Journal of Medicinal Chemistry
Brief Article
strings. Of these, 62000 were removed because they contained reactive
functional groups (e.g., acyl halides) or were unsuitable leads (e.g.,
nitro compounds). Compounds were then filtered based on “rule of
three” criteria which were modified to increase the number of resulting
compounds: molecular weight (MW) ≤ 350 Da, AlogP ≤ 3, hydrogen-
bond acceptors ≤3, hydrogen-bond donors ≤3, rotatable bonds ≤3,
and polar surface area ≤80. A principal component analysis and
neighborhood algorithm was applied to the 1522 remaining
compounds to produce 281 fragments with a 0.75 diversity index.
Then 100 of these compounds were initially selected based on
affordability and the ease of future analogue synthesis
version B.02.00. Maximum entropy deconvolutions were performed
with a mass step of 1, S/N threshold of 30, average mass at 90% of
peak height, and 5 charge states minimum.
Papain Activity Assays. Papain (4.8 μM) in 50 mM Na3PO4 and
2 mM EDTA was preactivated with 1 mM DTT for 30 min. Activated
papain (3.84 μM) in 4:1 mixture of 50 mM Na3PO4 and 2 mM EDTA
at pH 6.2 and acetronitrile was then preincubated for 1 h with varying
concentrations of the electrophilic fragment. Every 10 min, 10 μL of
the reaction mixture was added to a well of 96-well plate containing
100 μL of 4:1 mixture of 50 mM Na3PO4/2 mM EDTA/pH
6.2:acetronitrile with 400 μM Cbz-Gly-ONp. p-Nitrophenol product
formation was monitored by absorbance at 340 nM (ε: 6800 M−1
cm−1) with a Biotek Synergy 4 plate reader. All reactions were
performed in duplicate. Product concentration vs time was plotted
with GraphPad Prism software, and the initial slope was calculated to
determine enzymatic activity (E). The values of kinact/Ki for each
inhibitor were then determined according to the method of Kitz and
Synthesis of 6−108. The carboxylic acid fragment (0.2 mmol)
was dissolved in dimethylformamide (0.2 M, 1 mL), then 5 (46 mg,
0.2 mmol), HBTU (73.8 mg, 0.16 mmol), and HOBt (29.8 mg, 0.22
mmol) were added, followed by EtN(i-Pr)2 (100.7 μL, 0.6 mmol). The
reaction was stirred at 23 °C for 16 h. TLC at 16 h showed conversion
to product. The reaction was quenched with H2O (5 mL) and
extracted three times with CH2Cl2 (5 mL). The combined organic
layers were washed with 1 M HCl (10 mL), saturated aqueous
NaHCO3 (10 mL), and saturated aqueous NaCl (10 mL). The organic
layer was dried over MgSO4, filtered, and evaporated. Purified by flash
column chromatography with a CH3OH/CH2Cl2, CH3OH gradient
0−5% to yield compounds 6−108. Yields ranged from 11% to 100%,
with an average yield of 60%. Chemical structures of compounds 6−
108 are shown in SI. For initial library creation, compounds were
Wilson.23 Briefly, the slopes of the plots of ln(100 × Einhibited
/
Euninhibited) vs time were used to determine the pseudo-first-order
inhibition constant kobs for a given concentration of a given inhibitor.
The slope of the plot of kobs vs [Inhibitor] was then used to determine
the second-order inhibition constant kinact/KI (because [I] ≪ Ki, the
plots were linear at the concentrations tested).
ASSOCIATED CONTENT
* Supporting Information
■
1
characterized by H NMR and low resolution MS. All compounds
S
tested in enzymatic assays were also characterized by 13C NMR and
≥95% purity was confirmed by HPLC.
Synthetic procedures, characterization of the synthesized
chemical compounds, and supplementary figures. This material
NMR Rate Studies. N-Acetyl cysteine methyl ester was dissolved
in 2:1 deuterated PBS:DMSO-d6 (78 mM) with 10 mM CH2Cl2 as an
internal standard. The electrophile (10 mM) was then added
immediately prior to acquiring NMR spectra. H1 spectra were taken
every 30 s for 30 min (or every 4 s for 5 min for highly reactive
compounds 1c and 2a−c). The integrals of the vinyl peaks were used
to determine the concentration of the electrophile over time. The
natural logarithm of the concentration of the electrophile vs time was
then plotted using GraphPad Prism software. The linear slope of this
plot was used to determine the pseudo-first-order rate constant.
Deuterated PBS recipe: 20 mM Na3PO4, 50 mM NaCl in D2O was
adjusted to pD 8 with DCl solution.
AUTHOR INFORMATION
Corresponding Author
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Funding from Northwestern University is greatly acknowl-
edged. A.V.S. is a Pew Scholar in the Biomedical Sciences,
supported by the Pew Charitable Trusts. S.G.K. is supported by
the NIH training grant 1T32GM105538-01.
Irreversible Tethering Screening Assay. Papain (Sigma P4762,
10 μM), UbcH7 (recombinantly expressed, 10 μM), GST-264
HRV3C protease (recombinantly expressed, 10 μM), or USP08
(recombinantly expressed, 10 μM) in 50 mM HEPES, 150 mM NaCl,
and 0.1 mM EDTA pH 7.5 was treated with a mixture of 10 fragments
(SI Table S2) (10 mM DMSO stock solutions, final concentrations:
100 μM of each fragment, and 1% DMSO). The reaction mixture was
incubated for 1 h or 4 h at 23 °C before being passed through Zeba gel
filtration columns (Thermo, 7K MWCO) to remove unreacted
fragments. The protein solution was then immediately analyzed by
whole protein LC/ESI-MS.
LC/ESI-MS Protocol. Accurate-mass data were obtained on an
Agilent 6210A LC-TOF mass spectrometer in positive ion mode using
electrospray ionization. Samples were chromatographed on the LC-
TOF instrument using a Poroshell 120 EC-C18 HPLC column (2.1
mm × 50 mm, 2.7 μm), an Agilent Series 1200 HPLC binary pump,
and an Agilent Series 1200 autoinjector. The HPLC column was held
at 45 °C, and the autosampler was held at 8 °C. Mobile phase A was a
solution of 0.1% formic acid in water:acetonitrile (19:1). Mobile phase
B was a solution of 0.1% formic acid in acetonitrile. The flow rate was
set to 250 μL/min. The gradient used was 0% B for 2 min, ramping
linearly to 90% B from 2 to 5 min, holding at 90% B from 5 to 7 min,
and then returning to 0% B at 7.1 min. The column was allowed to
equilibrate for 2.7 min before the next injection was initiated. The
eluent from the column was diverted to waste for the first 2 min. The
spectra were acquired from 301 to 3200 Da using a gas temperature of
340 °C, a gas flow of 7 L/min, and the nebulizer gas at 35 psi. The
following voltages were used: capillary 4200 V, fragmentor 230 V,
skimmer 64 V, and octapole RF peak 250 V. Spectra were acquired at a
rate of 1 spectra/s. The data was processed using MassHunter software
REFERENCES
■
(1) Scott, D. E.; Coyne, A. G.; Hudson, S. A.; Abell, C. Fragment-
Based Approaches in Drug Discovery and Chemical Biology.
Biochemistry 2012, 51, 4990−5003.
(2) Erlanson, D. A.; Braisted, A. C.; Raphael, D. R.; Randal, M.;
Stroud, R. M.; Gordon, E. M.; Wells, J. A. Site-directed ligand
discovery. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 9367−9372.
(3) Erlanson, D. A.; Wells, J. A.; Braisted, A. C. Tethering: fragment-
based drug discovery. Annu. Rev. Biophys. Biomol. Struct. 2004, 33,
199−223.
(4) Miller, R. M.; Paavilainen, V. O.; Krishnan, S.; Serafimova, I. M.;
Taunton, J. Electrophilic fragment-based design of reversible covalent
kinase inhibitors. J. Am. Chem. Soc. 2013, 135, 5298−5301.
(5) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence of
covalent drugs. Nature Rev. Drug Discovery 2011, 10, 307−317.
(6) Zartler, E.; Shapiro, M. Fragment-Based Drug Discovery: A
Practical Approach; Wiley: New York, 2008.
(7) Cardoso, R.; Love, R.; Nilsson, C. L.; Bergqvist, S.; Nowlin, D.;
Yan, J.; Liu, K. K.; Zhu, J.; Chen, P.; Deng, Y. L.; Dyson, H. J.; Greig,
M. J.; Brooun, A. Identification of Cys255 in HIF-1α as a novel site for
development of covalent inhibitors of HIF-1α/ARNT PasB domain
protein−protein interaction. Protein Sci. 2012, 21, 1885−1896.
(8) Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.;
Dillon, M. B.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F.
E
dx.doi.org/10.1021/jm500345q | J. Med. Chem. XXXX, XXX, XXX−XXX