Journal of the American Chemical Society
Article
(11) Musse, A. A.; Li, Z.; Ackerley, C. A.; Bienzle, D.; Lei, H.; Poma,
R.; Harauz, G.; Moscarello, M. A.; Mastronardi, F. G. Dis. Model. Mech.
2008, 1, 229.
(12) Moscarello, M. A.; Wood, D. D.; Ackerley, C.; Boulias, C. J. Clin.
Invest. 1994, 94, 146.
implemented a substrate-based fragment discovery method for
identifying PAD inhibitors by screening a library of guanidines
to identify substrates, optimizing substrate structure for
cleavage efficiency and then conversion to inhibitors by
replacement of the guanidine by the chloroacetamidine
inhibitor pharmacophore. This method enabled the rapid
identification of three distinct classes of small molecule
inhibitors. Inhibitor 14b, with a kinact/KI of 15,600 toward
PAD3 had the optimal combination of potency and selectivity.
(13) (I) For the link between PAD4 and rhuematoid arthritis, see:
(a) Kinloch, A.; Lundberg, K.; Wait, R.; Wegner, N.; Lim, N. H.;
Zendman, A. J. W.; Saxne, T.; Malmstrom, V.; Venables, P. J. Arthritis
Rheum. 2008, 58, 2287. (b) Masson-Bessiere, C.; Sebbag, M.; Girbal-
Neuhauser, E.; Nogueira, L.; Vincent, C.; Senshu, T.; Serre, G. J.
Immunol. 2001, 166, 4177. (c) Schellekens, G. A.; de Jong, B. A. W.;
van den Hoogen, F. H. J.; van de Putte, L. B. A.; van Venrooij, W. J. J.
Clin. Invest. 1998, 101, 273. (II) For the link between PAD4 and
ulcerative colitis, see: (d) Chen, C.; Isomoto, H.; Narumi, Y.; Sato, K.;
Oishi, Y.; Kobayashi, T.; Yanagihara, K.; Mizuta, Y.; Kohno, S.;
Tsukamoto, K. Clin. Immunol. 2008, 126, 165. (III) For the link
between PADs and lupus, see: (e) Knight, J. S.; Zhao, W.; Luo, W.;
Subramanian, V.; O’Dell, A. A.; Yalavarthi, S.; Hodgin; Eitzman, D.;
Thompson, P. R.; Kaplan, M. J. Clin. Invest. 2013, 123, 2981.
(14) Monahan, S.; Cherrington, B. D.; Horibata, S.; McElwee, J. L.;
Thompson, P. R.; Coonrod, S. A. Biochem. Res. Int. 2012, 2012, 11.
(15) Lange, S.; Gogel, S.; Leung, K. Y.; Vernay, B.; Nicholas, A. P.;
Causey, C. P.; Thompson, P. R.; Greene, N. D. E.; Ferretti, P. Dev.
Biol. 2011, 355, 205.
(16) The inhibitor values reported here were performed in our lab
with 0.1% of Triton-X in the assay buffer to prevent micelle formation
and with 5% of DMSO, which was needed to solubilize some of the
inhibitors to determine isozyme selectivitry. For Cl-amidine, the
Thompson lab performed assays without Triton-X or DMSO and
observed a different kinact/Ki values with the largest difference for
PAD1. We established that while the presence of Triton-X had
minimal affect on a kinact/Ki values, as little as 1% DMSO caused a
pronounced reduction in Cl-amidine inhibitor potency for PAD1.
(17) An analog of Cl-amidine with comparable enzyme inhibitory
activity and good cell and animal efficacy has recently been reported:
Knight, J. S.; Subramanian, V.; O’Dell, A. A.; Yalavarthi, S.; Zhao, W.;
Smith, C. K.; Hodgin, J. B.: Thompson, P. R.; Kaplan, M. J. Ann.
Rheum. Dis. [Online early access]. DOI: 10.1136/annrheumdis-2014-
205365 Published Online: Aug 7, 2014.
(18) Willis, V.; Gizinski, A. M.; Banda, N. K.; Causey, C. P.;
Knuckley, B.; Cordova, K. N.; Luo, Y. A.; Levitt, B.; Glogowska, M.;
Chandra, P.; Kulik, L.; Robinson, W. H.; Arend, W. P.; Thompson, P.
R.; Holers, V. M. J. Immunol. 2011, 186, 4396.
(19) Causey, C. P.; Jones, J. E.; Slack, J. L.; Kamei, D.; Jones, L. E.;
Subramanian, V.; Knuckley, B.; Ebrahimi, P.; Chumanevich, A. A.;
Luo, Y.; Hashimoto, H.; Sato, M.; Hofseth, L. J.; Thompson, P. R. J.
Med. Chem. 2011, 54, 6919.
(20) F4-amidine and Cl4-amidine were previously reported by the
Thompson group as inhibitors that show PAD3 selectivity relative to
PAD1 and PAD4, although no inhibitory data was provided for PAD2
(see reference 4). An exact IC50 value of 65 μM for PAD3 was
reported for F4-amidine. The approximate IC50 values for F4-amidine
for PAD1 and PAD4 as provided in the chart in Figure 6A were ∼250
μM and ∼625 μM, respectively.
(21) Knuckley, B.; Jones, J. E.; Bachovchin, D. A.; Slack, J.; Causey,
C. P.; Brown, S. J.; Rosen, H.; Cravatt, B. F.; Thompson, P. R. Chem.
Commun. 2010, 46, 7175.
ASSOCIATED CONTENT
■
S
* Supporting Information
Complete experimental procedures and characterization data
for all compounds described as well as kinact/KI data for all
inhibitors. This material is available free of charge via the
AUTHOR INFORMATION
■
Corresponding Author
Present Addresses
†RA Pharmaceuticals, One Kendall Square #B7202, Cambridge,
MA, 02139
‡Melinta Therapeutics, 300 George Street, New Haven, CT
06511−663
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support has been provided by the National Institutes of Health
(R01-GM054051). H.J. also acknowledges support from the
NIH Chemical Biology training grant (5T32 GM7499-34).
Paul Thompson is also gratefully acknowledged for helpful
discussions and for providing constructs enabling recombinant
expression of PADs 1−4. Caroline Chandra Tjin’s assistance
with the synthesis and assay of selected inhibitors is also greatly
appreciated. Corey E. Perez is acknowledged for his assistance
with cloning and associated biological experiments.
REFERENCES
■
(1) Vossenaar, E. R.; Zendman, A. J. W.; van Venrooij, W. J.; Pruijn,
G. J. M. Bioessays 2003, 25, 1106.
(2) Bicker, K. L.; Thompson, P. R. Biopolymers 2013, 99, 155.
(3) Jones, J. E.; Causey, C. P.; Knuckley, B.; Slack-Noyes, J. L.;
Thompson, P. R. Curr. Opin. Drug Discovery Dev. 2009, 12, 616.
(4) Knuckley, B.; Causey, C. P.; Jones, J. E.; Bhatia, M.; Dreyton, C.
J.; Osborne, T. C.; Takahara, H.; Thompson, P. R. Biochemistry 2010,
49, 4852.
(5) Luo, Y.; Arita, K.; Bhatia, M.; Knuckley, B.; Lee, Y.-H.; Stallcup,
M. R.; Sato, M.; Thompson, P. R. Biochemistry 2006, 45, 11727.
(6) Arita, K.; Hashimoto, H.; Shimizu, T.; Nakashima, K.; Yamada,
M.; Sato, M. Nat. Struct. Mol. Biol. 2004, 11, 777.
(22) Knuckley, B.; Luo, Y.; Thompson, P. R. Bioorg. Med. Chem.
2008, 16, 739.
(23) Slack, J. L.; Causey, C. P.; Luo, Y.; Thompson, P. R. ACS Chem.
Biol. 2011, 6, 466.
(24) Wood, W. J. L.; Patterson, A. W.; Tsuruoka, H.; Jain, R. K.;
(7) Kearney, P. L.; Bhatia, M.; Jones, N. G.; Yuan, L.; Glascock, M.
C.; Catchings, K. L.; Yamada, M.; Thompson, P. R. Biochemistry 2005,
44, 10570.
(8) Ishida-Yamamoto, A.; Senshu, T.; Takahashi, H.; Akiyama, K.;
Nomura, K.; Iizuka, H. J. Invest. Dermatol. 2000, 114, 701.
(9) Raijmakers, R.; Vogelzangs, J.; Raats, J.; Panzenbeck, M.; Corby,
M.; Jiang, H. P.; Thibodeau, M.; Haynes, N.; Van Venrooij, W. J.;
Pruijn, G. J. M.; Werneburg, B. J. Comp. Neurol. 2006, 498, 217.
(10) Wood, D. D.; Ackerley, C. A.; van den Brand, B.; Zhang, L.;
Raijmakers, R.; Mastronardi, F. G.; Moscarello, M. A. Lab. Invest. 2008,
88, 354.
Ellman, J. A. J. Am. Chem. Soc. 2005, 127, 15521.
(25) For the potential for nonadditivity in substrate fragments, see:
Barelier, S.; Cummings, J. A.; Rauwerdink, A. M.; Hitchcock, D. S.;
Farelli, J. D.; Almo, S. C.; Raushel, F. M.; Allen, K. N.; Shoichet, B. K.
J. Am. Chem. Soc. 2014, 136, 7374.
(26) McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. J.
Med. Chem. 2002, 45, 1712.
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