ACS Medicinal Chemistry Letters
LETTER
Phe1032 required for efficient binding of these compounds.
Additionally, the nature of the substitution at the opposite end of
the inhibitors is also important. Although 21ꢀ27 all contain
either sulfonamides or nitriles, departure from a five-membered
azole ring at the opposite end of the molecule resulted in
significantly weaker (21, 22, 25, and 27) or total loss (23, 24,
and 26) of activity. We reasoned that both the size of the ring and
the position of ring heteroatoms are important for activity. This is
clearly seen with compound 27, which contains a furan ring
similar in size to imidazole, but the heteroatom at the 2-position
is unable to make a hydrogen bond to Thr1443 predicted to
interact with the imidazolyl moiety of 10 (Figure 2c).
To probe the selectivity of enzyme inhibition, compounds
found to be active in the E. coli RNAP assay were subjected to
specificity assays using malate dehydrogenase and chymotrypsin to
identify promiscuous activity,22 and inhibition against yeast RNA
Polymerase II (Pol II) to ascertain whether compounds were
selective for bacterial RNAP. Compounds 8ꢀ13 exhibited no
substantial inhibition of the three enzymes at 100 μM and, there-
fore, appear to be selective inhibitors of bacterial RNAP (Table 3).
MICs were determined for all of the compounds described
using standard susceptibility tests, in triplicate, against S. aureus,
Bacillus subtilis, and E. coli, strains according to the British Society
for Antimicrobial Chemotherapy guidelines (see the Supporting
Information for further details).23 None of the compounds were
found to possess antibacterial activity (>256 μg/mL in all cases),
even in the presence of the outer membrane permeabilizer
polymyxin B nonapeptide (PMBN) and E. coli deficient in the
AcrAB multidrug efflux pump component.
In summary, we have used a de novo molecular design
approach to identify a new series of inhibitors targeting bacterial
RNAP. To our knowledge, they are the first examples of designed
small molecule inhibitors predicted to bind to the MyxB binding
region of RNAP. Compounds 8ꢀ13 are of low molecular mass,
have improved potency and ligand efficiency over MyxB, are
selective for bacterial RNAP, and do not appear to be promis-
cuous inhibitors. The compounds may not possess the requisite
physicochemical properties required for antibacterial activity,
since antibacterial agents occupy a unique chemical property space
compared to that for other therapeutic drugs.24 Most likely, the lack
of antibacterial activity of these compounds is attributable to poor
cell penetration, and in this connection, further optimization of this
scaffold is in progress. This research advances our understanding of
bacterial RNAP inhibitors with binding sites in nonessential
prokaryotic domains that are distant from the catalytic site.
’ ACKNOWLEDGMENT
We thank the BBSRC and MRC for support. We also thank T.
Moy for the gift of MyxB, D. Bushnell for yeast Pol II, and K.
Simmons and M. Migliore for discussion.
’ REFERENCES
(1) Villain-Guillot, P.; Bastide, L.; Gualtieri, M.; Leonetti, J. P.
Progress in targeting bacterial transcription. Drug Discovery Today
2007, 12, 200–208.
(2) Darst, S. A. New inhibitors targeting bacterial RNA polymerase.
Trends Biochem. Sci. 2004, 29, 159–162.
(3) Ho, M. X.; Hudson, B. P.; Das, K.; Arnold, E.; Ebright, R. H.
Structures of RNA polymerase-antibiotic complexes. Curr. Opin. Struct.
Biol. 2009, 19, 715–723.
(4) Chopra, I. Bacterial RNA polymerase: A promising target for the
discovery of new antimicrobial agents. Curr. Opin. Invest. Drugs 2007,
8, 600–607.
(5) Barker, J. J. Antibacterial drug discovery and structure-based
design. Drug Discovery Today 2006, 11, 391–404.
(6) Chopra, I. Research and development of antibacterial agents.
Curr. Opin. Microbiol. 1998, 1, 495–501.
(7) Belogurov, G. A.; Vassylyeva, M. N.; Sevostyanova, A.; Appleman,
J. R.; Xiang, A. X.; Lira, R.; Webber, S. E.; Klyuyev, S.; Nudler, E.;
Artsimovitch, I.; Vassylyev, D. G. Transcription inactivation through local
refolding of the RNA polymerase structure. Nature 2009, 457, 332–335.
(8) Mukhopadhyay, J.; Das, K.; Ismail, S.; Koppstein, D.; Jang, M. Y.;
Hudson, B.; Sarafianos, S.; Tuske, S.; Patel, J.; Jansen, R.; Irschik, H.;
Arnold, E.; Ebright, R. H. The RNA Polymerase “Switch Region” Is a
Target for Inhibitors. Cell 2008, 135, 295–307.
(9) Irschik, H.; Gerth, K.; Hofle, G.; Kohl, W.; Reichenbach, H. The
Myxopyronins, New Inhibitors of Bacterial RNA-Synthesis from Myx-
ococcus-Fulvus (Myxobacterales). J. Antibiot. 1983, 36, 1651–1658.
(10) Doundoulakis, T.; Xiang, A. X.; Lira, R.; Agrios, K. A.; Webber,
S. E.; Sisson, W.; Aust, R. M.; Shah, A. M.; Showalter, R. E.; Appleman,
J. R.; Simonsen, K. B. Myxopyronin B analogs as inhibitors of RNA
polymerase, synthesis and biological evaluation. Bioorg. Med. Chem. Lett.
2004, 14, 5667–5672.
(11) Lira, R.; Xiang, A. X.; Doundoulakis, T.; Biller, W. T.; Agrios,
K. A.; Simonsen, K. B.; Webber, S. E.; Sisson, W.; Aust, R. M.; Shah, A. M.;
Showalter, R. E.; Banh, V. N.; Steffy, K. R.; Appleman, J. R. Syntheses of
novel myxopyronin B analogs as potential inhibitors of bacterial RNA
polymerase. Bioorg. Med. Chem. Lett. 2007, 17, 6797–6800.
(12) Mauser, H.; Guba, W. Recent developments in de novo design
and scaffold hopping. Curr. Opin. Drug Discovery Dev. 2008, 11, 365–374.
(13) Simmons, K. J.; Chopra, I.; Fishwick, C. W. G. Structure-based
discovery of antibacterial drugs. Nat. Rev. Microbiol. 2010, 8, 501–510.
(14) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L.
Drugs for bad bugs: Confronting the challenges of antibacterial dis-
covery. Nat. Rev. Drug Discovery 2007, 6, 29–40.
(15) Gillet, V.;Johnson, A. P.; Mata, P.; Sike, S.; Williams, P. Sprout—
A Program for Structure Generation. J. Comput.-Aided Mol. Des. 1993, 7,
127–153.
(16) Woon, E. C. Y.; Zervosen, A.; Sauvage, E.; Simmons, K. J.;
Zivec, M.; Inglist, S. R.; Fishwick, C. W. G.; Gobec, S.; Charlier, P.;
Lexen, A.; Schofield, C. J. Structure Guided Development of Potent
Reversibly Binding Penicillin Binding Protein Inhibitors. ACS Med.
Chem. Lett. 2010, 2, 219–223.
’ ASSOCIATED CONTENT
S
Supporting Information. Details of compound syntheses
b
and characterization and of the biological assay protocols. This material
’ AUTHOR INFORMATION
(17) Cowen, D.; Bedingfield, P.; McConkey, G. A.; Fishwick,
C. W. G.; Johnson, A. P. A study of the effects of substituents on the
selectivity of the binding of N-arylaminomethylene malonate inhibitors
to DHODH. Bioorg. Med. Chem. Lett. 2010, 20, 1284–1287.
(18) Agarwal, A. K.; Johnson, A. P.; Fishwick, C. W. G. Synthesis of
de novo designed small-molecule inhibitors of bacterial RNA polymer-
ase. Tetrahedron 2008, 64, 10049–10054.
Corresponding Author
* (CWGF) Tel: +44(0)113 3436510. Fax: +44(0)113 3436565.
E-mail: C.W.G.Fishwick@leeds.ac.uk; (APJ) +44(0)113 3436515.
Fax: +44(0)113 3436565. E-mail: P.Johnson@leeds.ac.uk.
Present Addresses
§Medicinal Chemistry and Chemical Biology Technology
Group, School of Chemistry.
(19) Mariner, K. R.; Trowbridge, R.; Agarwal, A. K.; Miller, K.;
O'Neill, A. J.; Fishwick, C. W. G.; Chopra, I. Furanyl-Rhodanines Are
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