Synergy Potential of Ursolic Acid-Based Hybrid Molecules

Abstract

Background
Ursolic acid (UA, 3β-hydroxy-urs-12-en-28-oic acid), a pentacyclic triterpenoid from various medicinal plants, has been blessed with proven biological effects, including anti-inflammatory, anticancer, antidiabetic, antioxidant and antibacterial, but its bioavailability and solubility limit its clinical application.

Objective
Synthesis of UA-based hybrid molecules to explore their antibacterial and synergy potential in combination with azithromycin (AZT) for the treatment of multidrug-resistant (MDR) bacterial infections.

Methods
Hybrid molecules of UA with menthol, eugenol, and nalidixic acid (NAL) along with some other ester derivatives were synthesized, and evaluated for their antibacterial and synergy potential in combination with AZT against the clinical isolate of Escherichia coli in terms of their minimum inhibitory concentration (MIC), fold reduction in MIC, fractional inhibitory concentration index (FICI) and type of interaction. In silico screening of pharmacokinetic parameters, docking affinity against efflux pump proteins AcrA, AcrB, and TolC was performed on the most potent derivative 7 (3-O-nalidixoyl UA).

Results
Derivative 7 showed MIC of 62.5 µg/mL and a strong synergistic effect with AZT reducing the MIC of AZT from 100 to 0.19 µg/mL (512-fold reduction) against E. coli at a concentration of 12.5 µg/mL. Other derivatives neither showed antibacterial activity of their own (MIC > 1000 µg/mL) nor any significant synergistic interaction in combination with AZT. The in silico studies on 7 revealed improved druggability parameters over the parent UA and NAL.

Conclusion
The findings highlight derivative 7 as strong synergistic agent in combination with AZT which may be further investigated to render its efficient use for the treatment of MDR bacterial infections.

Full text

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Letters in Drug Design & Discovery, XXXX, XX, 1-10 1
RESEARCH ARTICLE
1570-1808/XX $65.00+.00 © XXXX Bentham Science Publishers
Synergy Potential of Ursolic Acid-Based Hybrid Molecules
Krishna N. Mishra1, Sonam Singh1, Harish C. Upadhyay1,#,*, Swaroop Sanket2, Manoj Kumar3,
Umme Yashmeen4, Rajni Kant3 and Gaurav R. Dwivedi3,#
1Laboratory of Chemistry, Department of Applied Sciences, Rajkiya Engineering College (Affiliated with Dr. A.P.J. Ab-
dul Kalam Technical University, Lucknow), Churk, Sonbhadra-231206, India; 2Department of Microbiology, Utkal Uni-
versity, Bhubaneswar, Odisha- 751004, India; 3ICMR-Regional Medical Research Center, Gorakhpur-273013, India;
4Department of Biotechnology, Deen Dayal Upadhyaya Gorakhpur University, Gorakhpur-273009, India
Abstract: Background: Ursolic acid (UA, 3β-hydroxy-urs-12-en-28-oic acid), a pentacyclic triterpenoid
from various medicinal plants, has been blessed with proven biological effects, including anti-
inflammatory, anticancer, antidiabetic, antioxidant and antibacterial, but its bioavailability and solubility
limit its clinical application.
Objective: Synthesis of UA-based hybrid molecules to explore their antibacterial and synergy potential in
combination with azithromycin (AZT) for the treatment of multidrug-resistant (MDR) bacterial infec-
tions.
Methods: Hybrid molecules of UA with menthol, eugenol, and nalidixic acid (NAL) along with some
other ester derivatives were synthesized, and evaluated for their antibacterial and synergy potential in
combination with AZT against the clinical isolate of Escherichia coli in terms of their minimum
inhibitory concentration (MIC), fold reduction in MIC, fractional inhibitory concentration index (FICI)
and type of interaction. In silico screening of pharmacokinetic parameters, docking affinity against efflux
pump proteins AcrA, AcrB, and TolC was performed on the most potent derivative 7 (3-O-nalidixoyl
UA).
Results: Derivative 7 showed MIC of 62.5 µg/mL and a strong synergistic effect with AZT reducing the
MIC of AZT from 100 to 0.19 µg/mL (512-fold reduction) against E. coli at a concentration of 12.5
µg/mL. Other derivatives neither showed antibacterial activity of their own (MIC > 1000 µg/mL) nor any
significant synergistic interaction in combination with AZT. The in silico studies on 7 revealed improved
druggability parameters over the parent UA and NAL.
Conclusion: The findings highlight derivative 7 as strong synergistic agent in combination with AZT
which may be further investigated to render its efficient use for the treatment of MDR bacterial infections.
A R T I C L E H I S T O R Y
Received: October 28, 2021
Revised: April 01, 2022
Accepted: July 18, 2022
DOI:
10.2174/1570180819666220929143234
Keywords: Hybrid molecules, nalidixic acid, azithromycin, ursolic acid, antibacterial, multidrug-resistant, drug resistance re-
versal.
1. INTRODUCTION
The rapid spread of antimicrobial-resistant bacterial
pathogens marks the onset of a possible severe global health
crisis [1-3]. Antibiotics are becoming increasingly ineffec-
tive in treating infections because of the development of new
resistance mechanisms acquired by pathogenic bacteria [4,
5]. The situation of antimicrobial resistance (AMR) in de-
veloping countries is at an alarming rate due to gross abuse
*Address correspondence to this author at the Laboratory of Chemistry,
Department of Applied Sciences, Rajkiya Engineering College (Affiliated
with Dr. A.P.J. Abdul Kalam Technical University, Lucknow), Churk,
Sonbhadra-231206, India; Tel/Fax: +91-9415164409;
E-mail: harishcu@gmail.com
#These authors contributed equally to this work.
in the use of antimicrobials without any control and effective
monitoring [6, 7]. WHO has declared AMR as one of the top
ten global public health threats facing humanity which re-
quires urgent multisectoral action to achieve sustainable de-
velopment goals [8]. In view of drug resistance, the WHO
listed 12 human pathogenic bacteria (dirty dozen), including
Acinetobacter baumannii, Pseudomonas aeruginosa and
Enterobacteriaceae on which the existing antibiotics are
becoming critically ineffective [9, 10]. The current rate of
emergence and spread of AMR expects to reach 10 million
deaths with an estimated economic cost of $100 trillion
annually by the year 2050 [10, 11]. It is noticeable that the
current antibiotic pipeline is almost dry, also the molecules
under trial are not robust enough to address the current and
projected clinical needs [12, 13]. The study indicates that in

8 Letters in Drug Design & Discovery, XXXX, Vol. XX, No. XX Mishra et al.
Careful analysis of the results of in silico studies indi-
cates that 7 follows the Lipinski Rule of five with violations
for molecular weight and logP values. These violations do
not limit this molecule to being a potential drug candidate as
being a natural product-derived molecule, it may be the sub-
strate for biological transporters allowing it to bypass the
limits of the rule of five for intestinal wall permeability [52].
Further, the binding affinity of 7 with all three receptors is
higher than the parent compounds NAL and UA. The results
of in silico docking and ADMET studies are in good agree-
ment with the assumption that the hybrid molecules may
interact with the target receptors with higher affinity than an
individual bioactive molecule and hence may be a better
alternative to overcome the problem of MDR.
CONCLUSION
Ester derivatives of UA were semi-synthesized, affording
hybrid molecules of UA with menthol, eugenol and NAL in
mild yield. The derivatives were evaluated for their
antibacterial and drug resistance reversal potential in
combination with conventional antibiotic AZT against the
clinical isolates of Escherichia coli. The derivative 7, a
hybrid molecule of UA with NAL was found to be most
potent in reducing MIC of AZT by 512-fold. In further in
silico studies, the 7 showed the highest binding affinity with
receptors AcrA, AcrB and TolC involved in the efflux pump
mechanism responsible for MDR. Our study suggests that
after detailed investigations, derivative 7 may find its poten-
tial use as a co-drug in the treatment of multidrug-resistant
bacterial infections.
LIST OF ABBREVIATIONS
ADME = Absorption, Distribution, Metabolism and
Excretion
DCC = Dicyclohexylcarbodiimide
DMAP = 4-dimethylaminopyridine
FICI = Fractional inhibitory concentration index
IC50 = Half Maximal Inhibitory Concentration
MDR = Multidrug Resistant
MIC = Minimum Inhibitory concentration
MRSA = Methicillin-resistant Staphylococcus
aureus
NAL = Nalidixic acid
SAR = Structure-Activity Relationship
UA = Ursolic acis
USFDA = United States Food and Drug
Administration
WHO = World Health Organization
ETHICS APPROVAL AND CONSENT TO
PARTICIPATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the
basis of this research.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS!
The authors confirm that the data supporting the findings
of this study are available within the article and it’s
supplementary material.
FUNDING!
None.!
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
HCU thanks Dr. S.K. Srivastava, Scientist CSIR-Central
Institute of medicinal and Aromatic Plants, Lucknow (India),
for providing ursolic acid and helping in spectroscopic anal-
ysis of the samples and Director, Rajkiya Engineering
College, Sonbhadra for his constant encouragement and
providing all types of facilities for this study.
SUPPLEMENTARY MATERIAL
A detailed description of the receptor proteins AcrA,
AcrB and TolC and ligand-protein interaction profiles have
been provided as supporting information.
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