Interkingdom pharmacology of angiotensin-I converting enzyme inhibitor phosphonates produced by actinomycetes

Article abstract of DOI:10.1021/ml4004588

The K-26 family of bacterial secondary metabolites are N-modified tripeptides terminated by an unusual phosphonate analog of tyrosine. These natural products, produced via three different actinomycetales, are potent inhibitors of human angiotensin-I converting enzyme (ACE). Herein we investigate the interkingdom pharmacology of the K-26 family by synthesizing these metabolites and assessing their potency as inhibitors of both the N-terminal and C-terminal domains of human ACE. In most cases, selectivity for the C-terminal domain of ACE is displayed. Co-crystallization of K-26 in both domains of human ACE reveals the structural basis of the potent inhibition and has shown an unusual binding motif that may guide future design of domain-selective inhibitors. Finally, the activity of K-26 is assayed against a cohort of microbially produced ACE relatives. In contrast to the synthetic ACE inhibitor captopril, which demonstrates broad interkingdom inhibition of ACE-like enzymes, K-26 selectively targets the eukaryotic family.

Full text of DOI:10.1021/ml4004588

Letter
pubs.acs.org/acsmedchemlett
Interkingdom Pharmacology of Angiotensin‑I Converting Enzyme
Inhibitor Phosphonates Produced by Actinomycetes
Glenna J. Kramer,‡ Akif Mohd,†,§ Sylva L. U. Schwager,∥ Geoffrey Masuyer,§ K. Ravi Acharya,§
Edward D. Sturrock,∥ and Brian O. Bachmann*,‡
‡Vanderbilt University Department of Chemistry, 7300 Stevenson Center, Nashville, Tennessee 37204, United States
§University of Bath, Department of Biology & Biochemistry, Bath BA2 7AY, United Kingdom
∥University of Cape Town, The Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine,
Observatory 7925, South Africa
S
* Supporting Information
ABSTRACT: The K-26 family of bacterial secondary
metabolites are N-modified tripeptides terminated by an
unusual phosphonate analog of tyrosine. These natural
products, produced via three different actinomycetales, are
potent inhibitors of human angiotensin-I converting enzyme
(ACE). Herein we investigate the interkingdom pharmacology
of the K-26 family by synthesizing these metabolites and
assessing their potency as inhibitors of both the N-terminal
and C-terminal domains of human ACE. In most cases,
selectivity for the C-terminal domain of ACE is displayed. Co-
crystallization of K-26 in both domains of human ACE reveals the structural basis of the potent inhibition and has shown an
unusual binding motif that may guide future design of domain-selective inhibitors. Finally, the activity of K-26 is assayed against a
cohort of microbially produced ACE relatives. In contrast to the synthetic ACE inhibitor captopril, which demonstrates broad
interkingdom inhibition of ACE-like enzymes, K-26 selectively targets the eukaryotic family.
KEYWORDS: Angiotensin-I converting enzyme (ACE), K-26, bacterial dicarboxypeptidase, ACE inhibitor
selectivity within the K-26 family and the chemical ecological
significant number of secondary metabolites produced by
Abacteria are potent and selective inhibitors of enzymes and roles of these compounds across kingdoms.
receptor targets in organisms classified in other kingdoms.1
Whether these activities are the result of adaptive evolutionary
pressures or coincidence is unknown, but increasing evidence is
accumulating that microbial secondary metabolites play central
roles in interkingdom chemical ecological relationships.2 The
significance of this interkingdom pharmacology is also evident
in the role of bacterial natural products in clinically approved
therapies. A striking example of such interkingdom activity is
the microbial natural products, K-26 (1) and related
compounds (2−7), which are produced by actinomycetes
isolated from soil-based ecosystems3−6 (Figure 1). These
tripeptides contain a conserved terminal phosphonic acid
analog of tyrosine, (R)-1-amino-2-(4-hydroxyphenyl)-
ethylphosphonic acid ((R)-AHEP, 8). This nonproteinogenic
amino acid, unique to this family, is the zinc-binding
pharmacophore that renders these compounds among the
most potent reported natural product inhibitors of human
angiotensin-I converting enzyme (ACE). ACE is a central
player in mammalian physiology and a clinical target for the
modulation of blood pressure via the renin angiotensin
aldosterone system. The potency of the K-26 family against
ACE, produced by a soil microbe for unknown purposes, raises
questions pertaining to the structural origin of the potency and
The importance of ACE inhibitors to human health has been
emphasized by a World Health Organization World Health
Statistics 2012 report. Hypertension is directly responsible for
12.8% of deaths worldwide and is a high-risk condition for
stroke and coronary heart disease.7 ACE inhibitors were
successfully developed based on a paradigm of rational drug
design in the 1970s. However, current ACE-targeting therapies
for hypertension are not without deleterious side effects such as
cough and angioedema,8 which are likely a consequence of
elevated levels of bradykinin and substance P,9,10 along with off-
target effects of ACE inhibitors on tissue specific homologues
of ACE and other carboxypeptidases distributed throughout
mammalian cell biology. It was only after the discovery of the
first generation ACE inhibitors that human somatic ACE
(sACE) was shown to comprise two tandem homologous
domains, the N- and the C- domain, each with an active site
consisting of an HEXXH zinc binding sequence.11,12 These
domains differ in pharmacological inhibitor preference and
effectiveness at binding and cleaving biologically relevant
Received: November 12, 2013
Accepted: February 4, 2014
Published: February 4, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
publically available, and K-26 metabolites are generated at very
low production levels. The full complement of the six known
naturally occurring K-26 variants were synthesized, with the
exception of the compound SF2513 A (6) (Supporting
Information Scheme 1A), which was not readily accessible to
us using an array of standard peptide coupling conditions
(including HATU, PyBrop, EDC, and through generation of
the acid chloride). Therefore, a close structural analog of
SF2513 A (6) was generated lacking methylation on the
penultimate amide nitrogen, and designated SF2513 D (7)
(Supporting Information Scheme 1B). Both synthetic schemes
yielded the desired K-26 analogs as mixtures of two major
diastereomers, which were separated by preparative C18-HPLC.
1
Comparison of H and 13C NMR acquired from synthetic
(natural diastereomeric) natural products was consistent with
extant literature spectral data, confirming the originally
proposed structures and stereochemistries of these natural
compounds.
Chemically synthesized K-26 family phosponotripeptides
were initially assayed for mammalian ACE inhibition using the
benchmark chromogenic substrate furylacryloyl-phenylalanyl-
glycyl-glycine (FAPGG) and rabbit lung somatic ACE
extract16,18 (Table 1). Consistent with previous data from K-
Figure 1. K-26 analogs of interest in this study. K-26 (1), K-4 (2), 15-
B-2 (3), SF2513 B (4), SF2513 C (5), and SF2513 A (6) have been
isolated from actinomycete cultures and noted for their potent ACE
inhibitory activity. SF2513 D (7) is a synthetic analog used as a
replacement for SF2513A in this study. All analogs are characterized
by a nonproteogenic phosphonate analog of tyrosine, 1-amino-2-(4-
hydroxyphenyl)ethylphosphonic acid (AHEP) (8).
substrates.13,14 Furthermore, a tissue-specific isoform, testicular
ACE (tACE), has one active site and a single domain which,
aside from a unique N-terminal leader sequence, is identical to
the C-domain of sACE.15 Correspondingly, domain-selective
inhibitors have potential as biochemical and physiological
probes of ACE isoform function, and as improved therapies for
hypertension.
Table 1. ACE Inhibitory Activity of Synthesized K-26
Variants
IC50 data (nM)
Ki data (nM)
a
b
b
b
b
compd
K-26
sACE
C-dom
N-dom
C-dom
N-dom
d
d
e
f
d
25
370
140
100
35
11
110
11
NT
NT
21
75
NT
NT
33
e
d
c
g
K-4
590
220
e
e
e
d
e
c
15-B-2
54
52
Here we investigate the structural basis for K-26 inhibition of
human ACE and the domain selectivity within the K-26 family
of secondary metabolites. Chemical synthesis of five naturally
produced structural variants of K-26 and analysis of their
domain-selective inhibition properties facilitates the determi-
nation of the structure−activity relationships within the natural
product family. The cocrystallization of the most potent
member K-26, in both N- and C-domains, provides the first
structural data of a natural product inhibitor in human ACE and
reveals a new mode of inhibitor binding. To investigate
interkingdom structure−activity relationships, we also explore
the activity of K-26 and synthetic ACE inhibitors against a
panel of bacterial zinc metalloenzyme analogs of human ACE.
Structural departures from the K-26/hACE inhibitor complex,
engendered either within the inhibitor or the enzyme, resulted
in significantly decreased binding affinity, confirming previous
studies.16 Understanding the biochemical and structural basis
for K-26 inhibition provides an avenue for the future
development of domain-selective inhibitors for improved
ACE-targeting therapies and the groundwork for subsequent
studies to elucidate the biological mechanism for the selectivity
of K-26 for a mammalian enzyme.
Members of the K-26 family of natural products were
originally discovered using bioassay guided fractionation of
crude extracts via mammalian ACE inhibition. The tripeptide
K-26 is produced by Astrosporangium hypotensionis (NRRL
2379),5,17 the SF2513 series of characterized analogs are
produced by Streptosporangium nondiastiaticum SF2513,3 and
K-4 and 15-B-2 are produced by Actinomadura spiculosospora,4,6
all of which are genera within the taxonomically diverse
Streptosporangineae suborder of soil actinobacteria. With the
exception of A. hypotensionis, the producing organisms are not
d
c
e
f
d
d
SF2513 B
SF2513 C
SF2513 D
12
76
e
e
7
88
12
170
NT
5200
NT
NT
NT
a
Rabbit lung sACE was tested for inhibition with the substrate
b
FAPGG. Z-FHL was used as a substrate with purified N- and C-
domain ACE constructs. Standard deviations, reported in the
c
Supporting Information on pp S89−S93, are as follows: 1−10%.
d
e
f
g
10−25%. 25−50%. 50−75% of the mean. NT is not tested.
26 SAR studies, diastereomers terminated by the naturally
occurring (R)-AHEP amino acid were 10- to 400-fold more
potent than those terminated by (S)-AHEP (Supporting
Information Table 1). With the exception of SF2513 D
(desmethyl SF2513 A), all synthesized natural products were
potent nanomolar inhibitors of somatic rabbit lung ACE.
SF2513 A was previously reported to be a nanomolar inhibitor
of ACE, with the initial structure of SF2513 A being elucidated
3
1
using H NMR, 13C NMR, and chemical degradation. With
these results in mind, if the originally published structure of
SF2513 A is correct, the N-methyl substituent must have an
important role in promoting binding of SF2513 A in the active
site of ACE. In general, N-acetylated family members were
markedly more potent than their respective N-methylated
congeners using the substrate FAPGG. Of the N-acetylated
variants, K-26 was found to be the most potent inhibitor of
rabbit lung somatic ACE, with an IC50 of 25 nM, followed by
SF2513 C and then SF2513 B.
Each of the synthesized natural products that exhibited
nanomolar somatic ACE inhibition were also tested for domain
selectivity by measuring the IC50 of inhibition using the
substrate Cbz-Phe-His-Leu-OH (Z-FHL)19−21 with purified N-
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ACS Medicinal Chemistry Letters
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Figure 2. K-26 binding in both domains of ACE. (A) K-26 (green) bound in the N-domain of ACE. (B) K-26 (cyan) bound in the C-domain of
ACE. Residues involved in K-26 interactions are shown as gray sticks. The zinc ion is shown as an orange sphere and water molecules in red.
Potential hydrogen interactions and water mediated ones are presented with black and red dashed lines, respectively. Omit maps for K-26 bound to
the N- and C-domains are shown at the 1σ contour level.
domain and C-domain human ACE constructs. Analogs
showing promising domain selectivity were reevaluated via
measurement of the inhibition constant Ki. The N-methylated
analogs, K-4 and 15-B-2, did not exhibit a large degree of
domain-selectivity, and SF2513 B only showed a slight
preference for the C-domain, whereas N-acetylated analogs
K-26 and SF2513 C showed approximately 7- and 15-fold
higher affinities for the C-domain, respectively.
To further understand the structural basis for inhibition of
human ACE by the most potent member of this family of
bacterial natural products, K-26 was separately cocrystallized in
the N-domain and the C-domain. As shown in the resulting
crystal structures, K-26 adopts a similar conformation in both
domains (Figure 2A and B), conserving nearly identical
potential hydrogen bond networks between the two domains
(Supporting Information Table 2). K-26 has a unique binding
motif, as it occupies the “non-prime” binding pockets of this
mammalian ACE construct (Figure 3). The phosphonate
coordinates the zinc, the side chain of the AHEP sits in the S1
binding pocket, the tyrosine side chain fills the S2 binding
pocket, and the N-acetyl isoleucine occupies a S3 binding
pocket. Other known inhibitors of the enzyme, including the
clinically relevant captopril and lisinopril, and the synthetic
phosphinic inhibitor RXPA380, fill the “prime” binding
pockets.22−24 The binding pattern of K-26 in human ACE is
almost identical to that adopted by K-26 in the active site of
Drosophila ACE (AnCE).25
The shared activity of K-26 family metabolites is largely
determined by the terminal nonproteinogenic amino acid (R)-
AHEP, which has been demonstrated to be an essential
component for potent ACE inhibitory activity. On its own,
AHEP does not possess potent activity (IC50 > 1 μM);
however, replacement of AHEP by tyrosine causes a 1500-fold
decrease in ACE inhibitory activity.16 Alteration of stereo-
chemistry also decreases the activity 10-fold. The crystal
structure reveals the importance of the phosphonate for potent
binding, as this is shown to coordinate with the zinc in the
active site of the enzyme and form a hydrogen bond network
with several conserved amino acids in both domains of ACE.
The AHEP side chain fits into the S1 binding pocket, where the
aromatic group of AHEP may form hydrophobic interactions
with Val 518 (Thr 496 in N-ACE). Furthermore, the high
resolution of the crystallographic data allowed for the
Figure 3. Binding motifs of K-26, captopril, and RXPA380 in the
active site of ACE. K-26 is shown in cyan, captopril (PDB 1UZF) in
green, and RXPA380 in pink (PDB 2OC2). Zinc is shown as an
orange sphere. The binding pockets of the enzymes are labeled within
the surface-rendered catalytic channel of C-domain ACE.
identification of several key water molecules which may have
a role in inhibitor binding. The AHEP seems to be stabilized at
the active site by a network of water molecules anchoring K-26
at S1 in both crystal structures. Interestingly, additional water
molecules interacting with K-26 were visible in the C-domain.
In the S2 binding pocket, the tyrosine side chain may form
conserved aromatic stacking interactions with His 387 and His
410 (His 365 and His 388 in N-domain) and hydrophobic
interactions with Phe 391 (Tyr 369 in N-ACE). Interestingly,
synthesis of a phosphinic inhibitor library analogous to the
inhibitor RXPA380 (Figure 3) showed that a Phe or Tyr in the
S2 position of the inhibitor encouraged the most ACE binding
potency in both domains, suggesting the importance of these
interactions.24 Hydrogen bonds between the backbone of the
tyrosine in K-26 and the backbone of Ala 356 (Ala 334 in N-
ACE) are also evident. Additionally, the hydroxyl group of the
tyrosine side chain makes water-mediated interactions with the
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backbone of Arg 402 and Gly 404 (Figure 2B). These two
residues are conserved in both domains of ACE; however, Glu
403 (C-domain) is occupied by Arg 381 in the N-domain. The
long polar side chain of Arg 381 may thus prevent these water-
mediated interactions in the N-domain, decreasing the binding
affinity for K-26.
The remaining structural features of the K-26 class of natural
products are the third amino acid side chain and the N-terminal
substitution, which occupies the S3 position. The N-terminal
substitution has an effect on potency as shown with K-4 and 15-
B-2, which have an N-terminal methyl group and inhibit
somatic ACE with a 5−10-fold decrease in ACE inhibition, than
the corresponding N-acetyl analogs. The origin of this activity
difference likely derives from the ability of the N-acetyl
carbonyl to form a hydrogen bond with the backbone nitrogen
of Asp 358 (Asp 336 in N-domain) and the OH from Tyr 369
in the N-domain. These hydrogen bonding interactions with
the N-acetyl tail are likely important interactions for the
increased potency of the N-acetylated analogs in comparison to
cationic N-methylated congeners.
Figure 4. Phylogenetic tree comparing ACE and related bacterial
dicarboxypeptidases (DCP). Dicarboxypeptidases which have been
overexpressed and purified in this study are shown in bold text. The
optimal tree with the sum of branch length = 6.39523051 is shown.
Astrosporangium hypotensionis, the organism from which the
tripeptide K-26 was originally isolated. MR1DCP was selected
for the high similarity with human ACE, as it has a 43%
sequence identity with both the N- and C- domains of human
ACE. All three enzymes contain the characteristic HEXXH
zinc-binding domain, characteristic of the zincin family of zinc
metalloproteases. The dicarboxypeptidases EcDCP and the K-
26 producer K26DCP both are classified specifically as a M3
zinc metalloproteases due to the presence of two additional
glutamate residues, 30 and 37 amino acids C-terminal to the
HEXXH zinc-binding domain that are believed to aid in
Previous studies suggest that domain selectivity can be
influenced by amino acids side chains occupying the S1 and S2
binding pockets of the enzyme.24,26,27 As K-26 is shown in the
crystal structure to occupy the S1 and S2 pockets and a putative
S3 binding pocket, this S3 binding pocket and area extending
out further from the catalytic zinc is an unexplored area for
inhibitor design. Synthetic analogs could focus on the S3
position where potential aromatic interactions with Trp 357
(Trp 335 in N-domain ACE), might provide enhanced potency.
The crystal structures presented here have shown that the
preferential C-domain selectivity observed with K-26 is
provided by hydrophobic interactions at the S2 site along
with water-mediated interactions with S2 and the N-acetyl. The
nonconserved residues Glu 403 and Phe 391 (Arg 381 and Tyr
369 in N-domain ACE) seem again to be key positions to
enhance domain selectivity.26 Although the domain selectivity
of ACE inhibitors has been achieved through accessing peptidic
inhibitors capable of occupying both the prime and nonprime
binding pockets, the potential of a domain-specific inhibitor
using only the nonprime binding sites has not been studied.
As K-26 family natural products are of microbial origin, and
likely have not evolved to specifically inhibit human ACE, we
endeavored to assay their interkingdom ACE-like activity.
Biochemical characterization of several bacterial dicarboxypep-
tidases shows remarkable similarities in substrate and inhibitor
preferences in comparison to mammalian ACE.28,29 In support
of these observations, sequence alignment of several bacterial
dicarboxypeptidases reveals a high degree of sequence
homology between mammalian ACE and bacterial dicarbox-
ypeptidases (Supporting Information Table 3 and Figure 1).
This encouraged us to investigate the potential of the known
potent mammalian ACE inhibitor, K-26, as an inhibitor of
bacterial analogs of ACE. In order to explore the scope of
interkingdom activity of K-26, we have recombinantly ex-
pressed, purified, and assayed a panel of ACE-like enzymes
represented by phylogenetically distinct, bacterial zinc-depend-
ent dicarboxypeptidases for inhibition by captopril and K-26
(Figure 4).
coordinating the zinc in the active site of the enzyme.30
A
glutamate residue present 29 residues downstream from the
HEXXH zinc-binding domain, believed to be a ligand for zinc
coordination in both MR1DCP and mammalian ACE,
distinguishes these enzymes within the M2 subfamily of ACE
zinc metalloproteases.
All three bacterial dicarboxypeptidases were found to
efficiently hydrolyze the substrate FAPGG. Captopril was
shown to be a potent, low nanomolar inhibitor of all the
purified overexpressed dicarboxypeptidases (Table 2). How-
Table 2. Inhibition of Overexpressed Bacterial
Dicarboxypeptidases by K-26
dicarboxypeptidase
type
captopril IC50 (M)
K-26 IC50 (M)
somatic ACE
K26DCP
EcDCP
M3
M2
M2
M3
7.7 × 10−9
5.2 × 10−9
1.6 × 10−8
9.2 × 10−9
2.5 × 10−8
4 × 10−5
1.5 × 10−4
1.1 × 10−5
MR1DCP
ever, K-26 was found to be a potent inhibitor of the mammalian
ACE but a poor inhibitor of the bacterial ACE-like enzymes,
including the dicarboxypeptidase encoded in the genome of the
K-26 producer. Additionally, in previous studies, in the insect
homologue of ACE, AnCE (Drosophila melanogaster), captopril
has been shown to be a more potent inhibitor (Ki of 1.1 nM) of
the enzyme than K-26 (Ki of 160 nM).25,31 Sequence alignment
of human tACE with the most similar bacterial enzyme,
MR1DCP, reveals five mutations in residues near the K-26
binding site (Supporting Information Figure 2). In the binding
pocket, F391, G404, and F512 in tACE are mutated to Q396,
S409, and Y515, altering the polarity of the pocket. However,
the difference in potency between the mammalian and bacterial
enzyme may arise from the mutation of F391 to Q396, which
increases the net negative charge in the active site and likely
deleteriously effects binding of our negatively charged substrate,
K-26.
Three bacterial enzymes were chosen: dicarboxypeptidases
from the K-26 producing organism (K26DCP), from E. coli st.
K-12 sbstr. MG1655 (EcDCP),28 and from Schwanella onedesis
str. MR-1 (MR1DCP). K26DCP is the only putative zinc
metallopeptidase present in the genome of the actinomycete
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Letter
In conclusion, we have illuminated the structure−activity
relationships of the K-26 family of natural products in
mammalian ACE, the enzyme target that was originally used
in screening for their discovery from actinomycetes. These
results provide structural insight that can be applied toward
future development of potential domain-selective inhibitors
through improving binding in the unexplored nonprime
pockets of ACE. The inhibition data from bacterial ACE-like
dicarboxypeptidases, together with previous data from
Drosophila ACE, emphasize that, among all known groups of
ACE-like dicarboxypeptidases, mammalian ACE seems
uniquely inhibited by K-26. Indeed, the combined enzyme
and substrate structural activity data suggest that the natural
target for K-26 possesses an active-site architecture similar to
mammalian ACE, prompting questions regarding potential
interkingdom chemical ecological roles for the natural target of
K-26. Future studies will investigate the potential for generation
of improved domain selective analogs building from the AHEP
pharmacophore into the unique binding pose of this family of
natural products, and work to identify potential chemical
ecological targets for AHEP functional natural products.
26 dicarboxypeptidase; MR1DCP, Schwanella onedesis str. MR-
1 dicarboxypeptidase; sACE, somatic angiotensin converting
enzyme; tACE, testicular angiotensin converting enzyme; Z-
FHL, Cbz-Phe-His-Leu-OH
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic details of the K-26 family of natural products,
including NMR spectra for final products and synthetic
intermediates, overexpression and purification of ACE con-
structs and bacterial dicarboxypeptidases, experimental meth-
ods for IC50 and Ki measurements, sequence homology and
alignment for ACE-like enzymes of interest, primers used in the
study, IC50 curves, and Dixon plots. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: brian.o.bachmann@vanderbilt.edu.
■
Present Address
†(A.M.) Department of Biochemistry, University of Hyderabad,
India 500046.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by the University of Cape Town and
the South African National Research Foundation to E.D.S. The
crystallographic work reported here was supported by the
Medical Research Council (U.K.) through a project grant
(number G1001685) and a Wellcome Trust (U.K.) equipment
grant (number 088464) to K.R.A. G.J.K. was supported in part
by the D. Stanley and Ann T. Tarbell Endowment fund. Also,
K.R.A. wishes to thank the scientists at PX station I03,
Diamond Light Source, Didcot, Oxon (U.K.) for their support
during X-ray diffraction data collection. B.O.B. and G.J.K were
supported by National Institutes of Health Grant GM077189,
the Vanderbilt Institute of Chemical Biology, and the
Vanderbilt International Office. We thank Dr. Cody Goodwin
for high resolution mass spectral data acquisition.
ABBREVIATIONS
■
AHEP, 1-amino-2-(4-hydroxyphenyl)ethylphosphonic acid;
DCP, dicarboxypeptidase; EcDCP, E. coli dicarboxypeptidase;
FAPGG, furylacryloyl-phenylalanyl-glycyl-glycine; K26DCP, K-
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