are obvious structural alerts (toxicophores) and functionalities
that may be reactive in acidic conditions in the presence or
absence of reactive nitrogen species. The nonreplicating active
molecule can be incubated at 37
C in nonreplicating medium
Æ0.5 mM NaNO 2 and analyzed at 24–72 h by LC-MS to
determine what percentage of the starting material remains.
One can try to purify any new species (although it is possible a
new species does not ionize and may not be detectable) and test
its replicating and nonreplicating activity, as well as its toxicity
to HepG2 cells.
Acknowledgements
We are very grateful for the numerous contributions toward the
development of the nonreplicating assay and critical reading of this
chapter: Julia Roberts, Yan Ling, Selin Somersan-Karakaya, Landys
Lopez-Quezada, David Little, Maneesh Pingle, Kristin BurnsHuang (Weill Cornell Medical College, NY, USA) and Alfonso
Mendoza-Losana, Maria Martinez-Hoyos, and Manuel MarinAmieva (GSK, Tres Cantos, Spain). This work was supported by
the TB Drug Accelerator Program of the Bill and Melinda Gates
Foundation and by the Abby and Howard P. Milstein Program in
Translational Medicine. The Department of Microbiology and
Immunology was supported by the William Randolph Hearst
Foundation.
References
1. Nathan C (2012) Fresh approaches to antiinfective therapies. Sci Transl Med 4
(140):140–142. https://doi.org/10.1126/
scitranslmed.3003081
2. Gold B, Nathan C (2017) Targeting phenotypically tolerant Mycobacterium tuberculosis.
Microbiol Spectr 5(1). https://doi.org/10.
1128/microbiolspec.TBTB2-0031-2016
3. Nathan C (2017) Kunkel lecture: fundamental
immunodeficiency and its correction. J Exp
Med 214(8):2175–2191. https://doi.org/10.
1084/jem.20170637
4. Nathan C, Barry CE 3rd (2015) TB drug
development: immunology at the table. Immunol Rev 264(1):308–318. https://doi.org/10.
1111/imr.12275
5. Bryk R, Gold B, Venugopal A, Singh J, Samy R,
Pupek K, Cao H, Popescu C, Gurney M,
Hotha S, Cherian J, Rhee K, Ly L, Converse
PJ, Ehrt S, Vandal O, Jiang X, Schneider J,
Lin G, Nathan C (2008) Selective killing of
nonreplicating mycobacteria. Cell Host
Microbe 3(3):137–145. https://doi.org/10.
1016/j.chom.2008.02.003
6. Lin G, Chidawanyika T, Tsu C, Warrier T,
Vaubourgeix J, Blackburn C, Gigstad K,
Sintchak M, Dick L, Nathan C (2013) N,
C-capped dipeptides with selectivity for mycobacterial proteasome over human proteasomes:
role of S3 and S1 binding pockets. J Am Chem
Soc 135(27):9968–9971. https://doi.org/10.
1021/ja400021x
7. Lin G, Li D, de Carvalho LP, Deng H, Tao H,
Vogt G, Wu K, Schneider J, Chidawanyika T,
Warren JD, Li H, Nathan C (2009) Inhibitors
selective for mycobacterial versus human proteasomes.
Nature
461(7264):621–626.
https://doi.org/10.1038/nature08357
8. Nathan C, Gold B, Lin G, Stegman M, de
Carvalho LP, Vandal O, Venugopal A, Bryk R
(2008) A philosophy of anti-infectives as a
guide in the search for new drugs for
632
Ben Gold et al.
that may be reactive in acidic conditions in the presence or
absence of reactive nitrogen species. The nonreplicating active
molecule can be incubated at 37
C in nonreplicating medium
Æ0.5 mM NaNO 2 and analyzed at 24–72 h by LC-MS to
determine what percentage of the starting material remains.
One can try to purify any new species (although it is possible a
new species does not ionize and may not be detectable) and test
its replicating and nonreplicating activity, as well as its toxicity
to HepG2 cells.
Acknowledgements
We are very grateful for the numerous contributions toward the
development of the nonreplicating assay and critical reading of this
chapter: Julia Roberts, Yan Ling, Selin Somersan-Karakaya, Landys
Lopez-Quezada, David Little, Maneesh Pingle, Kristin BurnsHuang (Weill Cornell Medical College, NY, USA) and Alfonso
Mendoza-Losana, Maria Martinez-Hoyos, and Manuel MarinAmieva (GSK, Tres Cantos, Spain). This work was supported by
the TB Drug Accelerator Program of the Bill and Melinda Gates
Foundation and by the Abby and Howard P. Milstein Program in
Translational Medicine. The Department of Microbiology and
Immunology was supported by the William Randolph Hearst
Foundation.
References
1. Nathan C (2012) Fresh approaches to antiinfective therapies. Sci Transl Med 4
(140):140–142. https://doi.org/10.1126/
scitranslmed.3003081
2. Gold B, Nathan C (2017) Targeting phenotypically tolerant Mycobacterium tuberculosis.
Microbiol Spectr 5(1). https://doi.org/10.
1128/microbiolspec.TBTB2-0031-2016
3. Nathan C (2017) Kunkel lecture: fundamental
immunodeficiency and its correction. J Exp
Med 214(8):2175–2191. https://doi.org/10.
1084/jem.20170637
4. Nathan C, Barry CE 3rd (2015) TB drug
development: immunology at the table. Immunol Rev 264(1):308–318. https://doi.org/10.
1111/imr.12275
5. Bryk R, Gold B, Venugopal A, Singh J, Samy R,
Pupek K, Cao H, Popescu C, Gurney M,
Hotha S, Cherian J, Rhee K, Ly L, Converse
PJ, Ehrt S, Vandal O, Jiang X, Schneider J,
Lin G, Nathan C (2008) Selective killing of
nonreplicating mycobacteria. Cell Host
Microbe 3(3):137–145. https://doi.org/10.
1016/j.chom.2008.02.003
6. Lin G, Chidawanyika T, Tsu C, Warrier T,
Vaubourgeix J, Blackburn C, Gigstad K,
Sintchak M, Dick L, Nathan C (2013) N,
C-capped dipeptides with selectivity for mycobacterial proteasome over human proteasomes:
role of S3 and S1 binding pockets. J Am Chem
Soc 135(27):9968–9971. https://doi.org/10.
1021/ja400021x
7. Lin G, Li D, de Carvalho LP, Deng H, Tao H,
Vogt G, Wu K, Schneider J, Chidawanyika T,
Warren JD, Li H, Nathan C (2009) Inhibitors
selective for mycobacterial versus human proteasomes.
Nature
461(7264):621–626.
https://doi.org/10.1038/nature08357
8. Nathan C, Gold B, Lin G, Stegman M, de
Carvalho LP, Vandal O, Venugopal A, Bryk R
(2008) A philosophy of anti-infectives as a
guide in the search for new drugs for
632
Ben Gold et al.