Macrocyclic FKBP51 Ligands Define a Transient Binding Mode with Enhanced Selectivity

Article abstract of DOI:10.1002/anie.202017352

Subtype selectivity represents a challenge in many drug discovery campaigns. A typical example is the FK506 binding protein 51 (FKBP51), which has emerged as an attractive drug target. The most advanced FKBP51 ligands of the SAFit class are highly selective vs. FKBP52 but poorly discriminate against the homologs and off-targets FKBP12 and FKBP12.6. During a macrocyclization pilot study, we observed that many of these macrocyclic analogs have unanticipated and unprecedented preference for FKBP51 over FKBP12 and FKBP12.6. Structural studies revealed that these macrocycles bind with a new binding mode featuring a transient conformation, which is disfavored for the small FKBPs. Using a conformation-sensitive assay we show that this binding mode occurs in solution and is characteristic for this new class of compounds. The discovered macrocycles are non-immunosuppressive, engage FKBP51 in cells, and block the cellular effect of FKBP51 on IKKα. Our findings provide a new chemical scaffold for improved FKBP51 ligands and the structural basis for enhanced selectivity.

Full text of DOI:10.1002/anie.202017352

Angewandte
Research Articles
Chemie
How to cite:
International Edition: doi.org/10.1002/anie.202017352
German Edition: doi.org/10.1002/ange.202017352
Medicinal Chemistry
Macrocyclic FKBP51 Ligands Define a Transient Binding Mode with
Enhanced Selectivity
Andreas M. Voll, Christian Meyners, Martha C. Taubert, Thomas Bajaj, Tim Heymann,
Stephanie Merz, Anna Charalampidou, Jꢀrgen Kolos, Patrick L. Purder, Thomas M. Geiger,
Pablo Wessig, Nils C. Gassen, Andreas Bracher, and Felix Hausch*
Abstract: Subtype selectivity represents a challenge in many
drug discovery campaigns. A typical example is the FK506
binding protein 51 (FKBP51), which has emerged as an
attractive drug target. The most advanced FKBP51 ligands of
the SAFit class are highly selective vs. FKBP52 but poorly
discriminate against the homologs and off-targets FKBP12 and
FKBP12.6. During a macrocyclization pilot study, we ob-
served that many of these macrocyclic analogs have unantici-
pated and unprecedented preference for FKBP51 over
FKBP12 and FKBP12.6. Structural studies revealed that these
macrocycles bind with a new binding mode featuring a transient
conformation, which is disfavored for the small FKBPs. Using
a conformation-sensitive assay we show that this binding mode
occurs in solution and is characteristic for this new class of
compounds. The discovered macrocycles are non-immuno-
suppressive, engage FKBP51 in cells, and block the cellular
effect of FKBP51 on IKKa. Our findings provide a new
chemical scaffold for improved FKBP51 ligands and the
structural basis for enhanced selectivity.
functions. The larger homolog FKBP51[2] is a regulator of
glucocorticoid receptor (GR) signaling[3] and has emerged as
a potential target for depression,[4] obesity-induced diabetes[5]
and chronic pain.[6] In contrast, the homologous proteins
FKBP12, FKBP12.6 and FKBP52 are considered anti-targets
due to their important roles in cardiology, sexual develop-
ment and female infertility, emphasizing the need for selective
inhibition.[7]
The most advanced FKBP51 ligands are compounds of
the SAFit class (Selective Antagonists of FKBP51 by induced
fit),[8] which bind to a transient binding pocket unavailable to
FKBP52[1] and are up to 10000-fold selective for FKBP51
over FKBP52.[8,9] However, SAFit-like ligands still bind
FKBP12 and its isoform FKBP12.6 with substantial affinities.
These FKBPs are cofactors of the ryanodine receptor[10] and
play an important role in fine-tuning the excitability of
smooth or heart muscle. FKBP12 knockout or knockdown
lead to severe cardiac defects in mice,[11] underscoring the
importance of selectivity for FKBP51 over FKBP12/12.6 in
FKBP51-based therapies.
Introduction
Macrocyclization is a popular approach to improve drug-
like properties for compounds outside the rule-of-five
space[12] and is thought to be crucial for the unusually
beneficial properties of the clinically used natural products
FK506 (Figure 1B), Rapamycin and Cyclosporin.[13] Macro-
cyclization was key to enhance the affinities or physicochem-
ical properties of synthetic ligands for FKBP12[14] and cyclo-
philins.[15] In a pilot study on the macrocyclization of SAFit
analogs,[16] we surprisingly observed a rearrangement of the
FKBP51 binding pocket, which in turn allowed an unprece-
dented selectivity against the off-targets FKBP12 and
FKBP12.6.
Proteins often cluster in families with similar structure.
The discovery of selective ligands that can discriminate
between these close homologs remains a formidable chal-
lenge in chemical biology as well as in drug development.
Most proteins are flexible and differential dynamics have
been suggested as a way to distinguish between otherwise
very similar proteins.[1]
A typical example is the family of FK506-binding proteins
(FKBPs) that possess a highly conserved binding pocket
(Figure 1A) but have diverged to perform diverse biological
[*] A. M. Voll, Dr. C. Meyners, M. C. Taubert, T. Heymann, S. Merz,
A. Charalampidou, J. Kolos, P. L. Purder, T. M. Geiger,
Prof. Dr. F. Hausch
Dr. A. Bracher
Max-Planck-Institute of Biochemistry
Am Klopferspitz 18, 82152 Martinsried (Germany)
Department Chemistry and Biochemistry
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202017352.
Clemens-Schçpf-Institute, Technical University Darmstadt
Alarich-Weiss Strasse 4, 64287 Darmstadt (Germany)
E-mail: felix.hausch@tu-darmstadt.de
ꢂ 2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
T. Bajaj, Dr. N. C. Gassen
Research Group Neurohomeostasis
Department of Psychiatry and Psychotherapy
University of Bonn
Venusberg Campus 1, 53127, Bonn (Germany)
Prof. Dr. P. Wessig
Universitꢀt Potsdam, Institut fꢁr Chemie
Karl-Liebknecht-Strasse 24–25, 14476 Potsdam (Germany)
&&&&
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
These are not the final page numbers!

Angewandte
Research Articles
Chemie
Figure 1. A) Superimposition of FKBP12 (red, PDB-ID: 1FKJ), FKBP12.6 (blue, PDB-ID: 5HKG), FKBP51 (yellow, PDB-ID: 3O5R) and FKBP52
(magenta, PDB-ID: 4LAX), in complex with FK506 or Rapamycin (not shown for clarity). B) The chemical structure of the FKBP ligands FK506 and
SAFit1. The key interactions of SAFit1 with FKBP51 and the macrocyclization strategy are indicated. C) SAFit1-analog iFit4 in complex with the
FK1 domain of FKBP51 (PDB-ID: 4TW7) highlighting the key interactions with the amino acid residues I87, Y113 and F67 and the structural basis for
macrocyclization indicated by the black arrow.
Results and Discussion
separate both E and Z isomer (ratio in crude mixture = 89:11)
and for the larger macrocycle 8b we only observed and
isolated the E isomer. Unfortunately, none of these macro-
cycles showed detectable binding to FKBP51 in a fluorescence
polarization assay.[18] To introduce additional functionalities
into the linker, we further derivatized the E isomers of 8a/b
by Wacker oxidation, dihydroxylation or hydrogenation (9a–
g), which for 8a/b resulted in only one Wacker product (9b/f).
After dihydroxylation of the smaller macrocycle, we obtained
the diastereomers (9c/d) and an inseparable dihydroxylated
diastereomeric mixture (9g, dr= 1:1 by NMR). Gratifyingly,
the dihydroxylated derivative 9g of the larger macrocycle
bound to FKBP51 with a Ki of 1.2 mm, whereas for 9a–f no
Based on the structure–affinity relationship findings of
SAFit analogs[8,9] and the highly conserved binding mode
(Figure 1C), we chose to keep the pipecolate, the chalcone-
derived A/B rings, and cyclohexyl ring constant and to cyclize
between the latter two. The synthesis started from compound
1,[8] where the ketone was reduced by an asymmetric Noyori
catalyst to the chiral alcohol 2 and then coupled with allyl
bromide or linker 3 to the alcohols 4a/b (Scheme 1). After
coupling with Fmoc-S-pipecolate,[17] 5a/b were deprotected
and coupled with 6.[9b] The linear precursors 7a/b were
cyclized by RCM to yield 8a and b. For 8a we were able to
Scheme 1. First generation of SAFit-derived macrocycles. a) RuCl2[(S)-(DM-SEGPHOSꢃ)][(S)-DAIPEN], THF, 10 bar H2, KOtBu, rt; b) K2CO3, allyl
bromide or 3, MeCN, rt; c) DCC, DMAP, Fmoc-S-pipecolate, DCM; d) 20% 4-methylpiperidine in DMF; e) 6, HATU, HOAt, DIPEA, DMF;
f) Grubbs-2 cat., DCM; g) Wilkinson cat. or Pt/C, DCM/MeOH, 1 bar H2; h) PdCl2, p-benzoquinone, THF/H2O; i) OsO4, NMO, Ac/H2O.
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!

Angewandte
Research Articles
Chemie
Scheme 2. Second generation of SAFit-derived macrocycles. The steps e, f and g are optionally applied in case of N-methylation. a) 20% 4-
methylpiperidine in DMF, rt, 3ꢄ10 min; b) Fmoc-d-Chg-OH, HATU, HOAt, DIPEA, DMF; c) 5% 4-methylpiperidine in DMF, 08C, 3ꢄ5 min;
d) Fmoc-AA-OH, HATU, HOAt, DIPEA, DMF; e) NosCl, Collidine, DMF; f) PPh3, dry MeOH, DIAD, dry THF, rt, 3ꢄ10 min; g) 2-mercaptoethanol,
DBU, DMF, rt, 3ꢄ10 min; h) 20% HFIP in DCM; i) 1 mm in DMF, HATU, DIPEA.
Table 1: Affinities of the macrocycles with amino acid containing linker
determined by a competitive fluorescence polarization assay.
binding to FKBP51 could be detected. To our great surprise,
9g did not bind to FKBP12 or FKBP12.6.
Cmpd. Linker
FKBP51FK1
FKBP12
Ki [mm][a]
FKBP12.6
Therefore, we set out to investigate this finding in more
detail, resorting to amino acids to rapidly explore the effect of
the linker (Scheme 2). Using an Fmoc SPPS strategy, we
started from the immobilized SAFit1 precursor 10[19] to
introduce d-cyclohexyl glycine as the FKBP52-discriminating
moiety, followed by coupling with a variety of amino acids
yielding the immobilized intermediates. The deprotection of
11 had to be optimized to suppress diketopiperazine forma-
tion.[20] Prior to derivatization of the intermediates 12a an
optional N-methylation sequence was included to probe the
influence of the resulting amide groups (12a–c).[21] Finally, the
linear peptides 12a or 12c were cleaved from the resin and
cyclized by macrolactamization (13a–o).
SAFit1 No linker 0.004Æ0.001[b] 0.163Æ0.009[b] 0.019Æ0.002[b]
FK506 Figure 1A 0.104[c]
0.0006[c]
0.004[c]
13a
13b
13c
13d
2.30Æ0.05[d]
>80
>80
1.00
>80
>80
>80
>80
>80
>80
0.29Æ0.05[b]
0.40Æ0.05[b]
13e
13 f
0.40
>80
>80
>80
>80
1.30
All final compounds were screened for affinity towards
the FKBPs 12, 12.6, 51 and 52 in a competitive fluorescence
polarization assay (Table 1).[18,22]
13g
13h
3.10
0.37
>80
>80
>80
>80
Gratifyingly, most of the macrocycles with amino acid-
based linkers bound to FKBP51 in the low to submicromolar
range and as expected none to FKBP52 (not shown). The
glycine derivative 13a had an affinity of 2.3 mm, which
gradually increased with increasing substitution (13b (d-
Ala): 1.0 mm, 13c (Aib): 0.29 mm). For geminal cyclic amino
acids the affinity slightly decreased with size (13d: 0.40 mm,
13e: 0.40 mm, 13 f: 1.3 mm). N-methylation and N-cyclization
did not substantially affect affinity [13g (R2 = Me): 3.1 mm),
13h (Aib + R2 = Me), 13i (d-Pro): 0.8 mm].
In contrast, the l-Ala derivative 13j bound more weakly,
consistent with the substantially reduced affinity of the l-Pro
derivative 13k. Longer linkers such as b-Ala 13l and GABA
(no binding, not shown) displayed reduced affinity, which
could be compensated by appropriate rigidification as in 13m
(1.8 mm; other diastereomers were inactive, not shown).
Notably, none of the tested linear precursors bound to
FKBP51, underlining the significance of the macrocyclization.
Most importantly, however, none of the macrocycles did show
any affinity towards FKBP12 or FKBP12.6.
13i
13j
13k
13l
0.80
17
>80
>80
>80
>80
>80
>80
>80
>80
5.10
7.40
13m
1.80
>80
>80
[a] The value >80 mm indicates the highest measurable concentration
before compound precipitation; no binding to FKBP52FK1 was observed
for any of the tested compounds up to 80 mm. [b] Standard error from
three independent measurements. [c] Values derived from literature.[23]
[d] Error from two independent measurements.
affinity to FKBP51 (Kd = 45 Æ 7 nm) but poorly to FKBP12,
FKBP12.6 or FKBP52 (Figure 2B). The affinity of the tracer
was further confirmed in a FRET assay with a fluorescein-
labeled FKBP51FK1 domain (Kd = 80 Æ 10 nm; Figure S2).
To check if compounds of the new class of macrocycles
were able to engage FKBP51 inside cells, we performed
a NanoBRET assay using a transiently expressed FKPB51–
NLuc construct. The assay utilizes a fluorescent tracer for the
FKBP51–Nluc construct, which accepts the luminescent
energy to generate a BRET signal. If compounds engage
the FKBP–Nluc construct inside cells, the tracer is displaced,
reducing the BRET signal and allowing direct quantification
The affinity of 13a and d for FKBP51 was confirmed by
isothermal calorimetry (ITC), yielding an enthalpy-driven
Kd = 3.6 mm Æ 0.9 mm for 13a and Kd = 0.6 mm Æ 0.1 mm for
13d, respectively (Figure S1).
We also prepared the fluorescent analog 14 of the best
binding compound 13c (Figure 2A, synthesis see Scheme S1),
which bound in a fluorescence polarization assay with high
&&&&
www.angewandte.org
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
These are not the final page numbers!

Angewandte
Research Articles
Chemie
Figure 2. A) Structure of the TAMRA labelled tracer 14 [5/6-TAMRA regio isomer]. B) Binding of the TAMRA-tracer 14 to FKBPs 51, 52, 12 and
FKBP52FK1
12.6 determined by a fluorescence polarization assay (KdFKBP51FK1 =45Æ7 nm, KdFKBP12 =6Æ1.4 mm, KdFKBP12.6 =3.6Æ0.8 mm, Kd
>80 mm).
Error bars represent the standard deviation of three independent experiments. C) NanoBRET assay in HEK293T cells transiently expressing
FKBP51-NLuc. Data points represent means and standard deviations of three independent cellular assays per concentration. D) FK506, but not
13d, blocks the phorbol/ionomycin-induced IL8 secretion in Jurkat cells and 13d does not interfere with FK506-mediated immunosuppression.
E,F) Inhibition of IKKa phosphorylation by 13d and SAFit2 in murine microglia SIM-A9 cells. Bars below represent means and standard errors of
three and six independent cellular assays, respectively, quantified by Western blots (for full blots see Supporting Information). *: p<0.05, **:
p<0.01, ***: p<0.001.
of FKBP51–NLuc occupation. Representative macrocyclic
compounds 13c, 13e and 13h as well as SAFit1 and -2 all
dose-dependently competed with a fluorescent NanoBRET
tracer inside cells (Figure 2C). The lower potencies of the
macrocycles compared to SAFit1 and -2 are in line with the
lower affinities of the macrocycles.
FK506 works as an immunosuppressant by an FKBP12-
dependent gain-of-function mechanism. A cellular analysis
showed that compound 13d has neither immunostimulatory
nor immunosuppressive properties on its own (Figure 2D).
Importantly, unlike the pan-selective FKBP ligand
[4.3.1]16h[21] (Figure S6A) 13d also did not block the immu-
nosuppressive activity of FK506, in line with its selectivity
against FKBP12 (Figure 2D).
We next explored if the macrocyclic ligands could
interfere with the cellular functions of FKBP51. We therefore
treated SIM-A9 cells, which were recently discovered as
a SAFit-sensitive cellular model for stress-mediated secretory
autophagy.[24] Compound 13d (Figure 2E) as well as com-
pounds 13c, 13e, 13h and 13i (Figure S6B) all inhibited IKKa
phosphorylation, similar to SAFit1 and SAFit2[4f] (Figure 2F
and S6C). For compounds 13e, 13i, SAFit1 and SAFit2 a clear
dose-dependence was observed. For 13c, 13d and 13h, the
apparent maximal inhibition was already reached at the
lowest tested concentration of 1 mm, possibly reflecting the
higher affinities and/or improved cell permeability of 13c,
13d and 13h compared to 13e and 13i. Taken together, these
results show that the here discovered macrocycles can
penetrate human cells, intracellularly occupy FKBP51 and
interfere with its function.
To clarify the structural basis for this unprecedented
selectivity, we solved the cocrystal structures of 13a, 13d and
13h in complex with FKBP51 (Figure 3A and S7A/C; PDB-
ID: 7AOU, 7AOT, 7AWF).[25]
As intended, the interactions of the pipecolate, the A- and
B-rings, as well as the cyclohexyl group with FKBP51 were
completely conserved in comparison to previous FKBP51-
SAFit co-crystal structures (Figure 3A). This includes a dis-
placement of F67, which is responsible for the strong
selectivity vs. FKBP52 of SAFit-like ligands.[8,9] However,
the b3b strand, which contains F67 and which we and others
previously showed to display enhanced basal mobility,[1,26] was
substantially rearranged (Figure 3B and S7B/D). Strikingly,
we observed that the carbonyl group of 13a, 13d and 13h
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!

Angewandte
Research Articles
Chemie
Figure 3. A) Crystal structure of the FK1 domain of FKBP51 in complex with 13d (pink-colored sticks), key interactions with the residues I87, Y113
and Y57 indicated as orange broken line (distance annotated in ꢅ), PDB-ID: 7AOU. B) Crystal structure of the FK1 domain of FKBP51 (pale cyan)
in complex with 13d (pink-colored sticks) superimposed to the cocrystal structure of the SAFit1-analog iFit4 in complex with FKBP51FK1 (pale
green, PDB-ID: 4TW7, iFit4 has been omitted for clarity). The b3b strand is highlighted in cyan and green, respectively, and key residues Y57, D68
and H71 are shown as sticks. The key carbonyl of 13d displacing D68 is highlighted in magenta and the new hydrogen bond between Y57 and the
13d carbonyl is indicated as orange broken line (distance annotated in ꢅ). C) Side view of FKBP51 from cocrystal structures with 13d (pale cyan,
7AOU), with the SAFit analog iFit4 (pale green, 4TW7), and the conventional binding-mode ligand [4.3.1]-16h (pale magenta, 5OBK). The residues
F67, D68 and H71 are shown as pale sticks. K58, K60 and K65 as the attachment point for the environment-sensitive dye are highlighted as intense
colored sticks. D) Fluorescence life-time analysis of DBD-labeled FKBP51FK1K58C in the presence of the indicated ligands.
displaced D68 and replaced it as a hydrogen bond acceptor for
the e-hydroxy group of Y57. The rearrangement of the b3b
strand is stabilized by an inward flip of H71, which partially
replaces S70 and substitutes the former as a hydrogen bond
donor for the backbone carbonyl of Y57. A similar inward flip
of H71 has previously been observed for FKBP51 in complex
with Rapamycin and FRB (PDB-ID: 4DRH).[27] Intriguingly,
H71 is replaced in FKBP12 and FKBP12.6 by an arginine (R40
in FKBP12/12.6 numbering), which can be expected to be less
efficient in stabilizing the 13a-binding conformation, provid-
ing a molecular rationale for the discrimination vs. FKBP12/
12.6 observed for the macrocycles.
To clarify if the structural rearrangement of the b3b strand
was also stabilized in solution, we developed a set of
conformation-sensitive assays[28] that are responsive to alter-
ations of the b3b strand. Towards this end, we introduced
environment-responsive dyes selectively at positions 58, 60
and 65 in the b2 strand below the b3 strand or in the b2–b3b
loop (Figure 3C). Remarkably, all three sensors clearly
differentiated between ligands with canonical and a SAFit-
like binding mode (Figure 3D and S8A/B). When using the
K60C- and K65C-based sensors, compound 13d induced similar
changes in fluorescence lifetime as SAFit1, but with lower
potency in accordance with its lower affinity. However, the
K58C-based sensor clearly differentiated macrocycle 13d from
&&&&
www.angewandte.org
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
These are not the final page numbers!

Angewandte
Research Articles
Chemie
Smith, F. Holsboer, M. B. Muller, M. V. Schmidt, Neuropharma-
both SAFit1 as well as canonical FKBP ligands (Figure 3D).
This strongly suggests that the new peptide-based macro-
cycles stabilize a new conformation in solution that is
different from the known FK506-like or SAFit-like ligands.
cology 2012, 62, 332 – 339; d) S. Albu, C. P. Romanowski, M.
Letizia Curzi, V. Jakubcakova, C. Flachskamm, N. C. Gassen, J.
Hartmann, M. V. Schmidt, U. Schmidt, T. Rein, F. Holsboer, F.
Hausch, M. Paez-Pereda, M. Kimura, J. Sleep Res. 2014, 23, 176 –
185; e) J. Hartmann, K. V. Wagner, S. Gaali, A. Kirschner, C.
Kozany, G. Ruhter, N. Dedic, A. S. Hausl, L. Hoeijmakers, S.
Westerholz, C. Namendorf, T. Gerlach, M. Uhr, A. Chen, J. M.
Deussing, F. Holsboer, F. Hausch, M. V. Schmidt, J. Neurosci.
2015, 35, 9007 – 9016; f) A. S. Zannas, M. Jia, K. Hafner, J.
Baumert, T. Wiechmann, J. C. Pape, J. Arloth, M. Kodel, S.
Martinelli, M. Roitman, S. Roh, A. Haehle, R. T. Emeny, S.
Iurato, T. Carrillo-Roa, J. Lahti, K. Raikkonen, J. G. Eriksson,
A. J. Drake, M. Waldenberger, S. Wahl, S. Kunze, S. Lucae, B.
Bradley, C. Gieger, F. Hausch, A. K. Smith, K. J. Ressler, B.
Muller-Myhsok, K. H. Ladwig, T. Rein, N. C. Gassen, E. B.
Binder, Proc. Natl. Acad. Sci. USA 2019, 116, 11370 – 11379;
g) L. Konig, L. S. Kalinichenko, S. E. Huber, A. M. Voll, M.
Bauder, J. Kornhuber, F. Hausch, C. P. Muller, Addict. Biol.
2020, 25, e12758.
Conclusion
Macrocycles have repeatedly been discussed to impart
improved physicochemical properties. However, they have
rarely been associated with selectivity. Here we show that
macrocycles can also provide the basis for subtype selectivity.
In this particular case, the macrocycles provide the scaffold
for the proper positioning of the key carbonyl group that
displaces Asp68, which was not possible in the linear analogs.
With regards to FKBP51, our results provide the first ligands
that robustly discriminate between FKBP51 and FKBP12/
FKBP12.6 and provide a structural basis for the rational
design for further optimization regarding affinity, stability,
specificity and cellular activity.
[5] a) L. A. Stechschulte, B. Qiu, M. Warrier, T. D. Hinds, Jr., M.
Zhang, H. Gu, Y. Xu, S. S. Khuder, L. Russo, S. M. Najjar, B.
Lecka-Czernik, W. Yong, E. R. Sanchez, Endocrinology 2016,
157, 3888 – 3900; b) G. Balsevich, A. S. Hausl, C. W. Meyer, S.
Karamihalev, X. Feng, M. L. Pohlmann, C. Dournes, A. Uribe-
Marino, S. Santarelli, C. Labermaier, K. Hafner, T. Mao, M.
Breitsamer, M. Theodoropoulou, C. Namendorf, M. Uhr, M.
Paez-Pereda, G. Winter, F. Hausch, A. Chen, M. H. Tschop, T.
Rein, N. C. Gassen, M. V. Schmidt, Nat. Commun. 2017, 8, 1725.
[6] a) M. Maiarꢁ, K. K. Tochiki, M. B. Cox, L. V. Annan, C. G. Bell,
X. Feng, F. Hausch, S. M. Geranton, Sci. Transl. Med. 2016, 8,
325ra319; b) S. D. Linnstaedt, K. D. Riker, C. A. Rueckeis,
K. M. Kutchko, L. Lackey, K. R. McCarthy, Y. H. Tsai, J. S.
Parker, M. C. Kurz, P. L. Hendry, C. Lewandowski, E. Datner, C.
Pearson, B. OꢂNeil, R. Domeier, S. Kaushik, A. Laederach, S. A.
McLean, J. Neurosci. 2018, 38, 8407 – 8420; c) M. Maiarꢁ, O. B.
Morgan, T. Mao, M. Breitsamer, H. Bamber, M. Pohlmann,
M. V. Schmidt, G. Winter, F. Hausch, S. M. Geranton, Pain 2018,
159, 1224 – 1234.
Acknowledgements
This work was supported by the M4 Award 2015 (BIO-1601-
0003), the BMBF grant 51TaValP (16GW0290K), the DFG
grant (HA-5655-5/1) and the LOEWE cluster TRABITA,
and the Pioneer Fund (ENTEGA/TU Darmstadt). We thank
Andreas Rꢀder and Matthias Roth for preliminary experi-
ments and Prof. Franz-Josef Meyer-Almes for using the
fluorescence life time reader. Open access funding enabled
and organized by Projekt DEAL.
Conflict of interest
[7] J. C. Sivils, C. L. Storer, M. D. Galigniana, M. B. Cox, Curr.
Opin. Pharmacol. 2011, 11, 314 – 319.
[8] S. Gaali, A. Kirschner, S. Cuboni, J. Hartmann, C. Kozany, G.
Balsevich, C. Namendorf, P. Fernandez-Vizarra, C. Sippel, A. S.
Zannas, R. Draenert, E. B. Binder, O. F. Almeida, G. Ruhter, M.
Uhr, M. V. Schmidt, C. Touma, A. Bracher, F. Hausch, Nat.
Chem. Biol. 2015, 11, 33 – 37.
The authors declare no conflict of interest.
Keywords: FKBP51 · immunophilin · macrocycle ·
subtype selectivity · transient binding pocket
[9] a) S. Gaali, X. Feng, A. Hahle, C. Sippel, A. Bracher, F. Hausch,
J. Med. Chem. 2016, 59, 2410 – 2422; b) X. Feng, C. Sippel, A.
Bracher, F. Hausch, J. Med. Chem. 2015, 58, 7796 – 7806; c) X.
Feng, C. Sippel, F. H. Knaup, A. Bracher, S. Staibano, M. F.
Romano, F. Hausch, J. Med. Chem. 2020, 63, 231 – 240.
[10] a) Z. Yan, X. Bai, C. Yan, J. Wu, Z. Li, T. Xie, W. Peng, C. Yin, X.
Li, S. H. W. Scheres, Y. Shi, N. Yan, Nature 2015, 517, 50 – 55;
b) S. Dhindwal, J. Lobo, V. Cabra, D. J. Santiago, A. R. Nayak,
K. Dryden, M. Samso, Sci. Signaling 2017, 10, eaai8842;
c) T. W. E. Steele, M. Samso, J. Struct. Biol. 2019, 205, 180 – 188.
[11] a) W. Shou, B. Aghdasi, D. L. Armstrong, Q. Guo, S. Bao, M. J.
Charng, L. M. Mathews, M. D. Schneider, S. L. Hamilton, M. M.
Matzuk, Nature 1998, 391, 489 – 492; b) X. Sun, J. Wang, X. Yao,
W. Zheng, Y. Mao, T. Lan, L. Wang, Y. Sun, X. Zhang, Q. Zhao,
J. Zhao, R. P. Xiao, X. Zhang, G. Ji, Y. Rao, Cell Discovery 2019,
5, 10.
[1] P. K. A. Jagtap, S. Asami, C. Sippel, V. R. I. Kaila, F. Hausch, M.
Sattler, Angew. Chem. Int. Ed. 2019, 58, 9429 – 9433; Angew.
Chem. 2019, 131, 9529 – 9533.
[2] a) M. V. Schmidt, M. Paez-Pereda, F. Holsboer, F. Hausch,
ChemMedChem 2012, 7, 1351 – 1359; b) B. M. Dunyak, J. E.
Gestwicki, J. Med. Chem. 2016, 59, 9622 – 9644; c) J. M. Kolos,
A. M. Voll, M. Bauder, F. Hausch, Front. Pharmacol. 2018, 9,
1425.
[3] A. Hꢀhle, S. Merz, C. Meyners, F. Hausch, Biomolecules 2019, 9,
35.
[4] a) J. C. O’Leary III, S. Dharia, L. J. Blair, S. Brady, A. G.
Johnson, M. Peters, J. Cheung-Flynn, M. B. Cox, G. de Eraus-
quin, E. J. Weeber, U. K. Jinwal, C. A. Dickey, PLoS One 2011,
6, e24840; b) C. Touma, N. C. Gassen, L. Herrmann, J. Cheung-
Flynn, D. R. Bull, I. A. Ionescu, J. M. Heinzmann, A. Knapman,
A. Siebertz, A. M. Depping, J. Hartmann, F. Hausch, M. V.
Schmidt, F. Holsboer, M. Ising, M. B. Cox, U. Schmidt, T. Rein,
Biol. Psychiatry 2011, 70, 928 – 936; c) J. Hartmann, K. V.
Wagner, C. Liebl, S. H. Scharf, X. D. Wang, M. Wolf, F. Hausch,
T. Rein, U. Schmidt, C. Touma, J. Cheung-Flynn, M. B. Cox, D. F.
[12] a) F. Giordanetto, J. Kihlberg, J. Med. Chem. 2014, 57, 278 – 295;
b) A. K. Yudin, Chem. Sci. 2015, 6, 30 – 49; c) E. Marsault, M. L.
Peterson, J. Med. Chem. 2011, 54, 1961 – 2004.
[13] E. M. Driggers, S. P. Hale, J. Lee, N. K. Terrett, Nat. Rev. Drug
Discovery 2008, 7, 608 – 624.
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!

Angewandte
Research Articles
Chemie
[14] a) D. A. Holt, J. I. Luengo, D. S. Yamashita, H. J. Oh, A. L.
Konialian, H. K. Yen, L. W. Rozamus, M. Brandt, M. J. Bossard,
M. A. Levy, D. S. Eggleston, J. Liang, L. W. Schultz, T. J. Stout, J.
Clardy, J. Am. Chem. Soc. 1993, 115, 9925 – 9938; b) Z. Guo, S. Y.
Hong, J. Wang, S. Rehan, W. Liu, H. Peng, M. Das, W. Li, S. Bhat,
B. Peiffer, B. R. Ullman, C. M. Tse, Z. Tarmakova, C. Schiene-
Fischer, G. Fischer, I. Coe, V. O. Paavilainen, Z. Sun, J. O. Liu,
Nat. Chem. 2019, 11, 254 – 263.
[15] a) V. A. Steadman, S. B. Pettit, K. G. Poullennec, L. Lazarides,
A. J. Keats, D. K. Dean, S. J. Stanway, C. A. Austin, J. A.
Sanvoisin, G. M. Watt, H. G. Fliri, A. C. Liclican, D. Jin, M. H.
Wong, S. A. Leavitt, Y. J. Lee, Y. Tian, C. R. Frey, T. C. Appleby,
U. Schmitz, P. Jansa, R. L. Mackman, B. E. Schultz, J. Med.
Chem. 2017, 60, 1000 – 1017; b) R. L. Mackman, V. A. Steadman,
D. K. Dean, P. Jansa, K. G. Poullennec, T. Appleby, C. Austin,
C. A. Blakemore, R. Cai, C. Cannizzaro, G. Chin, J. C. Chiva,
N. A. Dunbar, H. Fliri, A. J. Highton, H. Hui, M. Ji, H. Jin, K.
Karki, A. J. Keats, L. Lazarides, Y. J. Lee, A. Liclican, M. Mish,
B. Murray, S. B. Pettit, P. Pyun, M. Sangi, R. Santos, J. Sanvoisin,
U. Schmitz, A. Schrier, D. Siegel, D. Sperandio, G. Stepan, Y.
Tian, G. M. Watt, H. Yang, B. E. Schultz, J. Med. Chem. 2018, 61,
9473 – 9499.
having a mass of 250 gmolÀ1 was confirmed via LCMS in the
deprotection solution. To prevent this, the reaction was per-
formed with 5% 4-methylpiperidine in DMF at 08C in 5 min.
This was only possible on solid phase and not in solution, due to
the fact that a longer reaction time also showed increased
amount of side product.
[21] J. Chatterjee, F. Rechenmacher, H. Kessler, Angew. Chem. Int.
Ed. 2013, 52, 254 – 269; Angew. Chem. 2013, 125, 268 – 283.
[22] S. Pomplun, C. Sippel, A. Hahle, D. Tay, K. Shima, A. Klages,
C. M. Unal, B. Riess, H. T. Toh, G. Hansen, H. S. Yoon, A.
Bracher, P. Preiser, J. Rupp, M. Steinert, F. Hausch, J. Med.
Chem. 2018, 61, 3660 – 3673.
[23] a) L. A. Banaszynski, C. W. Liu, T. J. Wandless, J. Am. Chem.
Soc. 2005, 127, 4715 – 4721; b) E. A. Blackburn, M. D. Walkin-
shaw, Curr. Opin. Pharmacol. 2011, 11, 365 – 371.
[24] S. Martinelli, E. A. Anderzhanova, S. Wiechmann, F. Dethloff,
K. Weckmann, T. Bajaj, J. Hartmann, K. Hafner, M. L.
Pçhlmann, L. Jollans, G. Maccarrone, F. Hausch, C. W. Turck,
A. Philipsen, M. V. Schmidt, B. Kuster, N. C. Gassen, bioRxiv
2020, 2020.2005.2013.090514.
[25] A. Bracher, C. Kozany, A. K. Thost, F. Hausch, Acta Crystallogr.
Sect. D 2011, 67, 549 – 559.
[16] M. Bauder, C. Meyners, P. L. Purder, S. Merz, W. O. Sugiarto,
A. M. Voll, T. Heymann, F. Hausch, J. Med. Chem. 2021, 64,
3320 – 3349.
[17] a) F. P. Jørgensen, D. Madsen, M. Meldal, J. V. Olsen, M.
Petersen, J. Granhoj, M. Bols, J. Med. Chem. 2019, 62, 5191 –
5216; b) F. P. Jørgensen, M. Bols, J. Org. Chem. 2018, 83, 6050 –
6055.
[26] a) S. M. Mustafi, D. M. LeMaster, G. Hernandez, Biochem. J.
2014, 461, 115 – 123; b) D. M. LeMaster, S. M. Mustafi, M.
Brecher, J. Zhang, A. Heroux, H. Li, G. Hernandez, J. Biol.
Chem. 2015, 290, 15746 – 15757.
[27] A. M. Marz, A. K. Fabian, C. Kozany, A. Bracher, F. Hausch,
Mol. Cell. Biol. 2013, 33, 1357 – 1367.
[28] R. Wawrzinek, J. Ziomkowska, J. Heuveling, M. Mertens, A.
Herrmann, E. Schneider, P. Wessig, Chemistry 2013, 19, 17349 –
17357.
[18] C. Kozany, A. Marz, C. Kress, F. Hausch, ChemBioChem 2009,
10, 1402 – 1410.
[19] R. Gopalakrishnan, C. Kozany, Y. Wang, S. Schneider, B.
Hoogeland, A. Bracher, F. Hausch, J. Med. Chem. 2012, 55,
4123 – 4131.
[20] The Fmoc deprotection under standard conditions (20%
piperidine in DMF, 15 min, r.t.) predominantly underwent a side
reaction in which the deprotected amine cleaved off the
piperidine and formed a diketopiperazine. The side product
Manuscript received: December 31, 2020
Revised manuscript received: March 14, 2021
Accepted manuscript online: April 11, 2021
Version of record online: && &&
,
&&&&
&&&&
www.angewandte.org
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
These are not the final page numbers!

Angewandte
Research Articles
Chemie
Research Articles
Medicinal Chemistry
Macrocyclic FKBP51 ligands reveal a new
transient binding pocket for the FK506-
binding protein 51 and enable the first
ligands that also discriminate against the
close homologs FKBP12 and FKBP12.6.
This macrocyclic motif represents a new
scaffold to develop drugs for depression,
obesity or chronic pain.
A. M. Voll, C. Meyners, M. C. Taubert,
T. Bajaj, T. Heymann, S. Merz,
A. Charalampidou, J. Kolos, P. L. Purder,
T. M. Geiger, P. Wessig, N. C. Gassen,
A. Bracher, F. Hausch*
&&&& — &&&&
Macrocyclic FKBP51 Ligands Define
a Transient Binding Mode with Enhanced
Selectivity
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!