Microbial Pathogenesis 179 (2023) 106108

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

A historical, economic, and technical-scientific approach to the current

crisis in the development of antibacterial drugs: Promising role of
antibacterial peptides in this scenario
Yeimer A.S. Guevara a, Maria H.C. Santos b, Francisco I.R. Gomes b, Sheheryar b, Felipe
P. Mesquita a, b, Pedro F.N. Souza a, b, *
a
Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Ceará, Brazil
b
Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, Ceará, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The emergence of antibiotic resistance (AMR) is a global public health problem. According to estimates, drug-
Antibacterial resistance resistant bacteria infect 2 million patients and perish 23,000 annually. To overcome this problem, antimicro­
Antibiotic crisis bial peptides became a potential solution based on a new mechanism of action against bacteria. This article
Multidrug-resistant pathogens
addresses the phenomenon of antibacterial resistance in most of its nuances, responding to historical, technical-
Synthetic antibacterial peptides
scientific, and economic aspects. Likewise, it explores new therapeutic approaches to combat multi-resistant
pathogens, specifically concerning antibacterial peptides, as a potential therapeutic tool to mitigate the cur­
rent crisis of antibacterial drugs. It is expected that, with technological advances, especially with the advent and
adoption of artificial intelligence, there will be an increase in diversified synthetic peptide production, which can
face the challenges that we have in terms of antibacterial drugs.

1. Introduction antibiotics as growth promoters in feed creates selective pressure that

According to the World Health Organization, antibiotic resistance is
a global public health problem [55]. According to estimates in the
United States, drug-resistant bacteria infect 2 million patients and perish
23,000 of them annually [26]. It is estimated that, if no action is taken,
by 2050, antimicrobial resistance (AMR) could result in 10 million
deaths per year worldwide and accumulate at least US$100 trillion in
hospital costs and lost productivity [17].
AMR can be innate or acquired. Thus, antibiotic administration
creates an environment where resistance strains have a selective
advantage over those not [2,37]. Mechanisms involved in AMR include
the production of inactivating enzymes, drug efflux and drug entry in­
hibition, target modification, metabolic pathway alteration and chro­
mosomal or plasmid-mediated removals [42,48].
The unrestricted/excessive misuse in humans and animals acceler­
ated the natural process of evolution that led to AMR, despite increasing
pressure on the animal production sector to limit or completely cease the
use of antimicrobials antibiotics [6,42]. Additionally, the use of
leads to the emergence of resistant bacteria in cattle that can subse­
quently colonize or infect humans [9].
One of the main concerns of the WHO is multi-resistant bacteria due
to their rapid spread and the difficulty of their treatment [13]. The so­
lution to the problem involves, in part, infection prevention and AMR
monitoring, the promotion of rational antimicrobial therapy, and the
development of new antimicrobials [17,42]. Unfortunately, in recent
decades, the discovery and development of new antibiotics have slowed
drastically to such an extent that no new class has been discovered since
the 1980s, only those obtained from the optimization or combination of
already known compounds being marketed [12,30,39]. According to
Spellberg et al. [43], there are 3 leading reasons for discovering new
antibiotics: first, there are scientific challenges for the discovery and
development of new agents; second, compared to other classes of drugs,
antibiotics are economically unattractive for investment in research and
development (R&D) and third, to the difficulties imposed by the Food
and Drug Administration (FDA) for conducting clinical trials with new
antibiotics [43]. Thus, while nearly 4000 immune-oncology agents are

* Corresponding author. Drug Research and Development Center, Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Ceará,
60020-181, Brazil.
E-mail addresses: pedrofilhobio@gmail.com, pedrofilhobio@ufc.br (P.F.N. Souza).

https://doi.org/10.1016/j.micpath.2023.106108
Received 10 March 2023; Received in revised form 4 April 2023; Accepted 5 April 2023
Available online 10 April 2023
0882-4010/© 2023 Elsevier Ltd. All rights reserved.
Y.A.S. Guevara et al. Microbial Pathogenesis 179 (2023) 106108

developing, only about 30–40 new antibacterial compounds are
currently in the clinical trial development phases. Less than 25% of the
current drugs in the clinical development pipeline represent a new class
or act through a novel mechanism and are not active against bacteria
from the ESKAPE group (Enterococcus, Staphylococcus aureus, Klebsiella
species, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter),
critically threatened pathogens according to the WHO [27,28].
Classically, most antibiotics were produced by screening for soil
microorganisms (Waxman Platform), but this limited resource of cul­
turable bacteria was superseded by the 1960s and synthetic approaches
to producing antibiotics. It has not been able to completely replace it,
except for teixobactin, a depsipeptide with excellent inhibitory potential
against Gram-positive pathogens, including resistant strains [22,25].
The combination of widespread multidrug resistance, disenchantment
with natural products as sources of new drugs, lack of success using
synthetic compounds, and changing economic and regulatory issues
conspire to move research investment away from the field of antibiotics.
The result is a growing crisis in antibiotic drug discovery that threatens
modern medicine [58].
Finding new antibiotics depends on the ability of scientists to explore
a new chemical space through novel screening of libraries of diverse and
distinct compounds, targeted synthesis or modification of compounds
with improved physicochemical properties, phenotypic assays, and
other methods that are explicitly adapted to bacterial pathogens [1].
Thus, the problem should be approached in several parallel ways,
requiring the discovery of novel chemical scaffolds that evade the
resistance mechanisms that circulate in pathogens today and the
exploration of non-traditional anti-infective strategies [44,58]. In this
context, the investigation and arrival on the scene of peptides with
antibacterial properties are emerging as an invaluable tool to mitigate
the current crisis of antibacterial resistance.
the 1960s–1970s, its dissemination in the hospital environment made it
urgent to look for new antibiotic compounds [15].
The clinical introduction of a variety of third-generation cephalo­
sporins characterized the 1980s. These agents (mainly cefotaxime, cef­
tizoxime, ceftriaxone, and ceftazidime) had activity against Enterobacter
spp, Citrobacter freundii, and, in the case of ceftazidime, P. aeruginosa,
which were characteristically resistant to β other-lactam strains avail­
able. The widespread use of these agents was associated with the
emergence and dissemination of Gram-negative bacilli, most commonly
Klebsiella pneumoniae, which were resistant to expressing β-lactamases of
extended-spectrum (ESBLs) [37]. Linezolid and daptomycin were
introduced in the 2000s, and their resistance was reported in five years
[24].
Added to the problem of AMR against the antibiotics mentioned
above is the abrupt drop in the production of new molecules with
antibacterial properties (Fig. 2). The discovery and development of an­
tibiotics in recent decades have decreased considerably; for example, 16,
14, 10 and 7 new antibiotics were approved during 1983–1987,
1988–1992, 1993–1997 and 1998–2002, respectively, while only 5 and
2 were approved during 2003–2007 and 2008–2012, respectively
(Fig. 2). This decline was due to the decrease in antibiotic research and
development in major pharmaceutical companies; investment in
developing new antibiotics has been hampered by uncertain life cycles
(associated with the development of antibiotic resistance) of new anti­
biotics [5,11].

2. Antibacterial resistance phenomenon and crisis in the

development of new antibiotic molecules

2.1. Timeline of antimicrobial resistance phenomena

After introducing sulfonamides and penicillin, their respective

resistance was reported in a few years, in mid-1945. Resistance to
tetracycline, streptomycin and chloramphenicol was found in the 1950s
(Fig. 1) [24,36].
Between the 1940’s and 1960’s, several antibiotics were discovered
through screenings of natural microbial products, most effective for
treating Gram-positive bacteria, such as β-lactams, aminoglycosides,
tetracyclines, and macrolides (Fig. 1). Methicillin was introduced in
1959 and methicillin-resistant S. aureus (MRSA) was identified in 1961
[15] (Fig. 1). With the advent of antimicrobial resistance increase from Fig. 2. Rate of production of new antibiotic molecules in the last decades.

Fig. 1. Timeline of antibacterial resistance phenomena.

2
Y.A.S. Guevara et al.
2.2. Why did new antibiotic production fall to the detriment of other
drugs? And what are the consequences of that fact?
In recent decades, the discovery and development of new antibiotics
have slowed dramatically due to scientific barriers, regulatory chal­
lenges, and declining returns on investment, leading major pharma­
ceutical companies to scale back or abandon drug procurement research
antibiotics and focus on research areas with better financial prospects
[26,35,39,58] such is the case of AstraZeneca in 2016 and Novartis and
Sanofi in 2018, which in certain places chose to invest in the research of
new drugs for chronic diseases such as diabetes and cancer that guar­
antee better dividends [12,35].
Finding novel antibacterial molecules is the challenge, and since
daptomycin and linezolid were launched in the 1980s, only five non-
derivative antibiotics have been approved by the FDA since 2000
(Fig. 1) [12,30]. In this context, most current antibiotic development
programs primarily focus on modifying existing classes of drugs
discovered decades ago to circumvent bacterial resistance [39]. An
important reason for the lack of innovation is the absence of a global
market that guarantees the sustainable income necessary to finance
research, commercialization, and production at scale [4,46]. In this
context, the preclinical stages of antibiotic R&D are the riskiest, ac­
counting for about 45% of total costs. Although the academic sector can
promote it, the association with external financiers, such as the phar­
maceutical industry [35]. Unfortunately, this only comes to fruition
after the nomination of extensively validated preclinical candidates, and
often even these companies require phase I clinical data [28].
In line with the causes of the “market failure” of antibiotics
mentioned above, bring up the scientific challenges for the discovery
and development of new agents and the regulations imposed by the FDA
during the last two decades in clinical trials, which, in the opinion of
experts, have been taken to irrational extremes based solely on statistical
considerations [43]. In this context, pharmaceutical companies have
stopped producing new antibiotics, a determination that has been
reinforced by the availability of multiple low-cost generic antibiotics
[38,57] and the adoption by control entities, from the principle of
administration, aimed at reducing the general use of antibiotics in the
hospital setting [26] which ends up impacting the sales of new antibi­
otics [3,43]. Also, if that new agent shows promising results but retains
some toxicity, it may never see enough patients in a trial, and the
therapeutic index may not be well known [58].
Classically, most antibiotics were produced by screening soil mi­
croorganisms Waxman platform. Still, this limited resource of cultivable
bacteria was outgrown in the 1960s, and synthetic approaches to pro­
ducing antibiotics have failed to replace it [25,54]. The pharmaceutical
industry responded by replacing the Waksman platform with a new
chemical modification strategy that unfortunately did not produce the
expected results, causing the need for antibiotics and, in particular, for
natural products to decrease [16,57], such disenchantment with natural
products as sources of new drugs, lack of success in the use of synthetic
compounds exacerbate the growing crisis in antibiotic drug discovery
that threatens modern medicine [50,58].
However, with advances in next-generation genome sequencing,
bioinformatics, and analytical chemistry, attempts are being made to
advance the renaissance of the antimicrobial industry to address the
current crisis [57]. Thus, more than 1200 antimicrobial peptides (AMPs)
of various origins have been discovered in recent years. Unfortunately,
none of them have been used as antibiotics [12]. Since the Infectious
Diseases Society of America applied for approval of 10 new systemic
antibiotics by 2020 in the United States, there have been six approvals:
ceftaroline, dalbavancin, oritavancin, tedizolid, ceftolozane-tazoba
ctam, and ceftazidime-avibactam [14,26], however, many of them
have not been commercialized in the main markets, due to the cost of
registration and commercialization, which are unaffordable by low- and
middle-income countries, which have the highest burden of AMR [5,31,
34]. The previous becomes especially concerning in the case of
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Microbial Pathogenesis 179 (2023) 106108
Gram-negative bacteria such as Pseudomonas aeruginosa and Acineto­
bacter baumannii, for which treatment options are already limited. This
has raised fears of a “post-antibiotic era,” as it has been estimated that
between 5 and 20 new antibacterial drugs must enter clinical develop­
ment to effectively address the current resistance problem [19].
Because of the abandonment by pharmaceutical companies in the
production of new antibiotics and the growing phenomena of AMR, it is
predicted to result in the death of 10 million people by 2050 and
cumulatively incur at least $100 trillion in hospital costs and produc­
tivity losses [8,17,52]. Another problem that arises from the misuse of
antibiotics is the selective pressure on bacteria, resulting in new modi­
fied strains that seem to reduce the chance that treatments are
adequately effective in patients, for example, compromised by HIV im­
munity, cancer, surgical procedures, and diabetes; causing profound
consequences that lead to morbidity and mortality or clinical compli­
cations [20,45]. Although this is considered a natural process, incom­
plete knowledge about such a catastrophe triggered the AMR rate [3,
29]. In this context, the emergence of the ESKAPE pathogens group, for
example, is the leading cause of nosocomial-resistant infections world­
wide, many of which lack clinical treatments and represent currently
deadly bacteria with fast-growing multidrug-resistant properties [30,
53].
2.3. Is the production of new classes of antibiotics economically viable?
One of the main problems when it comes to innovating in the pro­
duction of new classes of antimicrobial drugs is the economic return. The
challenge involved in the search for new molecules with the desired
pharmacological characteristics, added to the well-intentioned regula­
tory policies on using this type of pharmaceutical agent, among other
factors, paints a bleak picture for investors in large companies. Since the
new and effective antibiotics are left as a last resort to prevent the
development of AMR, combined with low market prices conditioned in
part by those established for generics, it leads to unsustainable profits
[4].
In clinical trials, antimicrobials are generally evaluated for non­
inferiority, rather than superiority, compared to existing treatments, so
they may not be cost-effective in traditional tests, where the new anti­
microbial is associated with additional cost and a comparable health
result, disregarding the beneficial impact at the population level in
terms of combating AMR [17]. On average, it takes $985 million and
10–15 years to bring a new drug to market, and the value of a new
antibiotic ranges from $486 million to $12 billion [26,30]. A 2017 es­
timate puts the cost of developing an antibiotic at around $1.5 billion;
meanwhile, industry analysts estimate that the average revenue gener­
ated from the sale of an antibiotic is approximately $46 million per year
[35].
These estimates are made using the so-called Net Present Value
(NPV), a standard method companies use to prioritize investment stra­
tegies to calculate the value of a drug over a given period. Recent data
put the NPV of an antibiotic at a negative $50 million, meaning losses to
developers, to which additional costs per patient must be added due to
the AMR phenomenon that ranges between US$5 and more than US
$55,000 [38].
This financial reality for antibiotic research largely explains the fact
that while there are nearly 4000 immuno-oncology agents in develop­
ment, only about 30–40 new antibacterial compounds are currently in
the development phases and clinical trials, and none of them is poten­
tially active against bacteria of the ESKAPE group [28].
Given this scenario, fiscal incentive policies are necessary from the
private or state sector [38]. Multiple academic studies have estimated
that the incentives to use a cost-based approach range from $1 billion to
$5 billion per new antibiotic [32].
Boluarte & Schulze (2022) [4], using data collected from other
antibiotic-specific models and refined with industry experts, calculates a
minimum incentive for the first antibiotic on the market between $2
Y.A.S. Guevara et al.
billion and $3 billion, paid over five to ten years. Fortunately, there are
signs that the industry is beginning to respond to the economic problem
of antibiotics. The International Federation of Pharmaceutical Manu­
facturers and Associations announced the AMR Action Fund involving
24 companies, which aims to bring two to four new antibiotics to market
by 2030 and has committed nearly $1 billion to support needed research
to achieve this goal [35].
2.4. What are the technical difficulties in producing new antibacterial
compounds?
When addressing the development issues of new antibiotic molecules
from a merely technical approach, scientific barriers emerge as pro­
tagonists, making it necessary to change some of the paradigms that lead
to more interdisciplinary fundamental research. The document: A sci­
entific road map for antibiotics discovery (2016) emphasizes the need
for specific training and experience and a mechanism to transfer
knowledge from industry to scientists to materialize the initiatives [1].
Improvements to FDA guidelines are also needed for the execution of
controlled clinical trials with antibiotics that, given the inconvenience of
being executed involving a placebo, entail the need to use extensive
samples and, consequently, high costs, discouraging research [57].
Microbial natural products, with their privileged properties of mi­
crobial penetration and affinity for bacterial targets, may lead to a new
era of antibiotic discovery [10,57]. A hallmark of natural antibiotic
products is their chemical diversity and complexity. They are based on
producing chemical scaffolds consisting of a peptide, polyketide, car­
bohydrate, alkaloid, or terpene backbone that serve as the core structure
[47]. Despite these characteristics, pharmaceutical companies have
gradually set aside natural compounds for obtaining antibiotics, given
that they may not be suitable as drugs once purified. In addition, the
chemical structure of the leading natural product requires
semi-synthesis, which increases the cost and detracts from the attrac­
tiveness of natural products [57].
Exploring non-cultured bacteria could revive the Waxman Platform
for discovering antibacterial of natural origin [25]. It is more interesting
to investigate, under rational design, synthetic compounds. The rational
design of drugs consists of the empirical synthesis of new molecules
designed according to several rules (Lipinski rules) so that they are well
absorbed, non-toxic and active against a specific target [12]. Although
Lipinski’s rule does not apply to antibiotics, most pharmaceutical
companies have not developed separate synthetic chemical libraries
with microbial physiology in mind, making it challenging to develop
similar rules for synthetic compounds that are effective as antibiotics
[57].
A more fundamental problem particular to antibiotic discovery is the
need for a better understanding of the scientific drive to build a suc­
cessful library. Pharmaceutical companies have extensive collections of
compounds that can be used as starting materials to find chemicals that
are active against newly identified biological targets. Given the unique
characteristics of antibiotics, the goal of this effort is not to find and
harvest new sources of natural products or build vast libraries of syn­
thetic compounds but rather to generate and selectively modify chem­
ical matter suited for the discovery of new antibiotics [1].
One of the main challenges of the antibiotic industry is the AMR
phenomenon. The last 20 years have seen a severe withdrawal of many
pharmaceutical companies from research and development of antimi­
crobial drugs targeting AMR strains [18]. A particular drawback is
Gram-negative bacteria, most of which have built-in abilities to evade
antibiotics and develop resistance; for example, they have two mem­
branes with orthogonal characteristics, which makes it difficult to
design drugs that can penetrate both barriers, along with a variety of
efflux pumps that push drugs out of the cell, making it difficult to design
new antibiotics [1]. Thus, understanding the orientation and binding of
LPS (lipopolysaccharide) molecules to the outside of the outer mem­
brane could facilitate the development of cationic molecules to break it.
4
Microbial Pathogenesis 179 (2023) 106108
In the same context, the drug’s ability to penetrate the outer mem­
brane and its susceptibility to efflux mechanisms must be tracked
throughout the drug optimization process to develop new antibiotics
successfully [19]. Currently, work is being done to target new cellular
mechanisms with no known resistance and develop structure-based
“rules” to improve the permanence of the antibiotic inside the path­
ogen, especially in Gram-negative bacteria [30,49].
Another way to reduce the limitations in the search for new antibi­
otics is to change the pharmaceutical industry’s main focus, concen­
trating on a relatively challenging task of hunting for broad-spectrum
novel antibacterial agents [57]. Expanding the scope of antibiotics to
include narrow-spectrum molecules, syncretic combinations, and viru­
lence inhibitors offers an exciting prospect for new drug discovery, along
with identifying new antibiotic chemical scaffolds that evade resistance
mechanisms and continue to develop, exploring non-traditional anti-­
infective strategies [58].
2.5. What are the advances and perspectives in developing new
antibacterial drugs?
Despite the limitations and drawbacks in recent years in obtaining
new antibiotic drugs, several approaches have been proposed and
implemented to overcome such limitations. Various methods for culti­
vating organisms that had not been cultivated until then, the rational
approach to drug design to improve already known molecules, and the
promotion of technology to obtain synthetic products; have resulted in
obtaining molecules with acceptable pharmacological characteristics.
These efforts have resulted in the achievement of drugs with proven
antimicrobial activity. Thus, for example, from the metabolites of acti­
nomycetes, walkmycin B, Validamycin and signamycin B were isolated,
with new mechanisms of action such as histidine kinase inhibitors; since
rational drug design, more than 10 million new molecules have been
synthesized. However, few active molecules have reached the market,
particularly anti-tuberculosis drugs [12] such as tubelactomicin A and
caprazamycins, and amycolamycin was identified as an antibiotic of
Staphylococcus aureus resistant to anti methicillin. These compounds’
discovery encourages the search for more natural products active
against antimicrobial-resistant species [18].
Developing new antibiotics to treat life-threatening infections with
bacteria resistant to existing antibiotics is highly prioritized [51]. In
2016, AMR became only the fourth health issue after HIV, non­
communicable diseases, and Ebola to be discussed by the United Nations
General Assembly [38]. Faced with the challenge of AMR, antibiotics
that use novel mechanisms to combat antimicrobial resistance are
needed.
Teixobactin represents a new class of antibiotics with a unique
chemical scaffolding and no detectable resistance, interfering with lipid
II, a peptidoglycan precursor [40]. The said molecule, obtained through
the cultivation of hitherto uncultivated organisms, inhibits peptido­
glycan synthesis with excellent activity against Gram-positive patho­
gens, including resistant strains [25]. Thus, these new methods to
activate silent biosynthetic gene clusters using co-culture and to grow
‘non-culturable’ bacteria have revitalized natural products as a source of
antibiotics [30]. On the other hand, in the race against the AMR phe­
nomenon, it is required that scientists working on the discovery of an­
timicrobials be allowed greater participation in decision-making
networks within commercial funding sources so that their appreciation
is hated and taken into consideration [28]. The following steps must
include joint action by the human and animal health sectors; addressing
antimicrobial resistance is complex. The formal tripartite of WHO, the
Food and Agriculture Organization, and the World Organization for
Animal Health must significantly identify and promote feasible strate­
gies to tackle AMR [21].
New genomic technologies, including rapid and low-cost DNA
sequencing and a growing understanding of the mechanical details of
natural product biosynthesis, along with innovation in chemical
Y.A.S. Guevara et al.
synthesis and analytical methods, can be combined to expand bioactive
chemical diversity in a fashion that is compatible with high-throughput
technology and begin to overcome the innovation gap in antibiotic
discovery [47]. Another critical approach implemented in obtaining
new antimicrobial products is the modification of already known mol­
ecules; thus, cefiderocol was developed from cephalosporins, and
Terramycin was obtained from modifying the aminoglycoside sisomicin,
both approved by the FDA for urinary infections [12].
The known antibiotic activity of natural products has generally been
identified by phenotypic screening campaigns that determine activity
against panels of test organisms in standardized assays. The basis for
bioactivity-guided isolation of natural products from complex mixtures,
these screens efficiently recover bioactive compounds when evaluating
crude extract libraries [28]. Consequently, using natural products and
their derivatives has evolved as a promising approach to combat
drug-resistant microbial strains. Furthermore, these natural products,
characterized by the presence of novel structures and mechanisms of
action, may guide the development of new potential chemotherapies
[18].
To fully exploit the privileged nature of natural products in antibiotic
drug discovery, the ability to rapidly identify scaffolds, tailor them using
a library of modifying enzymes and rapidly purify the resulting com­
pounds in sufficient quantity to generate libraries of molecules suitable
for high throughput screening. The development of synthetic biology
offers an excellent opportunity to address these problems [47]. Despite
these advances, less than 0.1% of the 28,000 natural product antibiotics
discovered in the last few decades are in clinical use. Many of the
remaining 99.9% will never be suitable as drugs due to intractable ef­
ficacy problems, toxicity, stability, etc. [58].
While a molecule may be active in a target-based assay, it will ulti­
mately fail to progress if it cannot reach its target in sufficient concen­
trations. Poor accumulation can be due to low drug cell wall penetration,
rapid removal of the drug from inside the cell via efflux pumps, or in the
worst-case a combination of both [30]. Intending to remedy the problem
of shortage of new antibiotics, faced with the AMR challenge, the in­
dustry responded by focusing on synthetics, and some high-tech ap­
proaches came together to form the new rational
design/genomics/combichem/HTS platform.
Conserved essential proteins identified through genomics were sup­
posed to serve as targets for HTS and rational design, producing new
antibiotics. However, it did not perform as expected, with oxazolidi­
nones being the only new class of synthetic compounds developed in
recent years [23]. Over the last decade, considerable progress has also
been made in identifying gene products that are essential for bacterial
physiology and pathogenic attributes. As a result, there have been
numerous suggestions for providing new targets or antibiotics. Howev­
er, there is a considerable gap between identifying an essential bacterial
factor and the inhibitors that can form the basis for developing a new
drug [19].
Various innovative and complementary technologies are required to
improve access to new product scaffolds for new antimicrobials. Thus,
computational tools could provide powerful help at different levels, with
artificial intelligence playing a revolutionary role in identifying new
drug candidates [28]. Fortuitously, the renewed interest in natural
products as drug leads is concurrent with the development of new
technologies in genomics, bioinformatics, analytical chemistry, and
chemical synthesis that enable a new assessment of the compatibility of
natural products with modern drug discovery. Synthetic biology can
play a leading role in this area. It holds real promise to help overcome
some challenges for increased integration of natural products in anti­
biotic drug discovery [47].
Together with the technical improvements in the research of new
antibiotics, a greater state and private investment is required. In this
sense, progress has been made, with the proposal of almost 50 incentive
strategies to increase the number of effective antibiotics. The two most
recent large-scale efforts in this area are in the final planning stages.
5
Microbial Pathogenesis 179 (2023) 106108
The first of these efforts, the Infectious Disease Finance Facility, is a
financing mechanism proposed by the European Investment Bank and
the European Commission and the second is a summary published by
Review on Antimicrobial Resistance, which seeks to identify the charac­
teristics of an optimal incentive strategy to promote the private devel­
opment of more antimicrobial compounds. Both potentially offer
valuable contributions to address the current crisis [17]. These and other
efforts that will consolidate future projects for the identification and
development of antibiotics require the implementation of a legal
framework for intellectual property that is agile and dynamic to reduce
the time needed to negotiate property agreements between the invent­
ing institutions so as not to discourage the industry from granting
licenses for the marketing of the new drug [28].
Finally, to determine our position concerning developing new anti­
microbials, as of November 2021, 80 products (46 antibiotics and 34
non-traditional antibacterial agents) were in clinical development,
including in the pre-registration phase [56]. They include 28 antibiotics
and 21 non-traditional antibacterial targeting priority pathogens, 13
antibiotics and 1 non-traditional antibacterial targeting M. tuberculosis,
and 5 antibiotics and 12 non-traditional antibacterial targets C. difficile
(Fig. 3). In the following link, you can find a table with detailed infor­
mation on these new antibiotics, discriminating by type and name of the
product, class of antibacterial, clinical trials, developer, therapeutic in­
dications, route of administration and study phase.
3. Antimicrobial peptides
These AMPs presented many biological activities in defense against
pathogens and thus medical applications based on antimicrobial and
anti-larvicidal activities [4], anti-HIV activities [17], inhibitory activity
against α-amylase and trypsin activity, redox activity [4],
ribosome-inactivating activity [19].
Molecules of natural origin have shown excellent characteristics as
antibacterial drugs; many are plant metabolites. They also offer other
possibilities as sources of molecules with antimicrobial potentials, such
as AMPs, mainly thionins, plant defensins, and cyclotides [62,63] rich in
cysteine and disulfide bonds subsequently used as their primary defense
against microbial infections [61]. AMPs are categorized according to
their structure, sequence, or mode of action, such as bacterial killing,
immunological regulation, inhibiting biofilm formation, or anti-cancer
and anti-viral function [64]. α-Helix, β sheets, and extended/random
coil peptides are three different forms of their secondary structures [65,
66].
3.1. Classes of AMPs
Antimicrobial peptides (AMPs) occur naturally in all living organ­
isms, such as bacteria, fungi, plants, and animals, and compose the first
line of defense innate immunity from animals and plants against path­
ogens [83,84]. AMPs are cationic amphipathic, have 10 to 50 residues,
and are classified into several groups according to sequence, 3D struc­
ture and function [59]. While in more complex organisms, AMPs play a
critical function in natural immunity and help protect the host from
diseases. Bacteria eliminate other bacteria that threaten their ecological
niche [60].
AMPs are classified in thionins [4,7,9,10], defensins [11,12],
hevein-like peptides [13–15], knottins [15–18], stable-like peptides [19,
20], lipid transfer proteins [21–23], snakins [24–28], and cyclotides
[29–32]. These peptides are mainly found in seeds, leaves, flowers,
fruits, and other plant tissues. These AMPs range from 29 to 54 amino
acid residues, the residues being the thionins, the larger and cyclotides,
the smaller ones. Additionally, all these molecules were classified into
subgroups and share many conserved cysteine residues involved in di­
sulfide bonds leading to higher stability due to temperature, pH, and
proteolytic degradation [11–18]. The three-dimensional is represented
by α-helix, parallel and antiparallel β-sheets, coiled, extended, and
Y.A.S. Guevara et al.
Fig. 3. Antibacterial products in clinical development for priority pathogens WHO (2022). The data visualization shows the numbers of antibacterial products
by type (chart A.1), category of non-traditional products (chart A.2), pathogen category and phase of clinical development (chart A.3), expected activity against
priority pathogens (chart B), innovativeness (chart C.1) and if a novel chemical entity is involved or not (chart C.2). accessed on Pipeline of antibacterial products in
clinical development (who.int).
disordered structures.
3.1.1. Thionins
Thionins are 45–48 amino acid residue peptides with and molecular
weight of around 5 kDa [4]. According to amino acid sequence homol­
ogy, 3D structural and disulfide bridge positions can be classified into
five types (I–V) [12]. Several studies have reported the broad-spectrum
antimicrobial activity of thionins, such as antibacterial [14,16]. Taveira
et al. [16] reported that NsW1 and NsW2 purified from Nigella sativa
presented an attractive antibacterial potential against Bacillus subtilis
and Staphylococcus aureus. NsW1 and NsW2 increased membrane
permeability, and atomic force microscopy revealed a change in the
ultrastructure of cells.
3.1.2. Defensins
Defensins are AMPs widely expressed in live organisms, and they are
nontoxic molecules extremely active against bacteria [19]. Defensins
have 45–54 amino acid residues and β-sheets. An α-helix 3D structure is
supported by four disulfide bridges, making them resistant to proteolysis
and stable to extremes of pH and temperature. These molecules have a
variety of biological functions, such as inhibiting microbial growth and
inhibiting α-amylase and trypsin activity [4].
Defensins are known based on their antimicrobial activity selectively
against bacteria at deficient concentrations. For example, The Dm-
AMP1, a defensin from Dahlia merckii, presented an IC50 at 15 μg
mL− 1 for Bacillus subtilis [21]. Other defensins, such as VuDef2 from
Vigna unguiculata, inhibit Staphylococcus aureus at 15 μg mL− 1 [22].
PaDef, a defensin from avocado (Persea americana var. drymifolia), had
potent antibacterial activity against multidrug-resistant Escherichia coli
and S. aureus. The mechanism of antibacterial action is based on the
destabilization of the bacterial membrane. The most important infor­
mation about the mechanism of defensin’s antibacterial action came
from Velivelli et al.’s study [23]. In vitro, MtDef5 induced membrane
permeability and inhibited bacteria’s DNA replication and gene
6
Microbial Pathogenesis 179 (2023) 106108
expression [25]. The authors used as a model the MtDef5, purified from
Medicago truncatula [23]. In this study, authors employed site-directed
mutagenesis in MtDef5 at the γ-core motif, changing the cationic resi­
dues. A defensin, as a model. The mutated MtDef5 lost antibacterial
activity, revealing this region is important for antibacterial activity.
3.1.3. Hevein-like peptides
Hevein-like peptides are basic 29–45 amino acid residues peptides.
These peptides were first isolated from Hevea brasiliensis [27]. This
group has the chitin-binding domain, antifungal activity and antibac­
terial activity. These peptides have a 3D structure comprising three
antiparallel beta-sheets and a short α-helix, stabilized by 3–5 disulfide
bonds [28]. Many studies focus on the antibacterial potential of
hevein-like peptides, all regarding antimicrobial activity against phy­
topathogens [30], which is not the focus of this review.
3.1.4. Knottins
Knottins are 30 amino acid residue peptides, including α-amylase,
carboxypeptidase, and trypsin inhibitors [31]. These molecules were
first discovered in the 1980s and have an interesting 3D structure with
disulfide bridges conferring high thermal stability. Knottins are named
“promiscuous peptides” because they present a lot of biological func­
tions, including antibacterial activity [31]. They can interact with ion
channels in membranes and cell membranes and acid-sense channels.
Those that target membranes can pass through and intracellular attack
targets, such as carboxypeptidases, promoting resistance. They also have
antimicrobial activity against bacteria, fungi, viruses and insects [32].
Knottins present an amphipathic ability with cationic and hydrophobic
portions, an essential feature to interact with membranes [33].
3.1.5. Stable-like peptides
Stable-like peptides are small peptides with a typical Cys motif
(XnC1X3C2XnC3X3C4Xn), where X is any amino acid residue except
cysteine) forming a helix-loop-helix structure [34]. These peptides
Y.A.S. Guevara et al.
present a highly conserved 3D structure in a staple-like form. Stable-like
peptides present a broad spectrum of biological activity, including
antibacterial [34]. Many studies of stable-like peptides focused on their
biotechnological application in clinics based on their antibacterial ac­
tivity. These peptides have high activity against E. coli [36]. The
mechanisms of action behind the stable-like peptides did not involve
damage to cell membranes. In contrast, the peptide binds to DNA,
inhibiting RNA and protein synthesis, producing a bactericidal effect
[36].
3.1.6. Lipid transfer proteins (LTPs)
LTPs are positive-charged peptides with a low molecular weight
(7–10 kDa) [37]. The 3D structures are pockets, with eight cysteines
forming four disulfide bonds stabilizing five α-helices [38]. The helices
provide a hydrophobic spot essential to bind to lipids, including fatty
acids, phospholipids, prostaglandin B2, hemolytic derivatives, and
acyl-coenzyme A, involved in antibacterial activity [39].
Regarding the activity of LTPs against human bacteria, CmLTP iso­
lated from Chelidonium majus was active against Campylobacter jejuni,
Listeria greyi and Clostridium perfringens [41–43]. The mechanism of
antimicrobial activity is based on the transfer of lipids in the membrane,
leading to pore formation and cell imbalance.
3.1.7. Snakins
Snakins are a class of peptides with cysteine residues involved in
disulfide bonds contributing to stability and biological activities. The
general 3D structure of snakins is composed of an N-terminal signal
peptide, a variable site, and the C-terminal region [44–47].
These peptides can inhibit the growth of bacteria such as Micrococcus
luteus and Staphylococcus cohnii. Snakins present prominent bactericidal
activity against Gram-negative bacteria. Furthermore, due to their
ability to agglutinate microorganism cells, studies have shown that
snakins can help to prevent the spread of pathogens in damaged tissues
[48].
3.2. What are antimicrobial peptides (AMPs)? and could they contribute
to the fight against AMR?
Multi-resistant bacteria have become a global public health problem,
and introducing new antibiotics in the clinical phases is necessary. In
this context, AMPs have gained attention as a potential future thera­
peutic candidate [67]. They play a crucial role in the body’s natural
defense and immune systems, which carry out a variety of efficient
methods to eliminate pathogens [68]. Numerous antimicrobial effects
demonstrated by AMPs include disruption of cell membranes, inhibition
of protein and DNA synthesis, and suppression of crucial cellular pro­
cesses such as protein folding, cell wall formation, and metabolic turn­
over [69].
Some studies have shown the beneficial effects of AMPs in treating
bacterial infections. For example, the VK25 peptide from the plasma of
the Komodo dragon (Varanus komodoensis) was used as a template to
create a novel, short AMP, DRGN1, effective against P. aeruginosa and
S. aureus biofilms of both single and mixed species. Pexiganan has
finished the phase 3 clinical trials for treating DFUs with bacterial in­
fections (NCT01590758). Additionally, several AMPs have been effec­
tively created for commercial and pharmaceutical applications [70].
On the other hand, synthetic AMPs (SAMPs) constitute another
promising alternative to treating bacterial infections. The two goals of
designing synthetic peptides from natural AMP sequences are to (1)
extract the better component, which has antimicrobial activity, and (2)
exclude the worse part, which is poisonous and has low proteolytic
resistance [71]. With the help of in silico technology, specific amino
acids can be modified, and new AMPs can be designed and tested for
enhanced antibacterial activity [72]. For improved antibacterial activ­
ity, some of these alterations include the addition of hydrophobic and
cationic residues; for increased in vivo stability, other modifications
7
Microbial Pathogenesis 179 (2023) 106108
include D-amino acids, cyclization, acetylation, and peptidomimetics
[73,74].
Synthetic peptides are typically more effective than natural peptides
since they exert more antimicrobial activity at lower concentrations
than their native natural peptides. For instance, the natural AamAP1
from Androctonus amoreuxi exhibits antimicrobial action against
Candida albicans, Escherichia coli, and Staphylococcus aureus [75]. The
synthesized form of AamAP1, AamAP1-Lysine, is 4–20 times more
effective than its natural form and has antimicrobial activity against the
same pathogens at concentrations as low as 5–7.5 μM [76]. In another
example, radish (Raphanus sativus L.) natural peptide Rs-AFP2 exhibits
action against Fusarium culmorum.
According to the Data Repository of Antimicrobial Peptides
(DRAMP) database, until now, only 77 peptides have been created by
pharmaceutical companies [77]. Other AMPs used in clinical settings to
treat bacteria include those that work against Gram-positive bacteria
like daptomycin, oritavancin, dalbavancin, teicoplanin, telavancin and
gramicidins, against Gram-negative bacteria like polymyxins (cyclic
peptides from Paenibacillus polymyxa) [78]. Successful SAMPs should
typically have apparent efficacy and be superior to conventional ther­
apies. However, SAMPs’ advantage of causing less microbial resistance
than traditional antibiotics is not considered, a feature that should not
be disregarded (reviewed by [79]).
3.3. Synergy between AMPs and antibiotics
In addition to killing multidrug-resistant (MDR) bacteria like MRSA
and P. aeruginosa, several previously identified AMPs are employed with
traditional antibiotics. Combining two antibacterial medicines results in
better efficacy than using either separately, suggesting a synergistic
interaction between them. Clinically, this combinational use is signifi­
cant because it offers more alternatives for treating challenging in­
fections [80]. Additionally, AMPs have a more comprehensive range of
targets than most commonly used antibiotics, making their synergistic
beneficial combination in various contexts [81]. As a result, there is a
significant therapeutic promise for the clinical use of AMPs in combi­
nation with other antimicrobial drugs, such as traditional antibiotics.
In some cases, combining AMPs with antibiotics enhances their
antibacterial and cytotoxic effects. For instance, when combined,
gentamicin and AMP PG-1 have a synergistic cytotoxic impact on human
neutrophils. Therefore, effective combinations should be carefully cho­
sen to find the best synergistic combination of AMPs and antibiotics to
boost the antibacterial effects [82].
4. Conclusion
The current antibacterial crisis reveals a bleak scenario that many
experts classify as one of the worst health catastrophes that humanity
can suffer. Even though we already face problems resulting from AMR,
which may worsen in the long run. For this reason, it is vital that science,
especially pharmaceuticals, lean towards research on new therapeutic
alternatives. In this context, antibacterial peptides, given their differ­
entiated characteristics, are emerging as a tool of particular interest in
combating multi-resistant bacteria. Both clinical and preclinical studies
conducted with these molecules show their antibacterial therapeutic
potential significantly when they are improved under a rational design
scheme to reduce their toxic effects. It is expected that, with techno­
logical advances, especially with the advent and adoption of artificial
intelligence, there will be an increase in diversified synthetic peptide
production, which can face the challenges that we have in terms of
antibacterial drugs.
CRediT authorship contribution statement
Yeimer A.S. Guevara: Writing – review & editing, Writing – original
draft, Funding acquisition, Formal analysis, Data curation,
Y.A.S. Guevara et al.
Conceptualization. Maria H.C. Santos: Writing – review & editing,
Writing – original draft, Data curation, Conceptualization. Francisco I.
R. Gomes: Writing – review & editing, Writing – original draft, Funding
acquisition, Formal analysis, Data curation, Conceptualization. She­
heryar: Writing – original draft, Formal analysis, Data curation,
Conceptualization. Felipe P. Mesquita: Writing – original draft, Visu­
alization, Validation, Data curation, Conceptualization. Pedro F.N.
Souza: Writing – review & editing, Writing – original draft, Visualiza­
tion, Supervision, Project administration, Methodology, Investigation,
Funding acquisition, Formal analysis, Data curation, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments and Funding
The authors are grateful to CNPq for providing funding for this work.
Pedro F. N. Souza is a senior researcher from CNPq at process number
305003/2022–4.
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