Journal of the American Chemical Society
Article
previously reported structure of BCA (6SKV), employing
free energies of binding of these macrocycles and BCA from the
6
2
Autodock Vina. In all but one docked binding pose, negatively
MD simulation, ΔGMM‑GBSA, correlated reasonably with the
63
charged benzenesulfonamide moieties were in the substrate
cleft, with <4 Å distances between S atoms of benzenesulfona-
experimental K values obtained with ITC studies (Figure 5F,
d
Table S5). The hydrophobic interactions observed in the pose
from MD calculations were consistent with experimentally
2
+
mide and Zn (Table S4). Next, we employed MD simulation
for 1 μs to examine the docked binding pose of BCA and sa-
SFCDTYC macrocycle complex. For one of the regio-isomers,
the macrocycle reoriented from the docked pose to a new pose
within the first 20 ns (Figure 5A−C, Figure S52A and SI video
observed 50-fold increase in K value upon Phe → Ala and Tyr
d
→ Ala substitutions in both macrocycles (Figure 4E). Changing
the Asp to an Ala in both the macrocycles also caused over an
100-fold increase in the Kd values. These experimental
observations were corroborated by MD simulations that yielded
the hydrogen bonding and ionic interactions between BCA and
Asp in the macrocycles (Figure 5F).
1
), while the other isomer took longer to reach a stable
conformation. Therefore, we moved forward with the isomer
that reoriented within a shorter time interval into a low energy
conformation. After reorientation, the macrocycle established
several favorable contacts. The S1 forms hydrogen bonds with
E69 (BCA) with its N-terminus and −OH groups for 68% of 1
μs trajectory (Figure S52A,B). The reorientation enabled a
formation of the salt bridge between D4 and R58 (BCA), and
formation of nonpolar contacts between F2 and Y6 to BCA
CONCLUSION
■
The broad substrate scope of Knorr pyrazole synthesis makes it
an attractive strategy for the diversification of macrocycles with
built in 1,3-diketones using a large range of commercially
available alkyl and aryl-hydrazine warheads. So far, we identified
only two substrates with suboptimal reactivity: reaction between
(
Figure 5B). Overall, the macrocycle reorientation process is
characterized by a decrease in its free energy of binding (Figure
D), with ΔG changing from the initial value of −7 kcal/
1
,3-diketone-macrocycle and N-acyl hydrazines and benzene-
5
MM‑GBSA
sulfonohydrazide in water was slow or incomplete. Even when
65
mol to a more favorable value of −20 kcal/mol within first 50 ns.
The overall peptide orientation in the binding pose shown in
Figure 5C remained similar throughout a 1 μs long trajectory,
with rearrangements of the side chains contributing to further
formed, N-acyl 1,2-pyrazoles can be readily cleaved by thiols
and other biological nucleophiles; such cleavage makes N-acyl
,2-pyrazoles not suitable for the stable grafting of functionalities
1
onto peptides. Further substrate scope profiling may uncover
further limitations; however, we foresee few problems in
reactions that employ simple alkyl and aryl hydrazine fragments.
A limitation of the Knorr-pyrazole ligation is formation of two
regioisomers of pyrazole: we observed the formation of a 1:1
mixture of two isomers in many LC-traces; interestingly, this
ratio was skewed toward one isomer in reaction with
perfluorophenyl hydrazine (Figure S10). The reason for this
regio-induction is presently not clear. The formation of isomers
is a trait of many contemporary modifications of polypeptides:
for example, reactions between Cys and maleimide yield
mixtures of stereoisomers. However, this reaction is used in
the manufacturing of FDA-approved antibody-drug conjugates
(ADC) such as Ketruda, Trodelvy, Enhertu, Polivy, Adcetris,
decreases and fluctuations in the value of ΔG
(Figure
MM‑GBSA
5
E).
We compared the MD simulations of the sa-SFCDTYC:BCA
complex to the conformational ensemble of free sa-SFCDTYC
macrocycles in aqueous solution. Within first several nano-
seconds, the peptide fragment changed from its initial helical
conformation to a random coil conformation (Figure S53A) that
fluctuated over time. We monitored the structure of the peptide
backbone over time with the root-mean-square deviations
(
RMSD) measured with respect to a reference state with the
helical peptide backbone (Figure S53B,D). The RMSD
increased within nanoseconds, as the peptide backbone
underwent stepwise conformational changes (certain conforma-
tions are preserved for tens of nanoseconds). The control
SFCDTYC macrocycle with a disulfide bond between C3 and
C7 amino acids also transitioned from helical conformation to
the random coil within nanoseconds. However, its RMSD plot,
evaluated with respect to the helical peptide backbone
conformation, fluctuated without stepwise conformational
changes observed with pyrazole formation, indicating that the
pyrazole modification increases the rigidity of the backbone of
sa-SFCDTYC compared to the disulfide peptide (Figure
S53C,D).
34,66,67
and Padcev. A ligation to dehydroalanine on polypeptides
yields two diastereomers and has been successfully translated to
33
68
the manufacture of ADCs. Pictet-Spengler and Hydrazino-
iso-Pictet-Spengler (HIPS) reactions that yield diastereomeric
linkages are employed in the manufacture of TRPH-222 ADC,
which is currently in a Phase 1 clinical trial. Many reagents for
the modification of protein, via strain-promoted cycloadditions
and inverse demand Diels−Alder reactions, form isomeric
6
9
linkages. Other bioorthogonal ligations of aldehydes to
4
5
44
hydrazines, oximes, 2-amino benzamidoximes, and Wittig
46
To juxtapose the results of the MD simulations of
BCA:macrocycle complexes and experimental observations,
several macrocycles were prepared in the binding pose of Figure
ylides form E and Z products. In mRNA- and phage-displayed
7,34
libraries, reactions that yield a mixture of stereo
or
have been employed as well. In such mixed-
isomer libraries, activity can be attributed to one synthetic
7
,70,71
regioisomers
5
B and examined in short MD simulations. The examined
7
,70,71
macrocycle sequences were SFCDTYC, SICFDYC, and
SICQSYC, and single alanine mutants of SFCDTYC sequence
in positions 2, 4, 5, and 6. The systems with the macrocycles
were prepared by extracting a relaxed binding pose from MD
simulations of BCA and sa-SFCDTYC and mutating the amino
acids to the desired sequence. Extended MD simulation of the
complexes was critical because we did not observe any
correlation between docking scores produced by Autodock
isomer postdiscovery.
Formation of two regioisomers,
thus, is not an impediment to a GE-discovery process: In this
report, the macrocyclic peptides with phenyl sulfonamide
fragments were discovered and synthesized postdiscovery as
mixtures of two regioisomers. It is likely that one isomer has
higher activity than the other, but we did not attempt to measure
the activities of separated isomers.
In this report, important advances were made to late stage
modifications of GE-macrocycles described in the pioneering
Vina and K values for macrocycles (Figure S54A), in line with
d
72
73
the acknowledged challenges in docking of static peptide
report of GE-FBD by Roberts and Dwyer et al. Simplicity
6
4
structures. In contrast to static docking scores, the calculated
and robustness of chemical modification make it simple to adapt
5
503
J. Am. Chem. Soc. 2021, 143, 5497−5507
Ekanayake, Arunika I.
