Exploiting ligand conformation in selective inhibition of non-ribosomal peptide synthetase amino acid adenylation with designed macrocyclic small molecules

Article abstract of DOI:10.1021/ja0721521

Macrocyclic aminoacyl-AMP analogs have been developed to inhibit non-ribosomal peptide synthetase amino acid adenylation domains selectively by mimicking a cisoid ligand binding conformation observed in crystal structures. In contrast, these macrocycles d

Full text of DOI:10.1021/ja0721521

Published on Web 06/02/2007
Exploiting Ligand Conformation in Selective Inhibition of Non-Ribosomal Peptide
Synthetase Amino Acid Adenylation with Designed Macrocyclic Small Molecules
Justin S. Cisar,†,‡ Julian A. Ferreras,§ Rajesh K. Soni,§ Luis E. N. Quadri,*,†,§ and Derek S. Tan*,†,‡
Tri-Institutional Training Program in Chemical Biology, Tri-Institutional Research Program and Molecular
Pharmacology & Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York AVenue, Box 422,
New York, New York 10021, and Department of Microbiology & Immunology and Molecular Biology Program,
Weill Medical College of Cornell UniVersity, 1300 York AVenue, Box 62, New York, New York 10021
Received March 27, 2007; E-mail: leq2001@med.cornell.edu; tand@mskcc.org
Recent evidence suggests that natural products are much more
than simply agents of “microbial warfare” and actually play critical
roles in bacterial pathogenesis and communication.1 In particular,
non-ribosomal peptide (NRP) natural products have been identified
as key players in bacterial iron uptake,2 biofilm formation,3 and
commensalism.4 Thus, small molecule inhibition of NRP biosyn-
thesis would provide a powerful means to study the biological roles
of these natural products and a potential avenue to develop novel
antibiotics. Detailed mechanistic insights into NRP biosynthesis,5
developed primarily from the perspectives of fundamental interest
and engineered biosynthesis, can be leveraged to design such
inhibitors. Along these lines, we recently reported a mechanism-
based inhibitor of NRP siderophore biosynthesis, salicyl-AMS
(5′-O-[N-salicylsulfamoyl]adenosine).6 This compound targets
salicylate adenylation enzymes by mimicking a key salicyl adenylate
Figure 1. (a) Reactions catalyzed by amino acid adenylation domains
(salicyl-AMP) reaction intermediate through replacement of the
during NRP biosynthesis (PCP-SH nucleophile) and by aminoacyl-tRNA
reactive phosphate group with a stable sulfamate moiety. Related
inhibitor design strategies have also been extended to other aryl
acid adenylation enzymes that have no human homologs.7 However,
application of this strategy to more widely distributed amino acid
adenylation domains is complicated by a major selectivity prob-
lem: aminoacyl-tRNA synthetases, which are used ubiquitously
in ribosomal protein translation, catalyze nearly identical reactions
(Figure 1a). Thus, simple aminoacyl-AMP analogs inhibit both
classes of enzymes,8,9 making them unsuitable for probing NRP
function or as antibiotics. Herein, we report a solution to this
problem using macrocyclic aminoacyl-AMP analogs (2a, 2b) that
exploit pronounced structural differences between these two protein
classes to inhibit an amino acid adenylation domain selectively.
To design inhibitors that selectively target NRP synthetase amino
acid adenylation domains, we compared the reported ligand-bound
crystal structures of a phenylalanine adenylation domain (PheA)
and a phenylalanyl-tRNA synthetase (PheRS).10 These enzymes
catalyze analogous reactions involving adenylation of phenylalanine
to form a phenylalanyl-AMP intermediate followed by transesteri-
fication to a peptidyl carrier protein thiol or tRNA hydroxyl,
respectively (Figure 1a). However, these proteins have unrelated
folds and bind their ligands in distinct conformations.11 In the PheA
structure, phenylalanine and AMP are bound in an overall “cisoid”
conformation (Figure 1b). Examination of closely related aryl acid
adenylation enzyme, fatty acyl-CoA ligase, and luciferase structures
suggests that this cisoid ligand conformation is conserved across
this family.11,12 In contrast, in the PheRS structure, a phenylalanyl-
AMP analog is bound in a “transoid” conformation (Figure 1c).
Examination of all available ligand-bound aminoacyl-tRNA syn-
synthetases during protein translation (tRNA-OH nucleophile). PCP )
peptidyl carrier protein. (b) Cisoid conformation of phenylalanine and AMP
ligands in a phenylalanine adenylation domain active site (PheA; PDB
1AMU). (c) Transoid conformation of a Phe-AMP analog in a phenylalanyl-
tRNA synthetase active site (PheRS; PDB 1B7Y). Adenine C8 to phenyl-
alanine Câ distances are indicated (gray).
thetase structures confirmed transoid ligand conformations in all
cases.11 On the basis of this analysis, we postulated that nonhy-
drolyzable aminoacyl-AMP analogs in which the cisoid conforma-
tion is promoted or enforced would inhibit NRP synthetase amino
acid adenylation domains selectively, without inhibiting the cor-
responding aminoacyl-tRNA synthetases.
Further examination of the PheA structure revealed an unob-
structed 4.1 Å space between C8 of adenine and Câ of phenyl-
alanine (Figure 1b), suggesting that a two- or three-atom linker
might be used to enforce the desired cisoid pharmacophoric
conformation in a macrocyclic inhibitor. Such compounds would
not be able to adopt the transoid conformation required to bind the
corresponding aminoacyl-tRNA synthetases. Thus, we designed
macrocycles 2a-e (Figure 2) as constrained analogs of alanyl-AMP.
We envisioned that such compounds might inhibit other amino acid
adenylation domains, such as the cysteine adenylation domain in
the Yersinia pestis siderophore biosynthesis enzyme HMWP2,13
provided that the missing â sidechain could be compensated by a
reduced entropic cost of binding and/or new favorable binding
interactions.14
After exploring several synthetic approaches, we arrived at
macrocycles 2a-e using the general strategy outlined in Figure
2.11 Briefly, 8-iodoadenosine 3 is functionalized at the adenine C8
position via cross-coupling reactions, then 5′-O-sulfamoylated.
Macrolactamization of 4 and global deprotection provides macro-
† Tri-Institutional Training Program in Chemical Biology.
‡ Memorial Sloan-Kettering Cancer Center.
§ Cornell University.
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J. AM. CHEM. SOC. 2007, 129, 7752-7753
10.1021/ja0721521 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
compounds and analogs thereof are ongoing. Given the high
structural homology among amino acid adenylation domains,11,12d
it will be of interest to determine whether such compounds can
inhibit other domains, thereby providing a broad spectrum means
to inhibit NRP biosynthetic pathways and to probe the biological
and therapeutic implications thereof. Broad inhibitors might also
synergistically inhibit multiple adenylation domains in an individual
pathway to afford increased potency15 and decreased susceptibility
to resistance conferring mutations.16
Acknowledgment. We thank Dr. George Sukenick, Hui Fang,
and Sylvi Rusli for mass spectral analyses. L.E.N.Q. is a Stavros
S. Niarchos Foundation Scholar. D.S.T. is a NYSTAR Watson
Investigator. Financial support from the NIH (R21 AI063384, P01
AI056293), William Randolph Hearst Foundation, William H.
Goodwin and Alice Goodwin and the Commonwealth Foundation
for Cancer Research, and MSKCC Experimental Therapeutics
Center is gratefully acknowledged.
Figure 2. Structures of adenylation domain inhibitors and general synthetic
approach to macrocycles.11 (R1, R2 ) Boc or H; Y ) NHBoc or H).
Supporting Information Available: Detailed experimental pro-
cedures and analytical data for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
Table 1. Inhibition of a Non-Ribosomal Peptide Synthetase Amino
Acid Adenylation Domain and of in Vitro Translation11
Kiapp
IC50 (in vitro
translation,
M)b
entry
compound
(HMWP2,
µ
M)a
µ
References
1
2
3
4
5
6
7
8
9
1a (L-Cys-AMS)
0.24 ( 0.02c 15.5 ( 0.3
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1b (L-Ala-AMS)
1c (D-Cys-AMS)
1d (D-Ala-AMS)
1e (Gly-AMS)
2.5
( 0.2
0.16 ( 0.03
0.37 ( 0.02
n.d.
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>250
>250
n.d.
34
16
1.7
5.4
150
400
( 3
( 5
2a (cyclo8C â-L-Ala-AMS)
( 0.1d
( 0.8e
( 60
( 100
( 30
2
2b (cyclo8C â-L-Ala-AMS)
3
2c (cyclo8C â-D-Ala-AMS)
2
2d (cyclo8C â-D-Ala-AMS)
n.d.
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10 2e (cyclo8C â-propionyl-AMS) 210
n.d.
3
a With 30 nM HMWP21-1491-His6, 1 mM [32P]-PPi, 3 mM ATP, 3 mM
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)
0.062 ( 0.002 µM.11 d KiCys ) 0.27 ( 0.03 µM; KiATP ) 0.179 (
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