promoted alkyne–azide cycloaddition reactions.
used in chemical probes and target a broad panel of amino acids. The in-gel fluorescence revealed that incubating the protein mixture
The required aldehydes R1–R9 were readily prepared from com- with acethydrazide L3-based reagents resulted in minimal labeling
mercially available building blocks in no more than four steps. of the proteins (Fig. 2D). The fluorescence intensity for Strp or CA-II
Considering that R1–R5 could decompose in the presence of increased considerably when we reacted the aldehydes R1–R3 either
a-nucleophiles, we first evaluated if in situ synthesis of the probes with biotin ligands L1 or L4, or benzenesulfonamide hydrazide
was feasible. 1H-NMR (Fig. S1, ESI†) and UPLC-MS (Fig. S2, ESI†) L2, respectively. At the same time the signal for OVA was left
experiments with acethydrazide and methoxyamine revealed that unchanged, which underlines that the target proteins only get
R1–R3 and R5 can be linked to ligands using imine chemistry. labeled efficiently in the presence of the appropriate ligand.
However, R4 is not suitable, as it readily decomposed, even in the Comparing the fluorescence intensity of the labeled proteins
absence of an a-nucleophile.
indicated that the in situ formed probes containing R1 or R2
Next, we assessed if the in situ formed probes could be used for label Strp and CA-II with a similar efficiency. The labeling with
protein labeling experiments on a mixture of Strp, CA-II and R3 is considerably weaker, even when the reaction time was
ovalbumin (OVA). We selected this model system, because Strp extended to 6 h. As was expected from the NMR studies, we only
and CA-II are readily modified with chemical probes that contain detected a fluorescent signal for R4 when functionalized with
a biotin or benzenesulfonamide targeting group, respectively.4h,9 The biotin alkoxyamine L4 (Fig. S3, ESI†).
included OVA serves as a first control for protein specificity. By
We then performed a similar experiment with R5–R9. These
reacting R1–R4 with an equimolar amount of biotin hydrazide L1, reagents lack an azide or alkyne handle. To detect the labeled
benzenesulfonamide hydrazide L2 or biotin alkoxyamine L4 over- proteins, we performed a transimination reaction with an excess of
night, we prepared a library of probes that target Strp and CA-II. We a hydroxylamine fluorophore as has been reported previously.8a
also prepared the corresponding non-targeted reagents from acet- Unfortunately, inefficient exchange of the imine hampered reliable
hydrazide L3 as a second control for ligand-independent labeling. detection of protein–probe adducts (Fig. S3, ESI†). We therefore
Fig. 2 Development and screening of the imine chemical probes. (A) Carbonyl-containing reactive groups. (B) Biotin and benzenesulfonamide
a-nucleophile ligands. (C) Example of an in situ generated chemical probe. (D) Probes prepared from ligands L1–L4 and reagents R1–R3 modify their
respective targets Strp and CA-II. (E and F) Incorporation of an azido reporter group allows labeling of Strp (E) and avidin (F) using probes prepared with
ligands L5–L6 and reagents R5–R9. The DMSO lanes are representative examples of background fluorescence.
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evaluated the suitability of R5–R9 for in situ probe formation on
Strp and avidin using azide-functionalized ligands L5 and L6.
These biotin-binding proteins only got labeled efficiently when
R5–R9 were bound to biotin ligand L5. Besides the ligand, also the
reactive group had a large effect on the outcome of the experiment,
which underscores the importance of screening different reactive
groups. Sulfonyl fluoride L5R5 labeled both target proteins
strongly, while aryl fluoride L5R7 modified neither of them
(Fig. 2 and Fig. S3, ESI†). The remaining probes did label Strp,
but labeled avidin only very weakly, despite the structural similarity
between these proteins.
These initial studies demonstrated that in situ probe for-
mation with R1–R3 and R5–R9 is feasible and that incomplete
probe formation does not cause off-target labeling. This was
further confirmed by extending the incubation time (Fig. S4–S6,
ESI†) and by lowering the concentration of the target protein,
while keeping the OVA and probe concentrations constant
(Fig. S7, ESI†). Neither resulted in increased labeling of the
Fig. 3 Profiling studies in cell lysate of BirA-overexpressing E. coli.
(A) In-gel fluorescence using BODIPY-azide or BODIPY-alkyne as read-out.
The CBB lane is a representative example of the total protein content in the
cell lysate. (B) Western blotting using streptavidin–horseradish peroxidase
as read-out. CBB: Coomassie Brilliant Blue, BCCP: biotin carboxy carrier
protein. For full gel images see Fig. S9 (ESI†).
control protein. Even when using multiple probes in a single probes, labeling of BirA reached a plateau after 2–4 hours
sample, we observed selective labeling of the targeted proteins (Fig. S11, ESI†). The labeling efficiency also demonstrated to
(Fig. S8, ESI†), suggesting that our method may be suitable to be concentration dependent. At low concentrations, all probes
screen multiple reactive groups in the same sample.
showed weak labeling of BirA. The signal intensity improved
To demonstrate that our modular probe synthesis strategy drastically when we increased the probe concentration.
can be extended to other proteins, we applied it on BirA from However, only L1R5, L4R5, L5R7 and L1R8 labeled cleanly at
E. coli. This essential biotin ligase has emerged as a target for higher concentrations (Fig. S12, ESI†). Of these probes, we
the development of antibacterial agents10 and we recently decided to determine the detection limit of L1R5 and L5R7.
showed that a biotin-diazotransfer reagent modifies purified Even though as little as 10 ng of BirA (275 fmol) can be
BirA.11 Unfortunately, this reagent did not label BirA efficiently visualized in E. coli lysate with these probes (Fig. S13, ESI†),
in more complex samples. To identify new probe leads, we BirA could not be detected in native lysates, presumably due
derivatized biotin ligands L1, L4 and L5 with reactive groups to the low copy number of BirA per cell (Fig. S14, ESI†).12
R1–R3 and R5–R9 and incubated cell lysate of BirA-overexpressing Nonetheless, these leads form a good starting point for the
E. coli with 22.2 mM of the resulting probes for 2 hours. Analysis of further development of more sensitive probes.
the labeling reaction by fluorescence scanning (for the probes that
Finally, we focused our attention on the off-target of L5R5.
contain a bioorthogonal handle) or by western blotting (for the Labeling of this protein is concentration- and time-dependent
probes that attach biotin covalently to their targets) showed that and could be abolished by heat-denaturing the protein (Fig. 4A,
acyl imidazoles L1R2 and L4R2, sulfonyl fluorides L1R5 and L4R5, B and Fig. S15, ESI†). Chemoproteomic analysis of the labeled
alkylating agent L5R7 and photocrosslinker L1R8, L4R8 and L5R8 protein identified chloramphenicol acetyl transferase 3 (CAT)
label BirA rather selectively (Fig. 3 and Fig. S9, ESI†). Interestingly, as the major hit (Fig. S15 and Table S2, ESI†). The codon-
biotin sulfonyl fluoride L5R5 labeled, besides BirA, an additional optimized E. coli strain that we used for the profiling studies
unidentified protein of approximately 25 kDa. Control probe L6R5 carries a pACYC-based plasmid that codes for the expression of
and biotin-based probes L1R5 and L4R5 labeled this off-target this enzyme. Competition experiments with CAT substrate
only weakly, suggesting that labeling is ligand-dependent. The chloramphenicol or known inhibitor fusidic acid confirmed
other probes either did not label BirA or showed the same that L5R5 indeed labeled CAT (Fig. 4C). Both compounds
labeling intensity as the control probes. Again, the reactive diminished the signal in a concentration-dependent fashion,
group preferences slightly differ from the other biotin-binding which suggests that L5R5 binds in the same binding pocket.
proteins. Molecular docking of the probes in the X-ray crystal A recent study demonstrated that CAT also can be labeled with
structure of BirA (PDB ID: 4WF2) provided some insight why pentafluoroaryl reactive groups similar to R7,13 but in our case
acyl imidazole R2, sulfonyl fluoride R5 and alkylating agent R7 we only observed good labeling when using sulfonyl fluoride
are preferred. Several lysine residues are suitably positioned L5R5. Interestingly, sulfonyl fluoride probes based on L1 and
near the entrance of the active site and these might react L4 did not. Furthermore, competition with biotin did not block
with these reactive groups (Fig. S10, ESI†). Moreover, the labeling of CAT, indicating that the azidolysine is important for
pentafluoroaryl group of L5R7 is in close proximity of a the targeting of CAT. To validate this hypothesis and to target
methionine residue that might be alkylated. This residue seems CAT selectively over BirA, we synthesized benzoyl- and butyryl-
to be out of reach of the other pentafluoroaryl probes.
azidolysine ligands L7 and L8. Furthermore, we also prepared
Next, we analyzed the leads of the screen further by varying azido-D-serine hydrazide L9 as a chloramphenicol mimic.
the incubation time and the amount of probe. For most of the Of the newly designed probes, only L7R5 and L8R5 labeled CAT
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grant number 723.015.004 (M. D. W., A. J. V. D. Z.); 723.014.008
(R. V. D. V.) and the Marie Sklowdoska Curie-CIG (CIG PCIG14-
GA-2013-631057 to M. D. W.) are acknowledged for funding.
Conflicts of interest
There are no conflicts of interest to declare.
References
1 (a) M. J. Niphakis and B. F. Cravatt, Annu. Rev. Biochem., 2014, 83,
341–377; (b) R. Kubota and I. Hamachi, Chem. Soc. Rev., 2015, 44,
4454–4471; (c) M. Garland, J. J. Yim and M. Bogyo, Cell Chem. Biol.,
2016, 23, 122–136.
2 S. Pan, H. Zhang, C. Wang, S. C. L. Yao and S. Q. Yao, Nat. Prod. Rep.,
2016, 33, 612–620.
3 (a) M. J. C. Long, J. R. Poganik and Y. Aye, J. Am. Chem. Soc., 2016,
138, 3610–3622; (b) K. Amaike, T. Tamura and I. Hamachi, Chem.
Commun., 2017, 53, 11972–11983.
4 (a) S. Fujishima, R. Yasui, T. Miki, A. Ojida and I. Hamachi, J. Am.
Chem. Soc., 2012, 134, 3961–3964; (b) J. Lohse, L. J. Y. M. Swier,
Fig. 4 (A) Time dependency of CAT labeling by L5R5 in the presence of
10 mM biotin. (B) Concentration dependency of BirA and CAT labeling by L5R5.
(C) Competition experiments with L5R5. Competitors: (À) no competitor,
(1) chloramphenicol (10 nmol), (2) fusidic acid (10 nmol), (3) biotin (50 nmol).
(D) Labeling of CAT and BirA with L7R5, L8R5, L9R5 and L5R5. (E) Competition
experiment between methyl 4-nitrobenzenesulfonate (MNBS) and L5R5.
(F) Structures of L7–L9. All samples in A, C–E contain 200 pmol probe, except
the mock, which does not contain probe. Typical sample size: 9 mL.
´
R. C. Oudshoorn, G. Medard, B. Kuster, D. J. Slotboom and M. D.
Witte, Bioconjugate Chem., 2017, 28, 913–917; (c) J. Ge, C.-J. Zhang, L. Li,
L. M. Chong, X. Wu, P. Hao, S. K. Sze and S. Q. Yao, ACS Chem. Biol.,
2013, 8, 2577–2585; (d) L. M. McGregor, M. L. Jenkins, C. Kerwin,
J. E. Burke and K. M. Shokat, Biochemistry, 2017, 56, 3178–3183;
(e) A. Narayanan and L. H. Jones, Chem. Sci., 2015, 6, 2650–2659;
( f ) Q. Zhao, X. Ouyang, X. Wan, K. S. Gajiwala, J. C. Kath, L. H. Jones,
A. L. Burlingame and J. Taunton, J. Am. Chem. Soc., 2017, 139, 680–685;
(g) O. Fadeyi, M. D. Parikh, M. Z. Chen, R. E. Kyne, A. P. Taylor,
I. O’Doherty, S. E. Kaiser, S. Barbas, S. Niessen, M. Shi, S. L. Weinrich,
J. C. Kath, L. H. Jones and R. P. Robinson, ChemBioChem, 2016, 17,
1925–1930; (h) S. Tsukiji, M. Miyagawa, Y. Takaoka, T. Tamura and
(Fig. 4D and Fig. S15, ESI†). As expected, replacing the biotin for a
benzoyl or butyryl group leads to reduced BirA labeling. Docking of
the probes in the X-ray crystal structure of CAT bound to fusidic
acid (PDB ID: 1Q23) showed that they may adopt a pose in which
the sulfonyl fluoride is in proximity to catalytic histidine residue
193 (Fig. S16, ESI†). Alkylation of this histidine with methyl
4-nitrobenzenesulfonate14 inhibited labeling of CAT, indicating
that this residue might be modified by our probes. However, several
of the sulfonyl fluorides that did not label CAT, including L9R5,
adopt a similar pose. While the docking helps to explain the
outcome, its predictive value is limited, once more underpinning
the importance of screening combinations.
In conclusion, we here describe a novel combinatorial approach to
prepare chemical probes. Readily available carbonyl-functionalized
protein labeling reagents can be reacted with a-nucleophiles to form
chemical probes that modify their targets selectively within a mixture
of proteins and cell lysates. The described methodology allows
straightforward screening of the selectivity of ligand-reactive group
combinations using reaction mixtures. The identification of novel
probes for CA-II and three different biotin-binding proteins, and the
serendipitous discovery of a probe for CAT underlines the power of
our methodology. Hydrazides are readily prepared from acids or
esters. Therefore, a large number of different scaffolds can be
converted into suitable ligands for our methodology. As a conse-
quence, our methodology should be applicable towards many differ-
ent protein targets. Thus, combined with the simplified development
of probes and the power of straightforward screening of reactive
groups, we envision that our modular probe synthesis methodology
will considerably simplify chemical probe development.
´
I. Hamachi, Nat. Chem. Biol., 2009, 5, 341–343; (i) P. Martın-Gago, E. K.
Fansa, M. Winzker, S. Murarka, P. Janning, C. Schultz-Fademrecht,
M. Baumann, A. Wittinghofer and H. Waldmann, Cell Chem. Biol., 2017,
24, 589–597; ( j) Z. Li, D. Wang, L. Li, S. Pan, Z. Na, C. Y. J. Tan and
S. Q. Yao, J. Am. Chem. Soc., 2014, 136, 9990–9998; (k) M. H. Wright,
C. Fetzer and S. A. Sieber, J. Am. Chem. Soc., 2017, 139, 6152–6159;
(l) H. Park, J. Y. Koo, Y. V. V. Srikanth, S. Oh, J. Lee, J. Park and
S. B. Park, Chem. Commun., 2016, 52, 5828–5831.
5 (a) A. Borodovsky, H. Ovaa, N. Kolli, T. Gan-Erdene, K. D. Wilkinson,
H. L. Ploegh and B. M. Kessler, Chem. Biol., 2002, 9, 1149–1159;
(b) S. M. Hacker, K. M. Backus, M. R. Lazear, S. Forli, B. E. Correia
and B. F. Cravatt, Nat. Chem., 2017, 9, 1181–1190.
6 (a) J. Ge, X. Cheng, L. P. Tan and S. Q. Yao, Chem. Commun., 2012,
48, 4453; (b) J. T. Bush, L. J. Walport, J. F. McGouran, I. K. H. Leung,
G. Berridge, S. S. van Berkel, A. Basak, B. M. Kessler and C. J. Schofield,
Chem. Sci., 2013, 4, 4115; (c) C. G. Parker, A. Galmozzi, Y. Wang,
B. E. Correia, K. Sasaki, C. M. Joslyn, A. S. Kim, C. L. Cavallaro,
R. M. Lawrence, S. R. Johnson, I. Narvaiza, E. Saez and B. F. Cravatt,
Cell, 2017, 168, 527–541.
7 M. Mondal and A. K. H. Hirsch, Chem. Soc. Rev., 2015, 44, 2455–2488.
8 (a) Y. Takaoka, H. Tsutsumi, N. Kasagi, E. Nakata and I. Hamachi,
J. Am. Chem. Soc., 2006, 128, 3273–3280; (b) H. Wakabayashi,
M. Miyagawa, Y. Koshi, Y. Takaoka, S. Tsukiji and I. Hamachi,
Chem. – Asian J., 2008, 3, 1134–1139.
9 L. N. Anderson, P. K. Koech, A. E. Plymale, E. V. Landorf, A. Konopka,
F. R. Collart, M. S. Lipton, M. F. Romine and A. T. Wright, ACS Chem.
Biol., 2016, 11, 345–354.
10 B. P. Duckworth, T. W. Geders, D. Tiwari, H. I. Boshoff, P. A. Sibbald,
C. E. Barry, D. Schnappinger, B. C. Finzel and C. C. Aldrich, Chem.
Biol., 2011, 18, 1432–1441.
11 J. Lohse, A. Schindl, N. Danda, C. P. Williams, K. Kramer, B. Kuster,
´
M. D. Witte and G. Medard, Chem. Commun., 2017, 53, 11929–11932.
12 (a) A. M. Femino, F. S. Fay, K. Fogarty, R. H. Singer, X. S. Xie, J. Hearn,
A. Emili and X. S. Xie, Science, 1998, 280, 585–590; (b) G.-W. Li,
D. Burkhardt, C. Gross and J. S. Weissman, Cell, 2014, 157, 624–635.
13 A. M. Embaby, S. Schoffelen, C. Kofoed, M. Meldal and F. Diness,
Angew. Chem., Int. Ed., 2018, 57, 8022–8026.
The authors thank N. Danda and C. P. Williams for produ-
cing lysates of BirA-overexpressing E. coli and A. K. H. Hirsch
for the useful discussions and support with docking. The
Netherlands Organisation for Scientific Research (NWO-VIDI) 14 A. Lewendon and W. V. Shaw, Biochem. J., 1993, 290, 15–19.
Chem. Commun.
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