Sulfur(VI) Fluoride Exchange (SuFEx)-Enabled High-Throughput Medicinal Chemistry

Article abstract of DOI:10.1021/jacs.9b13652

Optimization of small-molecule probes or drugs is a synthetically lengthy, challenging, and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstr

Full text of DOI:10.1021/jacs.9b13652

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Communication
SuFEx-enabled high-throughput medicinal chemistry
Seiya Kitamura, Qinheng Zheng, Jordan L. Woehl, Angelo Solania, Emily
Chen, Nicholas Dillon, Mitchell V. Hull, Miyako Kotaniguchi, John R. Cappiello,
Shinichi Kitamura, Victor Nizet, K. Barry Sharpless, and Dennis W. Wolan
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 01 Jun 2020
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SuFEx-enabled high-throughput medicinal chemistry
†
,††
‡,††
†
†
§
Seiya Kitamura , Qinheng Zheng , Jordan L. Woehl , Angelo Solania , Emily Chen , Nicholas Dil-
⊥
,¶
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∇
‡
∇
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lon , Mitchell V. Hull , Miyako Kotaniguchi , John R. Cappiello , Shinichi Kitamura , Victor Ni-
zet⊥ , K. Barry Sharpless and Dennis W. Wolan
,#,¶
‡*
†,ǁ,*
†
ǁ
‡
§
Department of Molecular Medicine, Department of Chemistry, California Institute for Biomedical Research, and
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.
Department of Pediatrics, Collaborative to Halt Antibiotic-Resistant Microbes (CHARM), Skaggs School of Pharmacy
⊥
#
¶
∇
and Pharmaceutical Sciences, UC San Diego, La Jolla, CA 92093, USA. Laboratory of Advanced Food Process Engineer-
ing, Osaka Prefecture University, 1-2, Gakuen-cho, Nakaku, Sakai, Osaka 599-8570, Japan.
Supporting Information Placeholder
tion (CuAAC) reaction has been used in proof-of-
concept studies on lead optimization, including the di-
rect evaluation of biological potency ; a recent break-
through on generating 1000-mer modular libraries of
organic azides from primary amines has greatly boosted
ABSTRACT: Optimization of small-molecule probes or
drugs is a synthetically lengthy, challenging and re-
source-intensive process. Lack of automation and reli-
ance on skilled medicinal chemists is cumbersome in
both academic and industrial settings. Here, we demon-
strate a high-throughput hit-to-lead process based on the
3
-8
9
its potential in drug discovery .
biocompatible SuFEx click chemistry.
throughput screening hit benzyl
thyl)carbamate (K = 8 µM) against a bacterial cysteine
protease SpeB was modified with a SuFExable imino-
sulfur oxydifluoride [RN=S(O)F ] motif, rapidly diversi-
A
high-
(cyanome-
i
2
fied into 460 analogs in overnight reactions, and the
products directly screened to yield drug-like inhibitors
with 480-fold higher potency (K = 18 nM). We showed
i
that the improved molecule is active in a bacteria-host
co-culture. Since this SuFEx linkage reaction succeeds
on picomole scale for direct screening, we anticipate our
methodology can accelerate the development of robust
biological probes and drug candidates.
Figure 1. Comparison of CuAAC- and SuFEx-based me-
dicinal chemistry campaigns. Lead molecules can be as-
sembled through an iminosulfur oxydifluoride hub (build-
ing block I, BB-I) and by reactions with a collection of
primary and secondary amines (BB-II) to generate a diver-
sified library.
The introduction of high-throughput screening (HTS)
robotics, liquid handler systems, and assay miniaturiza-
tion have revolutionized screening of bioactive mole-
cules. Relatively inexpensive HTS processes are now
routinely used in cell-based and in vitro assays against
biomedically relevant targets. Nevertheless, compound
optimization is typically necessary to improve target
specificity, potency, and stability. Lead optimization re-
lies heavily on medicinal chemists, and extensive time
and labor costs remain significant hurdles for probe and
drug development.
The sulfur(VI) fluoride exchange (SuFEx) represents
the most recent set of ideal click chemistry transfor-
10
mations . Specifically, aryl fluorosulfates (ArOSO
2
F)
) are readily
synthesized using two connective oxyfluoride gases,
sulfuryl fluoride (SO ) and thionyl tetrafluoride
and iminosulfur oxydifluorides (RN=S(O)F
2
2 2
F
Click chemistry has found broad applications in mate-
rials chemistry, chemical biology, and drug development
11
VI
4
(O=SF ), respectively . These two S −F motifs have
1
-2
been successfully used as connective linkers in polymer
since the concept was first introduced in 1999 . The
prototypic copper(I)-catalyzed azide-alkyne cycloaddi-
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Figure 2. SuFEx-enabled high-throughput medicinal chemistry. (a) SuFEx-enabled SAR from HTS hit 1 identified several
potent SpeB inhibitors. (b) Scatter plot of % inhibition of reaction crudes at 250 nM (derivatives of 3, ●) or 2 µM (deriva-
tives of 4, ●). (c) Correlation of SpeB inhibitory activity of reaction crudes in picomole and nanomole scale reaction.
synthesis and for construction of various functional
been explored in medicinal chemistry. Unlike the planar
1,4-disubstituted triazole formed in CuAAC, the sul-
fur(VI)-center projects its ligands outward into three-
dimensional space along the vertexes of a tetrahedron. In
1
2-14
molecules
2
. Sulfonyl fluoride (RSO F) and aryl
fluorosulfate moieties have been successfully introduced
into many bioactive molecules in chemical biology and
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drug discovery
, especially as covalently binding
addition, the S –N bonds formed in this embodiment of
1
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warheads . In addition, these moieties have been used
in protein research
to unite diverse modules using an O=SF
SuFEx reactivity are common in medicines; for exam-
ple, more than 150 sulfonamide drugs are available on
2
0-21
. However, the potential of SuFEx
hub has not
2
2
4
the market . Here, we present a one-step, overnight re-
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action, high-throughput hit-to-lead optimization process
based on iminosulfur oxydifluoride SuFEx click chemis-
try that can be performed on picomole scale.
The reaction products (see Supporting Information)
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were directly screened for SpeB inhibition with an es-
25-26
tablished kinetic fluorogenic substrate assay
. Scatter
plots of the screening results are shown in Figure 2b.
Notably, molecules derived from secondary amines
gives higher potency compared to those derived from
primary amines. All 460 amine structures and their cor-
responding SpeB inhibition are provided in Tables S4 &
S5. Additionally, the panel of amines alone (absence of 3
or 4 in the reactions), the reaction condition, and the
fluoride ion by-product were assessed for inhibition of
SpeB hydrolysis, with no appreciable effect on proteoly-
sis or the assay (Figures S1 & S2). Molecules selected
based on potency, lipophilicity, and molecular weight
were re-synthesized on milligram scale (Table S6), and
their potencies in the original screen were fully con-
firmed (Figure S3). Structures of representative SpeB
inhibitors with improved IC50 values and predicted
physicochemical properties of the molecules (i.e., LogP,
MW, number of hydrogen bond donors/acceptors) are
shown in Figures 2a and S4, respectively. Notably, no
correlation between SpeB inhibitor potency and the mo-
lecular properties is observed.
We rely on the SuFEx reaction between iminosulfur
oxydifluoride (RN=S(O)F ) cores and primary or sec-
2
ondary amines to create a focused library of compounds
united by sulfamide bonds (Figure 1). This series of ro-
bust and near perfect reactions was recently described
for bioconjugation and DNA-encoded library construc-
2
3
tion , and we posited that the biocompatible reaction
conditions would enable us to measure the activity of
products directly using in vitro enzyme assays to priori-
tize the molecules. Additional benefits with respect to
medicinal chemistry, include: 1) rapid and diverse com-
pound synthesis from chemistry’s most available and
useful connective functional groups (i.e., primary and
secondary amines); 2) three-dimensional substitutable
sulfur center hubs (Figure 1); 3) display of multiple hy-
drogen-bond donors/acceptors; 4) tunable lipophilicity;
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and 5) stability in aqueous buffers .
As a proof-of-concept, we started with a modest inhib-
itor (1, IC50 = 14 µM) of the cysteine protease SpeB, a
virulence factor secreted from the bacterial pathogen
Streptococcus pyogenes, previously identified in our
2
4-25
HTS campaign (Figure 2a)
. Although peptidic SpeB
2
6-27
inhibitors were reported, such as E-64
, potent small
molecule inhibitors have not been found against SpeB.
In preliminary SAR studies, introduction of an (S)-
benzyl moiety (2, Figure 2a) improved the potency to
2
5
1.8 µM. The SpeB:1 co-complex x-ray structure and
the initial SAR campaign (Table S1) suggested that ad-
ditional surface sites on SpeB were accessible for com-
pound optimization via extension of 2 from the meta
positions of both benzyl substituents. An iminosulfur
oxydifluoride SuFEx handle was therefore introduced at
the meta positions of each benzyl group in 2, generating
3 and 4, respectively. The installed and still reactive
SuFEx hubs in 3 and 4 were then coupled with a panel
of 230 amines to generate 460 analogs using
DMSO:PBS = 1:1 as solvent and overnight incubation at
37 °C. Representative reactions were monitored using
2
LC-CAD-MS are shown in Table S2 and Supplemen-
tary Data. It should be noted that the reactions between
iminosulfur oxydifluoride-containing molecules with the
amines showed improved rates and yield when PBS, pH
7.4 was added to DMSO (DMSO: PBS = 1:1, Table S3).
Primary amines and secondary, cyclic amines generated
the desired product in good yields, while acyclic sec-
ondary amines gave poorer conversion in our reaction
condition (Table S2). The reactions to generate SpeB
inhibitor analog libraries were performed at relatively
low concentrations (i.e., 50 µM compound 3 or 4 and
250 µM amine) and likely accounts for the lower prod-
uct yields than our previously reported reactions on the
Figure 3. X-ray structure of SpeB-5 (PDB ID 6UQD). (a) 5
yellow carbon) in complex with the SpeB active site
(
(green carbon) are shown as sticks (red oxygen, blue nitro-
gen, mustard sulfur, teal fluorine) with key interactions
highlighted. (b) Zoomed view of the potential hydrogen
bonds between SpeB and 5 near the catalytic Cys192 with
distances (Å). The absolute configuration of sulfur could
2
3
millimolar scale .
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Page 4 of 7
not be determined based on the electron density; thus, one
sulfur) of two configurations is depicted.
no similar drug effect of 7 occurred in the ΔSpeB mutant
strain (Figure 4b).
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(R
With significantly improved SpeB inhibitors in hand,
we also determined that miniaturization of the SuFEx
reaction was feasible using an Echo Acoustic liquid
2
8-29
handler
. A strong correlation in inhibitory potency
was observed between the picomole-scale (1536-well, 2
µL, 200 µM of iminosulfur oxydifluoride compound,
400 pmol) and nanomole-scale (96-well, 50 µL, 10
nmol) syntheses, thereby demonstrating the successful
miniaturization of the library construction via the crucial
sulfamide-forming linkage reaction between modules
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(Figures 2c & S5). These direct-screen, condensation
linked libraries thus promise to join those CuAAC tria-
9
zole fusion libraries . Importantly, unlike previously
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9
reported nanoscale medicinal chemistry attempts , our
sub-nanoscale SuFEx-based library synthesis does not
require specialized equipment, such as dry-boxed liquid
handlers and highly sensitive mass spectrometry for the
biological assay. This SuFEx-based format can be readi-
ly adapted in screening facilities with standard HTS ro-
botics and liquid handler systems.
Figure 4. Improved SpeB inhibitor 7 is drug-like and bio-
logically active in bacteria-neutrophil co-culture. (a) Physi-
cochemical properties and pharmacological parameters of
Enzyme kinetics established that 5 is a reversible,
competitive inhibitor with K = 18 ± 1 nM (Figures S6 &
i
4-35
36
7
. Solubility in PBS3 , caspase activity , microsomal
S7). Its binding affinity was further validated by differ-
ential scanning fluorometry (Figure S8). The x-ray crys-
tal structure of SpeB in complex with 5 sheds light on its
improved potency (Figures 3a & S9, Table S7). Interest-
ingly, 5 binds SpeB in a U-shaped conformation with an
3
7
stability and cellular toxicity were measured as described ,
LogP was predicted with ChemDraw Ultra 17.1. (b) The
inhibition of SpeB by 7 prevents S. pyogenes WT
GAS5448 from neutrophil-induced killing; however, no
effect is observed on the DSpeB strain. Vehicle (□), 7 (20
µM (■) or 40 µM (■)). Statistical analysis was performed
using one-way ANOVA with Dunnett's multiple compari-
sons test, * p ≤ 0.05.
3
0-31
intramolecular CH-π interaction
between the benzyl
moiety and a hydrogen on the piperidyl group that likely
contributes to the bent binding confirmation (Figure
S10). 5 binds within the SpeB active site whereby the
carbonyl oxygen is oriented toward the SpeB oxyanion
hole created by the main-chain H-N amide nitrogens of
residues Cys192 and Val193 (Figure 3b). It should be
noted that 5 consists of two diastereomers, i.e., fixed (S)-
configured benzyl attached carbon center and mixture of
In conclusion, we provide a proof-of-concept for an
expedited high-throughput SAR process to improve po-
tency of an HTS hit molecule to develop biologically
useful molecules. This study highlights the utility of
SuFEx chemistry for rapidly generating diversified mol-
ecules for hit-to-lead applications and shows the poten-
tial of click chemistry’s near perfect linkage step(s), and
then immediate biological testing. Efforts to improve
and expand the method are underway to develop an HT
medicinal chemistry platform applicable for routine
(Rsulfur)- and (Ssulfur)-configurations at the stereogenic
sulfur center.
Based on biological stability and solubility in PBS
(Table S8), 7 was selected for further biological charac-
terization. As shown in Figure 4a, 7 is stable against
human liver microsomes in vitro, soluble in PBS, selec-
tive for SpeB (over other cysteine proteases), and non-
cytotoxic to human Jurkat T lympohocytes. We tested
the effect of inhibitor 7 in an established neutrophil kill-
ing assay, wherein SpeB activity boosts relative re-
3
8
use . Molecules described here represent the first potent
and selective small molecule SpeB inhibitors and can be
used to address biological functions of this protease in
cellular and animal models and establish it as a potential
target for the development of treatments to combat
streptococcal infections.
3
2-33
sistance of S. pyogenes against human neutrophils
.
Wild-type (WT) S. pyogenes (M1 serotype strain 5448)
and a corresponding isogenic mutant strain lacking the
SpeB gene (∆SpeB) were preincubated with 7 prior to
introduction of freshly isolated neutrophils from human
blood. The presence of 7 decreased the viability of WT
S. pyogenes in a concentration-dependent manner, while
ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on
the ACS Publications website.
Additional texts, figures and tables (PDF)
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(
7) Mamidyala, S. K.; Finn, M. G., In situ click chemistry: probing
AUTHOR INFORMATION
1
2
3
4
5
6
7
8
9
the binding landscapes of biological molecules. Chem. Soc. Rev.
2010, 39 (4), 1252-1261.
(
son, J. C., Click and pick: Identification of sialoside analogues for
siglec-based cell targeting. Angew. Chem. Int. Ed. 2012, 51 (44),
11014-11018.
Corresponding Authors
8) Rillahan, C. D.; Schwartz, E.; McBride, R.; Fokin, V. V.; Paul-
*
Email: sharples@scripps.edu (KBS)
Email: wolan@scripps.edu (DWW).
*
Author Contributions
(
9) Meng, G.; Guo, T.; Ma, T.; Zhang, J.; Shen, Y.; Sharpless, K.
††SK and QZ contributed equally to this work. SK, QZ,
KBS, and DWW conceived the project. SK designed the
experiments. SK designed, synthesized and characterized
B.; Dong, J., Modular click chemistry libraries for functional screens
using a diazotizing reagent. Nature 2019, 574 (7776), 86-89.
(
fur(VI) Fluoride Exchange (SuFEx): Another good reaction for click
chemistry. Angew. Chem. Int. Ed. 2014, 53 (36), 9430-9448.
10) Dong, J.; Krasnova, L.; Finn, M. G.; Sharpless, K. B., Sul-
molecules. QZ and SK performed SOF reaction. SK, AS
4
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and JW performed the in vitro kinetics studies and analysis.
SK, EC, and MVH performed the high-throughput synthe-
sis and assay. SK and DWW performed the crystallography
and structure analysis. AS performed the toxicity screen.
ND and VN performed neutrophil assays. SK and MK per-
formed LC-CAD analysis. JRC proofread the manuscript.
The manuscript was written through contributions of all
authors. All authors have given approval to the final ver-
sion of the manuscript.
(
11) Li, S.; Wu, P.; Moses, J. E.; Sharpless, K. B., Multidimen-
sional SuFEx click chemistry: Sequential Sulfur(VI) Fluoride Ex-
change connections of diverse modules launched from an SOF hub.
4
Angew. Chem. Int. Ed. 2017, 56 (11), 2903-2908.
(12) Wang, H.; Zhou, F.; Ren, G.; Zheng, Q.; Chen, H.; Gao, B.;
Klivansky, L.; Liu, Y.; Wu, B.; Xu, Q.; Lu, J.; Sharpless, K. B.; Wu,
P., SuFEx-based polysulfonate formation from ethenesulfonyl fluo-
ride–amine adducts. Angew. Chem. Int. Ed. 2017, 56 (37), 11203-
11208.
(13) Dong, J.; Sharpless, K. B.; Kwisnek, L.; Oakdale, J. S.; Fokin,
V. V., SuFEx-based synthesis of polysulfates. Angew. Chem. Int. Ed.
2014, 53 (36), 9466-9470.
Funding Sources
(
14) Gao, B.; Zhang, L.; Zheng, Q.; Zhou, F.; Klivansky, L. M.;
The authors gratefully acknowledge financial support from
The Scripps Research Institute (to DWW), NIH grant R01
AI077780 (to VN), R01 GM117145 (to KBS), and the El-
len Browning Scripps Foundation (to QZ).
Lu, J.; Liu, Y.; Dong, J.; Wu, P.; Sharpless, K. B., Bifluoride-
catalysed sulfur(VI) fluoride exchange reaction for the synthesis of
polysulfates and polysulfonates. Nat. Chem. 2017, 9, 1083.
(15) Liu, Z.; Li, J.; Li, S.; Li, G.; Sharpless, K. B.; Wu, P., SuFEx
click chemistry enabled late-stage drug functionalization. J. Am.
Chem. Soc. 2018, 140 (8), 2919-2925.
(
16) Baranczak, A.; Liu, Y.; Connelly, S.; Du, W.-G. H.; Greiner,
Notes
E. R.; Genereux, J. C.; Wiseman, R. L.; Eisele, Y. S.; Bradbury, N.
C.; Dong, J.; Noodleman, L.; Sharpless, K. B.; Wilson, I. A.; Encala-
da, S. E.; Kelly, J. W., A fluorogenic aryl fluorosulfate for intraorga-
nellar transthyretin imaging in living cells and in Caenorhabditis
elegans. J. Am. Chem. Soc. 2015, 137 (23), 7404-7414.
(17) Chen, W.; Dong, J.; Li, S.; Liu, Y.; Wang, Y.; Yoon, L.; Wu,
P.; Sharpless, K. B.; Kelly, J. W., Synthesis of sulfotyrosine-
containing peptides by incorporating fluorosulfated tyrosine using an
Fmoc-based solid-phase strategy. Angew. Chem. Int. Ed. 2016, 128
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank I. Wilson, R. Stanfield, M. Elsliger, and X. Dai
for computational assistance, H. Rosen and A. Ward for
access to instrumentation, and the staff of beamline 12.2 at
the Stanford Synchrotron Radiation Lightsource.
(
5), 1867-1870.
18) Mortenson, D. E.; Brighty, G. J.; Plate, L.; Bare, G.; Chen,
(
W.; Li, S.; Wang, H.; Cravatt, B. F.; Forli, S.; Powers, E. T.; Sharp-
less, K. B.; Wilson, I. A.; Kelly, J. W., “Inverse drug discovery” strat-
egy to identify proteins that are targeted by latent electrophiles as
exemplified by aryl fluorosulfates. J. Am. Chem. Soc. 2018, 140 (1),
200-210.
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