Ent-Beyerane Diterpenes as a Key Platform for the Development of ArnT-Mediated Colistin Resistance Inhibitors

Article abstract of DOI:10.1021/acs.joc.0c01459

Colistin is a last-resort antibiotic for the treatment of multidrug resistant Gram-negative bacterial infections. Recently, a natural ent-beyerene diterpene was identified as a promising inhibitor of the enzyme responsible for colistin resistance mediated by lipid A aminoarabinosylation in Gram-negative bacteria, namely, ArnT (undecaprenyl phosphate-alpha-4-amino-4-deoxy-l-arabinose arabinosyl transferase). Here, semisynthetic analogues of hit were designed, synthetized, and tested against colistin-resistant Pseudomonas aeruginosa strains including clinical isolates to exploit the versatility of the diterpene scaffold. Microbiological assays coupled with molecular modeling indicated that for a more efficient colistin adjuvant activity, likely resulting from inhibition of the ArnT activity by the selected compounds and therefore from their interaction with the catalytic site of ArnT, an ent-beyerane scaffold is required along with an oxalate-like group at C-18/C-19 or a sugar residue at C-19 to resemble L-Ara4N. The ent-beyerane skeleton is identified for the first time as a privileged scaffold for further cost-effective development of valuable colistin resistance inhibitors.

Full text of DOI:10.1021/acs.joc.0c01459

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Article
Ent-beyerane diterpenes as a key platform for the
development of ArnT-mediated colistin resistance inhibitors
Deborah Quaglio, Maria Luisa Mangoni, Roberta Stefanelli, Silvia Corradi, Bruno
Casciaro, Valeria Vergine, Federica Lucantoni, Luca Cavinato, Silvia Cammarone,
Maria Rosa Loffredo, Floriana Cappiello, Andrea Calcaterra, Silvia Erazo, Francesca
Ghirga, Mattia Mori, francesco imperi, Fiorentina Ascenzioni, and Bruno Botta
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c01459 • Publication Date (Web): 27 Jul 2020
Downloaded from pubs.acs.org on August 3, 2020
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Ent-beyerane diterpenes as a key platform for the development of
ArnT-mediated colistin resistance inhibitors
‡
‡
†
#
Deborah Quaglio , Maria Luisa Mangoni , Roberta Stefanelli ±,‡, Silvia Corradi †,¥, Bruno Casciaro
†
¥ ,Valeria Vergine , Federica Lucantoni ±, Luca Cavinato ± , Silvia Cammarone †, Maria Rosa Loffre-
do , Floriana Cappiello #, Andrea Calcaterra , Silvia Erazo , Francesca Ghirga *, Mattia Mori *,
#
†
§
¥
^
Francesco Imperi ‡, Fiorentina Ascenzioni ± and Bruno Botta †*
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† Department of Chemistry and Technology of Drugs, “Department of Excellence 2018−2022”, Sapienza University of
Rome, P.le Aldo Moro 5, 00185 Rome, Italy
# Laboratory affiliated to Pasteur Italia-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza
University of Rome, P.le Aldo Moro 5, Rome 00185, Italy
± Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Laboratory affiliated to Pas-
teur Italia-Fondazione Cenci Bolognetti, Via dei Sardi 70, 00185 Rome, Italy
‡ Department of Science, Roma Tre University, Viale Guglielmo Marconi 446, 00146 Rome, Italy
¥ Center For Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Viale Regina Elena, 291, 00161 Rome, Italy
§ Department of Pharmacological and Toxicological Chemistry, Faculty of Chemical and Pharmaceutical Sciences, Uni-
versity of Chile, Santiago, Chile
^ Department of Biotechnology, Chemistry and Pharmacy, “Department of Excellence 2018−2022”, University of Si-
ena, via Aldo Moro 2, 53100 Siena, Italy
KEYWORDS: Natural products chemistry • Arnt inhibitors • Diterpenes • molecular modeling • Pseudomonas aeruginosa
ABSTRACT: Colistin is a last-resort antibiotic for treatment of multidrug resistant Gram-negative bacterial infections. Re-
cently, a natural ent-beyerene diterpene was identified as a promising inhibitor of the enzyme responsible for colistin re-
sistance mediated by lipid A aminoarabinosylation, in Gram-negative bacteria, namely ArnT (undecaprenyl phosphate-
alpha-4-amino-4-deoxy-L-arabinose arabinosyl transferase). Here, semi-synthetic analogs of hit were designed, synthetized
and tested against colistin-resistant Pseudomonas aeruginosa strains including clinical isolates, to exploit the versatility of
the diterpene scaffold. Microbiological assays coupled with molecular modeling indicated that for a more efficient colistin
adjuvant activity, likely resulting from inhibition of the ArnT activity by the selected compounds and therefore from their
interaction with the catalytic site of ArnT, an ent-beyerane scaffold is required along with an oxalate like group at C-18/C-
19, or a sugar residue at C-19 to resemble L-Ara4N. The ent-beyerane skeleton is identified for the first time as a privileged
scaffold for further cost-effective development of valuable colistin resistance inhibitors.
1
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The Journal of Organic Chemistry
Introduction. Control of infections has long been a serious
the lifetime of these antibacterial drugs and minimize the
impact of the appearance of resistance. Medicinal plants
are an extraordinary rich storehouse of bioactive second-
ary metabolites with a large-spectrum of enzyme inhibito-
ry potential.18-21 They can work as ligands and bind to an
enzyme blocking its activity with an irreversible or re-
versible process. Recently, a unique in house library of nat-
ural products available in our group was screened in silico
against the catalytic site of the ArnT enzyme to identify pu-
tative inhibitors of Ara4N-dependent colistin resistance
mechanism.22 This led to the selection of the ent-beyerene
diterpene 1 (formerly known as BBN149), isolated from
the leaves of Fabiana densa var. ramulosa, with a colistin
adjuvant activity versus colistin-resistant P. aeruginosa
strains, without any significant effect on colistin-
susceptible strains.22, 23 Here we exploit the versatility of
the diterpene scaffold by designing, synthesizing and test-
ing several analogs of 1. Through the combination of com-
putational modeling, organic synthesis and biological eval-
uations in a concerted multidisciplinary strategy, we ex-
plore structure-activity relationships (SAR) around the ini-
tial diterpene hit 1, and validate its scaffold for the produc-
tion of novel antibacterial agents for the treatment of col-
istin-resistant infections. Chemical analogs featuring a
structurally related diterpene core were synthesized and
screened in vitro against colistin-resistant P. aeruginosa
strains including clinical isolates, while the putative bind-
ing mode against the ArnT enzyme was investigated by
molecular modeling. Herein, the ent-beyerane skeleton is
identified for the first time as a privileged scaffold for fur-
ther development and optimization of valuable colistin re-
sistance inhibitors.
1
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clinical concern and the discovery of antibiotics during the
1930s to 1960s opened the door to current antimicrobial
drug discovery.1 Nevertheless, the excessive use of antibi-
otics in humans and in the livestock, the poor sanitation
and the release of non-metabolized antibiotics in the sur-
roundings, have threatened most of the recorded advanc-
es.2 Together with the unavailability of newer drugs these
factors have contributed to the genetic selection pressure
for the appearance and evolution of multidrug resistant
(MDR) bacteria with global spread in the last decades,
which stand for a serious public health emergency and a
current challenge with considerable economic impacts.3, 4
As an example of MDR microorganisms, the Gram-negative
bacterium Pseudomonas aeruginosa is one of the leading
causes of nosocomial and chronic infections, especially in
cystic fibrosis patients where it concurs to lung disease
which accounts for more than 85% mortality.5 P. aerugino-
sa has intrinsic resistance to a large number of antibiotics,
because of the low permeability of its outer membrane
(OM), the presence of active efflux pumps and the expres-
sion of antibiotic-modifying enzymes.6 Furthermore, at the
infectious site, it often lives within biofilm communities
that make bacteria recalcitrant to stressful environmental
conditions, antibiotic treatments and to the host immune
clearance.7 Currently, there are very few antipseudomonal
agents in clinical development, while the lack of treatment
options for MDR bacteria has contributed to reconsider
colistin as a last-line antimicrobial therapy, despite its tox-
icity for kidneys and neural tissues.8, 9 Colistin is a cationic
multicomponent lipopeptide that targets lipopolysaccha-
rides (LPS) in the OM of Gram-negative bacteria.10, 11 It ini-
tially interacts with the anionic phosphate headgroups of
the lipid A moiety of LPS, displacing divalent cations i.e.
Ca++ and Mg++ that stabilize adjacent LPS molecules. This is
then followed by destabilization of the OM with subse-
quent disruption of the inner membrane leading to cell
death. Unfortunately, resistance to colistin has been docu-
mented in several case reports.12, 13 This can have devastat-
ing effects if no other therapeutic strategies are uncovered
to combat infections, including those associated with P. ae-
ruginosa in cystic fibrosis lungs. One of the mechanisms of
resistance consists in the covalent modification of LPS by
the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) or
phosphoethanolamine groups to lipid A, which decrease
the overall charge of LPS and, as a result, the binding affini-
ty of the cationic lipopeptide.14 In P. aeruginosa these
changes are controlled by enzymes encoded by the arn op-
eron which is regulated by several two-component sys-
tems. One of these enzymes is the glycosyltransferase ArnT
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Results and discussion. Compound 1 is a tetracyclic ent-
beyerene diterpene which was recently discovered by our
group and patented for its novel colistin adjuvant
activity.23
It
was
isolated
from Fabiana
den-
sa var. ramulosa (Solanaceae), a native shrub of Chile, and,
to the best of our knowledge, this compound is not availa-
ble from other chemical sources than our own in house li-
brary.24, 25 It is the oxaloyl ester of the ent-beyer-15-en-18-
ol (4), which was identified in the same plant along with
other diterpene analogs i.e. the malonoyl (2) and succinoyl
(3) esters.25 (Chart 1) To validate the power of the diter-
pene scaffold as a key platform for further development of
ArnT-mediated colistin resistance inhibitors with im-
proved activity, a large variety of chemical analogs was
produced for SAR studies. In particular, different deriva-
tives of compound 1 were synthesized with the aim to in-
vestigate the role of (i) the length and flexibility of the alkyl
chain of the functional group at C-18; (ii) the chirality of C-
4; (iii) the presence of a sugar unit to mimic L-Ara4N, and
(iv) the unsaturation between C-15 and C-16, on the bio-
logical properties of the original diterpene scaffold. To as-
sess whether the expansion of the alkyl chain between the
carbonyl groups as well as its removal at C-18 could affect
the colistin adjuvant activity, the analogs 2-4 were repur-
posed and some of them prepared according to the semi-
synthetic procedure previously described by using alcohol
4 as starting material.25 However, the semisynthetic ap-
(undecaprenyl
phosphate-alpha-4-amino-4-deoxy-L-
arabinose arabinosyl transferase) which catalyzes the
transfer of L-Ara4N, provided by the lipid carrier un-
decaprenyl phosphate, to lipid A phosphate groups.15 Po-
tentiating the effect of existing antimicrobial compounds
represents a promising approach to address the current
antibiotics crisis and poor efficacy.16 In particular, inhibi-
tors of resistance enzymes offer an alternative avenue to
withstand this threat.17 The combination of such inhibitors
with clinically relevant antibiotics may effectively extend
2
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